We hypothesize that the frequent chromosomal aberrations found in treatment-related acute myeloid leukemia/myelodysplastic syndrome (t-AML/t-MDS) reflect dysregulation of DNA double strand break (DSB) repair. We performed functional studies of DSB repair using primary bone marrow cells from 15 t-AML/t-MDS patients and CD34+ cells from 5 normal donors. We evaluated DSB by measuring phosphorylated histone H2AX (pH2AX), a well-established marker for DSB, in myeloblasts (CD45 dim, low side scatter) and lymphocytes (a surrogate for normal cells). Baseline measurements of primary cells, coupled with a time course to measure pH2AX induction and decay after 2 Gy of irradiation (IR) were used to assess the basal DSB burden and response to acute damage, respectively.
We found that 4 of 15 t-AML/t-MDS patients had myeloblasts that displayed baseline and post-damage pH2AX levels similar to normal CD34+ controls, while 11/15 patients had abnormal pH2AX levels which fell into one of three major patterns. 1) The first subset of patients had impaired pH2AX induction compared to normal donor CD34+ cells (1.44 vs 2.97 fold increase in pH2AX over baseline, respectively, p<=0.01), suggesting a defect in detecting DSBs. 2) A second subset of patients had delayed resolution of pH2AX levels compared to CD34+ controls post IR either at 4 hours (mean 1.54 fold higher than CD34+ control cells, p<0.05) or delayed resolution over 24 hours compared to controls (p<0.001). 3) The final subset had myeloblasts in which baseline pH2AX levels were elevated compared to CD34+ cells (2.01 fold, p<=0.002) suggesting an increased basal DSB burden in these cells. This phenotype was unique to patients with trisomy 8 and was tumor specific, as their lymphocytes displayed pH2AX levels similar to those from normal controls. The neutral Comet assay confirmed the presence of significantly elevated DSB in myeloblasts from 5/5 t-AML/t-MDS patients with trisomy 8 as compared to CD34+ controls (mean percent DNA in tail, 31.9 vs 11.8 respectively; p=0.002). Furthermore, myeloblasts from 4/5 of these patients had elevated basal DSB compared to those from the t-AML/t-MDS patients with normal pH2AX kinetics (p=0.004). Elevated basal DSB may be due to increased cells in S phase (replication fork collapse), increased apoptotic cells, or increased spontaneous and/or persistent DNA damage in cells harboring trisomy 8. The percent of myeloblasts in S phase from patients with trisomy 8 was not significantly different than those with normal pH2AX kinetics, either at baseline (mean 21vs 25.7, respectively, p=0.42) or 24 hours after mock irradiation (mean 49.7 vs 48.1, respectively, p=0.92). Furthermore, the percent of apoptotic (caspase+PI+) myeloblasts from the patients with trisomy 8 was not significantly different than those with normal pH2AX kinetics at baseline (mean 2.57 vs 1.87, respectively, p=0.77) or 24 hours after mock irradiation (mean 0.32 vs 0.94, respectively, p=0.39). Although it is possible that caspase negative early apoptotic cells could contribute to the elevated DSBs, there is no attrition of trisomy 8 myeloblasts compared to those with normal pH2AX kinetics after 24 hours (mean fold increase in cells 1.72 vs 0.88, respectively, p=0.13) in the setting of similar S phase percentages; further, the pH2AX levels in unirradiated cells after 24 hours remain significantly elevated in trisomy 8 myeloblasts versus those with normal pH2AX kinetics (2.01 fold, p<0.001) and CD34 controls (2.11 fold, p<0.001). Collectively, these results are most consistent with spontaneous and/or persistent DNA damage existing in myeloblasts harboring trisomy 8 and suggest that these cells may tolerate an elevated DSB burden.
Of note, MYC is located on chromosome 8 and has been shown to induce DNA damage. Gene expression analysis showed that MYC is significantly overexpressed in samples with trisomy 8 as compared to CD34+ controls (p=0.01). To test whether overexpression of MYC in primary hematopoietic progenitor cells induces DSBs, we transduced mouse ckit+ cells with MSCV-MYC-ires-GFP or MSCV-ires-GFP retrovirus. MYC- overexpressing cells had elevated DSBs compared to control GFP+ cells (p<0.001), suggesting that MYC overexpression contributes to the spontaneous and/or persistent DNA damage in myeloblasts harboring trisomy 8. The ability of these cells to persist in the setting of elevated DSB may provide a mechanism for chemoresistance.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.