University of Barcelona, Spain

Abstract Title

A new small molecule binds directly to oncogenic KRAS increasing downstream KRAS signalling while reducing colorectal cancer cells (CRC) viability

KRAS is one of the best-known oncogenes with the highest mutation rate among all cancers and is associated with a series of highly lethal tumours, including pancreatic, lung and colorectal cancers. The identification of tumour driven genes and the development of specific inhibitors
have revolutionised cancer treatment strategies. Numerous clinical results have shown that targeted therapies significantly extend progression-free survival and are less toxic than standard chemotherapy. KRAS has always been considered as a therapeutic target in cancer but until now only two compounds that inhibit one specific KRAS mutation have been approved for clinical use.
By using molecular dynamics and docking analysis, we found a new compound (P14B) that stably binds to a druggable pocket near the α4-α5 helices of the allosteric domain of KRAS. This region had been previously identified to bind calmodulin (CaM). Using surface plasmon resonance and pulldown analyses, we proved that P14B binds directly to oncogenic KRAS thus competing with
CaM. Surprisingly, P14B favoured oncogenic KRAS interaction with BRAF and phosphorylated CRAF and increased downstream Ras signalling in CRC cells expressing oncogenic KRAS.
Interestingly, the viability of these tumoral cells, but not that of normal cells was impaired by P14B treatment. In order to analyse a potential relevance for cancer treatment, Patient Derived-CRC Organoids (PDCOs) were established with the aim to recapitulate the architecture, physiology, and underlying genetic signature of primary tissue. A preliminary result has obtained which showed that P14B reduced the viability of PDCOs while normal PDOs seems to maintain their viability upon P14B treatment. These data support the significance of the α4-α5 helices region of KRAS in the regulation of oncogenic KRAS signalling and demonstrate that drugs interacting with this site may drive CRC cells to death by increasing oncogenic KRAS downstream signalling.



The Christie NHS Foundation Trust, UK

Abstract Title

A Functional and Clinical Analysis of KRAS Codon 12 and 13 Mutations in Non-Small Cell Lung Cancer.

KRAS is the most commonly mutated oncogene in non-small cell lung cancer (NSCLC), with the majority of mutations found at codons 12 (G12), 13 (G13) and 61 (Q61). Newly developed KRAS-G12C and G12D inhibitors have highlighted the need to investigate unique molecular and functional mechanisms of specific allelic subtypes.

We developed a panel of doxycycline-inducible isogenic cell lines (MLE-12: alveolar type II cells), expressing FLAG-tagged KRAS WT, G12C, G12D, G13C and G13D. This model was characterised in 3D via proliferation, viability and signalling assays. To investigate KRAS-addicted cells a panel of “RASless” mouse embryonic fibroblasts (MEFs) with lentiviral transduction of KRAS WT, G12C, G12D and G13D was evaluated. For clinical validation, we developed an international clinical database and explored cBioPortal to interrogate co-mutational frequencies.

The KRAS initiation model (MLE-12) identified an increase in proliferation with KRAS-G12 expression vs. a plateau observed with KRAS-G13, similar changes in viability were identified. Signaling differences were found with a reliance on PI3K-AKT-MTOR signalling in KRAS-G12D. In KRAS addicted MEFs similar differences were detected in proliferation and signalling. Therapeutic vulnerabilities to a novel active-state selective RAS (‘ON’) inhibitor (RM-042) was assessed in the KRAS addicted model, where the most and least sensitive RAS mutant cells were those expressing G12C or G12D, respectively. Inhibitors targeting the PI3K-AKT-MTOR pathway demonstrated strongest reduction of viability in KRAS-G12D expressing cells. Analysis of clinical data sets revealed an increased co- mutational rate in KRAS-G13 mutant samples with increased rates of NF1, TP53, KEAP1, and STK11  co-mutation.

Our findings highlight increased oncogenic potency of KRAS-G12 mutations across two isogenic models, and distinct signalling differences and vulnerabilities to targeted inhibitors relative to G12C and G13 mutations. Clinical data revealed an increased co-mutational rate in KRAS-G13 samples consistent with weaker oncogenicity. Kinome profiling will allow us to further characterise these mutations in NSCLC.



Spanish National Cancer Research Centre (CNIO), Spain

Abstract Title

Identifying RAF1 degraders as a therapeutic approach in KRAS-driven lung tumors

RAF1 ablation in mouse models with KRAS-driven lung adenocarcinoma promotes tumor regression without arising into significant toxicities. Cryo-EM analysis of RAF1 has revealed that it forms a complex with HSP90 and CDC37, known as the RHC complex, which is crucial for RAF1 stability. Since RAF1 has emerged as a promising therapeutic target, both the purified protein and its atomic structure have proven to be invaluable tools for early drug discovery.

In order to identify molecules that can disrupt the RAF1-CDC37 interaction, in silico studies were conducted. These studies revealed a molecular pocket in the interaction site that could potentially be targeted by small molecules to prevent the interaction.

To experimentally test the potential compounds, an in vitro and in cellulo screening platforms were designed and developed. The first one is based on the homogenous time-resolved fluorescence (HTRF) technology which allows the detection of the interaction between the two proteins by adding a mixture of two labeled antibodies to the purified RHC complex. To detect both molecules capable of disrupting the complex and preventing RHC complex formation, an additional strategy based on separating the 3 components of the RHC complex with mild conditions of urea was designed. The second platform developed can be used in cells and involves the fusion of NanoLUC® protein to RAF1, allowing the detection of RAF1 degradation through luminescence measurements. This platform provides a more physiologically relevant environment for testing the potential compounds.

In summary, in silico studies have identified potential compounds that can impair the RAF1-CDC37 interaction. These compounds will be assesed using two screening platforms designed to test them in the purified complex and directly in cells. By targeting the RAF1-CDC37 interaction and promoting RAF1 degradation, these compounds hold promise as a strategy for treating KRAS-driven cancers.



Instituto de Biología Molecular y Celular del Cáncer, Spain

Abstract Title

RAS-PI3K signalling in CAFs promotes activation of mechanotransduction programs to promote lung cancer progression

Cancer-associated fibroblasts (CAFs) play a crucial role in cancer progression by modulating the tumor microenvironment and promoting tumorigenesis. The mechanical properties of CAFs, in particular, have emerged as essential factors in tumor development. Among the key signaling
pathways in CAFs, the YAP (Yes-associated protein) pathway has gained attention due to its involvement in mechanotransduction and cellular response to extracellular matrix (ECM) stiffness.

Our data indicate a pivotal role for RAS-PI3K signaling in regulating YAP expression within CAFs. Disruption of the RAS-PI3K interaction leads to alterations in YAP expression, impacting focal adhesion formation, stress fibers, and nucleus shape. Importantly, we have identified several
YAP target genes that are involved in ECM remodeling, which are significantly affected in CAFs with defective RAS-PI3K signaling. Notably, CAFs with impaired RAS-PI3K signaling produce thinner and more disorganized matrices, suggesting a critical link between this pathway and ECM remodeling.

To further elucidate the underlying molecular mechanisms, we are currently investigating how RAS-PI3K signaling regulates YAP expression in CAFs. Additionally, we aim to unravel the intricate
crosstalk between RAS-PI3K-YAP signaling and its role in dictating the formation and remodeling of the tumoral extracellular matrix, ultimately promoting cancer progression.

Understanding the importance of CAFs and their mechanical properties, along with the
regulatory role of YAP and RAS-PI3K signaling, could open up new avenues for therapeutic interventions targeting the tumor microenvironment. Disrupting the interactions between these pathways may present a promising strategy to inhibit ECM remodeling and thwart cancer progression. Our ongoing research endeavors hope to shed light on these intricate mechanisms
and provide potential novel targets for future anti-cancer therapies.


Arenas Molina

Universidad de Granada / GENYO, Spain

Abstract Title

Engineering large genomic deletions for the selective ablation of KRAS mutant alleles.

Lung cancer is the leading cause of cancer death worldwide, with lung adenocarcinoma
(LUAD) being the most prevalent subtype. KRAS is the most frequently altered oncogene in LUAD, with ~30% of LUAD patients harboring KRAS mutations, and most of these KRAS mutations are found in the 12th codon of exon 2. Codon 12 KRAS mutations are termed driver as they turn KRAS into a hyperactive protein, promoting cell proliferation and eventually leading to tumor development.

Given the high frequency of these mutations in lung cancer and the reported acquired resistance to recently approved KRASG12C inhibitors (i.e., sotorasib and adagrasib), we have developed a CRISPR/Cas9-based targeting strategy against two of the most frequent mutations of KRAS in LUAD patients (G12C for smokers, and G12D for non-smokers), aiming to overcome new KRAS-dependent resistance mechanisms. In this work, we tried to optimize the efficiency of the CRISPR-based targeted therapy by engineering a double cut strategy which targets both the KRAS mutation site and an intronic position with the aim to generate larger in-frame deletions in the KRASG12C/G12D alleles.

To selectively ablate mutant alleles without affecting wildtype, non-tumor KRAS, we built ribonucleoprotein particles (RNPs) using a high-fidelity version of Cas9 (HiFi-Cas9) coupled to a pair of guide RNAs (gRNAs): one mutation-specific (G12C/G12D) gRNA, and one intron-targeted gRNA. PCR-based amplifications of resulting deletions demonstrated high editing accuracy only in mutant KRAS, while leaving wildtype KRAS (KRASWT) untouched. Furthermore, the therapeutic potential of the system was assessed using KRASG12C/G12D and KRASWT LUAD cancer cell lines, showing a massive decrease in cell viability, and even outperforming our previous approach of inducing small indels in the mutation site, possibly due to a larger DNA damage leading to greater cytotoxicity.



The Francis Crick Institute, UK

Abstract Title

Role of RAS/CRAF interaction in RAS-driven lung cancer.

The RAF/MAPK pathway is a major RAS effector pathway implicated in RAS oncogenic properties, regulating a diversity of cellular processes. Recently, Sanclemente et al. demonstrated that complete ablation of CRAF in established advanced lung tumours (KrasG12V/Trp53) triggers sustainable tumour regression, despite CRAF kinase activity being dispensable. This approach validates CRAF as a therapeutic target for KRAS-driven lung cancer, but also suggests limitations to the use of CRAF kinase specific inhibitors in the clinic.

RAS-GTP strongly interacts with CRAF through its Ras Binding domain (RBD). This interaction is required to activate CRAF. However, disruption of this interaction has never been proven to be a therapeutic target in vivo. Using CRISPR/Cas9 technology, we engineered a new mouse model in which CRAF RBD is constitutively mutated to completely abolish its interaction with RAS GTPases (CRAFR89L). This mouse model recapitulates the embryonic lethal phenotype of the constitutive CRAF KO mouse. Using a combination of murine lung KP (KrasG12V/Trp53) cell lines, either constitutively or conditionally expressing the CRAFR89L allele (CRAFflox/R89L), we show that acute disruption of CRAF/RAS interaction slows cell proliferation. Interestingly, CRAF ablation or RBD disruption minimally affects ERK signalling. Disruption of RAS/CRAF interaction also sensitizes cells to anoikis. This loss of cell-to-cell contact is characterized by a significant decrease of E-cadherin at the plasma membrane. Moreover, our mutant cell lines display a PI3K/AKT positive feedback. Similarly to CRAF ablation, RBD disruption drastically reduces tumour growth in vivo in a syngeneic transplant model. KP lung tumour-bearing mice constitutively expressing one copy of the R89L allele (CRAFFlox/R89L) are partially protected against mutant KRAS-induced lung tumourigenesis, showing a dose-dependent effect. RBD disruption recapitulates CRAF ablation and triggers tumour regression in these animals. Our work highlights CRAF interaction with RAS as a therapeutic target for KRAS-driven lung cancer.



Instituto de Biología Molecular y Celular del Cáncer, Spain

Abstract Title

Capicua Is a Barrier to Lung Cancer Development Driven by KRAS Oncogenes

KRAS mutations are a well-established driver of lung adenocarcinomas. Yet, the mechanistic bases of lung cancer development driven by KRAS oncogenes remain poorly defined. Evidence from genetically-engineered mouse models indicates that signaling via the MAPK effector pathway is critical for tumor initiation. However, those factors or signaling pathways that promote KRAS-driven lung tumor formation beyond the MAPK pathway have not been established in detail. The transcriptional repressor Capicua (CIC) has recently been identified as a critical substrate of ERK kinases in development and disease. In the absence of KRAS/MAPK signaling, CIC binds to specific DNA sequences and represses transcription of its target genes. In contrast, when the KRAS/MAPK pathway is active, ERK directly phosphorylates CIC and triggers its inactivation leading to de-repression of its target genes.

Here, we have explored the role of CIC in KRAS-driven lung cancer formation. CIC inactivation using a conditional knock-out mouse model significantly reduced the survival of Kras+/LSLG12V;p53lox/lox (KP) mice. These mice, designated as KPCic, developed significantly more tumors, indicating that inactivation of CIC substantially facilitated lung tumor initiation. In addition, whereas tumors from KP mice showed much higher levels Kras of amplification, we observed that tumors grown in KPCic mice presented stringly reduced levels of Kras allelic imbalance. Thus, suggesting that gain of the mutant Kras allele occurs, at least in part, to effectively inactivate CIC. Mechanistically, CIC inactivation facilitated transformation of bronchiolar Club cells by converting them into tumor cells positive for AT2 cell markers, thereby contributing to increased lung tumor initiation and reduced survival of mice.

Given the impact of CIC inactivation in KRAS mutant lung cancer initiation, we set to explore whether loss of CIC can drive lung cancer development by itself. Inactivating mutations in CIC are found in at least 3% of all human lung adenocarcinomas and rarely co-occur with other known drivers such as KRAS or EGFR. Yet, systemic inactivation of CIC did not affect lung tissue in any detectable manner. Thus, we reasoned that, to act as a driver, loss of CIC may require specific co-occurring alterations. Indeed, two thirds of those human lung adenocarcinomas with inactivating CIC mutations display co-occurring mutations in TP53. In mice, CIC inactivation with concomitant deletion of Trp53 resulted in lung tumor development in 50% of the animals, indicating that inactivation of CIC could act as a driving mechanism in lung cancer under specific conditions.



Instituto de Biología Molecular y Celular del Cáncer, Spain

Abstract Title

Inactivation of Capicua confers resistance to MAPK pathway inhibition but exposes selective vulnerabilities in KRAS mutant lung cancer cells

Capicua (CIC) is a transcriptional repressor negatively regulated by KRAS/MAPK signaling that represses expression of multiple tumor-promoting genes including the pro-oncogenic PEA3 family of transcription factors. A subset of patients with KRAS mutant lung adenocarcinoma carry loss-of-function mutations in CIC that drive resistance to inhibitors of the MAPK pathway.

Here, we have explored several strategies to overcome resistance driven by inactivation of CIC in KRAS mutant lung adenocarcinoma. As a proof-of-concept of CIC reactivation in cancer cells, we have generated adenoviral vectors that ectopically express either wild-type CIC or CICS173A , a phosphorylation-insensitive mutant known to repress its target genes more effectively. Ectopic expression of CIC reduced tumor cell proliferation and restored sensitivity to the MEK inhibitor trametinib. These results were even more pronounced when expressing CICS173A , suggesting that CIC re-activation could be a valid strategy to overcome resistance.

Next, we performed RNA-seq to identify those MAPK-pathway regulated genes that are controlled by CIC and show constitutive de-repression in its absence. This approach revealed the PEA3 transcription factors ETV4 and ETV5 as the top candidates. Silencing of ETV4 and ETV5 reduced proliferation of KRAS mutant lung tumor cells that lack functional CIC, but also of tumor cells that carry wild-type CIC. Moreover, silencing of ETV4 and ETV5 also restored sensitivity to trametinib, indicating that resistance due to loss of CIC function is mainly mediated via de-repression of ETV4 and ETV5.

Finally, we reasoned that loss of CIC function may create selective vulnerabilities. Thus, we performed a screening with a library of known and well-characterized anti-cancer drugs to identify compounds that selectively inhibit tumor cells that have lost CIC function. We identified 2 drugs (PFK15 and Tx-1123) that more effectively affected proliferation of KRAS-mutant lung tumor cells when CIC repressor function was compromised. Combining these compounds with trametinib again restored sensitivity to this inhibitor. Together, our results show that CIC inactivation drives resistance to targeted inhibition of the MAPK pathway via constitutive de-epression of ETV4 and ETV5 and that this generates selective vulnerabilities that can be exploited.



Centro Nacional de Investigaciones Oncológicas (CNIO), Spain

Abstract Title

Design and Development of Novel Therapeutic Strategies Targeting K-RAS Driven Pancreatic Ductal Adenocarcinoma (PDAC)

Pancreatic Ductal Adenocarcinoma (PDAC) has a 5-year survival rate of 5% due to late diagnosis and lack of effective treatments. Recent studies demonstrate that precision medicine based on genetic and molecular profiling benefits PDAC patients. Our published results show that ablation of Egfr and Raf1 results in complete regression of a subset of mutant Kras/Trp53-driven mouse
PDACs. According to this, the tumors were classified as Responders (tumors that do not grow in the absence of Egfr/Raf1) and Non-Responders (tumors that grow in the absence of Egfr/Raf1). The response of the tumors to the dual ablation is determined by their transcriptional profiles (Blasco et al., 2019). Analysis of these profiles led to the design of a signature that predicts response to deletion of the targets in mouse tumors.

We aim to identify human tumors that regress in the absence of Egfr/Raf1 and to genetically and molecularly characterize them to distinguish them from those that will grow. In the concept of personalized therapy, we will validate the predictive value of our signature regarding the response to the therapeutic strategy of inhibition of EGFR and RAF1 in patient-derived organoids

We are characterizing the mutational and molecular landscape of our PDOs. Together, we are evaluating drug response in medium and high throughput in vitro assays. Through bioinformatic analysis, we will correlate genomic and transcriptomic profiles with drug responses of organoids. Future studies will focus on validating therapeutic options in patient-derived xenografts (PDX). Using PDOs and genetically engineered preclinical models that recapitulate the molecular mechanisms of PDAC progression will improve our investigation’s translation to clinical trials.



University of Turin, Italy

Abstract Title

Relevance of RAF kinases localization at the membrane as key feature to sustain oncogenic MAPK activity

RAF kinases are among the most relevant known RAS effectors and have an important role in the MAPK pathway. Their activation in the RAS signalosome requires direct contact with active RAS proteins localized at the membrane and leads to the initiation of a sequence of downstream phosphorylation events starting from MEK to different cellular targets, that will orchestrate a variety of biological responses. However, a detailed understanding of RAS-RAF membrane dynamics is not complete. This study investigates whether forced localization of
RAF kinases at the cell membrane can be per se an oncogenic trigger that leads to
hyperactivation of the MAPK signaling even in the absence of oncogenic mutations of KRAS. We evaluated the oncogenic potential of constitutive or inducible localization of RAF kinases at the cell membrane in vitro.

Our results showed that, by forcing RAF localization at the membranes, the MAPK pathway is activated by all individual isoforms, increasing pMEK and pERK activity. However, only in the case of ARAF, cells show high tolerance to its exogenous expression in both Ras-less or Raf-less MEFs without causing cellular toxicity. Forced membrane localization of BRAF and CRAF in Ras-less cells caused a dose-dependent toxicity due to excessive MAPK signaling, resulting in the selection of cells with lower lever of exogenous RAF-CAAX expression in the constitutive context. Most intriguingly, none of the isoforms have a cytotoxic effect in Raf-less cells, implying that RAS proteins can buffer RAF activity and/or expression fluctuation to prevent MAPK-dependent toxicity.

• We demonstrate that in the absence of RAS proteins, increased expression of specific RAF isoforms at the cell membrane is sufficient to activate the MAPK pathway and promote cell growth.

• Ras-less cells are particularly sensitive to excessive membrane-localized BRAF and CRAF expression.

• Raf-less cells are insensitive to variation of expression of membrane-localized RAF isoforms.



University of Salamanca, Spain

Abstract Title

C3G deletion in hematopoietic stem cells delays P210BCR-ABL1-induced chronic myeloid leukemia development

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm associated with a characteristic chromosomal translocation that gives rise to the so-called Philadelphia (Ph) chromosome, which harbors the BCR-ABL1 fusion oncogene, responsible for the disease. C3G is an essential and ubiquitous guanine nucleotide exchange factor (GEF) for Rap1 and R-Ras, two members of the Ras family of small GTPases. In CML cell lines and
primary cells from patients it is expressed a truncated isoform of C3G, p87C3G, which
directly interacts with BCR-ABL1, being also phosphorylated by this oncoprotein. 
RAPGEF1 (the gene encoding C3G) is also translocated together with c-ABL, being part
of the Ph chromosome. Therefore, we hypothesize that C3G might play a role in the development of CML.

To study in vivo the role of C3G in CML, we have used a murine model that reproduces human CML, in which Rapgef1 has been deleted in the same cells that express p210BCR-ABL1 oncogene, i.e., hematopoietic stem and progenitor cells (Tec-p210+/-; Rapgef1flox/flox , HSC-Scl-CreERT+/- , hereinafter p210/C3GHSC-Scl-KO). Thus, comparison of CML development in Tec-p210 mice expressing either wild-type C3G (p210/C3GHSC-Scl-wt, lacking the CreERT allele) or knocked-out C3G (p210/C3GHSC-Scl-KO) will clarify C3G function in this neoplasia.

Tec-p210 mice develop a CML-like disease, beginning at 6-8 months, characterized by a
gradual increase in myeloid cells, mainly neutrophils, in peripheral blood. Early results
showed that 6-7-month-old p210/C3GHSC-Scl -KO mice had significantly lower levels of peripheral blood neutrophils, compared to p210/C3GHSC-Scl -wt mice and exhibited features of late-onset or milder CML. Moreover, preliminary data showed that p210/C3GHSC-Scl -KO mice tended to die later due to the myeloproliferative disease compared with their wild-type counterparts. This suggests that deletion of C3G in hematopoietic stem cells could delay the development of the disease. However, it is necessary to increase the number of analyzed mice to confirm the observed tendency.


Boned del Rio

University College London (UCL), UK

Abstract Title

Overcoming resistance to SHOC2/MEK inhibition in RAS-mutant cancer cells

The RAS-MEK-ERK pathway is upregulated in most human cancers but has proved difficult to inhibit successfully in the clinic because of resistance and on-target toxicity. New strategies to inhibit it with better therapeutic margins are critically needed and targeting the SHOC2-MRAS-PP1 (SMP) complex may provide such a mechanism.

The SMP complex dephosphorylates the ‘S259’ inhibitory site in RAF kinases and contributes to ERK activation in a context-dependent manner, being preferentially required by oncogenic RAS.

SHOC2 was identified as one of the best targets against RAS-driven cancers in genome-wide fitness screens and inhibiting SHOC2 potently synergizes with MEKi to kill RAS mutant cells, leading to tumour
regressions in mouse models. However, our data show that a small proportion of cancer cells survive SHOC2/MEK inhibition and cause tumour relapse upon discontinuation of MEKi treatment. These cells regain drug sensitivity upon MEKi re-addition, consistent with the reversible and non-genetic resistance of drug tolerant persister cells (DTPs) that have been observed in response to different targeted- and chemo-therapeutics in multiple cancer types.

We have developed models of acute SHOC2 inhibition in human and mouse cancer cells using the dTAG-protein degradation system and used transcriptomics and proteomics to study these DTPs. The most striking feature observed was upregulation of the antigen processing and presentation machinery,
suggesting DTPs generated in response to SHOC2/MEK inhibition may be particularly vulnerable to immunotherapy. Consistently, using a KP orthotopic lung tumour model, we found that SHOC2/MEK inhibition promotes an immune response characterised by accumulation of effector CD8+ T-cells and that PD-1 blockade synergizes with SHOC2/MEK inhibition to drive potent durable tumour regressions
that persist after treatment interruption. 


Overall, this study identifies new vulnerabilities to eliminate cells resistant to SHOC2/MEK inhibition and highlights the potential of immune checkpoint blockade for combination therapies with SHOC2/MEK
inhibition for the treatment of RAS-driven cancers.



Novartis Institutes for BioMedical Research (NIBR), Switzerland

Abstract Title

Mechanisms of tumor resistance to clinically relevant KRASG12C and SHP2 inhibitor combination in preclinical models of non-small cell lung cancer

Although KRASG12C inhibitors have shown clear clinical activity in patients with KRAS G12C mutated NSCLC and other solid tumor malignancies, the depth and durability of responses is limited by multiple mechanisms of treatment-emergent resistance. The KRASG12C inhibitor JDQ443 shows enhanced preclinical antitumor activity combined with the SHP2 inhibitor TNO155. We evaluated the duration of response in a mouse model to JDQ443 ± TNO155 (± the PI3Kα inhibtor alpelisib and/or the CDK4/6 inhibitor ribociclib), and the genetic mechanisms associated with loss of response to a KRASG12C/SHP2 inhibitor combination. Antitumor responses to JDQ443 ± TNO155, with or without additional alpelisib and/or ribociclib, were assessed in cell-derived mouse xenograft models derived from the KRASG12C -mutant non-small cell lung cancer (NSCLC) line LU99.

Single-agent tumor regression by JDQ443 at clinical relevant doses of 100 mg/kg in the LU99 model was on average 2 weeks and was increasingly extended (but not prevented) by JDQ443 dual-agent, triple or quadruple combinations. Growth resumption was accompanied by increased KRASG12C protein, mRNA and gene copy number. Adaptive mechanisms of resistance to KRASG12C/SHP2 co-inhibition were explored by functional genome-wide CRISPR screening in KRASG12C-dependent NSCLC lines with distinct mutational profiles.

CRISPR screening in a panel of 5 KRAS G12C-mutated cell lines identified sensitizing and rescuing genetic interactions to a KRASG12C/SHP2 inhibitor combination; FGFR1 was the strongest sensitizer, and PTEN the strongest rescuer. Consistent with this, KRASG12C/SHP2 inhibitor antiproliferative activity was strongly enhanced by PI3K inhibitors. Amplification of KRAS G12C itself and alterations of the MAPK/PI3K pathway were the predominant mechanisms of resistance to KRASG12C/SHP2 co-inhibition in this model. Biological nodes identified by CRISPR screening might provide starting points for effective combination treatment approaches.

This study identifies mechanisms of resistance to KRASG12C/SHP2 co-inhibition and highlights the need of further combination therapy for NSCLC, and offers the basis for the development of more effective combination approaches.



Centro Nacional de Investigaciones Oncológicas (CNIO), Spain

Abstract Title

Exploring the role of KSR as a therapeutic target to overcome resistance to KRAS inhibition

KSR proteins have long been considered only as scaffold proteins required for optimal mitogen-activated protein kinase (MAPK) pathway signaling. However, recent evidence suggests that they play a more complex role within this pathway with profound implications for cancer therapy.

Here, we aimed to explore the activities of KSR, identify new protein interactors and evaluate the therapeutic potential of KSR targeting.

Results and discussion:
Ectopic expression of KSR1 or KSR2 was sufficient to activate the MAPK pathway and to induce cell proliferation in the absence of RAS proteins. KSR1 requires dimerization with at least one member of the RAF family to stimulate proliferation, resulting in translocation of the heterodimerized RAF protein to the cell membrane. This activity also required efficient ATP binding. We further show that Sotorasib, KRASG12C inhibitor, is less effective when KSR1 expression levels are elevated in human cancer cell lines. In agreement with these results, when we silence KSR the response upon Sotorasib treatment improves 

To get further insights into the mechanisms of KSR activity, we performed a BioID assay and identified known KSR interactors along with several subunits of the PP6 phosphatase never been described before that could play a key role in the modulation of KSR activity.

KSR induces RAS-independent proliferation by the activation of MAPK pathway and reduces the effectiveness of KRAS inhibition. Thus suggesting that increased levels of expression of KSR may make tumor cells less dependent on KRAS oncogenic signaling, providing an alternative strategy for targeting KRAS-driven tumors.



IPATIMUP, Portugal

Abstract Title

Exploring the role of mutant KRAS in mediating the crosstalk between colorectal cancer cells and fibroblasts: implications for invasion and metastatic potential

Oncogenic KRAS signaling has proved to exert numerous effects in the orchestration of the tumor microenvironment. Recognizing the tumor microenvironment’s significant role in determining the aggressiveness of cancer, extensive research in this domain has emerged as a compelling avenue to unveil novel targets involved in the interplay between cancer cells
and their microenvironment. Within the tumor microenvironment, cancer-associated
fibroblasts stand out as prominent stromal constituents, acknowledged for their capability to drive the invasive behavior of colorectal cancer (CRC) cells. Therefore, in this work, we aimed to explore and characterize the role of mutant KRAS in mediating CRC cells-fibroblasts crosstalk.

By challenging KRAS mutated cells with fibroblast-conditioned media, we observed that KRAS silencing decreased the invasive capacity of the cancer cells. Analysis of the
conditioned media revealed the presence of high levels of HGF. Neutralization and
supplementation experiments showed that HGF induced invasion in a KRAS-dependent
manner. Consequently, we observed that KRAS regulates the expression of the HGF
receptor, C-MET and that its silencing decreases the levels of fibroblast-induced invasion, suggesting a role of the HGF-C-MET axis in regulating the invasive properties of these cells.

Our data bestows upon fibroblasts a pivotal role in mediating KRAS oncogenic effects,
particularly in facilitating invasion. It provides valuable insights into the mechanisms underlying several clinical observations. For instance, we are presently devoted to exploring the possible connection between the invasive potential facilitated by the interaction between
KRAS and fibroblasts and the peritoneal dissemination commonly associated with mutant KRAS consensus subtype 4 CRC. Additionally, we aim to shed light on the mechanisms underlying the dismal prognosis of mutant KRAS CRC liver metastasis and the propensity for lung recurrence following resection of the liver metastasis.



University of Louisville, USA

Abstract Title

A First-in-Class RALGEF inhibitor to suppress RAS driven pancreatic cancer

Activated forms of RAS drive transformation by stimulating three main effector suites. These are the RAF/MAPK pathway, the PI3K pathway and the RALGDS/RAL pathway. The Raf and PI3K pathways have been extensively studied and multiple clinical agents targeting them have been developed. 
However, their clinical utility has proved somewhat disappointing. In contrast, no clinical agents have been reported for the third arm of RAS signaling: RALGDS.

Here we describe a novel small molecule that binds directly to RALGDS and inhibits the interaction of RAS with both RALGDS and RGL2. The agent preferentially suppresses 3D tumor cell growth and RAL pathway signaling. It promotes anoikis and can suppress the Cancer Stem Cell compartment. The agent is active in vivo and suppresses the development of pancreatic tumor cell lines and pancreatic pdx. It exhibits no apparent toxicity.


De Hita

Instituto de Biología Molecular y Celular del Cáncer, Spain

Abstract Title

Keeping balance: decoding the role of DUSP4 phosphatase in MAPK pathway activation during lung adenocarcinoma progression

In lung adenocarcinoma (LUAD), virtually all genetic alterations driving tumor progression are directly linked to the RAS-MAPK pathway, upregulating its activity to provide tumor cells with a proliferative advantage.
However, excessive MAPK signaling is detrimental. Through mechanisms that are far from being fully decoded, the tumor must undergo a process of selection for a set of both positive and negative regulators to
keep balance. This regulation has been validated both in vitro and in mouse cancer models but its clinical impact is currently unknown.

Using a transcriptional signature of 4 genes, we assessed MAPK activity levels in KRAS mutant tumors from TCGA patient data. Strikingly, high MAPK tumors had a better prognosis compared to those with lower
pathway output, implying moderate MAPK activation may confer greater aggressiveness, while highly active
lesions are associated with various stress phenotypes. 

Paradoxically, DUSP4, a dual specificity phosphatase originally considered part of the signature, as it forms a transcriptional negative feedback loop with ERK, exhibited anti-correlation with pathway activation in
KRAS-mutant patients.

Genomic analysis revealed susceptibility to copy number variations (CNVs) at the DUSP4 locus. Low MAPK patients, with poor survival, showed increased copy number gains of DUSP4, while high MAPK patients, with
better prognosis, exhibited increased copy number losses since early stages of the disease. Our working hypothesis postulates that DUSP4 loss initially confers a proliferative advantage but this turns into a
deleterious effect during later tumor stages likely due to sustained MAPK activity.

To validate this hypothesis we took advantage of an inducible mutant KRAS mouse strain (KrasLSLG12V). By infecting the mice with a dual viral system expressing Cre recombinase for KRAS activation and CRISPR/Cas9-mediated DUSP4 knockout, we studied how DUSP4 loss affects tumor initiation and progression. We confirmed that DUSP4 loss results in a proliferative advantage to early neoplastic lesions. However, long-term loss of DUSP4 increases MAPK activity and induces stress, for example through the activity of reactive oxygen species.

Furthermore, by studying differentially expressed genes between High and Low MAPK patients, we also identified several candidates coding for PP1, PP2, and PP4 phosphatases and their regulatory subunits as potential novel pathway regulators and we will address their implication in the future.


De la Puente Ovejero

Centro Nacional de Investigaciones Oncológicas (CNIO), Spain

Abstract Title

Uncovering Raf1 degradation and its tumorigenic role using the dTAG system

Genetic interrogation of the KRAS signalling pathway in genetically engineered mouse models has shown that RAF1 ablation promotes tumor regression without significant toxicities, highlighting it as
a promising therapeutic target. In this work, considering the unavailability of RAF1 specific degraders, we propose the use of the dTAG-system as a proof of concept to pharmacologically reproduce those effects observed upon RAF1 genetic ablation. This strategy allows the study of the immediate
consequences of protein loss and provides useful information about RAF1 degradation that could be used to develop a selective degron or PROTAC. To achieve this, we generated a homozygous knock-in mouse with a FKBP12F36V tag inserted into the RAF1 locus, where lung adenocarcinomas are driven by KRASG12V and loss of Trp35. Phenotypically, some differences were observed in comparison with RAF1wt mice. Not only they seemed weaker and smaller than wild type mice, but tumor formation after adenoviral infection was 5-fold lower than expected. Since the key genetic modification lies in the expression of FKBP12-RAF1 instead of the WT protein, some differences between them are suspected. Moving on to the validation of target degradation under dTAGv1 treatment, we demonstrate a rapid and efficient systemic elimination of FKBP12-RAF1 without associated toxicities. However, protein degradation did not correspond to the expected anti-tumor effect. These results could be correlated with the afore mentioned suspected differences.
Interestingly, based on our results, our model offers a new approach to gaining insights into the major role of RAF1 protein in tumor development and progression by exploring the differences between this kinase and FKBP12-RAF1 through an interactome analysis. Moreover, the generation of RAF1 independent tumors allows the study of underlying resistance mechanisms. Altogether, this work opened up new possible strategies to clarify the tumorigenic role of RAF1.


De Paz

Instituto de Biología Molecular y Celular del Cáncer, Spain

Abstract Title

Unraveling the Role of Oncogenic Drivers in Lung Cancer Microenvironment: A Quest for Novel Therapeutic Strategies

Lung cancer stands as the leading cause of cancer-related deaths globally, with EGFR and KRAS mutations being highly prevalent in lung adenocarcinoma (LUAD). Despite advancements in targeted therapies, patients often face relapse due to resistance mechanisms, necessitating improved
therapeutic approaches. Emerging evidence points to the critical impact of the extracellular matrix (ECM) and cancer-associated fibroblasts (CAFs) in cancer biology, influencing cancer cell behavior and
immune infiltration. However, there remains a dearth of knowledge concerning how various oncogenic drivers modulate CAF function and ECM remodeling, fostering cancer progression. This study delves
into the intricate interplay between oncogenic drivers and the tumor microenvironment, with a particular focus on ECM dynamics and CAF phenotype modulation. Our primary objective is to explore the influence of EGFR and KRAS mutations on ECM and CAF biology, with the ultimate goal of identifying innovative therapeutic strategies for lung cancer patients.

By mining available lung cancer databases, we have identified a set of differentially expressed ECM components in EGFR or KRAS mutated LUAD, significantly correlating with patient survival. Through
comprehensive biological and OMIC analyses, we are now uncovering the underlying molecular mechanisms governing this interplay, identifying specific markers for each oncogenic driver. These
markers hold immense potential as prognostic or predictive biomarkers and serve as novel therapeutic targets, ultimately improving lung cancer treatment outcomes.



Weill Cornell Medicine Graduate School of Medical Sciences, USA

Abstract Title

Uncovering resistance to targeted KRAS inhibition using CRISPR base editing sensor screens

Non-small cell lung cancer (NSCLC) is the leading cause of cancer related deaths
worldwide and mutations in KRAS are the most frequent gain-of-function alteration in this disease. Recent development of covalent KRAS inhibitors provide a promising tool to directly target oncogenic KRAS signaling in cancer and early clinical trial data are promising. However, evidence of resistance to targeted inhibition has been demonstrated both in laboratory studies and in human patients highlighting the necessity to understand mechanisms of resistance to better serve these patients. Our lab has developed a CRISPR base editing sensor screening platform and have designed custom libraries used in this study. The mouse base editing sensor library is comprised of sgRNAs to interrogate 
commonly occurring cancer variants of unknown significance (MBES) additionally, a
mouse Kras sgRNA tiling library (mKras). We established base editing lung tumor cell line
models derived from LSL-KrasG12C;p53fl/fl mice. KrasG12C mutant tumor lines were infected with each library and treated with a panel of targeted KRAS inhibitors at increasing doses over time. Our findings first validate our screening platform approach with detection of already published resistance mutations, and additionally this work has revealed novel missense mutations that contribute to targeted KRAS inhibition. We expect that this work will be instrumental in guiding the field to improved treatment of NSCLC patients with targeted inhibitors.


Entrialgo Cadierno

Foundation for Applied Medical Research, Spain

Abstract Title

The phospolipid transporter PITPNC1 links KRAS to MYC to prevent autophagy in lung and pancreatic cancer

Background. The discovery of functionally relevant KRAS effectors in lung
adenocarcinoma (LUAD) and pancreatic ductal adenocarcinoma (PDAC) may
yield novel molecular targets or mechanisms amenable to inhibition strategies.
Here, we studied the role of PITPNC1 and its controlled network in the development and progression of both mutant KRAS-driven cancers.

Methods. Genetic modulation of KRAS expression as well as pharmacological
inhibition of canonical effectors was done. PITPNC1 genetic depletion was performed in in vitro and in vivo LUAD and PDAC models. PITPNC1-deficient cells were RNA sequenced, and Gene Ontology and enrichment analyses applied to the output data. Protein-based biochemical and subcellular localization assays were run to investigate PITPNC1-regulated pathways. A drug repurposing approach was used to predict surrogate PITPNC1 inhibitors that were tested in combination with KRASG12C inhibitors in 2D, 3D and in vivo models.

Results. PITPNC1 expression was increased in human LUAD and PDAC and associated with poor patients’ survival. PITPNC1 was regulated by KRAS through MEK1/2 and JNK1/2. Functional experiments showed PITPNC1 requirement for cell proliferation, cell cycle progression and tumour growth. PITPNC1 controlled a transcriptional signature that highly overlapped with a KRAS-regulated one. Notably, PITPNC1 loss decreased MYC protein expression and prevented mTOR localization to lysosomes. A JAK2 inhibitor was predicted as putative PITPNC1 inhibitor and decreased MYC levels, induced autophagy and displayed
antiproliferative effect. Furthermore, its combination with a KRASG12C inhibitor
elicited a substantial antitumor effect in LUAD and PDAC.

Conclusions. Our data highlight the functional and clinical relevance of PITPNC1 in LUAD and PDAC. Moreover, PITPNC1 constitutes a new mechanism that regulates the KRAS downstream targets MYC and mTOR and controls a druggable transcriptional network for combinatorial treatments.


Fernández García

Centro Nacional de Investigaciones Oncológicas (CNIO), Spain

Abstract Title

Type I interferon signaling pathway enhances immune-checkpoint inhibition in KRAS mutant lung tumors.

Canonical RAS GTPases are three proteins with highly similar sequences and structure, but despite this similarity, there is a clear specificity of function at the physiological and pathological levels. Our previous “in vivo” analysis of their functions using mouse KO models, showed that only KRAS is indispensable for embryonic development, meanwhile HRAS and NRAS KO mice are
perfectly viable with only alterations in their immune response. Nevertheless, further studies on the HRAS;NRAS Double knockout mice (DKO) showed that combined elimination of these GTPases led to perinatal death of a 90% of the DKO pups in the first day of life, due to defective lung maturation and respiratory failure. In addition, the few DKO animals that reached adulthood
were smaller and had a patched lung with areas of atelectasis and emphysema.

In this work we have analyzed the adult HRAS;NRAS DKO mice, and in addition to the abovementioned phenotypes, the DKO animals show craniofacial dysmorphism resembling that observed in mouse models of RASopathies. This is observed in a CT-scan of their head that shows reduced interorbital width and cranial length. Other phenotypes observed in these DKO animals similar to human symptoms of RASopathies include engrossed heart walls, splenomegaly and
increased numbers of GR1+/CD11b+ myeloid-derived stem cells. Further analysis has shown that adult HRAS;NRAS DKO mice also have thrombocytopenia and reduced clotting capacity due to increased platelet apoptosis, a symptom that has been observed in some human RASopathy
patients. All these phenotypes are accompanied by an increase in KRAS activation in tissues and cells isolated from these mice, pointing to a compensatory hyperactivation of KRAS as the cause of the RASopathy symptoms in the HRAS;NRAS DKO mice.


Fernández Medarde

University of Salamanca/CSIC, Spain

Abstract Title

KRAS hyperactivation leads to a RASopathy like phenotype in the HRAS;NRAS Double Knockout mice

Canonical RAS GTPases are three proteins with highly similar sequences and structure, but despite this similarity, there is a clear specificity of function at the physiological and pathological levels. Our previous “in vivo” analysis of their functions using mouse KO models, showed that only KRAS is indispensable for embryonic development, meanwhile HRAS and NRAS KO mice are
perfectly viable with only alterations in their immune response. Nevertheless, further studies on the HRAS;NRAS Double knockout mice (DKO) showed that combined elimination of these GTPases led to perinatal death of a 90% of the DKO pups in the first day of life, due to defective lung maturation and respiratory failure. In addition, the few DKO animals that reached adulthood
were smaller and had a patched lung with areas of atelectasis and emphysema.

In this work we have analyzed the adult HRAS;NRAS DKO mice, and in addition to the abovementioned phenotypes, the DKO animals show craniofacial dysmorphism resembling that observed in mouse models of RASopathies. This is observed in a CT-scan of their head that shows reduced interorbital width and cranial length. Other phenotypes observed in these DKO animals similar to human symptoms of RASopathies include engrossed heart walls, splenomegaly and
increased numbers of GR1+/CD11b+ myeloid-derived stem cells. Further analysis has shown that adult HRAS;NRAS DKO mice also have thrombocytopenia and reduced clotting capacity due to increased platelet apoptosis, a symptom that has been observed in some human RASopathy
patients. All these phenotypes are accompanied by an increase in KRAS activation in tissues and cells isolated from these mice, pointing to a compensatory hyperactivation of KRAS as the cause of the RASopathy symptoms in the HRAS;NRAS DKO mice.



Instituto de Biología Molecular y Celular del Cáncer & CIBERONC, Spain

Abstract Title

Unraveling the Role of SOS RAS-GEF in Calcium Signaling in Non-Excitable Cells

Calcium signaling is a fundamental process involved in diverse cellular functions, including cell proliferation, differentiation, and gene expression. In non-excitable cells, calcium signaling relies on store-operated calcium entry (SOCE) and is tightly regulated by mitogen-activated protein kinase (MAPK) pathways. This study aimed to elucidate the role of SOS RAS GEFs in
calcium signaling and their interaction with MAPK pathways. These results revealed that the absence of SOS1 impaired calcium influx through SOCE in different cell types. Specifically, SOS1-deficient mouse embryonic fibroblasts (MEFs) and T cells exhibit impaired calcium entry, indicating the critical involvement of SOS1 in non-excitable cell calcium signaling. Furthermore, SOS1 downregulation in Jurkat cells resulted in reduced calcium influx, further supporting its role in SOCE-mediated calcium signaling. Additionally, SOS1- deficient MEFs displayed a delay in ERK activation following treatment with thapsigargin, a known inhibitor of endoplasmic reticulum calcium pumps. These findings suggest that SOS1 is essential for the activation of ERK pathway in response to calcium signaling. Overall, the loss of SOS1 led to the partial inhibition
of calcium entry triggered by thapsigargin and delayed ERK1/2 activation. This study also provides insights into the suppressive effect of SOS1 absence on store-operated calcium entry through the ERK pathway, offering a novel mechanism by which SOS1 influences calcium signaling in non-excitable cells. By unraveling the intricate interplay between the SOS1, calcium
signaling, and MAPK pathways, this study contributes to a better understanding of the intricate mechanisms underlying cellular processes regulated by calcium signaling in non-excitable cells.

Grants from ISCIII-MCUI (FIS PI19/00934 and PI22/01538), ISCIII-CIBERONC (CB16/12/00352), Fundación Memoria de Don Samuel Solórzano Barruso (FS/7-2022) and AECC Excellence Program “Stop RAS Cancer” EPAEC 22641CICS funds supported this research. The CIC is supported by the Programa de Apoyo a Planes Estratégicos de Investigación de Estructuras de
Investigación de Excelencia de Castilla y León (CLC-2017-01).


Gómez Zepeda

University of Barcelona, Spain

Abstract Title

KRAS phosphorylation at Ser181 regulates secretome protein composition and therefore, cell invasiveness in colorectal cancer

Colorectal cancer (CRC) is the third most common type of cancer worldwide and the second leading cause of cancer deaths. KRAS small GTPases are mutated in 40% of CRC cases. Oncogenic mutations in KRAS promote an active permanent GTP-bound state, independent of extracellular signals increasing proliferation, cell growth, and apoptosis avoidance. For this reason, KRAS is a key therapeutic target in CRC, however, efforts to inhibit KRAS have been mostly unsuccessful. In this work, we analyze how changes in oncogenic KRAS Serine 181
phosphorylation/dephosphorylation cycle impact the composition of the secretome of CRC cells, which might be affecting tumor properties.

First, by CRISPR technology we generated clones of SW480 cells endogenously expressing non-phosphorylatable oncogenic KRAS phosphomutant protein (S181A). Additionally, we obtained subclones of the wild type phosphorylatable SW480 cell line (S181) to use as control. Next, we collected secretome samples and performed the proteomic analysis by LC-MS/MS and validation by Western blot.

A total of 303 differentially secreted proteins were detected between the clones expressing the phosphorylatable and non-phosphorylatable oncogenic KRAS at position 181. Biological processes related to cell migration, cell adhesion, regulation of cell death, stem cell differentiation and MAPK cascade signaling were overrepresented. This suggests that the oncogenic KRAS Serine 181 phosphorylation/dephosphorylation cycle might regulate these functions through the control of protein secretion.

Specifically, phosphorylatable KRAS expressing clones secreted higher levels of proteases related to cell invasiveness: MMP2 and cathepsin B. In view of these results, we carried out invasion assays in vitro and metastasis studies in mice. A phosphomimetic CRISPR-generated KRAS S181D clone was also included in these experiments. Interestingly, non-phosphorylatable KRAS expressing clones showed less invasion capacity in vitro; and the phosphomimetic KRAS expressing clone displayed more metastatic potential in vivo. These data suggest that KRAS phosphorylation at Ser181 regulates these oncogenic functions in CRC cells.



University of Salamanca, Spain

Abstract Title

Characterization of cellular models of glioblastoma treated with PI3K and MAPK inhibitors

PI3K and MAPK are frequently dysregulated pathways in glioblastoma. Consequently, inhibitors against members of this network are actively pursued by pharmaceutical companies to treat this malignancy and other types of cancer. However, these pathways converge to regulate downstream functions and often compensate each
other, leading to drug resistance. Here by treating cellular models of glioblastoma with PI3K and MEK inhibitors individually we show different patterns of PI3K-MEK signalling network circuitry across the profiled cell lines. For example, we found an increase of activity markers for PI3K pathway after MEK inhibitor treatment, indicating that the PI3K pathway is inhibited by the MAPK in some models. These effects demonstrate that therapies based on
cotreatments with PI3K and MEK/MAPK inhibitors are necessary to combat compensation mechanisms and transient responses to therapy. In fact, clinical trials are evaluating combination therapies with inhibitors against PI3K and MAPK signalling members, which are approved to treat different cancers in combination with other agents. Our results will advance in the knowledge of this aggressive brain cancer that remains extremely difficult to treat.



University of Zurich, Switzerland

Abstract Title

Discovering Modulators of KRAS Nanoclustering

The understanding of how KRAS assembles into nanoclusters and what consequences this has for signal transduction is clearly still at the beginning. Being able to modulate KRAS nanoclustering would allow to identify factors involved and understanding structure-function relationships. Considering the ongoing research, KRAS oligomerization is a complex process involving numerous protein-protein interactions. This concept in principle provides multiple ways of intervention with some being particularly relevant in an oncogenic context.

In order to discover small molecules that could potentially affect RAS nanoclustering we have developed a cell-based BRET assay with the aim to enable high-throughput screening of the EU-OPENSCREEN chemical collection. We initially validated the assay with intracellular biologics targeting either the allosteric or the effector interface of KRAS, before developing stable cell lines that now allowed us to observe the effect of clinical stage KRAS (G12C) inhibitors.

The stable cell lines generated for KRAS (G12C) and KRAS (G12V) were validated for HTS by our partners at the University of Santiago de Compostela and subjected to a pilot screen. Here, a library of bioactive compounds is screened to discover compounds whose molecular targets are already reported, potentially revealing druggable protein-protein interactions relevant for KRAS nanoclustering. Initial hits, such as inhibitors of the PDEδ-KRAS interaction and of farnesyltransferase activity validate the assay and indicate the potential of the setup to discover such compounds.

We will subsequently screen the full EU-OS compound library with the aim to identify new inhibitors and activators of KRAS nanoclustering. Being able to modulate KRAS nanoclustering will allow to identify factors involved and understanding structure-function relationships. Being able to modulate KRAS nanoclustering would extend the mechanisms by which the KRAS oncogene could be targeted.



Uniformed Services University of the Health Sciences (USUHS), USA

Abstract Title

SOS1 inhibition enhances the efficacy of and delays resistance to G12C inhibitors in lung adenocarcinoma

KRAS G12C inhibitors (G12Cis) including adagrasib and sotorasib have demonstrated clinical benefit for patients with KRASG12C -mutated lung adenocarcinoma. Unfortunately, clinical effectiveness of G12Ci is limited both by intrinsic and acquired resistance, and the duration of clinical benefit is modulated by co-mutation of tumor suppressors including KEAP1. These data suggest that combination approaches are likely needed for a majority of patients. Survival of drug-tolerant persister cells within the heterogeneous tumor population and/or acquired mutations that reactivate RTK/RAS signaling can lead to outgrowth of tumor initiating cells (TICs) and drive therapeutic resistance. We found that targeting proximal RTK signaling using
the SOS1i BI-3406 both enhanced the efficacy of and delayed resistance to G12Ci treatment, but the extent of SOS1i effectiveness was modulated by both SOS2 expression and the specific mutational landscape.

SOS1i enhanced the killing effect of G12Ci under 3D culture conditions, but this effect was modulated by SOS2 protein levels. In cells expressing elevated SOS2, SOS2 deletion restored the synergistic effects of combined SOS1i:G12Ci. Unbiased assessment of signaling pathways modulated by combined SOS1i:G12Ci revealed that SOS1i regulated hypoxia/HIF1α pathways, which are a key regulator of TIC survival. TICs are a subset of drug-tolerant persister cells
hypothesized to drive therapeutic resistance. TICs can be functionally defined by their ability to proliferate in 3D culture from a single cell. Using this functional definition, we found that 72-h treatment with the G12Cis adagrasib or sotorasib enriched for functional TICs 2-3 fold in multiple KRASG12C -mutated cell lines, suggesting that TICs could act as a sanctuary population
of G12Ci resistant cells. We further isolated cells based on ALDH activity and found isolated ALDHhigh TICs are markedly recalcitrant to short-term G12Ci treatment. In both cases, we found that SOS1i and/or SOS2 KO inhibited the recalcitrance of TICs to G12Ci to synergistically inhibit TIC survival. Using isogenic cells, we found that combined KEAP1/STK11 KO markedly
increased TIC frequency, and these DKO cells were recalcitrant to SOS1i. SOS1i also delayed acquired G12Ci resistance in situ, but the effectiveness of SOS1i was blunted by combined KEAP1/STK11 DKO. These data suggest that SOS1i could be an effective strategy to both enhance G12Ci efficacy and prevent G12Ci resistance, but the effectiveness could be limited for patients whose tumors harbor KEAP1/STK11 mutations.

These studies were supported by funding from the NIH (CA255232) and a CRADA with
Boehringer Ingelheim.



Boehringer Ingelheim RCV GmbH & Co KG, Austria

Abstract Title

Combined KRASG12C and SOS1 inhibition enhances and extends the anti-tumor response in KRASG12C-driven cancers by delaying the emergence of intrinsic and acquired resistance

Recently approved KRASG12C inhibitors have achieved impressive results in the clinic. However, intrinsic or acquired resistance to therapy invariably develops leading to disease progression in the majority of treated patients. Therefore, intensive efforts are directed at identifying approaches to improve the responses to KRASG12C  inhibitors. Here, we used preclinical models of KRASG12C  non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) to compare the response to KRASG12Ci (adagrasib) monotherapy with the responses to KRASG12Ci combinations with SOS1i (BI-3406), SHP2i (TNO155) or anti-EGFR antibody (cetuximab). We found that in KRASG12C i-naïve in vivo models the combination of KRASG12C i and SOS1i leads to stronger anti-tumor responses than KRASG12C i alone and comparable to those seen with KRASG12Ci combination with SHP2i or anti-EGFR. This enhanced anti-tumor response is associated with a deeper and prolonged suppression of the RAS-MAPK signaling. Moreover, the combination treatment delays the emergence of resistance to KRASG12Ci treatment in both CRC and NSCLC models. We also found that in a KRASG12C  mutant CRC model acquired resistance to KRASG12Ci monotherapy is associated with increased expression of RAS superfamily gene MRAS, suggesting that the activation of MRAS-SHOC2-PP1C complex may compensate for the loss of KRASG12C-driven signaling. In this model combined KRASG12Ci and SOS1i treatment also allows to circumvent resistance to KRASG12Ci and leads to tumor regression. Overall, we envision that co-administering of SOS1i with KRASG12C inhibitors is a promising approach for treating both KRASG12Ci-naïve and resistant KRASG12C mutant cancer.



University of Turin, Italy

Abstract Title

Inhibition of oncogenic mutant KRAS via oxidation at Cysteine 118

Oncogenic mutant RAS is one of the main drivers of cancer. The redox-biology of RAS involves a redox-sensitive Cysteine, C118. By reacting with C118, free-radical oxidants, predominantly nitric oxide, activate RAS by promoting a nucleotide exchange. Recently we discovered in C. elegans that the ortholog of mutant KRAS is inhibited via oxidation at C118 by the non-radical oxidant hydrogen peroxide. Based on this finding, we investigated whether the mechanism of mutant KRAS inhibition through oxidation at C118 is maintained in mammalian cells in vitro and in vivo, and we explored the therapeutic value of KRAS oxidation.

Material and Methods
To investigate the effects of oxidation on KRAS activity, we generated a new model by transducing different oxidation-mimetic KRAS constructs into Ras-less cells (Drosten et al, EMBO J, 2010). To mimic a permanent oxidation of KRAS at C118 by hydrogen peroxide, we replaced Cysteine 118 with Aspartic acid in KRAS (C118D); to inhibit oxidation at C118, we replaced Cysteine 118 with Serine (C118S). These mutations were introduced either alone or in cis with the most common oncogenic mutations in KRAS (G12C/G12D/G12V).

Results and Discussion
We found that the C118D substitution inhibited the growth rate of KRAS-driven cells in vitro. Given the C118D substitution mimics the Cysteine Sulfinic acid oxidative modification (an intermediate state of Cysteine oxidation by hydrogen peroxide) without affecting the KRAS protein level, the GTP level, the effector binding and localization, and with minimal impairment to the MAPK pathway, our data indicates that human mutant KRAS is inhibited via oxidation at C118 by hydrogen peroxide. Moreover, treatment with pro-oxidants in combination with nitric oxide production inhibitor L-NAME inhibited mutant KRASG12V activity. Further proving that C118 is the target of this oxidation, we found that the C118S substitution rendered mutant KRAS insensitive to the inhibitory effect of the aforementioned combination treatment. In vivo, we confirmed that C118D in cis with oncogenic G12V mutation significantly impaired tumor growth and prolonged overall survival.

Mimicking oxidation by hydrogen peroxide at C118 inhibits mutant KRAS in vitro and in vivo. A combination of nitric oxide production inhibition and reactive oxygen species (ROS) production inhibits mutant KRAS by targeting C118. We conclude that oxidation at C118 presents a novel way to inhibit mutant KRAS, thereby paving the way to explore oxidation based anti-KRAS treatments in humans.



Centro Nacional de Investigaciones Oncológicas (CNIO), Spain

Abstract Title

Complete regression of pancreatic adenocarcinomas upon combined inhibition of EGFR, RAF1 and STAT3

Pancreatic cancer is one of the most lethal cancer types and it has been projected as the second highest in mortality by 2030. Currently, standard treatment only includes cytotoxic chemotherapy, with overall survival in the range of weeks to few months. Most of the therapeutic pre-clinical studies with genetically engineered mouse models, only
reveal the preventive role of genes, at the stages of pancreatic ductal adenocarcinoma
(PDAC) initiation. However, our lab has recently developed a PDAC therapeutic mouse model that enables the study of the curative function of genes, as it allows deletion of the targets in fully developed tumors. In this concept, we have demonstrated that genetic ablation of Egfr and Raf1, results in complete regression of a significant fraction of Kras/Trp53 driven tumors of small sizes. Yet, tumors of bigger sizes remained refractory to this combined deletion (Blasco et al., 2019).

In the presentstudy we have characterized by OMIC methodsthe molecular signaling of PDAC cells that respond differently to this combined elimination, Responder (R) and Non responder (NR) cells. More importantly, we identified the transcription factor STAT3 as the key mediator of the mechanism of resistance in NR cells. Indeed, combined in vivo and in vitro genetic elimination of the three genes leads to complete regression of tumors induced by Kras/Trp53 mutations. We have also recently validated the efficacy of this therapeutic strategy using combinations of pharmacologic compounds with minimal toxicities, such as EGFR inhibitors and a STAT3 PROTAC degrader. Therefore, we have developed a promising therapeutic strategy for NR mouse PDACs based on the simultaneous inhibition of EGFR, RAF1 and STAT3. We are currently validating our findings in our collection of human 3D cultures, the so-called patient-derived organoids (PDO), as well as in PDO-derived xenografts.



IPATIMUP, Portugal

Abstract Title

Tumor-Hematopoietic Communication in Colorectal Cancer Pathogenesis

The immunoediting process regulates tumor evolution under the guidance of the
immune system. It comprises an initial elimination phase targeting nascent tumor cells, followed by an equilibrium phase wherein the immune response and tumor cells enter a state of homeostasis, temporarily constraining tumor expansion, though imposing selective pressures that prompt molecular adaptations. Tumors eventually escape immunosurveillance, leading to the development of detectable benign tumors and escalating the risk of malignancy.

Our data pinpoints a novel immunosurveillance escape mechanism involved in intestinal tumorigenesis that implicates long-distance communication with hematopoietic organs. Using a genetically engineered mouse model of colorectal carcinogenesis
(Msh2Loxp/LoxpKrasLSL/+) we observed accelerated tumor growth compared with single mutants. Twenty-week-old Msh2/Kras mice displayed multiple tumors throughout the small intestine, enlarged spleens, signs of anemia, and histologic alterations in the bone marrow. The spleens of Msh2/Kras tumor-bearing mice displayed a significant decrease in CD45+ cells and an increase of F4/80+ compared with age-matched wild-type and tumor-free single mutant mice. Additionally, the number of tumors shows a positive correlation with spleen size and a negative correlation with the percentage of spleen CD45+ cells. We are now addressing how these alterations impact the tumor immune microenvironment. Nevertheless, Msh2Loxp/Loxp mice also presented similar histologic alterations in the spleen and bone marrow when they developed tumors (~40 weeks), suggesting a common immunoregulatory mechanism. Our ongoing investigation focuses on elucidating whether mutant KRAS facilitates escape from immunosurveillance by
differentially regulating the communication between tumors and the hematopoietic
organs, thereby accelerating tumorigenesis.

In summary, our study highlights a novel immunosurveillance escape mechanism
involved in intestinal tumorigenesis through long-distance communication with
hematopoietic organs. This knowledge is crucial for the development of novel therapeutic strategies aimed at bolstering the immune response against nascent tumors, impeding
their escape, and ultimately curbing the progression toward malignancy.



Instituto de Biología Molecular y Celular del Cáncer, Spain

Abstract Title

Tumour regression upon targeted protein degradation of KRasG12V in lung cancer.

Lung cancer (LC) is the leading cause of cancer related deaths globally. Lung adenocarcinoma (LUAD), the major histological subtype of LC, exhibits one of the highest mutation rates found in KRAS, a member of the canonical RAS family of genes involved in linking upstream cell
surface receptors to downstream proliferation and survival pathways such as RAF-MEK-ERK and PI3K-AKT-mTOR. Despite being one of the most frequent oncogenic drivers in cancer, KRAS mutations have been therapeutically pursued for nearly 40 years yielding little success.
However, the promising clinical benefits stemming from the recent discovery of covalent inhibitors targeting KRASG12C  mutant protein, have renewed the hope of inhibiting oncogenic KRAS through pharmacological tools. Unfortunately, these small molecule inhibitors end up failing due to the appearance of acquired on- and off-target resistance mechanisms, emphasising the need for additional strategies. Accordingly, proteolysis targeting chimeras (PROTACs) technology represents a powerful approach. PROTACs are heterobifunctional molecules able to degrade target proteins by hijacking the ubiquitin-proteasome system. To assess the therapeutic potential of upcoming KRAS targeting PROTACs and based on the recently described dTAG system, we induced murine dTAG-KRasG12V driven lung tumours from which we established LUAD cell lines susceptible to KRASG12V degradation by PROTAC based molecules. In vitro data reveals that degrader treated cells cease to proliferate rapidly.

Furthermore, KRASG12C  degradation is accompanied by an efficient MAPK pathway abrogation with a concomitant increase of cleaved caspase-3, cell cycle inhibitors and p-AKT expression levels. Interestingly, prolonged PROTAC treatment leads to the development of resistant cells that can be resensitized by alternating the use of different E3 ligase recruiting PROTACs.
Finally, LUAD cells intravenously injected into both immunocompetent and immunocompromised mouse models, result in a profound lung tumour burden reduction as soon as one week upon treatment initiation, underlining the high expectations placed on this novel therapeutic intervention.



300K Solutions SL, Salamanca, Spain

Abstract Title

Novel Dry Storage Approach for DNA Preservation at Room Temperature

In the era of precision oncology, many of the new targeted therapies under development are biomarker driven, therefore accurate and standardize assays are needed in clinical trials for high quality NGS data. Clinical research requires from high quality samples that are not always processed immediately after collection. Pre-analytical variations such as collection, processing, storage, and transportation need to be considered to minimize their influence on the outcome of data. Current gold standard method for DNA preservation is ultra-low temperature freezing at -80oC which implies high maintenance costs, large spaces, constant energy supply and safety measures for Biobanks.

We evaluated the quality and suitability of the genetic material processed and stored with an innovative solution based on precision drying of DNA samples for long term storage. DNA samples were stored in parallel under 3 conditions: -80C; dried and stored at 22C; dried and stored at 60C. We evaluated the integrity and purity of the DNA samples during the 18 months of real storage using TapeStation, Long Multiplex PCR and absorbance ratios as standard methods. The 21 years of RT-equivalent storage did not significantly affect the DNA integrity and the detection of the 17,5Kb band in multiplex Long PCR. Whole Exome Sequencing of the RT storage samples showed a recall of 99% in all time points analyzed, when compared with the -80C gold standard. Finally, we performed a Cytoscan 750K analysis of DNA samples just after its extraction and at the end of the storage period at different conditions, which revealed the same alterations, showing the same loss or gain patterns observed at extraction.

Based on the results obtained, the technology here evaluated demonstrated to be suitable for the long-term storage of DNA under RT conditions, preserving its integrity and purity and allowing its use with high demanding genomic methods, even after 21 years of RT-equivalent storage. A standardized methodology like this may enormously contribute to the safety and sustainability of sample storage.


Martín Martín

Universidad Austral de Chile, Chile

Abstract Title

Targeting AMD1 and KRASG12C for Novel Therapeutic Strategies in Non-Small Cell Lung Cancer

Non-small cell lung cancer (NSCLC) is the most deadly form of lung cancer. The G12C point mutation in the KRAS protein is found in 20-30% of NSCLC patients and is associated with a poor prognosis. Adagrasib and sotorasib are novel inhibitors of the KRASG12C protein that have shown efficacy in KRASG12C-driven NSCLC. KRASG12C inhibitors often face resistance, highlighting the need to explore combination treatments. Polyamines (putrescine, spermidine, and spermine), crucial
molecules for cancer growth and survival, are elevated in various cancer types,
including NSCLC. Adenosylmethionine decarboxylase-1 (AMD1) is an enzyme pivotal for spermidine and spermine synthesis and can be selectively inhibited by the
compound SAM486.The objective of this work was to study the levels of AMD1 in
KRAS-mutated NSCLC cells and evaluate the role of SAM486 in NSCLC cell proliferation alone or in combination with the KRASG12C inhibitors, adagrasib and sotorasib.

We used the NSCLC cell lines H358 (KRASG12C), A549 (KRASG12S), CORL-L23
(KRASG12V), and H1299 (KRASwt). AMD1 levels and KRAS-ERK pathway
phosphorylation were measured by immunoblotting. Cell proliferation was evaluated by BrdU incorporation and colony formation assay. Cell viability was measured by MTT reduction, and drug combination studies were analyzed using Combenefit and SynergyFinder software, employing the Loewe additivity model.

SAM486 reduced cell proliferation in NSCLC cells, independent of the KRAS mutation. Also, the KRASG12C inhibitors, sotorasib and adagrasib, reduced AMD1 levels and cell viability in H358 cells harboring the KRASG12C mutation. Additionally, high synergy scores were observed when using sotorasib and adagrasib in combination with SAM486 in the cells harboring the KRASG12C
mutation, as analyzed by Combenefit and SynergyFinder software, both employing
the Loewe additivity model as the analysis method.

These results suggest that the combination of a KRASG12C inhibitor and an AMD1
inhibitor could lead to a new therapeutic strategy against KRASG12C-mutated tumors.


Martín Vega

University of Texas Southwestern Medical Center, USA

Abstract Title

Mechanisms of ERK action in MEK inhibitor-insensitive lung cancers

Small cell lung cancer (SCLC) and other neuroendocrine (NE) lung tumors account for 25% of all lung cancers, the most aggressive types with poorest prognosis. The absence of driver oncogenes involved in
kinase regulation combined with acquired resistance limit the efficacy of the few available treatments resulting in relapse. Unlike in other tumor types, tumorigenic mutations in the RAS-ERK pathway are rare
in SCLC. Previous studies demonstrated that ERK activation interferes with SCLC growth. However, the mechanism is still unknown. The transcription factor ASCL1 is essential for survival in most SCLC and other NE tumors. ASCL1 increases DUSP6 transcription and consequently suppresses ERK activity.
Conversely, ASCL1 downregulation by ERK activation affects cell cycle progression and survival. DUSP6 inhibition with BCI sustains ERK activation, decreasing ASCL1. One concern is potential off-target effects of BCI. To assess its specificity, we compared its effects on viability and proliferation to DUSP6 KO cells.
Both pharmacological inhibition and genetic ablation of DUSP6 decreased cell viability and proliferation in ASCL1-high cells. BCI was not toxic in cells lacking the target. In addition to this negative relationship
between ASCL1 expression and ERK activity, high chromatin acetylation levels render low ASCL1 expression. Furthermore, a complete knockout of ERK1/2 is not achievable in these tumors as some ERK activity is required for their viability. However, each isoform can be deleted individually. Interestingly, ERK2 KO cells undergo an adaptation process leading to higher proliferation than the WT cells presumably attributed to constitutive Akt activation. Further studies are required to elucidate the implications of ERK activity in NE lung tumors and the potential mechanisms of resistance that may arise upon targeted therapies. A clearer perception of SCLC biology and the identification of the mechanisms underlying
therapy resistance will be crucial for developing effective treatment strategies for these recalcitrant tumors.



IPATIMUP, Portugal

Abstract Title

Chromatin remodeling as a new potential epigenetic mechanism of tolerance to KRAS loss

Although KRAS-targeted inhibition has yielded promising clinical responses, the development of resistance to these therapies has revealed that cancer cells can easily bypass the loss of the key oncogene to which they were previously addicted. Therefore, in this study, we aimed to
understand how mutant KRAS cancer cells tolerate the loss of KRAS, which would shed light on the mechanism of KRAS inhibition resistance.

We utilized siRNAs to silence KRAS expression in colorectal cancer (CRC) cell lines that carry KRAS mutations. We then used mass spectrometry to characterize the proteome of these cells, and the expression of significantly altered proteins was validated by western blotting. Additionally, we investigated chromatin states using both transmission electron and partial wave spectroscopic (PWS) microscopy.

Our results showed that upon KRAS inhibition, there was a significant reduction in the number of cells, accompanied by changes in the cell cycle, which supported KRAS-dependency. Proteomics analysis revealed that KRAS inhibition-tolerant cells upregulated several proteins associated with
the extracellular exosome or nuclear compartments. Molecular function and biological process gene ontology terms revealed an up-regulation of proteins mainly associated with binding activities (RNA, protein, nucleosome, and core promoter binding), as well as gene expression
regulation, mRNA splicing and processing, and nucleosome assembly and repositioning. Moreover, the proteomics data also revealed an upregulation of proteins associated with active chromatin states.

In addition, our findings showed that KRAS-silenced persister cells presented alterations in some histone post-translational modifications and in chromatin packing, which suggests that transcription is impacted.

These observations suggest an epigenetic mechanism underlying tolerance to KRAS inhibition, which involves chromatin structural changes and transcriptional alterations. We are currently pursuing this mechanism to gain a better understanding of the resistance to KRAS inhibition.



The Christie NHS Foundation Trust, UK

Abstract Title

Investigating KRAS mutant isoform-specific therapeutic vulnerabilities in non-small cell lung cancer

Direct inhibitors of KRASG12C and more recently KRASG12D are currently under clinical trial assessment and represent the beginning of a major translational breakthrough for non-small cell lung cancer (NSCLC) and cancer in general, targeting its most commonly mutated oncogene. However early reports of resistance with these small molecules have highlighted the pressing need for rationale combination partners. While combination therapies to enhance KRASG12D inhibition and combat innate resistance have already been reported for pancreatic and colorectal cancer, the application of KRASG12D inhibitors alongside combination partners to KRASG12D-driven NSCLC has not been investigated.

Here we compared KRASG12C– and KRASG12D -initiated lung tumorigenesis using genetically engineered mouse models andcell models of KRASG12C and KRASG12D -drivenNSCLC initiationin terms of oncopotency and signaling and examined whether signaling differences persist in available established tumors. We identified that these two mutant KRAS isoforms have contrasting potencies in driving tumor initiation which is accompanied by co-operation from distinct signaling programmes. This difference in oncogenicity is lost when tumors are established, which is reflected in the survival outcomes of internationally-recruited patients affected by advanced KRASG12C and KRASG12D NSCLC. However, the signaling differences are still preserved, offering therapeutically exploitable targets. Targeting our identified KRASG12D -specific vulnerability alongside KRASG12D inhibition (MRTX-1133) induced a robust synergistic apoptotic response in KRASG12D tumor cell lines of varying sensitivity to KRASG12D inhibition, highlighting a combinatorial approach to override intrinsic resistance to KRASG12D inhibitors.

Our data proposea unique combination treatment vulnerability that suggests patient selection strategies for combination approaches should be i) contextualised to individual RAS mutants, and ii) tailored to their downstream signaling programmes.



Medical Faculty and University Hospital Carl Gustav Carus, TU Dresden, Germany

Abstract Title

Efficient Correction of Oncogenic KRAS and TP53 Mutations through CRISPR Base Editing

KRAS is the most frequently mutated oncogene in human cancer, and its activating mutations represent long-sought therapeutic targets. Programmable nucleases, particularly the CRISPR-Cas9 system, provide an attractive tool for genetically targeting KRAS mutations in cancer cells. In this study, we show that cleavage of a panel of KRAS driver mutations suppresses growth in various human cancer cell lines, revealing their dependence on mutant KRAS. However, analysis of the remaining cell population after long-term Cas9 expression revealed the occurence of
oncogenic KRAS escape variants that were resistant to Cas9-cleavage. In contrast,
the use of a cleavage-deficient adenine base editor to correct a panel of KRAS mutations progressively depleted the targeted cells without the appearance of escape variants and allowed efficient and simultaneous correction of a cancerassociated TP53 mutation. Furthermore, base editing of a TP53 mutation restored the wildtype TP53 allele and function in patient-derived organoid model, demonstrating the
utility of genome editing for precision oncology. Finally, oncogenic KRAS and TP53 base editing was possible in cancer cell lines and in patient-derived cancer organoids, suggesting that base editor approaches to correct cancer mutations could be developed for the functional interrogation of patient-specific vulnerabilities for cancer therapy.


Mendonça Oliveira

IPATIMUP, Portugal

Abstract Title

Triple threat to therapy response: Fibroblasts, cancer stem cells and KRAS- inhibited persister cells

KRAS-targeted therapies are a promising approach for treating a subset of cancer patients with KRAS mutations, but response rates are lower than expected and many patients quickly develop resistance to
this treatment by molecular mechanisms still unknown.

Cancer stem cells (CSC) are thought to be responsible for resistance to therapy. Furthermore, both KRAS oncogenic activation and cancer associated fibroblasts (CAFs) have been shown to be crucial in generating and maintaining CSC. Moreover, we showed that 60% of CAF-induced alterations in the proteome of mutant KRAS colorectal cancer (CRC) cell lines occur independently of KRAS. Therefore, we hypothesize that CAF-derived factors may drive stem phenotype, and consequently induce resistance to KRAS-targeted inhibition.
We first analyzed the basal expression of stem cell markers (SCM) by flow cytometry in KRAS mutated CRC cell lines. The expression of SCM was heterogeneous, giving each cell line a unique stem marker signature. After KRAS silencing, CD24 was the only SCM up-regulated across all cell lines while integrins α6 and β4 were down-regulated. Furthermore, silencing KRAS led to a decreased colonosphere-forming efficiency (CFE), both suggesting a reduced stem cell potential across cell lines.

Then, we evaluated the CFE of CRC cell lines in the presence of conditioned media (CM) of fibroblasts activated or not. This led to only minor changes in the expression of some SCM but increased the CFE in all cell lines. After combinatory KRAS silencing and treatment with CM of CAFs, surprisingly, CRC cells were able to override the inhibitory effects of KRAS silencing and increase their CFE to values similar of the non-silenced cells.

RNAseq analysis suggests that cholesterol and NOTCH signaling pathways might play a role in CFE recovery. Altogether, our data highlights a novel mechanism of resistance to KRAS inhibition involving the induction of CSC activity mediated by CAFs-derived factors.



CRUK Beatson Institute, UK

Abstract Title

The characterisation and targeted treatment of mutant KRASG12C in genetically engineered mouse models of colorectal cancer

Intestinal tumorigenesis driven by loss of WNT suppressor gene Apc, co-occurring with a point mutation in the KRAS oncogene represents 40% of CRC cases. These mutations drive tumour growth via the MAPK/ PIK3ca pathways, rendering this subset of CRC patients ineligible for current targeted therapies. Mutant specific KRASG12C inhibitors offer novel treatment opportunities in epithelial cancers however only modest effects are observed in CRC. As an expanded repertoire of RAS targeting
molecules emerge, we aim to characterise the impact of direct RAS inhibition in CRC and identify durable combination treatments.

We generated a clinically relevant model of Apc deficient, KRASG12C mutant CRC in genetically engineered mice (VillinCreER Apcfl/+ KRASG12C/+). In analyzing the response of 3D intestinal organoids generated from tumours of the aforementioned model, we identified an additive effect of the clinically approved AKT inhibitor (Capivasertib – AZD5363) to KRASG12C inhibition by AZD4625 and determined such impact on proliferation and disease progression in vivo. Additionally, we generated models of allelic imbalance (VillinCreER Apcfl/+ KRASG12C/fl) which recapitulate a common genetic alteration, the loss of WT KRAS, that occurs during the progression of KRAS driven CRC. We examined the impact of KRASG12C inhibition in these models, given current literature indicates loss of WT KRAS renders KRAS mutants susceptible to targeting of the MAPK pathway. These models introduce a representative platform for understanding CRC with respect to mechanism and response of KRAS mutations.


Ming Ching

Institute of Pathology, Charité – Universitätsmedizin, Germany

Abstract Title

Concurrent inhibition of AXL and KRAS/ERK overcomes compensatory feedback mechanisms mediated by adaptive responses in pancreatic cancer

Pancreatic cancer is the third leading cause of cancer deaths in Germany. Mutational activation of the KRAS oncogene is found in ~95% of pancreatic ductal adenocarcinoma (PDAC). Thus, the development of effective anti-KRAS therapies will make a significant impact on the treatment of this deadly disease.
A major milestone in cancer drug discovery was the 2021/2 approval of KRAS inhibitors specific for the KRASG12C mutation in lung cancer. However, patients who respond initially relapse due to acquired resistance due to ERK activation. Furthermore, the KRASG12C mutation respresents less than 2% of KRAS mutations in PDAC. Our studies address both these limitations. First, since ERK inhibitors as monotherapy have shown limited/no clinical efficacy, in part due to treatment-induced onset of resistance mechanisms, and additionally limited by normal cell toxicity, we identified drug combinations that enhance ERK inhibitor clinical efficacy. We performed a 525-oncology drug library screen and discovered AXL receptor tyrosine kinase inhibitors as synergistic combination partners with the ERK inhibitor SCH772984 (ERKi). We determined that treatment with the AXL-selective inhibitor bemcentinib/BGB324 (AXLi) alone suppressed PDAC growth. However, AXLi treatment was associated
with a compensatory activation of ERK. Conversely, ERKi treatment was associated with a compensatory increase in AXL activation. To target these compensatory activities, we evaluated concurrent AXL and ERK inhibition and observed synergistic growth suppression. Our ongoing studies
are evaluating the combination of AXL and ERK inhibition in organoid models of PDAC. Second, we are evaluating the combination of AXL and KRAS inhibitors for KRASG12D -mutant PDAC cell lines. To better
understand the mechanistic for for the combination-associated synergy, are applying reverse phase protein arrays (RPPA) signaling activation profiling. Finally, we are applying CRISPR-Cas9 screens to define genes that modulate combination sensitivity. In summary, our observations support the promise of combination inhibition of AXL and ERK for PDAC treatment.



University of Turin, Italy

Abstract Title

Dissecting the cell state transition from persistence to resistance associated with KRAS G12C-specific inhibitor response in lung cancer

Drug resistance is a bottleneck in the clinical treatment of cancer, limiting durable therapeutic benefits. Cancer cells, rather than being either sensitive or resistant, can be dynamic and transient, highlighting a generalized adaptive mechanism of survival due to non-genetic variations and resumption of drug sensitivity upon drug removal. This particular state-lying between sensitivity and resistance-is termed the “drug-tolerant persister” (DTP) state. The recent development of direct inhibitors of KRASG12C (G12Ci) has initiated a new era in the clinical management of KRAS-mutant
lung adenocarcinoma. Despite high initial efficacy, tumors develop secondary resistance in almost all patients. Several
genomic alterations have been described in patients with acquired resistance to G12Ci, but adaptive mechanisms of resistance to G12Ci arising from persister cells have not been described in lung cancer yet. We used human and murine cell lines harbouring a KRASG12C mutation to study in vitro the formation of persister/resistant cells over time upon treatment with G12Ci, by alternating cycles “on” treatment and “off” treatment. The number of residual cells was
monitored over time and RNA and proteins were collected at different time points for molecular and gene expression analysis. Preliminary results suggest that lung cancer cells treated with G12Ci for one week undergo a G1-arrest and downregulation of cyclin D1 followed by apoptosis of sensitive cells, while two weeks treatment allows the emergence of drug-tolerant subclones with a slow-cycling phenotype that could be reverted upon drug removal. Long-term
schedules of intermittent treatment for up to 30 days give rise to resistant clones with activated ERK, AKT, CRAF and MEK and upregulation of different RAS isoforms. The molecular characterization of these drug-refractory cells before acquisition of permanent resistance is crucial to explain the onset of clinical relapse in those patients where acquired alterations are not detected and to identify new therapeutic vulnerabilities.



The Christie NHS Foundation Trust, UK

Abstract Title

Persisting RAS addiction- a therapeutic vulnerability in the context of KRAS G12C inhibitor resistance

Lung cancer is the most common cause of cancer related death. Mutations in KRAS are
found in ≈30% of lung adenocarcinomas, with G12C the most common. The development of KRAS G12C inhibitors provides a new targeted therapy option for patients who were previously dependent upon combinations of chemotherapy and immunotherapy. Initial results from clinical trials with G12C inhibitors are promising but the development of acquired resistance highlights limitations of these agents. It is highly likely that combinations will be
needed to derive the most benefit of these inhibitors.

To investigate mechanisms of acquired resistance a panel of KRAS G12C mutant lung adenocarcinoma cell lines were cultured with a G12C inactive state selective inhibitor for over 9 months until there were clear changes in sensitivity. Characterisation of these cell lines with bulk RNA-seq and proteomics shows differences in MAPK and AKT signalling between untreated and G12C inhibitor cultured cells. Immunoblotting suggests that in resistant cells, G12C inhibitor is bound to KRAS and remains bound at 48 hours suggesting that resistance is not driven by mutations affecting drug binding or drug efflux. Following G12C inhibitor treatment some cell lines underwent epithelial to mesenchymal transition whilst one cell line underwent mesenchymal to epithelial transition suggesting that plasticity may be more important in resistance rather than the specific morphological state. A single cell RNA-seq experiment was performed to better understand the evolution of drug resistance and to identify transcriptomic states that may be associated with resistance to G12C inhibitors.

Alongside characterisation of the cell lines, the resistant cell lines have been treated with a variety of targeted inhibitors. The resistant cell lines remain sensitive to active state selective RAS inhibition suggesting that resistant cell lines remain addicted to oncogenic RAS signalling. Future work is aimed at better understanding the mechanism behind this therapeutic vulnerability.


Muñoz Félix

University of Salamanca, Spain

Abstract Title

Vessel co-option can be inhibited by targeting BMP9-ALK1 pathway in experimental lung metastases

Primary tumors and metastases require oxygen and nutrients for their growth. For this purpose, vascularization strategies are developed, being angiogenesis the main process studied. Angiogenesis consists of the formation of blood vessels from pre-existing ones. However, many tumors or metastases can gorw through non-angiogenic vascularization strategies, where tumor cells take advantage of pre-existing blood vessels in a mechanism known as vessel co-option. These tumors do not respond to anti-angiogenic therapies. BMP9 is a circulating factor that induces blood vessel quiescence through ALK1 receptor and downstream signalling by Sma1/5/8.
This pathway interacts with many signalling pathways such as Ras-MAPK. We hypothesize co-opted vessels are quiescent and thus tumor vessel co-option can by inhibited by targeting this pathway.

In our laboratory we have developed experimental models of lung metástasis in BALB/c mice. We use a breast cancer cell line (4T1), which generates vessel co-option growth and another renal carcinoma cell line (RENCA), which generates tumor angiogenesis. In both models we inhibited the BMP9-ALK1 signalling pathway by using the inhibitor K02288.

In vitro studies demonstrate that K02288 induces proliferation in endothelial cells (EC) but not in tumor cells. K02288 treatment leads to a decrease in tumor burden indicating that the inhibition of cell quiescence leads to a reduction of lung metastases growing via vessel co-option and also in lung metastases undergoing angiogenesis. These changes are related with a decrease in tumor hypoxia and an increase of CD8+ cell infiltration.

These results indicate us that the inhibition of vessel quiescence can reduce tumor growth in metastases undergoing vessel co-option and promoting an immunopermissive tumor microenvironment. Moreover, the inhibition of vessel quiescence with K02288 also reduces tumor growth in angiogenic metastases in a mechanism that needs to be further studied.



NYU Langone Health, USA

Abstract Title

New Approaches to Targeting KRAS

KRAS mutations are major driver abnormalities in human cancer. Recently, two covalent KRAS (G12Ci) were approved for use in second line non-small cell lung cancer (NSCLC) patients, and inhibitors of other mutant alleles are in development. While G12Cis represent a landmark in cancer
therapeutics, responses are incomplete, resistance always develops, and cure remains elusive. Immune checkpoint therapy can also be effective in NSCLC, and long-term responses can occur.
Yet tumors fail to respond completely or at all. KRAS-mutant NSCLC with co-mutations in STK11 (LKB1) and/or KEAP1 is particularly refractory to conventional, targeted, and immune therapeutics. We took several approaches to augment G12Ci therapy. First, we carried out a genome-wide CRISPR screen for adagrasib synthetic lethality in four KRAS/STK11-mutant NSCLC lines, three of which also are KEAP1-mutant. Multiple recurrent synthetic lethal (SL) “hits” were identified, including three serine-threonine kinases, multiple tRNA biosynthetic enzymes, proteoglycan and glycosylation
enzymes, DNA damage proteins, and YAP/TAZ and/or TEAD transcription factors. We validated several hits using orthogonal shRNAs and also found that their over-expression conferred G12Ci resistance. Because TEAD inhibitors are in clinical development, we extensively validated the
YAP/TAZ/TEAD pathway, using cell-derived xenograft and genetically engineered mouse models (GEMMs) and genetic and pharmacologic modulation of TEAD activity. Analysis of GEMMs that acquired G12Ci resistance and resistant patient samples revealed strong overlap between pathways
enriched in SL hits and G12Ci resistance at the transcriptomic and proteomic levels. Mechanistic analysis indicated that G12Ci results in induction of multiple RHO pathway genes and increased nuclear translocation of YAP and TAZ. Interestingly ROCK inhibition partially blocks YAP/TAZ
translocation and increases G12Ci efficacy in vitro. We also performed similar SL screens and analysis of the same NSCLC lines treated with G12Ci +SHP2i.

In parallel, we developed a therapeutic strategy (“HapimmuneTM”) that exploits that hapten properties of covalent protein adducts formed when G12Cis react with KRAS. Using phage display technology, we identified scFvs that bind selectively to G12C peptides presented on multiple HLAs,
including those of different pseudotype. Combining these “front ends” with anti-CD3 “back-ends” yields hybrid molecules capable of directing selective, T-cell directed killing of G12Ci-treated cells. This strategy can generalize to multiple G12Cis and other covalent targeted therapies.




Abstract Title

AI-Driven Multi-Scale Investigation of the RAS/RAF Activation Lifecycle

The goal of the AI-Driven Multi-Scale  Investigation of the RAS/RAF Activation   Lifecycle (ADMIRRAL) Consortium is to elucidate the molecular details leading to activation of RAF kinase at the plasma
membrane. To achieve this, we enhanced our Multi-scale Machine-Learned Modeling Infrastructure (MuMMI) to capture biologically relevant time- and length-scales for RAS-RAF biology and the activation
process. The MuMMI architecture provides highly accurate simulations that are self-healing, have quantifiable uncertainties, and generate testable hypotheses. We iteratively parameterize, couple, and validate these multi-resolution simulations with advanced imaging, biochemical, biophysical, and
structural data to build a dynamic model of RAS-driven RAF activation in the context of a complex and fluctuating plasma membrane. Our previous studies, focused on KRAS and a realistic membrane mimetic, support the hypothesis that protein-lipid interactions are important determinants of KRAS clustering and generate local environments that are conducive to signaling through the binding and activation of RAF Kinase. Addition of the RAF kinase RBDCRD shows that RBDCRD induces unique RAS states, there is no strong interface preference between RAS or RAS-RBDCRD, and RAS-RBDCRD states
are correlated with distinct lipid fingerprints in a stable signaling platform precursor. This is consistent with a model where cooperative molecular interaction between lipids, RAS and RAF are necessary to form nanoclusters and initiate the assembly of signaling hub. We are now dissecting the molecular mechanism of RAF activation by incorporating autoinhibited RAF. Initial simulations and experiments suggest that RAF RBD in the autoinhibited complex is dynamic and available for RAS binding. We are also assessing the dynamics and energetics of the other autoinhibited RAF components (CRD, 14-3-3, CR2/CR3, and the kinase domain) to initiate simulations that will capture the molecular events during
the release of autoinhibition, including domain movement and rearrangement needed for the full activation of dimerized, kinase-active RAF.

This work was performed under the auspices of the US Department of Energy (DOE) by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344; and under the auspices of the NCI by FNLCR under Contract 75N91019D00024 and the NCI Center for Cancer Research. This work has been supported in part by the NCI-DOE Collaboration program established by the US DOE and the NCI of the NIH.


Nuevo Tapioles

NYU Langone Health. Perlmutter Cancer Center, USA

Abstract Title

KRAS4A promotes hexokinase 1 oligomerization on the outer mitochondrial membrane

RAS genes are the most frequently mutated oncogenes in human cancer and therefore are considered important targets for anticancer therapy. Among them, KRAS is mutated more often than the other two RAS genes. The KRAS pre-mRNA is alternatively spliced to generate two transcripts, KRAS4A and KRAS4B, that encode small GTPases that differ almost exclusively in their C-terminal hypervariable regions that control subcellular trafficking and membrane
association. Whereas KRAS4B is basal to all RAS genes, the KRAS4A isoform arose 475 million years ago in jawed vertebrates and has persisted in all vertebrates ever since, strongly suggesting non-overlapping functions of the splice variants. We recently found that depalmitoylated, GTP-bound KRAS4A binds to and directly regulates hexokinase I (HK1) by limiting the allosteric feedback inhibition physiologically mediated by glucose-6-phosphate. These findings provide the first evidence of a RAS isoform-specific regulation of a metabolic enzyme. HK1 forms dimers on the outer mitochondrial membrane (OMM), and this configuration is essential for its allosteric regulation. Therefore, we hypothesized that KRAS4A increases the activity of HK1 by regulating its oligomerization state on the OMM. Using co-immunoprecipitation, blue native gels, and FLIM-FRET assays, here we show that depalmitoylated, GTP-bound KRAS4A interacts with HK1 and promotes its oligomerization. Using subcellular fractionation and HK1 mutants that lack a mitochondrial targeting sequence, we demonstrate that while KRAS4A does not affect the abundance of HK1 on the OMM, it promotes its oligomerization exclusively on the surface of the organelle. Super-resolution microscopy and single molecule tracking (SMT) revealed domains on the OMM enriched for HK1 where oligomerization appears to take place. Expression of depalmitoylated, GTP-bound KRAS4A increased the dwell time of HK1 on the OMM as measured by SMT. These data support a model whereby KRAS4A regulates HK1 by influencing its oligomerization on the OMM.



AstraZeneca, UK

Abstract Title

Kinetic profiling of response to G12C inhibition in KrasG12C;Stk11null lung tumour models shows evidence of drug tolerance in vivo

Mutant KRAS inhibitors brought a new outlook for mutant KRAS cancers, but clinical data indicate that their efficacy can be limited by the early onset of resistance. To improve our understanding of mutant KRAS inhibition and resistance in lung tumours we generated KRASG12C ;Stk11null (KS) genetically engineered lung cancer mouse models1, as STK11 is frequently co-mutated with KRAS in lung adenocarcinoma and associated with chemo- and checkpoint inhibitor resistance. KS lung tumours were then treated with AZD4625 , a covalent allosteric inhibitor of KRASG12C , and acute and long-term responses evaluated.

AZD4625 treatment resulted in rapid pathway inhibition in tumours and strong induction of apoptosis and cell cycle arrest within 1-3 days of treatment. MRI-guided longitudinal analysis of individual tumours confirmed robust tumour inhibition by AZD4625, with 80% of tumours showing partial or total responses within 1 week of treatment. Importantly, G12C inhibition significantly improved survival and overall health (AZD4625: 100% survival; Vehicle: 40%, after 3 weeks of treatment). Of note, Stk11- and p53-null lung tumours responded similarly to G12C inhibition, in agreement with clinical data from sotorasib-treated patients.

Despite the high efficacy of AZD4625 in KS lung tumours, long-term treatment exposed heterogeneity in tumour responses. Fifty-six tumours were followed longitudinally by MRI and while all initially regressed, 17 (27%; variable initial size) remained visible, albeit stable, after 6 weeks of treatment, suggesting adaptive resistance to G12C-inhibition. Strikingly, when treatment was discontinued, most tumours rapidly regrew at their original location including those classed as complete responders, indicating the presence of drug tolerant cells in most original tumours. RNAseq analysis confirmed that long-term AZD4625 treatment drove the emergency of unique expression signatures in ~2/3 of surviving tumours. These putative signatures of resistance to G12C inhibitors are currently being validated to ultimately guide the development of improved therapies for mutant KRAS cancers.

1 Lundin et al., Nat Commun (2020)

2 Chakraborty et al., Mol Cancer Ther (2022)


Olarte San Juan

Instituto de Biología Molecular y Celular del Cáncer & CIBERONC, Spain

Abstract Title

SOS-RAS GEFs as potential therapeutic targets in liver cancer and metabolism

RAS mutations are frequent (about 30%) in various human cancers. The activity of RAS
proteins is positively regulated by guanine nucleotide exchangers (GEFs) of the SOS1/2
family that is homogeneously expressed in different tissues and stages of development.
Recent work in our laboratory has shown that SOS1-mediated signalling is involved in
controlling mitochondrial metabolism, skin homeostasis, organismal viability, and
carcinogenesis. In addition, SOS-RAS-MAPK signalling pathway plays an essential role
in metabolism. Since obesity and fat intake are known risk factors for cancer,
understanding the functional relevance of SOS1/2 GEFs in lipid metabolism under
physiological and pathological contexts is crucial.

Deletion of both SOS1/2 isoforms in MEFs caused a significant loss of their ability to
differentiate into adipocytes, whereas single SOS2KO cells kept a normal adipocytic
differentiation capacity. Interestingly, SOS2KO mice showed a substantial increase in body weight compared to WT or SOS1KO mice after prolonged feeding with HFD. The
HFD also caused significant fat accumulation in the livers of SOS2KO and WT mice, in sharp contrast to the livers of single SOS1KO mice fed with the same diet. Late-stage disruption of SOS1 decreased the aggressiveness of DEN-induced hepatocarcinoma in certain SOS1KO mice than WT and SOS2KO mice. Moreover, early-stage disruption showed greater improvement, indicating a relevant role of SOS1 in the early stages of carcinogenesis.

Our findings indicate SOS1 and SOS2 GEFs have distinct functions in the adipose tissue
and liver homeostasis of adult mice, which could help uncover potential therapeutic
targets for metabolic diseases and/or cancer.



Verastem Oncology, USA

Abstract Title

The RAF/MEK clamp avutometinib induces an immunogenic tumor microenvironment and potentiates the efficacy of anti-PD-1

The RAS/RAF/MEK/ERK (MAPK) pathway is one of the most commonly mutated oncogenic pathways in human cancers. Although RAS, RAF and MEK have been validated as anticancer targets with approval of KRAS G12C, BRAF and MEK inhibitors, combination strategies with chemotherapy, targeted therapies and/or immune checkpoint inhibitors may be optimal for deep and durable response.

Avutometinib is a unique RAF/MEK clamp that potently inhibits MEK kinase activity and induces dominant negative RAF/MEK complexes preventing phosphorylation of MEK by ARAF, BRAF and CRAF. Preclinically, avutometinib has shown strong anti- roliferative potency across tumor cell lines carrying various MAPK pathway alterations. Clinically, avutometinib monotherapy has shown responses in gynecological cancers and KRAS mutant non-small cell lung cancer. Here, we tested the immune modulatory effects of avutometinib on tumor cells and tumor-infiltrating immune cells and assessed anti-tumor efficacy in mice treated with avutometinib in combination with an anti-PD1 antibody.

In a panel of KRAS and BRAF mutant human tumor cell lines, avutometinib treatment increased the expression of MHC-I complex genes, including B2M, HLA-A and TAP1. In the CT26 KRAS G12D syngeneic colorectal cancer model, upregulation of B2M and TAP1 by avutometinib was confirmed in vivo, suggesting that avutometinib may increase antigen presentation. Furthermore, avutometinib upregulated the expression of markers of T cells(CD8, PDCD1), NK cell activation (NCR1) and interferon response (IFNG, IRF7, IL12) in the CT26 model. Interestingly, all these pro-immune changes observed with avutometinib were stronger than those observed with an equivalent dose level of the MEK-only inhibitor trametinib.
Avutometinib also increased expression of B2M, CD8A, PDCD1, NCR1 and IRF7 in a KPARG12C orthotopic lung cancer model. Flow cytometry analysis showed that avutometinib also significantly increased MHC- II expression by tumor cells and the numbers of CD8 T cells and M1 macrophages, and significantly decreased monocytic and granulocytic MDSCs. These immune changes indicate that avutometinib induces an immunogenic tumor microenvironment that may potentiate the efficacy of immuno-oncology agents such as anti-PD-1. Accordingly, in the CT26 model, whereas avutometinib and anti-PD-1 each delayed tumor growth, combination of avutometinib with anti-PD-1 increased antitumor efficacy and prolonged survival. Furthermore, all complete responders in the avutometinib + anti-PD-1 group were able to reject a re-challenge with CT26 tumor cells and showed increased CD8 and CD4 effector memory T cells relative to untreated naïve control mice, indicating that avutometinib + anti-PD-1 treatment induces durable immune memory.

These results support clinical evaluation of avutometinib in combination with an anti-PD-1 antibody for treatment of patients with solid tumors harboring MAPK pathway alterations such as KRAS or BRAF mutations.



University of Eastern Finland, Finland

Abstract Title

The dynamic characteristics of the druggable Switch-II pocket of KRAS – is it transferable to other RAS GTPases?

The KRAS switch-II pocket (SII-P) has proven to be one of the most successful tools for
targeting KRAS with small molecules to date. This has been demonstrated with several KRAS(G12C)-targeting covalent inhibitors, already resulting in two FDA-approved drugs. Several earlier-stage compounds have also been reported to engage KRAS SII-P with other position 12 mutants, including G12D, G12S, and G12R. The SII-P is very enigmatic in nature,
as it is enclosed by the highly dynamic  switch-II region [1]. Over 70 SII-P binder co-crystal structures are publicly available to date, which have considerably improved our understanding of this pocket.

In the contribution I will provide insights into what those structures have revealed about
the SII-P, including aspects related to the crucial Thr58-associated conserved water [2].
Furthermore, looking beyond the static crystal structures, I will illustrate what the classical molecular dynamics (MD) simulations suggest about the pocket behavior with the bound ligands [2,3], including two clinical candidates with 200 μs simulation data [4]. Finally, one of the important future questions will be addressed: can the SII-P strategy be transferred to other RAS GTPases beyond KRAS? Related to this I will disclose our key findings related to SII-P from our 1 millisecond MD simulations of MRAS, RRAS and RRAS2 GTPases [5].

[1] Pantsar T. The current understanding of KRAS protein structure and dynamics. Comput Struct Biotechnol J. 2020, 18, 189–198.

[2] Leini R & Pantsar T. In Silico Evaluation of the Thr58-Associated Conserved Water with
KRAS Switch-II Pocket Binders. J Chem Inf Model. 2023, 63, 5, 1490–1505.

[3] Pantsar T. KRAS(G12C)–AMG 510 interaction dynamics revealed by all-atom molecular dynamics simulations. Sci Rep 2020 10, 11992.

[4] Leini R & Pantsar T. Manuscript in preparation.

[5] Kurki M & Pantsar T. Manuscript in preparation.



Charité Berlin, Germany

Abstract Title

Deciphering Cell Cycle Plasticity to ERK Inhibition in KRAS-driven Pancreatic Cancer Cells at the Single-Cell Level

The KRAS oncogene is mutationally activated in ~95% of pancreatic ductal adenocarcinoma (PDAC) and represents an important therapeutic target. With current clinical KRAS inhibitors limited to the
KRASG12C mutant, the most promising therapeutic approach remains inhibition of the key KRAS effector pathway, the ERK mitogen-activated protein kinase (MAPK)-signaling cascade. However, treatment-induced acquired drug resistance will limit long-term anti-tumor efficacy.

To better understand ERK inhibitor-induced cell cycle arrest, we have applied a multiplex protein imaging approach. Multiplex imaging allowed us to monitor 55 antibody-specific expression levels, activities, and localizations of proteins involved in cell cycle regulation, cell signaling, and cancer stemness at the single-cell level. We screened nine KRAS-mutant PDAC cell lines treated for five days using five concentrations of the ERK1/2-selective inhibitor LY3214996 from 125 to 2000 nM.

Our analysis of 250.000 images allowed us to characterize 300 features in each of the identified 600.000 single cells. Applying manifold learning, we constructed cell cycle structures and respective trajectories towards cell cycle progression and arrest. We identified several cell cycle detours, that
appeared exclusively under ERK inhibition. Only small cell subpopulations utilized these new cell cycle routes to escape the ERK inhibitor-induced cell cycle arrest, making it almost impossible for total cell population analyses to identify these resistance-driving cells. Within these ERK inhibitor-induced cell
cycle detours we discovered novel as well as expected resistance mechanisms such as modulation of the RB tumor suppressor. Our current analyses are focused on evaluating the novel mechanisms of cell cycle arrest escape mechanisms shared between subsets of PDAC cells.

In summary, our multiplex imaging approach offers unique insights on cell cycle adaptation in response to targeted therapies. A better understanding of an adaptive cell cycle and its resistance-driving mechanisms will help to develop more effective treatment strategies to reduce the rapid onset of drug resistance.


Pericacho Bustos

Universidad de Salamanca & IBSAL, Spain

Abstract Title

Effect of the expression of Endoglin on the adherent junctions of VE-Cadherin in cultured endothelial cells

Objectives: Endoglin overexpression in tumor vasculature results in leaky
vessels that impairs blood flow and facilitates metastasis. The aim of this study
is to analyze whether endothelial endoglin levels affects the expression or
organization of VE-Cadherin, causing unstable junctions.

Methods: This study was performed on an established human endothelial cell line infected with a vector carrying the endoglin cDNA (ENG+ cells) or with an empty vector (Mock cells). The subcellular organization of VE-Cadherin and the structure of adherent junctions were studied by immunofluorescence. Gene and protein expression of VE-Cadherin and other key proteins of the signaling pathways were analyzed by qPCR and Western Blot.

Results: The results show that confluent cells present an increase in VE-Cadherin expression and this change is significantly higher in ENG+ cells. We observed that endoglin overexpression alters the stabilization of VE-Cadherin junctions, keeping them with a more active phenotype. Moreover, this overexpression impairs contact inhibition. On the other hand, our results indicate that phosphorylation levels of AKT, ERK and p38 decrease upon achieving confluence; being this decrease blocked by endoglin overexpression.

Conclusions: The overexpression of endoglin in endothelial cells upon achieving confluence causes an increase in the expression of VE-Cadherin that leads to the formation of more unstable or active junctions that prevent the correct stabilization and functioning of the adherent junctions and, ultimately, the generation of fenestrated blood vessels that may allow the intravasation of tumor cells into the blood, increasing the number of metastases.


Quirós Fernández

DKFZ/Universidad de Costa Rica, Costa Rica

Abstract Title

Identification and characterization of T cell receptors reactive to a KRAS G12V neoepitope

Approximately 90% of KRAS mutations are detected in exon 2 in codon 12, 1,2 which result in a single amino acid substitution.Less common are mutations in codon 13 (2-6%) and 61 (1%)Non–small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancers. Mutated KRAS is the most common oncogene in NSCLC, accounting for about 30% of cases, being the most frequent mutations G12C (42%), G12V (21%) and G12D (17%). We have developed an optimized and fast protocol for the identification of T cell receptors (TCRs), which are reactive to a given neoepitope presented by HLA molecules, from the CD8+ T cell repertoire of healthy individuals. Our approach is based on two rounds of isolation and expansion of CD8 T cells bound to peptide-HLA-multimers, followed by single-cell TRAV and TRBV sequencing. By this means, we identified five different TCRs that specifically recognize a KRAS5-14  peptide carrying the G12V mutation presented in the context of HLA-A*02:01. NK-92CD3/CD8 cell line derivatives transfected to express separately the TCRs were capable of binding KRAS G12V5-14-multimers with different affinities, but not KRAS WT5-14. The transgenic NK-92CD3/CD8 –TCR cells showed TCR-mediated cell activation when co-incubated with T2 cells loaded with low amounts of the target peptide, and cytotoxicity against SW480 (KRAS G12V) tumor cells. We are currently developing a co-stimulatory chimeric antigen receptor to improve the cytotoxic capacity and specificity of the effector cells expressing the KRAS TCRs as described elsewhere.3

1. Reita D, Pabst L, Pencreach E, Guérin E, Dano L, Rimelen V, Voegeli AC, Vallat L, Mascaux C, Beau-Faller M. Direct
Targeting KRAS Mutation in Non-Small Cell Lung Cancer: Focus on Resistance. Cancers (Basel). 2022 Mar 4;14(5):1321. doi: 

2. Cascetta P, Marinello A, Lazzari C, Gregorc V, Planchard D, Bianco R, Normanno N, Morabito A. KRAS in NSCLC: State of
the Art and Future Perspectives. Cancers (Basel). 2022 Nov 4;14(21):5430. doi: 10.3390/cancers14215430.

3. Quiros-Fernandez, I, Poorebrahim, M, Marmé, F, Burdach, SEG, Cid-Arregui, A (2023) A costimulatory chimeric antigen
receptor targeting TROP2 enhances the cytotoxicity of NK cells expressing a T cell receptor reactive to human papillomavirus
type 16 E7. Cancer Letters 566: 216242.



Instituto de Biología Molecular y Celular del Cáncer, Spain

Abstract Title

Functional screening of potential scaffold proteins controlling KRAS nancolustering

KRAS is a small GTPase protein bound to the plasma membrane that activates different
signaling pathways. It is one of the most frequent oncogenic drivers in cancers, such as lung or pancreatic adenocarcinoma. Targeting KRAS has been a challenge for decades and after the development of several covalent inhibitors against this protein, now we are facing a new problem: resistance to these drugs. The need for alternative strategies has led us to focus on
other molecular aspects of the activation of KRAS. The ability of this protein to organize itself into nanoclusters seems to be a well-established event that might be crucial to implement its biological activity. However, we still have to decipher the mechanism by which KRAS forms these complexed structures. Previous studies showed that scaffold proteins may help KRAS in the formation of these nanoclusters. To approach this issue, we have used the A549 lung adenocarcinoma cell line to develop an inducible tripartite fluorescent readout that depends on KRAS membrane clustering. Through the CRISPR Knock-out and CRISPR activation technique, we will test the role of a list of 24 scaffold proteins that might be potentially implicated in the formation and/or stabilization of KRAS assemblies. In parallel, we will be using total internal
reflection fluorescence (TIRF) microscopy to evaluate how interfering with the most promising scaffolds affects the dynamic of KRAS nanoclusters. Furthermore, we are currently using this cellular model to perform an unbiased chemical screen aimed to identify compounds that reduce KRAS membrane clustering. With this research, we hope to increase our mechanistic
knowledge of the regulation of KRAS clustering that may result in alternative treatments for patients with resistance to KRAS direct inhibitors.



Instituto de Biología Molecular y Celular del Cáncer, Spain

Abstract Title

Exploring the Role of SOS RAS-GEFs in Mitochondrial Metabolism in immortalized MEFs

RAS GTPases, oncogenes mutated in almost 30% of cancers, are signal transduction
regulators that cycle between their active (GTP-bound) and inactive (GDP-bound) states. The cycling process is regulated by GTPase activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs), being SOS proteins (SOS1/2) the most relevant RAS-GEFs due to their ubiquitous expression and functional relevance. To study their functional specificity or redundancy in physiological and pathological roles our laboratory developed a mouse model based on conditional SOS1 knockout (KO) and constitutive SOS2 KO. Thanks to this model different physiological abnormalities related to SOS1 ablation have been found, such as mitochondrial and redox balance defects, in primary Mouse Embryonic Fibroblasts (MEFs). Therefore, we hypothesized that these defects are caused due to a lack of SOS-activated RAS, and that restoring RAS signaling would in consequence reverse the reported defects.

To test our hypothesis, we generated immortalized WT, SOS1-KO, SOS2-KO and
SOS1/2-DKO MEFs (iMEFs), obtaining a genetically manipulable in vitro model. iMEFs were characterized at the level of signaling, proliferation and respiratory metabolism. SOS deletion was related to lower proliferation rates in our iMEFs. Regarding REDOX balance, we detected that SOS1/2-DKO iMEFs produced higher Reactive Oxygen Species. Additionally, SOS2-KO and SOS1/2-DKO iMEFs showed differences in mitochondrial morphology, whereas SOS1-devoid genotypes displayed respiratory defects. Moreover, different active RAS isoforms were transfected to iMEFs, observing a partial rescue of the mitochondrial respiratory phenotype on SOS1-ablated genotypes.

As a whole, our results confirm the importance of SOS proteins in mitochondrial
metabolism, glimpsing the key role of SOS/RAS signaling in mitochondrial respiratory metabolism and suggesting that RAS isoforms might have functional specificity in this context.



Boehringer Ingelheim, Austria

Abstract Title

Relative Cycling Rates of KRAS Mutant Alleles and Selectivity of Response to KRAS Inhibitor Treatment

KRAS cycles between the GDP-bound (OFF state) and the GTP-bound (ON state) state (Kim et al., 2020). Only the ON state of KRAS allows interaction with downstream RAF kinases and signaling output of the MAP
kinase pathway. Most inhibitors of KRAS target the OFF state of KRAS (Kim et al., 2020) and consequently shifting the balance towards the ON state has been observed as a key resistance pathway for KRAS G12C
inhibitors (Hallin et al., 2020; Kim et al., 2020; Lito et al., 2016; Xue et al., 2020). Therefore, it is important to understand the allele specific balance between the OFF and the ON state of KRAS mutants to understand
opportunities and potential limitations for OFF state inhibitors targeting other alleles. We here probed the kinetic changes in signaling output of the KRAS node across various alleles in an isogenic background to
evaluate the mutant-specific cycling rates. We find that the several KRAS mutants show wildtype-like cycling rates (and should therefore be responsive to KRAS OFF inhibitors), while others are significantly impaired in their cycling rates compared to G12C and are therefore likely more challenging to drug with OFF state
inhibitors. We also find that the approach can be used to determine allele-specificity of a certain inhibitor class. Together, the approach can be used to assess the efficacy and specificity of KRAS inhibitors on the ON state of various KRAS mutant alleles.



IIS Aragón, Spain

Abstract Title

Minnelide chemosensitizes pancreatic ductal adenocarcinoma by preferentially targeting the NRF2 pathway in cancer stem cells

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest tumors, partly due to the intrinsic aggressiveness and chemoresistance of resident subpopulations of cancer cells, the Cancer Stem Cells (CSCs). We have previously proved that pancreatic CSCs are especially sensitive to redox imbalance, induced by either overproduction of mitochondrial ROS or
depletion of the antioxidant glutathione. Here, we demonstrate that the potent anti-tumor activity shown for the compound Triptolide and its water-soluble analogue Minnelide can be largely attributed to its ability to induce oxidative stress in PDAC cells. Using a variety of Patient-Derived
Xenografts (PDXs) models, this effect was most strongly confined to the contained CSCs and their tumor-promoting functionality. Specifically, Triptolide abrogated colony and sphere
formation abilities, as well as in vivo tumorigenicity. Mechanistically, Triptolide inhibited the expression of NRF2 and its downstream target GCLC, the rate-limiting enzyme implicated in glutathione synthesis, thus inducing intracellular glutathione depletion. Interestingly, in several
public databases we found that high NRF2 expression was not only related to K-Ras signaling but also to stemness in PDAC patients. Triptolide-induced oxidative stress caused mitochondrial malfunction, which translated into decreased mitochondrial respiration and ATP production, most
likely inducing an energy crisis and subsequent cell death preferentially in the CSC subpopulation. Intriguingly, in vivo treatment with Minnelide resulted in effective chemosensitization for 16 out of 20 tested PDX models, translating into stable disease or even partial remission. Response to Minnelide was positively correlated with NRF2 expression in the tumors, suggesting its value as predictive biomarker for identifying responsive phenotypes. In
summary, our data indicate that Minnelide treatment effectively reverses tumor progression by inhibiting the antioxidant response via NRF2 in a significant population of PDAC PDXs, and that NRF2 expression may predict response to the compound in vivo.


Sanjuan Hidalgo

Centre for Genomics and Oncological Research (GENyO), Spain

Abstract Title

High-Fidelity Cas 9-mediated targeting of KRAS driver mutations restrains lung cancer in preclinical models.

Lung cancer is the subtype of cancer with the highest mortality worldwide. The most
mutated oncogene in lung cancer patients (~30%) is KRAS (G12C mutation for smoker
patients, and G12D for non-smokers). During the last decade, KRAS was considered
an undruggable target for lung cancer therapy until the development of two novel drugs (sotorasib and adagrasib), recently approved by the FDA for the treatment of KRASG12C lung cancer patients. However, the performance of these two compounds has fallen short of expectations, mainly due to the acquisition of resistance in the patients a short time after the treatment. Thus, in this work we propose a proof-of-concept for a CRISPR/Cas9-based therapeutical strategy against mutant KRAS G12C and G12D (KRASMut ) lung cancer. By using a high-fidelity version of Cas9 (HiFi-Cas9) and designing single guide RNAs (sgRNAs) against KRASMut , we were able to specifically target mutant KRAS cells, while leaving wildtype KRAS cells untouched. Our CRISPR- KRAS therapy was able to impair tumor growth of KRASMut  cells both in 2D and 3D cultures in vitro, as well as in in vivo cell-derived xenografts (CDX). Moreover, our CRISPR-KRAS therapy reduced tumor growth in preclinical models of patient-derived xenografts (PDX) when delivered in the form of intratumorally-injected adenoviral particles, and further cell viability experiments in cell lines showed that our HiFi-Cas9 therapeutic strategy is comparable to KRASG12C inhibitor sotorasib.

In conclusion, our CRISPR-KRAS strategy presents a proof-of-concept for specifically and efficiently disrupting KRASG12C/G12D in tumor cells without affecting, KRASWT  cells. This shows greater versatility and could potentially circumvent the problems with resistances to KRASG12C inhibitors like sotorasib. However, further research is still necessary to overcome the off-target and delivery issues that hinder the clinical use of CRISPR/Cas9-based therapies in cancer.


Santos Ramos

Instituto de Investigación Hospital 12 de Octubre (i+12) & CNIO, Spain

Abstract Title

Combination of KRASG12C and pan-KRAS inhibitors as a therapeutic strategy to overcome resistances

KRAS is the most frequent mutated driver oncogen in lung adenocarcinoma (LUAD)
nevertheless, it was considered undruggable for a long time. Recently, the first direct
inhibitors against KRASG12C (G12Ci) have been approved by FDA/EMA, however, ~60%
of patients do not respond because they are intrinsic resistants, and the ~40% of responder patients develop acquired resistance. Some resistance mechanisms are the appearance of new RAS alterations and upstream hyperactivation. Thus, the use of new pan-KRASi in combination with G12Ci could result as an optimal therapeutic strategy to overcome resistances.

A panel of 8 KRASG12C LUAD cell lines plus 8 KRASG12C LUAD PDX-derived Organoids
(PDXDO) were tested for KRASG12Ci sensitivity and defined as responder or resistant. We also generated 11 acquired resistant LUAD cell linesto G12Ci. We tested the efficacy of the combination of KRASG12C and pan-KRAS inhibitors in all intrinsic and acquired resistant models, previously characterize at the proteomic (phosphor-Array and WB), genomic (WES) and transcriptomic (WTS) levels. We evaluated the KRAS pathway inhibition with the combination by Western-Blot.

We found 2 cell lines and 3 PDXDO intrinsically resistant to KRASG12Ci. In addition, we generated 11 acquired resistant cell lines. In resistant models we found molecular alterations that could explain resistance, such as KRAS amplifications, other commutations, EMT activation or different levels of activation of RTKs signaling pathways. KRASG12C and pan-KRAS inhibitors combination treatment showed efficacy in some of intrinsic and acquired resistant models. Comparing the models depending on the response to the treatment, we observed differences in the activation or expression of signaling pathways of RTKs which could explain the response/non-response to combination. In conclusion, this combination strategy could resensitizes the intrinsic resistance or delay the appearance of acquired resistance, but it would be necessary to validate some biomarkers that would help us to predict the response in patients.



University of Turin, Italy

Abstract Title

EML4-ALK mediates resistance to KRAS G12C inhibition

KRAS is one of the most frequently mutated oncogenes in cancer and has been considered undruggable for decades, untill the development of two KRASG12C  inhibitors, sotorasib and adagrasib, that were approved by FDA between 2021 and 2022. The response rate in KRASG12C-tumors represented a major clinical breakthrough, nevertheless therapy resistance invariably occurs. Secondary on-target mutations have been found in patients, as well as potential by-pass mechanisms that, however, remain to be validated.

Among patients enrolled in the KRYSTAL-1 clinical trial, two relapsed patients showed acquired EML4-ALK fusion. This work investigates whether EML4-ALK drives the resistance to adagrasib via the reactivation of the MAPK pathway and we validated EML4-ALK as a therapeutic target in adagrasib-resistant tumors.

By CRISPR-Cas9 approach, we engineered the EML4-ALK fusion in KRASG12C NSCLC cell lines that were tested in vitro and in vivo. We kept these cells in constant presence of adagrasib to mimic the clinical scenario where acquired alterations are selected upon treatment. 

In vitro
, the acquisition of EML4-ALK in KRASG12C  NSCLC cell lines conferred resistance to adagrasib compared to parental cell lines. The reactivation of the MAPK pathway, triggered by an increase of GTP-KRAS, was blocked upon concomitant treatment with adagrasib and the ALKi alectinib. In vivo, double mutant KRASG12C/EML4-ALK xenografts were resistant to adagrasib as single agent, but sensitive to adagrasib in combination with ALKi. When cells were subjected to constant treatment with adagrasib in vitro, their oncogenic signaling rewired towards dependency on EML4-ALK signaling and became sensitive to ALKi alone while remaining resistant to adagrasib. Mechanistically, the MAPK pathway remained active with high levels of GTP-KRAS in presence of adagrasib, while it was inhibited by ALKi treatment, alone or in combination with adagrasib. Likewise, ALKi both as single agent and in combination with adagrasib showed significant therapeutic activity in vivo.



University of Turin, Italy

Abstract Title

Impact of microbiota in anticancer therapies: a study on Lacticaseibacillus rhamnosus GG in KRAS G12C cellular models.

KRAS mutations are common in lung adenocarcinomas (LUADs) and are associated with poor prognosis in patients. Although new targeted therapies for G12C mutations have been approved in the clinic, resistance has already emerged as a major challenge. The role of microbiota in cancer
research has developed in this context, as the formation of a tumoral microenvironment (TME) leads to microbial dysbiosis, which may worsen the pathological setting and alter therapeutic responses. KRAS-driven LUAD in mice generated a peculiar TME that disrupted the microbiota by favoring certain species and exacerbating tumor severity. Also, the aerosolization of Lacticaseibacillus rhamnosus GG (GG) in the lungs prevented metastasis and improved response to standard chemotherapy. Our overall aim is decoding both the reciprocal relationship between microbial-dependent patterns and tumor cells and the influence of certain bacterial species on response and resistance to therapy. We describe here a preliminary study on the effects of GG on KRAS G12C cellular models, both in naïve conditions and resistant to the targeted inhibitor sotorasib (AMG). GG activated the MAPK pathway to different extents in the cell lines suggesting that a heterogeneous background can impact the responses to an environmental stimulus. In addition, when AMG resistant cells were treated with AMG and GG, the drug sensitivity was restored. Thus, we have demonstrated in vitro that in KRAS-driven tumor cells a diverse biological setting results in different responses of lung cancer cells to probiotics, which had also a promising impact on the resistance to therapies. This preliminary observation poses the basis for unraveling novel strategies for reversing therapy resistance and harnessing the relationship between microbiota and KRAS-driven tumor cells in LUADs. The ultimate objective of our research is not only to develop more effective treatments for KRAS-driven lung cancer but also to comprehend the complex scenario of microbiota-cancer interactions.



IIS Aragón, Spain

Abstract Title

Development nanobody-based imaging agents for non-invasive diagnosis and monitoring of pancreatic ductal adenocarcinoma.

The revolution in cancer genomics has uncovered clinically relevant alterations that have yet to be integrated into the clinical management of patients, in part due to the lack of non-invasive imaging biomarkers. An innovative option is termed “immunotargeted imaging”. By merging the
high target specificity of antibodies with the high spatial resolution, sensitivity, and quantitative capabilities of molecular imaging techniques (e.g. PET/SPECT), it is possible to conduct a non-invasive diagnosis and monitoring of patients over time using in vivo, integrated, quantifiable, 3D, full body “immunohistochemistry”.

The limitations of current imaging techniques for pancreatic ductal adenocarcinoma (PDAC), including challenges in accessing clinicopathologic samples and underestimating tumor extension, coupled with the low resectability rate and limited post-surgical survival, highlight the potential of ImmunoPET/SPECT to enhance molecular diagnostics, reduce the need for incomplete biopsies, and improve patient outcomes.

Applying bioinformatics on patient datasets, we have identified suitable molecular targets for the development of immunoPET/SPECT agents for PDAC whose expression is associated with poor survival and impaired response to chemotherapy. To generate imaging agents based on these biomarkers, we used camel derived nanobodies (Nb) from a novel Nb-library obtained by immunization of camels with human tumor cells over-expressing our targets of interest followed by a novel E. coli display system.

For clinical development we will humanize the selected Nbs and we will label them with PET isotopes such as 68Gallium, which advantageously can be produced in a generator rather than a cyclotron. We are exploiting this approach to label multiple imaging tracers to the same pretargeted Nb in vitro as well as multi-modal and multifunctional imaging and theranostics. These imaging agents could be used for other tumor types and pathologies and may have a major impact on the diagnosis and monitoring of patients.



The Christie NHS Foundation Trust, UK

Abstract Title

The E3 Ubiquitin Ligase HUWE1 is required for KRAS-driven NSCLC Tumorigenesis

Ubiquitination is often dysregulated in cancer, reshaping the cellular proteome to promote behaviours associated with tumour initiation and malignant progression – such as increased cell survival and proliferation.
In RAS mutant cancers the proteasome is the only hit to arise from multiple synthetic lethality screens [1]. Whilst use of general proteasome inhibitors has shown some promise in KRAS mutant Non-Small Cell Lung Cancer (NSCLC)[2], their use is hampered by severe dose-limiting toxicity. Instead, we pose that specific E3 ligases or
ubiquitination events could be manipulated to selectively target cancer cells. Across patient datasets the ubiquitin-proteasome system is dysregulated in NSCLC, and the E3 ubiquitin ligase HUWE1 is known to regulate
tumorigenesis in a range of cancer contexts through ubiquitination of a variety of substrates [3].

Here we show that HUWE1 is essential for the formation of mutant KRAS induced lung tumours in vivo, but is not required for the maintenance or function of untransformed alveolar type II (AT2) cells in the normal, healthy mouse lung. We identify that this phenotype is independent of known regulation of p53 [4]. Consistent with this,
NSCLC cells suffer growth aberrations following HUWE1 depletion in vitro and we have employed multiple proteomics approaches to interrogate the molecular mechanism by which HUWE1 contributes to NSCLC tumorigenesis. From available clinical datasets we have identified that HUWE1 is commonly upregulated in LUAD tumour samples compared to matched normal lung tissue, and higher expression correlates with poorer patient survival. This is corroborated by IHC staining of a LUAD tissue microarray (TMA) for HUWE1. Together, these data demonstrate a cancer-specific requirement for HUWE1 in the alveolar epithelia in the context of KRAS mutant NSCLC.

[1] Downward J., Clin Cancer Res., (2015)
[2] Drilon A, et al., Cold Spring Harb Mol Case Stud., (2019)
[3] Kao SH, et al., J Biomed Sci, (2018)
[4] Yang D, et al., Theranostics, (2018)



University of Turin, Italy

Abstract Title

Deciphering cellular vulnerabilities to mutant RAS and RAF expression

Therapies targeting mutated RAS and RAF proteins and signalling pathways controlled by them are becoming increasingly important for the treatment of oncogene-driven cancers. However, the tumour cell populations develop a variety of resistance mechanisms, which limit long-lasting therapy success. Therefore, it is necessary to identify specific vulnerabilities that could inform the future design of combination therapies or other downstream measures after initial direct anti-RAS or anti-RAF therapy. To this end, we used tumour model cells conditionally expressing KRASG12V, HRASG12V or BRAF V600E and identifed synthetically lethal factors as well as up-regulated targets, whose expression is necessary for the expression of oncogenic phenotypes. We describe the results of synthetic lethality screens in colorectal cancer cells with lentiviral shRNA libraries representing previously identified MAPK targets. Furthermore, we report a multi-omics analysis of the time-resolved effects of RAS expression in pre-neoplastic cells. We identified the DNA replication licensing factor minichromosome maintenance complex component 7 (MCM7), whose knockdown induces DNA damage and replication stress in KRASG12V-expressing Caco2 cells, but not in Caco2 wild-type cells. In addition, we found splicing factor proline and glutamine rich (SFPQ) and Claspin (CLSPN), whose ablation leads to collision between replication and transcription in BRAFV600E-expressing cells. Among the mutant HRASG12V-responsive transcription factors, non-histone chromatin factor high mobility AT-hook2 (HMGA2) and FOS-like 1 (FOSL1), a member of the AP1 transcription factor family, were particularly prominent. Both HMGA2 and FOSL1 ablation leads to significant reversion of mutant RAS-mediated transcription and, at the phenotypic level, to loss of anchorage independence and epithelial-mesenchymal transition. In the future, small molecule inhibitors directed against these factors could be interesting candidate compounds to limit oncogene effects alone or in combination with direct RAS or RAF inhibitors.



Systems Biology Ireland, University College Dublin, Ireland

Abstract Title

A combination of conformation-specific RAF inhibitors synergises to inhibit tumour proliferation in Pancreatic Ductal Adenocarcinoma cell lines.

Pancreatic ductal adenocarcinoma (PDAC), a very aggressive form of cancer(1) displays KRASmutation in 95% of cases leading to the hyperactivation of downstream pathways such as RAF/MAPK and PI3K/AKT, which promote cell proliferation and survival (2,3). Before the discovery of a targetable pocket in KRAS-G12C mutant (4), RAS has long been considered undruggable and the research mainly focused on downstream targets with the development of RAF inhibitors (RAFi) (5). Those RAFi showed a very poor response in RAS tumours due to the paradoxical activation of ERK signalling caused by the
asymmetrical binding of the drugs to RAF dimers (6). However, a recent study based on structure modelling demonstrated that a combination of RAFi targeting different RAF conformations(CI/DO and CO/DI) synergise in RAS mutant melanoma cell lines to effectively inhibit cell proliferation (7). This
work aimed to determine if a combination of RAFi could synergise to inhibit cell growth in the context of PDAC. We determine the sensitivity of 3 PDAC cell lines (PSN1, PANC1 and Capan2) to a panel of RAFI: CO/DI (Vemurafenib and Encorafenib) or CI/DO (Sorafenib and TAK-632) and to MEK inhibitors (MEKi) (Trametinib and Cobimetinib). We observed that PANC1 was the most resistant cell line to both RAFi and MEKi, while PSN1 was the most sensitive to those drugs. These results were observed both for growth inhibition and MAPK signalling with a strong linear correlation between the GI20 (cell growth) and the pERK inhibition IC20. Using high doses of RAFi and MEKi, we have grown resistant
PSN1 cell lines. We observed that Vemurafenib-Sorafenib and Encorafenib-TAK-632 combinations efficiently synergized to inhibit both pERK signal and cell proliferation in a cell line-dependent manner. Those results suggest that a combination of RAFi could be a good strategy to inhibit MAPK-mediated
cell proliferation in the context of PDAC.


1. Hosein AN, Dougan SK, Aguirre AJ, Maitra A. Translational advances in pancreatic ductal adenocarcinoma therapy. Vol. 3, Nature Cancer. Nature Research; 2022. p. 272–86.

2. Hobbs GA, Der CJ, Rossman KL. RAS isoforms and mutations in cancer at a glance. J Cell Sci. 2016;129(7):1287–92.

3. Gimple RC, Wang X. RAS: Striking at the Core of the Oncogenic Circuitry. Front Oncol. 2019;9(September):1–16.

4. Ostrem JM, Peters U, Sos ML, Wells JA, Shokat KM. K-Ras(G12C) inhibitors allosterically control GTP affinity and effector interactions. Nature. 2013;503(7477):548–51.



Department of Biotechnology, Motilal Nehru National Institute of Technology Allahabad, U.P. INDIA

Abstract Title

CTNND1-RAB6A a novel RAS GTPase fusion transcript identified in CRC may modulate migratory potential of colon cancer cells

The interchange of DNA sequences between genes may occur as a result of structural chromosomal rearrangements; an alternative chimeric gene may be formed as a result of this. These fusion transcripts have been linked to carcinogenesis in various cancer types and have accumulated
significant interest in recent times. We used paired end RNA-seq. data of four CRC and one normal sample generated from our previous study. The STAR-Fusion pipeline was used to identify the fusion genes unique to CRC. The in silico identified fusion gene(s) were explored for their diagnostic/ therapeutic potential and validated through PCR and sequencing in our previously collected CRC sample cohort. Further, cell line-based studies were performed to gain functional insights of the novel fusion transcript. Two fusion transcripts (CTNND1-RAB6A, and
CASTOR2(GATSL1)-GTF2I) were identified. CTNND1-RAB6A fusion was amplified from one CRC sample. Sequencing revealed that there was a total loss of the CTNND1 gene, whereas RAB6A retained its coding sequence. Further, RAB6A was functionally characterized for its oncogenic potential using HCT116 cell line. RAB6A under expression was found to be significantly associated with increased cell migration and is proposed to be regulated via RAB6A-ECR1-Liprin-α axis. Our results suggest that the RAB6A gene may play a significant role in CRC oncogenesis and could be used as a potential biomarker and therapeutic target for better management of CRCs in future.

Keywords: Colorectal cancer, Fusion gene, CTNND1, RAB6A, STAR-Fusion, Cell Migration assay, ERC1/PPFIA1.



Centro Nacional de Investigaciones Oncológicas (CNIO), Spain

Abstract Title

Elimination of Hyaluronic acid in Pancreatic Ductal Adenocarcinoma (PDAC) as a potential Strategy for immunotherapyn

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related
deaths, with a five-year survival rate around 5–7%. In PDAC, stromal cancer-associated
fibroblasts (CAFs) play a vital role in promoting the desmoplastic and immunosuppressive tumor microenvironment (TME), as well as tumor growth and malignancy, and have emerged
as cancer targets (1). Has proteins are responsible for the production of hyaluronic acid (HA).
Has overexpression results in accumulation of HA which leads to high pressure on
neighboring structures as well as elevated interstitial fluid-pressure (IFP) within PDAC,
which can interfere with drug delivery (2) and has also been linked with tumor escape from immune surveillance. Previous results from our lab showed that Has1 and Has2 was
differentially expressed between PDGFRa+ CAFs and PDGFRα+ normal pancreatic
fibroblasts (NPFs) (3).

We have developed developed genetically modified mouse models (GEMMs) of Has triple knocked-out (Has1/2/3 TKO) based on Has2 conditional KO mice (4) to study the role of HA in PDAC development and progression. We aim to explore the influence of HA in tumor development and in the desmoplastic and immunosuppressive TME. In allograft studies, the TKO mice environment significantly inhibited proliferation of tumor cells competent for
these genes. These studies may help to design future therapeutic strategies.

(1) Sahai E et al. A framework for advancing our understanding of cancer-associated fibroblasts. Nat Rev Cancer 20, 174-186 (2020).

(2) Z. Luo, Y. Dai, H. Gao, Development and application of hyaluronic acid in tumor targeting drug delivery. Acta Pharm Sin B 9, 1099-1112 (2019).

(3) M. Djurec et al. Saa3 is a key mediator of the protumorigenic properties of cancer-associated fibroblasts in pancreatic tumors. Proceedings of the National Academy of Sciences of the United States of America 115, E1147-E1156 (2018).

(4) Matsumoto K et al. Conditional inactivation of Has2 reveals a crucial role for hyaluronan in skeletal growth, patterning, chondrocyte maturation and joint formation in the developing limb. Development. 136, 2825-35.(2019)



Imam Abdulrahman Bin Faisal University, Saudi Arabia

Abstract Title

In silico Drug Repurposing Approach of some FDA approved drugs against KRAS G12C for cancer therapy

RAS protein is one of the interesting cancer targets. RAS is a key upstream mediator of
several pathways that control cell growth, differentiation, and survival and it is the most frequently mutated oncogene in human cancer. RAS mutation is usually found in non-small cell lung cancer, colorectal cancer, and pancreatic cancer all of which have poor prognosis. Ever since discovered it was considered “undruggable “ till recently when a pocket was discovered in
KRAS G12C mutation that led to the discovery and approval of two inhibitors: Sotorasib and Adagrasib in 2021 and 2022, respectively. Unfortunately, resistance to these inhibitors was developed and started to become a significant issue. We aim to repurpose some FDA-approved drugs to target KRAS G12C mutation using in silico drug repurposing to find drugs that could
bind covalently with the Cys12 nucleophile of KRAS G12C.

A total of 1650 FDA-approved drugs were retrieved from the Zinc 15 database then
prepared and filtered using Maestro software resulting in 1590 unique compounds. Then, molecular docking was performed on the crystal structures of the human KRAS G12C protein retrieved from the Protein data bank (PDB code: 6OIM). High Throughput Virtual Screening (HTVS) provided us with 64 drugs that can be covalently bound to the KRAS G12C. A further Standard-precision (SP) screening results in 8 hits. Of the 8 hits, 2 were excluded and the resulting 6 hits (Capreomycin, Ertapenam, Cefdinir, Cefadroxil, Cortisone acetate, Natamycin)
were subjected to Shape Screening, Induced Fit Docking, and Covalent Docking to validate the binding.

Our study suggests that the 6 final hits have the potential to be a candidate for repurposing as an anticancer drug against KRAS G12C mutation. Future research will focus on performing molecular dynamics investigations and in vitro studies of the 6 potential agents against cell lines with KRAS G12C mutation.




Abstract Title

Ras and Lipidomics

Keywords: Palmitoylation, Ras, lipidomics

Posttranslational lipidation (farnesylation and palmitoylation) controls the subcellular trafficking, localization and oncogenicity of multiple Ras proteins [1,2]. As a result, there is a growing interest in understanding the basis of these modifications and the enzymes involved in their regulation. Moreover, recent evidence suggested that palmitoylation may not be restricted to the saturated C16 palmitate but also involves shorter, longer or unsaturated fatty acids. In addition, the functional implications of this heterogeneous lipidation and the possibility of representing a new layer of regulation remain largely unexplored.

Hydroxylamines at neutral pH are known to selectively cleave thioesters, releasing the corresponding fatty acids hydroxamates. We previously developed a fluorescently labelled hydroxylamine that enabled the assessment of the S-linked lipid species using lipidomics techniques. This approach revealed that the cellular S- acylome and an overexpressed N-Ras contain different fatty acids. Two N-Ras proteins bearing a palmitate and a palmitoleate were then prepared by ligation of a truncated protein with a lipidated C-terminal peptide and its different behavior was assessed using atomic force microscopy and FRET-based kinetics assays. [3]

In a step forward, we have also adapted this technique to use commercially available hydroxylamine. The optimized method has been applied to both cell lysates and animal tissues, disclosing significant differences in the lipid composition of their S-acylomes. In future studies we plan to apply this methodology to explore the lipidation pattern of endogenous S-acylated proteins.

1. Zimmermann G, Papke B, Ismail S, Vartak N, Chandra A, Hoffmann M, Hahn SA, Triola G, Wittinghofer A, Bastiaens PI, Waldmann H. Small molecule inhibition of the KRAS-PDEδ interaction impairs oncogenic KRAS signalling. Nature. 2013, 497(7451):638-42.

2. Weise K, Triola G, Brunsveld L, Waldmann H, Winter R. Influence of the lipidation motif on the partitioning and association of N-Ras in model membrane subdomains. J Am Chem Soc. 2009,131(4):1557-64.

3. Schulte-Zweckel, J., Dwivedi, M., Brockmeyer, A., Janning, P., Winter, R., and Triola, G. A hydroxylamine probe for profiling S-acylated fatty acids on proteins. Chem. Commun. 2019, 55 (75), 11183–11186.



Laboratory of Cellular Oncology, USA

Abstract Title

The pro-oncogenic non-canonical activity of a RAS-GTP:RAN-GAP complex facilitates nuclear protein export

Most RAS oncogenic activity is thought to be mediated by its canonical signaling, which results mainly from RAS-GTP interactions with its effectors at the plasma membrane. The best studied effectors are RAF and PI3K, which respectively activate MEK/ERK and AKT/mTOR pathways.
In this study, we have identified a fundamental non-canonical activity of RAS-GTP that increases the export of nuclear proteins into the cytoplasm and is independent of RAF/MEK and PI3K/AKT signaling. Nuclear protein export machinery is known to depend on a trimeric protein
complex composed of RAN-GTP, XPO1, and nuclear protein cargo. Surprisingly, we have found that RAS-GTP forms a stable complex with RAN GTPase-Activating Protein 1 (RAN-GAP), which facilitates the release of nuclear protein cargo into the cytoplasm by hydrolyzing RAN- GTP to RAN-GDP. Remarkably, the RAS-GTP:RAN-GAP complex has been identified in primary human tumors, patient derived xenografts (PDXs), and human lung cancer lines. RAS inhibition suppresses RAN-GTP hydrolysis and prevents protein cargo release. Conversely, high RAS activity increases RAN-GTP hydrolysis and promotes protein cargo release. Importantly, combined inhibition of PI3K and MEK does not inhibit RAS-GTP:RAN-GAP complex formation or affect its stimulation of nuclear protein export, which we find contributes to the oncogenic activity of RAS-GTP. One critical pro-oncogenic function of the RAS-GTP:RAN- GAP complex is to increase the cytoplasmic EZH2 methyltransferase, whose nuclear export leads to cytoplasmic Lysine-678 methylation of the DLC1 tumor suppressor protein and its subsequent ubiquitin-dependent proteasomal degradation. Inhibiting KRAS alone, which prevents nuclear export of EZH2 methyltransferase, thereby stabilizing the DLC1 protein, is more anti-oncogenic than combined inhibition of PI3K and MEK, which does not stabilize DLC1. This biological difference is abolished in isogenic lines from which the DLC1 gene has been knocked out. Thus, these findings establish nuclear protein export as a critical non-canonical pro-oncogenic RAS function that is mediated by the RAS-GTP:RAN-GAP complex.



Fundación para la Investigación Biomédica del Hospital Universitario 12 de octubre, Spain

Abstract Title

Effect of Fra-2 expression on tumour immunogenicity and prognosis in KRAS-mutant lung adenocarcinoma

Targeted therapies are available for lung adenocarcinoma (LUAD); however, more than 50% of patients either cannot benefit from them or become resistant, highlighting the need for alternative strategies. Our project focuses on FRA-2 (encoded by FOSL2 gene), a member of the AP-1 family that acts downstream of KRAS to control the expression of genes related to proliferation, migration and differentiation. Computational analyses of public databases have revealed a correlation between high FOSL2 expression and poor survival in KRAS-mutant LUAD.
Consistently, tumour cell expression of Fra-2 has been found to correlate with shorter survival and higher tumour burden in a genetically engineered mouse model of LUAD driven by KRasG12V mutation and Tp53 loss. Cell viability was assessed in primary cells cultured from KRasG12V/Tp53-
null mouse tumours with Fra-2 gain- and loss-of-function, confirmed that overexpression of Fra-2 increases tumour cell viability. However, the absence of Fra-2 did not affect cell growth, suggesting that in vivo observations in Fra-2-deficient tumours could depend on anti-tumoral mechanisms that are extrinsic to tumour cells.

In samples prospectively collected from LUAD patients subjected to tumour resection (N=27; 9 KRASmut), we found that the percentage of tumour cells expressing FRA-2 correlates with FRA-2 expression in immune cells as well as in cancer-associated fibroblasts (αSMA). FRA-2 protein expression tends to show a positive correlation with proliferation (KI-67) and a negative 
correlation with T cell infiltration. A more detailed analysis by flow cytometry revealed that FRA-2 expression was related to an increased tumour density of regulatory T cells and decreased PD-1 expression, particularly in KRAS mutant tumours.

Our results suggest that FRA-2 could play a significant role as a mediator in the tumorigenic effects driven by KRAS mutations in LUAD, by promoting both tumour cell proliferation and immune
evasion. These findings provide a new therapeutic opportunity in a clinically relevant setting.



Boehringer Ingelheim, Austria

Abstract Title

Dissecting the clonal evolution of resistance to KRAS inhibitors through genetic barcoding and scRNAseq

Acquired resistance to KRAS inhibitors (KRASi) limits their efficacy and poses as significant issue in the treatment of cancer patients.

In many instances this is due to the emergence of resistant cancer cell clones. To improve treatments, it is essential to understand how these resistant clones evolve and if they share vulnerabilities that
can be targeted. Therefore, their identification and characterization
is key. As resistance can originate from transient cell states of rare clones, an immediate functional readout through single-cell RNAseq (scRNAseq) analyses is necessary to identify these states.

Furthermore, lineage tracing approaches such as genetic barcoding allow to trace their contribution to resistance over time.
We have previously applied the barcoding tool CaTCH which additionally enables the retrospective isolation of resistance-founding clones from heterogeneous cell populations prior to their evolutionary
selection. By combining genetic barcoding and scRNAseq we aim to identify new entry points for improving therapies.



Catalan Institute of Oncology and Germans Trias i Pujol Research Institute (IGTP), Spain

Abstract Title

Overexpression of CD73 is dependent on KRAS mutations in colorectal cancer and can be targeted by MRTX1133

CD73 (NT5E) has been studied as a new cancer therapeutic target due to its role in producing extracellular adenosine from AMP, promoting immunosuppression. Its expression has been associated with RAS, BRAF and EGFR mutations in lung cancer. However, the role of CD73 in colorectal cancer (CRC) is little known. This work aimed to study CD73 expression in CRC, its
association with RAS mutations, and its possible targeting by KRAS inhibitors.

Materials and Methods
CRC cell lines: SW48 KRAS-WT and SW48 KRAS(G12D)-mutant (Horizon Discovery), Difi, LS513, LS174T, LoVo, and HCT116 (ATCC). CD73, ERK and p-ERK protein expression were measured by western blot (Cell Signaling), and CD73 also by flow cytometry (BioLegend). CD73 activity was measured by the 5’-Nucleotidase (CD73) Activity Assay (Abcam). CD73 IHC staining was performed in TMAs from 80 CRC samples. Associations of NT5E expression with RAS mutations
and the Consensus Molecular Subtypes (CMS) were performed by using RNA-seq data from the TCGA project (COAD=456 and READ=166).

CD73 expression was correlated with the presence of RAS mutations both at RNA (Wilcoxon p-value<0.001), and protein levels [72.5% of CD73-positive samples were RAS-mutant, compared to 46.2% within CD73-negative samples (Fisher’s p-value=0.04)]. Only KRAS-mutant cell lines expressed CD73, and perfectly correlated with activity in SW48-WT (0mU/mg) and -G12D cells (47.97mU/mg). AB680 (CD73 inhibitor), abrogated its activity. NT5E expression was higher in CMS1 and 3 (Wilcoxon p-value<0.001).

Trametinib (MEK inhibitor) decreased CD73 protein levels in SW48-G12D cells, while Capivasertib (AKT inhibitor) did not. MRTX1133 (KRAS-G12D inhibitor) decreased CD73 expression in KRAS G12D-mutant cells.

Our results suggest that RAS-mutant CRC is enriched in CD73 and that treatment with KRAS (or MEK) inhibitors, may induce its down-regulation. Further experiments are needed to elucidate if KRAS-mutant CRC patients may benefit from KRAS inhibitors, which can be potentially combined with cetuximab and/or CD73 inhibitors.


Vietti Michelina

University of Turin, Italy

Abstract Title

Sequential treatment with direct KRAS inhibitors: how to improve the management of KRAS G12C drugs

Introduction: lung cancer is responsible for 12% of cancer-related deaths worldwide (
Mutant KRAS, particularly KRASG12C , is the driver mutation in more than 25% of cases. Recently, two KRAS direct inhibitors, sotorasib (AMG510) and adagrasib (MTRX849), have been FDA approved for KRASG12C LUAD treatment. Even if they are effective, patients develop resistance to sotorasib or adagrasib monotherapy, leaving limited possibility for further clinical treatment with targeted therapies. Our research aims to determine whether treating in a sequential mode KRASG12C  cells who are resistant to sotorasib with adagrasib –and viceversa– can be successful in prolonging the response in vitro and in vivo.

 Results: Proliferation assays on adaptive-resistant cells revealed that, while MRTX-R cells were resistant to AMG, AMG-R cells were still sensitive to MRTX treatment. Afterwards, we studied the effects on MAPK pathway, which showed that both models were characterized by lower pERK levels upon sequential treatment with the reciprocal drug.

Constitutively resistant models showed different drug-response profile dependent on the mutation: KRAS G12C/Y96D and G12C/R68S were resistant to both drugs, whereas KRAS G12C/H95R and G12C/H95Q were resistant to MRTX849 but still slightly sensitive to AMG510. In this situation, combination therapy appeared to be more effective.

Adaptive resistant in vivo models confirmed that AMG-R tumors were partially responsive to MRTX treatment whereas MRTX-R tumors were resistant to AMG treatment.

-MRTX-R models are resistant to sotorasib treatment whereas AMG-R cells are still partially sensitive to adagrasib

-G12C/Y96D and G12C/R68S cells are resistant to both drugs

-G12C/H95Q and G12C/H95R are resistant to adagrasib, but slightly sensitive to sotorasib

in vivo, MRTX-R tumors are resistant to sotorasib treatment whereas AMG-R tumors are still partially sensitive to adagrasib



The Christie NHS Foundation Trust, UK

Abstract Title

Profiling copy number mutational signatures in KRAS mutant non-small cell lung cancer

Background: Lung cancer is the 3rd most common cancer in the UK and leading cause of cancer mortality worldwide. KRAS mutant cancers are the largest molecular subset of non-small cell lung cancer (NSCLC). Immunotherapy is standard 1st line therapy for advanced stage NSCLC, but reliable biomarkers for patient selection are lacking. Mutational signatures are a genetic imprint of mutational processes that have occurred during carcinogenesis, and preliminary evidence suggests a role in predicting immunotherapy response.

Methods: Tumour whole genome sequencing (WGS) data is available through the 100 000 Genomes Project (Genomics England). Copy number (CN) signature extraction was performed using SigProfiler (COSMIC version 3.3), and KRAS mutations profiled using Ensembl Variant Effect Predictor (VEP).

Results: 1058 NSCLC patients were included; 734 KRAS wild type (69%) and 324 KRAS mutant (31%). Common KRAS variants were present, including G12C (38%), G12V (16.4%) and G12D (14.1%). Nine CN signatures were extracted and present in both KRAS mutant and wild type cohorts. KRAS mutant cancers had lower CN signature activity when compared to wild type, except for the normal diploid signature (CN1, p = 0.017, OR 1.38). Within the KRAS mutant group 10 variants were analysed alongside wild type, with significant variation in CN signature expression seen across subtypes. KRAS G12D was positively associated with diploidy (CN1, p<0.05) and negatively associated with loss of heterozygosity (CN9, 13, p<0.05).

Discussion: KRAS mutant NSCLC is more chromosomally stable when compared to KRAS wild type. G12D is commonly associated with never smokers, and the more chromosomally stable phenotype observed could be explained by the association between tobacco smoke and chromosomal instability. Further work is ongoing to comprehensively profile the full set of mutational signatures, including known smoking signatures. Using reverse translation and clinical data integration, mutational signatures could provide prognostic information and inform predictive models.



University of Turku, Finland

Abstract Title

RAS-mediated regulation of ribosomal RNA processing by DEAD-box RNA helicases

High protein translation activity is one of the hallmarks of cancer and it requires efficient
ribosomal RNA (rRNA) processing. Consequently, dysregulated abundance of pre-rRNA transcripts correlates with malignancy and poor prognosis in several cancers. However, it is very poorly understood how rRNA processing is regulated by oncogenic signaling and by RAS. We recently identified RAS-mediated phospho-regulation of multiple proteins involved in rRNA processing. Among these are eight different DEAD-box helicases in which altogether twelve phosphorylation sites were found significantly regulated by either RAS or its antagonist phosphatase PP2A. Together these findings strongly suggest a coordinated effort of RAS/PP2A in
increased rRNA biogenesis in human cancers. Our preliminary data indicates innate defects during rRNA processing in RAS driven cancers (NSCLC and PDAC). Additionally, gene expression analysis of the eight helicases under hyper-activated RAS and PP2A inhibited conditions showed
interesting trends. Finally, our initial findings suggest these helicases to be important for cell viability. We are currently evaluating the effect of the twelve phosphorylation site changes on cellular fitness using a CRISPR dropout screen. We next aim to dissect the contribution of individual DEAD-box helicases in alterations of rRNA biogenesis or processing eventually driving the malignancy of these cancers.