Ryota Amachi

Pim-2 kinase is an important target of treatment for tumor progression and bone loss in myeloma.

Pim-2 kinase is overexpressed in multiple myeloma (MM) cells to enhance their growth and survival, and regarded as a novel therapeutic target in MM. However, the impact of Pim-2 inhibition on bone disease in MM remains unknown. We demonstrated here that Pim-2 expression was also upregulated in bone marrow stromal cells and MC3T3-E1 preosteoblastic cells in the presence of cytokines known as the inhibitors of osteoblastogenesis in MM, including interleukin-3 (IL-3), IL-7, tumor necrosis factor-α, transforming growth factor-β (TGF-β) and activin A, as well as MM cell conditioned media. The enforced expression of Pim-2 abrogated in vitro osteoblastogenesis by BMP-2, which suggested Pim-2 as a negative regulator for osteoblastogenesis. Treatment with Pim-2 short-interference RNA as well as the Pim inhibitor SMI-16a successfully restored osteoblastogenesis suppressed by all the above inhibitory factors and MM cells. The SMI-16a treatment potentiated BMP-2-mediated anabolic signaling while suppressing TGF-β signaling. Furthermore, treatment with the newly synthesized thiazolidine-2,4-dione congener, 12a-OH, as well as its prototypic SMI-16a effectively prevented bone destruction while suppressing MM tumor growth in MM animal models. Thus, Pim-2 may have a pivotal role in tumor progression and bone loss in MM, and Pim-2 inhibition may become an important therapeutic strategy to target the MM cell–bone marrow interaction.

Effective impairment of myeloma cells and their progenitors by blockade of monocarboxylate transportation.

Cancer cells robustly expel lactate produced through enhanced glycolysis via monocarboxylate transporters (MCTs) and maintain alkaline intracellular pH. To develop a novel therapeutic strategy against multiple myeloma (MM), which still remains incurable, we explored the impact of perturbing a metabolism via inhibiting MCTs. All MM cells tested constitutively expressed MCT1 and MCT4, and most expressed MCT2. Lactate export was substantially suppressed to induce death along with lowering intracellular pH in MM cells by blockade of all three MCT molecules with α-cyano-4-hydroxy cinnamate (CHC) or the MCT1 and MCT2 inhibitor AR-C155858 in combination with MCT4 knockdown, although only partially by knockdown of each MCT. CHC lowered intracellular pH and severely curtailed lactate secretion even when combined with metformin, which further lowered intracellular pH and enhanced cytotoxicity. Interestingly, an ambient acidic pH markedly enhanced CHC-mediated cytotoxicity, suggesting preferential targeting of MM cells in acidic MM bone lesions. Furthermore, treatment with CHC suppressed hexokinase II expression and ATP production to reduce side populations and colony formation. Finally, CHC caused downregulation of homing receptor CXCR4 and abrogated SDF-1-induced migration. Targeting tumor metabolism by MCT blockade therefore may become an effective therapeutic option for drug-resistant MM cells with elevated glycolysis.

Synergistic targeting of Sp1, a critical transcription factor for myeloma cell growth and survival, by panobinostat and proteasome inhibitors

Panobinostat, a pan-deacetylase inhibitor, synergistically elicits cytotoxic activity against myeloma (MM) cells in combination with the proteasome inhibitor bortezomib. Because precise mechanisms for panobinostats anti-MM action still remain elusive, we aimed to clarify the mechanisms of anti-MM effects of panobinostat and its synergism with proteasome inhibitors. Although the transcription factor Sp1 was overexpressed in MM cells, the Sp1 inhibitor terameprocol induced MM cell death in parallel with reduction of IRF4 and cMyc. Panobinostat induced activation of caspase-8, which was inversely correlated with reduction of Sp1 protein levels in MM cells. The panobinostat-mediated effects were further potentiated to effectively induce MM cell death in combination with bortezomib or carfilzomib even at suboptimal concentrations as a single agent. Addition of the caspase-8 inhibitor z-IETD-FMK abolished the Sp1 reduction not only by panobinostat alone but also by its combination with bortezomib, suggesting caspase-8-mediated Sp1 degradation. The synergistic Sp1 reduction markedly suppressed Sp1-driven prosurvival factors, IRF4 and cMyc. Besides, the combinatory treatment reduced HDAC1, another Sp1 target, in MM cells, which may potentiate HDAC inhibition. Collectively, caspase-8-mediated post-translational Sp1 degradation appears to be among major mechanisms for synergistic anti-MM effects of panobinostat and proteasome inhibitors in combination.

Pim-2 is a critical target for treatment of osteoclastogenesis enhanced in myeloma.

Alexandrakis, M.G., Passam, F.H., Kyriakou, D.S. & Bouros, D. (2004) Pleural effusions in hematologic malignancies. Chest, 125, 1546–1555. Banwait, R., Aljawai, Y., Cappuccio, J., McDiarmid, S., Morgan, E.A., Leblebjian, H., Roccaro, A.M., Laubach, J., Castillo, J.J., Paba-Prada, C., Treon, S., Redd, R., Weller, E. & Ghobrial, I.M. (2015) Extramedullary Waldenstr€ om macroglobulinemia. American Journal of Hematology, 90, 100–104. Kavuru, M.S., Tubbs, R., Miller, M.L. & Wiedemann, H.P. (1992) Immunocytometry and gene rearrangement analysis in the diagnosis of lymphoma in an idiopathic pleural effusion. The American Review of Respiratory Disease, 145, 209–211. Mansoor, A., Wagner, R.P. & DePalma, L. (2000) Waldenstrom macroglobulinemia presenting as a pleural effusion. Archives of Pathology & Laboratory Medicine, 124, 891–893. Poulain, S., Boyle, E.M., Roumier, C., Demarquette, H., Wemeau, M., Geffroy, S., Herbaux, C., Bertrand, E., Hivert, B., Terriou, L., Verrier, A., Pollet, J.P., Maurage, C.A., Onraed, B., Morschhauser, F., Quesnel, B., Duthilleul, P., Preudhomme, C. & Leleu, X. (2014) MYD88 L265P mutation contributes to the diagnosis of Bing Neel syndrome. British Journal of Haematology, 167, 506–513. Treon, S.P., Xu, L., Yang, G., Zhou, Y., Liu, X., Cao, Y., Sheehy, P., Manning, R.J., Patterson, C.J., Tripsas, C., Arcaini, L., Pinkus, G.S., Rodig, S.J., Sohani, A.R., Harris, N.L., Laramie, J.M., Skifter, D.A., Lincoln, S.E. & Hunter, Z.R. (2012) MYD88 L265P somatic mutation in Waldenstr€ om’s macroglobulinemia. The New England Journal of Medicine, 367, 826–833. Treon, S.P., Cao, Y., Xu, L., Yang, G., Liu, X. & Hunter, Z.R. (2014) Somatic mutations in MYD88 and CXCR4 are determinants of clinical presentation and overall survival in Waldenstrom macroglobulinemia. Blood, 123, 2791–2796. Treon, S.P., Xu, L. & Hunter, Z. (2015) MYD88 mutations and response to Ibrutinib in Waldenstr€ om’s Macroglobulinemia. The New England Journal of Medicine, 373, 584–586. Xu, L., Hunter, Z.R., Yang, G., Zhou, Y., Cao, Y., Liu, X., Morra, E., Trojani, A., Greco, A., Arcaini, L., Varettoni, M., Varettoni, M., Brown, J.R., Tai, Y.T., Anderson, K.C., Munshi, N.C., Patterson, C.J., Manning, R.J., Tripsas, C.K., Lindeman, N.I. & Treon, S.P. (2013) MYD88 L265P in Waldenstr€ om macroglobulinemia, immunoglobulin M monoclonal gammopathy, and other B-cell lymphoproliferative disorders using conventional and quantitative allele-specific polymerase chain reaction. Blood, 121, 2051–2058. Xu, L., Hunter, Z.R., Tsakmaklis, N., Cao, Y., Yang, G., Chen, J., Liu, X., Kanan, S., Castillo, J.J., Tai, Y.-T., Zehnder, J.L., Brown, J.R., Carrasco, R.D., Advani, R., Sabile, J.M., Argyropoulos, K., Lia Palomba, M., Morra, E., Trojani, A., Greco, A., Tedeschi, A., Varettoni, M., Arcaini, L., Munshi, N.M., Anderson, K.C. & Treon, S.P. (2015) Clonal architecture of CXCR4 WHIM-like mutations in Waldenstr€ om Macroglobulinaemia. British Journal of Haematology, 172, 735–744.

A vicious cycle between acid sensing and survival signaling in myeloma cells: acid-induced epigenetic alteration.

Myeloma (MM) cells and osteoclasts are mutually interacted to enhance MM growth while creating acidic bone lesions. Here, we explored acid sensing of MM cells and its role in MM cell response to acidic conditions. Acidic conditions activated the PI3K-Akt signaling in MM cells while upregulating the pH sensor transient receptor potential cation channel subfamily V member 1 (TRPV1) in a manner inhibitable by PI3K inhibition. The acid-activated PI3K-Akt signaling facilitated the nuclear localization of the transcription factor Sp1 to trigger the expression of its target genes, including TRPV1 and HDAC1. Consistently, histone deacetylation was enhanced in MM cells in acidic conditions, while repressing a wide variety of genes, including DR4. Indeed, acidic conditions deacetylated histone H3K9 in a DR4 gene promoter and curtailed DR4 expression in MM cells. However, inhibition of HDAC as well as either Sp1 or PI3K was able to restore DR4 expression in MM cells suppressed in acidic conditions. These results collectively demonstrate that acid activates the TRPV1-PI3K-Akt-Sp1 signaling in MM cells while inducing HDAC-mediated gene repression, and suggest that a positive feedback loop between acid sensing and the PI3K-Akt signaling is formed in MM cells, leading to MM cell response to acidic bone lesions.

Expansion of Th1-like Vγ9Vδ2T cells by new-generation IMiDs, lenalidomide and pomalidomide, in combination with zoledronic acid.

Expansion of Th 1 -like Vγ 9 Vδ 2 T cells by new-generation IMiDs, lenalidomide and pomalidomide, in combination with zoledronic acid

TAK1 inhibition subverts the osteoclastogenic action of TRAIL while potentiating its antimyeloma effects

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) agonists induce tumor-specific apoptosis indicating that they may be an attractive therapeutic strategy against cancers, including multiple myeloma (MM). Osteoclastogenesis is highly induced in MM, which in turn enhances MM growth, thereby forming a vicious cycle between MM tumor expansion and bone destruction. However, the effects of TRAIL on MM-enhanced osteoclastogenesis remain largely unknown. Here, we show that TRAIL induced apoptosis in MM cells, but not in osteoclasts (OCs), and that it rather facilitated receptor activator of NF-κB ligand-induced osteoclastogenesis along with upregulation of cellular FLICE inhibitory protein (c-FLIP). TRAIL did not induce death-inducing signaling complex formation in OCs, but formed secondary complex (complex II) with the phosphorylation of transforming growth factor β-activated kinase-1 (TAK1), and thus activated NF-κB signaling. c-FLIP knockdown abolished complex II formation, thus permitting TRAIL induction of OC cell death. The TAK1 inhibitor LLZ1640-2 abrogated the TRAIL-induced c-FLIP upregulation and NF-κB activation, and triggered TRAIL-induced caspase-8 activation and cell death in OCs. Interestingly, the TRAIL-induced caspase-8 activation caused enzymatic degradation of the transcription factor Sp1 to noticeably reduce c-FLIP expression, which further sensitized OCs to TRAIL-induced apoptosis. Furthermore, the TAK1 inhibition induced antiosteoclastogenic activity by TRAIL even in cocultures with MM cells while potentiating TRAILs anti-MM effects. These results demonstrated that osteoclastic lineage cells use TRAIL for their differentiation and activation through tilting caspase-8-dependent apoptosis toward NF-κB activation, and that TAK1 inhibition subverts TRAIL-mediated NF-κB activation to resume TRAIL-induced apoptosis in OCs while further enhancing MM cell death in combination with TRAIL.