Session: 604. Molecular Pharmacology and Drug Resistance in Myeloid Diseases: Poster II
Hematology Disease Topics & Pathways:
apoptosis, Biological, Therapies, Biological Processes, genomics, TKI, molecular interactions
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.
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