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Background: The FMS-like tyrosine kinase 3 (FLT3) is mutated in approximately 30% of acute myeloid leukemia (AML) patients either by internal tandem duplication (ITD) or by point mutations in the tyrosine kinase domain (TKD). PHI-101 is a Type I FLT3 inhibitor developed to overcome resistance in AML. PHI-101 showed potent cellular activity in vitro and in vivo for both FLT3 ITD mutations and a wide variety of FLT3 point mutations in TKD. In preclinical studies utilizing primary human FLT3-ITD leukemia cells, PHI-101 showed increased anti-leukemic activity compared to gilteritinib based on increased survival in mice transplanted with these samples. The Phase 1a and Phase 1b global clinical trial with PHI-101 (NCT04842370) is currently underway to assess the overall safety and efficacy of PHI-101 in refractory and relapsed AML. Study Design and Methods: The overall design of the AML clinical trial consists of two parts, phase 1a, the dose-escalation, and phase 1b, the dose-expansion trial utilizing oral PHI-101 tablets. Up to 5 dose levels are planned for phase 1a and a single subject enrolled per dose level until the occurrence of at least one subject with more than Grade 2 toxicity according to CTCAE5.0 criteria during the 28-day evaluation period. The dose escalation will then convert to a standard 3+3 scheme, with 3 to 6 subjects per dose level cohort over the 28-day cycle. If there are no dose-limiting toxicities (DLT), dose escalation to the next higher dose levels will proceed upon the recommendation of the Safety Monitoring Committee. Subjects with FLT3 mutations or FLT3 wild-type will be enrolled in the dose-escalation cohort, and blood samples are collected for the primary endpoint pharmacokinetics (PK) and for the exploratory pharmacodynamic (PD) endpoint evaluation, including plasma inhibitory assay and biomarker analysis. Results: The Phase 1a clinical trial of PHI-101 was initiated in June 2020 at a daily dose of 40mg for level 1. As of July 31, 2021, 8 patients with relapsed or refractory AML have been enrolled in the trial, all of whom had prior anti-leukemic treatments with intensive chemotherapy, hypomethylating agents and/or other FLT3 inhibitors. To date, 5 enrolled patients were available for safety assessment at three dose levels and have not experienced any dose-limiting toxicities (DLTs) from repeating doses of the 28-day DLT-assessment window. The leukemic blasts in marrow or peripheral blood were dramatically reduced by up to 98% with one cycle (28 days). The pharmacokinetic data of plasma PHI-101 varied in a dose-proportional manner and peak plasma concentrations (Cmax) were reached between 4 and 6 hours after once-daily dosing. The pharmacodynamic evaluation by plasma inhibitory assay showed that plasma from patients receiving at daily doses of PHI-101 inhibited more than 90% of the phosphorylation of the FLT3-ITD receptor. PHI-101 was well tolerated with these dose levels. This study is currently recruiting FLT3 mutation or wild-type AML patients at multiple sites in Korea and Australia. Conclusion: PHI-101 is a next-generation FLT3 inhibitor that showed a potent anti-leukemic activity and improved efficacy in primary AML samples harboring FLT3/ITD and FLT3/TKD mutations in preclinical studies. The current analysis of this trial indicated that PHI-101 is a very effective FLT3 inhibitor for both refractory and relapsed AML patients, including those that have relapsed on other FLT3 TKI. The assessments of safety, tolerability, and PK of PHI-101 to determine the recommended dose for expansion are ongoing in the Phase 1a clinical trial. Disclosures Lee: Astellas Pharma, Inc.: Consultancy, Honoraria, Other: Advisory board; AbbVie: Honoraria, Other: Advisory board; Korean Society of Hematology: Membership on an entity's Board of Directors or advisory committees. Im: Pharos iBio Co., Ltd.: Current Employment. Nam: Pharos iBio Co., Ltd.: Current holder of individual stocks in a privately-held company. Kim: Pharos iBio Co., Ltd.: Current holder of individual stocks in a privately-held company. Han: Pharos iBio Co., Ltd.: Current holder of individual stocks in a privately-held company. Yoon: Pharos iBio Co., Ltd.: Current holder of individual stocks in a privately-held company. Small: Pharos iBio Co., Ltd.: Consultancy, Other: Scientific Advisory Board. The arrangement has been reviewed and approved by Johns Hopkins University in accordance with its conflict-of-interest policies, Research Funding; InSilico Medicine: Other: Scientific Advisory Board.

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.

Background: FMS-like tyrosine kinase 3 (FLT3), a member of the class III receptor tyrosine kinase family, plays a pivotal role in regulating cell growth and differentiation of hematopoietic cells. FLT3 is mutated in approximately 30% of AML patients either by internal tandem duplication (ITD) within the juxtamembrane portion or by point-mutations in the kinase domain (TKD). Thus, FLT3 inhibitors including quizartinib (AC220), midostaurin (PKC412) and gilteritinib (ASP2215) have been developed and undergone clinical testing for the treatment of AML. However, patients frequently relapse due to intrinsic and extrinsic resistance to these FLT3 inhibitors in spite of the initial clinical efficacy. We have developed a potent third-generation FLT3 inhibitor, PHI-101, capable of overcoming some of these mechanisms of resistance. Methods: Biochemical kinase assays for PHI-101 have been performed on 9 different FLT3 mutants and wild type FLT3. Cellular potencies of PHI-101 have also been assessed using various patient-derived AML cells as well as MV4-11, MOLM14 and BaF3 cell lines transformed with human FLT3 mutants including single mutations [FLT3(ITD), FLT3(D835Y)], double or triple mutations [FLT3(ITD/D835Y), FLT3(ITD/F691L), FLT3(ITD/F691L/D835Y)]. In order to evaluate in vivo efficacies of oral administration of PHI-101, xenograft mouse models and in vivo bioluminescence imaging have been utilized. Results: PHI-101 possessed excellent enzymatic potencies against FLT3 potential resistant mutants such as ITD/D835V and the gatekeeper ITD/F691L mutation, as well as against FLT3 single activating mutants that include ITD, D835V, D835H, and D835Y. PHI-101 inhibited the phosphorylation of FLT3 and downstream STAT5 and ERK1/2 more effectively than quizartinib and gilteritinib at equivalent doses. Moreover, oral administration of PHI-101 induced tumor regression in the xenograft mouse models developed by injection of BaF3 cells transformed with FLT3-ITD, FLT3-TKD or FLT3-ITD/TKD mutants in a dose-dependent fashion with no appreciable toxicities. In luciferase-bearing blood circulating mouse models with FLT3-double mutants (ITD/D835Y and ITD/F691L) or triple mutants (ITD/D835Y/F691L), oral administration (30 mpk, QD) of PHI-101 diminished more than 89% of bioluminescent intensity and reduced leukemic burden. PHI-101 also showed increased efficacy in extending the lifespan of xenograft mice compared to quizartinib. PHI-101 strongly suppressed proliferation and induced apoptosis in primary AML samples harboring FLT3/ITD and FLT3/TKD mutations with little effect on wild-type FLT3 samples. Conclusion: PHI-101, an orally bioavailable novel small molecule, is a potent third-generation FLT3 inhibitor able to overcome resistance to several resistance mutations based on in vitro and in vivo experiments. PHI-101 possesses excellent in vitro and in vivo activities against not only FLT3 single activating mutations (ITD or TKD mutants) but also FLT3 double (ITD/D835Y or ITD/F691L) and triple (ITD/D835Y/F691L) resistant mutations with no pronounced toxicities. Preclinical evaluation of PHI-101 showed clear evidence of antileukemic activity and improved efficacy in both in vitro and in vivo models. PHI-101 is currently under investigation in first-in-human clinical trials with relapsed or refractory AML patients. Disclosures Nam: Pharos I&BT Co., Ltd.: Current equity holder in private company. Im:Pharos I&BT Co., Ltd.: Current Employment. Han:Pharos I&BT Co., Ltd.: Current equity holder in private company. Kim:Pharos I&BT Co., Ltd.: Current Employment. Yoon:Pharos I&BT Co., Ltd.: Current equity holder in private company. Cho:Pharos I&BT Co., Ltd.: Research Funding. Choi:Pharos I&BT Co., Ltd.: Research Funding. Young:Pharos I&BT Co., Ltd.: Research Funding. Nguyen:Pharos I&BT Co., Ltd.: Research Funding. Zhu:Pharos I&BT Co., Ltd.: Research Funding. Li:Pharos I&BT Co., Ltd.: Research Funding. Small:Pharos I&BT Co., Ltd.: Consultancy, Research Funding. Sim:Pharos I&BT Co., Ltd.: Research Funding.

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.