TBK1/Ikkε Inhibitor Amlx Blocks Multiple Myeloma Cell Growth in Vitro and In Vivo

Multiple myeloma (MM) is the most frequent cancer to involve the skeleton and remains incurable for most patients, thus novel therapies are needed. MM bone disease is characterized by osteolytic lesions that contribute significantly to patient morbidity and mortality. We showed that TBK1 signaling is a novel pathway that increases osteoclast (OCL) formation in Paget's disease, an inflammatory bone disease. Therefore, we hypothesized that TBK1 plays a similar role in MM induction of OCL. We found that MM conditioned media (MM-CM) dose-dependently increased bone marrow monocyte (BMM) expression of activated TBK1 protein and enhanced RANKL-driven OCL formation. TBK1 knockdown by shRNA transduction into BMM significantly attenuated the ability of MM-CM to increase OCL differentiation without altering OCL differentiation in control media. We found that the TBK1/IKKε inhibitor Amlexanox (Amlx) blocked normal and MM-enhanced OCL formation. Importantly, TBK1 mRNA expression in CD138+ plasma cells (PC) isolated from MM or PC leukemia patients is significantly higher as compared to PC from Monoclonal Gammopathy of Undetermined Significance (MGUS) patients.

Therefore, we tested whether targeting the TBK1/ IKKε signaling pathways would also affect MM cells. We found that Amlx strongly decreased the viability of several MM cell lines and primary MM cells via induction of apoptosis. Amlx treatment of MM cell lines also induced a G1/S blockade, decreased activated ERK1/2, and increased translation of the dominant-negative C/EBPb-LIP isoform in several MM cell lines. The positive-acting C/EBPb-LAP isoform was previously shown to be a critical transcription factor for MM viability. Importantly, Amlx also enhanced the effectiveness of the proteasome inhibitors bortezomib and carfilzomib to kill MM cells in culture. Further, Amlx sensitized MM1.S cells to the induction of apoptosis by the autophagic inhibitor Bafilomycin A. Amlx dose-dependently inhibited tumor growth in a syngeneic MM mouse model in which 5TGM1 MM cells expressing secreted GLuc were injected subcutaneously into immunocompetent C57Bl/KaLwRij. Tumor growth was assessed by measuring tumor volumes and by the levels of secreted GLuc in the blood. Further, OCL formation ex vivo from bone marrow monocytes obtained from AMLX-treated mice versus controls was decreased. Amlx did not affect the viability of primary BMM, bone marrow stromal cells (BMSC), or splenocytes. Further, Amlx treatment of primary BMSC from MM patients or normal donors decreased expression of TNFα, IL-6 and RANKL, thereby decreasing BMSC support of MM survival and OCL differentiation. Amlx pretreatment of BMSC and murine pre-osteoblast MC4 cells also decreased VCAM1 expression and reduced MM cell adhesion, another mechanism for Amlx reduction of bone microenvironmental MM support.

These data suggest that targeting TBK1/IKKε signaling may decrease MM bone disease by slowing MM growth, directly and indirectly, and preventing MM-induced osteolysis.