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Introduction: Acute myeloid leukemia (AML) with FMS-like tyrosine kinase 3 (FLT3) mutation is associated with poor prognosis with a high risk of relapse after therapy and reduced overall survival. Currently, FLT3 inhibitors have shown clinical benefits in the corresponding AML patients. Activating mutations within internal tandem duplication (ITD) and tyrosine kinase domain (TKD) point mutations of FLT3 have been reported as oncogenic driver mutations in about 30% of AML. The acquired D835Y and F691L point mutations of FLT3-TKD are associated with resistance to FLT3-targeted AML therapy. In this study, we have characterized HM43239, a novel FLT3 inhibitor, and assessed its potential as a novel therapy in overcoming resistance for AML patients. Materials and Methods: In vitro site-directed competition binding assay was performed to measure interactions between HM43239 and FLT3 mutations. Standard proliferation assay, immunoblotting, and apoptosis analysis were carried out to validate the potency of HM43239 in AML resistance cell lines. In vivo study, HM43239 was evaluated in Ba/F3 cells expressing FLT3 ITD/F691L or FLT3 ITD/D835Y xenograft mice models. Combination studies were evaluated in Acute Myeloid Leukemia xenograft mice models. Results: HM43239 potently inhibited both FLT3 ITD/D835Y and FLT3 ITD/F691L mutations in preclinical evaluation. It showed high in vitro binding affinity to both mutations, and exhibited potent inhibitory activity in in vitro and in vivo models using Ba/F3 cells expressing FLT3 ITD/D835Y or FLT3 ITD/F691L. Moreover, HM43239 could overcome the FL-induced drug resistance with a higher cytotoxic potency in MOLM-14 cells harboring FLT3 ITD. In KG-1a cells, HM43239 potently inhibited phosphorylation of SYK, STAT3 and STAT5. In addition, it inhibited the proliferation and induced the apoptosis of leukemic stem cell (LSC) marker-expressing KG1a cells (CD34+/CD38- cells), suggesting the possibility of targeting LSC. Also, HM43239 significantly inhibited p-FLT3 and p-STAT5 under normal human plasma milieu in a dose-dependent manner in Ba/F3 and MOLM-14 cell line harboring FLT3 ITD. Furthermore, the combination treatment of HM43239 with various reagents (e.g., IAP inhibitor, chemotherapy, etc) demonstrated synergistic efficacy in mouse models, xenografted with both MV-4-11 and MOLM-13 cell lines without any significant toxicity. Conclusion: Taken together, HM43239 demonstrated the potential therapeutic efficacy for the treatment of AML patients, and implicated the mechanism of overcoming resistance and preventing relapse. Citation Format: JiSook Kim, InHwan Bae, JaeYul Choi, MinJeong Kim, JooYun Byun, MiJin Moon, EunYoung Lee, Yu-Yon Kim, Hyun Jeong Kang, Eunyoung Kim, SunYoung Jung, YoungGil Ahn, YoungHoon Kim, Kwee Hyun Suh. HM43239, a novel FLT3 inhibitor in overcoming resistance for acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1293.

KRAS is a well-known oncogene with the highest mutation rate among various cancers. KRAS cycles between GDP-loaded ‘off’ and GTP-loaded ‘on’ states induce downstream signal transduction to promote cell proliferation and survival. Interconversion between ‘on’ and ‘off’ states is assisted by SOS (Son of sevenless), a binary molecular switch of KRAS. SOS family as a guanine-nucleotide exchange factor (GEF) is composed of SOS1 and SOS2, but SOS1 is a node in the negative feedback regulation of the KRAS pathway while SOS2 is not. Since SOS1 is a direct upstream of KRAS, SOS1 inhibitor has the potential to be a pan-KRAS inhibitor affecting various cancers harboring diverse KRAS mutations. Here, we suggest HM99462 as a novel SOS1 inhibitor for KRAS regulation and present its promising druggability. We developed HM99462 for an orally bioavailable SOS1 inhibitor which abrogates GTP-binding to KRAS and the profile of drug potency and mechanism of action was examined with in vitro and in vivo models. Biochemical activity on SOS1 was evaluated by a GTP-exchange assay on KRAS G12C, D, and V, and HM99462 showed excellent inhibitory activity, while SOS2 action was not affected. Moreover, HM99462 decreased ERK phosphorylation in various cancer cell lines harboring different major KRAS mutation statuses such as KRAS G12C, G12V, G12D, or G13D. Growth inhibition assay was performed using a three-dimensional (3D) conformation culture that mimics the complexity and heterogeneity of tumors and has elevated p-ERK. In this 3D spheroid growth inhibition assay, HM99462 demonstrated significantly effective GI50 against various KRAS mutant cell lines. HM99462 also had almost no inhibition of seven CYP isozymes guided by the FDA, and a low plasma protein binding rate around 80%, offering high unbound drug concentration. Additionally, HM99462 was evaluated in cancer cell xenograft mouse models harboring various KRAS mutations. Reasonable tumor growth inhibition activity was shown within tolerable doses when administered alone. Also, the combination of HM99462 with KRAS G12C or MEK inhibitors led to synergistic anti-tumor activity in both in vitro and in vivo models with several KRAS mutations. Through our exploratory study, a novel SOS1 inhibitor, HM99462 could be suggested as an appropriate therapeutic agent for diverse inhibitory activity against cancers causing by the hyperactivation of oncogenic KRAS signaling. And it has the potential to overcome the limitations of KRAS G12C or MEK inhibitor through combination. HM99462 is currently in IND enabling GLP-toxicity studies, planned to initiate clinical study in early 2024. Citation Format: Seung Hyun Jung, Jaeyul Choi, Wongi Park, Jooyun Byun, Semi Lim, Youngjoo Lee, Yu-Yon Kim, Hyunjin Park, So-Ye Jeon, Taehun Song, Kyungjin Choi, Tae-yeon Kong, Heecheol Kim, Wook Jang, EunYoung Lee, Minhwa Kim, Young Gil Ahn, Young Hoon Kim, Kwee Hyun Suh. A novel SOS1 inhibitor, HM99462 demonstrates antitumor activity against KRAS-mutant cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1625.

Acute myeloid leukemia (AML) with FMS-like tyrosine kinase 3 (FLT3) mutations is associated with poor prognosis with a high risk of relapse after therapy and reduces overall survival. Approximately 30 % of AML patients carry FLT3 internal tandem duplication (ITD) or tyrosine kinase domain (TKD) mutations. Sustained FLT3 inhibition can result in the emergence of resistance-conferring genetic alteration in TKD domain, usually at residues D835 and F691. Therefore, acquired TKD mutations has become a critical therapeutic target in AML therapy. On the other hand, SYK is one of kinases deeply implicated in many hematologic malignancies and highly activated in FLT3 mutation AML. SYK overexpression is known to promote over transformation of FLT3 driven AML and induce resistance to FLT3 targeted therapy. In this study, we characterized HM43239, a novel FLT3 inhibitor with SYK inhibitory activity, and assessed its potential as a novel therapeutic agent to overcome the resistances against AML therapy using current FLT3 inhibitors. HM43239 is an orally active small molecule inhibitor and it exhibited sub-nanomolar potency on binding affinity for FLT3 wild type, ITD, TKDs and ITD/TKDs mutants. HM43239 potently inhibited phosphorylation of FLT3 and its downstream such as p-STAT3/p-STAT5 dose dependently in both MOLM-14 cells harboring FLT3 ITD/F691L and FLT3 ITD/D835Y. In KG-1a cells, HM43239 potently inhibited phosphorylation of SYK, STAT3 and STAT5. Moreover, it inhibited the proliferation and induced the apoptosis of leukemic stem cell (LSC) marker-expressing KG-1a cells (CD34+/CD38- cells), suggesting the possibility of targeting LSC. Furthermore, HM43239 showed to be exhibited good inhibitory activity against FLT3 mutated AML cell lines and effectively regress the tumors in MOLM-14 cells expressing FLT3 ITD/F691L or FLT3 ITD/D835Y xenograft mice models. On the other hand, it was confirmed that HM43239 strongly inhibited the phosphorylation of SYK in the medium of co-culture of stromal cells and AML cells. As a result, HM43239 alone more effectively induced tumor regression and prolonged the survival duration of animals than an approved FLT3 inhibitor (e.g. gilteritinib) in resistant FLT3 ITD/D835Y or ITD/F691L mutated MOLM-14 xenograft mice models. These results suggest that HM43239 could overcome the resistance induced by bone marrow microenvironment in AML patients. Taken together, HM43239 showed strong anticancer activity through various in vitro and in vivo preclinical models of AML, implicating the mechanism of overcoming resistance and preventing relapse. The effect of HM43239 in human would be demonstrated in ongoing Phase I/II clinical trials (NCT03850574) to develop promising therapeutics for patients with AML. Citation Format: Inhwan Bae, Jaeyul Choi, Jiyoung Song, Joo-Yun Byun, Eunyoung Lee, Taehun Song, Yu-Yon Kim, Yesol Bak, Young Hoon Kim, Young Gil Ahn, Kwee Hyun Suh. HM43239, a novel FLT3 inhibitor, has the potential to inhibit mutations resistant to FLT3 inhibitors [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 1257.