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

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 < 0.05). Ingenuity Pathway Analysis (IPA) highlighted the activation of transcription factors NFKBIA (IκBα), RELA (NFκB subunit), HIF1α, EP300 (co-activator of HIF1α), TP73, PRDM1 (regulator of TP53 activity), NUPR1 (regulator of p21 relocalization), and FOXO3 (post-transcriptional regulators of MYC) in FLT3 /ITD-D835 mutant primary patient samples compared to FLT3 /ITD primary samples (Z score >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 < 0.05). IPA indicated activation of HIF1α and MYC and inhibition of TP53 in FLT3 /ITD-D835 compared to FLT3 /ITD cells. Immunoblot analysis confirmed higher expression of c-Myc, IκBα and HIF1α and lower expression of TP53 in FLT3 /ITD-D835 compared to FLT3 /ITD cells. In turn, FLT3 downstream targets phospho- (p-) ERK, p-AKT, and p-STAT5 were significantly lower in FLT3 /ITD-D835 than in FLT3 /ITD cells. These results indicate that secondary FLT3 -D835 mutations activate oncogenic transcription factors c-Myc, IκBα and HIF1α, inhibit tumor suppressor TP53, and downregulate FLT3 downstream signaling pathways ERK, AKT, and STAT in FLT3 /ITD mutated AML cells.

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 < 0.05). IPA analysis highlighted TP53, NUPR1 and CDKN2A (ARF; stabilizer of TP53) as the top three activated transcription factors by selinexor both in FLT3 /ITD and FLT3 /ITD-D835 cells. Immunoblot assay detected that selinexor significantly upregulated TP53, and downregulated IκBα and HIF1α both in FLT3 /ITD and FLT3 /ITD-D835 Ba/F3 cells. Selinexor treatment suppressed c-Myc and increased cleaved caspase 3 more prominently in FLT3 /ITD-D835 cells, but also upregulated p-ERK, p-AKT, p-STAT5 and p-STAT3, more notable in FLT3 /ITD cells. MCL1 and mTOR downstream p-4EBP1 and p-pS6 were repressed by selinexor in FLT3 /ITD-D835 but not in FLT3 /ITD cells.

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.