XPO1 Inhibition Targets Transcriptional Vulnerability of FLT3-ITD+D835 Double Mutant AML through p53 Accumulation and Inhibition of Oncogenic Transcription Factors: Lesson Learned from Cage Sequencing of Primary AML Cells

Abstract Acute myeloid leukemia (AML) with Fms-like tyrosine kinase 3 / the internal tandem duplications (FLT3 /ITD mutations) present a major clinical challenge, because of short remission duration and higher relapse rates. FLT3-targeted therapies using tyrosine kinase inhibitors (TKIs) often promote acquisition of additional point mutations in the tyrosine kinase domains (TKD) mutations, most commonly at D835 within the activation loop. To elucidate the alterations of transcriptome signatures of FLT3 /ITD-D835 mutants in AML, we performed cap analysis of gene expression (CAGE) sequencing of 14 AML patient samples with FLT3 /ITD and 12 patient samples with FLT3 /ITD-D835 mutations. CAGE identifies and quantifies the 5' ends of capped mRNA transcripts, which enables the identification of transcription start sites (TSS) necessary for gene expression. The TSS of genes altered in AML samples with FLT3 /ITD-D835 compared with FLT3 -ITD were mapped. CAGE detected upregulation of 378 and downregulation of 77 genes, respectively (false discovery rate; FDR 1.9). To validate these transcriptional changes, we utilized paired isogenic FLT3 /ITD or FLT3 /ITD-D835Y transfected Ba/F3 cell lines. CAGE detected upregulation of 523 and downregulation of 373 genes, respectively, in FLT3 /ITD-D835 compared to FLT3 /ITD cells (FDR Exportin 1 (XPO1) mediates the nucleo-cytoplasmic transport, and is overexpressed in AML cells with FLT3 /ITD mutations (Kojima, Blood, 2013) . TP53 and IκBα are XPO1 cargos, and XPO1 inhibitors are known to upregulate TP53 and block c-MYC and NFκB signaling. We therefore examined the anti-leukemia activity of clinically available XPO1 inhibitor selinexor in FLT3 /ITD and FLT3 /ITD-D835 Ba/F3 cells. As expected, selinexor effectively inhibited cell proliferation in both FLT3 /ITD and FLT3 /ITD-D835 mutated cells at IC50 of 0.3 μM and 0.1 μM, respectively (48 hrs). CAGE detected upregulation of 1,590 and 2,220 genes and downregulation of 1,238 and 2,640 genes, respectively by selinexor treatment in FLT3 /ITD and FLT3 /ITD-D835 Ba/F3 cells, (FDR Collectively, the primary mechanism underlying the sensitivity to XPO1 inhibition of FLT3 /ITD-D835 cells is intolerance to the accumulation of nuclear TP53 and inhibition of multiple oncogenic transcription factors, including c-Myc, IκBα and HIF1α, which confer pro-survival activity independent of FLT3 downstream signaling. These findings indicate that XPO1 (CRM1) inhibition is a promising therapeutic strategy for AML patients with FLT3 /ITD and secondary acquired D835 mutations. Disclosures Harada: Celgene: Research Funding; NIPPON SHINYAKU CO.: Speakers Bureau; NOVARTIS: Research Funding. Andreeff: Daiichi Sankyo: Consultancy.