signature=7a9af9bc8059ad018b006cc518187b69,Abstract 2701: Characterization of acquired EPZ-5676 resi...

摘要：

DOT1L inhibitor EPZ-5676 is currently under Phase 1 clinical trial investigation in relapsed/refractory patients with acute leukemia, including those with an MLL-rearrangement (MLL-r). Early clinical results, including complete remissions, support ongoing clinical development and preclinical investigation into mechanisms precipitating EPZ-5676 treatment induced resistance. MLL-r cell lines KOPN-8 (MLL-ENL) and NOMO-1 (MLL-AF9) were exposed to an EPZ-5676 concentration above the pre-determined 14 day proliferation assay IC. Initial treatment of the cell lines led to the expected inhibition of H3K79 dimethylation (H3K79me2) and MLL-r target genes HOXA9 and MEIS1 as outlined in previous work (Daigle et al, Cancer Cell 2011). Resistance to EPZ-5676 in both cell lines emerged following three weeks of continued treatment with EPZ-5676 and was defined by increased growth rates in the presence of inhibitor. Mechanisms of resistance for both cell lines were investigated using RNASeq and ChIPSeq on parental and resistant cell line pools. Our analysis identified common characteristics between the resistant cell lines, but mechanisms by which they became resistant differed. Global H3K79me2 inhibition was maintained in both refractory cell lines, yet ChIP-seq analysis of resistant pools identified specific loci with H3K79me2 recovery in KOPN-8 cells. In resistant KOPN-8 cells recovery of H3K79me2 was concentrated at the HOXA locus and other MLL-r target genes (e.g. MEIS1 and RUNX2), with the remainder of actively transcribed genes maintaining H3K79me2 inhibition at levels observed in parental cells. In contrast, resistant NOMO-1 cells did not recover H3K79me2 at any actively transcribed genes, including those of the MLL-r signature. Only resistant KOPN-8 cells regained expression of the MLL-r target genes HOXA9 and MEIS1. Of note both resistant NOMO-1 and KOPN-8 cell lines had 8 and 40 fold upregulation of the ABCB1 (MDR1, P-gp) tranporter respectively when compared to a matched control cell line. To explore the role of drug efflux transporter ABCB1 on resistance, we treated cells with Valspodar, a known inhibitor of ABCB1. Following treatment with 1 μM Valspodar, KOPN-8 cells showed decreased cell growth similar to the naïve control cell line. Supporting the presence of an alternative resistance mechanism in NOMO-1 cells in addition to MDR1 upregulation, NOMO-1 cells remained resistant upon Valspodar treatment. Detailed gene expression and pathway analysis will be presented supporting mechanisms of treatment emergent resistance to EPZ-5676. In summary, we have identified two mechanisms of EPZ-5676 resistance in MLL-r cell lines, one mechanism dependent on and the other independent of ABCB1. Further refinement of these mechanisms will aid in providing hypotheses for testing mechanisms of EPZ-5676 treatment emergent resistance in patients and may support designing future rational clinical combinations.

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