AML-173: BCL2A1: A Novel Target in Refractory/Relapsed AML with FLT3-ITD/D835 Double Mutations

Abstract

<h3>Context:</h3> BCL2A1 exerts a pro-survival function in <i>FLT3-</i>ITD/D835-double mutant acute myeloid leukemia (AML) cells and may be a putative novel therapeutic target in FLT3-mutant AML. The combination of gilteritinib, which reduced BCL2A1 with venetoclax, was highly synergistic in FLT3-ITD/D835-double mutant cells. <h3>Objective:</h3> While FLT3 tyrosine kinase inhibitors (TKI) emerged as a new therapeutic option in AML with internal tandem duplications in the juxtamembrane domain of <i>FLT3</i> (FLT3-ITD), the prognosis of FLT3-mutated AML is still poor because heterogeneous clonal populations with additional acquired <i>FLT3</i> and <i>Ras</i> mutations emerge and cause drug resistance. <i>FLT3</i>-ITD plus a point mutation at residue D835 in the tyrosine kinase domain (<i>FLT3</i>-ITD/D835) often cause resistance or decrease sensitivity to TKIs. In this study, we investigated new drug targets in AML with FLT3-ITD and TKD double mutations. <h3>Design:</h3> We carried out cap analysis of gene expression (CAGE) in primary AML cells from 26 patients to characterize transcriptome differences between <i>FLT3</i>-ITD-positive and <i>FLT3</i>-ITD/D835 double-positive primary AML cells. Selected genes were confirmed and validated with immunoblotting and RT-qPCR using FLT3-ITD-positive and FLT3-ITD/D835-positive MV4-11 cells. Furthermore, we generated <i>FLT3</i>-ITD-postive cells with overexpression of the selected genes using lentivirus vectors. <h3>Results:</h3> CAGE demonstrated that AML cells harboring <i>FLT3-</i>ITD/D835 double mutations expressed increased <i>BCL2A1</i> gene transcripts compared to those with <i>FLT3</i>-ITD alone. RT-qPCR and immunoblot analysis also revealed that <i>FLT3</i>-ITD/D835-positive MV4-11 cells expressed higher transcript and protein levels of BCL2A1 than MV4-11 cells with FLT3-ITD mutation alone. Overexpression of BCL2A1 attenuated sensitivity to venetoclax and decreased sensitivity to quizartinib in AML cells with a heterozygous FLT3-ITD mutation (FLT3-wildtype/FLT3-ITD) but not in cells with a homozygous mutation (<i>FLT3</i>-ITD/<i>FLT3</i>-ITD). Gilteritinib targets BCL2A1 through inactivation of STAT5, and combinatorial gilteritinib and venetoclax were highly synergistic in <i>FLT3</i>-ITD/D835 double-positive cells. Furthermore, inhibition of BRD4 with the BET inhibitor CPI-0610 exhibited anti-tumor activity in <i>FLT3</i>-ITD/D835 double-positive AML and suppressed BCL2A1 expression in these cells. <h3>Conclusions:</h3> This study elucidates overexpression of BCL2A1 as a novel survival mechanism in <i>FLT3-</i>ITD/D835 mutant AML cells and identifies BCL2A1 as a putative novel therapeutic target in FLT3-mutant AML.