RAS Pathway Inhibitors Research Articles

Abstract C026: Concurrent inhibition of the RAS ERK-MAPK pathway and PIKfyve as a therapeutic strategy for pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is characterized clinically by poor survival and mechanistically by KRAS- and autophagy-dependent growth. We and others previously demonstrated that inhibition of KRAS signaling via downstream inhibition of the RAF-MEK-ERK pathway enhanced autophagic flux and dependency of PDAC on autophagy. Furthermore, we demonstrated that concurrent treatment with the nonspecific autophagy inhibitor chloroquine (CQ) and ERK MAPK inhibitors synergistically blocked PDAC growth. These findings provided rationale for our initiation of Phase I/II clinical trials evaluating the combination of MEKi (binimetinib; NCT4132505) or ERKi (LY3214996; NCT04386057) with HCQ in PDAC. However, a limitation of this approach is that CQ/HCQ is limited in terms of specificity and potency. Thus, we aimed to identify alternative anti-autophagy strategies. We performed a CRISPR-Cas9 loss- of-function screen in PDAC cell lines that identified the lipid kinase PIKfyve as a growth- promoting gene. PIKfyve is a lipid kinase that is essential for the generation of PI(3,5)P2 and critical for the dynamic regulation of lysosomal recycling. Because PIKfyve has been implicated in regulation of autophagy in other cellular contexts, we evaluated the effects of PIKfyve inhibition on growth and autophagic flux in PDAC cell lines and tumors. We demonstrated that PIKfyve inhibition by the small molecule apilimod resulted in durable growth suppression, with much greater potency than CQ treatment. PIKfyve inhibition potently reduced autophagy as indicated by decreased autophagic flux and the robust accumulation of autophagy-related proteins. Additionally, PIKfyve inhibition resulted in the accumulation of LAMP1 positive vacuoles that exhibited impaired cargo degradation, likely due to a lack of required acidity. Next, we hypothesized that PIKfye inhibition would sensitize PDAC cells to RAS pathway inhibition. We demonstrated that PIKfyve inhibition blocked the compensatory increases in autophagic flux associated both with MEK inhibition and with direct RAS inhibition. Accordingly, combined inhibition of PIKfyve and either the RAS ERK-MAPK pathway or RAS itself showed robust growth suppression across a panel of KRAS-mutant PDAC models. Growth suppression was due, in part, to potentiated cell cycle arrest and induction of apoptosis following loss of IAP proteins. These findings implicate PIKfyve as an effective anti-autophagy target when paired with RAS or ERK-MAPK pathway inhibition in pre-clinical models of PDAC. Citation Format: Jonathan M DeLiberty, Mallory K Roach, Clint A Stalnecker, Ryan Robb, Elyse G Schechter, Noah L Pieper, Runying Yang, Scott Bang, Khalilah E Taylor, Kristina Drizyte-Miller, John P Morris IV, Channing J Der, Adrienne D Cox, Kirsten L Bryant. Concurrent inhibition of the RAS ERK-MAPK pathway and PIKfyve as a therapeutic strategy for pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C026.

Abstract IA-06: Elucidation of metabolic resistance mechanisms to RAS inhibition

Abstract Pancreatic ductal adenocarcinoma (PDAC) is characterized by KRAS- and autophagy-dependent growth. We previously determined that concurrent inhibition of autophagy, using the lysosomal inhibitor chloroquine (CQ), and of ERK, using a small molecule ERK inhibitor (ERKi), synergistically suppressed the growth of pancreatic ductal adenocarcinoma (PDAC) cell lines and patient xenograft-derived (PDX) organoids in vitro and PDX tumors in vivo. Our findings provided the rationale for our initiation of Phase I and Phase I/II clinical trials evaluating the combination of MEKi (binimetinib; NCT04132505) or ERKi (LY3214996; NCT04386057) with hydroxychloroquine (HCQ) in PDAC. We have extended these findings and determined that the combined inhibition of KRASG12C or KRASG12D and autophagy was effective in KRAS-mutant cancers. Our ongoing studies are centered on developing additional combinations for targeting metabolic resistance mechanisms to KRAS or ERK MAPK targeted therapies. First, we performed a CRISPR/Cas-9 mediated genetic loss-of-function screen in the presence of CQ to determine additional sensitizers to autophagy inhibition. Top sensitizers included multiple facilitators of the DNA damage response, mTOR pathway components, and genes involved in the upstream regulation of the autophagy pathway. Additionally, we identified PIKfyve, a lipid kinase critical for the recycling dynamics of lysosomes, as an essential autophagy-related gene in PDAC cells. PIKfyve inhibition with apilimod resulted in a potent reduction of autophagic flux and growth. Importantly, PIKfyve inhibition blocked the compensatory increases in autophagic flux associated both with MEK inhibition and with direct RAS inhibition. Accordingly, combined inhibition of PIKfyve and either the RAS ERK-MAPK pathway or RAS itself showed robust growth suppression across a panel of KRAS-mutant PDAC models. Growth suppression was due, in part, to potentiated cell cycle arrest and induction of apoptosis following loss of IAP proteins. We conclude that concurrent inhibition of RAS and PIKfyve is a synergistic, cytotoxic combination that may represent a novel therapeutic strategy for PDAC. In a parallel line of investigation, we determined that autophagic signaling is temporally regulated following RAS pathway inhibition, peaking at 48-72 hours, and returning to basal levels by 7 days. Interestingly, we found that as autophagic upregulation subsides, another nutrient scavenging pathway, macropinocytosis, increases. We hypothesize that further dissection of the dynamic regulation of autophagy and macropinocytosis will improve current anti-nutrient scavenging treatment strategies. We conclude that concurrent suppression of multiple metabolic processes, to block compensatory rebound activities, will be needed for effective PDAC treatment. Citation Format: Jonathan M. DeLiberty, Ryan Robb, Mallory K. Roach, Sarah E. Ackermann, Runying Yang, Noah L. Pieper, Khalilah E. Taylor, Scott Bang, Elisa Baldelli, Emanuel F. Petricoin III, John P. Morris IV, Clint A. Stalnecker, Kirsten L. Bryant. Elucidation of metabolic resistance mechanisms to RAS inhibition [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr IA-06.

Epigenetic and oncogenic inhibitors cooperatively drive differentiation and kill KRAS-mutant colorectal cancers.

Current treatments for KRAS-mutant colorectal cancers (CRCs) are often limited by cellular plasticity and rewiring responses. Here we describe a promising therapeutic strategy that simultaneously targets epigenetic and oncogenic signals. Specifically, we show that inhibitors of the histone methyltransferase, EZH2, synergize with various RAS pathway inhibitors and promote dramatic tumor regression in vivo. Together these agents cooperatively suppress WNT-driven transcription and drive CRCs into a more differentiated cell state by inducing the Groucho/TLE corepressor, TLE4, along with a network of WNT pathway inhibitors and intestinal differentiation proteins. However, these agents also induce the pro-apoptotic protein BMF, which subsequently kills these more differentiated cells. Accordingly, cell death can be prevented by activating β-catenin, blocking differentiation, or by ablating BMF expression. Collectively, these studies reveal a new therapeutic approach for treating KRAS-mutant CRCs and illustrate a critical convergence of EZH2 and RAS on oncogenic WNT signals, intestinal differentiation, and apoptosis.

Mechanisms of Response and Tolerance to Active RAS Inhibition in KRAS-Mutant Non-Small Cell Lung Cancer.

Resistance to inactive state-selective RASG12C inhibitors frequently entails accumulation of RASGTP, rendering effective inhibition of active RAS potentially desirable. Here, we evaluated the antitumor activity of the RAS(ON) multiselective tricomplex inhibitor RMC-7977 and dissected mechanisms of response and tolerance in KRASG12C-mutant non-small cell lung cancer (NSCLC). Broad-spectrum reversible RASGTP inhibition with or without concurrent covalent targeting of active RASG12C yielded superior and differentiated antitumor activity across diverse comutational KRASG12C-mutant NSCLC mouse models of primary or acquired RASG12C(ON) or RASG12C(OFF) inhibitor resistance. Interrogation of time-resolved single-cell transcriptional responses established an in vivo atlas of multimodal acute and chronic RAS pathway inhibition in the NSCLC ecosystem and uncovered a regenerative mucinous transcriptional program that supports long-term tumor cell persistence. In patients with advanced KRASG12C-mutant NSCLC, the presence of mucinous histologic features portended poor response to sotorasib or adagrasib. Our results have potential implications for personalized medicine and the development of rational RAS inhibitor-anchored therapeutic strategies. Significance: Our work reveals robust and durable antitumor activity of the preclinical RAS(ON) multiselective inhibitor RMC-7977 against difficult-to-treat subsets of KRASG12C-mutant NSCLC with primary or acquired RASG12C inhibitor resistance and identifies a conserved mucinous transcriptional state that supports RAS inhibitor tolerance. See related commentary by Marasco and Misale, p. 2018.

Prognosis and immunotherapy in melanoma based on selenoprotein k-related signature

Selenium and selenoproteins are closely related to melanoma progression. However, it is unclear how SELENOK affects lipid metabolism, endoplasmic reticulum stress (ERS), immune cell infiltration, survival, and prognosis in melanoma patients. Transcriptome data from melanoma patients was used to investigate SELENOK levels and their effect on prognosis, followed by an investigation of SELENOK’s effects on immune cell infiltration. Furthermore, a risk model based on ERS, lipid metabolism, and immune-related genes was constructed, and its utility in melanoma prognosis was evaluated. Finally, the drug sensitivity of the risk model was analyzed to provide a reference for melanoma therapy. The results showed that melanoma with a high SELENOK level had a greater degree of immune cell infiltration and a better prognosis. Additionally, SELENOK was found to regulate ERS, lipid metabolism, and immune cell infiltration in melanoma. The risk model based on SELENOK signature genes successfully predicted the prognosis of melanoma, and the low-risk group exhibited a favorable immunological microenvironment. Furthermore, high-risk patients with melanoma were candidates for chemotherapy with RAS pathway inhibitors, whereas low-risk patients were more susceptible to routinely used chemotherapy medicines. In summary, SELENOK was shown to regulate ERS, lipid metabolism, and immune cell infiltration in melanoma, and SELENOK was positively associated with the prognosis of melanoma. The risk model based on SELENOK signature genes was valuable for melanoma prognosis and therapy.

Targeting RAS/RAF/PI3K pathway for CD40 induction in melanoma cells to overcome resistance to anti-PD1 immunotherapies

Abstract The first-line treatment for metastatic melanoma is immune checkpoint blockade (ICB), but it fails in many patients and may have serious adverse events. We showed that rigosertib (RGS), a RAS-pathway inhibitor, promotes CD40 upregulation on melanoma cells and synergizes with ICB in preclinical melanoma models. However, residual disease still occurred with uncontrolled ERK activation. Our present study explores the efficacy of RGS plus trametinib (T), a MEK1/2 inhibitor, to overcome ICB resistance. The concurrent RGS+T+αPD1 resulted in an additive effect to suppress ICB-resistant NRASmut 1014 tumor growth, and 44.4% (4/9) tumors completely regressed after 2 weeks of treatment. The RGS+T regimen promoted CD8+ T cell responses in the tumor microenvironment (TME) and extended the time to αPD1 resistance (p<0.0001). Combining RGS+T with agonist CD40 (aCD40) resulted in increased CD8+ T cells, natural killer cells, and M1 macrophages in the TME, with a reduction of myeloid-like CD11b+PD-L1+ regulatory B cells in the tumors (~70%, p<0.0001) and tumor-draining lymph nodes (~40%, p<0.0001). CRISPR/dCas9-based overexpression of CD40 (CD40-OE) in 1014 melanoma cells reduced in vivo tumor growth and successfully turned the ICB-resistant tumors into responders to αPD1 (p=0.025), which further regressed with aCD40+αPD1 (p<0.001). Our preclinical data support the therapeutic use of RAS/RAF/PI3K inhibition plus CD40 agonism for metastatic melanoma patients who do not respond to ICB.

Abstract 3884: Artificial intelligence (AI)-enabled discovery of GLX1546, a novel and potent inhibitor of ULK1/2 kinases and autophagy

Abstract Background: Autophagy is an evolutionarily conserved process, critical for the recycling of damaged or excess cellular components, which promotes cell survival and growth under stress conditions. Cancer cells, especially those with RAS mutations, can activate the ULK1/2 (UNC-51-like kinase 1/2)-initiated autophagy pathway for survival and diminish RAS pathway inhibitors’ efficacy. The combination of RAS pathway and ULK1/2 kinase inhibitors may provide synergistic or additive effects for RAS mutated solid tumors. We have identified a structurally small molecule, GLX1546, which shows strong inhibition against ULK1/2, as well as autophagy induced by RAS pathway inhibitors in cells. Methods: Multiple dual-target activity predictive models were constructed and trained using extensive protein-ligand interaction data. These models, in conjunction with in-house built physicochemical property predictive models, were integrated into an AI-driven computational pipeline to perform the in silico screening on both ULK1/2 inhibitory activities and drug-like properties. ULK1/2 in vitro activities were assessed by enzymatic assays and cellular assays based on the phosphorylation of ULK substrate. The in vivo anti-tumor efficacy of GLX1546 was evaluated in A549 CDX mouse model both as monotherapy and in combination with trametinib. Results: An AI-driven high-throughput virtual screening strategy using deep learning models was implemented for the discovery of novel and potent ULK1/2 small-molecule inhibitors. GLX1546 was developed as a potent ULK1/2 inhibitor with IC50 values of 1.13 nM for ULK1 and 4.75 nM for ULK2. Moreover, to address trametinib-induced activation of ULK1/2 in KRAS and BRAF mutated solid tumors, GLX1546 provided IC50 values of 54 nM in A549, 21 nM in HCT 116, 67 nM in MIA PaCa-2, and 76 nM in A375 cells against both basal and trametinib-induced pATG13. Furthermore, GLX1546 exhibited good exposure, sound safety, and high oral bioavailability (~100%) in mice. The combination of trametinib (PO, QD) with GLX1546 (PO, BID) in 21-day cycles also led to inhibition of ATG13 phosphorylation and greater tumor growth inhibition than trametinib alone in A549 CDX mouse model. Conclusions: These results indicate that GLX1546 can inhibit ULK-mediated autophagy induced by trametinib. The combination of GLX1546 with RAS pathway inhibitors may offer a novel cancer therapy for RAS mutated cancers, highlighting AI as an effective tool for drug discovery. Keywords: AI-driven drug discovery, autophagy, small-molecule inhibitors, ULK Citation Format: Guonan Cui, Xulun Sun, Wenyu Zhu, Qi Wang, Jinchen Li, Zhongzong Pan, Chengtao Li. Artificial intelligence (AI)-enabled discovery of GLX1546, a novel and potent inhibitor of ULK1/2 kinases and autophagy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3884.

Abstract 1230: MTX-531, a first-in-class pan-PI3K inhibitor spares hyperinsulinemia yielding durable tumor regressions and resilience to adaptive resistance

Abstract Adaptive resistance mechanisms compromise the long-term effectiveness of kinase-targeted agents dictating the need for strategic combination approaches. MTX-531 was computationally designed to selectively target both PI3K and wild type EGFR. MTX-531 exhibits low nanomolar potency against the PI3K isoform family and EGFR with a high degree of specificity predicted by co-crystal structural analyses. Full kinome screening confirmed that MTX-531 is exquisitely selective for its intended targets. We evaluated the pharmacological profile of MTX-531, whereupon single agent treatment with this lead molecule elicited a high incidence of durable tumor regressions in a broad panel of PIK3CA mutant CDX and PDX squamous head and neck (HNSCC) models. The combination of MTX-531 with RAS pathway inhibitors, including agents directed against KRAS G12C, led to durable regressions of KRAS mutant colorectal and pancreatic xenografts, resulting in striking increases in median survival. MTX-531 is exceptionally well tolerated in mice and uniquely does not lead to hyperglycemia and hyperinsulinemia commonly seen with predecessor pan PI3K inhibitors. MTX-531 acts as an agonist of PPARγ, a structural feature of the molecule that is thought to mitigate hyperglycemia induced by PI3K inhibition. This unique attribute of MTX-531 confers a favorable therapeutic index not typically seen with PI3K inhibitors. To the best of our knowledge, MTX-531 is the first reported pan-PI3K inhibitor that does not lead to hyperglycemia. The pharmaceutical profile of MTX-531 includes favorable cross-species oral bioavailability, microsomal stability, and a facile 3 step chemical synthesis. MTX-531, which has the unique capability of concurrently and selectively inhibiting PI3K and EGFR, illustrates the power of rational computational drug design to target multiple adaptive resistance mechanisms in a single molecule. The versatility of MTX-531 in both the single agent and combination settings offers a breadth of development strategies that continue to evolve as new combination candidates become available. MTX-531 is currently undergoing advanced preclinical development for anticipated first in human clinical trials in late 2024. Citation Format: Christopher E. Whitehead, Elizabeth K. Ziemke, Christy L. Frankowski-McGregor, Rachel A. Mumby, June Chung, Jinju Li, Nathaniel Osher, Oluwadara Coker, Michelle Norris, Scott Kopetz, Veera Baladandayuthapani, Melinda Hollingshead, Sharad Verma, Judith S. Sebolt-Leopold. MTX-531, a first-in-class pan-PI3K inhibitor spares hyperinsulinemia yielding durable tumor regressions and resilience to adaptive resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1230.

Abstract 657: Discovery of a novel brain penetrant SHP2 allosteric inhibitor with anti-tumor effects

Abstract KRAS is the most frequently dysregulated oncogene with high prevalence in non-small cell lung cancer (NSCLC), colorectal cancer (CRC), and pancreatic ductal adenocarcinoma (PDAC). Currently, FDA-approved sotorasib and adagrasib provide ground breaking through therapies for cancer patients harboring KRAS G12C mutation. However, there is still high unmet medical needs for inhibitors targeting broader KRAS-driven tumor, especially a substantial unmet medical need for agents that can penetrate BBB to control the brain metastases. SHP2, a key activator of KRAS located in the upstream of RAS pathway, facilitates the conversion of GDP-bound KRAS (off) state to GTP-bound KRAS (on) state serving as a key mechanism of KRAS activation. Therefore, SHP2 could be a promising therapeutic target to shut down the oncogenic RAS pathway as a broad combination partner with RAS/RAF/MEK/ERK pathway inhibitors. Here we report a highly potent and selective brain penetrant SHP2 inhibitor that is designed to target solid tumors metastazied to brain. KT-01766 showed excellent potency against SHP2 protein in various biochemical and cellular assays and showed the robust synergistic effect with RAS pathway inhibitors. KT-01766 as a mono therapy and in combination with KRAS G12C inhibitors demonstrated great anti-tumor efficacy with no apparent toxicity in mouse xenograft models harboring KRAS mutations. Furthermore, robust anti-tumor acitivty was confirmed in intracranial brain tumor model, supporting its potential to control brain-metastasized tumors. The present data suggest that KT-01766 is a potential candidate for combination therapy with agents targeting RAS pathway, as it is able to effectively suppress tumor growth both systemically and within the brain in KRAS mutated cancers. Citation Format: Miyeon Kim, Dongsu Kim, Yeejin Jeon, Kyeong Jin Yoon, Anna Jang, Mijung Lee, Dahye Jeon, Soyeon Jang, Jinhwan Kim, Eunji Kim, Jihoon Park, Victor Hong, Hayoun Jung, Sungpil Choi. Discovery of a novel brain penetrant SHP2 allosteric inhibitor with anti-tumor effects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 657.

Abstract 3628: Combined mek and jak inhibition in vitro is an effective strategy in overcoming jak inhibitor resistance in primary myelofibrosis (PMF)

Abstract Introduction Primary myelofibrosis (PMF) is a myeloproliferative neoplasm arising from various mutations in myeloid-committed hematopoietic progenitor cells. Treatment of PMF is challenging as Jak inhibitor resistance is common, making the expansion of molecular characterization and novel therapeutic target identification in PMF essential. The main goals of this study are to determine both the presence of Ras activity in PMF and the impact on Ras/MAP Kinase pathway targeting in the disease. Methods Integrated genomic-transcriptomic sequencing analyses were performed on peripheral blood mononuclear cells (PBMCs) purified from 27 patients with PMF at a single institution; protein was then obtained from whole cell lysates. Ras activity was then assessed by applying Ras binding domain (RBD) to sample in an ELISA and was compared between patients with PMF, essential thrombocythemia (ET), polycythemia vera (PV) and healthy donors. Statistical analyses were conducted using two-way unpaired t-tests. The next set of in vitro studies aimed to evaluate the effects of MEK inhibition in PMF. Cell viability studies were first performed on human erythroleukemia (HEL) cells to identify IC50 values. Colony forming assays in methylcellulose-enriched media formulated for the growth of CD34+ cells were then performed in 4 conditions: Ruxolitinib and Cobimetinib monotherapy, combined therapies and untreated sample. Results As previously reported in our initial study, Ras activity in 21 patients with MF was 1.74 fold higher than healthy donors (13 volunteers, p=0.0304) and 1.64 fold higher than in PV/ET (12 patients, p=0.0462). We also reported previously on Ras activity from differential enrichment of myeloid and lymphoid cells from PBMC samples, where a key observation was that myeloid contribution to Ras activity in PMF patients with RAS/MAP Kinase pathway mutations was 3.8 times higher in Ras/MAP Kinase WT patients (p=0.0012), potentially suggesting that RAS pathway mutations have a predominance in myeloid cells. In HEL cell viability assays, cobimetinib and ruxolitinib, when utilized in tandem, resulted in more cell growth inhibition than either agent individually. This same finding was recapitulated in colony forming assays in 4 different patients with varying levels of sensitivity to Jak inhibitors, in all of whom ruxolitinib and cobimetinib lead to a greater reduction in colony formation than ruxolitinib monotherapy. Discussion Data from colony forming assays of bone marrow from patients with PMF identifies at least an additive effect of combined Jak and Ras/MAP Kinase (specifically Mek) inhibition on CD34+ colony formation. The specific observation that Ras pathway inhibition may be utilized as a means of overcoming Jak inhibitor resistance in PMF is a novel finding in this field and may be a valuable avenue for future early-phase clinical trials. Citation Format: Samuel Benjamin Reynolds, Sho Matono, Malathi Kandarpa, Kristen Pettit, Mark Ribick, Moshe Talpaz, Qing Li. Combined mek and jak inhibition in vitro is an effective strategy in overcoming jak inhibitor resistance in primary myelofibrosis (PMF) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3628.

Quando pensare alla sindrome di Noonan

Noonan syndrome is a pleomorphic genetic disorder that causes cardiovascular involvement in a high percentage of affected individuals. Care includes attentiveness to several comorbidities, some directly impacting cardiac management (bleeding diatheses and lymphatic anomalies). More than 50% of patients with Noonan syndrome harbour PTPN11 pathogenic variation, which results in hyperactivation of RAS/ mitogen-activated protein kinase signalling. Molecular diagnosis with gene resequencing panels is now widely available, but phenotype variability and, in some cases, subtlety still make Noonan syndrome difficult to be diagnosed. Until genetic testing becomes universal for patients with congenital heart disease, alertness to the broad clinical presentations of Noonan syndrome remains crucial. Genotype-phenotype associations for Noonan syndrome enable better prognostication for affected patients when a molecular diagnosis is established. Noonan syndrome-specific treatment is still lacking; however, newly developed anticancer RAS pathway inhibitors might fill that gap if safety and efficacy can be established for indications such as pulmonary valve stenosis.

Abstract PR010: Preliminary safety and pharmacokinetic profiles of RMC-6236, a first-in-class, RAS-selective, tri-complex RASMULTI(ON) inhibitor in patients with KRAS mutant solid tumors on the Phase 1 trial RMC-6236-001

Abstract Background: RMC-6236 is an oral RASMULTI(ON) tri-complex inhibitor that is selective for the active, GTP-bound state of both mutant and wild-type variants of the canonical RAS isoforms. RMC-6236 binds to cyclophilin A, which is abundantly expressed in normal tissues and tumors, resulting in a binary complex that potently binds to RAS(ON) to form a tri-complex, blocking downstream signaling. Preclinical studies with RMC-6236 demonstrated deep and sustained regressions across multiple RASMUT tumor types at well tolerated doses, particularly tumors harboring KRAS glycine-12 substitutions (KRASG12X). Methods: Patients with previously treated, advanced KRASG12X mutant solid tumors were enrolled at escalating doses of single agent RMC-6236 administered once daily (QD). Results: As of May 11, 2023, 54 patients received RMC-6236 at dose levels from 10 to 220 mg QD. The majority of patients had pancreatic cancer (26 [48%]), non-small cell lung cancer (13 [24%]), or colorectal cancer (10 [19%]). The most common (≥10% of patients) treatment-related adverse events (TRAEs) were rash and gastrointestinal (GI)-related toxicities that were all Grade 1 or 2 in severity. Most rashes were acneiform (35%) or maculopapular (13%), consistent with known on-target rash presentation from other RAS pathway inhibitors. Rash frequency, but not severity, was dose-dependent. One patient required a dose reduction due to Grade 2 acneiform rash. The most common GI-related toxicities were nausea (32%), diarrhea (19%), and vomiting (15%) that were manageable with standard supportive care. There were two Grade ≥3 TRAEs in patients with pancreatic cancer: Grade 4 large intestine perforation at the site of an invasive tumor that reduced in size on treatment, and Grade 3 pancreatitis reported at the time of disease progression. Thirty-one patients (57%) remain on treatment, and 23 (43%) discontinued, including 17 (31%) due to progressive disease (PD) per RECIST v1.1, 2 (4%) due to clinical progression, and other reasons due to adverse event (Grade 4 large intestine perforation), death (due to PD), investigator’s decision, or patient withdrawal (1 patient each). RMC-6236 exhibited dose-dependent increases in exposure in patients with no apparent accumulation in the blood following repeated daily dosing. At 220 mg QD, exposure to RMC-6236 was within range of the preclinical exposures in mice that demonstrated significant tumor regressions. Preliminary evidence of anti-tumor activity, including partial responses per RECIST v1.1, was observed in patients across several tumor types and dose levels. Molecular responses were observed with reduction in circulating tumor DNA of the KRAS-mutated allele and other somatic mutations consistent with inhibition of mutant RAS by RMC-6236. Conclusions: RMC-6236 is safe and well tolerated at doses that induce anti-tumor activity in patients with tumors harboring KRASG12X mutations and has favorable pharmacokinetics with oral dosing. A recommended phase 2 dose has not yet been determined and dose escalation is ongoing. Citation Format: Alexander I Spira, Alexander N Starodub, Kathryn C Arbour, David Sommerhalder, Brian M Wolpin, Minal Barve, Ignacio Garrido-Laguna, Salman Punekar, Meredith Pelster, Sumit Kar, Jamie Wong, Tong Lin, Rakesh Raman, Lin Tao, Zeena Salman, Xiaolin Wang, W. Clay Gustafson, David S Hong. Preliminary safety and pharmacokinetic profiles of RMC-6236, a first-in-class, RAS-selective, tri-complex RASMULTI(ON) inhibitor in patients with KRAS mutant solid tumors on the Phase 1 trial RMC-6236-001 [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr PR010.

Secondary Lesions and Sensitivity to Signaling Inhibitors of Pediatric iAMP21 B-Cell Precursor Acute Lymphoblastic Leukemia

Introduction Intrachromosomal amplification of chromosome 21 (iAMP21) B-cell precursor acute lymphoblastic leukemia (BCP-ALL) in children is a high-risk subtype for which targeted drugs are lacking. In this study we aimed to determine the frequency of secondary lesions and investigated the cellular sensitivity for candidate targeted drugs. Methods We performed total RNA sequencing on 28 iAMP21 and 28 B-other (negative for sentinel fusion genes) pediatric samples to determine the frequency of secondary lesions in newly diagnosed patients. A panel of 18 patient derived xenografts (PDX) of 8 primary iAMP21 ALL samples was generated, and secondary lesions were validated by PCR, RT-PCR, and whole exome sequencing. To test sensitivity, primary or PDX cells were exposed ex vivo to a concentration range of gilteritinib (FLT3 inhibitor), trametinib (MEK1/2 inhibitor), or ruxolitinib (JAK1/2 inhibitor). Results Secondary lesions in the cytokine receptor gene FLT3 were enriched in iAMP21 compared with B-other ALL including internal tandem duplications (ITD) and other activating lesions (50.0% vs. 10.7%, p=0.003). Lesions in genes encoding cytokine receptors CRLF2 and IL7R had a similar frequency between iAMP21 and B-other cases (25% vs. 17.9%, p=0.75 and 7.1% vs. 0%, p=0.49 respectively). Inactivating lesions in SH2B3, the downstream negative regulator of JAK/STAT and FLT3 signalling, were more frequent in iAMP21 cases vs. B-other (46.4% vs. 7.1%, p=0.002), while the frequency of JAK1 and JAK2 mutations did not differ (3.6 vs. 0% and 10.7 vs. 10.7% p = 1 for both). All SH2B3, CRLF2 and JAK lesions were retained in PDX samples, whereas in contrast FLT3-ITD was retained in only 2 of 5 PDX. Gilteritinib sensitivity did not differ between iAMP21 and B-other cases (median LC50 1.24 µM, range 0.33 to 4.85 µM vs. median LC50 1.21 µM, range 1.20 to 1.3 µM, p=0.95). Grouping iAMP21 cases by FLT3 and SH2B3 status, samples with both FLT3-ITD and SH2B3 lesion had the highest sensitivity to gilteritinib (median LC50 0.39 µM, range 0.35 to 0.43 µM). Samples with only FLT3 or SH2B3 lesion did not show increased sensitivity compared to those without a lesion (p>0.5 for both). Median ruxolitinib sensitivity did not statistically differ between iAMP21 and B-other cases (median LC50 8.38 µM, range 1.00 to >10 µM, vs. median LC50 0.48, range 0.21 to >10 µM; p=1) although extreme resistance to ruxolitinib seemed more frequent in iAMP21 cases (6 out of 12 cases) and was not related to FLT3 or SH2B3 status. CRLF2-rearranged ( CRLF2r) iAMP21 cases were slightly more sensitive to ruxolitinib than those lacking CRLF2r, although this difference did not reach significance (median LC50 4.72 µM, range 1.0-4.85 vs. median LC50 >10 µM, range 2.66 to >10 µM; p=0.08). The highest sensitivity was found in the only case with both a CRLF2r and a JAK1 mutation (LC50 of 1.00 µM). A large variation was present in trametinib sensitivity amongst both iAMP21 and B-other cases (median LC50 0.11 µM, range 0.017 to >5 µM vs. median LC50 0.18, range 0.018 to >5 µM; p=0.72). More than half of iAMP21 cases were sensitive irrespective of RAS-pathway lesion status. Conclusion iAMP21 leukemias are enriched in FLT3 and in SH2B3 lesions which, when co-occurring, affect sensitivity to FLT3 inhibition by gilteritinib but do not affect JAK-inhibition by ruxolitinib. This suggests these lesions act synergistically and might bypass downstream JAK/STAT signalling. This might also explain the observed sensitivity to RAS-pathway inhibition irrespective of secondary lesions. These results suggest that further research into FLT3 and RAS signalling inhibitors might lead to better treatment options for pediatric iAMP21 BCP-ALL.

Clinical and genomic landscape of RAS pathway mutations in gynecologic cancers.

5611 Background: We aimed to describe RAS mutations in gynecologic cancers as they relate to clinicopathologic features, subtypes, co-mutations, and implications for therapy. Methods: Patients with gynecologic cancers who had next-generation sequencing at our institution between Feb 2010 and Aug 2022 were included. Clinical, histopathologic, and sequencing data were collected and analyzed. Overall survival was estimated using the Kaplan-Meier model. Results: Of the 3310 gynecologic cancer patients tested, 522 (15.8%) harbored a RAS mutation. For patients with mutation, median age was 56 (18-87) years, with cancer types including ovarian (43.5%), endometrial (43.4%), cervical (9.2%), and vulvovaginal (2.9%). Most patients (92.2%) had recurrence. The most common histology for each cancer included endometrioid in endometrial (60.8%), low grade serous in ovarian (34.4%), adenocarcinoma in cervical (60.4%), and melanoma in vulvovaginal (66.7%). Mucinous and clear cell differentiations were overrepresented (12.5% each). Median overall survival for all RAS-mutated patients was 69 months (95%CI 59-82), which was significantly better than in wildtype control of 56 months (95%CI 52-59, p = 0.022). Mutations observed were in KRAS (85.8%), NRAS (12.8%), and HRAS (3.3%). The most common mutant alleles for each were KRAS G12D (34.5%), KRAS G12V (31.7%), NRAS Q61R (34.3%), and HRAS G12S (17.6%). Of note, the incidence of KRAS G12C was 5.6% (0.8% overall). The most common co-mutations for RAS were PIK3CA (35.7%), PTEN (33.3%), TP53 (32.4%), and ARID1A (20.8%). This trend held for each mutation and cancer type. Of these, 64.7% were potentially actionable alterations, including 91.1% of PIK3CA, 83.7% of PTEN, and 50.0% of ARID1A. RAS pathway targeted therapies were administered to 62 patients (57 MEK, 5 BRAF, 5 KRAS G12C, 4 SHP2). The median overall survival for these patients was significantly improved (101 months, 95%CI 78-160) compared to RAS-mutated patients who did not receive a RAS pathway inhibitor (66 months, 95%CI 53-79, HR 0.7, p = 0.031). Of interest, novel KRAS G12C inhibitors showed durable benefit in 3 ovarian (high grade serous, low grade serous, clear cell) and 2 endometrial (mixed endometrioid, clear cell; mixed endometrioid, serous, mucinous) cancers with a clinical benefit rate of 100%, duration of objective response of 22 months, and ongoing responses at 5 and 16 months. Conclusions: Gynecologic cancers with RAS mutations have distinct histopathologic distribution and portend improved overall survival. The most commonly actionable co-mutations were PIK3CA, PTEN, and ARID1A, which may aid in combination therapy design. RAS pathway targeted therapy is associated with improved overall survival in this cohort and should be considered. [Table: see text]

Abstract IA26: Exploiting altered metabolism as a therapeutic strategy for RAS-driven cancers

Abstract We recently determined that concurrent inhibition of autophagy, using the lysosomal inhibitor chloroquine (CQ), and of ERK, using a small molecule ERK inhibitor (ERKi), synergistically suppressed the growth of pancreatic ductal adenocarcinoma (PDAC) cell lines and patient xenograft-derived (PDX) organoids in vitro and PDX tumors in vivo (Bryant et al., 2019, Nat Med 25:628). Our findings, together with similar observations by McMahon, Kinsey and colleagues (Kinsey et al., 2019, Nat Med 25:620), provided the rationale for our initiation of Phase I and Phase I/II clinical trials evaluating the combination of MEKi (binimetinib; NCT04132505) or ERKi (LY3214996; NCT04386057) with hydroxychloroquine (HCQ) in PDAC. We have extended these findings and determined that combined inhibition of the ERK MAPK pathway and autophagy is effective in additional cancers driven by KRAS/NRAS, BRAF, or EGFR. Additionally, we have also shown that the combined inhibition of KRASG12C or KRASG12D and autophagy was effective in KRAS-mutant cancers. Our ongoing studies are centered on developing additional combinations for targeting metabolic resistance mechanisms to KRAS or ERK MAPK targerted therapies. First, we performed a CRISPR/Cas-9 mediated genetic loss-of-function screen in the presence of CQ to determine additional sensitizers as well as mediators of resistance to autophagy inhibition. Top sensitizers included multiple facilitators of the DNA damage response, mTOR pathway components, and genes involved in the upstream regulation of the autophagy pathway. Second, we determined that autophagic signaling is temporally regulated following RAS pathway inhibition and hypothesize that further dissection of this regulation will improve current anti-nutrient scavenging treatment strategies. We conclude that concurrent suppression of multiple metabolic processes, to block compensatory rebound activities, will be needed for effective PDAC treatment. Citation Format: Jonathan M. DeLiberty, Ryan Robb, Claire E. Gates, Noah L. Pieper, Runying Yang, Clint A. Stalnecker, Kirsten L. Bryant. Exploiting altered metabolism as a therapeutic strategy for RAS-driven cancers [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr IA26.

Abstract B035: Targeting transcriptional elongation kinases prevents adaptation to KRASG12C inhibitors in both MAPK-dependent and -independent models of acquired resistance

Abstract The development of covalent inhibitors targeting KRASG12C mutations offers a precision medicine approach to a large cohort of lung cancer patients previously lacking opportunities for targeted therapy. Despite promising initial responses ranging from disease control to partial response in most patients undergoing treatment, the efficacy of these inhibitors has been limited by a short duration of response. Several strategies have been advanced to delay or prevent acquired drug resistance in lung cancer patients treated with KRASG12C inhibitors. Many of these involve co-administration of other targeted therapies to prevent activation of Ras-dependent mitogen activated protein kinase (MAPK) signaling, which is a hallmark response to KRASG12C inhibitors. While this approach has proven effective in some patients, combination clinical trials and postmortem analysis of tumors that progressed on KRASG12C inhibitors demonstrate that non-MAPK-dependent mechanisms of resistance remain a major challenge. We have developed a panel of lung cancer cell lines with acquired resistance to the KRASG12C inhibitor sotorasib. While Erk phosphorylation is observed in all resistant models, treatment with a panel of Ras and MAPK pathway inhibitors demonstrates differential dependence on MAPK pathway activity, including complete MAPK independence in a resistant model derived from the H358 cell line. Mutation, transcription and phosphoproteomic analysis revealed that these resistant models exhibit metabolic reprogramming and activation of a variety of DNA damage response pathways. Based on differential phosphorylation of the transcriptional elongation kinases CDK12 and CDK13 in KRASG12C-treated cells, we hypothesized that inhibiting these kinases might prevent or revert acquired drug resistance. Our prior studies demonstrate that CDK12/13 inhibition reduces expression of a broad spectrum of DNA damage response and metabolic genes, and RNA sequencing confirms that these pathways are sensitive to the CDK12/13 inhibitor SR-4835 in both sotorasib-sensitive and -resistant cells. Resistant cells show differential responses to specific DNA damage-directed therapies and cell-line specific metabolic reprogramming, but up-front combinatorial treatment with SR-4835 prevents the development of acquired resistance to sotorasib across these models. Combined treatment with sotorasib and SR-4835 does not prevent Erk phosphorylation rebound; thus, the mechanism of SR-4835 in suppressing acquired resistance is independent of MAPK pathway reactivation. In vivo combination of sotorasib and SR-4835 potentiates drug response and prevents drug resistance in the H358 cell line, which can develop both MAPK-dependent and -independent drug resistance. These results demonstrate that targeting transcriptional elongation kinases in combination with sotorasib may benefit a broader spectrum of patients than MAPK-directed combination therapies and provide rationale for suppressing the DNA damage response and metabolic reprogramming as an approach to extend the efficacy of KRASG12C inhibitors. Citation Format: Yaakov E. Stern, Pompom Ghosh, Hitendra S. Solanki, Denis Imbody, Liznair Bridenstine, Hannah L. Walker-Mimms, John W. Mosior, Andrii Monastyrskyi, Derek R. Duckett, Eric B. Haura. Targeting transcriptional elongation kinases prevents adaptation to KRASG12C inhibitors in both MAPK-dependent and -independent models of acquired resistance [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B035.

Abstract B024: Combined inhibition of farnesyltransferase and MEK is effective in fusion-negative HRAS-mutant rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood, and RAS pathway mutations are the known driver mutations in the majority of fusion-negative (FN) RMS. Studies have demonstrated that HRAS mutations are enriched in infants with FN-RMS and can be associated with inferior outcomes. Using HRAS-mutant RMS models, we have demonstrated that tipifarnib (farnesyl transferase inhibitor, FTI) decreases ERK signaling and decreases in vitro and in vivo tumor growth. The effects of tipifarnib can be incomplete, and limitations may be due to adaptive or acquired resistance, or the presence of concurrent mutations that limit the response to single agents. These features suggest that HRAS-mutated FN-RMS may be sensitive to inhibition with combination therapy that prevents or delays the emergence of resistance. We examined the transcriptional effects of tipifarnib in FN-RMS cell lines using bulk RNA-sequencing to identify therapeutic vulnerabilities that may be exploited by RAS-directed therapies. Analysis of RNA sequencing data revealed downregulation of ERK transcriptional output genes upon treatment with tipifarnib, confirming the role of the MEK-ERK pathway in mediating the response to FTI. Trametinib (MEKi) inhibits tumor growth in xenograft models of FN-RMS but has only modest activity as a single agent. The efficacy of inhibition with FTI and MEKi has not been previously explored in RAS-driven FN-RMS. We therefore tested tipifarnib in combination with trametinib in HRAS-mutant FN-RMS cell lines and utilized in vitro assays to evaluate the effects of the combination on cell growth, differentiation and signaling via RAS effector pathways. In HRAS-mutant cells, we observed additive dose-dependent 2D and 3D growth inhibition in response to the combination. Further, tipifarnib and trametinib more potently reduced ERK phosphorylation than either drug individually, indicating effective RAS pathway inhibition. Additionally, we found that the combination of tipifarnib and trametinib induced myosin heavy chain expression, suggesting both inhibition of proliferation and promotion of myogenic differentiation. In an in vivo model, the combination of tipifarnib and trametinib led to tumor regression which was not seen with either drug alone. Our data suggest that the combination of tipifarnib and trametinib is active in models of HRAS-driven FN-RMS and may represent an effective therapeutic strategy for a genomically-defined subset of patients with FN-RMS. Citation Format: Patience Odeniyide, Alla Lisok, Elana J. Fertig, Alyza Skaist, Christine A. Pratilas. Combined inhibition of farnesyltransferase and MEK is effective in fusion-negative HRAS-mutant rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B024.

Abstract 515: Discovery of potent and orally available small molecule inhibitors of the SOS1-KRAS interaction

Abstract Abnormal activating mutation of KRAS is frequently found in many human cancers, including pancreatic ductal adenocarcinoma (PDAC), colorectal cancer (CRC), and non-small cell lung cancer (NSCLC). KRAS activating mutations lead to hyperactivation of the MAPK/ERK signaling pathway, resulting in promotion of cell proliferation and growth. SOS1 is one of the major guanine nucleotide exchange factors (GEFs) that regulates RAS proteins including KRAS. Since SOS1 plays a critical role in converting the GDP-bound inactive KRAS “off” state to the GTP-bound active KRAS “on” state, disruption of SOS1 and KRAS protein-protein interaction would be effective to block KRAS-driven oncogenic signaling regardless of its mutation status. Importantly, SOS1 activity is crucial during the reactivation of the KRAS/MAPK signaling upon the treatment of RAS/MEK/ERK inhibitors, thus a SOS1 inhibitor would be an effective therapeutic option to treat KRAS-driven tumors in combination with RAS pathway inhibitors. We developed potent, selective, and orally available small molecules that effectively disrupt the interaction between SOS1 and KRAS. Current lead compounds originated from a virtual screening displayed excellent ADME and PK profiles. In cellular assays, a robust reduction of phospho-ERK level and cancer cell growth were shown. We also observed excellent in vivo antitumor activity in the mouse xenograft models. Moreover, combination with Sotorasib synergistically inhibited tumor cell growth both in vitro and in vivo. Overall, our SOS1 inhibitors demonstrate great therapeutic potential for cancer patients with KRAS mutations. Citation Format: Ha Na Yu, Dong Hyuk Ki, Joonwoo Nam, Eun-Jung Kim, Sungeun Kim, Hunmi Choi, Jieun Kim, Jihyun Yu, Donggeon Kim, Dohyun Park, Kyeong Jin Yoon, Seongin Jo, So-Hyeon Hwang, Sang Kyun Lim, Young Sook Shin, Wooseok Han. Discovery of potent and orally available small molecule inhibitors of the SOS1-KRAS interaction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 515.

Abstract 2624: Assay toolkit for the discovery of new RAS pathway inhibitors

Abstract The small GTPase KRAS is a known oncogene that is very frequently mutated in a variety of cancers. Over the past several years, there has been a resurgence of interest in KRAS drug discovery following the identification of new inhibitor target sites and several molecules entering clinical trials. We have produced proteins and set up assays for a large collection of Ras variants, guanine nucleotide exchange factors and effectors to facilitate drug discovery activities targeting KRAS/MAPK pathway. RBC’s extensive kinase assay portfolio enables identification of kinase inhibitors downstream of Ras. This poster will summarize our most recent assay development efforts for biochemical and biophysical assays for these targets. Citation Format: Ekaterina Kouznetsova, Lauren S. McCauley, Rebecca Eells, Elaine Steinke-Neal, Timothy Sullivan, Louis Primavera, Charles Longen, Haiching Ma. Assay toolkit for the discovery of new RAS pathway inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2624.

Abstract 2623: Identification of KRAS suppressor genes: an ORFeome library screening

Abstract The RAS family genes encode small GTP-binding cytoplasmic proteins that are important signaling molecules. They regulate cell growth, survival and differentiation by coupling receptor activation to downstream effector pathways. Activating mutations of oncogenic RAS pathway genes are frequently detected in human cancers. KRAS is the most frequently mutated oncogene in human cancers. Most of these tumors are addicted to KRAS signaling. However, tumors can become resistant to RAS signaling pathway inhibitors through the activation of suppressor mechanisms. A possible explanation is that these tumors carry a second genomic perturbation that can compensate for the detrimental effects of the RAS signaling inhibitors, a phenomenon referred to as suppression. In model organisms, suppression interactions are generally divided into two classes: genomic suppressors which are secondary mutations in the genome that bypass a mutant phenotype, and dosage suppression interactions in which overexpression of a suppressor gene rescues a mutant phenotype. We decided to use the latter strategy in human cancer cells to identify KRAS suppressor genes. KRAS suppressor genes can be involved in resistance mechanisms of RAS pathway inhibitors and this approach could identify new targets for drug research programs and/or novel drug combinations. In this study we performed a screen on a human Open Reading Frame library (hORFeome collection) to identify potential KRAS suppressor genes. First, we engineered a SW620 cell line with a conditional expression of KRAS G12V by replacing the KRAS endogenous promoter by the doxycycline regulated TREG3 promoter. Second, we transduced this conditional cell line with a human ORFeome collection, and we looked for suppressors among the transduced cells that lead to cell proliferation in absence of KRAS G12V protein. Then candidate suppressors have been identified by NGS and validated after individual cloning and expression in SW620 KRAS conditional cells. Finally, two transcription factors and two orphan G protein-coupled receptors have been validated as KRAS suppressor genes when they are highly expressed. Citation Format: Christophe Marcireau, Philippine Furge, Fanny Sennechael, Alice Willart, Anne-Marie Blanchet, Veeranagouda Yaligara, Michel Didier, Frederic Lacroix, Melina Meunier, Laurent Debussches. Identification of KRAS suppressor genes: an ORFeome library screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2623.