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Drug combinations with novel PDE6D inhibitors release differentiation block of oncogenic KRAS
Several RAS pathway genes are somatically mutated in cancer, while their mutation in the germline is associated with developmental diseases summarized as RASopathies. An overarching mechanistic model that would explain these different diseases is currently missing. It is however plausible to assume that the common denominator of these distinct diseases is an altered differentiation trajectory
of stem/ progenitor cells.
During asymmetric divisions of stem/ progenitor cells, the primary cilium disassembles and resolves into the mother centrosome, which is typically segregated to the stemness retaining cell. We found that amongst the cancer associated RAS isoforms, KRAS localizes most abundantly to the membrane of the primary cilium at the apical face of C2C12 myoblasts. Intriguingly, the differentiation of C2C12 cells is altered by modulating ciliary targeting of KRAS. Ciliary localization of KRAS is facilitated by the trafficking chaperone PDE6D and binding of KRAS to PDE6D is suppressed by phosphorylation of Ser181 by PKG2. We therefore developed novel nanomolar PDE6D inhibitors and explored, whether they can synergize in combination with PKG2 activators, such as approved Sildenafil, to release the differentiation block imposed by oncogenic KRAS variants. Our data suggest that a stemness driving function of KRAS is emanating from the primary cilium and that this activity is perturbed by pathogenic variants of KRAS.
Biological relevance of KRAS complexes interaction at the cell membrane
Clustering of RAS molecules at the cellular membrane is of paramount importance for
their active signaling function within cells. The debate currently centers around the
mechanisms underlying this clustering phenomenon. Specifically, the discussion
focuses on whether the exclusive trigger of clustering is through the membrane
interaction of its C-terminal hypervariable region, whether contributions stem from the
G-domain of RAS, or if secondary effectors also play a role. This topic has garnered
significant attention in recent months.
Various experimental factors could be influencing these outcomes. These factors include the levels of protein expression, the cellular models being employed, the zygosity of RAS, and the potential role of secondary interactions. These variables have been observed to modulate the effects mediated by mutations that regulate the G- domain interactions, and consequently, the interactions between RAS molecules. By addressing these variables, we aim to shed light on the role of the G-domain mutations and, by extension, the intricate interactions between RAS molecules that drive cellular signaling.
Proteomic profiling of KRAS signaling; Context, CAFs and Combinations
KRAS mutations drive a wide range of cancers. Proteomic approaches help understanding signal transduction downstream of KRAS mutations in different disease contexts such as lung, colorectal and pancreatic cancer can give insights into drug response. Further, proteomic analysis of the secretome of cancer
associated fibroblasts (CAFs) lead to understanding of the influence of stroma on
drug sensitivity. Finally, machine learning algorithms using dynamic proteomic data allow us to predict emerging mechanisms of drug resistance. Examples of clinical trials based on this data will be discussed.
Disruption of Immune Homeostasis Circuits in Mutant KRas Tumors
Mutations in ras genes are highly prevalent in human cancers where they play a key role
in tumor initiation, progression, and maintenance. Mutant Ras proteins are endowed with a diverse set of biological capabilities that impinge on homeostatic mechanisms that promote cell growth and survival. We have been interested in defining the molecular basis of these capabilities with the goal of leveraging this information to
identify new therapeutic intervention strategies. Specifically, our current efforts focus
on investigating how mutant Ras tumors escape the host immune mechanisms. The
presentation will provide updates pertaining to these efforts highlighting the contribution of tissue specific determinants to immune evasion, and their relevance to the development of immunotherapeutic approaches.
Targeting KRAS signalling
Although KRAS oncogenes were identified in human tumors more than four decades ago, the first KRAS selective inhibitor could not be approved until 2021. Despite this long-awaited breakthrough, KRAS inhibitors approved so far only block one of the multiple oncogenic isoforms, KRASG12C.
Moreover, tumor resistance is becoming a significant issue in the clinic, limiting the beneficial effect of these inhibitors on the overall survival of treated patients. Therapeutic strategies to target KRAS mutant tumors should also include those that block KRAS signaling pathways. Previous studies in our laboratory have shown that ablation of RAF1 in Kras/p53 driven-lung adenocarcinoms induce effective tumor regression without significant toxicities (Sanclemente et al. Cancer Cell, 2018; Esteban-Burgos et al., PNAS 2020). Likewise, concomitant ablation of RAF1 and EGFR in Kras/p53 driven-pancreatic ductal adenocarcinomas (PDAC) resulted in the complete regression of a limited fraction of tumors (Blasco et al, Cancer Cell 2019). We have now identified STAT3 activation as the main mechanism for the resistance of PDAC tumors to RAF1/EGFR ablation.
Indeed, combined ablation of RAF1/EGFR/STAT3 completely blocked proliferation of mouse tumor cell lines and organoids. Moreover, tumors induced in syngeneic orthotopic models regressed completely with no evidence of tumor progression. These results open the door to the development of pharmacologically strategies, that in combination with forthcoming KRAS inhibitors, should have a significant impact on the treatment of KRAS mutant tumors in the clinic.
Targeting RAS-Driven PI3Kα Activation in Human Tumors
RAS and PI3Kα are the two most mutated oncogenes in human tumors. The co-activation of their respective pathways is often observed in transformed cells and is sufficient to drive a malignant phenotype. Early evidence to support their cooperation showed that transformation with HRAS is ineffective in the absence of PI3Kα signaling. More elegant studies later established that this cooperation may rely on the physical interaction between RAS and PI3Kα as point mutations in the RASbinding domain (RBD) of PI3Kα that abrogate this interaction strongly inhibited KRASG12D-driven tumor growth. Although targeted therapeutics have been developed against key nodes of both pathways, none
has yet exploited the importance of the cooperation driven by this physical interaction.
Here, we describe the discovery and characterization of novel small molecules, referred to as “breakers”, that potently and selectively abrogate the interaction between RAS and PI3Kα in tumor cells. These agents 1) selectively bind to the RAS-binding domain (RBD) of PI3Kα, 2) block the interaction of PI3Kα with all three oncogenic isoforms of RAS, (KRAS, HRAS, and NRAS), and 3) inhibit RAS-driven AKT activation in RTK-driven, KRAS-mutant and PI3Kα-mutant cancer cell lines. Breakers with drug-like properties and oral bioavailability show strong pharmacodynamic effects (>90% pAKT inhibition) in tumor-bearing mice without any changes in glucose metabolism. Lastly, efficacy studies show that models of cancer driven by RTK amplification, KRAS mutations, or PIK3CA mutations (or any combination
thereof) display a high degree of sensitivity to breaker treatment with monotherapy and/or combination with KRAS inhibitors, resulting in significant tumor regression.
In conclusion, we have identified a novel approach to inhibit the PI3Kα signaling pathway by blocking its interaction with, and activation by RAS. This approach can achieve strong pAKT inhibition in tumor cells without changes in glucose metabolism. Combination of breakers with novel KRAS inhibitors provide an exciting opportunity to simultaneously inhibit the RAS and PI3Kα pathways in human tumors without dose-limiting toxicities.
New ideas to target RAS
Inhibition of oncogenic signaling using targeted cancer drugs frequently results in
drug resistance through re-activation of the inhibited pathway by secondary mutations. There is increasing evidence that further activation of signaling pathways that are already activated by oncogenic mutations can be as lethal to cancer cells as their inhibition. This lethality is thought to result from overloaded stress response pathways that are unable to compensate for the increased mitogenic activity. We used small molecule inhibition of protein phosphatase 2A (PP2A) to hyperactivate both WNT and MAPK signaling in colon cancer cells and used genome scale CRISPR screens and compound screens to find the vulnerabilities of cells that experience hyperactivation of oncogenic signaling. Our data indicate that inhibition of PP2A combined with inhibition of the WEE1 kinase to perturb the mitotic stress response results in DNA replication stress followed by mitotic catastrophe and cell death. Cells that have developed resistance to this drug combination have downregulated oncogenic signaling in the absence of drugs and consequently fail to form tumors in vivo. Our data suggest that paradoxical activation of oncogenic signaling results in tumor suppressive resistance caused by downregulation of oncogenic signaling.
«Out of the box» functions of RHOA mutations in peripheral T cell lymphoma
Classical RAS genes and, to a much lesser extent RHO family genes (e.g., RAC1) undergo gain-of-function mutations in human cancer. However, the mutations found in the RHOA gene in peripheral T cell lymphoma (PTCL) are more complex, since they can lead to the expression of either loss- (mostly G17V) or gain-of-function proteins in different patients and disease subtypes. The rationale for this rather perplexing and antagonistic pattern of RHOA mutations in PTCL remains unclear. In this talk, we will show that all the gain- and loss-of-function RHOA mutants found in PTCL are characterized by the common acquisition of a neomorphic pathway that leads to the stimulation of the nuclear factor of activated T cells (NFAT) in a T cell receptor-, LAT-, PLCγ-, and SLP76-dependent manner. This noncanonical function is RHOA mutant-specific, since wild-type RHOA or analogous RAC1 and CDC42 mutants cannot engage this pathway. It is also PTCL-specific, since most RHOA mutants found in other tumor types do not exhibit this neomorphic feature. The primary effector responsible for the engagement of this new pathway has been identified and validated using proteomic and genetic techniques, respectively. We will also provide genetic and pharmacological data demonstrating that this neomorphic function is essential for RHOA mutant-driven T cell lymphomagenesis.
Progress on selective targeting of KRAS mutant alleles and emerging clinical implications.
RAS mutation tropism
Despite over 50 possible oncogenic RAS mutations, each cancer type has a specific and often unique subset of these mutations. As RAS mutations typically occur early during tumorigenesis, if not being the initiating mutation, this ‘mutation tropism’ ostensibly reflects fundamental biology underlying the process of tumor initiation.
Elucidating the mechanisms giving rise to RAS mutation patterns would therefore address a foundational question in cancer biology- how cancer originates. However, trying to backtrack to catch one random mutagenic event in one gene from one cell that initiates cancer decades before manifesting as a disease is challenging in humans. On the other hand, urethane carcinogenesis is particularly well suited to interrogate how RAS mutation tropism arises. Namely the moment of tumor initiation can be precisely defined – the moment mice are exposed to urethane – and urethane mimics the RAS mutation tropism observed in humans – giving rise to pulmonary tumors initiated by a single Q61L/R mutation in Kras. We therefore adopted an ultra-sensitive sequencing approach to capture mutations in mice, engineered with different genetic backgrounds, days after urethane exposure and thereafter to follow the process of RAS mutation tropism. Results from these experiments will be presented that support a narrow window of oncogenic RAS signaling, potent enough to initiate promotion without
eliciting an oncogene-induced stress response in normal cells, selects for specific RAS
mutations to initiate cancer.
Three Rs: RNA Regulates RAS-ERK signals
LncRNAs are long known to play important roles in the regulation of gene expression and as architectural components of chromatin organization and other nuclear molecular assemblies.
As such, lncRNAs participation in signal transduction was mostly indirect, via the regulation of signaling molecules expression levels. Unveiling a hitherto unprecedented function for LncRNAs as direct regulators of signaling modules, we will present our latests findings showing that a LncRNA can directly interact with different constituents of the RAS-ERK pathway, thereby regulating RAS-ERK pathway signal flux, and evoking profound alterations in cellular processes implicated in carcinogenesis.
Targeting the KRAS-ERK-MYC signaling network for the treatment of KRAS-mutant pancreatic cancer
KRAS is mutationally activated in 95% of pancreatic ductal adenocarcinomas (PDAC) and essential for maintenance of PDAC tumorigenic growth. The recent clinical evaluation of inhibitors of one KRAS mutant (KRASG12C ) supports the therapeutic value of direct targeting of KRAS for PDAC treatment. However, this mutant comprises less than 2% of KRAS mutations in PDAC. Therefore, we have evaluated direct inhibitors of the predominant KRAS mutations (G12D, G12V and G12R) in PDAC. Additionally, both innate and treatment-associated resistance mechanisms will limit the effectiveness of direct KRAS inhibitors. We applied CRISPR screens and identified genes that modulate sensitivity to KRAS inhibitors that included components of the Hippo-YAP tumor suppressor pathway. Genomic sequencing analyses of patients who relapsed on KRASG12C inhibitors suggest that reactivation of KRAS effector signaling comprises one major mechanism of resistance. We determined that activation of
MEK-ERK but not AKT activation alone is sufficient to drive resistance to KRASG12C/D inhibitors.
However, our recent clinical evaluation of direct ERK inhibition in PDAC found that toxicity limited the effectiveness of this therapeutic approach. These observations prompted our studies to complete a system-wide determination of the molecular mechanisms by which ERK signaling supports KRAS-dependent PDAC growth. First, we determined the KRAS-ERK regulated transcriptome, kinome, total proteome and phosphoproteome in KRAS-mutant PDAC. We identified KRAS-dependent gene signature (677 KRAS- ependent and 1,051 KRAS-suppressed genes) that diverged significantly from the classical Hallmark KRAS gene signature. Our KRAS-ERK gene signature verified target inhibition in PDAC patients treated with the ERK inhibitor ulixertib that correlated with PDAC tumor regression. We identified 5,117 ERK-dependent phosphosites on 2,252 proteins, of which 88% and 67%, respectively, were not previously associated with ERK. Thus, we identified a considerably more complex ERK-regulated kinome signaling network than identified in previous studies. Second, since a major consequence of ERK signaling involves activation of MYC-regulated gene transcription, we also determined the MYC-dependent transcriptome (2,086 MYC-dependent and 2,403 MYC-suppressed genes) that include 80 protein kinases. Together, these analyses define a highly complex and dynamic ERK-regulated signaling network that deregulates cell cycle progression, RHO GTPase function and metabolism to support KRAS-dependent PDAC growth.
Developing rational approaches to targeting RAS in combination with immune regulators
The approval of two KRAS G12C mutant-specific inhibitory drugs, sotorasib and adagrasib, has markedly improved the treatment of lung cancer patients harbouring KRAS G12C oncogenic mutations. However, development of acquired drug resistance is very common after initial responses, and impact on overall survival is small. Additional therapies will therefore need to be combined with KRASG12C inhibitors if long-term cures are to be achieved. Due to the immunosuppressive nature of the signaling network controlled by oncogenic KRAS, targeted KRAS G12C inhibition can indirectly affect anti-tumour immunity.
This has served as a rationale for combination with immune checkpoint blockade (ICB),
which we have shown leads to additional therapeutic benefit in immunogenic/hot, but not immune evasive/cold, pre-clinical lung tumour models. However, in clinical trials in KRAS G12C mutant lung cancer, combination of KRAS G12C inhibitors with PD-(L)1 blockade has yet to show improved outcome, either due to toxicities or lack of efficacy, possibly linked to prior progression on immunotherapy. Study of the response of KRAS G12C mutant lung
tumours to KRAS G12C inhibitory drugs using spatial and multi-omic technologies has
highlighted the role of particular populations of immune cells in the tumor
microenvironment in impeding immune attack on the tumor. In immune evasive lung cancer, following KRAS G12C inhibitor treatment a community of cells is identified within the tumor containing dendritic cells, CD8+ T cells and dying tumor cells, which appears to function as an antigen presentation hub. This community is restrained by T reg cells, whose coordinate targeting markedly improves the impact of KRAS G12C inhibitors. In an immunogenic/hot mouse model of KRAS G12C mutant lung cancer, tertiary lymphoid structures are seen along with tumor-binding antibodies, which target endogenous retrovirus (ERV) envelope glycoproteins on the tumor cells. ERV-targeting B cell responses are amplified by ICB in both humans and mice, and by targeted KRAS G12C inhibition in the murine model. ERV-reactive antibodies exert anti-tumour activity that extend survival in the murine model and ERV expression predicts the outcome of ICB in human lung adenocarcinoma. Rational, mechanism-based exploitation of the impact of KRAS inhibition on tumor immune evasion provides opportunities for successful combination therapy of RAS mutant cancers.
Enhancing immunotherapy in Kras driven lung cancer
Lung cancer is the 3rd leading cause of cancer related deaths worldwide. Here we provide
data demonstrating the efficacy of targeting avb3-integrin in overcoming resistance to
immuno checkpoint blockade in mouse models of non small cell lung cancer.
Additionally, treatment with the avb3 targeting agent is sufficient to alter the cxc-subfamily cytokine profiles corresponding to responders to immunotherapy in human lung cancers.
Using Tri-Complex RAS(ON) Inhibitors to Target RAS Addicted Cancers
KRAS mutations at codons 12, 13, or 61 stabilize the active GTP-bound form of RAS (RAS(ON)) by disrupting GAP stimulated GTP hydrolysis. Revolution Medicines is pursuing the discovery and development of a series of potential therapeutic agents with a novel mechanism of action, specifically designed to target RAS(ON). Inspired by natural products like cyclosporine and rapamycin, these compounds enter a tumor cell, bind initially to an abundant immunophilin (cyclophilin A) and form a
binary complex. This binary complex presents a new protein binding surface that is optimized for a selective high-affinity interaction with a particular RAS(ON) target. Formation of the tri-complex rapidly
inactivates RAS signaling by sterically preventing binding and activation of downstream RAS pathway effectors. These tri-complex inhibitors bind near the common oncogenic RAS mutations at codons 12,
13 and 61, enabling us to engineer mutant selectivity, or to identify compounds with activity across multiple RAS mutant proteins (RASMULTI(ON)). In preclinical models, RASMULTI(ON) inhibitors are active across a broad range of RAS-dependent genotypes, including oncogenic KRAS, NRAS, HRAS,
EGFR, MET, and type II and type III BRAF mutations. Genetic analysis of RAS-dependent genotypes reveals that KRASG12X (where X is A, S, V, C, D, R) mutated cell lines are highly RAS-addicted and these are also very sensitive to inhibition with RAS(ON) targeted inhibitors.
Ras-effector networks in (patho)physiological contexts
Ras proteins are key cellular on-off switches. In the on-state they can interact with different
effector proteins, thereby controlling downstream networks and ultimately cell behavior and phenotypes. Protein-protein interaction (PPI) databases to date list more than 1300 proteins that are part of complexes with Ras proteins. However, we still lack an understanding of how PPI downstream of Ras are (re)wired in specific conditions or cell types and how they link to cell phenotypes. The approach that we take in my lab is to combine bioinformatics, experimental, quantitative computational modelling, and protein engineering approaches to deliver a framework that describes networks in the context of complex biology (phenotypes)
and disease. I will present our recent work about context-specific Ras-mediated networks.
Back to the future: the role of RAF kinases in RAS signaling and transformation
The RAF-MEK-ERK pathway is a main effector of oncogenic RAS signaling. Disappointingly, the strategy to block RAF for treating RAS driven cancers was largely ineffective to date. However, it has revealed a wealth of unexpected insights into RAS and RAF biology specifically, and network biology in general. A
main lesson was that network adaptations were added to the main mechanisms of drug resistance. Here, we will discuss what we have learned from these lessons through computational models focusing on network adaptations, how they confer drug resistance and how we can break them. The results put RAF kinases back into the limelight as potentially effective targets for RAS driven cancers albeit with new ways to target them. We also will introduce approaches to control and revert the malignant state of cancer cells rather than trying to destroy them. This strategy aims to make cancer a manageable
condition rather than a life or death decision. Finally, we will discuss our efforts to generate Digital Twin models of cancer patients to enable precise diagnostics and personalized treatments.
Applied biobanking to validate clinical vulnerabilities in RAS lung cancer variants
A number of reports have demonstrated fundamental biochemical, structural and signalling differences of RAS conferred by variation of its mutant allele, codon, or isoform. Despite this preclinical evidence, there has been no clear description of how these biological differences manifest in the lung cancer clinic, with most studies suggesting that lung cancers affected by different RAS point mutations will have similar survival outcomes. Using clinical information from a Trans-Atlantic precision medicine partnership, allied with focused biobanking of RAS-mutant epithelial cancer samples, we have generated a number of models, datasets and research samples that will inform a more detailed clinical understanding of RAS vulnerabilities according to its point mutations. This presentation will focus on differences we
have identified between i) KRAS codon 12 and 13 variants, and ii) KRAS G12C and KRAS
G12D lung cancers.
Challenges and opportunities in treating KRAS mutant tumors
Covalent inhibitors of KRAS G12C have shown antitumor activity against advanced/metastatic KRAS G12C-mutant cancers. JDQ443 is a structurally novel covalent KRAS G12C inhibitor with a unique binding mode that demonstrates potent and selective antitumor activity in cell lines and in vivo models. In preclinical models and patients with KRAS G12C-mutated malignancies, JDQ443 shows potent antitumor activity as monotherapy and in combination with the SHP2 inhibitor TNO155. Because resistance to KRAS inhibitors emerges, we are investigating known and differentiated strategies to targeting mutant KRAS, including complementary and orthogonal drug combinations to maximize patient response
The End of KRAS cancers?
Activating mutations in KRAS occur frequently in lung adenocarcinoma, pancreatic cancer and colorectal cancer. These mutations are usually at codons 12 or 13, and render the KRAS protein resistant to GTP Activating Proteins (GAPs). As a result, they accumulate in their active, GTP bound states and signal persistently through the RAF-MEK-ERK and the PI 3’ Kinase pathways.
In 2021, the FDA approved the first drug targeting KRAS directly. Sotorasib binds covalently to GDP-bound form of KRAS G12C and so traps it in the inactive state. Second-generation KRAS G12C inhibitors that engage the GTP-bound form of the KRAS G12C protein are already being tested in the clinic. The mechanism of action of one of these, BBIO 8520, will be discussed.
KRAS G12D and V mutations are more common than KRAS G12C, especially in pancreatic cancer. Non-covalent inhibitors will likely need to be developed. These could either trap the GDP-off state or target the active GTP-state directly. Such compounds, along with Pan-KRAS inhibitors, are also being developed. These compounds engage unique residues in the KRAS G-domain and therefore spare HRAS and NRAS.
In addition to drugs that bind directly to the KRAS protein and block its function, attempts are underway to promote KRAS degradation, using synthetic techniques such as PRO-TAC, or by using endogenous degradation pathways such as the LZTR1-CUL3 pathway. Other ways of targeting KRAS include blocking the process by which RAS activates RAF kinase, and the process by which RAS activates PI3 Kinase.
The relative merits of each of these processes will be discussed, as well as future prospects for long term clinical benefit to patients suffering from KRAS driven cancers.
Current and future challenges in the clinical development of KRAS G12C inhibitors in non-small cell lung cancer
KRAS mutations have been considered undruggable for years. The discovery of the
KRASG12C switch II pocket and the clinical development of direct covalent KRASG12C inhibitors move forward the field. During the last years, several KRASG12Cinhibitors have been studied in patients with solid tumors and the FDA approved sotorasib and adagrasib for patients with advanced NSCLC KRASG12C, based on the efficacy and safety data seen in phase I/II clinical trials. However, there is room for improvement taking into consideration the lower magnitude of benefit in terms of progression-free survival compared to the efficacy results observed with other actionable alterations in NSCLC. The results of the first randomized phase III clinical trial (CodeBreak-200) comparing sotorasib versus docetaxel were indeed somehow disappointing. This is
probably due to multiple reasons such as higher heterogeneity within the KRAS mutant
patients, the presence of intrinsic mechanisms of resistance or the emergence of adaptive mechanisms of resistance. Most direct KRASG12C inhibitors are currently being
studied in the frontline setting, generally in combination with other treatment strategies
(immunotherapy, chemotherapy, SHP2 inhibitors). The combinatorial approach is well
supported by preclinical data but could be compromised by toxicity issues and we are
still far from understanding which combination is the most appropriate for any patient. A challenge of greatest importance in the clinic is to define which should be the treatment sequence for patients with NSCLC harboring KRASG12C mutations and whether the established paradigm of frontline chemoimmunotherapy could be surpassed. Another important clinical challenge is the high frequency of brain dissemination in this patient population, which will require innovative and coordinate clinical efforts. On the other hand, multiple research opportunities will come out (e.g. design of clinical trials with KRAS inhibitors within earlier stages, longitudinal monitoring using liquid biopsy during the treatment) that could improve our understating of this complex disease and also make progress the patients’ life expectancy and quality of life.
Targeting KRAS via SOS1: Parallel Fragment Screening and HTS Approach
Since the late 1980s, mutations in the RAS genes have been recognized as major oncogenes with a high occurrence rate in human cancers. Such mutations reduce the ability of the small GTPase RAS to hydrolyze GTP, keeping this molecular switch in a constitutively active GTP-bound form that drives, unchecked, oncogenic downstream signaling. One strategy to reduce the levels of active RAS is to target guanine nucleotide exchange factors, which allow RAS to cycle from the inactive GDP-bound state to the
active GTP-bound form. Here, we describe the identification of potent and cell-active small-molecule inhibitors which efficiently disrupt the interaction between KRAS and its exchange factor SOS1, a mode of action confirmed by a series of biophysical techniques. The binding sites, mode of action, and selectivity were elucidated using crystal structures of KRASG12C–SOS1, SOS1, and SOS2. By preventing formation of the KRAS–SOS1 complex, these inhibitors block reloading of KRAS with GTP, leading to antiproliferative activity. The final compound 23 (BAY-293) selectively inhibits the KRAS–SOS1 interaction
with an IC50 of 21 nM and is a valuable chemical probe for future investigations.
Targeting kras and it effectors in both normal and transformed colorectal epithelium.
The question of how same-gene mutations can drive both cancer and neurodevelopmental disorders (NDDs) has been puzzling. It has also been puzzling why those with NDDs have a high risk of cancer. Ras, MEK, PI3K, PTEN, and SHP2 are among the oncogenic proteins that can harbor mutations that encode diseases other than cancer. Understanding why some of their
mutations can promote cancer, whereas others promote neurodevelopmental diseases, and why even the same mutations may promote both phenotypes, has important clinical ramifications. The talk will address these tantalizing questions. It will also suggest that NDDs, immunity, and cancer are connected, and that in all, there appears an almost invariable involvement of small GTPases (e.g., Ras, RhoA, and Rac) and their pathways. Although there are signaling similarities, decisive differentiating factors are timing windows, cell type specific perturbation levels, and signal strength.
How can same-gene mutations promote both cancer and developmental disorders?
Nussinov R, Tsai CJ, Jang H. Sci Adv. 2022 Jan 14;8(2):eabm2059. doi: 10.1126/sciadv.abm2059.
Neurodevelopmental disorders, immunity, and cancer are connected.
Nussinov R, Tsai CJ, Jang H. iScience. 2022. PMID: 35712080
How PTEN mutations degrade function at the membrane and life expectancy of carriers of
mutations in the human brain. Jang H, Chen J, Iakoucheva LM, Nussinov R. bioRxiv. 2023 Jan
27:2023.01.26.525746. doi: 10.1101/2023.01.26.525746. Preprint. PMID: 36747841
Ras variant abundance and biology
Activating mutations of Ras genes are often observed in cancer. The protein products of the three Ras genes are almost identical. However, for reasons that remain unclear, KRAS is far more frequently mutated than the other Ras isoforms in cancer and RASopathies. We have quantified HRAS, NRAS, KRAS4A and KRAS4B protein abundance across a large panel of cell lines and healthy tissues. Our data reveal new features of Ras biology and challenge and refine models explaining the pattern of Ras mutations in cancer and isoform-specific Ras contributions to development. New tools that will assist with
exploring Ras variant biology will also be discussed.
The SHOC2/MRAS/PP1 complex as a therapeutic target for RAS-driven cancers
Despite the well established key role of the RAF-MEK-ERK kinase cascade (i.e. ERK-MAPK
pathway) downstream of oncogenic RAS and the availability of potent inhibitors, it has proved difficult to target this pathway successfully in the clinic. In addition to the resistance mechanisms that invariable arise with targeted therapies, on-target toxicity precludes the deep inhibition that is required for clinical efficacy.
The SHOC2-MRAS-PP1 (SMP) complex provides a node in the regulation of the ERK-MAPK pathway with attractive properties as a therapeutic target. The SMP complex dephosphorylates the ‘S259’ inhibitory site in RAF kinases which is a crucial step for BRAF-CRAF heterodimerization. Intriguingly, this regulatory step appears to contribute to ERK pathway activity in a context-dependent manner, being dispensable in many contexts (where other regulatory mechanisms provide redundancy) but important in others, including in the context of oncogenic RAS. Consistent with this, gene essentiality studies show that SHOC2 is a top synthetic lethality of oncogenic (but not wt) RAS, both alone and in the context of MEK inhibition. Thus, SHOC2 is now widely accepted to be among the best targets for anti-RAS therapies with multiple drug discovery programs in development.
Our studies show that despite the potential of a vertical pathway inhibition strategy targeting
both SMP complex and MEK against RAS-driven tumours, additional strategies of horizontal inhibition will also be needed for lasting responses. Data using mouse models of systemic genetic inhibition to anticipate potential toxicity liabilities will also be discussed.
Understanding the role of endogenous and mutated KRAS in homeostasis and transformation of the colon
For many years, KRAS mutant colorectal cancer (CRC) has been a large clinical problem. KRAS is mutated in approximately 40% of CRC. KRAS mutation act a negative predictive biomarker for EGFR inhibition meaning there is very little options for KRAS mutant CRC
patients when chemotherapy was unsuccessful. Preclinical work from our laboratory and others, have also shown that KRAS mutation also confers resistant to many molecular targeted agents when combined with the most common mutation in CRC: APC
(Adenomatous Polyposis Colis) mutation e.g. MTOR, PI3K and MAPK signalling. Given we are in the midst of a revolution in the effective therapeutic targeting of RAS across multiple
cancers, it is now more important than ever to understand the physiological role of KRAS in
the intestinal tract, how different KRAS mutants cooperate with APC mutation and what are the responses of these KRAS mutant tumours to KRAS inhibition.
I will discuss our recent work examining the impact of KRAS deletion on intestinal homeostasis, the differing cooperating potential of KRAS mutants with APC loss and the impact of KRAS inhibition in a set of KRAS mutant CRC models. If time, I will also highlight the lack of a transcriptional classifier for KRAS mutant CRC and the need for us to stratify human CRC differently and how metabolic stratifiers may be more effective.
A perfect storm—formation of Ras-Raf signalosome and its implications
The protein K-Ras functions as a molecular switch in signaling pathways regulating
cell growth. In the MAPK signaling multiple Raf kinase-bound K-Ras proteins form clusters
at the cell membrane. It is increasingly clear that K-Ras and Raf proteins, together with
scaffold proteins Galectin-3 and 14-3-3σ, form a higher-order complex that underlies these
clusters. Our atomistic structural model for such an assembly (Ras-Raf signalosome)
suggests a network of positive feedbacks connecting Ras membrane localization,
dimerization, and Raf dimerization. Such a network suggests that disruption Ras
dimerization and preclusion of the formation of the Ras-Raf signalosome is essential for drug discovery.
Strategies for Drugging Undruggable Targets in Oncology With New Covalent Warheads
Somatic mutations in the small GTPase K-Ras are the most common activating lesions found in human cancer, and are generally associated with poor response to standard therapies. Oncogenic mutations result in functional activation of Ras family proteins by impairing GTP hydrolysis. I will discuss the recent development of small molecules that irreversibly bind to the somatic oncogenic mutant, K-RasG12D, which commonly occurs in pancreatic cancer. These compounds rely on the mutant aspartate for binding and therefore do not affect the wild type protein (WT). I will also discuss immunologic strategies to exploit the presentation of covalently modified Ras-neoantigens for targeted therapy.
KRAS dependencies in Pancreatic Cancer
KRAS is an oncogenic dependency in the majority of patients with pancreatic ductal carcinoma; and, therefore, methods to effectively target KRAS are under intensive evaluation. We have investigated how the oncogenic KRAS pathway promotes cellular transformation, and have identified a role for KRAS in tissue invasion, the extracellular matrix, and the regulation of cholesterol metabolism. All of these observations represent additional means to disrupt the
oncogenic KRAS pathway, and reveal new therapeutic possibilities for pancreatic cancer.
A clinical update on AMGEN's KRAS G12C-selective inhibitor Sotorasib and pre-clinical studies on a novel multi-KRAS inhibitor
Sotorasib is a specific, irreversible inhibitor of the KRAS G12C GTPase. This mutation is a key oncogenic driver in ~13% of patients with non-squamous non-small-cell lung cancer (NSCLC), and Sotorasib is currently approved as 2L monotherapy for this indication. In my talk, I will present key 2023 clinical highlights for Sotorasib, both as a monotherapy and in combination with other inhibitors. Additionally, I will detail new preclinical studies with AMGEN’s novel multi-KRAS inhibitors.
Drug repurposing strategies for the treatment of mutant KRAS cancers
The systematic study of transcriptomic changes featured in the oncogenic KRAS phenotype may lead to the identification of new molecular targets and serve as the basis for developing novel therapeutic strategies. To date, most of the therapeutic strategies for cancers driven by oncogenic KRAS based on
MEK1/2 or KRASG12C inhibitors have incorporated abrogation of KRAS proximal effectors involved in oncogenesis or treatment resistance. However, the antitumor effect is highly determined by compensatory mechanisms specifically arising in certain cell types or tumor subgroups. We propose that a
potential strategy to find drug combinations spanning a large percentage of mutant KRAS lung cancers may capitalize on the common, distal gene expression output elicited by oncogenic KRAS or key downstream effectors. In this presentation, we will provide examples of novel dual treatment strategies which build upon this approach and incorporate clinically approved drugs that could be repurposed for the treatment of KRAS-driven tumors in combination with inhibitor to the KRAS pathway.
Single and dual SOS1/SOS2 inhibition: evaluation of therapeutic effects and toxicity
Recent evidence suggests that genetic deletion, degradation, or pharmacological inhibition of SOS1 may be promising therapeutic strategies for KRAS-driven and other RAS-dependent malignancies. The highly homologous GEF SOS2 limits the efficacy of a SOS1 inhibitor to partial pathway blockade and may compensate for the loss of SOS1 activity. Concomitant SOS1/SOS2
inhibition could enhance the therapeutic effect, although potential toxicities would have to be carefully evaluated in that event. Here, we provide in vivo genetic and pharmacologic evidence supporting a potential benefit of single and/or dual SOS1/SOS2 inhibition in mutant KRAS
transformed cells. In addition, we also studied whether systemic side-effects occur following inhibition of SOS1 in SOSKO mice.
In vivo functional impact of genetic (Tamoxifen-induced SOS1 depletion) or pharmacologic (BI3406 treatment) inhibition of SOS1 was evaluated in SOS2KO mice. Immortalized MEFs harboring different KRAS mutations were used to compare the potential therapeutic effect of genetic or pharmacologic inhibition of SOS1, in vitro and in vivo, using xenograft models. Furthermore, a mouse model of KRASG12D-driven lung adenocarcinoma (LUAD) was used to characterize the in vivo effect(s) of pharmacological inhibition of SOS1.
In contrast to the lethality resulting from genetically-mediated disruption of both SOS1 and SOS2, BI3406 administration did not show any significant toxicity or deleterious biological effects in SOS2KO mice. Both genetic and pharmacologic inhibition of SOS1 in KRASmut cells resulted in reduced RAS/MAPK pathway signaling and significantly decreased tumor progression in KRASmut-driven xenografts models. Moreover, BI3406 administration in a KRASG12D LUAD model strongly reduced lung tumor burden and volume, correlating with reduced pERK levels.
In conclusion, blocking SOS1 GEF activity results in reduced tumor burden and identifies a
therapeutic window for BI3406-mediated SOS1 inhibition in presence of SOS2KO, supporting the consideration of the SOS GEFs as bona fide therapy targets for KRASmut LUAD and other RAS-dependent diseases.
Combination of the novel SHP2 inhibitor RMC-4550 and the novel ERK inhibitor LY3214996 for the treatment of KRAS mutant pancreatic cancer
Introduction: Mutant KRAS is present in over 90 % of pancreatic as well as 30-40 % of lung and colorectal cancers and is one of the most common oncogenic drivers. Despite decades of research and the recent
emergence of isoform-specific KRASG12C– and KRASG12D-inhibitors, most mutant KRAS isoforms, including the ones frequently associated with pancreatic ductal adenocarcinoma (PDAC), cannot be targeted directly. We and others have shown that targeting single RAS downstream effectors like MEK or ERK alone induces adaptive mechanisms leading to tumor recurrence or resistance. We report here on an alternative way of targeting mutant KRAS by the combined use of RMC-4550, targeting the non-receptor tyrosine phosphatase SHP2 upstream of KRAS, and LY3214996, targeting ERK, a key serine/threonine kinase downstream of KRAS.
Methods: We investigated in vitro growth inhibition, apoptosis induction, and MAPK-pathway inhibition of RMC-4550 and LY3214996 in murine and human PDAC cells. Wild-type littermates from in-house breedings and NSG mice were used for an in vivo MTD (maximum tolerated dose). We then used several models to evaluate in vivo efficacy and tolerability of RMC-4550 and LY3214996 as monotherapy or in combination and in different regimens.
Results: RMC-4550 + LY3214996 shows synergistic anticancer activity, superior disruption of the MAPK pathway, and significantly increased apoptosis induction compared to single-agent treatments in PDAC cells. We also demonstrate good in vivo tolerability and efficacy of the combination, with significant tumor regression in multiple PDAC mouse models. We also show that 18F-FDG PET can be used to detect and predict early drug responses in animal models.
Conclusion: The combination of SHP2 and ERK inhibition was shown to be highly effective in KRAS mutant pancreatic cancer models and is currently under investigation in a clinical trial (SHERPA, SHP2 and ERK inhibition in pancreatic cancer, NCT04916236) in patients with KRAS-mutant NSCLC, CRC and PDAC.
Targeting the RAS Pathway through a First-in-Class DOCK5 Degrader
KRAS is mutated in roughly 20% of all tumors making it the most prevalent oncogenic driver in cancer, with particularly high penetrance in Pancreatic, Colorectal and Lung adenocarcinomas. 2021 and 2022 saw the FDA approval of KRAS-G12C covalent small molecule inhibitors; Sotorasib from Amgen, followed by Adagrasib from Mirati, for 2L lung cancer patients whose tumors carry a G1C mutation. G12C selective molecules have shown compelling yet limited clinical efficacy with PFS of ≈6- onths. This is in part due to the acquisition of on-target mutations in KRAS itself, as well as on-pathway mutations, emphasizing the continued dependency of these tumors on ERK-MAPK signaling.
DOCK5 has been revealed through genetic dependency screens to be a fitness gene selectively for KRAS mutant cancers. Internal oncogenic network analysis also emphasizes DOCK5 as a co-occurring lineage specific dependency of Pancreatic tumors. Moreover, DOCK5 was revealed as a top sensitizer to KRAS G12C inhibitors across multiple KRAS mutant cell models in compound anchored CRISPR screens.
A DEL enabled drug discovery effort by Pfizer has resulted in the generation of a highly potent and selective degrader of DOCK5. Targeted protein degradation of DOCK5 demonstrates anti-tumor activity in
KRAS mutant PDAC cell line models both in vitro, and in vivo in flank xenograft studies. In vivo efficacy correlates with tumor DOCK5 protein levels and PD biomarker responses.
DOCK5 degradation and KRAS inhibition converge on regulating an overlapping ERK-MAPK transcription factor network. Consistent with this we show that combining our DOCK5deg with direct KRAS (G12C/ D) targeting agents, demonstrates superior efficacy in KRAS mutant cell line models.
Combining KRAS-targeted agents with secondary targeted therapies that overcome adaptive resistance and delay acquired resistance will be required to provide greater patient responses. Here Pfizer proposes
a first-in Class DOCK5 PROTAC as a candidate KRAS targeted therapy combination opportunity.
Exploring mutation-specific differences of KRAS: conformational dynamics and implications for effector-protein preferences
Different oncogenic KRAS mutations are not equal. Our earlier simulation work suggested that conformational dynamics of KRAS G12X mutants is mutation-specific . Since, it has
been demonstrated that the mutants may exhibit, for instance, unique effector protein
preferences even in the clinical context, as observed with the impaired binding of
KRAS(G12R) to PI3Kα . However, the underlying mechanisms for the observed
discrepancies between the mutants are still somewhat unclear.
To gain better understanding of these discrepancies among KRAS mutants, we decided to expand our study of KRAS conformational dynamics . The enhanced force fields and increased computational capacity enabled us to improve the accuracy of our molecular dynamics simulations while taking them to longer timescales, resulting in nearly millisecond simulation data. This time, we also considered in greater detail the effect of different GTP- bound states, state 1 and state 2. These factors ensured the improved quality of our dataset to assess potential discrepancies between KRAS mutants and their effector protein binding preferences. In our simulations, we focused on five oncogenic mutants in addition to wild- type KRAS (total simulation time of 400 µs) and investigated the conformational dynamics of these mutants in complex with effector proteins (simulation time >500 µs). In the contribution, we will discuss our key findings and their potential implications on effector protein binding.
 Pantsar T. et al. Assessment of Mutation Probabilities of KRAS G12 Missense Mutants and Their Long-Timescale Dynamics by Atomistic Molecular Simulations and Markov State Modeling. PLoS Comput Biol 2018, 14 (9), e1006458.
 Hobbs G. A. et al. Atypical KRASG12R Mutant Is Impaired in PI3K Signaling and Macropinocytosis in Pancreatic Cancer. Cancer Discov 2020, 10 (1), 104–123.
 Kurki M. et al. Manuscript in preparation.
Assessment of KRAS mutant allele binding by covalent and non-covalent inhibitors through a cellular Switch-I/II and Switch-II pocket target engagement assay
KRAS mutations are highly prevalent in several cancer types, most commonly affecting codons 12, 13 and 61. As of now, only KRAS G12C mutations can be selectively targeted with clinically approved small-molecule inhibitors, while development for other allele-selective as well as pan(K)RAS small molecule inhibitors is actively pursued. Currently, most approaches focus on targeting the Switch-II pocket covalently or non-covalently. To characterize the binding of candidate (K)RAS inhibitors to different KRAS mutant alleles directly in cells, we used a bioluminescence resonance energy transfer (BRET) competition assay relying on (i) transfected, labeled KRAS monomers that then form multimeric complexes in cells, thereby generating the BRET donor signal, and (ii) bifunctional fluorescent probes based on Boehringer Ingelheim Switch-I/II and Switch-II pocket inhibitors that act as BRET acceptors unless competed away by test molecules.
Using this assay, we profiled a panel of diverse reversible and irreversible KRAS inhibitors for target engagement of KRAS WT, G12C, G12D, G12V, G13D and Q61H. These studies allow for hypothesis generation on inhibitor binding mode and engagement of different KRAS mutant alleles with different biochemical properties, which could help guide the development of future inhibitors.
Avutometinib and FAK inhibitor concomitant treatment enhances antitumor efficacy of sotorasib in mice harboring KRASG12C;p53 mutant lung adenocarcinomas
Lung cancer is the leading cause of cancer mortality worldwide, accounting for up to 18% of all cancer-related deaths. Lung adenocarcinoma (LUAD) is the most common subtype representing ~40% of all lung cancer cases. Most LUADs are diagnosed in advanced stages, which in most cases are already incurable. At the molecular level, oncogenic KRAS mutations occur in a third of the LUAD cases with KRAS G12C being mutated in 13% of patients with LUAD. Recently, sotorasib and adagrasib, covalent KRAS G12C inhibitors (G12Ci) have demonstrated antitumor activity in patients with KRASG12C LUAD and received FDA approval. However, treatment with G12Ci rapidly induces resistance due to acquired mutations in the MAPK pathway occurring
clinically upon progression. Altogether, these data support the need for clinical combinations with G12Ci to simultaneously target multiple nodes in the MAPK pathway. Avutometinib (VS-6766) 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 RAF1.
Here, we tested whether addition of avutometinib ± the FAK inhibitor VS-4718, to sotorasib could improve antitumor efficacy in a G12Ci-naïve setting. Avutometinib potentiated efficacy of sotorasib including complete responses in 25% of the mice in a KRASG12C; p53null LUAD model, whereas
addition of avutometinib and FAK inhibitor to sotorasib promoted a significant increase in the survival of mice. To model G12Ci resistance, avutometinib + FAK inhibitor were added following progression on sotorasib in the KRASG12C; p53null LUAD mouse model, and this triplet restored
antitumor response in most of the tumor lesions. These results support ongoing clinical studies of avutometinib in combination with sotorasib (NCT05074810) and adagrasib (NCT05375994) for patients with KRASG12C LUAD and suggest that addition of the FAK inhibitor defactinib may further increase antitumor efficacy in both G12Ci-naïve and G12Ci-progression settings.
Dissecting The Tissue-Specific Oncogenic Activity Of K-RasG12D
KRAS is the most commonly mutated oncogene in human cancers, but its mutational pattern is restricted to tumors originating in a subset of tissues. Notably, K-Ras expression levels do not correlate with mutation frequency. A better understanding of the biological mechanisms underlying the tissue-specific mutational pattern of KRAS could uncover novel therapeutic avenues.
Utilizing a variety of GEMMs carrying the Cre-dependent KrasLSL-G12D allele allowed us to force expression of activated K-Ras in the entire animal and study the response at the phenotypic and molecular level in ten tissues. We used EdU incorporaKon to assess changes in proliferation and a ddPCR-based assay to measure the fraction of recombined Kras-LoxP-G12D/WT cells in tissues over time. To idenKfy correlaKons between the baseline cell circuitry and K-RasG12D permissivity we acquired TMT-based (phospho-)proteomic measurements from wild-type mouse tissues. In addition, we generated a MS-based targeted assay for 180 members of the murine Ras pathway. This tool enabled us to obtain precise relative abundance measurements in unfractionated tissue samples using low amounts of starting material and reduced MS-instrument time.
As expected, only a subset of tissues displayed histological changes in response to oncogenic K- Ras expression. Strikingly, these did not always correlate with changes in proliferation. While K-RasG12D increased proliferation in some tissues it reduced it in others. Notably, many tissues displayed sex-dependent proliferation responses. Importantly, KRasG12D expression activated MAPK signaling in most tissues independently of its effects on proliferation. Consistent with an overall fitness advantage the fraction of KRasG12D-expressing cells increased over time in permissive tissues. Surprisingly, KRasG12D-expressing cells were not eliminated from non-permissive tissues and instead remained present at fixed proportions. Furthermore, KRasG12D did not induce senescence or apoptosis in non-permissive tissues.
Lastly, we identified cases of strong correlation between the expression of nodes in the Ras signaling pathway and a tissue’s K-RasG12D permissivity profile.
RAS-PI3K signaling: a master regulator of CAF phenotype to promote lung tumor progression
Lung cancer remains a clinical unmet need and a significant cause of death. KRAS is mutated in 30% of non-small cell lung cancer (NSCLC), the most frequent type of lung cancer. Recently, the FDA approved the first RAS-targeted therapies for NSCLC with RASG12C mutations, but patients harboring other mutations are not eligible for treatment.
Within the tumor microenvironment, cancer-associated fibroblasts (CAFs) play a crucial role in shaping tumor behavior. Among these, two distinct subtypes, inflammatory CAFs (iCAFs) and myofibroblastic CAFs (myoCAFs), exhibit differing functions. iCAFs are characterized by their involvement in immune response modulation, secreting pro-inflammatory cytokines, and contributing to an immunosuppressive environment that aids tumor immune evasion. On the other hand, myoCAFs possess contractile properties and contribute to tissue remodeling,
angiogenesis, and extracellular matrix deposition, thereby facilitating tumor growth and metastasis. These differing functions of iCAFs and myoCAFs contribute to heterogeneous tumor behavior, influencing factors such as tumor invasion, angiogenesis, and immune cell infiltration. The distinct phenotypes of CAFs can thus lead to varying therapeutic responses and clinical outcomes, highlighting the importance of understanding their roles in the context of tumor biology.
Here we show that in NSCLC models, CAFs rely on RAS-PI3K signaling for normal function and acquisition of an iCAF or myoCAF phenotype. Furthermore, genetic disruption of RAS-PI3K interaction, or pharmacological inhibition of PI3K p110α activity with BYL719 in murine and human CAFs, results in a shift towards an iCAF phenotype characterized by heightened IL6 production and diminished α-smooth muscle actin (α-SMA) levels. As a consequence, CAFs defective in RAS-PI3K form thinner and more disorganized extracellular matrices that are defective in components such as collagen or fibronectin, profoundly impacting macrophage and lymphocyte function. These defective ECMs also compromise proliferation, activation of epithelial to mesenchymal programs (EMT) and migration potential of several KRAS mutant lung cancer cell lines. Additionally, experiments in mice with fibroblast-specific disruption of RAS- PI3K interaction (Pik3caRBD/Lox/Col1a2CreER/WT)show that disrupting this interaction in CAFs causes a significant delay in KRAS-driven lung tumor growth and changes in CAF activation, ECM composition and immune modulation, further demonstrating the key role of RAS-PI3K interaction in CAFs for cancer progression.
In summary, this study highlights the potential of targeting the tumor microenvironment, specifically cancer-associated fibroblasts (CAFs), as a novel mutation-agnostic approach. The identification of RAS-PI3K signaling’s pivotal role in CAF phenotype acquisition further emphasizes its significance in orchestrating tumor progression. These findings provide valuable insights for developing innovative therapeutic strategies to address the complexity of NSCLC, addressing the urgent need for more effective treatment options.
Targeting NUPR1-dependet Stress Granules Formation as an Efficient Strategy to Induce Synthetic Lethality In KrasG12D -Dependent Tumors
Stress granules (SGs) are membrane-less organelles formed by liquid-liquid phase separation (LLPS) that regulates RNA metabolism and protein synthesis during stress responses. They are also crucial for tumor initiation and progression. Here, we report a novel subtype of SG induced by the overexpression of the stress-associated protein NUPR1, in response to KRASG12D
oncogene. NUPR1 is an Intrinsically Disordered Protein (IDP) able to induce LLPS and promote the formation of NUPR1- dependent SGs. Here, we provide preclinical evidences supporting the use of NUPR1-dependent SG inhibition as an effective strategy for targeting KRASG12D-dependent PDAC initiation, using pharmacological or genetic NUPR1 inhibition.
In this study, we show that NUPR1 can produce droplets through LLPS, but this capacity is absent in the presence of its inhibitor (ZZW-115). LLPS induced by NUPR1 is essential for NUPR1- dependent SG formation since genetic or pharmacological inhibition of NUPR1 activity prevents
SG formation in PDAC cells. In addition, we found that the oncogenic stress driven by KRASG12D mutation induced a strong overexpression of NUPR1 and the promotion of NUPR1-dependent SGs formation as a protective mechanism for cell survival. Notably, forced KRASG12D expression in
pancreatic cells became them highly sensitive to NUPR1 inactivation suggesting that these NUPR1-dependent SGs are essential for KRASG12D transformation process. Finally, ZZW-115-treatment of Pdx1-Cre;LSL-KRASG12D mice blocks the transformation process, contrary to vehicle- treated, by inducing cell death by apoptosis thought caspase-3 activation specifically in cells with activated KRASG12D, indicating that NUPR1-dependent SGs formation is necessary for PDAC development in vitro and in vivo. More interesting, when preneoplastic lesions PanIN were established in Pdx1-Cre;LSL-KRASG12D mice daily treatment with ZZW-115 for one week block SGs formation starting the reversion process of transformation.
This study provides a preclinical proof of concept suggesting that targeting NUPR1-dependent SGs formation by inhibiting NUPR1 could be a promising synthetic lethality therapeutic strategy for KRASG12D -dependent tumors.
Convergence of RAS and PP2A activity on the phosphoregulation of chromatin repressor complexes
Protein phosphatase 2A (PP2A) is a tumor suppressor while RAS proteins are potent oncogenes in different cancer types. It is well established that RAS-mediated oncogenic transformation requires simultaneous inhibition of PP2A, but the molecular basis of their interaction is poorly understood. To address the target mechanisms co- regulated by RAS and PP2A we performed
a phosphoproteomics screen upon RAS or PP2A manipulation. Overall the results indicated that RAS and PP2A regulate overlapping cellular processes, and that their activities might in particular converge at epigenetic machineries. To study the RAS and PP2A synergy on transcriptional and epigenome regulation we performed multi-omics analysis of cancer cells in which RAS and PP2A activities were modulated. To investigate the DNA methylation changes
and chromatin remodeling effects upon PP2A modulation we used RRBS (Reduced
representation bisulfite sequencing) & ATAC-seq (Assay for Transposase-Accessible
Chromatin using sequencing) respectively. The study reveals that RAS and PP2A regulate common phosphosites on epigenetic proteins such as HDAC1/2, KDM1A, MTA1/2, RNF168, and TP53BP. Either pharmacological or siRNA mediated RAS or PP2A modulation significantly affected HDAC1/2 recruitment to the chromatin while mutagenesis of the co- regulated phosphosite on RNF168 affected its interaction with TP53BP1. Further RAS activation and PP2A inhibition resulted in derepression of a GFP reporter indicative of epigenetic silencing and global gene expression leading to oncogenic transcription. PP2A inhibition further resulted in global DNA hypomethylation and an open state of chromatin confirming its role as a global repressor of epigenetic processes. Collectively these results identify a novel phosphorylation switch on chromatin repressor complex as a converge point of KRAS and PP2A activities in cancer. Generally the results provide first indications to global importance of PP2A-mediated phosphorylation regulation in epigenetic gene regulation in cancer.