Dual Inhibition Of Flt3 And Src Pathways By On150030, A Type 1 Inhibitor, As A Novel Strategy For Relapsed And Refractory Aml Therapy

Approximately one third of patients suffering from Acute Myeloid Leukemia harbor a FLT3 Internal tandem duplication mutation (FLT3-ITD). When mutated, this receptor tyrosine kinase increases the activity of pathways for proliferation and blocks apoptosis. Quizartinib is a 2 nd generation FLT3 inhibitor that inhibits FLT3-ITD in AML, but has a median duration response of 12.1 weeks. Studies revealed a secondary mutation in FLT3 at the aspartate of codon 835 (D835X) is responsible for relapse. The D835 substitution renders FLT3 constitutively active. Type 2 inhibitors like Quizartinib bind to FLT3 in its inactive state and fail to inhibit FLT3-ITD harboring a D835 mutation. Here, we tested the utility of ON150030, developed by our group, as a novel therapeutic agent to treat AML. Structural studies suggest ON150030 binds to the active form of FLT3 (Type 1 inhibitor) so mutations such as D835X do not affect the inhibitory activity of the compound. In vitro kinase assays demonstrate that ON150030 potently inhibits Wildtype and FLT3-D835Y forms, while Quizartinib fails to inhibit FLT3-D835Y. Additionally, ON150030 demonstrated time and temperature dependent inhibition of FLT3, suggesting that the compound is an irreversible inhibitor of FLT3. Biological studies reveal that ON150030 specifically inhibits the growth of MV4-11 cells harboring the FLT3-ITD mutation (GI50: 10nM). Western blot analysis demonstrates that MAPK and PI3K/AKT pathways in these cells are inhibited with increasing dose of ON150030. The JAK independent phosphorylation of STAT5 seen in the context of FLT3-ITD is also reduced in response to ON150030. Future goals are to introduce FLT3 and its various mutant isoforms into the mouse myeloid cells (32Dcl3) and examine how it affects proliferation and differentiation, and then compare the effects of ON150030 and Quizartinib. In addition to strongly inhibiting FLT3, ON150030 inhibits SRC, which was shown to induce resistance to targeted therapies in several leukemias including AML. We will introduce SRC into the 32D-FLT3-ITD cell lines sensitive to ON150030 and test whether these cells retain their sensitivity to the drug. Next, we will perform cytotoxicity and biochemical assays on patient-derived primary AML cells using ON150030. Mouse xenograft models will be used to determine if ON150030 synergizes with standard chemotherapy agents and inhibits cancer progression in vivo. At the conclusion of this project, we hope to demonstrate that ON150030 can be used in combination therapies in all AML patients harboring a FLT3 mutation, and result in sustained remission of disease. Citation Format: Helya Ghaffari, M.V. Ramana Reddy, Stephen C. Cosenza, Rodrigo Vasques del Carpio, E. Premkumar Reddy. Dual inhibition of FLT3 and Src pathways by ON150030, a type 1 inhibitor, as a novel strategy for relapsed and refractory AML therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2094. doi:10.1158/1538-7445.AM2017-2094