Abstract 3174: GCN5 regulates ATM mediated DNA repair responsible for onset of acquired resistance in leukemia

Abstract Introduction- Resistance to therapeutics targeting topoisomerase 2 is a major problem in the treatment of leukemia. Cells which survive and give rise to relapsed leukemia are known to modulate different pathways like downregulation of drug target, reduced drug accumulation, and improved DNA repair; ultimately leading to survival of drug resistant cells. Here, we wanted to identify the earliest detectable changes occurring when cells become resistant to topoisomerase 2 inhibitors. Methodology- We generated early and late stage drug (doxorubicin) resistant leukemic sub cell line from K562 and THP-1 parent cells, HL-60/MX2 resistant sub cell line of HL60 was also used for this study. Molecular changes were analyzed by electron microscopy, quantitative PCR, western blotting, immunofluorescence, flow cytometry, molecular inhibitors and MTT assay. Results were further confirmed in blast cells of AML (n=44) patient samples collected for this study. Furthermore, meta-analysis for survival was done from microarray expression data of 221 patient samples using PrognoScan. Results- We mapped molecular changes acquired by leukemic cells during evolution from Early Drug Resistant Population (EDRP) to Late Drug Resistant Population (LDRP). We found unlike LDRP, EDRP cells do not possess known bona fide drug-resistance mechanisms namely limited drug accumulation, reduced DNA damage or expression of drug target. Instead they survive by acquiring ‘poised epigenetic state’ that enhanced their DNA repair. Mechanistically, GCN5, a histone acetyl transferase get selectively upregulated in EDRP cells mediating higher H4K16 acetylation levels and consequent euchromatin state of EDRP. Upon Doxorubicin treatment, H4K16ac facilitate higher ATM recruitment and activation causing efficient activation of H2AX, NBS1, BRCA1, Chk2 and Mcl-1, accelerating DNA repair and survival of EDRP cells. Inhibition of GCN5 with Doxorubicin treatment significantly reduces H4K16ac levels, ATM recruitment and cell survival of EDRP cells. Similarly, ATM inhibition along with Doxorubicin completely ablates EDRP but not LDRP. Furthermore, baseline AML patient samples (n=44) showed significantly higher GCN5 expression in MRD positive compared to MRD negative samples. Additionally, meta-analysis of 221 AML patients showed increased GCN5 expression associates with poor survival of AML patients. Conclusion- We identify GCN5 expression as marker that defines onset of resistance in leukemia and GCN5 med