BCL2A1: A Novel Target in Refractory Acute Myeloid Leukemia with FLT3-ITD/D835 Dual Mutations

Internal tandem duplications in the juxtamembrane domain of FMS-like tyrosine kinase 3 gene (FLT3-ITD) and missense mutations in the gene’s tyrosine-kinase domain (FLT3-TKD) play critical roles in the pathophysiology of acute myeloid leukemia (AML). Recent study revealed that AML cells resistant to quizartinib, a type II TKI, consist of heterogeneous clonal populations harboring wild-type FLT3 as well as FLT3-ITD, FLT3-TKD and FLT3-ITD/TKD mutations, and on- and off-target mechanisms may contribute to the resistance to TKIs. To overcome the heterogeneous resistance mechanisms of FLT3-ITD and TKD mutations, various combinatorial therapies have been investigated. For example, BCL-2 inhibition in the presence of TKIs increased survival a murine FLT3-ITD AML model, and a phase Ib/II clinical trial of a combination of quizartinib with venetoclax, a BCL2 inhibitor is ongoing in relapsed/refractory FLT3-mutant AML patients (NCT03735875).

To dissect underlying mechanisms of drug-resistance and exploring new targets in refractory AML with FLT3-ITD and TKD mutations, we investigated alterations of transcriptome signatures by analyzing AML samples with FLT3-ITD/D835 dual mutations. Previously, we reported BCL2A1 transcriptomes were upregulated in primary AML cells with FLT3-ITD/D835 dual mutations compared to cells with FLT3-ITD mutations only. This was recapitulated in the MV4-11 cells harboring FLT3-ITD/D835 dual mutations after 6 month-exposure to quizartinib. The MV4-11 cells with the FLT3-ITD/D835 dual mutations became resistant to quizartinib, and the cells also became resistant to venetoclax, a BCL2 inhibitor (Yamatani et al. ASH 2019).

In this study, we further investigated BCL2A1 as new target in refractory AML with FLT3-ITD/D835 dual mutations. First, we examined whether overexpression of BCL2A1 induces drug-resistance in MV4-11 and Molm13 cell lines with FLT3-ITD. While parental MV4-11 and Molm13 cells are sensitive to venetoclax and quizartinib, MV4-11 and Molm13 cells transfected with lentivirus carrying BCL2A1 became resistant to venetoclax (IC₅₀: MV4-11 with BCL2A1 over-expression >1000 nM vs. mock vector 0.71 nM; Molm13 with over-expression >1000 nM vs. mock vector 0.38 nM, 72h). In contrast, the sensitivity to quizartinib was retained in the BCL2A1 overexpressing MV4-11 and Molm13 cells. These findings indicate that the overexpression of BCL2A1 could play a role in the acquired resistance to venetoclax, but not to quizartinib.

Bromodomain-containing protein 4 (BRD4), a family member of bromodomain and extra-terminal motif (BET) is known to transcriptionally modulate BCL2A1 gene expression. Next, we examined effects of CPI-0610, a BET inhibitor, on MV4-11 cells with FLT3-ITD or the FLT3-ITD/D835 dual mutation. CPI-0610 inhibited cell growth of MV4-11 cells by inducing apoptosis irrespective of co-existing FLT3 mutations (IC₅₀: FLT3-ITD/D835, 255 nM vs. FLT3-ITD, 191 nM, 72h). Immunoblotting analyses confirmed that BET inhibition by CPT-0610 decreased the expression of BCL2A1 in MV4-11 cells FLT3-ITD/D835.

In conclusion, transcriptome analysis and molecular pharmacological approaches identified alterations in the anti-apoptotic BCL2 family proteins in double-mutant FLT3 leukemias. BCL2A1 upregulation might be involved in drug resistance of FLT3-ITD/D835 dual mutant AML cells, and could be a promising new target in refractory AML with FLT3-ITD/D835 dual mutations.