Abstract 3098: Leukemia stem cells demonstrate enhanced DNA damage repair and chemoresistance in AML

Abstract

Abstract Leukemia stem cells demonstrate enhanced DNA damage repair and chemoresistance in AML Relapse of acute myeloid leukemia (AML) is common and the main cause of mortality in this disease. Leukemia stem cells (LSCs) contribute to relapse however the mechanisms underpinning chemoresistance are not well understood. Here, we explored the DNA damage response in AML LSCs to investigate if enhanced DNA repair contributes to relapse and chemoresistance. We discovered that expression of DNA repair pathway genes were up-regulated in functionally defined LSC vs bulk AML cells (n= 227, accession number GSE76008), NES = 1.74, FDR = 0.057, and undifferentiated (n= 7) vs committed AML samples (n = 4, Princess Margaret Cancer Centre cohort), NES = 2.041, FDR = 0.000. Next, we investigated the expression of DNA damage repair genes and response to DNA damage in FACs sorted stem and bulk fractions of 8227 cells. 8227 cells are low passage primary AML cells that maintain a hierarchical organization with functionally defined stem cells in the CD34+CD38- fraction. Compared to bulk cells, the stem cell fraction of 8227 cells had increased expression of genes associated with DNA repair through homologous recombination (RAD51, XRCC2, XRCC3) and non-homologous end joining (XRCC4, XRCC5, PRKDC). Sorted 8227 cells were treated with daunorubicin, an intercalating anthracycline that causes double stranded breaks. DNA damage and repair were evaluated by measuring foci of 53BP1, RAD51 and γH2AX by fluorescent microscopy and quantified using image J. Compared to bulk cells, 8227 stem cells demonstrated increased DNA damage repair with increased foci of 53BP1 and RAD51 and decreased γH2AX foci. We recently discovered that the metabolic enzyme hexokinase 2 (HK2) localizes to the nucleus to maintain stem cell number and function. Therefore, to understand how stemness impacts DNA damage repair, we selectively over-expressed HK2 in the nucleus of 8227 and NB4 cells by tagging HK2 with a nuclear localizing sequence. Over-expressing nuclear HK2 increased stem cell function as shown by clonogenic growth assays and engraftment into mouse marrow. We then treated these cells with daunorubicin and measured DNA damage repair. Increasing stem cell number and function by over-expressing nuclear HK2, increased 53BP1 and RAD51 foci and decreased γH2AX foci, similar to the phenotype observed in LSCs. As measured by the COMET assay, these cells had decreased levels of double strand DNA breaks under basal conditions and increased repair of DNA breaks after treatment with daunorubicin treatment. Over-expression of nuclear HK2 also conferred resistance to daunorubicin as measured by clonogenic growth assays. In summary, LSCs have increased levels of DNA repair genes and increased rates of DNA damage repair after exposure to chemotherapy. The accelerated DNA damage repair seen in LSCs may contribute to chemoresistance and subsequent disease relapse. Citation Format: Grace Egan, G.E. Thomas, Parasvi Patel, Veronique Voisin, Rose Hurren, Neil MacLean, Razq Hakem, Aaron D. Schimmer. Leukemia stem cells demonstrate enhanced DNA damage repair and chemoresistance in AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3098.