Phosphoinositide 3-kinase alpha (PI3Kα) is the most frequently mutated kinase in solid tumors. Traditionally, the development of PI3Kα inhibitors has focused on the active, or orthosteric site. The therapeutic index of orthosteric inhibitors is limited by the lack of clinically meaningful selectivity for mutant versus wild-type (WT) PI3Kα and off-isoform activity. Alpelisib, the only approved orthosteric PI3Kα inhibitor, is emblematic of the class with toxicity related to inhibition of WT PI3Kα and other PI3K isoforms resulting in sub-optimal inhibition of mutant PI3Kα with reductions in dose intensity and frequent discontinuation. To overcome these limitations, we designed RLY-2608, the first allosteric, mutant, and isoform-selective PI3Kα inhibitor. We solved the full-length cryo-EM structure of PI3Kα, performed long time-scale molecular dynamic simulations to elucidate conformational differences between WT and mutant PI3Kα, and leveraged these insights to enable the design of RLY-2608. RLY-2608 does not compete with orthosteric inhibitors for binding and associates 8x faster with mutant PI3Kα relative to WT. In biochemical assays, RLY-2608 inhibits both kinase domain (H1047R) and helical domain (E542K, and E545K) mutant PI3Kα activity with <10nM potency and 8-12x selectivity relative to WT PI3Kα. RLY-2608 is > 1000-fold selective over the β, δ, and γ PI3K isoforms in biochemical assays and demonstrates exquisite selectivity across a panel of 322 kinases, with no other kinases showing > 50% inhibition. In MCF10A cells engineered to express only mutant or WT PI3Kα, RLY-2608 inhibited phosphorylated AKT (pAKT) in a mutant-selective manner. Furthermore, pAKT and viability were significantly inhibited across a panel of cancer cell lines carrying hotspot PIK3CA mutations. RLY-2608 showed anti-tumor activity in both kinase and helical domain PIK3CA mutant in vivo xenograft models with marked regressions or stasis observed in all models. RLY-2608 was well tolerated, with pharmacodynamic modulation and efficacy observed in a dose dependent manner. Insulin levels measured as an indicator of glucose homeostasis were significantly lower when compared to orthosteric inhibitors, suggesting that RLY-2608 can achieve maximum efficacy by maintaining PI3Kα mutant target coverage throughout the dosing interval with significantly reduced impact on WT PI3Kα. In higher species, dosing of RLY-2608 for 28 days resulted in exposures exceeding mutant PI3Kα cellular PD IC90 throughout the dosing interval without elevated glucose levels or histopathological or ophthalmic findings associated with hyperglycemia. Compared to orthosteric inhibitors, RLY-2608 demonstrates preferential binding and inhibition of mutant PI3Kα, is highly selective across the kinome, and achieves in vivo efficacy without dysregulating glucose homeostasis. These results support clinical investigation of RLY-2608 as a differentiated mechanism for inhibition of oncogenic PI3Kα in patients with PIK3CA mutant tumors. Citation Format: Ermira Pazolli, Randy Kipp, Alessandro Boezio, Hakan Gunaydin, Amanda Iskandar, Matthew Zubrowski, Bret Williams, Kelley Shortsleeves, Alexandre Larivee, Tom McLean, Klaus Michelsen, Hongtao Zeng, Jonathan LaRochelle, Joe Manna, Lucian DiPietro, Mary Mader, Bindu Bennet, Jeremy Wilbur, Qi Wang, Levi Pierce, Iain Martin, James Watters, Pascal Fortin, Donald Bergstrom. Discovery and characterization of RLY-2608: The first allosteric, mutant, and isoform-selective inhibitor of PI3Kα [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P251.
The non-receptor protein tyrosine phosphatase SHP2 (PTPN11) plays an important role in the regulation of RAS/MAPK signal transduction downstream of growth factor receptor activation. Loss of SHP2 activity suppresses tumor cell growth, making SHP2 a potential target for cancer therapy. Here we report the discovery of GDC-1971 (formerly RLY-1971), a highly potent, selective, and orally bioavailable small-molecule SHP2 inhibitor that stabilizes SHP2 in a closed, auto-inhibited conformation. GDC-1971 potently inhibits both wild-type SHP2 (IC50 <1nM) and the E76K activating mutant (IC50 <250nM) in biochemical assays. In standard 2-dimensional and anchorage-independent growth conditions, GDC-1971 inhibits cellular proliferation in models harboring receptor tyrosine kinases (RTKs), SHP2, NF1, KRAS, or BRAF mutations in a dose-dependent manner. GDC-1971 potently inhibits the proliferation of cellular models harboring KRAS G12C or G12A mutations (median IC50 <80 nM) compared to models harboring other KRAS G12, G13 or Q61 mutations (median IC50 >1 uM), indicating a link between KRAS GTP hydrolysis and SHP2 dependency. Despite this trend, some non-KRAS G12C or G12A cell lines harboring other KRAS mutations responded to GDC-1971 in vitro, suggesting some heterogeneity in RTK/SHP2 signaling dependence in subsets of other KRAS mutants. In vivo, GDC-1971 demonstrates dose-dependent RAS/MAPK pathway inhibition and induces significant tumor-growth inhibition in human xenograft models harboring EGFR and KRAS alterations at continuous daily doses that are well tolerated. Given the reported role of SHP2 as a critical mediator of resistance to targeted therapies, we assessed the activity of GDC-1971 combinations in multiple contexts. We observed increased suppression of the MAPK signaling cascade and anti-proliferation synergy when combining GDC-1971 with EGFR, ALK, and KRAS G12C inhibitors in vitro. The observed in vitro synergy translated to dramatic anti-tumor growth effects in vivo. GDC-1971 in combination with the KRAS G12C covalent inhibitor GDC-6036 resulted in significant regressions at doses well below those required for single agent activity in a KRAS G12C-mutant NSCLC xenograft model. In rodent and dog toxicology studies, GDC-1971 is well tolerated at exposures above those required to induce regression in xenograft models. The biochemical and cellular potency and favorable pharmaceutical properties of GDC-1971 support the further clinical development in RTK/MAPK pathway altered tumors using continuous daily dosing alone and in combination with other targeted agents, including the KRAS-G12C inhibitor GDC-6036 (clinical trial NCT04449874). Citation Format: Bret Williams, Alexander Taylor, Olivia Orozco, Christopher Owen, Elizabeth Kelley, Andre Lescarbeau, Kelley Shortsleeves, Randy Kipp, Vy Nguyen, Erin Brophy, Jeremy Wilbur, Yong Tang, David Lanzetta, Nigel Waters, Sherri Smith, Fabrizio Giordanetto, Paul Maragakis, Jack Greismann, Lindsay Willmore, Eric Therrien, Yang Xiao, Marie Evangelista, Luca Gerosa, Eva Lin, Mark Merchant, Alfonso Arrazate, Emily Chan, Pablo Sáenz-López Larrocha, Stefan Chun, Thomas Hunsaker, Gauri Deshmukh, Christine M. Bowman, David E. Shaw, Mark Murcko, Mahesh Padval, W Patrick Walters, James Watters, Donald A. Bergstrom. Discovery and characterization of the potent, allosteric SHP2 inhibitor GDC-1971 for the treatment of RTK/RAS driven 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 3327.
Inhibition of CDK4/6 combined with the estrogen receptor (ER) degrader fulvestrant significantly improves progression free survival and overall survival in advanced hormone receptor positive (HR+) breast cancer patients and is now the standard of care (SOC) in this disease. Up to 40% of HR+ breast cancers harbor PIK3CA mutations leading to activation of phosphoinositide 3-kinase alpha (PI3Kα), which has been associated with resistance to CDK4/6 inhibitors and fulvestrant. Therefore, PI3Kα inhibitor combinations with CDK4/6 inhibitors and/or fulvestrant are of high interest in HR+, PIK3CA mutant breast cancer. The therapeutic index of active site (orthosteric) inhibitors of PI3Kα has been limited by the dual issues of no clinically meaningful selectivity for mutant versus wild-type (WT) PI3Kα and off-isoform inhibitory activity. Alpelisib, the only approved orthosteric PI3Kα inhibitor, is emblematic of the class with toxicity related to inhibition of wild type PI3Kα and other PI3K isoforms resulting in sub-optimal inhibition of mutant PI3Kα, frequent discontinuation, and challenges in combining with CDK4/6 inhibitors. To overcome these limitations, we designed RLY-2608, the first allosteric, mutant, and isoform-selective inhibitor of PI3Kα. We solved the full-length cryo-EM structure of PI3Kα, performed long time-scale molecular dynamic simulations to elucidate conformational differences between WT and mutant PI3Kα, and leveraged these insights to enable the design of RLY-2608. RLY-2608 does not compete with orthosteric inhibitors for binding and associates 8x faster with mutant PI3Kα relative to WT PI3Kα. In biochemical assays, RLY-2608 inhibits kinase domain (H1047R) and helical domain (E542K, and E545K) mutant PI3Kα activity, demonstrating <10nM potency with 8-12x selectivity relative to WT. RLY-2608 is > 1000-fold selective over the β, δ, and γ PI3K isoforms in biochemical assays and demonstrates exquisite selectivity across a panel of 322 kinases, with no other kinases showing >50% inhibition. We performed in vitro combinations in two HR+ PIK3CA mutant cell lines (MCF7: E545K; T47D: H1047R) and observed synergy between RLY-2608 and fulvestrant or CDK4/6 inhibitors. In vivo, we tested combinations of RLY-2608 with fulvestrant and/or the CDK4/6 inhibitor abemaciclib in the MCF7 xenograft model. Oral administration of RLY-2608 in combination with fulvestrant led to improved efficacy compared to either agent alone in a dose-dependent manner, with regressions observed in the combination arms at all doses. Furthermore, the triple combination of RLY-2608, fulvestrant, and abemaciclib resulted in superior efficacy compared to either the RLY-2608 + fulvestrant or RLY-2608 + abemaciclib doublets, with deep regressions observed in the triple combination arm. In addition, in vivo combination efficacy with fulvestrant and CDK4/6 inhibitors (palbociclib or abemaciclib) was assessed in patient-derived xenografts harboring the PIK3CA H1047R or E545K mutation along with a second site PIK3CA minor mutation. In these studies, combination benefit was observed with doses of RLY-2608 significantly lower than the dose required for maximum efficacy as a single agent. RLY-2608 synergizes in vitro with both anti-estrogen and CDK4/6 inhibitors in cell models of HR+/PIK3CA mutant breast cancer. RLY-2608 can be combined with fulvestrant and CDK4/6 inhibitors in vivo with tumor regressions observed in both cell- and patient-derived xenograft models. The pre-clinical profile of RLY-2608 supports the clinical development of RLY-2608 both in single agent and combination clinical trials in patients with PIK3CA mutant tumors, including HR+/PIK3CA mutant breast cancer. Citation Format: Ermira Pazolli, Randy Kipp, Alessandro Boezio, Hakan Gunaydin, Amanda Iskandar, Matthew Zubrowski, Bret Williams, Kelley Shortsleeves, Alexandre Larivee, Tom McLean, Klaus Michelsen, Hongtao Zeng, Jonathan LaRochelle, Joe Manna, Lucian DiPietro, Andre Lescarbeau, Mary Mader, Bindu Bennet, Jeremy Wilbur, Qi Wang, Levi Pierce, Iain Martin, James Watters, Pascal Fortin, Donald Bergstrom. RLY-2608: The first allosteric mutant- and isoform-selective inhibitor of PI3Kα, is efficacious as a single agent and drives regressions in combination with standard of care therapies in PIK3CA mutant breast cancer models [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-16-10.
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