Mutant-selective Inhibitor Research Articles

145 (PB133): Preclinical characterization of a novel PI3Kα H1047R mutant-selective inhibitor

145 (PB133): Preclinical characterization of a novel PI3Kα H1047R mutant-selective inhibitor

Abstract IA-04: Targeting the oncogenic state of RAS with tri-complex inhibitors

Abstract KRAS oncogenic driver mutations occur in approximately 30% of all human cancers, and in particular, in over 90% of patients with pancreatic ductal adenocarcinoma (PDAC). Small molecule inhibitors that selectively target the inactive, GDP-bound state of KRAS G12C mutant proteins have demonstrated clinical efficacy in non-small cell lung cancer (NSCLC) as monotherapies, and in the small subset of PDAC patients that harbor this mutation. However, there are no currently approved RAS-targeted therapy options for the majority of patients with non-G12C RAS mutant PDAC. We have designed a series of tri-complex inhibitors that specifically target the GTP-bound, active state of RAS (RAS(ON)). These small molecule inhibitors bind non-covalently to an abundant intracellular protein, cyclophilin A (CypA). The resulting binary complex selectively engages RAS(ON), forming a tri-complex that sterically inhibits RAS interaction with downstream effectors. We have generated mutant-selective inhibitors that covalently engage RAS(ON) G12C (RMC-6291) or RAS(ON) G12D (RMC-9805). In addition, RMC-6236 is a RAS(ON) multi-selective inhibitor that non-covalently inhibits the active, GTP-bound state of mutant and wild-type variants of the canonical RAS isoforms (KRAS, NRAS, and HRAS). Here we will describe the mechanism of action of these investigational agents and the preclinical profiles that support their clinical evaluation in patients with PDAC as monotherapies and in combination therapy regimens with standard-of-care agents or as novel RAS(ON) inhibitor doublets. Citation Format: Mallika Singh. Targeting the oncogenic state of RAS with tri-complex inhibitors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr IA-04.

Abstract ED10-01: PI3K past, present, and future

Abstract The PI3K pathway has long remained a fertile ground for drug discovery in cancer, including in estrogen receptor positive metastatic breast cancer. It has also remained a pathway difficult to drug effectively due to on target toxicities. New strategies to overcome these toxicities have manifested in novel drug types and management strategies for patients. Here I discuss the PI3K pathway in signaling and oncogenesis, current therapies inhibiting PI3K, mechanisms limiting efficacy, and increasing the PI3K therapeutic window with a focus on mutant selective PI3K inhibitors. Finally, I will expound on the translational future of the PI3K field, highlighting advances in biomarker discovery, tumor correlative analysis, and patient advocacy. Citation Format: N. Vasan. PI3K past, present, and future [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr ED10-01.

Abstract PO3-26-01: Preclinical in vitro and in vivo characterization of a novel, wild-type-sparing, PI3Kα H1047R mutant-selective inhibitor

Abstract The PI3K pathway is a key cell cycle regulating pathway that has an established role in tumor growth and development. Specifically, the H1047R and helical domain mutations E542K/E545K of the p110α subunit of PI3K are known activating mutations that are targeted by inhibitors under clinical investigation as well as by approved drugs. PI3Kα mutations are prevalent in patients with breast, colorectal, lung, endometrial, and numerous other cancers. The approved PI3Kα inhibitor, alpelisib, shows promise for this targeted class with improvements in progression-free survival in HR+/Her2- breast cancer in combination with fulvestrant. However, tolerability concerns such as hyperglycemia, gastrointestinal issues, and skin reactions have emerged related to on-target inhibition of wild type PI3Kα. These issues likely limit dose administration leading to exposure which is suboptimal for maximal efficacy and creates an opportunity to identify an inhibitor with an improved tolerability profile that targets oncogenic mutations while sparing wild type PI3Kα. Herein, we present preclinical in vitro and in vivo activity of a novel, wild type sparing PI3Kα inhibitor which is potent against the oncogenic H1047R mutation. Citation Format: Aaron C. Smith, Ben Arwood-Levine, Alexandra Born, Richard Brizendine, Payal Chatterjee, Mark J. Chicarelli, Michael L. Conner, Brad Fell, Jennifer Fulton, Anna Guarnieri, Ravi Jalluri, Hailey Knox, Keith Koch, Daniel Krischlunas, Vijay Kumar, Colin McHugh, Brent Mclean, Kelsey Nassar, Brad Newhouse, Rob Rieger, John Robinson, Marelí Rodriguez, Leah Salituro, Lee Stunkard, Francis Sullivan, Roy Turton, Shannon Winski, Yeyun Zhou. Preclinical in vitro and in vivo characterization of a novel, wild-type-sparing, PI3Kα H1047R mutant-selective inhibitor [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-26-01.

Abstract PO3-25-09: OKI-219, a PI3KαH1047R-mutant-selective inhibitor demonstrates efficacy as a single agent and drives combination responses with standard of care therapies in pre-clinical PI3KαH1047R mutant breast cancer models

Abstract Mutations in PI3Kα lead to constitutive activation of the PI3K/AKT/mTOR pathway and are highly prevalent in cancer. PI3KαH1047R is the most common amino acid substitution mutation and represents approximately one-third of all PI3Ka mutations, with 15% of breast cancers having a PI3KαH1047R mutation. In breast cancer, PI3KαH1047R mutations are associated with resistance to targeted therapies, including HER2- and estrogen receptor (ER)- targeted agents. Although targeting PI3Kα in cancer is a therapeutically proven strategy, with the currently approved drug alpelisib showing clinical efficacy alone or in combination with other therapies, treatment with non-mutant selective PI3Kα inhibitors such as alpelisib is associated with significant toxicities such as hyperglycemia, due to on-target inhibition of the wild-type enzyme. This often leads to dose modification or discontinuation. Therefore, there is a significant need to develop new PI3Kα-targeted therapies that can avoid or minimize on-target toxicity and improve the safety and clinical benefit for patients. OKI-219 is an orally bioavailable, PI3KαH1047R mutant-selective inhibitor, with ~100-fold cellular selectivity for the H1047R mutation over wild-type. OKI-219 demonstrates selectivity across a panel of 412 kinases, with no other kinases showing >30% inhibition at 1 µM. In vitro, OKI-219 drives decreased phosphorylated AKT (pAKT) and decreased proliferation specifically in PI3KαH1047R mutant cell lines across several tumor types, including breast cancer. In vivo, as a monotherapy, OKI-219 demonstrates dose dependent antitumor activity in multiple human tumor xenograft models, including the H1047R mutant breast cancer CDX models T47D and HCC1954 and several PDX models, with regression rates up to 100% at doses that show no sign of the metabolic dysfunction (increased glucose or insulin) associated with wild-type protein inhibition. The in vivo antitumor activity correlates with pathway inhibition, measured by decreased pAKT in tumors. Moreover, OKI-219 dosed in combination with the selective estrogen receptor degraders (SERDs) fulvestrant, elacestrant or camizestrant showed significant combination benefit leading to tumor regressions of up to 100% in the ER+ H1047R mutant breast cancer model xxT47D, at doses where no regressions were observed with single agent treatment. Dosing OKI-219 in combination with HER2-inhibitors, such as tucatinib, led to tumor regressions in the ER-HER2+ and H1047R mutant breast cancer CDX model HCC1954, also at doses where no regressions were observed with single agent treatment. These data indicate that OKI-219 offers improved efficacy and a wider therapeutic window compared to non-mutant selective PI3Kα inhibitors. OKI-219 is advancing into clinical trials. Citation Format: Molly A. Taylor, Qian Zhao, David Mareska, Maria Hoh, Yevgeniy Izrayelit, Kevin Litwiler, Mark L Boys, Rich Woessner, Duncan Walker, James D Winkler, Jennifer Diamond. OKI-219, a PI3KαH1047R-mutant-selective inhibitor demonstrates efficacy as a single agent and drives combination responses with standard of care therapies in pre-clinical PI3KαH1047R mutant breast cancer models [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-25-09.

Abstract ND03: Discovery of RMC-9805, an oral, covalent tri-complex KRASG12D(ON) inhibitor

Abstract RASG12D mutant cancers represent a significant unmet medical need with 55,000 new diagnoses annually in the US. The RASG12D mutation increases the abundance of the active, GTP-bound state of RAS (RASG12D(ON)) and occurs commonly in multiple tumor histotypes, including about 20%, 29% and 17% of RAS mutant colorectal, pancreatic, and non-small cell lung cancers, respectively. RMC-6236, an investigational RASMULTI(ON) inhibitor currently in clinical testing, selectively targets the active, GTP-bound state of both mutant and wild-type RAS variants, including RASG12D(ON) and has shown clinical anti-tumor activity against tumors harboring KRASG12D. Efforts to find a mutant-selective RASG12D(ON) inhibitor led to the discovery of RMC-9805.The investigational agent RMC-9805 is a first-in-class, orally bioavailable, mutant selective covalent inhibitor of RASG12D(ON) that forms a tri-complex between the abundant intracellular chaperone cyclophilin A (CypA) and the “ON” state of RASG12D, enabling selective covalent engagement of Asp-12 and disrupting downstream RAS signaling by steric occlusion of effector binding. In contrast to other examples in the literature of covalent inhibitors of KRASG12D that rely on highly reactive, low stability warheads to overcome the low intrinsic reactivity of aspartic acid, RMC-9805 employs a warhead with low intrinsic reactivity and high stability under biological conditions. Rapid, selective, covalent modification of RASG12D by RMC-9805 is enabled by our tri-complex technology. We used structure-guided design to position the warhead in the CypA-RAS interface in an optimal orientation that enhanced crosslinking of Asp-12 allowing us to use a warhead that showed no intrinsic reactivity to a model aspartic acid system and was stable to enable oral dosing. RMC-9805 drove selective and persistent covalent modification of KRASG12D in human cancer cell lines in vitro, leading to deep and durable suppression of RAS pathway activity, inhibition of cell proliferation, and apoptosis. RMC-9805 monotherapy induced tumor regressions at well-tolerated doses in a majority of preclinical PDAC and NSCLC models harboring KRASG12D. Though a more heterogeneous response was observed in KRASG12D CRC models, combinations of RMC-9805 with either RMC-6236 (RASMULTI(ON) inhibitor) or an anti-EGFR antibody improved the depth of response and delayed the onset of resistance in vivo. In addition, RMC-9805 promoted cancer-associated neoantigen recognition and synergized with immunotherapy in preclinical models. RMC-9805 is also CNS penetrant and drove regressions in intracranial xenograft models of human KRASG12D PDAC. RMC-9805 is a first-in-class orally bioavailable, mutant selective and covalent RASG12D inhibitor currently in Phase 1 clinical trial (NCT06040541). Citation Format: John E. Knox, G. Leslie Burnett, Caroline Weller, Lingyan Jiang, Dongyu Zhang, Nicole Vita, Abby Marquez, Kyle J. Seamon, Andrea Gould, Marie Menard, Elsa Quintana, Zhe Chen, Zhican Wang, Zhengping Wang, Elena S. Koltun, Malika Singh, Jingjing Jiang, David Wildes, Jacqueline A.M. Smith, Adrian L. Gill. Discovery of RMC-9805, an oral, covalent tri-complex KRASG12D(ON) inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr ND03.

Abstract LB449: Strategies to overcome resistance to PI3Kalpha-selective inhibitors mediated by acquired alterations in the PI3K/AKT pathway

Abstract PIK3CA is among the most frequently mutated kinases in multiple types of cancer, including in 40% of hormone receptor-positive, HER2-negative breast cancers. The PI3Kα-selective orthosteric inhibitor alpelisib is the only drug targeting PIK3CA currently approved in this patient population, in combination with fulvestrant. As commonly observed with other targeted therapies, resistance often develops during treatment with PI3Kα-selective inhibitors. In one of the largest patient cohorts to date, including serial liquid biopsies and rapid autopsies in 39 patients, our group has observed genomic alterations within the PI3K/AKT pathway as one of the major mechanisms of resistance to these agents. Among them, we have identified for the first-time secondary mutations in PIK3CA (W780R and Q859K) that decrease the affinity of the PI3Kα-selective inhibitors alpelisib and inavolisib, leading to acquired resistance. Multiple small molecule inhibitors designed to suppress signaling through the PI3K/AKT axis are currently in pre-clinical development. These include AKT inhibitors, isoform-selective as well as pan-PI3K inhibitors, and a novel subclass of allosteric PI3Kα-selective inhibitors designed to preferentially inhibit both kinase (H1047R) and helical (E545K) domain mutant PI3Kα activity. We have introduced all the emergent alterations observed in our patient cohort into breast cancer models and screen how they modify the effect of a structurally diverse array of PI3K/AKT inhibitors. While some acquired mutations compromise the effect of specific drugs, we have determined that the W780R mutation could drive universal resistance to ATP-competitive PI3K inhibitors that bind the catalytic pocket. Importantly, vertical pathway inhibition or allosteric inhibition with pan-mutant-selective PI3Kα inhibitors such as RLY-2608 or STX-478 could overcome resistance induced by all emerging secondary PIK3CA mutations.Dose-limiting toxicity in the form of hyperglycemia, rash or gastrointestinal issues are common in patients treated with orthosteric PI3Kα inhibitors. While mutant selective PI3Kα inhibitors induce less adverse effects in patients, they have a decreased potency targeting wildtype PIK3CA. We have identified wildtype PIK3CA-mediated feedback pathway reactivation as a potential mechanism of resistance to this type of inhibitors. Our work suggests that combining orthosteric and allosteric PI3Kα inhibitors as well as downstream pathway deactivation with AKT inhibitors could result in an increased therapeutic index and delay the emergence of resistance in patients. This work provides an insightful characterization of the current knowledge on clinical acquired resistance to PI3Kα inhibitors and proposes a detailed strategy to overcome resistance mediated by most PI3K/AKT alterations described to date. Citation Format: Ferran Fece de la Cruz, Andreas Varkaris, Elizabeth E. Martin, Bryanna L. Norden, Nicholas Chevalier, Allison M. Kehlmann, Ignaty Leshchiner, Haley Barnes, Sara Ehnstrom, Parasvi Patel, Janice S. Kim, Haley Ellis, Ioannis Sanidas, Kayao T. Lau, Aditya Bardia, Laura M. Spring, Steven J. Isakoff, Jochen K. Lennerz, Gad Getz, Ryan B. Corcoran, Dejan Juric. Strategies to overcome resistance to PI3Kalpha-selective inhibitors mediated by acquired alterations in the PI3K/AKT pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB449.

Abstract LB173: Discovery of ALTA-2618, the first allosteric, mutant-selective targeted therapy for AKT1 E17K driven cancers

Abstract BACKGROUND: AKT1 E17K is a clinically validated oncogenic driver mutated in ~1-2% of all cancers and is enriched in several solid tumors including breast (~5%), endometrial (~3%) and prostate (~1.5%) cancer. Over half of AKT1 E17K mutant breast and gynecologic cancers are homozygous for the mutant oncogene with loss of the wildtype (WT) AKT1 allele via copy-neutral loss-of-heterozygosity (CN-LOH), suggesting uniquely potent oncogene addiction in these tumor types. While inhibitors of WT PI3K or AKT have demonstrated clinical efficacy in HR+/HER2- metastatic breast cancers with PI3K/AKT pathway mutations, on-target adverse events - most commonly hyperglycemia, diarrhea, and rash - have hindered the tolerability and durability of these agents. We aimed to exploit the mutant lysine of the E17K variant to develop a covalent, highly mutant-selective inhibitor that would avoid the toxicities associated with WT AKT inhibition. METHODS: Structure-based design was guided by proprietary co-crystal structures, enabling a refined pharmacophore consensus model, which fueled novel small molecule covalent design iterations using an in-house computational chemistry cluster. Lead molecules were identified from cellular signaling assays assessing pAKT S473 suppression and selectivity over WT AKT1 and AKT2. Rapid PK assessments were performed in mice, rats, dogs, and cynomolgus monkeys. Molecules with low clearance and high oral bioavailability were advanced directly to breast and endometrial PDX models driven by AKT1 E17K mutations. RESULTS: We identified ALTA-2618, a potent (7nM EC50) inhibitor of AKT1 E17K with 22x selectivity over WT AKT1 and 140x selectivity over WT AKT2. ALTA-2618 and related analogues demonstrated covalent attachment to the AKT1 E17K protein via intact mass spectrometry, and electron density analysis of co-crystal structures of highly similar family members of ALTA-2618 reveals unambiguous covalent attachment to the K17 side chain. Based on favorable exposure-response relationships and ideal cross-species PK, ALTA-2618 was tested in once daily oral dosing and induced tumor regressions in AKT1 E17K mutant HR+/HER2low breast cancer, TNBC, and endometrial cancer PDXs at doses as low as 10 mg/kg/d. Complete responses were observed with 30 mg/kg/d in the HR+/HER2low breast PDX model that were sustained for 60 days of dosing without significant body weight loss or hyperglycemia. CONCLUSIONS: ALTA-2618 is a potent, highly mutant-selective AKT1 E17K inhibitor that demonstrates favorable PK and tolerability with oral once-daily dosing that enables prolonged tumor regressions in multiple AKT1 E17K-mutant PDX models. These data support a mid-2024 IND submission to investigate ALTA-2618 in patients with AKT1 E17K mutant cancers. Citation Format: Eric A. Murphy, Michael D. Bartberger, Maureen Ibanez, Troy T. Smith, Nicole Alvarez, Noel Timple, Xuefeng Zhu, Kevin Fan, Kevin Yu. Discovery of ALTA-2618, the first allosteric, mutant-selective targeted therapy for AKT1 E17K driven cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB173.

3D molecular generative framework for interaction-guided drug design

Deep generative modeling has a strong potential to accelerate drug design. However, existing generative models often face challenges in generalization due to limited data, leading to less innovative designs with often unfavorable interactions for unseen target proteins. To address these issues, we propose an interaction-aware 3D molecular generative framework that enables interaction-guided drug design inside target binding pockets. By leveraging universal patterns of protein-ligand interactions as prior knowledge, our model can achieve high generalizability with limited experimental data. Its performance has been comprehensively assessed by analyzing generated ligands for unseen targets in terms of binding pose stability, affinity, geometric patterns, diversity, and novelty. Moreover, the effective design of potential mutant-selective inhibitors demonstrates the applicability of our approach to structure-based drug design.

Abstract 3319: The MTA-cooperative PRMT5 inhibitor, MRTX1719, demonstrates increased anti-tumor activity in combination with KRAS mutant-selective inhibitors in MTAP del,KRAS-mutant cancers

Abstract MRTX1719 is an MTA-cooperative PRMT5 inhibitor that leverages the increased concentration of the metabolite MTA in cancer cells harboring a homozygous deletion of the MTAP gene (MTAP del) under investigation in clincial trials. MRTX1719 preferentially binds to the PRMT5•MTA complex to selectively inhibit PRMT5, an essential gene for all cells, in MTAP del cancer cells while sparing PRMT5 activity in normal MTAP-wildtype cells. MTAP del occurs in ~10% of all cancers, and these patients exhibit remarkably poor survival. Among pancreatic cancer, MTAP del occurs in ~ 25% of patients and of these ~30% have co-occurring KRAS G12D mutations. In addition, ~15% of lung adenocarcinoma patients harbor MTAP del and of these ~13% harbor KRAS G12C. While recently approved KRAS G12C mutant selective inhibitors demonstrate anti-tumor activity, strategies to address drug resistance and maximize clinical benefit are warranted. Combination of MRTX1719 with adagrasib or the KRAS G12D inhibitor MRTX1133 in CDX models harboring both a KRAS mutation (G12C or G12D) and homozygous MTAP deletion resulted in increased anti-tumor activity compared to either single agent. This includes the pancreatic KP4 model in which initial tumor regression in response to single agent MRTX1133 was observed, followed by adaptative resistance and tumor progression with monotherapy while co-administration with MRTX1719 continued to result in marked tumor regression. Mechanistically, combination treated animals demonstrated strong inhibition of both PRMT5 and RAS pathway signaling and combinatorial targeting of these pathways converged on enhanced inhibition of RB-1 phosphorylation. We also characterized this combination treatment in the context of resistance to KRAS selective inhibitors or MRTX1719 to evaluate the benefit of combination over sequential treatment. Finally, an NF1 mutant, MTAP del malignant peripheral nerve sheath tumor (MPNST) PDX model was treated with MRTX1719, the SOS1 inhibitor MRTX0902, or the combination, and a similar increase in anti-tumor activity was observed in the combination treatment compared to either monotherapy alone. These results suggest that the combination of an MTA-cooperative PRMT5 inhibitor with a KRAS mutant selective or KRAS pathway inhibitor may lead to deeper and more durable responses in MTAP del cancer patients with co-alterations in the KRAS pathway. Citation Format: Laura Waters, Ruth Aranda, Xousaen Helu, Laura Vegar, Krystal Moya, Andrew Calinisan, Allan Hebbert, Jill Hallin, Darin Vanderpool, David M. Briere, James G. Christensen, Peter A. Olson, Lars D. Engstrom. The MTA-cooperative PRMT5 inhibitor, MRTX1719, demonstrates increased anti-tumor activity in combination with KRAS mutant-selective inhibitors in MTAP del,KRAS-mutant cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3319.

Abstract 4588: Combination of MTA-cooperative PRMT5 inhibitor and direct mutant-selective KRAS inhibitors as a novel therapeutic approach for MTAP-deficient pancreatic cancer

Abstract KRAS is mutationally activated in 95% of pancreatic ductal adenocarcinoma (PDAC) patients. Direct KRAS inhibitors are under intense preclinical and clinical development, with two KRASG12C mutant-selective inhibitors (G12Ci) now approved. However, treatment-associated resistance to KRAS inhibitors has been reported in the clinic highlighting an urgent need to identify both additional therapeutic targets and novel combination treatment strategies with KRAS inhibitors. To that end, we performed a CRISPR/Cas9 loss-of-function screen using a library targeting ~2,500 druggable genes in KRASG12C/D-mutant PDAC cell lines. We identified multiple genes that were essential for PDAC cell growth and selected PRMT5, a protein arginine methyltransferase, to assess as a novel therapeutic target for pancreatic cancer. We found that suppression of PRMT5 activity using two distinct first-generation clinical candidate small molecule inhibitors (JNJ-64619178 and GSK3326595) demonstrated single agent activity and reduced PDAC cell growth. We further assessed a mechanistically distinct MTA-cooperative clinical candidate PRMT5 inhibitor, MRTX1719, that is selective for MTAP-deleted tumors, which occur in ~25% of PDAC patients. We determined that MRTX1719 exhibited low nanomolar GI50 activities in MTAP-deficient but not MTAP wild-type KRAS-mutant cell lines. Unexpectedly, PDAC cells treated with MRTX1719 exhibited a paradoxical activation of ERK, suggesting that combined inhibition of PRMT5 and KRAS might be therapeutically advantageous. In support of our hypothesis, we demonstrated that combination treatment with MRTX1719 and mutant selective KRAS inhibitors (G12Ci/MRTX849 and G12Di/MRTX1133) further sensitized KRASG12C/D-mutant PDAC cells to KRAS inhibition in short- and long-term growth assays as well as in vivo xenograft studies. Our ongoing studies are evaluating the consequences of co-targeting PRMT5 and KRAS on gene expression changes using RNA-Seq and cancer cell signaling pathways using RPPA analyses. In summary, our data support concurrent inhibition of PRMT5 and KRAS as a promising therapeutic strategy for MTAP-deficient KRAS-mutant pancreatic cancer. Citation Format: Kristina Drizyte-Miller, Wen-Hsuan Chang, Andrew M. Waters, Clint A. Stalnecker, Junning Wang, Andrew J. Aguirre, Adrienne D. Cox, Channing J. Der. Combination of MTA-cooperative PRMT5 inhibitor and direct mutant-selective KRAS inhibitors as a novel therapeutic approach for MTAP-deficient pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4588.

Abstract 1956: Discovery of a novel and potent mutant-selective inhibitor of PI3Kα

Abstract Gain-of-function mutations in hotspots of PI3Kα including H1047R, E545K, and E542K are well-recognized oncogenic drivers in human cancers. Prevalence of these mutations is enriched in many human malignancies, particularly in hormone receptor-positive/human epidermal growth factor receptor 2-negative (HR+/HER2−) breast cancer. PI3Kα inhibition showed clear clinical benefit in treating HR+/HER2− breast cancer and has been approved in combination with fulvestrant. First generation PI3Kα inhibitors such as alpelisib and inavolisib, however, have limited selectivity against wild-type (WT) PI3Kα; therefore, are associated with a variety of on-target adverse effects including grade 3 hyperglycemia, skin rash, and gastrointestinal toxicities. Discovery of a highly-selective inhibitor of mutant PI3Kα could enable deeper target coverage while reducing undesired effects of inhibiting WT PI3Kα. SNV-002 is a novel, proprietary, and mutant-selective inhibitor of PI3Kα. In cellular assays, SNV-002 inhibited Ser473 phosphorylation of AKT (pAKT) with single digit nanomolar potency in PI3Kα H1047R-mutant T47D cells and displayed greater than 50-fold selectivity over SKBR3 WT control. Similar inhibition of pAKT was also observed in other cell lines carrying PI3Kα hotspot mutations, such as CAL33, NCI-H1048, EFM19, and HCC1954. SNV-002 maintained selectivity in helical domain E545K-mutant cell lines including MCF7 and MDA-MB-361. This selectivity was further confirmed using MCF10A isogenic cell lines carrying either WT or mutant PI3Kα (H1047R and E545K). In cellular assays to assess inhibition of other isoforms of PI3K (PI3Kβ, PI3Kγ, and PI3Kδ), the IC50 of SNV-002 was over 10,000 nM. Similarly, a broad kinome screen showed no inhibition of activity of 371 kinases. Compared with alpelisib, SNV-002 showed improved potency and prolonged residence time in T47D breast cancer cells. Importantly, assays using H1047R-mutant cells in the presence of human whole blood showed SNV-002 potency to be less than 100 nM, which was over 10-fold more potent than that of alpelisib. Treatment of breast cancer cells with SNV-002 suppressed PI3K-AKT-mTOR pathway, as evidenced by reduced levels of phospho-S6 ribosomal protein and associated with loss of cell viability in mutant lines. The pharmacokinetic profile of SNV-002 was excellent across multiple species. Oral administration of HCC1954 tumor-bearing mice with SNV-002 resulted in dose-proportional exposure and >90% pharmacodynamic inhibition of pAKT in the tumor with doses of 10 mg/kg, and no sign of glucose dysregulation in contrast to alpelisib. Together, these data show that SNV-002 is a highly-selective and potent inhibitor of mutant PI3Kα with good drug-like properties that support further development of SNV-002 for treatment of cancers driven by oncogenic PI3Kα mutations. Citation Format: Mingming Gao, Chao Qi, Zhentian Li, Qipeng Fan, Liyang Wang, Fei Zhou, Lin You, Hewen Zheng, Yu Li, Liangxing Wu, Wenqing Yao, Phillip C. Liu. Discovery of a novel and potent mutant-selective inhibitor of PI3Kα [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1956.

Discovery of Pyridopyrimidinones that Selectively Inhibit the H1047R PI3Kα Mutant Protein.

The H1047R mutation of PIK3CA is highly prevalent in breast cancers and other solid tumors. Selectively targeting PI3KαH1047R over PI3KαWT is crucial due to the role that PI3KαWT plays in normal cellular processes, including glucose homeostasis. Currently, only one PI3KαH1047R-selective inhibitor has progressed into clinical trials, while three pan mutant (H1047R, H1047L, H1047Y, E542K, and E545K) selective PI3Kα inhibitors have also reached the clinical stage. Herein, we report the design and discovery of a series of pyridopyrimidinones that inhibit PI3KαH1047R with high selectivity over PI3KαWT, resulting in the discovery of compound 17. When dosed in the HCC1954 tumor model in mice, 17 provided tumor regressions and a clear pharmacodynamic response. X-ray cocrystal structures from several PI3Kα inhibitors were obtained, revealing three distinct binding modes within PI3KαH1047R including a previously reported cryptic pocket in the C-terminus of the kinase domain wherein we observe a ligand-induced interaction with Arg1047.

G2019S selective LRRK2 kinase inhibitor abrogates mitochondrial DNA damage

Pathogenic mutations in LRRK2 cause Parkinson’s disease (PD). The G2019S variant is the most common, which results in abnormally high kinase activity. Compounds that target LRRK2 kinase activity are currently being developed and tested in clinical trials. We recently found that G2019S LRRK2 causes mitochondrial DNA (mtDNA) damage and treatment with multiple classes of LRRK2 kinase inhibitors at concentrations associated with dephosphorylation of LRRK2 reversed mtDNA damage to healthy control levels. Because maintaining the normal function of LRRK2 in heterozygous G2019S LRRK2 carriers while specifically targeting the G2019S LRRK2 activity could have an advantageous safety profile, we explored the efficacy of a G2019S mutant selective LRRK2 inhibitor to reverse mtDNA damage in G2019S LRRK2 models and patient cells relative to non-selective LRRK2 inhibitors. Potency of LRRK2 kinase inhibition by EB-42168, a G2019S mutant LRRK2 kinase inhibitor, and MLi-2, a non-selective inhibitor, was determined by measuring phosphorylation of LRRK2 at Ser935 and/or Ser1292 using quantitative western immunoblot analysis. The Mito DNADX assay, which allows for the accurate real-time quantification of mtDNA damage in a 96-well platform, was performed in parallel. We confirmed that EB-42168 selectively inhibits LRRK2 phosphorylation on G2019S LRRK2 relative to wild-type LRRK2. On the other hand, MLi-2 was equipotent for wild-type and G2019S LRRK2. Acute treatment with EB-42168 inhibited LRRK2 phosphorylation and also restored mtDNA damage to healthy control levels. We further investigated the relationship between LRRK2 kinase activity, mtDNA damage and mitophagy. Levels of mtDNA damage caused by G2019S LRRK2 were fully re-established within 2 h of a LRRK2 inhibitor wash out and recovery experiment, indicating the mtDNA damage phenotype is highly dynamic. G2019S LRRK2 mitophagy defects were not alleviated with LRRK2 kinase inhibition, suggesting that mitophagy is not mechanistically regulating LRRK2 kinase-mediated reversal of mtDNA damage in this acute timeframe. Abrogation of mtDNA damage with the mutant selective tool inhibitor EB-42168 demonstrates the potential of a precision medicine approach for LRRK2 G2019S PD. Levels of mtDNA damage may serve as a potential pharmacodynamic biomarker of altered kinase activity that could be useful for small molecule development and clinical trials.

Abstract B137: Selective inhibition of the active state of KRASG12V with the non-covalent, tri-complex inhibitor RM-048

Abstract The KRASG12V mutation is the second most common oncogenic RAS mutation and is frequently observed in pancreatic, lung, and colorectal cancers. Developing a selective inhibitor of KRASG12V presents significant drug discovery challenges. The intrinsic GTP hydrolysis rate of KRASG12V is about 12-fold lower than that of KRASG12C, further biasing the cellular KRASG12V pool to the active, GTP-bound (“RAS(ON)”) state and emphasizing the importance of targeting the KRASG12V(ON) state for maximal suppression of this oncogenic driver. Additionally, achieving selectivity over wild-type RAS with small molecules is difficult because the valine residue introduced by KRASG12V is neither amenable to covalent inhibition nor to formation of polar, non-covalent interactions. We have previously applied our tri-complex inhibitor platform, which uses chemical re-remodeling of the cellular chaperone cyclophilin A (CypA) in order to bind to undruggable surfaces, to design mutant-selective inhibitors targeting KRASG12C, KRASG13C, KRASG12D, and KRASQ61H, in addition to the RASMULTI inhibitor RMC-6236. Here, we describe RM-048, a potent, selective, and oral tri-complex inhibitor of KRASG12V(ON). RM-048 binds to CypA with high affinity to form a binary complex. Binding to CypA creates a neomorphic interface that forms a selective, high-affinity, non-covalent interaction with KRASG12V(ON). The resulting tri-complex sterically blocks effector binding to KRASG12V(ON), thereby inhibiting downstream signaling. RM-048 potently suppressed ERK phosphorylation and proliferation in KRASG12V mutant cancer cells. In preclinical species, RM-048 showed good bioavailability, dose-proportional exposure, and low clearance, allowing for oral dosing. In human xenograft tumors harboring KRASG12V mutations, a single dose of RM-048 induced dose-dependent, deep, and durable suppression of RAS pathway signaling in vivo. Repeated daily oral administration of RM-048 was well tolerated and demonstrated profound anti-tumor activity, driving tumor regressions across a panel of KRASG12V xenograft models, including NSCLC, PDAC, and CRC models. RM-048 complements the mutant-selective tri-complex KRAS(ON) inhibitors RMC-6291 (KRASG12C) and RMC-9805 (KRASG12D) by potentially providing a mutant-selective inhibitor option for patients with tumors harboring one of the most frequent RAS mutations. Preclinical combination studies with RAS companion inhibitors, including the RASMULTI inhibitor RMC-6236, are ongoing. Citation Format: Bianca J Lee, Jim Cregg, Anne Edwards, Jerry Chen, Andre Eriksson, Emily Tonogai, Aidan Tomlinson, Kyle Seamon, Mariela Moreno Ayala, Nataliya Tovbis Shifrin, Nilufar Montazer, Kate Sanders, Jun Huang, Kang-Jye Chou, Benjamin Madej, Yang Liu, Zhican Wang, Zhengping Wang, John Knox, Elena Koltun, Jingjing Jiang, Adrian Gill, Jacqueline A.M. Smith. Selective inhibition of the active state of KRASG12V with the non-covalent, tri-complex inhibitor RM-048 [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 B137.

Abstract A090: Preclinical efficacy of BDTX-4933, a brain-penetrant, orthosteric RAF inhibitor, targeting oncogenic RAF conformation shared by groups of BRAF and upstream driver mutations

Abstract Alterations in the RAS-MAPK pathway, such as mutations in NF1, RAS, and BRAF, often lead to oncogenic signaling and result in aberrant cell proliferation and tumor growth. Oncogenic BRAF mutations may be activated as either monomers or dimers, while oncogenic RAS mutations or loss-of-function mutations in NF1, a negative regulator of RAS, drive activation of RAF dimers to transduce MAPK signaling. Currently approved BRAF inhibitors are selective against monomeric BRAF mutations and largely inactive against dimeric RAF mutants. Additionally, these agents may also promote deleterious paradoxical activation of RAF in select contexts. Moreover, FDA-approved KRAS G12C mutant-selective inhibitors are ineffective for cancer patients who harbor other (non-G12C) KRAS, NRAS, and NF1 mutations which all lead to active RAF dimer. There remains a high unmet clinical need for a highly CNS penetrant RAF inhibitor that targets a broad spectrum of oncogenic RAF conformations in the context of RAF, RAS, and other upstream driver mutations. BDTX-4933 is a next generation MasterKey RAF inhibitor designed to address the shortcomings of previous generation RAF inhibitors. BDTX-4933 is an orthosteric inhibitor that potently targets the oncogenic RAF conformation shared by groups of RAF driver mutations and induces an inactive conformation of RAF. As a MasterKey inhibitor, BDTX-4933 potently inhibits a broad spectrum of BRAF mutations and active RAF dimers promoted by upstream oncogenic MAPK pathway alterations, such as KRAS, NRAS, and NF1 mutations. In vivo, once daily oral dosing of BDTX-4933 resulted in robust and sustained target engagement, inhibiting ERK phosphorylation without inducing paradoxical activation. In a mouse breadth of efficacy study with patient derived xenografts (PDX), BDTX-4933 demonstrated tumor growth inhibition and regression as a single agent across models expressing a variety of targeted BRAF or MAPK pathway oncogenic mutations. Additionally, BDTX-4933 as a single agent and in combination with a MEK inhibitor, demonstrates anti-tumor efficacy in KRAS mutant lung PDX models. In a PDX model of BRAF V600E positive tumor that progressed following BRAF and MEK combination therapy, BDTX-4933 achieved robust tumor growth regression. Furthermore, BDTX-4933 exhibits high CNS exposure leading to dose-dependent tumor growth inhibition, and survival benefit in mice implanted intracranially with xenograft BRAF mutant tumors. BDTX-4933 has a best-in-class RAF inhibitor profile to treat cancer patients harboring BRAF mutations or RAF dimer-promoting upstream genetic alterations. BDTX-4933 is currently in a Phase I clinical study in patients with solid tumor harboring sensitive BRAF and RAS alterations (NCT05786924). For more information on this active Phase 1, please contact: ClinicalTrials@bdtx.com. Citation Format: Yoon-Chi Han, Pui Yee Ng, Luisa Shin Ogawa, Shao Ning Yang, Miao Chen, Darlene Romashko, Tai-An Lin, Elizabeth Buck. Preclinical efficacy of BDTX-4933, a brain-penetrant, orthosteric RAF inhibitor, targeting oncogenic RAF conformation shared by groups of BRAF and upstream driver mutations [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 A090.

Abstract B163: Discovery and characterization of OKI-219, an orally bioavailable H1047R-mutant-selective inhibitor of PI3Kα

Abstract Mutations in PI3Kα lead to constitutive activation of the PI3K/AKT/mTOR pathway and are found in approximately 13% of human cancers, with the PI3KαH1047R mutation being the most common. Targeting PI3Kα in cancer is a therapeutically proven strategy, with the currently approved drug alpelisib showing clinical efficacy alone or in combination with other therapies. However, treatment with non-mutant selective PI3Kα inhibitors, such as alpelisib, is associated with significant toxicities such as hyperglycemia due to on-target inhibition of the wild-type enzyme, which often leads to dose modification or discontinuation. Therefore, there is a significant need to develop new PI3Kα-targeted therapies that can avoid or minimize on-target toxicity and improve the safety and clinical benefit for patients. OKI-219 is a PI3KαH1047R mutant-selective inhibitor, with ~100-fold cellular selectivity for the H1047R mutation over wild-type and biochemical selectivity across a 412 kinase panel, with no other kinases showing >30% inhibition at 1 µM. In vitro, OKI-219 drives decreased phosphorylated AKT (pAKT) and decreased proliferation selectively in PI3KαH1047R mutant cell lines across several tumor types. In vivo, as a monotherapy, OKI-219 demonstrates dose-dependent antitumor activity in multiple human CDX and PDX tumor xenograft models, with regression rates up to 100% in PI3KαH1047R heterozygous (T47D) and homozygous (SUM185PE) CDX models, at doses that show no sign of the metabolic dysfunction (increased glucose or insulin) that is associated with wild-type protein inhibition. The in vivo antitumor activity correlates with pathway inhibition, measured by decreased pAKT in tumors. In addition to the monotherapy efficacy observed, OKI-219 in combination with the selective estrogen receptor degrader (SERD) fulvestrant showed significant combination benefit leading to tumor regressions in the H1047R-mutated ER+HER2- breast cancer model xxT47D, at doses where no regressions were observed with single agent treatment. These data support the hypothesis that OKI-219 will offer improved efficacy and a wider therapeutic window compared to non-mutant selective PI3Kα inhibitors. This hypothesis will be tested, as OKI-219 is moving into clinical investigation. Citation Format: Molly A Taylor, Qian Zhao, David Mareska, Maria Hoh, Yevgeniy Izrayelit, Kevin Litwiler, Mark L Boys, Rich Woessner, Duncan Walker, Jennifer Diamond, James D Winkler. Discovery and characterization of OKI-219, an orally bioavailable H1047R-mutant-selective inhibitor of PI3Kα [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 B163.

Discovery and Clinical Proof-of-Concept of RLY-2608, a First-in-Class Mutant-Selective Allosteric PI3Kα Inhibitor That Decouples Antitumor Activity from Hyperinsulinemia.

Treatments for PIK3CA-mutant cancers are limited by toxicities associated with the inhibition of WT PI3Kα. Molecular dynamics, cryo-electron microscopy, and DNA-encoded libraries were used to develop RLY-2608, a first-in-class inhibitor that demonstrates mutant selectivity in patients. This marks the advance of clinical mutant-selective inhibition that overcomes limitations of orthosteric PI3Kα inhibitors. See related commentary by Gong and Vanhaesebroeck, p. 204 . See related article by Varkaris et al., p. 227 . This article is featured in Selected Articles from This Issue, p. 201.

Phase 1 study to evaluate the effects of rifampin or itraconazole on the pharmacokinetics of limertinib (ASK120067), a novel mutant-selective inhibitor of the epidermal growth factor receptor in healthy Chinese subjects

ABSTRACT Background Limertinib is a novel mutant-selective and irreversible inhibitor of the epidermal growth factor receptor under development. A phase 1 open, two-period, single-sequence, self-controlled, two-part study was initiated to characterize the effects of a strong CYP3A4 inducer (rifampin) or inhibitor (itraconazole) on the pharmacokinetics of limertinib. Research design and methods Twenty-four healthy subjects in each part received a single dose of limertinib alone (160 mg, Part A; 80 mg, Part B) and with multiple doses of rifampin 600 mg once daily (Part A) or itraconazole 200 mg twice daily (Part B). Results Coadministration of rifampin decreased exposure (area under the plasma concentration-time curve from time 0 to infinity, AUC0-inf) of limertinib and its active metabolite CCB4580030 by 87.86% (geometric least-squares mean [GLSM] ratio, 12.14%; 90% confidence interval [CI], 9.89–14.92) and 66.82% (GLSM ratio, 33.18%; 90% CI, 27.72–39.72), respectively. Coadministration of itraconazole increased the AUC0-inf of limertinib by 289.8% (GLSM ratio, 389.8%; 90% CI, 334.07–454.82), but decreased that of CCB4580030 by 35.96% (GLSM ratio, 64.04%; 90% CI, 50.78–80.77). Conclusions Our study demonstrates that the concomitant use of limertinib with strong CYP3A inducers or inhibitors is not recommended. A single dose of limertinib, administered with or without rifampin or itraconazole, is generally safe and well tolerated in healthy Chinese subjects. Clinical trial registration www.clinicaltrials.gov identifier is NCT05631678.

CBF-Beta Mitigates PI3K-Alpha-Specific Inhibitor Killing through PIM1 in PIK3CA-Mutant Gastric Cancer.

Loss of either NEDD9 or BCL-XL confers hypersensitivity to PI3K-alpha inhibition whereas loss of CBFB confers resistance through a CBFB/PIM1 signaling axis.