TAK1 is a pivotal therapeutic target for tumor progression and bone destruction in myeloma

Along with tumor progression, the bone marrow microenvironment is skewed in multiple myeloma (MM), which underlies the unique pathophysiology of MM and confers aggressiveness and drug resistance in MM cells. TGF-b-activated kinase-1 (TAK1) mediates a wide range of intracellular signaling pathways. We demonstrate here that TAK1 is constitutively overexpressed and phosphorylated in MM cells, and that TAK1 inhibition suppresses the activation of NF-κB, p38MAPK, ERK and STAT3 in order to decrease the expression of critical mediators for MM growth and survival, including PIM2, MYC, Mcl- 1, IRF4, and Sp1, along with a substantial reduction in the angiogenic factor VEGF in MM cells. Intriguingly, TAK1 phosphorylation was also induced along with upregulation of vascular cell adhesion molecule-1 (VCAM-1) in bone marrow stromal cells (BMSC) in cocultures with MM cells, which facilitated MM cell-BMSC adhesion while inducing IL-6 production and receptor activator of nuclear factor κ-B ligand (RANKL) expression by BMSC. TAK1 inhibition effectively impaired MM cell adhesion to BMSC to disrupt the support of MM cell growth and survival by BMSC. Furthermore, TAK1 inhibition suppressed osteoclastogenesis enhanced by RANKL in cocultures of bone marrow cells with MM cells, and restored osteoblastic differentiation suppressed by MM cells or inhibitory factors for osteoblastogenesis overproduced in MM. Finally, treatment with the TAK1 inhibitor LLZ1640-2 markedly suppressed MM tumor growth and prevented bone destruction and loss in mouse MM models. Therefore, TAK1 inhibition may be a promising therapeutic option targeting not only MM cells but also the skewed bone marrow microenvironment in MM.

In order to improve therapeutic efficacy, we need to disrupt the MM niche that confers drug resistance. Therefore, we looked for novel molecules upregulated in the MM niche to be targeted, and found that proviral integrations of Moloney virus 2 kinase (PIM2) is constitutively overexpressed as an anti-apoptotic mediator in MM cells. 5 PIM2 expression has been demonstrated to be higher in hematologic malignancies than solid cancers or their normal tissue counterparts, and highest in MM. 6,7 Importantly, PIM2 can be further upregulated in MM cells in cocultures with BMSC as well as OC. 5 Although multiple soluble inhibitors for osteoblastogenesis have been reported to be overproduced in MM, including IL-3, 8 IL-7, 9 TNF-α, 10 TGF-b, 11 and activin A, 12 PIM2 was found to be upregulated in BMSC by these inhibitory factors acting as a common intracellular mediator to suppress their osteoblastogenesis. 13 Moreover, we subsequently reported that PIM2 is induced in osteoclastic lineage cells by receptor activator of nuclear factor κ-B ligand (RANKL) to act as a critical mediator of RANKLinduced osteoclastogenesis in MM. 14 Therefore, PIM2 appears to play a versatile role in tumor progression and bone destruction and bone loss in MM, and is regarded as an important therapeutic target.
TGF-b-activated kinase 1 (TAK1) is a member of the mitogen-activated protein kinase kinase (MAP3K) family, also known as MAP3K7. 15,16 It was originally identified as a key kinase in transducing TGF-b signaling down to p38 mitogen-activated protein kinase (MAPK) and c-Jun and N-terminal kinase (JNK). 15 Subsequently, TAK1 has been demonstrated to be associated with the activation of a wide range of intracellular signaling pathways important for various cellular functions, including the activation of nuclear factor-κB (NF-κB) and extracellular signal-regulated kinase (ERK). 15 Therefore, TAK1 appears to be a gate keeper to facilitate the multiple important intracellular signaling pathways. Therapeutic efficacy of TAK1 inhibition has been preclinically demonstrated in different types of cancers, including mantle cell lymphoma, 17 breast cancer, 18 and colon cancer. 19 We demonstrate here that TAK1 is constitutively overexpressed and phosphorylated in MM cells and that TAK1 acts as an upstream regulator responsible for multiple signaling pathways critical for MM growth and survival. 1 TAK1 phosphorylation is also induced in BMSC in cocultures with MM cells, which facilitates MM cell adhesion to BMSC between very late antigen-4 (VLA-4) and vascular cell adhesion molecule-1 (VCAM-1), thereby inducing IL-6 production and RANKL expression by BMSC. Importantly, TAK1 inhibition was able to effectively induce MM cell death, and alleviate bone destruction though suppression of osteoclastogenesis and restoration of osteoblastogenesis. Therefore, TAK1 appears to be a pivotal therapeutic target in MM to disrupt the key signal transduction pathways responsible for tumor progression and bone destruction.

Ethics
All procedures involving human samples from healthy donors and patients were performed with written informed consent in accordance with the Declaration of Helsinki and a protocol approved by the Institutional Review Board for human protec-tion at the University of Tokushima (Permission number: 240). All animal experiments were conducted under the regulation and permission of the Animal Care and Use Committee of Tokushima University, Tokushima, Japan (toku-dobutsu 13094).

Reagents
Reagents used in this manuscript are described in the Online Supplementary Appendix.

Cells and cell culture
Details of the cells and cell culture procedures are available in the Online Supplementary Appendix.

Western blotting
Cells were collected and lysed in RIPA lysis buffer (Santa Cruz, Dallas, TX, USA). For cytosolic and nuclear preparation, cells were lysed in NE-PER extraction reagent (Thermo Fisher Scientific, Waltham, MA) in accordance with the manufacturer's protocol. Western blot analysis was done with equal protein amounts of cell lysate, as described previously. 13

Cell viability
Cell viability was determined using the Cell Counting Kit-8 assay (Dojindo, Kumamoto, Japan) in accordance with the manufacturer's instructions. The absorbance of each well was measured at 450-655 nm using an iMark microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). In order to assess apoptotic cells, cells were stained with an annexinV-FITC and propidium iodide labeling kit (MEBCYTO Apoptosis Kit; MBL, Nagano, Japan) in accordance with the manufacturer's instruction, and analyzed by flow cytometry.

Small interfering RNA transfection
Small interfering RNA (siRNA) transduction was performed as described previously. 5,20 Human and mouse TAK1 siRNA were purchased from Santa Cruz. Human TAK1 siRNA was transfected into MM cells using a Human Nucleofector Kit (Lonza, Basel, Switzerland). Mouse TAK1 siRNA was transfected into mouse BMSC or RAW264.7 cells using siRNA Transfection Reagent (Santa Cruz) in accordance with the manufacturer's protocol.

Real-time reverse transcription polymerase chain reaction
RNA isolation and quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) were performed as described previously. 20 The following primer sequences were used: human RANKL F: TCGTTGGATCACAGCACATCA and R: TATGGGAACCAGATGGGATGTC, human IL-6 F: TCT-GAGGCTCATTCTGCCCTCGAGC and R: AACTGGACC-GAAGGCGCTTGTGGA, human GAPDH, used as an endogenous control to normalize each sample, F: TGTCTTCACCAC-CATGGAGAAGG and R: GTGGATGCAGGGATGAT-GTTCTG

Adhesion assays
Adhesion assays were performed as described previously. 21 Human BMSC (2×10 4 cells/well) were expanded in 96-well culture plates. MM cells were labeled with BCECF-AM (Dojindo) for 2 hours at 37°C and 5% CO 2 as described previously. 22 BMSC were washed, and the fluorescence-labeled MM cells were added onto the BMSC and incubated for 4 hours. Nonadherent BCECF-AM-labeled cells were removed by gently pipetting four times. Adherent cells were quantitated in a fluorescence multi-well plate reader (Infinite ® 200 PRO, TECAN, Männedorf. Switzerland).

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 previously. 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 oneway 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).

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 mononuclear cells (PMBC) only weakly expressed TAK1 ( Figure  1A). IRF4 and MYC have been regarded as master regulators 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][26][27] 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 confirmed 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.

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 overproduced in the bone marrow microenvironment in MM, and these elicit the signaling pathways responsible for MM cell growth and survival. 28 As we demonstrated previously, 5 PIM2 was substantially upregulated in MM cell 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 presence 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 cocultures 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 partially 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 cocultures 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 presence 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 adhesionmediated drug resistance (CAM-DR) in MM, 22,29 while enhancing the production of IL-6 30 and RANKL, an critical osteoclastogenic factor in MM. 31 In addition to TAK1 activation in MM cells, TAK1 was found to be phosphorylated along with PIM2 upregulation in BMSC, when cocultured 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 upregulation 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   Figures 4D); however, treatment with LLZ dose-dependently suppressed the RANKL-induced expression of NFATc1 and c-fos, and OC formation. TAK1 knockdown by siRNA also abolished the induction of NFATc1 and c-fos expression ( Figure 4E) and osteoclastogenesis ( Figure  4F) by RANKL. Furthermore, MM cells potently induced TRAP-positive multinucleated OC formation from bone marrow cells; however, treatment with LLZ suppressed OC formation ( Figure 4G). These results demonstrate that TAK1 inhibition is able to suppress osteoclastogenesis enhanced by MM cells.

TAK1 inhibition restores osteoblastogenesis suppressed by multiple myeloma cells as well as major inhibitors for osteoblastogenesis in multiple myeloma
In contrast to the enhanced osteoclastogenesis, osteoblastogenesis or bone formation is suppressed in MM. Conditioned media (CM) from MM cell lines as well as inhibitory factors for osteoblastogenesis overproduced in MM, including IL-3, IL-7, TNF-α, TGF-b, and activin A, [8][9][10][11][12] induced the phosphorylation of TAK1 ( Figure 5A) and suppressed mineralized nodule formation ( Figure 5B) in MC3T3-E1 preosteoblastic cells. However, treatment with the TAK1 inhibitor LLZ restored mineralized nodule formation ( Figure 5B). Osterix is an essential transcription factor for osteoblastogenesis, known as a downstream target of BMP-2. The upregulation of Osterix by BMP-2 was reduced in MC3T3-E1 cells in the presence of CM from MM cell lines or TNF-α; however, treatment with LLZ restored the upregulation of Osterix by BMP-2 ( Figure  5C).
TGF-b inhibits the terminal stage of OB differentiation or bone mineralization, whereas BMP-2 is a stimulator for osteoblastogenesis. 11,[35][36][37][38] We and others demonstrated that TGF-b plays a significant role in bone destruction in MM, and that the inhibition of the TGF-b signaling restored bone formation in MM animal models. 11,39-41 Treatment with TGF-b induced the phosphorylation of Smad2 and Smad3 in MC3T3-E1 cells ( Figure 5D). However, TAK1 inhibition with LLZ as well as TAK1 knockdown by siRNA abolished the phosphorylation of these factors. TGF-b has been shown to counteract the BMP-2 signaling to suppress the terminal differentiation of OB in part through the upregulation of Smad6, an inhibitory regulator for BMP-2 signaling. 42 Treatment with LLZ for 24 hours dose-dependently reduced Smad6 protein levels in MC3T3-E1 cells ( Figure 5E, upper). Moreover, LLZ inhibited TGF-b-induced upregulation of Smad6 in MC3T3-E1 cells ( Figure 5E, lower). In contrast, treatment with LLZ as well as TAK1 knockdown by siRNA enhanced the phosphorylation of Smad1/5 in MC3T3-E1 cells by BMP-2 ( Figure 5F). These results collectively suggest that TAK1 inhibition may resume osteoblastogenesis suppressed in MM.

TAK1 inhibition suppresses vascular endothelial growth factor secretion by multiple myeloma cells
Angiogenesis also plays an important role in the pathogenesis and progression of MM. Vascular endothelial growth factor (VEGF) appears to be the most critical angiogenic factor in MM. 43,44 VEGF has been demonstrated to be overproduced downstream of the signaling mediator ERK in MM cells. 45 As expected, TAK1 inhibition with LLZ as well as TAK1 knockdown with siRNA substantially reduced VEGF production by MM cells (Online Supplementary Figure S6). These results suggested that TAK1 inhibition can impair angiogenesis in MM to retard MM progression.

TAK1 inhibition suppresses multiple myeloma tumor progression and prevents bone destruction in vivo
We next examined the in vivo effects of the TAK1 inhibitor LLZ using MM mouse models by intratibial inoculation of mouse 5TGM1 MM cells. Mice were treated with LLZ every other day for 2 weeks from day 6, the day on which 5TGM1 MM cell-derived IgG2b levels started to increase in mouse sera. Vehicle-treated mice showed at day 21 large tumor masses around the tibiae where MM cells were inoculated (Figure 6A), and a progressive increase in serum IgG2b levels over time ( Figure 6B). Bone destruction in the tibiae was observed at day 21 in plain X-ray as well as m-computed tomography (m-CT) images ( Figure 6C). Treatment with LLZ substantially suppressed tumor sizes ( Figure 6A) with almost no increase in serum IgG2b ( Figure 6B), and prevented bone destruction of the tibiae ( Figure 6C). Cathepsin K-expressing OC increased in number on the surface of bone in 5TGM1-inoculated tibiae; however, treatment with LLZ reduced the OC numbers ( Figure 6D). These results demonstrate that TAK1 inhibition is able to suppress MM tumor growth while preventing bone destruction in vivo.
In order to further clarify the effects of TAK1 inhibition  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 inhibition on pathological bone loss without tumor cells, we investigated the effects of LLZ on bone loss in ovariectomized (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 OVXinduced 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 microenvironment 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 therapeutic target to disrupt the key signal transduction pathways responsible for tumor progression and bone destruction in MM. TAK1 activation appears to govern the expression of PIM2 in MM cells and osteoclastic as well as osteoblastic lineage cells; the TAK1-PIM2 signaling pathway is critical for MM tumor expansion and bone destruction. In addition to PIM2 upregulation, TAK1 activation induced Sp1 expression in MM cells. Sp1 is a ubiquitous zinc-finger transcription factor that binds guanine-cytosine-rich elements in the promoter region of its target genes and upregulates various important genes for cancer initiation 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 expression with Sp1 siRNA markedly induced apoptosis in MM cells, indicating that Sp1 as a novel therapeutic target for MM. [25][26][27] Our results showed that TAK1 activation contributes 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 cells 29,48,49 and osteoclastogenesis, 50 these results suggested the therapeutic impact of TAK1 inhibition on CAM-DR as well as osteoclastogenesis induced by the MM-bone marrow interaction.
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 phosphorylation 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 cocultures with MM cells ( Figure 4G). Together with suppression of the TAK1-dependent induction of RANKL expression in BMSC ( Figure 3G), TAK1 inhibition can reduce osteoclastogenesis enhanced in MM through blockade of RANKLmediated 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 preosteoblastic cells; however, TAK1 inhibition restored their osteoblastogenesis ( Figures 5A,B). Taken together, these results underscored the value of TAK1 inhibition for preventing progression of bone destruction and restoring bone formation in MM. Finally, we validated the therapeutic effects of TAK1 inhibition in vivo. Treatment with LLZ markedly suppressed 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 serious unmet issue in patients with MM; bone formation is hard to be restored in MM bone lesions by clinically available 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, targeting 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 compounds with better specificity for TAK1 with less toxicity. Further translational research will elucidate the dividends they may yield in improved clinical outcomes. JT and MA designed the research and conceived the project;  PCR was performed by JT, HT, AO and SS; flow cytometry by  AO, MH and TH; immunoblotting by JT, HT, MH, AO, AB,  TH, SN, MA, SS and MI; transfection by JT, HT, MH, AO and  TH; and cell cultures by JT, HT, MH, AO, AB, TH, SN, MA,  SS, MI, KS, MO, SF, KK and HM, JT, HT, MH, AO, TH,  SN, MH, IE, TH, TM,