Combined Targeting of Bcl-2 and XPO1 Overcomes Acquired Resistance to Tyrosine Kinase Inhibitors in the FLT3-ITD/TKD Double Mutant AML
Authors: Tabe Yoko , Yamatani Kotoko , Yang Haeun , Saito Kaori , Murakami-Tonami Yuko , Suzuki Koya , Zhang Weiguo , Kinjo Sonoko , Ikeo Kazuho , Hosoya Masaki , Mogushi Kaoru , Yamaguchi Shigeo , Harada Hironori , Miida Takashi , Shah Neil , Konopleva Marina Y. , Hayashizaki Yoshihide , Andreeff Michael 
Published: October 2019

Abstract
FMS-like tyrosine kinase 3 with internal tandem duplication (FLT3-ITD), the most frequent mutation in acute myeloid leukemia (AML), results in the constitutive ligand-independent activation of FLT3 receptor downstream signaling. FLT3-targeted therapies using tyrosine kinase inhibitors (TKIs) demonstrate single agent activity in these patients but long-term efficacy is limited by emerging resistance in part due to acquired point mutations in the tyrosine kinase domains (TKD) of the FLT3 gene, most commonly found at D835 within the activation loop.
Exportin 1 (XPO1) mediates the nucleo-cytoplasmic transport, and is overexpressed in AML cells with FLT3-ITD mutations (Kojima, Blood, 2013). We have reported that the clinically available XPO1 inhibitor selinexor effectively inhibits cell proliferation in both FLT3-ITD and FLT3-ITD/D835 mutated cells through upregulation of TP53 and blockade of c-Myc signaling (Tabe, ASH, 2017).
In this study, we aimed at developing novel mechanism-based combination therapies for TKI-resistant FLT3-ITD cells with acquired TKD mutation. First we investigated the transcriptional changes associated with XPO1 and FLT3 inhibition in FLT3-ITD and FLT3-ITD/D835 mutated cells. We utilized paired isogenic FLT3-ITD or FLT3-ITD/D835Y transfected Ba/F3 cell lines and performed the cap analysis of gene expression (CAGE) that identifies and quantifies gene expression at the transcription start sites (TSS). FLT3-ITD cells are sensitive and FLT3-ITD/D835 cells are resistant to FLT3 inhibitor quizartinib. CAGE detected the shared upregulation of 3833 TSS genes induced by selinexor in FLT3-ITD and FLT3-ITD/D835 cells (FDR <0.05, EdgeR) with the most prominent upregulation of the negative regulator of BCL-2, Ddit3, and the tumor suppressing transcriptional activator Klf6. Selinexor downregulated 4196 shared TSS genes in FLT3-ITD and FLT3-ITD/D835 cells with the top 2 downregulated hits being the transcriptional activator Foxm1 and the cell cycle control transcription factor E2f. In contrast, quizartinib induced TSS gene expression changes only in FLT3-ITD cells (upregulated 7295, downregulated 6335) but not in FLT3-ITD/D835 cells; similar to selinexor, quizartinib upregulated Ddit3 and Klf6 and downregulated E2f in FLT3-ITD cells. The previously reported upstream activation of TP53 and CDKN2A and inhibition of Myc and estrogen receptor 1 were consistently observed in selinexor-treated FLT3-ITD and FLT3-ITD/D835 cells and in quizartinib-treated FLT3-ITD cells (Ingenuity Pathway Analysis). CAGE also demonstrated higher basic TSS transcription levels of Bcl-2 and Myc in FLT3-ITD/D835 cells than in FLT3-ITD cells, which were confirmed by Western blot.
Based on these findings, we focused on Bcl-2 as a novel therapeutic target in FLT3-ITD/D835 cells, and investigated the anti-leukemia efficacy of combined inhibition of Bcl-2 and XPO1. We utilized the clinically available selective Bcl-2 inhibitor venetoclax and observed synergistic anti-proliferative and pro-apoptotic effects of venetoclax and selinexor in FLT3-ITD/D835 (Combination Index, CI: 0.39 and 0.45). Simultaneous inhibition of Bcl-2 by venetoclax and FLT3 by quizartinib showed no synergistic or additive effects in FLT3-ITD/D835 cells. Western blot analysis showed that the combination of venetoclax / selinexor reduced Bcl-2 and Mcl-1 in FLT3-ITD/D835 cells, and activated cleaved caspase3. In FLT3-ITD cells, the venetoclax / selinexor combination induced only small decrease of Bcl-2, did not change Mcl-1 and Bcl-xL, and increased cleaved caspase3. These changes were not observed in wt-FLT3 transfected Ba/F3 cells.
Collectively, CAGE analysis of transcription start sites identified the primary mechanism underlying the synergistic activity of concomitant inhibition of Bcl-2 and XPO1 in FLT3-ITD/D835 cells as related to the regulation of Bcl-2, Mcl-1, Bcl-xL and c-Myc. These findings suggest that Bcl-2 inhibition by venetoclax combined with XPO1 inhibition by selinexor could be developed into a promising therapeutic strategy for TKI-resistant AML patients with FLT3-ITD and secondary acquired TKD mutations.

Disclosures
Konopleva: Stemline Therapeutics: Research Funding. Andreeff:AstraZeneca: Research Funding.