ABSTRACT

Glycogen synthase kinase-3b (GSK-3b) has been identified as an important regulator of stem cell function acting through activation of the wingless (Wnt) pathway. Here, we report that treatment with an inhibitor of GSK-3b, 6-bromoindirubin 3 0 -oxime (BIO) delayed cell cycle progression by increasing cell cycle time. BIO treatment resulted in the accumulation of late dividing cells enriched with primitive progenitor cells retaining the ability for sustained proliferation. In vivo analysis using a Non-obese diabetic/ severe combined immunodeficient (NOD/SCID) transplantation model has demonstrated that pretreatment with BIO promotes engraftment of ex vivo-expanded hematopoietic stem cells. BIO enhanced the engraftment of myeloid, lymphoid and primitive stem cell compartments. Limiting dilution analysis of SCID repopulating cells (SRC) revealed that BIO treatment increased human chimerism without increasing SRC frequency. Clonogenic analysis of human cells derived from the bone marrow of transplant recipient mice demonstrated that a higher level of human chimerism and cellularity was related to increased regeneration per SRC unit. Gene expression analysis showed that treatment with BIO did not modulate the expression of canonical Wnt target genes upregulated during cytokine-induced cell proliferation. BIO increased the expression of several genes regulating Notch and Tie2 signaling downregulated during ex vivo expansion, suggesting a role in improving stem cell engraftment. In addition, treatment with BIO upregulated CDK inhibitor p57 and downregulated cyclin D1, providing a possible mechanism for the delay seen in cell cycle progression. We conclude that transient, pharmacologic inhibition of GSK-3b provides a novel approach to improve engraftment of expanded HSC after stem cell transplantation.

Clinical trials of CD19-specific chimeric antigen receptor (CAR19) T cells have demonstrated remarkable efficacy against relapsed and refractory B cell malignancies. The piggyBac transposon system offers a less complex and more economical means for generating CAR19 T cells compared to viral vectors. We have previously optimized a protocol for the generation of CAR19 T cells using the piggyBac system, but we found that CAR19 T cells had poor in vivo efficacy and persistence, probably due to deleterious FcγR interactions with the CAR's IgG1 Fc-containing spacer domain. We therefore designed three CD19-specifc CARs that lacked the IgG1 Fc region, and we incorporated combinations of CD28 or 4-1BB transmembrane and co-stimulatory domains. PiggyBac-generated CAR19 T cells expressing these re-designed constructs all demonstrated reactivity in vitro specifically against CD19 cell lines. However, those combining CD28 transmembrane and co-stimulatory domains showed CD4 predominance and inferior cytotoxicity. At high doses, CAR19 T cells were effective against B-ALL in a xenograft mouse model, regardless of co-stimulatory domain. At diminishing doses, 4-1BB co-stimulation led to greater potency and persistence of CAR19 T cells, and it provided protection against B-ALL re-challenge. Production of potent CAR T cells using piggyBac is simple and cost-effective, and it may enable wider access to CAR T cell therapy.