Targeting BTK As a Treatment For Multiple Myeloma Stem Cells

Abstract

BackgroundMultiple myeloma stem cells (MMSCs), characterized by drug-resistance and self-renewal, are very likely responsible for relapse. Though the phenotypic features of MMSCs are still controversial, it is believed that MMSCs are enriched in plasma cell progenitors. Bruton's tyrosine kinase (BTK) plays a central role in B-cell development and plasma cell differentiation. BLAB/c. CBA/N mice carrying the defective BTK gene are resistant to pristine-induced plasmacytomagenesis suggesting activation of BTK is essential for plasma tumor initiation. In this study, we explore the role of BTK in maintaining MMSCs. Materials and methodsReal-time-PCR was used to detect the BTK expression in MMSCs and compared to the bulk cells. BTK-related self-renewal and drug resistance were evaluated by clonogenic formation assays, using 10,000 MM cells from the BTK over-expressing ARP1 and OPM2 MM cell lines, treated with bortezomib (1nM, 10nM), as well as doxorubicin (50nM, 100nM), etoposide (50nM, 100nM), and verapamil (50nM). Flow cytometry was performed to detect the side population (SP) fraction and apoptosis in MM cells. Co-immunoprecipitation assay and western blot were utilized for the mechanistic BTK studies. Effect of BTK on MM progression in vivo was assessed in the 5TGM1 myeloma mice and NOD/SCID mice models. ResultsOur results revealed that BTK, iPS genes, Wnt and Hedgehog signaling pathways were significantly increased in MMSCs (CD138- and SP fraction) compared to bulk myeloma cells in both MM cell lines and primary MM cells. We also demonstrated that over-expression of BTK in the low-expressing MM cell lines, ARP1 and OPM2 increased the SP fraction, clonogenic potential and drug-resistance, while knockdown of BTK in high-expressing MM cell lines, OCI-MY5 and H929 abrogated these characteristics. Our in vivo study indicated that over-expression of BTK in ARP1 cells accelerates tumor formation in NOD/SCID mice compared to the EV control cells. Targeting BTK by shRNA or BTK inhibitor, CGI-1747 delayed MM tumor formation and MM progression in NOD/SCID mice and 5TGM1 model, respectively. Mechanistic studies identified that BTK activates the AKT pathway, inducing the drug-resistant genes, p-Bcl-2 and ABCB1, and inhibiting GSK3β leading to activation of the Wnt/ β-Catenin pathway. Inhibition of AKT by a specific inhibitor decreased Wnt and Hh proteins by western blot. ConclusionBTK plays an important role on maintaining MM stemness and drug-resistance through activating AKT, Wnt and Hh signaling pathways. Targeting BTK in MM may be a promising strategy to eliminate MMSCs. Disclosures:No relevant conflicts of interest to declare.