Self-Renewal and BM Niche Occupancy Defects In NHEJ Deficient HSCs.

Abstract 1455

Maintenance of hematopoietic stem cells (HSC) requires proper interaction between HSC and the bone marrow niche. DNA repair proteins, especially proteins involved in nonhomologous end joining (NHEJ), are critical for HSC maintenance. We hypothesize that NHEJ deficient HSCs have a BM niche occupancy defect.

NHEJ pathway is the major mechanism for double strand break repair in mammalian cells, several proteins participate in the process. Ku70 recognizes and binds to the DNA DSB ends with Ku80, recruits DNA activated catalytic polypeptide (Prkdc) to form DNA dependent protein kinase complex, and further recruits Artemis to process the ends and DNA ligase 4 to rejoin the broken ends. NHEJ deficient cells are hypersensitive to irradiation, and NHEJ deficient mice display SCID phenotypes.

We have previous shown that Ku70 deficient HSCs are defective in repopulation as well as BM niche occupancy. Prkdc is another key factor in the NHEJ pathway, and mice homologous for a spontaneous nonsense mutation (Prkdc^scid, commonly referred to as scid) are characterized by an absence of functional T cells and B cells due to the inability of V(D)J recombination. Though scid mice has been widely used for human HSCs engraftment studies and the nonobese diabetic/severe combined immune deficiency (NOD/scid) xenotransplantation model is now the “gold standard” for assaying human HSC activity, the function and BM niche occupancy capacity of HSCs of scid mice origin has not been characterized.

scid mice show comparable HSC frequency and similar apoptosis rates compared to HSC from WT mice. To assess scid HSC function in vivo, single and competitive serial transplantation was performed. BM from scid mice were able to reconstitute the myeloid lineage (Mac1+) but not lymphoid lineages (CD3+ or B220+) in primary recipients due to the deficiency in V(D)J recombination. Serial transplantation was hampered by a high frequency of donor-derived thymic lymphomas in primary recipients. Competitive repopulation assays between WT and scid BM cells showed that when scid BM were mixed with WT competitor BM at a 1:1 ratio, scid BM cells were completely outcompeted by the WT BM cells.

To investigate the BM niche occupancy ability of scid HSCs, we used the hematopoiesis niche occupancy assay, in which 5×10⁶ WT congenic BM cells were transplanted into WT and scid recipients without any recipient conditioning and evaluated for blood and marrow cell origins at 16–24 weeks. Remarkably, transplanted WT BM made a long-term multi-lineage (Mac1+, CD3+ and B220+) contribution to hematopoiesis as well as HSC engraftment in scid recipients, (18.4+ 3.8% Mac1+, all the CD3+, B220+ cells, and 8.2+ 2.6% of HSC are donor derived). By contrast, less than 1% stem cell engraftment occurred in WT recipients as expected. Thus, have a long term niche occupancy defect impacting hematopoiesis.

NHEJ is involved in both DNA double strand break repair and V(D)J recombination in lymphocytopoiesis. To determine whether lymphocytopoiesis is required for HSC function and BM niche occupancy, RAG1 deficient mice were used in the competitive repopulation assay and the hematopoiesis niche occupancy assay. Our results showed that RAG1-/- HSCs competed with WT HSC did not have defects in competitive repopulation or BM niche occupancy.

These data demonstrate that Prkdc, a key component of NHEJ, is required for HSC function. Prkdc deficiency in the HSC compartment results in loss of competitive repopulation ability and loss of long term BM niche occupancy. Together with our previous results obtained from Ku70-/- mice, we conclude that NHEJ is critical for HSC self-renewal and BM niche occupancy, a function independent of its role in lymphogenesis. These data also explains the nature of the conducive marrow niche environment of scid mice for xenotransplantation.