In a healthy adult, approximately 1011-1012 new blood cells are produced daily in order to maintain steady state levels in the peripheral circulation. Blood cells of all lineages are generated from small pools of long-lived hematopoietic stem cells (HSCs) that have the ability to continually replenish, self-renew, and generate differentiated progeny. DNA damage accrual is the principal factor that contributes to the functional decline in HSC renewal and in the immune system during ageing. In any cell, some DNA damage may remain despite the action of repair processes. DNA double strand breaks (DSBs) are the most deleterious form of DNA damage, caused by clastogens as well as natural cellular processes. Non-homologous DNA end-joining (NHEJ) is a major pathway that repairs DSBs in mammalian cells. It is critical for the repair of DSBs generated by V(D)J recombination (VDJ) and IgH class switch recombination (CSR), and general DSBs in all cell types, including those that suppress the capacity of HSCs to self-renew during ageing. Whether NHEJ repairs the DNA breaks required for the capacity of long-lived HSCs to replenish during ageing remains unclear. Lig4 is an ATP-dependent DNA ligase that has no known functions outside of NHEJ. In mice, a deficiency in Lig4 is embryonic lethal, and in humans, mutations in Lig4 underlie the rare genetic DNA repair disorder, DNA Lig4 Syndrome. The Lig4 Syndrome, among several defects, manifests in variable degrees of immunodeficiency associated with incomplete defects in VDJ and CSR, and bone marrow abnormalities that implicate potential HSC defects. We generated a knock-in mouse model of the Lig4 Syndrome R278H disease allele, a recessive hypomorphic mutation that significantly impairs the end-ligation function of the protein. Paralleling the disease, the young mice manifest incomplete defects in VDJ, CSR and age-dependent lymphopenia that implicate potential HSC defects, and predispose to malignant T cell lymphomas. Whereas long-lived HSCs are quiescent and are continually replenished, we find that these HSCs in the R278H mutant knock-in mice are substantially reduced, to have more accumulated DNA damage, and to produce elevated levels of reactive oxygen species. Together, these effects implicate a defect in their capacity to revert to quiescence, which could explain the defect in HSC homeostasis and the age-dependent lymphopenia in the mice. These and other observations we have made implicate a critical role for Lig4 in HSC homeostasis, and in regulations impacting the DNA damage response-signaling cascade and anti-oxidant defense system. Our proposed studies seek to further define the HSC and B cell defects in the R278H knock-in mice, to reveal the mechanisms underlying the defects in HSC homeostasis, VDJ and CSR, if they are related, and how they contribute to immunodeficiency. We anticipate the results of our studies will extend our knowledge of the normal functions of Lig4 protein and provide new insights into the pathobiology of LIG4 mutations with which to develop therapies relevant the DNA Lig4 Syndrome, to leaky SCID, and other immunodeficiency disorders.
The proposed project, which seeks to elucidate mechanisms that underlie defects associated with a rare recessive mutation (R278H) associated with the human LIG4 Syndrome has the potential to offer major breakthroughs towards understanding how mutations in DNA repair impacts on genomic stability and leaky severe combined immunodeficiencies in human disease. Elucidating impacts of this LIG4 mutation will extend our knowledge of how DNA damage response pathways function in producing a functional immune system and the suppression of genomic instability, and provide new insights into the pathobiology of DNA damage response mutations with which to develop new therapies relevant the DNA Lig4 Syndrome, and other immunodeficiency syndromes.
|Kang, Youn-Jung; Balter, Barbara; Csizmadia, Eva et al. (2017) Contribution of classical end-joining to PTEN inactivation in p53-mediated glioblastoma formation and drug-resistant survival. Nat Commun 8:14013|
|Park, Jihye; Welner, Robert S; Chan, Mei-Yee et al. (2016) The DNA Ligase IV Syndrome R278H Mutation Impairs B Lymphopoiesis via Error-Prone Nonhomologous End-Joining. J Immunol 196:244-55|
|Newton, Ryan H; Lu, Yu; Papa, Antonella et al. (2015) Suppression of T-cell lymphomagenesis in mice requires PTEN phosphatase activity. Blood 125:852-5|