Supplementary MaterialsbloodBLD2020005895-suppl1. deletion of EGFR in hematopoietic stem and progenitor cells (HSPCs) significantly decreased DNA-PKcs activity following irradiation, causing improved HSC DNA damage and stressed out HSC recovery over time. Systemic administration of epidermal growth factor (EGF) advertised HSC CAPN2 DNA restoration and quick hematologic recovery in chemotherapy-treated mice and experienced no effect on acute myeloid leukemia growth in vivo. Further, EGF treatment drove the recovery of human being HSCs capable of multilineage in vivo repopulation following radiation injury. Whole-genome sequencing analysis revealed no increase in coding region mutations in HSPCs from EGF-treated mice, but improved intergenic copy quantity variant mutations were detected. These studies demonstrate that EGF promotes HSC DNA restoration and hematopoietic regeneration in vivo via augmentation Ethyl dirazepate of NHEJ. EGF offers therapeutic potential to promote human being hematopoietic regeneration, and further studies are warranted to assess long-term hematopoietic effects. Visual Abstract Open in a separate window Intro Ionizing radiation (IR) and chemotherapy cause DNA damage in hematopoietic stem and progenitor cells (HSPCs), therefore contributing to a risk for hematopoietic stem cell (HSC) dysfunction, accelerated ageing, and malignancy over time.1-5 Eukaryotic cells repair DNA damage primarily through homologous recombination (HR) and nonhomologous end-joining (NHEJ) repair mechanisms.1,2 HSCs, which are largely quiescent in the steady-state, primarily undergo NHEJ in response to IR, whereas proliferating HSCs and progenitor cells are able to undergo HR.1 NHEJ is considered a more error-prone mechanism than HR, potentially resulting in increased deletions, insertions, translocations, and genomic instability.2,3 Mohrin et al reported that NHEJ in quiescent HSCs was associated with increased genomic rearrangements that persisted in vivo.1 Because IR and chemotherapy induce genomic instability in HSCs and increase the risk of malignant transformation, the development of therapies capable of reducing DNA damage or increasing DNA restoration in HSCs could be highly beneficial. Recently, de Laval et al shown that thrombopoietin stimulated DNA restoration in HSCs via Ethyl dirazepate augmentation of DNA-dependent protein kinase (DNA-PK)Cdependent NHEJ, and this DNA-PK activation was dependent on Erk and NF-B pathway activation.6,7 The broader part of extrinsic signals in regulating DNA restoration in HSCs remains poorly understood.6,7 We previously showed that high-dose total body irradiation (TBI) depletes bone marrow (BM) HSCs and encourages myeloid skewing and immune cell depletion in mice.8 Systemic administration of epidermal growth element (EGF), which is indicated by BM endothelial cells (ECs), mitigated these effects of TBI and promoted hematopoietic regeneration in vivo.8 However, the precise molecular mechanisms through which EGF advertised hematopoietic regeneration remained unclear. In tumor cells, epidermal growth element receptor (EGFR) can promote DNA restoration via activation of DNA-dependent protein kinaseCcatalytic subunit (DNA-PKcs).9-11 Here, we display that EGF treatment promotes HSC recovery and hematopoietic regeneration via augmentation of DNA-PKcs activity and NHEJ restoration in HSCs. EGFR is vital for activation of NHEJ fix in HSPCs and hematopoietic regeneration in vivo pursuing TBI. EGF treatment also boosts NHEJ fix in individual HSCs pursuing irradiation and promotes the recovery of individual HSCs with in vivo repopulating capability. Strategies Stream cytometry BM cells from tibia and femurs had been gathered in Iscove improved Dulbecco moderate, 10% fetal bovine serum, and 1% penicillin-streptomycin, pursuing red blood cell lysis with ACK Buffer (MilliporeSigma, Burlington, MA). Cells were stained with V450 Mouse Lineage Antibody (BD Biosciences, San Jose, CA), c-kit (CD117) PE Rat Anti-Mouse (BD Biosciences), and Sca-1 APC-Cy7 Rat Anti-Mouse (BD Biosciences) to measure the percentage of ckit+sca-1+lin? (KSL) cells. Cells were also stained with Alexa Fluor 488 Anti-Mouse CD41 Antibody (BioLegend, San Ethyl dirazepate Diego, CA), FITC Hamster Anti-Mouse CD48 (BD Biosciences), and Alexa Fluor 647 Rat Anti-Mouse CD150 (BD Biosciences) to measure the percentage of CD150+CD48?CD41? KSL HSCs.12,13 For hematopoietic engraftment analysis, Brilliant Violet 605 Anti-Mouse CD45.1 Antibody (BioLegend), FITC-CD45.2, PE-Mac-1 (CD11b), PE-Gr-1 (Ly-6G and Ly-6C), V450-CD3, and APC-Cy7-B220 (CD45R) (BD Biosciences) were used. For analysis of phosphorylated.