Covalent KRASG12C Inhibitor Research Articles

Abstract 2675: Discovery of potent CNS-penetrant covalent KRAS G12C inhibitors

Abstract The RAS family of GTPases, which include KRAS, NRAS, and HRAS, are key regulators of cellular signaling, acting as molecular switches downstream of receptor tyrosine kinases (RTKs) and as key regulators of the RAS/MAPK pathway. RAS cycles between inactive (GDP) and active (GTP) states to regulate the activity of the RAS/MAPK pathway. In many cancers, activating KRAS mutations enable KRAS to persist in the active GTP-bound state, resulting in hyperactive RAS/MAPK pathway signaling that drives cell growth and survival. The KRAS G12C mutation occurs in 14% of lung adenocarcinoma, a cancer type that frequently metastasizes to the brain (40%). To address the high prevalence of KRAS G12C mutant CNS metastases in lung adenocarcinoma, we are developing CNS-penetrant covalent KRAS G12C inhibitors (“ERAS G12Ci’s”) to treat both CNS and systemic disease. ERAS G12Ci’s rapidly form covalent adducts with the KRAS G12C protein in its inactive GDP state and exhibit low nanomolar IC50’s in a nucleotide exchange assay. ERAS G12Ci’s demonstrate high selectivity in a cell-based proteome selectivity assay and inhibit the growth of RAS Initiative KRAS G12C mutant cells, and not RAS Initiative KRAS WT cells. ERAS G12Ci’s potently inhibit cell proliferation in 3-dimensional Cell-Titer Glo (3D-CTG) assays in KRAS G12C mutant lung and pancreatic cell lines (NCI-H1373, NCI-H2122, and MIA PaCa-2). Mechanistically, ERAS G12Ci’s blocked RAS-RAF complex formation and inhibited ERK1/2 phosphorylation. This in vitro activity translates in vivo where ERAS G12Ci’s induce pharmacodynamic modulation in the pancreatic cancer MIA PaCa-2 model. ERAS G12Ci’s significantly inhibit tumor growth in NCI-H1373 and NCI-H2122 CDX lung adenocarcinoma and MIA PaCa-2 PDAC models. No meaningful body weight loss or clinical adverse events were observed with any of these compounds. Human efflux transporter substrate assessments indicate that ERAS G12Ci’s are either not substrates or weak/modest substrates of P-gp, a crucial efflux transporter that can limit CNS penetration. In rat CNS studies, these inhibitors exhibit good CNS penetration performance as measured by brain-to-plasma partition coefficients, which are comparable to those of approved CNS-active small molecule inhibitors. Daily oral administration demonstrates dose-dependent tumor regression in intracranial and intra-carotid injection (ICA) KRAS G12C CDX models. We are optimizing multiple covalent CNS-penetrant KRAS G12C inhibitors that exhibit both CNS and systemic activity in vivo. Citation Format: Jae Hyun Bae, Erin D. Lew, Jun Feng, Marcos Gonzalez-Lopez, Joanne Oh, Patrick Fagan, Matt Salie, Nick Isley, Richard Lam, Adriana Irimia, Robin Nevarez, Bingzhen Lin, Taylor Congdon, Jingchuan Zhang, Dawei Xuan, Ping Chen, Jean-Michel Vernier, Robert Shoemaker. Discovery of potent CNS-penetrant covalent KRAS G12C inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2675.

Abstract CT242: KRYSTAL-16: A phase I/Ib trial of adagrasib (MRTX849) in combination with palbociclib in patients with advanced solid tumors with KRASG12C mutation

Abstract Background: KRAS is a key mediator of the RAS/MAPK signaling cascade and promotes cellular growth and proliferation. It is the most frequently mutated oncogene in cancer, and KRASG12C mutations occur in 3-4% of colorectal cancers (CRC) and in up to 14% of non-small-cell lung cancers (NSCLC). The investigational agent adagrasib, a potent, covalent inhibitor of KRASG12C, irreversibly and selectively binds to KRASG12C and locks it in its inactive state. Adagrasib was optimized for favorable pharmacokinetic (PK) properties, including long half-life (~24 h), dose-dependent PK, and central nervous system penetration. Initial results have shown encouraging antitumor activity and tolerability of adagrasib monotherapy or combination therapy in patients with CRC or NSCLC harboring KRASG12C mutations. Cyclin D1 and cyclin-dependent kinases (CDK) 4 and 6 are downstream of signaling pathways that lead to cellular proliferation. Palbociclib, a CDK4/6 inhibitor, disrupts cancer cell proliferation by inducing cell cycle arrest by preventing the G1 to S phase transition. Preclinical data in KRASG12C mouse models suggest enhanced antitumor activity when combining adagrasib and palbociclib compared with either agent as a monotherapy. Trial Design: KRYSTAL-16 (NCT05178888) is a multicenter phase I/Ib trial evaluating the safety, PK, pharmacodynamics, and preliminary clinical activity of adagrasib in combination with palbociclib in patients with solid tumors harboring a KRASG12C mutation previously treated with at least 1 standard therapy. The first stage of the trial will comprise a PK lead-in to evaluate potential drug-drug interactions. Exploration of the dose and regimen will continue for the combination of adagrasib (starting dose 400 mg BID) and palbociclib, with expansion cohorts implemented as applicable to further evaluate the safety and preliminary clinical activity of the combination. Decisions regarding dose escalation, expansion, or de-escalation will be determined using the modified Toxicity Probability Interval (mTPI-2) design. Study endpoints include safety, maximum tolerated dose (MTD) and/or recommended Phase II combination dose regimen (RP2D), plasma PK parameters, and preliminary clinical activity (objective response rate [RECIST v1.1], duration of response, progression-free survival, and overall survival). Patients will receive treatment until disease progression, unacceptable adverse events, patient withdrawal, or death. This study will be enrolling at sites in the United States. Citation Format: David Sommerhalder, Jeena Thevathasan, Xenophon Ianopulos, Weining Volinn, David Hong. KRYSTAL-16: A phase I/Ib trial of adagrasib (MRTX849) in combination with palbociclib in patients with advanced solid tumors with KRASG12C mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT242.

Abstract 3600: DCC-3116, a first-in-class selective inhibitor of ULK1/2 kinases and autophagy, synergizes with the KRASG12C inhibitor sotorasib resulting in tumor regression in KRAS mutant NSCLC xenograft models

Abstract Mutationally-activated RAS oncoproteins are detected in approximately 19% of newly diagnosed human cancers and have been targets for drug discovery for over 40 years. Recently, the FDA approved sotorasib, a covalent inhibitor of KRASG12C, for the treatment of non-small cell lung cancer (NSCLC). Although sotorasib demonstrated clear clinical benefits, the emergence of drug resistance has led to drug combination approaches with the goal of deepening and sustaining the durability of patient responses. One potential mechanism of drug resistance is the induction of autophagy, which cancer cells use to survive during periods of stress such as treatment with pathway-targeted therapies. Indeed, KRAS-mutated cancer cells have been shown to exhibit constitutive autophagy for survival. Additionally, such cells further increase autophagy when treated with pathway-targeted inhibitors of RAS>RAF>MEK>ERK MAP kinase signaling as a resistance mechanism. Hence, the combination of a KRASG12C inhibitor with a specific and potent autophagy inhibitor could lead to deeper and more sustained clinical responses. DCC-3116 is an investigational, potent and selective pharmacological inhibitor of the protein kinases ULK1 and ULK2, which are critical initiating components of the autophagy pathway. Treatment of KRASG12C non-small cell lung cancer (NSCLC) cell lines with sotorasib induced autophagy by ~2-fold via activation of ULK kinases as measured by an increase in ULK-mediated phosphorylation of ATG13. Sotorasib-mediated ULK kinase activation, and resulting autophagic flux, was inhibited by DCC-3116 in a dose-dependent manner with IC50 values of 81-160 nM in NSCLC cell lines. These effects translated to in vivo efficacy. In the Calu-1 and H358 KRASG12C NSCLC xenograft models, the combination of DCC-3116 and sotorasib resulted in tumor regression whereas single treatment arms afforded only inhibition of tumor growth. A NSCLC KRASG12C patient derived xenograft (PDX) model further supported the DCC-3116 and sotorasib combination with increased tumor growth inhibition compared to sotorasib alone. These data demonstrate a compelling rationale to study DCC-3116 in combination with KRASG12C inhibitors such as sotorasib in NSCLC patients. DCC-3116 is currently in a Phase 1 clinical trial in patients with advanced solid tumors with documented KRAS, NRAS or BRAF mutations (NCT04892017). Citation Format: Martin McMahon, Madhumita Bogdan, Mary J. Timson, Hikmat Al-Hashimi, Phaedra Ghazi, Yu Zhan, Bryan D. Smith, Conan G. Kinsey, Daniel L. Flynn. DCC-3116, a first-in-class selective inhibitor of ULK1/2 kinases and autophagy, synergizes with the KRASG12C inhibitor sotorasib resulting in tumor regression in KRAS mutant NSCLC xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3600.

Abstract 3091: Patient-derived organoid drug responses corroborate known target-drug interactions for selected anticancer agents

Abstract Patient-derived organoids (PDOrgs) are heterogeneous three-dimensional cellular clusters that have been shown to recapitulate the tumor histology and genetic alterations of their originating tissue. Numerous studies suggest the in vitro drug responses of tumor organoids align with in vivo responses. In this study, we evaluated fourteen anticancer agents against a cohort of PDOrgs from three disease histologies: colon, pancreatic, and non-small cell lung adenocarcinoma. The PDOrgs were obtained from the National Cancer Institute’s Patient-Derived Models Repository (https://pdmr.cancer.gov): a resource that offers clinically annotated and molecularly characterized models. The PDOrg models were selected for specific genetic variants of KRAS and BRAF, or different RNA levels of ABCB1, an ATP-dependent efflux pump. The approved and investigational agents were selected to target specific genetic variants and pathways: KRAS G12C covalent inhibitors (sotorasib and MRTX-1257), RAS pathway inhibitors (BAY-293, BI-3406 and TNO-155), BRAF V600E/K inhibitors (dabrafenib and encorafenib), ABCB1 substrates (paclitaxel, doxorubicin, 5-FU, AZD-1775, and SN-38), and ABCB1 non-substrates (gemcitabine and trametinib). The goal of the study was to assess whether the sensitivities of PDOrgs to therapeutic agents matched these genetic profiles under standard in vitro conditions. PDOrgs were seeded into 384-well microplates, in a semi-automated fashion, and exposed to nine concentrations of each anticancer agent for six days followed by cell viability assessment by CellTiter-Glo 3D. Data analysis was performed using GRmetrics, an R package for calculation and visualization of concentration-response metrics based on growth rate inhibition (https://git.bioconductor.org/packages/GRmetrics). These data demonstrated that PDOrgs harboring a KRAS G12C variant were uniquely sensitive to sotorasib and MRTX-1257 and were, overall, more sensitive to the other RAS pathway targeting agents. Conversely, PDOrgs harboring wild type RAS and other KRAS variants were largely unresponsive to these targeted agents. Likewise, only PDOrgs harboring the BRAF V600E variant were sensitive to dabrafenib and encorafenib. For the majority of PDOrgs, the pharmacological responses to agents that are ABCB1 substrates inversely correlated with ABCB1 RNA expression. This study demonstrates the ability of organoids to serve as useful models for evaluating therapeutic responses to anticancer agents, including identifying known target-drug associations. Moreover, the technical conditions, as well as the selected PDOrgs and therapeutic agents, may be used as a reference set for the validation of a fully automated PDOrg screening system. This project was funded in part with federal funds from the NCI, NIH, under contract no. HHSN261201500003I. Citation Format: Curtis Hose, Erik Harris, John Connelly, Petreena S. Campbell, Mariaestela Ortiz, Eric Jones, Dianne Newton, Yvonne A. Evrard, Melinda Hollingshead, Ralph Parchment, Beverly A. Teicher, Nathan P. Coussens, James H. Doroshow, Annamaria Rapisarda. Patient-derived organoid drug responses corroborate known target-drug interactions for selected anticancer agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3091.

Abstract 2664: Development of covalent KRASG12C inhibitor APG-1842 for the treatment of solid tumors

Abstract The KRAS gene, which encodes a guanosine triphosphate (GTPase) protein, is frequently mutated in solid cancers. The KRASG12C mutation aberrantly attenuates GTPase activity, leading to accumulation of the GTP-bound activated form of the KRAS protein and activation of downstream signaling pathways. This genotype occurs in approximately 13% of non-small-cell lung cancers (NSCLCs) and 1% to 3% of colorectal cancers and other solid tumors. In this study, investigational agent APG-1842 was characterized as a potent, selective, and covalent KRASG12C inhibitor that irreversibly and specifically blocks KRASG12C in an inactive guanosine diphosphate (GDP)-bound state and downregulates KRAS-dependent signaling. A RAS-GTP pull-down assay demonstrated that APG-1842 (at a concentration as low as 12 nM) completely blocked GDP-GTP nucleotide exchange, which stabilized an inactive GDP-bound form of KRASG12C and thereby inhibited RAS and RAF binding in KRASG12C-mutant human NSCLC NCI-H358 cells. In these cells, APG-1842 dose dependently inhibited KRAS-dependent signaling target proteins, including phosphorylated extracellular signal-regulated kinase (ERK), S6, and serine/threonine protein kinase Akt (protein kinase B), with a half-maximal inhibitory concentration (IC50) value of 4 nM for ERK1/2 phosphorylation. Using a panel of 18 cancer cell lines representing multiple cancer types in cell viability assays, we found that APG-1842 selectively inhibited cell proliferation in KRASG12C-mutated, but not non-KRASG12C or KRAS wild-type, cell lines. Oral APG-1842 in mice carrying cell-derived NCI-H358 NSCLC subcutaneous xenografts exerted significant antitumor efficacy at doses ranging from 3 to 100 mg/kg, resulting in tumor regression at 30 mg/kg and above. Consistent with the cellular results, APG-1842 dose dependently inhibited ERK1/2 and S6 phosphorylation in xenografts after 6 and 24 hours of treatment, correlating with systemic and tumor drug exposures. Significant antitumor activity of APG-1842 was also confirmed in a panel of 8 KRASG12C-mutated patient-derived xenograft models derived from patient samples of lung, colorectal, and gastric cancers. Collectively, these preclinical results suggest that APG-1842 is a potent, bioavailable, and highly selective KRASG12C inhibitor, laying a foundation for clinical development of this agent for patients with KRASG12C-mutant solid tumors. Citation Format: Shaoulai Gu, Qiang Li, Chao Li, Qixin Wang, Douglas D. Fang, Shaomeng Wang, Dajun Yang, Yifan Zhai. Development of covalent KRASG12C inhibitor APG-1842 for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2664.

Abstract LB529: Oncogenic KRas plays a significant role in regulating expression of IDO1

Abstract Ras proteins are among the most widely studied proto-oncogene products. Ras proteins are membrane-anchored GTPases that participate in multiple signaling cascades regulating crucial cellular processes including cell survival, proliferation, and differentiation. Ras mutations and/or deregulated Ras activities frequently lead to inflammation and malignant transformation. Recent studies have also shown that Ras mutations are associated with immuno-resistance by positive regulation of PD-L1 expression. Indoleamine 2,3-dioxygenases 1 (IDO1) is a heme-containing enzyme that catalyzes the rate limiting step of conversion of tryptophan into kynurenine (Kyn). Extensive research in the past has shown that IDO1 and its catalytic product Kyn mediate immune tolerance, thus promoting tumorigenesis in vivo. IDO1 expression is activated by immune cytokines including interferon-γ (IFN-γ)and interleukin-6 (IL-6). However, whether there is a mechanistic link between oncogenic KRas and IDO1 expression and how IDO1 expression contributes to immune invasion of transformed cells with KRas mutations remain unclear. Here we report that oncogenic KRas significantly enhanced IFN-γ-induced IDO1 expression. In H358 lung carcinoma cells, IDO1 expression induced by IFN-γ was at least in part dependent on ERK activation, and treatment with ARS-1620, a covalent KRasG12C inhibitor, suppressed IDO1 expression induced by IFN-γ in a concentration-dependent manner. IDO1 expression was also induced by IFN-γ in HCT116 colon carcinoma cells that harbored a KRasG12D mutant allele, but not in HCT116 cells with wild-type KRas, suggesting that IFN-γ induced IDO1 expression requires Ras activation. In addition, KRas downregulation by specific siRNAs decreased IFN-γ-induced IDO1 expression in A549 lung cancer cells. Taken together, our study strongly suggests that Ras/ERK signaling pathway plays an important role in promoting IDO1 expression induced by IFN-γ. Citation Format: Eunji Lee, Felicia He, Byeong Hyeok Choi, Wei Dai. Oncogenic KRas plays a significant role in regulating expression of IDO1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB529.

Abstract 3596: RM-036, a first-in-class, orally-bioavailable, Tri-Complex covalent KRASG12D(ON) inhibitor, drives profound anti-tumor activity in KRASG12D mutant tumor models

Abstract KRASG12D mutant cancers represent a significant unmet medical need with 55,000 new diagnoses annually in the US. The KRASG12D mutation occurs commonly in multiple tumor histotypes, including about 20%, 29% and 17% of KRAS mutant colorectal, pancreatic, and non-small cell lung cancers, respectively. However, there are no directly targeted inhibitors of KRASG12D in clinical development. Mutant RAS proteins exist predominantly in the GTP-bound RAS(ON) state, leading to excessive downstream signaling via interaction with effectors such as RAF kinases. Highly selective inhibitors that covalently target the KRASG12C(OFF) state and inhibit conversion to KRASG12C(ON) have been shown to be active and well tolerated in the clinic, and we have described a novel, covalent tri-complex KRASG12C(ON) inhibitor that has robust anti-tumor activity and is well tolerated in preclinical studies. Developing a covalent inhibitor of KRASG12D presents several challenges. The Asp residue in KRASG12D is significantly less reactive toward electrophiles than is Cys. And most reported covalent chemical warheads capable of reacting with Asp exhibit high nonspecific reactivity. The intrinsic GTP hydrolysis rate of KRASG12D is about 2.6-fold lower than that of KRASG12C, further biasing the cellular KRASG12D pool to the RAS(ON) state and emphasizing the importance of targeting the KRASG12D(ON) state for maximal suppression of this oncogenic driver. Based on these considerations we designed RM-036, a potent, selective, orally bioavailable, covalent KRASG12D(ON) inhibitor with attractive drug-like properties. It forms a tri-complex between the abundant intracellular chaperone cyclophilin A (CypA) and the active state of KRASG12D, positioning the warhead relative to KRASG12D and enabling selective covalent engagement of Asp. In cellular models, RM-036 exhibited efficient and selective covalent binding to KRASG12D, with no detectable reactivity toward the adjacent Asp residue in cells with the KRASG13D mutation. RM-036 showed low off-target reactivity in cell-based proteomic screens. RM-036 potently inhibited growth and induced apoptosis in KRASG12D mutant cancer cells in vitro but not in BRAFV600E-dependent cells. RM-036, as a single agent, produced deep, durable, and dose-dependent suppression of tumor RAS pathway activation with covalent target engagement in vivo following repeat oral administration. Profound tumor regressions, including complete regressions, were observed in various KRASG12D mutant xenograft models upon treatment with RM-036. All treatments regimens tested with RM-036 were well tolerated in vivo. These preclinical findings provide a strong foundation for advancing RM-036 toward clinical evaluation in patients with tumors bearing the KRASG12D mutation. Citation Format: John E. Knox, Jingjing Jiang, G. Leslie Burnett, Yang Liu, Caroline E. Weller, Zhican Wang, Laura McDowell, Shelby L. Steele, Shook Chin, Kang Jye Chou, Fang Wang, Mengqi Zhong, Elena S. Koltun, David Wildes, Mallika Singh, Adrian L. Gill, Jacqueline A. Smith. RM-036, a first-in-class, orally-bioavailable, Tri-Complex covalent KRASG12D(ON) inhibitor, drives profound anti-tumor activity in KRASG12D mutant tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3596.

Abstract 4029: Combination of KRASG12C(ON) and SHP2 inhibitors overcomes adaptive resistance and enhances anti-tumor immunity

Abstract Oncogenic mutations in KRAS are frequent in non-small cell lung cancer and have been associated with poor prognosis. The recent development and approval of KRASG12C mutant-specific inhibitors could change the clinical practice of lung cancer patients harboring KRASG12C mutations. However, early clinical data indicate the development of acquired resistance after initial responses. All of the KRASG12C inhibitors being tested in clinical trials to date target GDP-bound KRAS (OFF state), which makes them vulnerable to upstream pathway reactivation, as this will increase KRAS in a GTP-bound (ON) state and therefore reduce drug efficacy. In this study we use a covalent tri-complex KRASG12C(ON) inhibitor which targets KRASG12C in the active state. In vitro, the KRASG12C(ON) inhibitor RM-029 exhibited higher efficacy than the KRASG12C(OFF) inhibitor MRTX849 (adagrasib). However, treatment with RM-029 also showed adaptive RAS pathway reactivation after 24 hours of treatment, which can be blocked using the SHP2 inhibitor RMC-4550, suggesting that pathway reactivation is mediated by signaling from RTKs to RAS proteins. In vivo, KRASG12C(ON) treatment led to strong regression of KRASG12C tumors and addition of a SHP2 inhibitor significantly increased anti-tumor responses. Single treatments with KRASG12C(ON) or SHP2 inhibitors led to complete responses in some animals and immunological memory in an immune responsive KRASG12C lung tumor model, with all tumors showing complete responses when the inhibitors were combined. Consistent with this finding, both KRASG12C(ON) and SHP2 inhibition resulted in a profound remodeling of the lung tumor microenvironment (TME), with increased infiltration and activation of T cells accompanied by a reduction of tumor-promoting myeloid cells. Interestingly, similar changes were obtained in an immune evasive KRASG12C lung cancer model. Besides acting upstream RAS, SHP2 has also specific roles in immune cells, including regulation of T cell function. As comparison, responses to SOS1 inhibition, which acts only upstream RAS, have also been analyzed. Overall, our preclinical results show that the anti-tumor activity of the combination of KRASG12C(ON) with SHP2 inhibition is the result of different mechanisms. First, a KRASG12C(ON) inhibitor potently inhibits oncogenic RAS and addition of a SHP2 inhibitor blocks RAS pathway reactivation increasing tumor responses. Second, both inhibitors have beneficial effects in the TME which can be potentiated when combined. Finally, addition of a SHP2 inhibitor could potentially overcome intrinsic resistant populations by directly targeting cells resistant to G12C inhibition and/or by increasing anti-tumor immunity. Citation Format: Panayiotis Anastasiou, Edurne Mugarza, Jesse Boumelha, Sareena Rana, Christopher Moore, Miriam Molina-Arcas, Julian Downward. Combination of KRASG12C(ON) and SHP2 inhibitors overcomes adaptive resistance and enhances anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4029.

Abstract ND11: Discovery of GDC-6036, a clinical stage treatment for KRAS G12C-positive cancers

Abstract Mutations in KRAS are the most common oncogenic driver mutations in human cancers. The KRAS G12C mutation is one of the most prevalent KRAS mutations, present in approximately 12% of non-small cell lung cancer, 4% of colorectal cancer, and up to 4% of other cancer types. The recent discovery and development of covalent KRAS G12C inhibitors provides an opportunity to inhibit what has historically been considered to be an “undruggable” target. GDC-6036 is an orally bioavailable, highly potent and selective KRAS G12C inhibitor, with a median IC50 in the sub-nanomolar range and greater than 18,000-fold selectivity for G12C versus non-G12C cell lines. GDC-6036 demonstrates greater potency and selectivity compared with other KRAS G12C inhibitors in vitro, and complete tumor growth inhibition in multiple KRAS G12C-positive cell lines and in xenograft mouse models. We will highlight the research program that led to the discovery and optimization of GDC-6036, which is currently in clinical development. Citation Format: Hans Purkey. Discovery of GDC-6036, a clinical stage treatment for KRAS G12C-positive cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr ND11.

Abstract 3595: RMC-6291, a next-generation tri-complex KRASG12C(ON) inhibitor, outperforms KRASG12C(OFF) inhibitors in preclinical models of KRASG12C cancers

Abstract The KRASG12C mutation is found in 11% of non-small cell lung cancers, 4% of colorectal cancers, and 2% of pancreatic cancers in the U.S., and drives these cancers by shifting the cellular equilibrium of KRAS towards the GTP-bound, active state, KRASG12C(ON). The resulting increased levels of KRASG12C(ON) in turn increase signaling output to initiate and support the oncogenic state. In recent years, a class of KRASG12C(OFF) inhibitors has transformed the treatment landscape for patients with cancers bearing KRASG12C. These inhibitors work via sequestration of the GDP-bound, inactive state, KRASG12C(OFF), starving cancer cells of their oncogenic driver, KRASG12C(ON). Recent reports on the nature of resistance to KRASG12C(OFF) inhibitors suggest this class of drugs can be overcome through reactivation of KRASG12C to the ON form. Direct inhibition of KRASG12C(ON) with a first in class, potent, orally bioavailable, selective, tri-complex inhibitor RMC-6291, represents a more robust approach and presents the possibility that RMC-6291 will be a ‘best-in-class’ inhibitor of tumors harboring KRASG12C. RMC-6291 is a potent covalent inhibitor of KRASG12C(ON) that forms a tri-complex within tumor cells between KRASG12C(ON) and cyclophilin A (CypA), a highly abundant immunophilin. The assembled tri-complex prevents KRASG12C(ON) from signaling via steric blockade of RAS effector binding. In cells, kinetic analyses demonstrate near-immediate disruption of RAS effector binding and extinction of KRASG12C(ON) signaling. Oral administration of RMC-6291 produces deep and durable suppression of RAS pathway activity in KRASG12C tumor models and drives profound tumor regressions in vivo at well-tolerated doses. In a mouse clinical trial consisting of multiple patient- and cell line-derived xenograft models of KRASG12C NSCLC, RMC-6291 outperformed adagrasib, a KRASG12C(OFF) inhibitor, by increasing the number of responses, the depth of tumor regressions, and the durability of responses. Combination treatment with RMC-6291 and SHP2 or SOS1 inhibitors was well tolerated in preclinical models and further increased anti-tumor activity, likely by preventing reactivation of wild-type RAS proteins that cooperate with KRASG12C to fuel cancer growth. RMC-6291 also combined well with immune checkpoint inhibitors, sensitizing KRASG12C-bearing cancer models to anti-tumor immunity. RMC-6291 is a next-generation, mutant-selective inhibitor of KRASG12C(ON) that overcomes limitations of first-generation KRASG12C(OFF) inhibitors in preclinical models by directly targeting the active form of this important oncogenic driver. Citation Format: Robert J. Nichols, Y.C. Yang, Jim Cregg, Chris J. Schulze, Zhican Wang, Richa Dua, Jingjing Jiang, Lindsay S. Garrenton, Nicole Nasholm, Alun Bermingham, John E. Knox, Kyle Seamon, Michael Longhi, Kang-Jye Chou, Shaoling Li, David P. Wildes, Mallika Singh, Elena S. Koltun, Adrian L. Gill, Jacqueline A.M. Smith. RMC-6291, a next-generation tri-complex KRASG12C(ON) inhibitor, outperforms KRASG12C(OFF) inhibitors in preclinical models of KRASG12C cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3595.

Abstract 413: WT RAS signaling is an essential therapeutic target in RAS mutated cancers

Abstract Single-agent targeting of RAS-mutated cancers using RAF/MEK/ERK or PI3K/AKT pathway inhibitors is ineffective; blocking one pathway relieves negative feedback control of RTK signaling and hyperactivates parallel effector pathways. While combined MEK and PI3K inhibition is effective in preclinical models, toxicity of this combination prevents its clinical use. Alternative strategies for treating RAS-mutated cancers are essential. Therapeutics directly targeting mutated RAS proteins have the potential to spare normal cellular function thereby lessening overall toxicity. Unfortunately, direct RAS inhibition as a monotherapy does not fully block RAS effector signaling. RAS proteins show differential activation of RAF and PI3K pathways: KRAS potently activates RAF but poorly activates PI3K, whereas HRAS potently activates PI3K but poorly activates RAF. Further, mutant RAS inhibition relieves negative feedback controls leading to rapid hyperactivation of RTK−WT RAS signaling. A more comprehensive understanding of the interplay between mutant RAS and RTK−WT RAS signaling is essential to developing rational therapeutic approaches to treat RAS-mutated cancers. We found that WT RAS enhanced mutant RAS-driven transformation by activating RAS effectors poorly engaged by mutated RAS. Further, inhibition of RAS effectors activated poorly by mutant RAS synergized with and limited resistance to mutant HRAS and KRAS inhibitors. The mutant HRAS inhibitor tipifarnib blocked PI3K signaling and synergized with MEK inhibitors in HRAS-mutated cancer cell lines; covalent KRASG12C inhibitors blocked MEK signaling and synergized with PI3K inhibitors in KRASG12C-mutated cell lines. Synergy between inhibitors of mutant RAS and RAS effectors was dependent on intact RTK/WT RAS signaling and was lost in both RASless or SOSless MEFs. Upstream of RAS, the RASGEFs SOS1 and SOS2 showed unique and overlapping functions to promote mutant RAS-driven transformation. SOS1 was critical for mutant RAS (G12V) activation and SOS1 inhibition augmented the efficacy of mutant RAS inhibitors. RTK−SOS2−PI3K signaling protected cells from anoikis and mediated mutant KRAS-driven transformation. These results should inform the design of clinical trials for patients with RAS-mutated cancers. Citation Format: Nancy E. Sealover, Robert L. Kortum. WT RAS signaling is an essential therapeutic target in RAS mutated cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 413.

D3S-001, a highly potent, selective, and differentiated covalent inhibitor of KRAS G12C: Human dose prediction and first‐in‐human (FIH) trial design.

e15087 Background: Effective inhibition of G12C mutated KRAS is believed to be critical for anti-tumor efficacy in KRAS-driven cancers. D3S-001 is a potent and highly differentiated KRAS G12C inhibitor, which demonstrates rapid and near complete target inhibition and strong anti-tumor activity in preclinical cell line and animal models [Preclinical characterization of D3S-001, a highly potent, selective, and differentiated covalent inhibitor of KRAS G12C, ASCO 2022 Abstract #378099]. The current study utilizes pharmacokinetic (PK) modeling to predict the extent of KRAS G12C target inhibition at the proposed clinical dose range. Methods: Allometric scaling and non-linear fixed effect modeling were used to predict human PK profiles from DMPK data in animals. Human in vivo target inhibition was calculated based on human PK predictions, free drug fraction and results from in vitro target engagement assay. A First-in-Human (FIH) study was designed to investigate D3S-001 monotherapy in subjects with advanced solid tumors having a KRAS G12C mutation. Six dose escalation cohorts will be investigated, starting from 50mg QD. Results: Based on a GLP toxicology study in dogs, the highest non-severely toxic dose (HNSTD) was determined to be 20 mg/kg/day. Per ICH S9, a 20 mg/kg/day dose in dog supports a FIH starting dose of 110 mg. To ensure an additional safety margin, a lower starting dose of 50 mg was proposed. At 50 mg QD, the predicted human exposure (Cmax 60 nM, Caverage 49 nM, Ctrough 31 nM) is expected to achieve ̃70% target inhibition at 2 hours after dosing. In NCI-H358 mouse xenograft model, this exposure is associated with 94% tumor growth inhibition (TGI). Safety margins based on Cmax and AUC comparing to dog HNSTD are 17-fold and 9-fold, respectively. At 100 mg QD, the predicted human exposure (Cmax 120 nM, Caverage 98 nM, Ctrough 63 nM) is expected to achieve ̃95% target inhibition. This exposure is associated with tumor regression of 72% in the NCI-H358 mouse xenograft model. At dose levels higher than 100 mg QD, predicted human exposures are expected to consistently achieve >95% target inhibition starting 1 hour after dosing. These exposures are associated with deeper levels of tumor regression of 97%. Moreover, even at 400 mg QD, predicted human plasma concentrations are still below dog plasma concentration of 443 nM, Caverage at HNSTD. Conclusions: D3S-001 is predicted to achieve rapid and consistently efficacious KRAS G12C target inhibition in humans at proposed clinical dose range. This high degree of KRAS G12C inhibition over the dosing interval may translate into greater clinical benefit to patients with KRAS G12C mutations comparing with other clinical-stage KRAS G12C inhibitors. Clinical data from the FIH study will be evaluated and compared to preclinical predictions.

Preclinical characterization of D3S-001, a highly potent, selective, and differentiated covalent inhibitor of KRAS G12C.

e15102 Background: KRAS G12C mutation is an oncogenic driver most frequently found in non-small cell lung cancer (NSCLC), colorectal cancer (CRC), and other tumor types. Covalent small molecule inhibitors such as sotorasib, recently FDA-approved, and others in clinical trials that target G12C-altered protein by irreversibly binding to the GDP-bound inactive form of KRAS are clinically active in NSCLC patients carrying this somatic mutation. However, in in vitro assays we have developed to evaluate cellular GTP-bound KRAS G12C level, these molecules did not achieve maximal KRAS G12C target inhibition at the minimum concentration (Cmin) of their respective clinically doses. We therefore hypothesize that the potency of KRAS G12C inhibitors may be further improved through medicinal chemistry optimization. Methods: A series of in vitro and in vivo studies were conducted to assess the preclinical pharmacological activity of D3S-001. The biochemical potency of D3S-001 was evaluated by SPR binding analysis. The kinetics of KRAS G12C target engagement was measured by ELISA method. The inhibitor effect of D3S-001 on phospho-ERK1/2 was determined by HTRF assay, and the anti-proliferation effect on cancer cell lines was evaluated by CTG luminescence assay. For in vivo efficacy studies, several human cancer xenograft models and a mouse syngeneic model were employed. Results: D3S-001 demonstrated an exceptionally high Kinact/KI value of 1.58^106 M-1 s-1 by SPR binding analysis. In target engagement assays, D3S-001 achieved rapid and near complete (> 95%) KRAS G12C inhibition within 2 hours at 5 nM. This rapid and effective target engagement correlated with its substantially enhanced cellular potency in both in vitro and in vivo efficacy studies. D3S-001 demonstrated single-digit nanomolar IC50 values in inhibiting cellular phospho-ERK1/2 and cell proliferation in a panel of cancer cell lines harboring the KRAS G12C mutation. D3S-001 was also high selective with no anti-proliferation effects on non-KRAS G12C mutant cell lines. In in vivo studies, D3S-001 demonstrated robust anti-tumor effects as monotherapy in KRAS G12C xenograft models with tumor regression observed at 10 mg/kg and a 30% durable complete remission (CR) rate in CT26, a KRAS G12C syngeneic model, at 30 mg/kg. When combined with an anti-PD-1 antibody, a 70% durable CR rate was achieved with no tumor re-growth observed in CR mice after rechallenging with CT26 tumor, suggesting a memory T cell-induced response. Conclusions: D3S-001 is a highly potent, selective KRAS G12C inhibitor that is differentiated by its rapid and effective target engagement. Human PK modeling predicts > 95% constant target blockage using a 200 mg QD dosing regimen, which may be important and relevant to the induction of deeper and more durable clinical response. D3S-001 will enter a first-in-human Phase 1 clinical trial in 2022.

Bcl-xL and association with apoptosis following KRASG12C inhibition in KRASG12C mutant colorectal cancer.

3557 Background: Novel covalent inhibitors of KRASG12C have shown modest response rates in KRASG12C mutant (MT) colorectal cancer (CRC) patients. Thus, novel KRASG12C inhibitor combination strategies that can achieve deep and durable responses are needed. Methods: The small molecule KRASG12C inhibitors AZ’1569 and AZ’8037 were employed. To identify novel candidate combination strategies for AZ’1569, we performed RNA sequencing, siRNA and high-throughput drug screening. Top hits were validated in a panel of KRASG12CMT CRC cells and in vivo xenograft models. AZ’1569 acquired resistant CRC models were generated and characterised. Results: Response to AZ’1569 was heterogeneous across the KRASG12CMT models. AZ’1569 was ineffective at inducing apoptosis when used as a single agent or combined with chemotherapy or agents targeting the EGFR/KRAS/AKT axis. Using a systems biology approach, we identified the anti-apoptotic BH3-family member BCL2L1/Bcl-xL as top hit mediating resistance to AZ’1569. Further analyses identified acute increases in the pro-apoptotic protein BIM following AZ’1569 treatment. ABT-263 (Navitoclax), a pharmacological Bcl-2 family-inhibitor that blocks the ability of Bcl-xL to bind and inhibit BIM, led to dramatic and universal apoptosis when combined with AZ’1569 in a panel of KRASG12C MT CRC cells. Furthermore, this combination also resulted in dramatically attenuated tumour growth in KRASG12C MT CRC xenografts. Finally, AZ’1569 acquired resistant KRASG12C MT CRC cells showed amplification of KRASG12C, EphA2/c-MET activation, increased pro-inflammatory chemokine profile and cross-resistance to standard-of-care chemotherapy and several targeted agents. Importantly, the KRAS amplification and AZ’1569-resistance were reversible upon drug withdrawal, arguing strongly for the use of drug holidays in the case of KRAS amplification. Conclusions: Combinatorial targeting of Bcl-xL and KRASG12C is highly effective, suggesting a novel therapeutic strategy for KRAS G12CMT CRC patients. The cross-resistance to other targeted therapies and importantly conventional chemotherapy in the AZ’1569 acquired resistant cells poses a challenge, with implications for the optimal use of KRASG12C inhibitors as a second or third line option.

A phase I/II study of first-in-human trial of JAB-21822 (KRAS G12C inhibitor) in advanced solid tumors.

3089 Background: KRAS G12C mutation occurs in approximately 4% of non-small cell lung cancer (NSCLC), 1-2% of colorectal cancer (CRC) and other solid tumors in China. JAB-21822 (Jacobio, Beijing, PRC) is a highly selective, covalent oral inhibitor of KRAS G12C. Methods: NCT05009329 is an ongoing first-in-human, open label phase I/II study of JAB-21822 in patients with advanced solid tumors. The primary objective is to evaluate the safety and tolerability of JAB-21822. Other objectives include preliminary efficacy, pharmacokinetics, and biomarkers. Here we report the results from the dose escalation phase of the trial. Results: As of January 28th, 2022, 53 patients with a median age of 62 years (39-79) were enrolled in 5 different dose levels: 200mg QD, 400mg QD, 800mg QD, 400mg BID and 400mg TID. Most patients (55%) had ≥ 2 prior lines of therapy. No dosing-limiting toxicities were observed. Two treatment-related adverse events (TRAEs) were G4 neutropenia (1 in 400mg BID and 1 in 400mg TID). The most common TRAEs (≥ 10%) included anemia (24.5%), total bilirubin increase (20.8%), direct bilirubin increase (15.1%), proteinuria (13.2%) and indirect bilirubin increase (11.3%). Only Grade 1 and 2 TRAEs were observed in the QD cohorts. A total of 33 patients (22 NSCLC, 9 CRC and 2 pancreatic cancer) had at least 1 post-baseline tumor assessment; in the 800mg QD cohort, overall response rate (ORR) and disease control rate (DCR) were 50% (5/10) and 100% (10/10), respectively, including 4 non-confirmed partial response (PR); in the 400mg QD cohort had an ORR and DCR of 80% (4/5) and 100% (5/5) respectively, including 2 non-confirmed PR. Patients with NSCLC (400mg QD and 800mg QD), the ORR and DCR were 70% (7/10) and 100% (10/10), respectively, including 5 non-confirmed PR. With respect to the pharmacokinetics analysis, JAB-21822 was rapidly absorbed with an average Tmax of 2 hr and reached higher plasma exposures (Cmax and AUC0-24h) after a single dose and multiple doses at C1D8. Conclusions: JAB-21822 was well tolerated with impressive preliminary efficacy in patients with heavily treated solid tumors harboring KRAS G12C mutation. The study is enrolling patients in the expansion phase. Multiple JAB-21822-based combination trials are also ongoing. Clinical trial information: NCT05009329.

Phase I dose-escalation study of IBI351 (GFH925) monotherapy in patients with advanced solid tumors.

3110 Background: IBI351(GFH925) is an irreversibly covalent inhibitor of KRASG12C. In this first-in-human dose-escalation study, we report the preliminary safety and anti-tumor activity of IBI351 (GFH925) in patients (pts) with advanced solid tumors harboring the KRAS p.G12C mutation. Methods: Pts with locally advanced, recurrent or metastatic solid tumors with KRASG12C mutation for whom standard therapy had failed were enrolled. Phase I dose escalation had an accelerated titration design for dose level 250mg QD and a BOIN design with 450/700/900mg QD. The primary end points were safety and tolerabilityThe secondary end points were pharmacokinetics (PK) and anti-tumor activity of IBI351(GFH925) monotherapy per RECIST v1.1. Results: As of Feb 07 2022, 15 pts (13 men, 2 women; median age: 62 yrs, range: 48–74 yrs) were enrolled, among whom 12 had non-small cell lung cancer (NSCLC), and 3 had colorectal cancer (CRC). 4 pts had ≥3 prior lines of treatment (tx). Median tx duration was 66.5 ds (range: 21–98 ds). No dose-limiting toxicity (DLT) or any ≥grade 3 treatment-related adverse events were observed in any dose cohorts. A total of 12 patients (80.0%) had treatment-related adverse events (grade 1, n = 6; grade 2, n = 6). By investigator-assessment, tumor response was evaluated in 9 pts (4 with ≥2 assessments); 6 pts had not reached their first assessment. 2 pts had PR (1 NSCLC at wks 12, 450mg, tx ongoing; 1 CRC at wks 6, 700mg, tx ongoing), 4 pts (NSCLC) had SD, and 3 pts had PD (1 NSCLC at wks 12, 2 CRC at wks 6). As data cut-off date, 11 pts were continuing to receive IBI351 (GFH925). Conclusions: IBI351(GFH925) was well-tolerated without unanticipated adverse events across all doses explored in pts with advanced solid tumors harboring the KRAS p.G12C mutation. The data also demonstrated the preliminary efficacy signal of IBI351 (GFH925) in previously treated advanced NSCLC and CRC. Clinical trial information: NCT05005234.

Expression of IDO1 is regulated via Ras signaling pathways

Ras proteins are among the most widely studied proto‐oncogene products. Ras proteins are membrane‐anchored GTPases that participate in multiple signaling cascades regulating crucial cellular processes including cell survival, proliferation, and differentiation. Ras mutations and/or deregulated Ras activities frequently lead to inflammation and malignant transformation. Recent studies have also shown that Ras mutations are associated with immuno‐resistance by positive regulation of PD‐L1 expression. Indoleamine 2,3‐dioxygenases 1 (IDO1) is a heme‐containing enzyme that catalyzes the rate limiting step of conversion of tryptophan into kynurenine (Kyn). Extensive research in the past has shown that IDO1 and its catalytic product Kyn mediate immune tolerance, thus promoting tumorigenesis in vivo. IDO1 expression is activated by immune cytokines including interferon‐γ (IFN‐γ)and interleukin‐6 (IL‐6). However, whether there is a mechanistic link between oncogenic KRas and IDO1 expression and how IDO1 expression contributes to immune invasion of transformed cells with KRas mutations remain unclear. Here we report that oncogenic KRas significantly enhanced IFN‐γ‐induced IDO1 expression. In H358 lung carcinoma cells, IDO1 expression induced by IFN‐γ was at least in part dependent on ERK activation, and treatment with ARS‐1620, a covalent KRasG12C inhibitor, suppressed IDO1 expression induced by IFN‐γαμμα in a concentration‐dependent manner. IDO1 expression was also induced by IFN‐γ in HCT116 colon carcinoma cells that harbored a KRasG12D mutant allele, but not in HCT116 cells with wild‐type KRas, suggesting that IFN‐γ induced IDO1 expression requires Ras activation. In addition, KRas downregulation by specific siRNAs decreased IFN‐γ‐induced IDO1 expression in A549 lung cancer cells. Taken together, our study strongly suggests that Ras/ERK signaling pathway plays an important role in promoting IDO1 expression induced by IFN‐γ.

Targeting Mutant Kirsten Rat Sarcoma Viral Oncogene Homolog in Non-Small Cell Lung Cancer: Current Difficulties, Integrative Treatments and Future Perspectives.

In the past few decades, several gene mutations, including the anaplastic lymphoma kinase, epidermal growth factor receptor, ROS proto-oncogene 1 and rat sarcoma viral oncogene homolog (RAS), have been discovered in non-small cell lung cancer (NSCLC). Kirsten rat sarcoma viral oncogene homolog (KRAS) is the isoform most frequently altered in RAS-mutated NSCLC cases. Due to the structural and biochemical characteristics of the KRAS protein, effective approaches to treating KRAS-mutant NSCLC still remain elusive. Extensive recent research on KRAS-mutant inhibitors has made a breakthrough in identifying the covalent KRASG12C inhibitor as an effective agent for the treatment of NSCLC. This review mainly concentrated on introducing new covalent KRASG12C inhibitors like sotorasib (AMG 510) and adagrasib (MRTX 849); summarizing inhibitors targeting the KRAS-related upstream and downstream effectors in RAF/MEK/ERK pathway and PI3K/AKT/mTOR pathway; exploring the efficacy of immunotherapy and certain emerging immune-related therapeutics such as adoptive cell therapy and cancer vaccines. These inhibitors are being investigated in clinical trials and have exhibited promising effects. On the other hand, naturally extracted compounds, which have exhibited safe and effective properties in treating KRAS-mutant NSCLC through suppressing the MAPK and PI3K/AKT/mTOR signaling pathways, as well as through decreasing PD-L1 expression in preclinical studies, could be expected to enter into clinical studies. Finally, in order to confront the matter of drug resistance, the ongoing clinical trials in combination treatment strategies were summarized herein.

Discovery, Preclinical Characterization, and Early Clinical Activity of JDQ443, a Structurally Novel, Potent, and Selective Covalent Oral Inhibitor of KRASG12C.

JDQ443 is a structurally novel covalent KRASG12C inhibitor with a unique binding mode that demonstrates potent and selective antitumor activity in cell lines and in vivo models. In preclinical models and patients with KRASG12C-mutated malignancies, JDQ443 shows potent antitumor activity as monotherapy and in combination with the SHP2 inhibitor TNO155. This article is highlighted in the In This Issue feature, p. 1397.

76TiP RMC-4630 and sotorasib for advanced KRASG12C NSCLC after failure of prior standard therapies: A phase II trial

Sotorasib is a selective, irreversible, oral small molecule covalent inhibitor of KRASG12C (OFF) recently approved in the U.S. for previously treated patients with KRASG12C NSCLC. RMC-4630 is a potent, selective, orally bioavailable allosteric inhibitor of SHP2, a protein tyrosine phosphatase functioning downstream of multiple RTKs as a convergent node in RAS signaling. RMC-4630 has demonstrated acceptable tolerability with an intermittent dosing schedule (Day 1 and Day 2 of each week) and single agent activity in KRASG12C NSCLC tumors in a phase 1b study.