Abstract 2056: Combined FLT3 and Pim kinase inhibitor treatment downregulates c-Myc early in apoptosis induction in acute myeloid leukemia with FLT3-ITD

Abstract

Abstract Background: fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is present in acute myeloid leukemia (AML) in 30 % of patients, with poor treatment outcomes due to rapid relapse. FLT3 inhibitors have limited activity and rapid onset of resistance. The oncogenic serine/threonine kinase proviral integration site for Moloney murine leukemia virus 1 (Pim-1) is upregulated downstream of FLT3-ITD and phosphorylates and stabilizes FLT3 in a positive feedback loop in cells with FLT3-ITD. Dual inhibition of FLT3 and Pim kinases is therefore a promising treatment strategy for AML with FLT3-ITD. The proto-oncogene c-Myc, upregulated in many cancer types, drives proliferation and causes resistance to apoptosis. We previously showed that concurrent FLT3 and Pim inhibition in cells with FLT3-ITD increases apoptosis through downregulation of the anti-apoptotic protein Mcl-1. Here we demonstrate downregulation of c-Myc upstream of Mcl-1. Methods: Ba/F3-ITD and MV4-11 cells, with FLT3-ITD, were cultured with the FLT3 inhibitors gilteritinib (15 nM) or quizartinib (1 nM) and the pan-Pim inhibitor AZD1208 (1 μM) alone and in combination, and with DMSO control. c-Myc and Mcl-1 protein expression was measured by immunoblotting and c-Myc mRNA by qPCR using SybrGreen. Cells were cultured with cycloheximide (100 μg/mL) with and without the proteasome inhibitor MG-132 (20 μM). Ba/F3-ITD cells were stably transfected with a lentiviral c-Myc plasmid or a retroviral Mcl-1 plasmid and corresponding empty vectors. Apoptosis was detected by Annexin V and propidium iodine staining, measured by flow cytometry. Results: Combined FLT3 and Pim inhibitor treatment decreased c-Myc protein expression in Ba/F3-ITD and MV4-11 cells within 30 minutes, preceding Mcl-1 downregulation. c-Myc mRNA levels did not change, but c-Myc protein half-life decreased with combination treatment, and this decrease was abrogated by proteasome inhibitionMcl-1 overexpression did not abrogate c-Myc downregulation by combination treatment, and c-Myc overexpression decreased apoptosis induction by combination treatment from 65% to 45%. Conclusion: Combined FLT3 and Pim kinase inhibitor treatment enhances apoptosis in FLT3-ITD cells through c-Myc downregulation, which precedes Mcl-1 downregulation. c-Myc downregulation is post-translational, via increased proteasomal degradation. Citation Format: Jonelle K. Lee, Mario Scarpa, Shivani Kapoor, Maria R. Baer. Combined FLT3 and Pim kinase inhibitor treatment downregulates c-Myc early in apoptosis induction in acute myeloid leukemia with FLT3-ITD [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 2056.