UC San Diego

Leukemia stem cells (LSC) play a pivotal role in therapeutic resistance and progression of chronic myeloid leukemia (CML) to blast crisis (BC). Although effective for treating CML, BCR-ABL targeted tyrosine kinase inhibitors (TKIs) fail to expunge quiescent, niche- resident LSC that drive blastic transformation and relapse. While resistance to therapy occurs through diverse molecular mechanisms, cumulative evidence suggests that anti-apoptotic BCL2 family genes may contribute to CML progression and TKI resistance as well as to normal hematopoietic stem and progenitor cell survival. However, the role of BCL2 family genes in human BC LSC maintenance has not yet been elucidated. This dissertation investigates the importance of BCL2 family genes for the survival of CML LSC both in vitro and in vivo and in the context of the cyto-protective bone marrow niche. Our investigations begin with an examination of BCL2 family gene expression in purified, primary human CML and normal myeloid progenitors (CD34⁺CD38⁺lin⁻). Next, we examine the role of BCL2 expression and quiescence in promoting bone marrow niche-dependent cyto-protection of LSC in a mouse xenograft model of CML. We then investigate whether LSC are susceptible to BCL2 inhibition using pharmacological inhibitors as well as shRNA. Finally, the dissertation concludes with investigations into whether niche-dependent LSC protection LSC may be overcome by using a BCL2 inhibitor in combination with standard TKI-treatment. CML LSC are found to upregulate multiple pro-survival BCL2 family genes upon progression to BC. Moreover, bone marrow niche-engrafted LSC are quiescent, upregulate BCL2, and are resistant to dasatinib, a potent TKI, compared to LSC in other hematopoietic niches. Notably, in both stromal co -culture and xenotransplantation experiments LSC are significantly inhibited with a novel, small molecule pan- BCL2 inhibitor, sabutoclax, at doses that spare normal hematopoietic progenitors. Sabutoclax also sensitizes marrow-niche LSC to dasatinib, and combination treatment delays CML relapse in serially transplanted mice. These data underscore the importance of pro-survival BCL2 genes in microenvironmental maintenance of malignant stem cells and suggest that niche targeted inhibition of BCL2 family proteins may serve as a vital component of a combined treatment strategy to eliminate quiescent, TKI-resistant LSC in human BC CML