Conditional Deletion of STAT5 in Non-Myeloablated Recipient Mice Promotes Donor Hematopoietic Stem Cell Engraftment.
Authors: Wang Zhengqi , Li Geqiang , Haviernikova Eleonora , Tse William , Bunting Kevin D. 
Published: April 2020

Currently there is a major need in stem cell transplantation to develop innovative conditioning regimens that allow a wider range of patients to recieve therapy. Signal transducer and activator of transcription-5 (STAT5) plays major roles in hematopoietic stem and progenitor cell engraftment. Our previous studies have reported that one injection of 1 x 107 wild-type bone marrow (BM) cells into unconditioned newborn STAT5-deficient mice resulted in high levels of donor engraftment. Here we describe studies to develop novel non-myeloablative conditioning approaches based on suppression of STAT5 in vivo followed by donor cell injection (1 x 107 cells, newborns; 5 x 106 cells, adults). We have used two genetic models for STAT5 deficiency in order to test feasibility; true null STAT5 knockout mice containing deleted STAT5a and STAT5b loci or a conditional knockout mouse with STAT5ab loci flanked by loxP sites. These allowed us to examine engraftment of donor cells into STAT5 deficient hosts. We found that STAT5ab+/null hosts could be engrafted to very high levels (9/13 mice engrafted at 10–33%) when injected with BM cells as newborn pups but not surprisingly engraftment was much lower in adult 4–6 week old STAT5ab+/null mice (5/6 mice engrafted at 1–3%). In both cases, the levels of engraftment were significantly higher than in wild-type control mice. Next, we utilized conditional Mx1-Cre/STAT5abflox/null mice to determine the effects of de novo loss of STAT5 in adult mice. Adult 4–6 week old mice were treated with 16 mg/kg pI:pC on days 1, 3, 5 to bring down STAT5 levels further. pI:pC treatment did not present toxicity and resulted in complete deletion in circulating Gr-1+ and B220+ cells. Notably, the CD4+ T-lineage remained ∼50% undeleted indicating a strong selection for the undeleted T cell progenitors. In sorted primitive c-Kit+LinnegSca-1+ cells, deletion was &gt;95%. STAT5 deletion and reduced circulating B cells and myeloid cells were sustained up to 16 weeks after the initial round of treatment without a re-emergence of the undeleted cells in non-T cell lineages. When mice treated with this regimen were injected with donor BM on day 7 we found that treatment lead to high level sustained multilineage engraftment of primary and secondary hosts at times up to 16 weeks following injection (4/4 mice engrafted with donor Gr-1+, B220+, Ter119+, and CD4+ cells between 12–78%). These data demonstrate that STAT5 can be conditionally knocked out in transplant recipients and this leads to sustained donor reconstitution in the absence of any myeloablative conditioning. Since the extremely high levels of deletion of STAT5 might be difficult to achieve therapeutically, we have also begun exploring whether there is a benefit to the combined blockage of Gab2 signaling and/or PI3-kinase in STAT5ab+/null hosts based on our observations of STAT5/Gab2 genetic interaction during hematopoiesis (Li et al., ASH 2007 abstract). In this very stringent model of a more clinically applicable situation, we have found that STAT5ab+/nullGab2−/− mice (2/8 mice engrafted at &gt;4%) are more highly and frequently engrafted than STAT5ab+/nullGab2+/+ mice (0/8). Overall, these studies provide strong genetic proof-of-principle for modulation of STAT5 as an adjuvant for transplantation conditioning. New methods for non-myeloablative hematopoietic stem cell engraftment through combinatorial signaling inhibition may ultimately be applicable in combination with cell and gene therapies for hematologic disorders.