3053 – DEFINING THE GENE REGULATORY NETWORKS TARGETED BY RUNX1 AND FLT3 INHIBITORS

Abstract

FLT3-ITD is a common activating receptor mutation in AML. We used genome-wide profiling to show that genes upregulated in FLT3-ITD+ AML had open chromatin regions (DHSs) enriched in binding sites for AP-1 and RUNX1 (1), within a gene regulatory network downstream of MAPK. We showed that MAPK inhibitors (i) suppress genes upregulated by FLT3-ITD, and (ii) inhibit growth of FLT3-ITD+ cells. Furthermore, concurrent RUNX1 mutations result in a suppression of genes normally activated by FLT3-ITD. We compared responses to two inhibitors targeting the FLT3-ITD gene regulatory network: (1)Gilteritinib (FLT3i) which targets FLT3-ITD, and (2)AI-14-91 (CBFbi) which blocks interactions between RUNX1 and CBFb (2). Overall, the responses to the 2 inhibitors were similar, suggesting that AP-1 and RUNX1 are in the same pathway. Treatment of FLT3-ITD+ AML cells with CBFbi or FLT3i led to a modest reduction in intensity of a subset of DHSs enriched in AP-1 and RUNX1 motifs. However, many pre-existing DHSs not normally bound by AP-1 acquired binding of FOS, at genes upregulated after either treatment. CBFbi suppressed FLT3-ITD AML cell cultures, but not healthy CD34+ blood stem cells, or AML cells with mutated RUNX1, suggesting that CBFbi plus FLT3i could form an AML therapy. To investigate CBFbi further, we performed cell culture assays on AML cells from a FLT3-ITD+ AML patient on AC220 FLT3 inhibitor therapy, who relapsed with a FLT3 D835H mutation. AML cells from this patient remained sensitive to culture in CBFbi, suggesting that CBFbi and MAPK inhibitors might form a second line of therapy in FLT3-ITD AML. (1)Assi et al. Subtype-specific regulatory network rewiring in acute myeloid leukemia. Nat Genet 51, 151-162 (2019). (2)Illendula et al. Small Molecule Inhibitor of CBFbeta-RUNX Binding for RUNX Transcription Factor Driven Cancers. EBioMedicine 8, 117-131 (2016). FLT3-ITD is a common activating receptor mutation in AML. We used genome-wide profiling to show that genes upregulated in FLT3-ITD+ AML had open chromatin regions (DHSs) enriched in binding sites for AP-1 and RUNX1 (1), within a gene regulatory network downstream of MAPK. We showed that MAPK inhibitors (i) suppress genes upregulated by FLT3-ITD, and (ii) inhibit growth of FLT3-ITD+ cells. Furthermore, concurrent RUNX1 mutations result in a suppression of genes normally activated by FLT3-ITD. We compared responses to two inhibitors targeting the FLT3-ITD gene regulatory network: (1)Gilteritinib (FLT3i) which targets FLT3-ITD, and (2)AI-14-91 (CBFbi) which blocks interactions between RUNX1 and CBFb (2). Overall, the responses to the 2 inhibitors were similar, suggesting that AP-1 and RUNX1 are in the same pathway. Treatment of FLT3-ITD+ AML cells with CBFbi or FLT3i led to a modest reduction in intensity of a subset of DHSs enriched in AP-1 and RUNX1 motifs. However, many pre-existing DHSs not normally bound by AP-1 acquired binding of FOS, at genes upregulated after either treatment. CBFbi suppressed FLT3-ITD AML cell cultures, but not healthy CD34+ blood stem cells, or AML cells with mutated RUNX1, suggesting that CBFbi plus FLT3i could form an AML therapy. To investigate CBFbi further, we performed cell culture assays on AML cells from a FLT3-ITD+ AML patient on AC220 FLT3 inhibitor therapy, who relapsed with a FLT3 D835H mutation. AML cells from this patient remained sensitive to culture in CBFbi, suggesting that CBFbi and MAPK inhibitors might form a second line of therapy in FLT3-ITD AML. (1)Assi et al. Subtype-specific regulatory network rewiring in acute myeloid leukemia. Nat Genet 51, 151-162 (2019). (2)Illendula et al. Small Molecule Inhibitor of CBFbeta-RUNX Binding for RUNX Transcription Factor Driven Cancers. EBioMedicine 8, 117-131 (2016).