Epidermal Growth Factor Receptor - Mediated DNA Repair Regulates Hematopoietic Stem Cell Regeneration

Hematopoietic myelosuppression is the principal dose-limiting toxicity of curative chemotherapy and radiotherapy in patients with cancer. Both chemotherapy and radiotherapy deplete hematopoietic stem cells (HSCs) via induction of DNA damage. DNA damage contributes to both HSC dysfunction and risk for malignant transformation over time. Extrinsic signals capable of promoting DNA repair in HSCs following injury can potentially improve HSC function and may decrease risk for dysplasia and leukemia over time. Here, we show that treatment with epidermal growth factor (EGF) decreases DNA damage in murine HSCs following irradiation via activation of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and nonhomologous end joining (NHEJ) repair. Specifically, we observed that in vitro treatment of irradiated bone marrow (BM) ckit+sca-1+lin- (KSL) hematopoietic stem/progenitor cells (HSPCs) with 100 ng/ml EGF decreased HSPC DNA damage at 1 hour as measured by gamma-H2AX foci and Comet assay (p<0.0001, p<0.0001). Further, EGF treatment increased phosphorylation and nuclear localization of DNA-PKcs, which is integral to NHEJ repair, and increased phosphorylation of Akt in irradiated HSPCs. Inhibition of Akt or DNA-PKcs completely abrogated EGF - mediated DNA repair and EGF - mediated recovery of HSPCs following irradiation in vitro. In irradiated (500 cGy) C57BL/6 mice, subcutaneous administration of 10 μg/day of EGF significantly increased recovery of peripheral blood white blood cells and lymphocytes (p=0.03, p=0.04), as well as BM SLAM+KSL cells (HSCs) at day +14 (p=0.0004). Treatment with the DNA-PKcs inhibitor, NU7441, abrogated EGF - mediated recovery of peripheral blood WBCs in irradiated mice (p=0.007), suggesting that EGF - mediated mitigation of radiation injury in vivo was dependent on DNA-PKcs - mediated NHEJ repair. Importantly, EGF treatment of irradiated mice also substantially increased the survival of irradiated mice compared to irradiated control mice (p=0.009) and caused the recovery of long-term HSCs capable of 20 week competitive repopulation in congenic recipient mice (p=0.01, 20 weeks).

Conversely, doxycycline inducible, cell - specific suppression of EGFR in hematopoietic cells using SCL-tTA;EGFR-DN mice caused a significant depressed regeneration of BM SLAM+KSL cells and KSL cells at day +10 after irradiation (p=0.005, p=0.02, respectively). SCL-tTA;EGFR-DN mice also displayed significantly decreased survival at day +30 compared to EGFR wild type mice (23.1% survival vs. 58.3% survival, p=0.04). Consistent with our hypothesis, BM HSPCs from EGFR-DN mice displayed increased DNA damage in response to total body irradiation, as measured by gamma H2AX foci and the Comet assay (p<0.0001, p=0.04). Coordinately, BM HSPCs from EGFR-DN mice displayed decreased phosphorylation of Akt and DNA-PKcs in response to irradiation in vitro (p<0.0001, p=0.004).

These studies suggest that EGFR - mediated DNA repair is necessary for HSC and progenitor cell recovery following irradiation and that EGF treatment accelerates hematopoietic regeneration via augmentation of NHEJ repair in HSCs. EGF treatment following high dose chemotherapy and/or radiotherapy may increase HSC function and lessen near - term hematologic toxicities and morbidities.