The introduction of BCR-ABL tyrosine kinase inhibitors has revolutionized the treatment of chronic myeloid leukemia (CML). A major clinical aim remains the identification and elimination of low-level disease persistence, termed "minimal residual disease". The phenomenon of disease persistence suggests that despite targeted therapeutic approaches, BCR-ABLindependent mechanisms exist which sustain the survival of leukemic stem cells (LSCs). Although other markers of a primitive CML LSC population have been identified in the preclinical setting, only CD26 appears to offer clinical utility. Here we demonstrate consistent and selective expression of CD93 on a lin − CD34 + CD38 − CD90 + CML LSC population and show in vitro and in vivo data to suggest increased stem cell characteristics, as well as robust engraftment in patient-derived xenograft models in comparison with a CD93 − CML stem/progenitor cell population, which fails to engraft. Through bulk and single-cell analyses of selected stem cell and cell survival-specific genes, we confirmed the quiescent character and demonstrate their persistence in a population of CML patient samples who demonstrate molecular relapse on TKI withdrawal. Taken together, our results identify that CD93 is consistently and selectively expressed on a lin − CD34 + CD38 − CD90 + CML LSC population with stem cell characteristics and may be an important indicator in determining poor TKI responders.

Execution of the intrinsic apoptotic pathway is controlled by the BCL-2 proteins at the level of the mitochondrial outer membrane (MOM). This family of proteins consists of prosurvival (e.g., BCL-2, MCL-1) and proapoptotic (e.g., BIM, BAD, HRK) members, the functional balance of which dictates the activation of BAX and BAK. Once activated, BAX/BAK form pores in the MOM, resulting in cytochrome c release from the mitochondrial intermembrane space, leading to apoptosome formation, caspase activation, and cleavage of intracellular targets. This pathway is induced by cellular stress including DNA damage, cytokine and growth factor withdrawal, and chemotherapy/drug treatment. A well-documented defense of leukemia cells is to shift the balance of the BCL-2 family in favor of the prosurvival proteins to protect against such intra- and extracellular stimuli. Small molecule inhibitors targeting the prosurvival proteins, named ‘BH3 mimetics’, have come to the fore in recent years to treat hematological malignancies, both as single agents and in combination with standard-of-care therapies. The most significant example of these is the BCL-2-specific inhibitor venetoclax, given in combination with standard-of-care therapies with great success in AML in clinical trials. As the number and variety of available BH3 mimetics increases, and investigations into applying these novel inhibitors to treat myeloid leukemias continue apace the need to evaluate where we currently stand in this rapidly expanding field is clear.

Dysregulation of the BCL-2 family is implicated in protecting chronic myeloid leukemia (CML) cells from intracellular damage and BCR::ABL1-inhibition with tyrosine kinase inhibitors (TKIs) and may be a viable therapeutic target in blast phase (BP-)CML, for which treatment options are limited. BH3 mimetics, a class of small molecule inhibitors with high-specificity against the prosurvival members of the BCL-2 family, have displayed clinical promise in the treatment of chronic lymphocytic and acute myeloid leukemia as single agents and in combination with standard-of-care therapies. Here we present the first comparison of inhibition of BCL-2 prosurvival proteins BCL-2, BCL-xL and MCL-1 in combination with a second or third generation TKI, crucially with comparisons drawn between myeloid and lymphoid BP-CML samples. Co-treatment of four BP-CML cell lines with the TKIs nilotinib or ponatinib and either BCL-2 (venetoclax), MCL-1 (S63845) or BCL-xL (A-1331852) inhibitors resulted in a synergistic reduction in cell viability and increase in phosphatidylserine (PS) presentation. Nilotinib with BH3 mimetic combinations in myeloid BP-CML patient samples triggered increased induction of apoptosis over nilotinib alone, and a reduction in colony-forming capacity and CD34+ fraction, while this was not the case for lymphoid BP-CML samples tested. While some heterogeneity in apoptotic response was observed between cell lines and BP-CML patient samples, the combination of BCL-xL and BCR::ABL1 inhibition was consistently effective in inducing substantial apoptosis. Further, while BH3 mimetics showed little efficacy as single agents, dual-inhibition of BCL-2 prosurvival proteins dramatically induced apoptosis in all cell lines tested and in myeloid BP-CML patient samples compared to healthy donor samples. Gene expression and protein level analysis suggests a protective upregulation of alternative BCL-2 prosurvival proteins in response to BH3 mimetic single-treatment in BP-CML. Our results suggest that BH3 mimetics represent an interesting avenue for further exploration in myeloid BP-CML, for which alternative treatment options are desperately sought.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.