Lnk/Sh2b3 Deficiency Increases DNA Damage Tolerance to Promote Hematopoietic Stem Cell Fitness

Abstract

DNA repair deficiency depicted by the syndrome Fanconi Anemia (FA), results in progressive bone marrow failure (BMF), increased leukemia incidence and cancer susceptibility. Out of the 23 causative genes for FA, FANCD2 is crucial for its central role in the regulation of DNA repair and replication. The FA pathway plays an important role in maintaining hematopoietic stem and progenitor cell (HSPC) homeostasis and genome integrity. Our previous studies revealed that loss of the adaptor protein LNK (SH2B3), a negative regulator of cytokine signaling, restores hematopoietic stem cell (HSC) function and genome integrity in Fancd2 deficient FA mouse models and primary human FA progenitors. Notably, mice deficient in Lnk harbor a remarkable 14-fold expansion of HSCs with superior self-renewal capability. However, the protective mechanisms that LNK controls to prevent stem cell attrition and increase stem cell fitness are not established. Here, we show that Lnk deficiency reduced DNA damage in Fancd2-/- HSCs at the steady-state in vivo, as well as in forced proliferation upon transplantation. Lnk deficiency reduced HSPC DNA damage in replication stress upon cytokine-induced proliferation in ex vivo culture, accompanied by reduced p53 activation. One injection of pIpC induces HSPC into the cell cycle within 24hr, thus we used this as a model to study replication stress in vivo. We found that Lnk deficiency reduced ATR but not ATM pathway activation in Fancd2-/- HSCs in pIpC-induced proliferation in vivo. ATR is activated by single-strand DNA breaks (SSBs) during replication stress; thus we directly examined the amount of single-strand DNA (ssDNA) and the level of ssDNA binding protein RPA (Replication Protein A) in HSCs during the cell cycle. Of note, Lnk deficiency reduced ssDNA as well as chromatin-bound RPA2 in Fancd2-/- HSCs in replication in vivo. Lnk deficiency also reduced RPA2 phosphorylation in HSPCs under Camptothecin (CPT) and hydroxyurea (HU) induced replication stress conditions. Mechanistically, loss of Lnk protected stalled replication fork from degradation, suppressed ssDNA gaps, and promoted replication fork restart in Fancd2-/- HSPCs upon HU-induced replication stress. In light of our data suggesting a new role for LNK in replication stress-induced DNA damage, we examined the DNA damage tolerance (DDT) mechanism that enables DNA replication to circumvent the lesions, thereby allowing the completion of DNA replication, increasing the survival of HSPCs and preventing genome instability. One of the major DDT pathways is translesion synthesis (TLS) in which the replicative DNA polymerase is temporarily replaced by special TLS polymerases pol ζ or η that can replicate across DNA lesions. Indeed, we found that Lnk deficient HSCs have increased chromatin-bound TLS polymerase eta (Pol ƞ) and were resistant to shRNA-mediated knockdown of PolH (the gene encodes polymerase eta) compared to wildtype HSCs in transplant assays. Strikingly, Lnk deficient HSCs were more resistant to the inhibitor of Rev1 that disrupts Rev1/ pol ζ interaction than wildtype HSCs in transplanting assays. These findings highlight a previously-unappreciated role of LNK in limiting DNA damage tolerance mechanisms in both normal and diseased replicative states. The novel link between LNK-controlled cytokine signaling and TLS sheds light on our understanding of replication stress mitigation to promote HSC fitness that can be explored for future regeneration medicine.