KRASG12C Inhibitor Research Articles

Abstract A099: Best in class, potent, SOS1 inhibitors demonstrate single agent activity in preclinical models of KRAS driven tumors

Abstract KRAS is an oncogene implicated in a wide variety of tumors (~21% of solid tumors harbor KRAS mutations). Interaction of KRAS with its predominant Guanine Exchange Factor (GEF), SOS1, is crucial for activation of KRAS and further KRAS mediated oncogenic signaling. Thus, pharmacological inhibition of SOS1 is expected to be effective in treating RAS driven cancers. Biochemical potency of compounds was confirmed in a SOS1 mediated FRET based guanine exchange factor (GEF) assay. Multiple potent SOS1 inhibitors were identified with potencies in the low double digit nanomolar range. Selectivity was determined against SOS2 and a host of other kinases in the RAS/RAF/MEK or RAS/PI3K/AKT pathways. Anti-proliferative activity across a panel of WT and mutant KRAS cell lines revealed IC50 between 50-200 nM with synergism observed when combined with MAPK pathway inhibitors as well as the KRAS G12C inhibitor, Sotorasib, in a 3D anchorage independent assay format. Correspondingly, significant reduction in PD biomarkers (>50% at 100 nM), pERK and pAKT, was demonstrated in KRAS mutant as well as EGFR/PI3K mutant cell lines. PK-PD correlation was also established in a tumor bearing mice model, with dose-dependent reduction of both pERK and pAKT. Compounds demonstrated excellent ADME properties with significant tissue distribution upon oral dosing. One of the lead molecules demonstrated significant single agent efficacy with growth inhibition of approximately 70% in a KRAS G12C bearing NSCLC xenograft model. No adverse signs including reduction in food intake or body weight loss were reported during the study period. IND-enabling studies are currently ongoing for the lead compounds. Citation Format: Balakrishna Lakhavath, Manoj Pothuganti, Githavani Kummari, Hemasankar Pathange, Rajan Sekar, Srikant Viswanadha. Best in class, potent, SOS1 inhibitors demonstrate single agent activity in preclinical models of KRAS driven tumors [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 A099.

Abstract B173: The anti-tumor activity of CDK2 inhibition alone or in combination with other anti-cancer agents in human cancers

Abstract Cyclin-dependent kinase 2 (CDK2) plays a crucial role in the regulation of the cell cycle and is involved in a variety of biological processes. It is responsible for phosphorylating proteins involved in G1 and S phase cell cycle progression, DNA damage response, intracellular transport, protein degradation, signal transduction, and DNA and RNA metabolism. CDK2 activity is particularly significant in cells exhibiting CCNE1 amplification or overexpression. Additionally, CDK2 activity has been linked to potential resistance against chemotherapy or targeted therapy. Several studies have indicated that abnormal activation of CDK2 may contribute to resistance against targeted therapies, such as CDK4/6 inhibitors and KRAS inhibitors. To investigate this hypothesis, we conducted experiments testing the combined effect of ARTS-021, a highly selective and potent CDK2 inhibitor, with Palbociclib, a CDK4/6 inhibitor, or AMG510, a KRAS-G12C inhibitor, in various xenograft tumor models. The combination of CDK2 inhibition with CDK4/6 inhibition demonstrated superior efficacy compared to either agent alone. This combination induced tumor regression in both Palbociclib-sensitive T47D parental cell line models and Palbociclib-resistant T47D cells overexpressing CCNE1. Similar benefits were also observed when CDK2 inhibition was combined with KRAS G12C inhibition in multiple KRAS-G12C lung cancer cell line xenografts. Importantly, these combination treatments were well tolerated. These findings suggest that CDK2 may play a crucial role in cancer cell resistance to chemotherapy or targeted therapy, and a CDK2 inhibitor may have broader applications beyond CCNE1-amplified cancers, as it can be combined with different treatment modalities. Citation Format: Yaoyu Chen, Jinhong Chen, Yali Guo, Wei Pang, Tingru Zhou, Xipan Liu, Nathan Schomer, Fang Li, Jiaqi Liang, Qing Sheng. The anti-tumor activity of CDK2 inhibition alone or in combination with other anti-cancer agents in human cancers [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 B173.

Abstract A127: Identification of therapeutic drug combinations targeting KRAS

Abstract KRAS is the most frequently mutated oncogene and increased development efforts targeting KRAS mutations have yielded recent FDA approvals of two KRAS G12C inhibitors. However, for some indications, there are other KRAS mutations more common than G12C. For Pancreatic ductal adenocarcinoma (comprising more than 90% of pancreatic cancer cases), 94% harbor KRAS mutations, where G12D mutations account for around 45% while G12C mutations are rare (1%). Hence, there is a need to develop methods to target a broader range of KRAS mutations. Beyond direct gene mutation, activation of oncogenic KRAS and downstream MAPK signaling through other genomic alterations may benefit from KRAS-specific therapeutic strategies, especially combination therapy. We have developed a platform, Optim.AI™, that rationally converges upon optimal drug combinations within a defined drug search space, without any prior knowledge of the background of the models. In this study, we utilized the platform to identify efficacious drug combinations that contain a histone deacetylase 4/6 inhibitor, KYAN-001 in the context of KRAS mutations. 7 cell lines (6 prostate and pancreatic cancer with normal, epithelial THLE-2 cell line) were evaluated with Optim.AI. The cancer cells and THLE-2 were seeded into 384-well plates and treated with 155 different combinations consisting of indication-specific drugs the following day. Post-drug treatment cell viability was used for Optim.AI™ analysis, which maps experimental data points to a second-order quadratic function to predict and rank cancer cell killing efficacies for all other permutations of drug combinations. In androgen resistant prostate cancer, Optim.AI identified synergistic interaction between KYAN-001 and a MEK inhibitor. With MEK being downstream of the KRAS pathway, we wanted to evaluate the efficacy of this combination on pancreatic cancer. Interestingly, this combination was demonstrated to be top ranked against PANC-1 and AsPC-1 cell lines, both harboring G12D KRAS mutation, as compared to a cell line with wild-type KRAS, BxPC-3. This was further validated via half-maximal inhibitory curves (IC50), where synergism was also observed for the combination on AsPC-1, but not BxPC-3. KYAN-001 was also shown to exhibit monotherapy sensitivity against pancreatic cancer patient-derived organoids (PDOs), with a mean of 4 µM. This study has successfully demonstrated the ability of Optim.AI™ to identify novel, optimal drug combinations for both prostate and pancreatic cancers, without any prior background knowledge on these cell lines. With MEK being downstream of the KRAS pathway and 94% of those diagnosed with pancreatic cancer have KRAS mutations, the differential sensitivities of this combination across the cell lines presents the utility of the platform in precision medicine, especially for undruggable targets like KRAS. With promising and demonstrated monotherapy efficacy of KYAN-001 on PDOs, further experiments could be done to evaluate the clinical relevance of both KYAN-001 and MEK inhibition on other relevant ex vivo models. Citation Format: Masturah Mohd Abdul Rashid, Lisa Chow, Edward Kai-Hua Chow. Identification of therapeutic drug combinations targeting KRAS [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 A127.

Abstract B084: Sotorasib/SHP2 inhibitors combo, KRASG12C(ON)I and RASMULTI(ON)I effectively target KRASG12C tumors developing secondary resistance to Sotorasib via KRASG12C amplification

Abstract Background: Amplification of KRASG12C mutant allele has been reported as one of the resistance mechanisms in tumors progressed to Sotorasib, the FDA approved KRASG12C(OFF) inhibitor. An identical mechanism was observed in one of our H358 xenograft tumors relapsed to Sotorasib. The cell line generated from relapsed tumor represents an appropriate in vitro model to study therapeutic opportunities in KRASG12C tumors developing secondary resistance to Sotorasib via KRASG12C amplification. Methods: We generated five Sotorasib resistant cell lines using relapsed tumors of Sotorasib-treated H358 xenografts (MR1-MR5) and two vehicle-treated tumors (MV1 and MV2). Whole exome sequencing (WES), Taqman copy number analysis and digital PCR to study mutant allelic fractions were performed to investigate acquired mechanisms of resistance. We have also generated a Dox-inducible system for H358 cells to overexpress KRASG12C protein and mimic KRAS amplification. Results: We observed loss of sensitivity to Sotorasib in models MR1-MR5, when compared to MV1 and MV2. Western blot analysis show EMT-associated signatures in all resistant lines except MR2. The WES of resistant lines shows higher allelic frequency of KRASG12C mutation in MR2, which is homozygous (through loss of heterozygosity). The dPCR analysis further confirmed higher mutant allelic frequency in MR2. Copy-number analysis revealed the presence of more than 10 copies of KRAS in MR2. MR2 model represents the best model to investigate therapeutic options in KRASG12C tumors developing secondary resistance to Sotorasib via KRASG12C amplification. Both RAS-RAF Proximity Ligation and Raf-RBD pull down assays confirm enhanced RAS signaling in MR2 cells. The combination of KRASG12C(OFF) and SHP2 inhibitors, or single agent KRASG12C(ON) or RASMULTI(ON) inhibitors suppressed enhanced MAPK signaling in MR2 cells and drove tumor suppressions in MR2 mice xenografts. Conclusions: The stronger inhibition of KRAS pathway is necessary to suppress enhanced MAPK signaling in KRASG12C mutated lung cancer that developed secondary resistance to Sotorasib treatment through KRASG12C amplification. These preclinical data suggest that KRASG12C(OFF)/SHP2 inhibitors combo, or single agent KRASG12C(ON)I or RASMULTI(ON)I represent potential therapeutic strategies for these relapsed tumors. Citation Format: Hitendra S Solanki, Denis Imbody, Bina Desai, Ryoji Kato, Paul A Stewart, Yaakov Stern, Anurima Majumder, Liznair Bridenstine, Bhaswati Sarca, Daria Miroshnychenko, Ida Aronchik, Andriy Marusyk, Eric B Haura. Sotorasib/SHP2 inhibitors combo, KRASG12C(ON)I and RASMULTI(ON)I effectively target KRASG12C tumors developing secondary resistance to Sotorasib via KRASG12C amplification [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 B084.

Abstract B088: VT3989, the first-in-class and first-in-human TEAD auto-palmitoylation inhibitor, enhances the efficacy and durability of multiple targeted therapies of the MAPK and P13K/AKT/mTOR pathways

Abstract TEAD transcription factors are the major effectors of the Hippo-YAP/TAZ-TEAD pathway involved in the regulation of cell proliferation, survival, and cell migration. TEAD auto-palmitoylation has been shown to be required for TEAD interaction with coactivator YAP/TAZ and hence activation of transcriptional activity. There are multiple reports that YAP/TEAD activation provides the essential survival signal in drug-tolerant persister/dormant cells in cancer in response to treatment with targeted therapies, leading to drug resistance and cancer relapse in BRAF-mutant, KRAS-mutant, EGFR-mutant, and ALK-rearranged non–small cell lung cancers (NSCLC). The TEAD inhibitor VT3989, currently in clinical testing, was evaluated for effect on emerging drug tolerant persister/dominant cancer cells and drug resistance. VT3989, with similar biological activity in vitro and in vivo as the potent small molecule TEAD auto-palmitoylation inhibitors reported (Tang et al, 2021, Mol Cancer Ther. 20(6):986-998), disrupts YAP/TAZ-TEAD protein interaction, suppresses TEAD transcriptional activity, and selectively blocks proliferation of NF2-deficient mesothelioma in vitro and inhibit NF2 mutant xenograft tumor growth in vivo. In in vitro assays as well as in vivo studies using cell line derived (CDX) and patient derived (PDX) xenograft models, VT3989 enhanced the efficacy and durability of clinically approved EGFR inhibitors (osimertinib, lazertinib), MET inhibitors (savolitinib, capmatinib), EGFR-MET bispecific antibody (amivantinib), KRAS-G12C inhibitors (sotorasib, adagrasib), BRAF inhibitors (encorafenib, dabrafenib), MEK inhibitors (trametinib, cobimetinib), and mTORC inhibitor (everolimus). With the addition of VT3989, combination anti-tumor effect was observed and/or tumor regrowth was delayed compared to monotherapy alone. Therefore, in addition to Hippo-YAP/TAZ-TEAD pathway driven cancers, such as mesothelioma, where VT3989 can show monotherapy efficacy, future VT3989 clinical studies will include combination therapy with approved targeted therapies to enhance the efficacy and durability of treatment. Citation Format: Tracy T Tang, Leonard Post. VT3989, the first-in-class and first-in-human TEAD auto-palmitoylation inhibitor, enhances the efficacy and durability of multiple targeted therapies of the MAPK and P13K/AKT/mTOR pathways [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 B088.

Abstract B023: The next-generation farnesyltransferase inhibitor KO-2806 constrains compensatory signaling reactivation to deepen responses to KRASG12D inhibition

Abstract The landmark approval of KRASG12C inhibitors toppled the decades-held notion that KRAS was undruggable, fueling the design and development of small molecules targeting other oncogenic KRAS mutants, including the most prevalent, KRASG12D. MRTX1133 is the first KRASG12D-selective inhibitor to reach the clinic, providing a much-needed therapeutic option for patients with KRASG12D-mutant tumors. Nevertheless, both preclinical MRTX1133 data and early clinical results from KRASG12C inhibitor trials suggest that responses to KRASG12D inhibition will vary widely, at least in part due to incomplete and/or transient inhibition of MAPK and mTOR signaling. We previously demonstrated that farnesyltransferase inhibitors (FTIs) can prevent adaptive resistance to PI3K inhibition by blunting compensatory reactivation of these signaling nodes. We therefore hypothesized that farnesyltransferase inhibition may similarly enhance the antitumor activity of MRTX1133 through synergistic blockade of mitogenic and survival signaling. In this study, we leveraged a panel of in vivo cell line (CDX) and patient-derived xenograft (PDX) models, in parallel with in vitro mechanistic studies, to assess the efficacy of combined KO-2806, a FTI, and MRTX1133, in controlling KRASG12D-mutant tumor growth. KO-2806 is a next-generation FTI with increased potency and improved pharmacokinetic properties relative to earlier FTI candidates and is poised to enter a first-in-human clinical trial. Given that reactivation of phospho-ERK and phospho-S6 is linked to diminished response to MRTX1133, we first probed the impact of KO-2806 on cell signaling in vitro. In KRASG12D mutant cell lines, MRTX1133 transiently inhibited both ERK and mTOR, as evidenced by decreased ERK, p90 RSK, p70 S6K, S6, and 4EBP1 phosphorylation. However, phosphorylation of these proteins rebounded partially within one day and completely by 48 hours post treatment. In contrast, when cells were co-exposed to MRTX1133 and KO-2806, robust inhibition of these pathways was maintained at 48 hours and corresponded with greater induction of apoptosis (PARP and caspase cleavage) and cell cycle arrest (ablation of Rb phosphorylation) compared to MRTX1133. These results are consistent with prior reports suggesting that durable MAPK and mTOR inhibition is required for optimal response to KRAS inhibitors. The mechanistic synergy we observed in vitro provided a strong rationale for evaluating combined MRTX1133 and KO-2806 in vivo. Across a panel of KRASG12D-mutant CDX and PDX solid tumor models, the MRTX1133/KO-2806 doublet led to deeper tumor growth inhibition compared to the single agents while remaining well tolerated in mice. We posit that KO-2806 augments the MAPK-mTOR inhibitory effects of MRTX1133 by blocking compensatory pathway reactivation, resulting in superior antitumor efficacy of the combination without amplifying toxicity. Based upon these results, combined farnesyltransferase and KRASG12D inhibition represents a promising therapeutic strategy for the treatment of KRASG12D0-mutant solid tumors. Citation Format: Alison Smith, Hetika Patel, Stacia Chan, Francis Burrows, Shivani Malik. The next-generation farnesyltransferase inhibitor KO-2806 constrains compensatory signaling reactivation to deepen responses to KRASG12D inhibition [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 B023.

Abstract C108: Enhanced growth inhibition in lung cancer cells through NBF-006 combination therapy with chemotherapy or KRAS inhibitors

Abstract Background: Glutathione-S-transferase P (GSTP) is strongly up-regulated in many cancer types, specifically KRAS-mutant lung and is also a known regulator of oncogenic MAPK and PI3K pathways. In cancer cells, overexpression of GSTP regulates apoptosis by inhibiting Jun-p53 complex in JNK pathway. NBF-006 is a novel lyophilized lipid nanoparticle formulation, encapsulating a small interfering ribonucleic acid (siRNA) designed to inhibit the expression of GSTP in lung tumors. NBF-006 has been very well tolerated with early signs of efficacy in NSCLC patients in a phase 1 dose escalation and expansion study. As NBF-006 is well tolerated and unlikely to have drug-drug interactions, combination strategies are currently being developed. Due to the detoxifying role of GSTP, combination with chemo agents used to treat NSCLC may increase the sensitivity of cancer cells resulting in apoptosis and/or reduced resistance. As GSTP and KRAS enhance tumor growth and survival, inhibition of GSTP and KRAS may be synergistic. Methods: Anti-tumor activity of GSTP siRNA (NDT-05-1040), when combined with chemo agents, KRAS inhibitors or immune cells was assessed in a panel of KRAS mutant NSCLC lines. For chemo agents and KRAS inhibitors, NDT-05-1040 combinations was evaluated using Combenefit software and Loewe model. Results: Combination of NDT-05-1040 with chemotherapy drugs including paclitaxel, gemcitabine, cisplatin, and pemetrexed controlled tumor cell growth significantly better than either NBF-006 alone or the chemo treatment alone. In A549, H1373, H358, and H1792 lines, at low doses paclitaxel combined with NDT-05-1040 and resulted in synergistic cell killing (Loewe scores >20). Gemcitabine & Cisplatin combination with NDT-05-1040 in H1373 cells further improved cell killing by 5-fold (Loewe scores >20). Pemetrexed combined with NDT-05-1040 and led to additive cell killing in A549 (Loewe score >10). Combination of NDT-05-1040 with KRASG12C inhibitors, Sotorasib & Adagrasib were conducted in H358, H1222, and MiaPaca-2. Cell viability assays demonstrated that the combination resulted in strong synergistic, anti-proliferative effects at low doses in H358 cells (the greatest Loewe scores for Adagrasib and Sotorasib: 53 & 26, respectively). Conclusion: Results from the current studies suggest that NBF-006, a very well tolerated monotherapy, can be used in combination with other drugs currently indicated for KRAS-mutant NSCLC to achieve more potent antitumor effects. Citation Format: Cima Cina, Akinori Sugiyama, Murali Kuracha, Nazia Abbasi, Dominic Medina, Hiroyuki Tanaka, Sonya Zabludoff. Enhanced growth inhibition in lung cancer cells through NBF-006 combination therapy with chemotherapy or KRAS inhibitors [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 C108.

Abstract B177: The discovery and preclinical characterization of AMG 193, a first-in-class MTA-cooperative PRMT5 inhibitor with broad activity against MTAP-null cancers

Abstract Homozygous deletion of the methylthioadenosine phosphorylase (MTAP) gene occurs in approximately 15% of all cancers due to its proximity to the commonly deleted tumor suppressor gene CDKN2A. Elevated methylthioadenosine (MTA) levels, driven by loss of MTAP, compete with the methyl donor S-adenosylmethionine (SAM) for binding to protein arginine N-methyltransferase 5 (PRMT5), placing the methyltransferase in a hypomorphic state, and vulnerable to further PRMT5 inhibition. The screening of a DNA-encoded library, designed to identify small molecules that preferentially bind to PRMT5 in the presence of MTA, resulted in the identification of AM-9934 as a promising hit. Using structure-based drug design, potency and cooperativity were enhanced to deliver AM-9747, an MTA-cooperative PRMT5 inhibitor suitable for in vivo proof-of-concept studies. Further optimization led to AMG 193, an orally bioavailable and MTA-cooperative PRMT5 inhibitor with potent biochemical and cellular activity, and an improved pharmacokinetic profile. Insights into the binding mode of AMG 193 were elucidated by the crystal structure of AMG 193 bound to the MTA-PRMT5 complex. AMG 193 activity is enriched in MTAP-null cells in vitro, as illustrated in the HCT116 isogenic pair; AMG 193 is ~40X selective for MTAP-null cells (IC50 = 0.1 mM) over MTAP WT cells (IC50 > 4 mM) in viability assays and >100X selective in an assay evaluating symmetric dimethylarginine (SDMA) levels. AMG 193 was profiled in a panel of >850 cancer cell lines, and response demonstrated preferential sensitivity in MTAP-null cells compared to WT across multiple cancer lineages, including pancreatic, lung, biliary tract, and lymphoma. Response strongly correlated with both MTAP and CDKN2A copy number loss, and cell lines that were dependent on PRMT5, as shown by RNAi and CRISPR knockdown, were most sensitive to AMG 193 treatment. In vitro mechanism-of-action studies demonstrated that PRMT5 inhibition induced DNA damage, cell cycle arrest in G2/M, and an increase in alternative mRNA splicing in MTAP-null cells. In mice oral, once-daily administration of AMG 193 selectively inhibited SDMA in tumor versus normal tissue and was well tolerated with no hematologic perturbations. Importantly, AMG 193 demonstrated robust anti-tumor activity across a broad range of MTAP-null CDX and PDX tumor models derived from NSCLC, pancreatic, and esophageal cancers. In vitro synergy was observed with the combination of AMG 193 and several chemotherapeutic agents, including carboplatin and paclitaxel, or the KRASG12C inhibitor, sotorasib. In vivo, these combinations led to enhanced anti-tumor activity relative to single agent in MTAP-null NSCLC tumor models. In February 2022, AMG 193 became the first MTA-cooperative PRMT5 inhibitor to enter clinical development and is currently being evaluated in subjects with advanced MTAP-null solid tumors in the ongoing FIH study (NCT05094336). Citation Format: Brian Belmontes, Katherine Slemmons, Siyuan Liu, Antonia Policheni, Jodi Moriguchi, Hong Tan, Fang Xie, Famke Aeffner, Matthew G Rees, Melissa M Ronan, Jennifer A Roth, Mikkel Vestergaard, Sanne Cowland, Jan Andersson, Ian Sarvary, Patricia Lopez, Nuria Tamayo, Liping H Pettus, Susmith Mukund, Jennifer R Allen, Sanne Glad, Matthew P Bourbeau. The discovery and preclinical characterization of AMG 193, a first-in-class MTA-cooperative PRMT5 inhibitor with broad activity against MTAP-null cancers [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 B177.

Abstract B083: Secondary KRAS mutations lead to acquired resistance to KRASG12D inhibitor in colorectal cancer

Abstract KRAS is the most frequently mutated oncogene, with high prevalence in tumor types with a significant unmet clinical need, such as colorectal cancer (CRC). Activating mutations in the KRAS gene leading to amino acid exchange at position 12 from Glycine to Asparagine, G12D, occur in approximately 12.5% of CRC. The approval of mutant specific KRASG12C inhibitors MRTX849 and AMG510 by the FDA raised the question if KRAS targeted therapy approaches could provide benefit to cancer patients with other mutant alleles. Several KRASG12D inhibitors have recently been described and MRTX1133, a selective KRASG12D inhibitor from Mirati Therapeutics, has now entered clinical trial, recruiting patients with advanced solid tumors harboring a KRAS G12D mutation. Alongside the great hopes placed in KRAS targeting therapy, resistance is likely to occur, as it was observed in patients relapsing in response to KRASG12C inhibitors. We aimed to use this pre-clinical study to predict and understand potential resistance mechanisms that may arise during targeted treatment against KRASG12D in the CRC setting. To this aim, the colorectal cancer cell line GP2d was continuously treated with MRTX1133 to generate resistance and individual outgrowing clones were subsequently profiled in a series of assays to identify potential mechanism(s). These assays included analysis of gene expression changes, mutation calling, as well as qPCR and protein-based assays. Interestingly, we identified two individual secondary on-target mutations in the KRAS gene, which could affect binding of MRTX1133 to the pocket in KRAS. These findings were confirmed in an orthogonal assay, by screening for resistance in Ba/F3 cells transduced with the corresponding KRAS double mutant. Combination assays were then performed in parental and resistant cells to study whether additional vertical or alternative pathway inhibition could address the resistance. Collectively our pre-clinical study identified secondary KRAS mutations in the compound binding pocket as a mechanism of resistance to KRAS G12D inhibition by MRTX1133 in colorectal cancer cells. Citation Format: Simone Lieb, Sabine Jurado, Sergej Nowoshilow, Krzysztof M. Zak, Marco H. Hofmann. Secondary KRAS mutations lead to acquired resistance to KRASG12D inhibitor in colorectal cancer [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 B083.

Abstract B113: Discovery of PRT3789, a first-in-class potent and selective SMARCA2 degrader in clinical trials for the treatment of patients with SMARCA4 mutated cancers

Abstract SWI/SNF complexes play an important role in controlling gene expression by remodeling chromatin. SMARCA2 (BRM) and SMARCA4 (BRG1) are the core catalytic subunits of the SWI/SNF complexes. SMARCA4 expression is lost in some cancers due to its gene mutations, and SMARCA4-deficient cancer cells are highly dependent on its paralog gene SMARCA2 for their survival. SMARCA4 mutation is associated with worse patient prognosis and reduced responsiveness to inhibitors of KRAS G12C or immune checkpoint, compared to patients with SMARCA4 WT cancers. Furthermore, SMARCA4 mutation is often mutually exclusive with other targetable oncogenic alterations such as EGFR, ALK, MET, RET and ROS1, making SMARCA4 mutated cancers a clinically unmet disease. We have identified PRT3789, a potent and selective SMARCA2 degrader, that selectively targets SMARCA4-deficient cancer cells. In the present studies, we discuss the mechanism and activity of PRT3789 in SMARCA4-deficient tumor models as well as our clinical methods to assess target engagement in PRT3789 treated patients. PRT3789 is a bifunctional small molecule comprised of a novel SMARCA2-bromodomain binder linked to a VHL E3 ubiquitin ligase-binding moiety. PRT3789 has been shown to effectively catalyze the polyubiquitination of specific lysine residues of SMARCA2, resulting in SMARCA2 selective degradation over SMARCA4. The selectivity of PRT3789-induced SMARCA2 degradation was confirmed by cellular assays, protein mass spectrometry as well as ex vivo treatment of human PBMCs. PRT3789 selectively inhibits cell proliferation of SMARCA4-deficient cancer cells, but not SMARCA4 WT cells, when tested in a panel of cancer cell lines. Cell lines with specific SMARCA4 missense mutations also responded to PRT3789, suggesting that they are LOF mutations. Mechanistically, degradation of SMARCA2 leads to disruption of SWI/SNF complexes, dysregulation of chromatin remodeling and gene expression, resulting in inhibition of oncogenic pathways such as cell-cycle and DNA replication gene signatures. After treatment with PRT3789, SMARCA4-deleted cancer cells undergo G1 cell-cycle arrest and apoptosis. In vivo efficacy studies were conducted with genomically selected cell line-derived and patient-derived NSCLC xenograft models in mice. PRT3789 administration significantly inhibits the growth of SMARCA4-deleted NSCLC but demonstrates no effects on SMARCA4 WT tumor growth. In preclinical models, the effects on tumor growth were synergistic when PRT3789 was combined with other SOC therapies including gemcitabine, docetaxel, KRAS G12Ci as well as investigational drug candidates such as MCL1i, CDK4/6i, and CDK9i. PRT3789 also showed combination effects with anti-PD1 mAb in efficacy models in vivo. In the first-in-human Phase I dose-escalation study, PRT3789 target engagement will be assessed by determining the levels of SMARCA2 protein in human PBMCs following treatment with PRT3789. We aim to further investigate the safety, pharmacokinetics (PK) as well as anti-tumor activity of PRT3789 in the ongoing clinical trial (NCT05639751). Citation Format: Koichi Ito, Michael Hulse, Anjana Agarwal, Jack Carter, Monisha Sivakumar, Komali Vykuntam, Min Wang, Miles Cowart, Joy Cote, William Gowen-MacDonald, Brian Vidal Torres, Justin Kurian, Neha Bhagwat, Norman Fultang, Alexander Grego, Andrew Moore, Ashley Schwab, Jessica Burtell, Olusola Peace Osinubi, Jacob Spruance, Liang Lu, Philip Pitis, Corey Basch, Klare Bersch, Chaofeng Dai, Raul Leal, Artem Shvartsbart, Ganfeng Cao, Bo Shen, Patrick Wen, Joseph Rager, Ross Kuskovsky, Bob Landman, Tom Emm, Stefan Ruepp, Chunhua Qin, Gina Paris, Jennifer Xavier, Rachel Chiaverelli, Sang Hyun Lee, Sandy Geeganage, Hong Lin, Diane Heiser, Bruce Ruggeri, Naveen Babbar, Andrew Combs, Peggy Scherle. Discovery of PRT3789, a first-in-class potent and selective SMARCA2 degrader in clinical trials for the treatment of patients with SMARCA4 mutated cancers [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 B113.

Abstract B081: Aurora kinase A inhibition overcomes adaptive resistance to KRAS G12C inhibitor by G1-checkpoint induced apoptosis in KRAS non-small cell lung cancer

Abstract Cancer cells gain drug-tolerant states and evade therapy. In response to KRAS G12C inhibitor (G12Ci), KRAS mutant non-small cell lung cancer (NSCLC) cells maintain a drug-tolerant state by aurora kinase A (AURKA). AURKA can phosphorylate c-Raf to maintain new KRAS signaling, however, how AURKA becomes activated to cause KRAS G12Ci resistance is unclear. We show here that KRAS G12C + AURKA inhibition cause synthetic lethality in KRAS G12C NSCLC cells. LY3499446 (KRAS G12Ci) and LSN3321213 (aurora kinase A inhibitor) induced apoptosis that is independent of inhibited MAPK reactivation. Using high-content imaging that tracks single-cell fate during treatment, we observed that single-agent KRAS G12Ci induces G1 arrest in a sub-population of cells. Upon co-treatment with G12C + AURKAi, cells that are halted in G1 phase undergo early apoptosis, while those that initially evade G1 arrest and proceed through G2/M undergo apoptosis in subsequent G1. These data suggest the hypothesis that AURKA inhibition may increase the probability of G1 checkpoint-induced apoptosis by facilitating chromosomal misalignment and genomic instability in G2. In summary, we provide pre-clinical rationale for clinical testing of KRAS G12C + AURKA inhibitors. We also suggest a novel mechanism explaining the dependency of KRAS G12C resistant subpopulations on AURKA, leading to the opportunity to investigate the role of genomic instability in conferring KRAS G12Ci adaptive resistance. Citation Format: Chendi Li, Jeremy Chang, Christopher J Graser, Usman M Syed, Anahita Nimbalkar, Yi Shen, Steve Altschuler, Lani Wu, Franziska Michor, Xueqian Gong, Aaron N Hata. Aurora kinase A inhibition overcomes adaptive resistance to KRAS G12C inhibitor by G1-checkpoint induced apoptosis in KRAS non-small cell lung cancer [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 B081.

Abstract B080: Non-genetic determinants driving sub-clonal resistance to KRAS G12C combination therapies in KRAS mutant non-small cell lung cancer

Abstract The FDA approval of the KRAS G12C inhibitor (G12Ci) sotorasib and the advancement of similar drugs into the clinic marks a major milestone in treating KRAS G12C non-small cell lung cancer (NSCLC). However, not all patients respond (sotorasib – ORR = 37.1%, adagrasib - 43%, JDQ443 – 35%), motivating preclinical and clinical investigation into mechanisms of intrinsic and acquired resistance. For instance, clinical studies have reported on-target KRAS mutations and preclinical studies have demonstrated mitogen-activated protein kinase (MAPK) feedback reactivation including EGFR, SHP2, and WT RAS signaling. In response to targeted therapies, sub-populations of cells can enter quiescence or specific epigenetic-driven states that confer drug tolerance. However, epigenetic states defining drug-tolerant persister populations and contributing to adaptive resistance to KRAS G12Ci have not been reported. Using a high-complexity DNA-barcoding system and mathematical modeling, we observed that pre-existing resistant clones to KRAS G12Ci gain clonal fitness during treatment independent of growth rate. Using single-cell profiling and RNA-lineage tracing barcoded systems, we identified distinct subpopulations of cells defined by specific chromatin and transcriptional states, including activation of interferon-gamma genes (ISG) and increased chromatin accessibility for pioneer transcription factors, which may contribute to MAPK reactivation-driven resistance. These results suggest a potential role of pioneer transcription factor-activated interferon-gamma response in driving MAPK feedback reactivation in KRAS G12Ci- resistant tumor subpopulations. Citation Format: Chendi Li, Christopher J Graser, Qian Qin, Usman M Syed, Barbara Karakyriakou, Sarah E Clark, Anne Y Saiki, Paul E Hughes, Christopher Ott, Luca Pinello, Franziska Michor, Aaron N Hata. Non-genetic determinants driving sub-clonal resistance to KRAS G12C combination therapies in KRAS mutant non-small cell lung cancer [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 B080.

Abstract B029: Investigating therapeutic strategies to promote immune rejection of KRAS G12C inhibitor-resistant sub-populations in lung cancer

Abstract Oncogenic KRAS signaling not only instigates aberrant growth in cancer cells but can further induce inhibitory effects on anti-tumor immune responses. Hence, recently developed KRAS G12C inhibitors (G12Ci) have the potential to alleviate immune suppression in the tumor microenvironment (TME) and reshape the therapy responses for patients with a KRAS G12C driver mutation. However, rewiring of oncogenic signaling circuits and acquisition of further mutations allows cancer cells to rapidly evolve drug resistance. In fact, clinical trial data from the recently approved G12Ci, adagrasib and sotorasib, showed a median progression-free survival in non-small cell lung cancer of just 7 months. In this project, we investigate how to combat G12Ci-resistant subpopulations in lung tumors by engaging an anti-tumor immune response. We mimic the development of drug resistance by combining traced isogenic G12C- and G12D-mutant lung cancer cells, where the KRAS G12D mutant cells are inherently resistant to G12Ci. By alleviating RAS-driven immune suppression and potentially inducing immunogenic cell death of G12Ci-responsive cells, we aim to further potentiate an adaptive immune response targeting the drug-resistant population. We found that G12Ci as monotherapy drives therapy resistance by giving the drug-resistant subpopulation a proliferative advantage. On the other hand, combining G12Ci with immunotherapy or a SHP2 inhibitor potentiated an adaptive immune response to induce complete eradication of both G12C and G12D KRAS mutant cancer cell populations and also immune memory towards drug-resistant KRAS G12D cells in mice. However, when repeating the combination treatment in Rag1 −/− mice, which lack B- and T-cells, there were no complete responders. Histopathological analysis of tumor sections in immune-competent mice revealed a strong T-cell infiltration in the TME of tumors treated with combination therapies. Although understanding the mechanistic changes in the TME induced by combination therapies is ongoing, these results indicate a role for the adaptive immune response in targeting G12Ci drug-resistant subpopulations. Understanding these underlying mechanisms will contribute to combating the development of drug resistance in patients by enhancing treatment regimens that promote immune-mediated destruction of drug-resistant tumor cell sub-populations. Citation Format: Mona Tomaschko, Miriam Molina-Arcas, Christopher Moore, Sareena Rana, Sophie de Carne, Panayiotis Anastasiou, Claire Pillsbury, Andrea de Castro, Julian Downward. Investigating therapeutic strategies to promote immune rejection of KRAS G12C inhibitor-resistant sub-populations in lung cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B029.

Abstract LB_C09: QTX3034, a potent and selective multi-KRAS inhibitor, synergizes with EGFR inhibitors and enhances anti-tumor activity

Abstract KRAS, one of the most frequently mutated oncogenes in cancer, was considered undruggable until the development of covalent KRASG12C inhibitors. While these KRASG12C-targeted therapies exhibit clinical benefit, they do not address the most common KRAS mutant variants. QTX3034 is a potent, highly selective, orally bioavailable allosteric multi-KRAS inhibitor that is efficacious against a broad spectrum of the more common KRASG12D- and KRASG12V-mutated cancers in vitro and in vivo. In an effort to enhance anti-tumor activity of QTX3034 and to minimize the development of possible resistance mechanisms, we explored the potential synergy of combining QTX3034 with EGFR inhibitors in multiple KRAS-mutated tumor models. Cell viability assays over a 7-day period revealed that QTX3034 synergized with three different EGFR inhibitors (afatinib, cetuximab, erlotinib) to profoundly inhibit proliferation across multiple KRASG12D, KRASG12V, and KRASWT Amp mutant cell lines with strong ZIP synergy scores (>10). Combination of QTX3034 and afatinib exhibited a markedly greater inhibitory effect on KRAS-mutant colony formation compared to the respective single agents. Analysis of RAS downstream signaling revealed that the combination of QTX3034 and afatinib produced a more potent suppression of the MAPK pathway than either single agent alone in KRAS-mutant cells. Moreover, QTX3034 monotherapy induced a dose-dependent increase in EGFR phosphorylation in the KRASG12D-mutated cell line GP2D, while co-administration with afatinib robustly inhibited this EGFR activation. Combining QTX3034 and cetuximab elicited durable tumor regressions in KRASG12D-mutant cell line-derived xenograft models. Furthermore, tumor regressions in these combination studies were obtained using meaningfully lower doses of QTX3034 than needed to achieve tumor regression by monotherapy. Similar combinations improved QTX3034 anti-tumor activity in patient-derived xenograft models that exceeded monotherapy with either agent. Taken together, these data support the rationale to co-administer QTX3034 and EGFR inhibitors to optimize therapeutic benefit for patients with KRAS-mutated cancers. Citation Format: Yang J. Zhang, Jillian M. Silva, Yang W. Zhang, Elizabeth Donohue Vo, John M. Micozzi, Douglas Krauth, Arghyotri Sinha, Ben Reid, Brooke McDonough, Gregory M. Lee, Christopher C. Pan, Audrey Hospital, Juan I. Luengo, Cameron Pitt, Hong Lin. QTX3034, a potent and selective multi-KRAS inhibitor, synergizes with EGFR inhibitors and enhances anti-tumor activity [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 LB_C09.

Abstract A087: BI KRASmulti, a first-in-class, orally bioavailable and direct inhibitor of diverse oncogenic KRAS variants drives tumor regression in KRAS G12V-driven preclinical models

Abstract Alterations of KRAS are found in approximately one in seven of all human tumors, making KRAS one of the most prevalent oncogenic drivers. Gain-of-function missense mutations in KRAS, leading to its aberrant activation, are found in ~90% of pancreatic cancer, ~40% of colorectal cancer and ~30% of lung adenocarcinoma, with KRASG12V mutations accounting for ~28%, 9% and 6% of the cases, respectively. The poor outcome associated with these tumor types, particularly pancreatic cancer, as well as the lack of targeted inhibitors for KRASG12V calls for the urgent need to identify therapies able to effectively address the KRASG12V mutant allele. BI 3706674 is a novel, potent and orally available small molecule inhibitor of the KRAS oncogene. BI 3706674 binds non-covalently to multiple KRAS mutant alleles, including KRASG12V, in the GDP-bound state, and blocks downstream oncogenic signalling. Here, we show that BI 3706674 has a strong anti-proliferative activity in a panel of KRASG12V mutant cancer cell lines in vitro, along with significant pharmacodynamic (PD) biomarker modulation (including inhibition of ERK1/2 phosphorylation and down-regulation of DUSP6 mRNA).In vivo, a twice daily oral dose of 30 mg/kg was well-tolerated, while inducing tumour regression in several mutant KRASG12V cell line-derived and patient-derived xenograft (CDX and PDX) models across multiple different human tumor types. We have selected different non-small cell lung cancer xenograft models for extensive PK/PD/efficacy analysis in vivo. Results of the ongoing analysis will be shared. Feedback activation of upstream signalling pathways including receptor tyrosine kinase, such as the EGFR, has been suggested to be able to limit the efficacy of GDP-KRAS targeting compounds in preclinical studies. These findings have been supported by clinical combination trials involving KRASG12C inhibitors such as Adagrasib and Sotorasib and anti-EGFR modalities. We demonstrate for the first time that combination with anti-EGFR antibodies (e.g., Cetuximab) potently enhances the response observed with BI 3706674 in settings of KRASG12V mutant pancreatic, colorectal, and lung cancer. The deeper response observed upon combination of BI 3706674 with Cetuximab across multiple xenograft models provides a strong rationale for the clinical investigation of this combination therapy. Moreover, we utilize an ex vivo organoid platform to rapidly identify novel drug combinations with BI 3706674 that can potentially translate to clinical trials. The KRASmulti inhibitor BI 3706674 is currently undergoing IND enabling studies. Single agent dose escalation will include patients with cancers harbouring KRASG12V mutations. Citation Format: David H Peng, Antonio Tedeschi, Lorenz Herdeis, Fabio Savarese, Francesca Rocchetti, Popow Johannes, Heinrich J Huber, Nicola Melillo, Jake Dickinson, Hitesh B Mistry, Birgit Wilding, Matthias Treu, Julian Fuchs, Joachim Broker, Tobias Wunberg, Fiorella Schischlik, Jesse Lipp, Vandhana Ramamoorthy, Joseph R Daniele, Scott Kopetz, Michael Kim, Don L Gibbons, Christopher P Vellano, Joseph R Marszalek, Timothy P Heffernan, Darryl McConnell, Mark Pearson, Norbert Kraut, Dorothea Rudolph. BI KRASmulti, a first-in-class, orally bioavailable and direct inhibitor of diverse oncogenic KRAS variants drives tumor regression in KRAS G12V-driven preclinical models [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 A087.

Oncogenic KRAS effector USP13 promotes metastasis in non-small cell lung cancer through deubiquitinating β-catenin

KRAS mutations are frequently detected in non-small cell lung cancers (NSCLCs). Although covalent KRASG12C inhibitors have been developed to treat KRASG12C-mutant cancers, effective treatments are still lacking for other KRAS-mutant NSCLCs. Thus, identifying a KRAS effector that confers poor prognosis would provide an alternative strategy for the treatment of KRAS-driven cancers. Here, we show that KRAS drives expression of deubiquitinase USP13 through Ras-responsive element-binding protein 1 (RREB1). Elevated USP13 promotes KRAS-mutant NSCLC metastasis, which is associated with poor prognosis in NSCLC patients. Mechanistically, USP13 interacts with and removes the K63-linked polyubiquitination of β-catenin at lysine 508, which enhances the binding between β-catenin and transcription factor TCF4. Importantly, we identify 2-methoxyestradiol as an effective inhibitor for USP13 from a natural compound library, and it could potently suppress the metastasis of KRAS-mutant NSCLC cells invitro and invivo. These findings identify USP13 as a therapeutic target for metastatic NSCLC with KRAS mutations.

Targeting Mutant KRAS with Novel Mutant-Specific Pharmacological Inhibitors: Activity Against Myeloma and Other Lymphoid Malignant Cells and Molecular Mechanisms of Resistance

Oncogenic RAS mutations are frequent genomic drivers in hematologic neoplasias such as multiple myeloma (MM) but are traditionally considered non-“druggable”. Novel specific inhibitors of G12C and G12D KRAS mutants are clinically active in solid tumors, but their impact on KRAS-mutant lymphoid malignancies has not been explored. Here, we examined the activity of these inhibitors against G12C/D KRAS-mutant cells from MM, B-ALL and T-ALL; the functional sequelae of pharmacological KRAS inhibition; and the potential mechanisms of resistance to these agents. We assessed the activity of novel G12C- (MRTX1257) and G12D- (MRTX1133) specific pharmacological KRAS inhibitors against 2 MM lines with G12C (KHM1B, XG7) and 2 with G12D (KARPAS620, KP6) KRAS mutations; as well as the G12D mutant KOPN-8 (B-ALL) and CCRF-CEM (T-ALL) lines. KRAS inhibitors were tested at concentration ranges achievable in patients with solid tumors. Potent and mutant-specific activity was achieved with both of these KRAS inhibitors in their respective lines, with IC50 in the range of 5-150 nM after 3-7d treatment. This level of in vitro activity was similar to KRAS-mutant lines (e.g., in our in vitro studies and also in the literature) from solid tumors that are clinically responsive to these agents. Furthermore, the KRAS inhibitors effectively abrogated downstream MAPK and AKT pathway activation (e.g. phosphorylation of ERK or AKT by immunoblotting). MRTX1133 led to potent induction of cell death of KARPAS620, KP6 and KOPN8 cells (flow cytometry for Annexin V/PI staining) while MRTX1257 induced cell death in KHM1B and had a predominantly cytostatic effect on XG7 cells. Combinations of MRTX1257 with clinically established anti-MM drugs (incl. melphalan, bortezomib, pomalidomide, trametinib) caused in some cases supra-additive effects, but - more importantly - showed no antagonism with any combination. RNA sequencing revealed that KRAS inhibitor treatment downregulated RAS effector genes (e.g., ETV4, ETV5), and negative regulators of RAS signaling (e.g., DUSP6, SPRY4 reflecting a RAS-driven negative feedback loop controlling expression of these genes), and molecules involved in cell cycle regulation (e.g., D-type cyclin), anti-apoptosis, and DNA replication/repair. Conversely, KRAS inhibition upregulated genes involved in cell cycle arrest and stress responses. Importantly, we performed genome-scale CRISPR activation studies in 4 MM lines with G12C/G12D mutant KRAS to identify genes whose gain-of-function (GOF) enhances or suppresses MM cell response to the respective KRAS inhibitors. Top “hits” from these screens were validated with individual sgRNAs. CRISPR activation of mutant KRAS itself promotes MM cell escape from treatment, likely due to stoichiometric reasons and decreased target saturation at a given drug concentration. Notably, CRISPR activation of positive regulators of RAS-MAPK signaling (e.g., SHOC2), and upstream surface receptors (e.g., EGFR) were among top genes mediating KRAS inhibitor resistance. Beyond KRAS itself, these top “hit” genes are typically not recurrently mutated, amplified, differentially expressed (e.g., compared with normal plasma cells) in MM and are not associated with “high-risk” clinical behavior. Some (e.g., EGFR) of the genes identified from these CRISPR studies are not typically expressed in MM cells at diagnosis, thus these results outline how ectopic expression (e.g., EGFR) or overexpression of these “hit” genes could represent candidate biomarkers for decreased response or early relapse in future clinical studies of KRAS inhibitors in KRAS-mutant MM. Ongoing studies are addressing how these genomic perturbations may affect the in vivo response of KRAS-mutant cells to KRAS inhibitors alone or in combinations with established therapies. Our study documents that novel KRAS G12C and G12D inhibitors exhibit potent and mutant-specific activity against MM and other lymphoid malignant cells at dose levels that are clinically achievable in solid tumor patients. This study also provides functional evidence about genes and molecular pathways that regulate MM cell sensitivity vs. resistance to KRAS inhibitors. These results create a framework for ongoing and future efforts to translate the use of KRAS inhibitors into clinical studies for MM and other lymphoid malignancies.

Correction: The KRAS-G12C inhibitor: activity and resistance

Correction: The KRAS-G12C inhibitor: activity and resistance

EXTH-45. DEVELOPMENT OF COMBINATION THERAPIES OF KRASG12C INHIBITOR ADAGRASIB IN PRECLINICAL MODELS OF BRAIN METASTASIS

Abstract BACKGROUND The highly selective KRASG12C inhibitor adagrasib (MRTX849) is FDA-approved for KRASG12C+ advanced non-small cell lung cancer (NSCLC). We recently provided preclinical evidence of MRTX849 brain penetration and activity, allowing MRTX849 inclusion in a national genomically-guided multicenter clinical trial (NCT03994796) for patients with brain metastasis (BM). However, multiple genetic alterations such as CDKN2A/B deletions, a common driver of NSCLC BM, can limit MRTX849 efficacy. Furthermore, although MRTX849 can sensitize extracranial tumors to immune checkpoint inhibitors (ICI), its intracranial immune benefit is unknown. Our objective was to improve MRTX849 monotherapy, evaluating combination therapies with CDK4/6 inhibitor abemaciclib or anti-PD-1 in preclinical models of BM. METHODS We tested in vitro sensitivity of MRTX849 and abemaciclib, using KRASG12C+, CDKN2A-deleted human lung cancer cell lines SW1573 and H2122. The efficacy of a 5-week treatment of MRTX849 and abemaciclib was evaluated in an intracranial NSCLC BM model in nude mice. Additionally, we utilized a syngeneic murine KrasG12C colorectal cancer BM model (CT26G12C) to test different MRTX849 dosages and assess its therapeutic effects in combination with anti-PD-1 antibody. RESULTS MRTX849 and abemaciclib demonstrated mild synergy in reducing cell viability in vitro. Combination treatment of MRTX849 and abemaciclib significantly improved both survival and intracranial tumor control relative to controls in SW1573 NSCLC BM model. In a syngeneic colorectal BM model, we identified benefit of a 2-week MRTX849 single treatment at 100 mg/kg (BID) in controlling brain tumor growth. In a dual subcutaneous intracranial BM model with CT26G12C, MRTX849 alone and combination therapy mediated complete regression of extracranial tumors. Although monotherapies reduced intracranial tumor growth, MRTX849 combination with anti-PD-1 resulted in a greater benefit. Both MRTX849 monotherapy and the combination therapy extended animal survival. CONCLUSIONS MRTX849 combination therapies with abemaciclib or anti-PD-1 demonstrate intracranial activity, inhibiting BM progression and prolonging survival, in preclinical models of KRASG12C+ BM.

CRISPR Screening Identifies Mechanisms of Resistance to KRASG12C and SHP2 Inhibitor Combinations in Non-Small Cell Lung Cancer.

Identification of resistance mechanisms to KRASG12C/SHP2 coinhibition highlights the need for additional combination therapies for lung cancer beyond on-pathway combinations and offers the basis for development of more effective combination approaches. See related commentary by Johnson and Haigis, p. 4005.