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TAK1 inhibition in myeloma
on bone metabolism in MM, we histomorphometrically analyzed bone lesions in the mouse models. In vehicle- treated mice, bone volume over total volume (BV/TV), tra- becular thickness (Tb.Th), trabecular numbers (Tb.N), number of osteoblast surface over bone surface (N.Ob/BS), osteoid surface over bone surface (OS/BS), and bone formation rate over bone surface (BFR/BS) were decreased, whereas trabecular separation (Tb.Sp) and the number of OC surface over bone surface (N.Oc/BS) were increased (Figure 6E). However, treatment with LLZ improved these changes in the 5TGM1-inoculated tibiae. These results demonstrate that the TAK1 inhibition not only suppresses osteoclastic bone destruction but also restores osteoid and bone formation in MM bone lesions. In order to further clarify the direct roles of TAK1 inhibi- tion on pathological bone loss without tumor cells, we investigated the effects of LLZ on bone loss in ovariec- tomized (OVX) mice. In vehicle-treated OVX mice, bone loss was revealed in m−CT; and BV/TV, Tb.Th and Tb.N were decreased, whereas Tb.Sp was increased in bone morphometric analysis (Online Supplementary Figure S7). However, treatment with LLZ was able to prevent OVX- induced pathological changes, suggesting a protective action of TAK1 inhibition on non-malignant bone loss.
Discussion
Although MM cells perturb bone metabolism with bone destruction, crosstalk between MM cells and the microen- vironment in bone lesions leads to a progressive vicious cycle of tumor growth and bone destruction. The present study demonstrated that TAK1 plays a critical role in the vicious cycle, and suggested that TAK1 is a pivotal thera- peutic target to disrupt the key signal transduction path- ways responsible for tumor progression and bone destruc- tion in MM. TAK1 activation appears to govern the expres- sion of PIM2 in MM cells and osteoclastic as well as osteoblastic lineage cells; the TAK1-PIM2 signaling path- way is critical for MM tumor expansion and bone destruc- tion. In addition to PIM2 upregulation, TAK1 activation induced Sp1 expression in MM cells. Sp1 is a ubiquitous zinc-finger transcription factor that binds guanine–cyto- sine-rich elements in the promoter region of its target genes and upregulates various important genes for cancer initia- tion and progression.46,47 Sp1 is known to be constitutively overexpressed in many cancers and is associated with poor prognosis.46 Sp1 expression and its DNA binding activity has been also demonstrated to be upregulated in MM cells.25 We and others reported that inhibition of Sp1 expres- sion with Sp1 siRNA markedly induced apoptosis in MM cells, indicating that Sp1 as a novel therapeutic target for MM.25-27 Our results showed that TAK1 activation con- tributes to Sp1 over-expression in MM cells, and that TAK1 inhibition reduces Sp1 expression to impair MM cell growth and survival. TAK1 inhibition was found to reduce Sp1-mediated IRF4 expression in MM cells. As IMiD have been reported to downregulate IRF4 expression though degradation of IKZF1/3,23,24 TAK1 inhibition may synergize the downregulation of IRF4 expression in combination with IMiD. TAK1 was also demonstrated to play a critical role in facilitating MM cell-BMSC adhesion via VLA-4- VCAM-1 interaction. TAK1 inhibition reduced VCAM-1 expression in BMSC upregulated by MM cells or TNF-α, and impaired MM cell adhesion to BMSC. MM cell-BMSC
adhesion induced IL-6 production and RANKL expression in BMSC in a manner dependent on TAK1 activation. Given that the adhesion of MM cells to BMSC via VLA-4- VCAM-1 interaction confers CAM-DR in MM cells29,48,49 and osteoclastogenesis,50 these results suggested the thera- peutic impact of TAK1 inhibition on CAM-DR as well as osteoclastogenesis induced by the MM-bone marrow inter- action.
RANKL plays an important role in osteoclastogenesis enhanced in MM. As reported previously,34 RANKL induced the phosphorylation of TAK1 in RAW264.7 preosteoclastic cells in parallel with the degradation of IκBα and phospho- rylation of p38MAPK and ERK (Figure 4A). However, TAK1 inhibition abolished these changes in the RANKL-mediated intracellular signaling and suppressed the formation of TRAP-positive OC from bone marrow cells upon treatment with exogenous RANKL (Figures 4D, F) as well as in cocul- tures with MM cells (Figure 4G). Together with suppression of the TAK1-dependent induction of RANKL expression in BMSC (Figure 3G), TAK1 inhibition can reduce osteoclasto- genesis enhanced in MM through blockade of RANKL- mediated signaling in osteoclastic lineage cells. As for osteoblastogenesis, major inhibitors for osteoblastogenesis in MM, including IL-3, IL-7, TNF-α, TGF-b, and activinA, as well as MM cell CM-induced TAK1 phosphorylation while suppressing osteoblastogenesis in MC3T3-E1 preosteoblas- tic cells; however, TAK1 inhibition restored their osteoblas- togenesis (Figures 5A,B). Taken together, these results underscored the value of TAK1 inhibition for preventing progression of bone destruction and restoring bone forma- tion in MM. Finally, we validated the therapeutic effects of TAK1 inhibition in vivo. Treatment with LLZ markedly sup- pressed MM tumor growth and prevented bone destruction in mouse MM models with intra-tibial injection of 5TGM1 MM cells (Figure 6). Although various bone-modifying agents have been developed, bone loss still remains a seri- ous unmet issue in patients with MM; bone formation is hard to be restored in MM bone lesions by clinically avail- able anti-resorptive agents, namely zoledronic acid and denosumab. In contrast to these agents, TAK1 inhibitors appear to be bone anabolic and anti-resorptive agents with tumor-suppressing activity, which may bring considerable benefits for patients with malignant diseases exhibiting bone loss, such as MM patients. Moreover, because TAK1 inhibition is a novel mechanism of action, combination with TAK1 inhibition can improve the therapeutic efficacy of currently available anti-cancer agents while preventing cancer-related and cancer treatment-induced bone loss. Therefore, TAK1 inhibition may be a promising therapeutic option with anti-tumor and bone modifying action, target- ing the interaction between MM cells and their surrounding microenvironment. The present results warrant further study for development of novel TAK1 inhibitors useful for MM treatment. We are currently synthesizing novel com- pounds with better specificity for TAK1 with less toxicity. Further translational research will elucidate the dividends they may yield in improved clinical outcomes.
Dislosures
MA received research funding from Chuagai Pharmaceutical, Sanofi K.K., Pfizer Seiyaku K.K., Kyowa Hakko Kirin, MSD KK, Astellas Pharma, Takeda Pharmaceutical, Teijin Pharma and Ono Pharmaceutical, and honoraria from Daiichi Sankyo Company. The other authors have no conflicts of interest to declare.
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