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TAK1 inhibition in myeloma
Multiple myeloma animal model and histological analyses
Multiple myeloma (MM) mouse models were prepared by intra-tibial inoculation of mouse luciferase-transfected 5TGM1 MM cells (a gift from Dr. Gregory R. Mundy [Vanderbilt Center for Bone Biology, Vanderbilt University, Nashville, TN, USA]) into ICR nu/nu mice (CLEA Japan) at 4–6 weeks old as described pre- viously.13,14 The assessment of tumor growth and bone volume, and bone histomorphometric and immunohistochemical analyses are described in the Online Supplementary Appendix.
Statistical analysis
Statistical analysis was performed using Student's t-test or one- way analysis of variance (ANOVA). P<0.05 was considered as a significant difference. All statistics were performed using the Statistical Package for Social Sciences (SPSS 13.0 for Windows; Chicago, IL, USA).
Results
TAK1 is activated to mediate growth and survival of multiple myeloma cells
We first examined the expression and activation status of TAK1 in MM cells. TAK1 is highly overexpressed and phosphorylated in MM cell lines and primary MM cells from patients, whereas normal peripheral blood mononu- clear cells (PMBC) only weakly expressed TAK1 (Figure 1A). IRF4 and MYC have been regarded as master regula- tors for MM cell survival and function, which MM cells are addicted to, and their reduction is a major mechanism for the anti-MM activity of immunomodulatory drugs lenalidomide and pomalidomide.23,24 Notably, the TAK1 inhibitor LLZ dose-dependently reduced MYC and IRF4 in parallel with the reduction in PIM2 expression and the phosphorylation of the PIM2 substrate 4E-BP1 as well as the anti-apoptotic factor Mcl-1 in MM cells (Figure 1B, left). We and others reported that transcription factor Sp1 is constitutively overexpressed in MM cells, which can serve as an important therapeutic target for MM.25-27 TAK1 inhibition also substantially reduced Sp1 (Figure 1B, left). The effects of TAK1 inhibition were confirmed with TAK1 knockdown using siRNA (Figure 1B, right).
TAK1 inhibition with LLZ dose-dependently induced cell death in all MM cell lines tested (Figure. 1C, left), and TAK1 knockdown also reduced the viability of MM cells (Figure 1C, right). The induction of apoptosis was con- firmed using annexinV and propidium iodide dual staining (Figure 1D) with activation of caspase-8, caspase-9, and caspase-3 (Figure 1E) in MM cells, indicating activation of the extrinsic as well as the intrinsic caspase-mediated apoptotic pathways. LLZ at the concentrations up to 10 mM did not apparently impair the viability of normal PBMC (Online Supplementary Figure S1). Therefore, TAK1 appears to be a good therapeutic target to effectively induce cell death in MM cells through suppression of PIM2 plus other pro-survival mediators.
lines in cocultures with BMSC as well as in the presence of IL-6 or TNF-α (Figure 2A). However, treatment with LLZ was able to abolish PIM2 upregulation in MM cells by BMSC as well as IL-6 or TNF-α. We further examined the effects of TAK1 inhibition on the signaling pathways in MM cells activated by IL-6 and TNF-α. After starving without serum, phosphorylation of TAK1 was reduced in RPMI8226 cells, but IL-6 (Figure 2B) and TNF-α (Figure 2C) promptly induced the phosphorylation of TAK1 and activation of their corresponding downstream signaling molecules in serum-depleted media. Treatment with LLZ abolished IL-6-induced phosphorylation of STAT3 (Figure 2B), and TNF-α-induced phosphorylation and degradation of IκBα and phosphorylation of p38MAPK and ERK (Figure 2C) in the MM cells. Analyses of nuclear extracts from MM cells treated with TNF-α showed the nuclear accumulation of p65; however, treatment with LLZ reduced the p65 content in the nuclear extracts in the pres- ence of TNF-α (Figure 2D). Consistent with the notion that PIM2 is transcriptionally upregulated in MM cells by the NF-κB signaling pathway,5 the PIM inhibitor SMI-16a showed only marginal effects on the nuclear accumulation of p65 induced by TNF-α (Figure 2D). Of note, the TAK1 inhibitor LLZ as well reduced MM cell viability in cocul- tures with BMSC (Figure 2E). TAK1 knockdown in MM cells mostly reduced their viability even in cocultures with BMSC (Figure 2F). Also, TAK1 knockdown in BMSC par- tially but significantly reduced their supportive activity for MM cell growth (Figure 2G). Therefore, TAK1 activation in both MM cells and BMSC is suggested to play an important role in MM cell growth and survival in cocul- tures with BMSC. Furthermore, although cytotoxic effects of doxorubicin on MM cells were blunted in cocultures with BMSC, TAK1 inhibition with LLZ was able to resume MM cell death by doxorubicin even in the pres- ence of BMSC (Online Supplementary Figure S2). These results suggested that TAK1 inhibition impairs MM cell growth and survival supported by the bone marrow microenvironment.
TAK1 inhibition impairs multiple myeloma cell adhesion to bone marrow stromal cells
MM cell adhesion to BMSC through the interaction between VLA-4 and its corresponding ligand, VCAM-1, is among the predominant mechanisms for cell adhesion- mediated drug resistance (CAM-DR) in MM,22,29 while enhancing the production of IL-630 and RANKL, an critical osteoclastogenic factor in MM.31 In addition to TAK1 acti- vation in MM cells, TAK1 was found to be phosphorylat- ed along with PIM2 upregulation in BMSC, when cocul- tured with MM cells (Figure 3A). Furthermore, VCAM-1 expression was substantially upregulated in BMSC after coculturing with MM cells, which was abolished by TAK1 inhibition with LLZ (Figure 3B). TNF-α is known as a potent inducer of VCAM-1 in BMSC through activation of the NF-κB signaling pathway.32,33 Treatment with LLZ as well as TAK1 knockdown by siRNA abrogated the upreg- ulation of VCAM-1 expression in BMSC by TNF-α (Figures 3B and 3C). Treatment with TNF-α promptly phosphorylated TAK1 and degraded IκBα, and induced the phosphorylation of p38MAPK and ERK in BMSC (Figure 3D). However, TAK1 inhibition with LLZ as well as TAK1 knockdown abolished the degradation of IκBα and reduced the phosphorylation of p38MAPK and ERK in the presence of TNF-α, indicating efficacious suppression
TAK1 inhibition is able to abolish IL-6 and TNF-α-induced signaling in multiple myeloma cells
IL-6 and TNF-α are predominant paracrine factors over- produced in the bone marrow microenvironment in MM, and these elicit the signaling pathways responsible for MM cell growth and survival.28 As we demonstrated pre- viously,5 PIM2 was substantially upregulated in MM cell
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