Leukemia stem cells (LSCs) play a pivotal role in the resistance of chronic myeloid leukemia (CML) to tyrosine kinase inhibitors (TKIs) and its progression to blast crisis (BC), in part, through the alternative splicing of self-renewal and survival genes. To elucidate splice-isoform regulators of human BC LSC maintenance, we performed whole-transcriptome RNA sequencing, splice-isoform-specific quantitative RT-PCR (qRT-PCR), nanoproteomics, stromal coculture, and BC LSC xenotransplantation analyses. Cumulatively, these studies show that the alternative splicing of multiple prosurvival BCL2 family genes promotes malignant transformation of myeloid progenitors into BC LSCS that are quiescent in the marrow niche and that contribute to therapeutic resistance. Notably, sabutoclax, a pan-BCL2 inhibitor, renders marrow-niche-resident BC LSCs sensitive to TKIs at doses that spare normal progenitors. These findings underscore the importance of alternative BCL2 family splice-isoform expression in BC LSC maintenance and suggest that the combinatorial inhibition of prosurvival BCL2 family proteins and BCR-ABL may eliminate dormant LSCs and obviate resistance.
Bibliographical noteFunding Information:
We thank Dennis Carson for his continuous advice and mentorship, Dennis Young for his expert assistance on all FACS experiments, Ida Deichaite for her excellent assistance with material transfer and industry relations, Jennifer Black and Rusty Wall for their help with sample preparation and mouse experiments, Isabel Newton for her advice and help with DiR studies, Derrick Duarte for his assistance with immunohistochemistry studies, Jerry Wu for his assistance with FACS cell-cycle experiments, and Kimberly Wilson for her assistance with grant and manuscript preparation and submission. This work was generously supported by the Ratner Family Fund and by the California Institute for Regenerative Medicine (CIRM; grants RN2-00910-1, RS1-00228-1, TR2-01789, and DR1-01430). D.J.G. was supported by the CIRM UCSD Stem Cell Training Grant II and the UCSD Cancer Training Grant. This work was also funded by the National Cancer Institute (NCI; no. CA-55164) and the National Institutes of Health (NIH; no. CA-149668), and supported by the Ontario Institute for Cancer Research, through generous support from the Ontario Ministry of Research and Innovation and the Cancer Stem Cell Consortium with funding from the Government of Canada through Genome Canada and the Ontario Genomics Institute (OGI-047), and through the Canadian Institute of Health Research (CSC-105367). J.C.R., J.W., and M.P. are coinventors of sabutoclax and related compounds, licensed by the SBMRI to Oncothyreon (Seattle). The Oncothyreon license entitles J.C.R. to milestone and royalty payments for studies advancing to clinic. He does not receive consulting fees or lab support, nor does he have stock or stock options.