While most patients with AML achieve remission with standard induction chemotherapy, the majority ultimately relapse. Relapsed AML is due, at least in part, to the persistence of chemoresistant leukemia stem cells (LSCs). The mechanisms of chemoresistance in LSCs are not fully understood. Here, we explored DNA damage repair in LSCs.
8227 cells are low passage primary AML cells that maintain a hierarchical organization with functionally defined stem cells in the CD34+CD38- fraction. We FACS sorted 8227 cells into stem and bulk fractions and measured expression of DNA repair genes. LSCs were primed for DNA repair with increased expression of genes associated with homologous recombination (RAD51, XRCC2, XRCC3) and non-homologous end joining (XRCC4, XRCC5, PRKDC). Next, we treated the cell fractions 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 enhanced DNA damage repair with increased foci of 53BP1 and RAD51 and decreased γH2AX foci, compared to their basal levels. Similar findings were noted after exposing the stem and bulk cells to radiation.
We recently discovered that the metabolic enzyme hexokinase 2 (HK2) localizes to the nucleus to maintain stem cell number and function. Therefore, we selectively over-expressed HK2 in the nucleus of 8227 and NB4 cells by tagging HK2 with a nuclear localizing sequence (PAAKRVKLD). We confirmed selective over-expression of HK2 in the nucleus by immunoblotting and confocal microscopy. Over-expressing 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. Over-expression of nuclear HK2 increased 53BP1 and RAD51 foci with decreased γH2AX foci, similar to the phenotype observed in LSCs. In addition, over-expression of nuclear HK2 conferred resistance to daunorubicin as measured by clonogenic growth assays.
In summary, LSCs appear to be primed for DNA repair with increased levels of DNA damage repair genes. After exposure to chemotherapy and radiation, LSCs have increased repair of double strand DNA breaks compared to more differentiated blasts. This accelerated DNA damage repair may partly explain the increased chemoresistance seen in LSCs.
Schimmer:Takeda:Honoraria, Research Funding;Novartis:Honoraria;Jazz:Honoraria;AbbVie Pharmaceuticals:Other: owns stock ;Otsuka:Honoraria;Medivir AB:Research Funding.
Asterisk with author names denotes non-ASH members.