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PI3Kα is highly mutated in cancer resulting in hyperactivation of lipid kinase activity and downstream AKT signaling. H1047 is the most common site of oncogenic mutation and occurs in ~14% of all breast cancers. Initial therapeutic benefit of targeting PI3Kα was established with alpelisib, an alpha-selective PI3K inhibitor that is equipotent against wild-type and mutant forms. However, wild-type PI3Kα inhibition results in frequent dose-limiting toxicities including hyperglycemia, restricting the full potential of this drug. Selective targeting of H1047X-mutant PI3Kα is expected to both improve anti-tumor activity and reduce toxicity. STX-478 is an allosteric, CNS-penetrant, selective PI3Kα H1047X inhibitor, having excellent drug-like properties and exceptional kinome selectivity. STX-478 demonstrated minimal inhibition of CYP enzymes in vitro, supporting the potential for combinations with a wide range of therapeutics in breast cancer and a variety of other tumor types. STX-478 selectivity extended to the inhibition of other activating kinase domain mutations in biochemical assays. In a diverse panel of PI3Kα H1047X mutant cell lines, STX-478 selectively reduced the cellular levels of pAKT (S473) with a strong correlation between pAKT inhibition and cell viability (R = 0.8). In a high-throughput viability screen of 467 cancer cell lines, the presence of PIK3CA H1047X and other kinase domain mutations were the single strongest predictor of STX-478 sensitivity with potency superior to alpelisib. STX-478 also selectively inhibited the proliferation of cell lines with PI3Kα helical domain mutations, potentially due to the selective dependency of these cells on mutant PI3Kα. When combined with fulvestrant, lapatinib, or abemaciclib, STX-478 demonstrated synergistic anti-proliferative activity in cell lines with relevant ER/HER2 status. Unlike alpelisib, STX-478 did not impair glucose metabolism or cause insulin resistance at efficacious doses. In the T47D (PI3Kα H1047R) breast cancer model, STX-478 (100 mg/kg) monotherapy caused tumor regression whereas alpelisib caused only stasis. STX-478 combination with fulvestrant was well-tolerated, with more consistent and deeper tumor regression. Similar results were observed in a PI3Kα H1047R mutant ER+/HER2- PDX model, where fulvestrant monotherapy showed minimal activity, while combination with STX-478 yielded tumor regressions. In an ER+/HER2+ PDX model (PI3Kα H1047R/R108H), palbociclib and STX-478 (100 mg/kg) monotherapy resulted in similar efficacy while the combination was well tolerated and yielded tumor regression. Together these data indicate robust STX-478 monotherapy activity that was well tolerated and improved when dosed in combination with fulvestrant or CDK4/6 inhibitors. Finally, we investigated the effect of STX-478 treatment in an ER+ PDX model carrying a helical domain mutation. STX-478 treatment resulted in tumor growth inhibition at doses that did not result in metabolic dysfunction, suggesting that STX-478 may also be efficacious in treating PIK3CA mutant tumors with helical domain mutations. In summary, STX-478 efficacy was superior to alpelisib at a dose level that exceeds the clinically relevant exposure in mice without causing metabolic dysfunction. STX- 478 has a predicted low human dose, CNS exposure, low risk of DDI, and a predicted long half-life with minimal variation in peak-to-trough plasma concentrations which further supports a favorable therapeutic index. STX-478 has the potential to provide a best-in-class profile to improve outcomes in patients harboring tumors with prevalent PI3Kα H1047X mutations as well as other kinase and helical domain mutant tumors. The significant CNS exposure of STX-478 is expected to enable this treatment for patients with brain tumors and brain metastases not afforded by existing options. STX-478 is currently in IND enabling studies and is expected to enter human clinical trials in 2023. Citation Format: Leonard Buckbinder, David J. St. Jean, Brendon Ladd, Trang Tieu, Philip Jonsson, Jacob Alltucker, Samantha Manimala, Weixue Wang, Angel Guzman-Perez, Darrin D. Stuart, Gregory Dowdell. STX-478, a mutant-selective PI3Kα H1047X inhibitor clinical candidate with a best-in-class profile: Pharmacology and therapeutic activity as monotherapy and in combination in breast cancer xenograft models [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-07-04.

Myeloperoxidase (MPO) is a highly oxidative, pro-inflammatory enzyme involved in post-myocardial infarction (MI) injury and is a potential therapeutic target. While multiple MPO inhibitors have been developed, the lack of an imaging reporter to select appropriate patients and assess therapeutic efficacy has hampered clinical development. Thus, a translational imaging method to detect MPO activity non-invasively would help to better understand the role MPO plays in MI and facilitate novel therapy development and clinical validation. Interestingly, many MPO inhibitors affect both intracellular and extracellular MPO, but previous MPO imaging methods can only report extracellular MPO activity. In this study, we found that an MPO-specific PET imaging agent (18F-MAPP) can cross cell membranes to report intracellular MPO activity. We showed that 18F-MAPP can track the treatment effect of an MPO inhibitor (PF-2999) at different doses in experimental MI. The imaging results were corroborated by ex vivo autoradiography and gamma counting data. Furthermore, extracellular and intracellular MPO activity assays revealed that 18F-MAPP imaging can report the changes induced by PF-2999 on both intracellular and extracellular MPO activities. These findings support 18F-MAPP as a translational candidate to noninvasively report MPO activity and accelerate drug development against MPO and other related inflammatory targets.

Oncogenic PIK3CA mutations are found in 14% of all cancers and most frequently occur at amino acids E542/E545 and H1047, involving the helical and kinase domains, respectively (Zhang 2017). Alpelisib is a PI3Kα isoform-selective, orthosteric kinase inhibitor, having equivalent activity on wild-type (wt) and mutant (mt) forms and was approved to treat PI3Kα mt, HR+/HER2- breast cancer in combination with fulvestrant, nearly doubling progression-free survival (Andre 2019). Hyperglycemia with insulin resistance is an on-target result of wt PI3Kα inhibition, leading to frequent alpelisib dose reductions, interruptions, and discontinuations. H1047X is the most common PIK3CA alteration, found in ~14% of all breast cancer patients (Martínez-Sáez 2020). We considered that selectively targeting this mutant could improve patient outcomes by sparing metabolic dysfunction related to wt inhibition. We have validated this therapeutic concept preclinically with the discovery of ST-814, an allosteric, CNS-penetrant, mutant selective PI3KαH1047X inhibitor, displaying excellent drug-like properties. ST-814 was 14-fold selective for PI3KαH1047R vs PI3Kαwt in biochemical assays (IC50 11 vs 146 nM) with exquisite isoform and kinome selectivity. Cell-based activity and selectivity were characterized in T47D (PI3KαH1047R) and SKBR3 (PI3KαWT) tumor cells grown in 10% FBS, with 8-fold mut-selectivity observed in target engagement (pAKT EC50 38 vs 304 nM) and 11-fold selectivity in viability (GI50 153 vs 1683 nM). ST-814 has robust anti-tumor activity in PI3KαH1047X in xenografts, with efficacy similar or superior to alpelisib dosed at 50 mg/kg (Table 1). To achieve this level of mouse efficacy, alpelisib exposure exceeded that in patients >200% (mouse vs human AUC0-24, 74,000 vs 33,224 ng*hr/mL). As such, repeat dosing of alpelisib at 50 mg/kg, caused profound elevations in serum insulin 1 hr post-dose (*p Table 1. CAL33 H1047R Oral Sq Cell Carcinoma Xenograft Alpelisib TGI% 50 mg/kg ST-814 TGI% 100 mg/kg Alpelisib ΔInsulin ng/mL ST-814 ΔInsulin ng/mL 79% 82% 4.42* 0.40 Citation Format: Leonard Buckbinder, David J. St. Jean, Trang Tieu, Weixue Wang, Gregory Kryukov, Philip Jonsson, Jacob Alltucker, Samantha Manimala, Brendon Ladd, Angel Guzman-Perez, Darrin Stuart. Discovery and characterization of a mutant selective PI3KαH1047Xinhibitor with a best-in-class profile [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 LB194.