Targeting Cancer Stem Cell Survival in Plasma Cell Leukemia with a Pan-BCL2 Inhibitor

INTRODUCTION Representing ten percent of all hematologic malignancies, multiple myeloma (MM) is typified by clonal plasma cell proliferation in the bone marrow (BM) and may progress to a therapy-resistant stage characterized by circulating malignant plasma cells, which is termed plasma cell leukemia, (PCL). Notably, copy number amplification involving the myeloid cell leukemia (MCL)-1 locus, and translocations at the BCL2 locus have been described in high-risk MM. Moreover, BCL2 family members, including both MCL1 and BCL2, are highly expressed in relapsed MM. Finally, downregulation of BCL2 family members was reported to increase sensitivity to lenalidomide, a standard treatment for MM, suggesting that BCL2-targeted treatments may obviate therapeutic resistance.Sabutoclax, a potent small molecule inhibitor of 6 anti-apoptotic BCL2 family proteins, was shown to inhibit cancer stem cell (CSC) survival in chronic myeloid leukemia (CML) (Goff et al Cell Stem Cell 2013). Thus, we investigated BCL2 pro-survival splice isoform expression in therapy-resistant MM and PCL and evaluated whether Sabutoclax reduces malignant plasma cell burden in humanized primagraft assays. METHODS AND RESULTS BCL2 pro survival expression in primary myeloma samples Mononuclear cells from MM or PCL samples were isolated by Ficoll-Paque density gradient separation and collected for RNA extraction or FACS Aria purified into CD138+ or CD34-/CD138- cancer stem cells (CSC) subsets. To evaluate BCL2 pro-survival family member expression, splice isoform-specific quantitative PCR was performed to measure expression of pro-survival long isoforms compared with short splice isoforms, which are pro-apoptotic. Interestingly, MM and PCL patients displayed higher levels of BCL2-L, MCL1-L, BCLX-L and BLF1-L compared to normal controls. Moreover, prolonged lenalidomide exposure increased BCL2-L and MCL1-L expression in the myeloma cell line H929, compared to untreated cells. Sabutoclax Treatment of a Novel Humanized Plasma Cell Leukemia Primagraft Model Mononuclear cells from three primary PCL patient samples were stably transduced with a GFP-luciferase lentiviral vector and transplanted intrahepatically in newborn RAG2γ-/- c-/- mice. Engraftment was monitored by peripheral blood free light chain ELISA assays. Flow cytometric analyses revealed robust engraftment of PCL cells in bone marrow, spleen, liver and peripheral blood. Once transplanted mice displayed significant tumor burden above background free light chain levels, animals were randomized by ELISA values in vehicle versus Sabutoclax groups. Sabutoclax was selected because this pan-BCL2 targeted compound, unlike related BCL2 inhibitors such as ABT-199, also inhibits MCL1. Sabutoclax (10mg/kg) was administered intravenously twice weekly for two to four doses. Sabutoclax treated PCL mice showed reduced human plasma cell burden in bone marrow and spleen tissues compared to vehicle controls. CONCLUSION Expression of pro-survival BCL2 splice isoforms, including BCL2-L and MCL1-L, portends PCL engraftment in immunocompromised mice. Treatment of human PCL engrafted mice with Sabutoclax reduces malignant plasma cell survival in hematopoietic tissues. Thus, selective targeting of pro-survival isoform expressing CSC with a pan-BCL2 inhibitor may abolish BCL2 and MCL1-dependent therapeutic resistance in MM and PCL.