PB1776: ABC TRANSPORTERS AFFECT THE SENSITIVITY OF ACUTE MYELOID LEUKEMIA BLASTS TO CHEMOTHERAPY

Abstract

Background: Approximately 40 % of acute myeloid leukemia (AML) patients does not achieve a complete hematologic remission (CR) after the first cycle of 3 + 7 induction therapy and thus face an adverse prognosis. Mechanisms of chemoresistance are still poorly understood, but drug export from cytosol plays a crucial role. 7 ABC transporters (ABCA2, ABCA5, ABCB1, ABCB6, ABCC1, ABCC3, ABCG2) were selected from literature for expression, methylation, genotype and efflux activity analysis. Aims: The aim of this study is to clarify the role of selected ABC transporters in reduced sensitivity to chemotherapy in AML. Methods: Peripheral blood was collected at diagnosis (day 0) and at the third day of 3 + 7 therapy (day 3). Leukemic blasts were separated on MACS (CD34+ or CD117+, Miltenyi Biotec). Gene expression was measured on StepOne Plus (Applied Biosystems) using TaqMan assays, B2M and YWHAZ were used as reference genes (n=85). Promotor, exons and splicing regions were sequenced on MiSeq (V2, Illumina) using Roche reagents (n=109). Methylation analysis was performed on Pyromark Q24 (Qiagen) for 85 CpGs in promoters of ABCA2, ABCC1 and ABCG2 (n=51). Efflux activity was assessed with Efflux-ID kit (Enzo) (n=12). For data analysis, we divided patients by primary response status into responding and refractory group based on a CR achievement after the first cycle of 3 + 7 therapy. Data analysis was performed in GraphPad Prism 9.1.2 and R software 4.0.0. Results: Higher expression of ABCA2 (p=0.005), ABCA5 (p=0.016), ABCC1 (p<0.0001) and ABCG2 (p=0.001) at day 0 was associated with primary treatment failure. ABCA2 (p=0.018) and ABCC1 (p=0.008) remained significant in the Cox regression analysis adjusted to age, leukocyte count at diagnosis and ELN risk stratification. Moreover, higher number of concurrently overexpressed ABC transporters (threshold defined by Cutoffinder) was predictive of failure in CR achievement (p=0.006) by Cox regression. We detected a significant rise of expression at day 3 in ABCA5 (p=0.003), ABCB6 (p=0.001) and ABCC3 (p<0.0001). Significantly higher rise of ABCC3 (p=0.029) expression was detected in responders compared to refractory patients. In terms of a CR achievement, we identified 6 unfavorable DNA variants (rs2301837, rs8077268, rs17563146, rs1373970189, rs113369901, 9:137022522-137022522), and 3 favorable DNA variants (rs556735807, rs2235048, rs1407267536). The risk variant rs113369901 correlated with higher expression of ABCC1 (p=0.039), which was also associated with chemoresistance. Cox regression analysis confirmed prognostic relevance for four variants: rs2301837 (p=0.032), rs8077268 (p=0.033), 9:137022522-137022522 (p=0.003) and rs1373970189 (p=0.039). Higher methylation of ABCG2 promoter correlated with its lower expression (p=0.0001). Promoters of ABCA2 and ABCC1 were basically unmethylated, especially ABCC1, which had the highest expression among the studied genes. The greatest contribution to cell efflux had transporters inhibited by MK-571 inhibitor, following by Verapamil. These influence activity of ABCC1 and ABCB1. Summary/Conclusion: We described differences in expression levels and genetic variants of selected ABC transporters in association with treatment response in AML. ABCC1 seems to contribute substantially as illustrated by expression, genotyping, DNA methylation as well as activity data. With such detailed knowledge of chemoresistance mechanisms in leukemic cells and an emergence of new drugs, a different therapeutic strategy for chemoresistant patients may be considered in the future. Supported by MHCR (00023736, IHBT).