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Purpose: Development of the potent and selective checkpoint kinase 2 (CHK2) inhibitor to overcome limiting clinical utility of poly(ADP-ribose) polymerase 1 (PARP1) inhibitors. Experimental Procedure: Preclinical evaluation of PHI-101 for cellular and molecular potency in ovarian and breast cancer cell lines and patient derived primary cells; it includes biochemical binding assay, cellular assays, animal efficacy studies, combination study, signaling pathway effect examination, and cell cycle analysis. Summary: CHK2 is a serine/threonine kinase and a cell cycle checkpoint regulator involved in the ATM-mediated DNA repair upon replication blocks and DNA damage. It has been proposed that Chk2 functions as a barrier to tumorigenesis by maintaining genomic stability, and this DNA damage induction is thought to prevent or delay genetic instability and tumorigenesis. The inhibition of CHK2 is a promising approach to achieve synthetic lethality of cancer cells when combined with PARP1 inhibitors. Solid cancer indications of PHI-101 was identified by the Chemiverse Network module which is an AI and Big data-based in-house drug discovery platform. Biochemical kinase assays for PHI-101 showed stronger affinity to CHK2 over CHK1 more than 5-fold. PHI-101 treatment of ovarian and breast cancer cells for 72 hrs elicit a synergistic lethal response in combination with PARP1 inhibitor Olaparib regardless of functional BRCA and P53 in the cells. PHI-101 also potentiates a countermeasure to dose-limiting toxicity triggered by genotoxic agents such as cisplatin and topotecan. The present results from in vivo and in vitro preclinical testing do demonstrate that PHI-101 is a highly potent inhibitor of CHK2 and may exert mono- and combinational therapeutic activity in ovarian and breast cancer model. Conclusion: The preclinical evaluation of PHI-101, a novel CHK2 inhibitor, showed clear evidence of anticancer activity for refractory ovarian and breast cancer cells and improved efficacy in both in vitro and in vivo models. Consequently, PHI-101 is currently under investigation in Phase 1 clinical trials for relapsed or refractory ovarian cancer patients. Citation Format: June H-J Han, Kyu-Tae Kim, Jeejin Im, Sojung Park, Min Kyung Choi, Inki Kim, Ky-Youb Nam, JeongHyeok Yoon. PHI-101, a potent and novel inhibitor of CHK2 in ovarian and breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1461.

Purpose: Develop a next generation FLT3 TKI to overcome previous resistance mechanisms. Description: Preclinical testing of PHI-101 for efficacy against FLT3 mutations both in vitro and in vivo in cell lines and primary FLT3 mutant leukemia samples. Summary: Mutations in the FLT3 gene are common in AML, occurring in approximately 1/3 of patients and result in poor prognosis for AML patients with FLT3 internal tandem duplication (ITD) mutations. Thus, it is a well-validated target for the development of tyrosine kinase inhibitors (TKI). A number of FLT3 TKI have been developed and the latest generation have proven increasingly successful at achieving thorough inhibition of FLT3 kinase activity in vivo, a necessary requirement for clinical efficacy against FLT3 mutant AML. Major reasons for the failure of some FLT3 TKI to achieve thorough inhibition include tight binding to alpha 1 acid glycoprotein (AGP) resulting in inadequate free levels of the drug and selection for resistance mutations within FLT3 itself. Resistance mutations within FLT3 have included those at D835 and F691 among others. We present here the initial preclinical data on a new FLT3 TKI, PHI-101, developed to overcome some of these mechanisms of resistance. PHI-101 is a potent small molecule inhibitor of both FLT3/ITD and FLT3/D835Y mutations with an IC50 of 5 nM and 11.1 nM, respectively, in cell-based assays. It is active against a number of FLT3/ITD resistance mutations including FLT3/ITD/A627T, FLT3/ITD/N676D, FLT3/ITD/F691L, with IC50s of 0.1nM, 2.3nM, 14.6nM, respectively. It is also active against FLT3 kinase domain (FLT3/KD) resistance mutations, with IC50s against FLT3/D835A and FLT3/840GS of 5.3nM and 9.6nM, respectively. Furthermore, it is active against the FLT3/ITD/KD and FLT3/ITD/F691 resistance mutations. We also demonstrate its effectiveness in inhibiting FLT3 activity by Western blot analysis. Importantly, when analyzed in the human plasma inhibitory assay (PIA), its IC50 shifts only 8.5-fold, unlike several other FLT3 TKI with shifts of >200-fold, rendering them unable to sufficiently inhibit FLT3 mutations in vivo. PHI-101 monotherapy induces dose-dependent regression of tumor growth measured with bioluminescence in FLT3-ITD mutant xenograft models. In addition, PHI-101 significantly inhibits proliferation and induces apoptosis in primary AML samples expressing FLT3/ITD and FLT3/KD mutations, but not wild-type FLT3. These effects suggest it is selective for primary AML with FLT3 mutations, comparable to that of Giltertinib, the newly-approved FLT3 TKI. PHI-101 will soon undergo first-in-human clinical trials in FLT3 mutant AML patients. Conclusion: PHI-101 is a new FLT3 TKI with characteristics that warrant entry into human trials. Citation Format: Ky-Youb Nam, Jeejin Im, JeongHyeok Yoon, David Young, Li Li, Ruiqi Zhu, Bao Nguyen, Donald Small. PHI-101, a next generation FLT3 inhibitor for acute myeloid leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4226.