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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.

4. Alcón-Perez

Marta

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

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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.

5. Arenas Molina

Alberto

Universidad de Granada / GENYO, Spain

Abstract Title

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

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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 12^thcodon 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 KRAS^G12Cinhibitors (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 KRAS^G12C/G12Dalleles.

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 (KRAS^WT) untouched. Furthermore, the therapeutic potential of the system was assessed using KRAS^G12C/G12Dand KRAS^WT 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.

6. Baer

Romain

The Francis Crick Institute, UK

Abstract Title

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

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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 (Kras^G12V/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 (CRAF^R89L). This mouse model recapitulates the embryonic lethal phenotype of the constitutive CRAF KO mouse. Using a combination of murine lung KP (Kras^G12V/Trp53) cell lines, either constitutively or conditionally expressing the CRAF^R89Lallele (CRAF^flox/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.

7. Ballesteros

Irene

Instituto de Biología Molecular y Celular del Cáncer, Spain

Abstract Title

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

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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.

8. Ballesteros

Irene

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

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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.

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

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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 p210^BCR-ABL1oncogene, i.e., hematopoietic stem and progenitor cells (Tec-p210^+/-; Rapgef1^flox/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/C3G^HSC-Scl-KO mice had significantly lower levels of peripheral blood neutrophils, compared to p210/C3G^HSC-Scl-wt mice and exhibited features of late-onset or milder CML. Moreover, preliminary data showed that p210/C3G^HSC-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.

12. Boned del Rio

Isabel

University College London (UCL), UK

Abstract Title

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

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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.

13. Brachmann

Saskia

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

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Although KRAS^G12Cinhibitors 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 KRAS^G12Cinhibitor 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 KRAS^G12C/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 KRAS^G12C-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 KRAS^G12Cprotein, mRNA and gene copy number. Adaptive mechanisms of resistance to KRAS^G12C/SHP2 co-inhibition were explored by functional genome-wide CRISPR screening in KRAS^G12C-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 KRAS^G12C/SHP2 inhibitor combination; FGFR1 was the strongest sensitizer, and PTEN the strongest rescuer. Consistent with this, KRAS^G12C/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 KRAS^G12C/SHP2 co-inhibition in this model. Biological nodes identified by CRISPR screening might provide starting points for effective combination treatment approaches.

Significance
This study identifies mechanisms of resistance to KRAS^G12C/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.

14. Brehey

Oksana

Centro Nacional de Investigaciones Oncológicas (CNIO), Spain

Abstract Title

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

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Introduction:
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.

Objective:
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
considerably.

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.

Conclusion:
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.

15. Carvalho

Joana

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

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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.

16. Clark

Geoffrey

University of Louisville, USA

Abstract Title

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

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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.

17. De Hita

Sergio

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

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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.

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

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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.

22. Fernández García

Fernando

Centro Nacional de Investigaciones Oncológicas (CNIO), Spain

Abstract Title

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

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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.

23. Fernández Medarde

Alberto

Abstract Title

Inhibition of oncogenic mutant KRAS via oxidation at Cysteine 118

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Introduction
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.

Conclusions
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.

31. Liaki

Vasiliki

Centro Nacional de Investigaciones Oncológicas (CNIO), Spain

Abstract Title

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

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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.

32. Machado

Ana Luisa

IPATIMUP, Portugal

Abstract Title

Tumor-Hematopoietic Communication in Colorectal Cancer Pathogenesis

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Abstract Title

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

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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 KRAS^G12C;Stk11^null(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 2 , a covalent allosteric inhibitor of KRAS^G12C, 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)

50. Olarte San Juan

Andrea

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

Abstract Title

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

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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.

51.

Pachter

Jonathan A.

Verastem Oncology, USA

Abstract Title

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

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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.

52. Pantsar

Tatu

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?

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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.

53. Papke

Bjoern

Charité Berlin, Germany

Abstract Title

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

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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.

54. Pericacho Bustos

Miguel

Universidad de Salamanca & IBSAL, Spain

Abstract Title

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

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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.

55. Quirós Fernández

Isaac

DKFZ/Universidad de Costa Rica, Costa Rica

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.

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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.

Bibliography

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.

Abstract Title

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

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Introduction
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).

Results
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.

Conclusions
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.

76. Vietti Michelina

Sandra

University of Turin, Italy

Abstract Title

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

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Introduction: lung cancer is responsible for 12% of cancer-related deaths worldwide (https://www.wcrf.org).
Mutant KRAS, particularly KRAS^G12C, 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 KRAS^G12CLUAD 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 KRAS^G12Ccells 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.

Conclusions:
-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

77. Woodhouse

Laura

The Christie NHS Foundation Trust, UK

Abstract Title

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

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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.

78. Yellapragada

Venkatram

University of Turku, Finland

Abstract Title

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

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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.