Genome-Wide shRNA Screen for DNA Damage Response Regulators in Human Hematopoietic Stem and Progenitor Cells

Abstract 1289

Hematopoietic stem cells due to their life-long function should protect genome integrity to avoid accumulation of genetic aberrations leading to malignant transformation or bone marrow failure. We reported recently that human HSC of cord blood origin are exquisitely sensitive to DNA damage-induced apoptosis. Thus, following 3Gy of ionizing radiation HSC show evidence of persistent DNA damage response and greater p53-dependent apoptosis in comparison with commited myeloid progenitors. To elucidate the molecular basis of these observations we carried out a genome-wide loss-of-function genetic screen using a library of 80 000 shRNA vectors targeting more than 16 000 human genes. A screen was performed on immortalized (by TLS-ERG infection) cord blood cells (TEX), which radio-sensitivity is similar to early hematopoietic cells. To find out the radio-protective hits we exposed infected cells to four rounds of 4Gy irradiation in three independent experiments. TEX cells exhibited steady increase of their proliferative potential after each irradiation exposure. Cells were gathered after each irradiation round and their DNA was subjected to sequencing to determine protective hits. Upon the analysis we recognized known regulators of DNA damage response (e.g. p53) and identified many genes that previously were not connected to genotoxic stress response. Thus, the knockdown of these genes was at least as effective as p53 knockdown in protection of TEX cells against gamma-irradiation. The validation of the chosen hits on TEX cells showed that about half of them indeed mediate the protection against irradiation. Further validation included real-time PCR of infected cells to ensure the absence of off-target effect along with Western blot analysis of affected protein. We followed up the investigation of chosen hits on primary human lineage-negative cord blood cells and observed that only few candidates were mediating the same effect on HSC/progenitors cells as on TEX cells. To further validate the effect of the most prominently effective hits we employed several shRNA vectors for the same target gene. Following this step of validation, we choose to investigate the knockdown of CHEK2, the gene with reported role in DNA damage response in several murine tissues. Our preliminary studies in long-term cytokine-supplemented cultures demonstrated that CHEK2 is involved in DNA damage response of human HSC and progenitors. The further investigation of the effect of CHEK2 knockdown on repopulating human HSC is currently underway. An integrated analysis of our observations revealed several putative CHEK2-centered molecular networks, which connect DNA damage response to HSC function.