Ex vivo expansion and activation of Vγ9Vδ2 T cells by CELMoDs in combination with zoledronic acid.

As multiple myeloma (MM) progresses, immune effector cells decrease in number and function and become exhausted. This remains an insurmountable clinical issue that must be addressed by development of novel modalities to revitalize anti-MM immunity. Human Vγ9Vδ2 T (Vδ2+ γδ T) cells serve as the first line of defense against pathogens as well as tumors and can be expanded ex vivo from peripheral blood mononuclear cells (PBMCs) upon treatment with amino-bisphosphonates in combination with IL-2. Here, we demonstrated that next-generation immunomodulators called cereblon E3 ligase modulators (CELMoDs), as well as lenalidomide and pomalidomide, expanded Th1-like Vδ2+ γδ T cells from PBMCs in the presence of zoledronic acid (ZA). However, the expansion of Th1-like Vδ2+ γδ T cells by these immunomodulatory drugs was abolished under IL-2 blockade, although IL-2 production was induced in PBMCs. BTN3A1 triggers phosphoantigen presentation to γδ T-cell receptors and is required for γδ T-cell expansion and activation. ZA but not these immunomodulatory drugs upregulated BTN3A1 in monocytes. These results suggest that immunomodulatory drugs and ZA have cooperative roles in expansion of Th1-like Vδ2+ γδ T cells, and provide the important knowledge for clinical application of human Vδ2+ γδ T cells as effector cells.

Acute accumulation of PIM2 and NRF2 and recovery of ß5 subunit activity mitigate multiple myeloma cell susceptibility to proteasome inhibitors.

Resistance to proteasome inhibitors (PIs) has emerged as an important clinical issue. We investigated the mechanisms underlying multiple myeloma (MM) cell resistance to PIs. To mimic their pharmacokinetic/pharmacodynamic (PK/PD) profiles, MM cells were treated with bortezomib and carfilzomib for 1 h at concentrations up to 400 and 1,000 nM, respectively. Susceptibility to these PIs markedly varied among MM cell lines. Pulsatile treatments with PIs suppressed translation, as demonstrated by incorporation of puromycin at 24 h in PI-susceptible MM.1S cells, but not PI-resistant KMS-11 cells. Inhibition of ß5 subunit activity decreased at 24 h in KMS-11 cells, even with the irreversible PI carfilzomib, but not under suppression of protein synthesis with cycloheximide. Furthermore, the proteasome-degradable pro-survival factors PIM2 and NRF2 acutely accumulated in MM cells subjected to pulsatile PI treatments. Accumulated NRF2 was trans-localized into the nucleus to induce the expression of its target gene, HMOX1, in MM cells. PIM and Akt inhibition restored the anti-MM effects of PIs, even against PI-resistant KMS-11 cells. Collectively, these results suggest that increased synthesis of ß5 proteasome subunit and acute accumulation of PIM2 and NRF2 reduce the anti-MM effects of PIs.

Therapeutic efficacy of the resorcylic acid lactone LL-Z1640-2 for adult T-cell leukaemia/lymphoma.

Adult T-cell leukaemia/lymphoma (ATL) remains incurable. The NF-κB and interferon regulatory factor 4 (IRF4) signalling pathways are among the critical survival pathways for the progression of ATL. TGF-ß-activated kinase 1 (TAK1), an IκB kinase-activating kinase, triggers the activation of NF-κB. The resorcylic acid lactone LL-Z1640-2 is a potent irreversible inhibitor of TAK1/extracellular signal-regulated kinase 2 (ERK2). We herein examined the therapeutic efficacy of LL-Z1640-2 against ATL. LL-Z1640-2 effectively suppressed the in vivo growth of ATL cells. It induced in vitro apoptosis and inhibited the nuclear translocation of p65/RelA in ATL cells. The knockdown of IRF4 strongly induced ATL cell death while downregulating MYC. LL-Z1640-2 as well as the NF-κB inhibitor BAY11-7082 decreased the expression of IRF4 and MYC at the protein and mRNA levels, indicating the suppression of the NF-κB-IRF4-MYC axis. The treatment with LL-Z1640-2 also mitigated the phosphorylation of p38 MAPK along with the expression of CC chemokine receptor 4. Furthermore, the inhibition of STAT3/5 potentiated the cytotoxic activity of LL-Z1640-2 against IL-2-responsive ATL cells in the presence of IL-2. Therefore, LL-Z1640-2 appears to be an effective treatment for ATL. Further studies are needed to develop more potent compounds that retain the active motifs of LL-Z1640-2.

Mechanisms of preferential bone formation in myeloma bone lesions by proteasome inhibitors.

Proteasome inhibitors (PIs) can preferentially restore bone in bone-defective lesions of patients with multiple myeloma (MM) who respond favorably to these drugs. Most prior in vitro studies on PIs used continuous exposure to low PI concentrations, although pharmacokinetic analysis in patients has shown that serum concentrations of PIs change in a pulsatile manner. In the present study, we explored the effects of pulsatile treatment with PIs on bone metabolism to simulate in vivo PI pharmacokinetics. Pulsatile treatment with bortezomib, carfilzomib, or ixazomib induced MM cell death but only marginally affected the viability of osteoclasts (OCs) with F-actin ring formation. Pulsatile PI treatment suppressed osteoclastogenesis in OC precursors and bone resorption by mature OCs. OCs robustly enhanced osteoblastogenesis in cocultures with OCs and MC3T3-E1 pre-osteoblastic cells, indicating OC-mediated coupling to osteoblastogenesis. Importantly, pulsatile PI treatment did not impair robust OC-mediated osteoblastogenesis. These results suggest that PIs might sufficiently reduce MM cell-derived osteoblastogenesis inhibitors to permit OC-driven bone formation coupling while suppressing OC differentiation and activity in good responders to PIs. OC-mediated coupling to osteoblastogenesis appears to be a predominant mechanism for preferential occurrence of bone regeneration at sites of osteoclastic bone destruction in good responders.

Myeloma bone disease: pathogenesis and management in the era of new anti-myeloma agents.

INTRODUCTION: Multiple myeloma (MM) is a malignancy of plasma cells with characteristic bone disease. Despite recent great strides achieved in MM treatment owing to the implementation of new anti-MM agents, MM is still incurable and bone destruction remains a serious unmet issue in patients with MM. APPROACH: In this review, we will summarize and discuss the mechanisms of the formation of bone disease in MM and the available preclinical and clinical evidence on the treatment for MM bone disease. CONCLUSIONS: MM cells produce a variety of cytokines to stimulate receptor activator of nuclear factor-κB ligand-mediated osteoclastogenesis and suppress osteoblastic differentiation from bone marrow stromal cells, leading to extensive bone destruction with rapid loss of bone. MM cells alter the microenvironment through bone destruction where they colonize, which in turn favors tumor growth and survival, thereby forming a vicious cycle between tumor progression and bone destruction. Denosumab or zoledronic acid is currently recommended to be administered at the start of treatment in newly diagnosed patients with MM with bone disease. Proteasome inhibitors and the anti-CD38 monoclonal antibody daratumumab have been demonstrated to exert bone-modifying activity in responders. Besides their anti-tumor activity, the effects of new anti-MM agents on bone metabolism should be more precisely analyzed in patients with MM. Because prognosis in patients with MM has been significantly improved owing to the implementation of new agents, the therapeutic impact of bone-modifying agents should be re-estimated in the era of these new agents.

The Xanthine Oxidase Inhibitor Febuxostat Suppresses Adipogenesis and Activates Nrf2.

Xanthine oxidoreductase (XOR) is a rate-limiting enzyme in purine catabolism that acts as a novel regulator of adipogenesis. In pathological states, xanthine oxidoreductase activity increases to produce excess reactive oxygen species (ROS). The nuclear factor erythroid 2-related factor 2 (Nrf2) is a critical inducer of antioxidants, which is bound and repressed by a kelch-like ECH-associated protein 1 (Keap1) in the cytoplasm. The Keap1-Nrf2 axis appears to be a major mechanism for robust inducible antioxidant defenses. Here, we demonstrate that febuxostat, a xanthine oxidase inhibitor, alleviates the increase in adipose tissue mass in obese mouse models with a high-fat diet or ovariectomy. Febuxostat disrupts in vitro adipocytic differentiation in adipogenic media. Adipocytes appeared at day 7 in absence or presence of febuxostat were 160.8 ± 21.2 vs. 52.5 ± 12.7 (p < 0.01) in 3T3−L1 cells, and 126.0 ± 18.7 vs. 55.3 ± 13.4 (p < 0.01) in 10T1/2 cells, respectively. Adipocyte differentiation was further enhanced by the addition of hydrogen peroxide, which was also suppressed by febuxostat. Interestingly, febuxostat, but not allopurinol (another xanthine oxidase inhibitor), rapidly induced the nuclear translocation of Nrf2 and facilitated the degradation of Keap1, similar to the electrophilic Nrf2 activator omaveloxolone. These results suggest that febuxostat alleviates adipogenesis under oxidative conditions, at least in part by suppressing ROS production and Nrf2 activation. Regulation of adipocytic differentiation by febuxostat is expected to inhibit obesity due to menopause or overeating.

Novel antimyeloma therapeutic option with inhibition of the HDAC1-IRF4 axis and PIM kinase.

Multiple myeloma (MM) preferentially expands and acquires drug resistance in the bone marrow (BM). We herein examined the role of histone deacetylase 1 (HDAC1) in the constitutive activation of the master transcription factor IRF4 and the prosurvival mediator PIM2 kinase in MM cells. The knockdown or inhibition of HDAC1 by the class I HDAC inhibitor MS-275 reduced the basal expression of IRF4 and PIM2 in MM cells. Mechanistically, the inhibition of HDAC1 decreased IRF4 transcription through histone hyperacetylation and inhibiting the recruitment of RNA polymerase II at the IRF4 locus, thereby reducing IRF4-targeting genes, including PIM2. In addition to the transcriptional regulation of PIM2 by the HDAC1-IRF4 axis, PIM2 was markedly upregulated by external stimuli from BM stromal cells and interleukin-6 (IL-6). Upregulated PIM2 contributed to the attenuation of the cytotoxic effects of MS-275. Class I HDAC and PIM kinase inhibitors cooperatively suppressed MM cell growth in the presence of IL-6 and in vivo. Therefore, the present results demonstrate the potential of the simultaneous targeting of the intrinsic HDAC1-IRF4 axis plus externally activated PIM2 as an efficient therapeutic option for MM fostered in the BM.

Vestigial-Like 3 Plays an Important Role in Osteoblast Differentiation by Regulating the Expression of Osteogenic Transcription Factors and BMP Signaling.

Our previous gene profiling analysis showed that the transcription cofactor vestigial-like 3 (VGLL3) gene expression was upregulated by mechanical tension in the mouse cranial suture, coinciding with accelerated osteoblast differentiation. Therefore, we hypothesized that VGLL3 plays a significant role in osteogenic differentiation. To clarify the function of VGLL3 in osteoblasts, we examined its expression characteristics in mouse bone tissue and the osteoblastic cell line MC3T3-E1. We further examined the effects of Vgll3 knockdown on osteoblast differentiation and bone morphogenetic protein (BMP) signaling. In the mouse cranial suture, where membranous ossification occurs, VGLL3 was immunohistochemically detected mostly in the nucleus of osteoblasts, preosteoblasts, and fibroblastic cells. VGLL3 expression in MC3T3-E1 cells was transient and peaked at a relatively early stage of differentiation. RNA sequencing revealed that downregulated genes in Vgll3-knockdown cells were enriched in gene ontology terms associated with osteoblast differentiation. Interestingly, most of the upregulated genes were related to cell division. Targeted Vgll3 knockdown markedly suppressed the expression of major osteogenic transcription factors (Runx2, Sp7/osterix, and Dlx5) and osteoblast differentiation. It also attenuated BMP signaling; moreover, exogenous BMP2 partially restore osteogenic transcription factors' expression in Vgll3-knockdown cells. Furthermore, overexpression of Vgll3 increased the expression of osteogenic transcription factors. These results suggest that VGLL3 plays a critical role in promoting osteoblast differentiation and that part of the process is mediated by BMP signaling. Further elucidation of VGLL3 function will increase our understanding of osteogenesis and skeletal disease etiology.

TGF-ß-activated kinase-1 inhibitor LL-Z1640-2 reduces joint inflammation and bone destruction in mouse models of rheumatoid arthritis by inhibiting NLRP3 inflammasome, TACE, TNF-α and RANKL expression.

OBJECTIVES: Aberrant NLRP3 inflammasome activation has been demonstrated in rheumatoid arthritis (RA), which may contribute to debilitating inflammation and bone destruction. Here, we explored the efficacy of the potent TGF-ß-activated kinase-1 (TAK1) inhibitor LL-Z1640-2 (LLZ) on joint inflammation and bone destruction in collagen-induced arthritis (CIA). METHODS: LL-Z1640-2 was administered every other day in CIA mice. Clinical and histological evaluation was performed. Priming and activation of NLRP3 inflammasome and osteoclastogenic activity were assessed. RESULTS: NLRP3 inflammasome formation was observed in synovial macrophages and osteoclasts (OCs) in CIA mice. TACE and RANKL were also overexpressed in synovial macrophages and fibroblasts, respectively, in the CIA joints. Treatment with LLZ mitigated all the above changes. As a result, LLZ markedly suppressed synovial hypertrophy and pannus formation to alleviate pain and inflammation in CIA mice. LLZ could block the priming and activation of NLRP3 inflammasome in RAW264.7 macrophage cell line, primary bone marrow macrophages and OCs upon treatment with LPS followed by ATP, thereby suppressing their IL-1ß production. LLZ also suppressed LPS-induced production of TACE and TNF-α in bone marrow macrophages and abolished IL-1ß-induced production of MMP-3, IL-6 and RANKL in synovial fibroblasts. In addition, LLZ directly inhibits RANKL-mediated OC formation and activation. CONCLUSION: TAK1 inhibition with LLZ may become a novel treatment strategy to effectively alleviate inflammasome-mediated inflammation and RANKL-induced osteoclastic bone destruction in joints alongside its potent suppression of TNF-α and IL-6 production and proteinase-mediated pathological processes in RA.

Myeloma-Bone Interaction: A Vicious Cycle via TAK1-PIM2 Signaling.

Multiple myeloma (MM) has a propensity to develop preferentially in bone and form bone-destructive lesions. MM cells enhance osteoclastogenesis and bone resorption through activation of the RANKL-NF-κB signaling pathway while suppressing bone formation by inhibiting osteoblastogenesis from bone marrow stromal cells (BMSCs) by factors elaborated in the bone marrow and bone in MM, including the soluble Wnt inhibitors DKK-1 and sclerostin, activin A, and TGF-ß, resulting in systemic bone destruction with loss of bone. Osteocytes have been drawn attention as multifunctional regulators in bone metabolism. MM cells induce apoptosis in osteocytes to trigger the production of factors, including RANKL, sclerostin, and DKK-1, to further exacerbate bone destruction. Bone lesions developed in MM, in turn, provide microenvironments suited for MM cell growth/survival, including niches to foster MM cells and their precursors. Thus, MM cells alter the microenvironments through bone destruction in the bone where they reside, which in turn potentiates tumor growth and survival, thereby generating a vicious loop between tumor progression and bone destruction. The serine/threonine kinases PIM2 and TAK1, an upstream mediator of PIM2, are overexpressed in bone marrow stromal cells and osteoclasts as well in MM cells in bone lesions. Upregulation of the TAK1-PIM2 pathway plays a critical role in tumor expansion and bone destruction, posing the TAK1-PIM2 pathway as a pivotal therapeutic target in MM.

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

Along with the 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-ß-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 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 (BMSCs) in cocultures with MM cells, which facilitated MM cell-BMSC adhesion while inducing IL-6 production and receptor activator of nuclear factor κ-Β ligand (RANKL) expression by BMSCs. TAK1 inhibition effectively impaired MM cell adhesion to BMSCs to disrupt the support of MM cell growth and survival by BMSCs. 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.

The Roles of ROS Generation in RANKL-Induced Osteoclastogenesis: Suppressive Effects of Febuxostat.

Receptor activator of NF-κB ligand (RANKL), a critical mediator of osteoclastogenesis, is upregulated in multiple myeloma (MM). The xanthine oxidase inhibitor febuxostat, clinically used for prevention of tumor lysis syndrome, has been demonstrated to effectively inhibit not only the generation of uric acid but also the formation of reactive oxygen species (ROS). ROS has been demonstrated to mediate RANKL-mediated osteoclastogenesis. In the present study, we therefore explored the role of cancer-treatment-induced ROS in RANKL-mediated osteoclastogenesis and the suppressive effects of febuxostat on ROS generation and osteoclastogenesis. RANKL dose-dependently induced ROS production in RAW264.7 preosteoclastic cells; however, febuxostat inhibited the RANKL-induced ROS production and osteoclast (OC) formation. Interestingly, doxorubicin (Dox) further enhanced RANKL-induced osteoclastogenesis through upregulation of ROS production, which was mostly abolished by addition of febuxostat. Febuxostat also inhibited osteoclastogenesis enhanced in cocultures of bone marrow cells with MM cells. Importantly, febuxostat rather suppressed MM cell viability and did not compromise Dox's anti-MM activity. In addition, febuxostat was able to alleviate pathological osteoclastic activity and bone loss in ovariectomized mice. Collectively, these results suggest that excessive ROS production by aberrant RANKL overexpression and/or anticancer treatment disadvantageously impacts bone, and that febuxostat can prevent the ROS-mediated osteoclastic bone damage.

Osteoclast-derived IGF1 is required for pagetic lesion formation in vivo.

We report that transgenic mice expressing measles virus nucleocapsid protein (MVNP) in osteoclasts (OCLs) (MVNP mice) are Paget's disease (PD) models and that OCLs from patients with PD and MVNP mice express high levels of OCL-derived IGF1 (OCL-IGF1). To determine OCL-IGF1's role in PD and normal bone remodeling, we generated WT and MVNP mice with targeted deletion of Igf1 in OCLs (Igf1-cKO) and MVNP/Igf1-cKO mice, and we assessed OCL-IGF1's effects on bone mass, bone formation rate, EphB2/EphB4 expression on OCLs and osteoblasts (OBs), and pagetic bone lesions (PDLs). A total of 40% of MVNP mice, but no MVNP/Igf1-cKO mice, had PDLs. Bone volume/tissue volume (BV/TV) was decreased by 60% in lumbar vertebrae and femurs of MVNP/Igf1-cKO versus MVNP mice with PDLs and by 45% versus all MVNP mice tested. Bone formation rates were decreased 50% in Igf1-cKO and MVNP/Igf1-cKO mice versus WT and MVNP mice. MVNP mice had increased EphB2 and EphB4 levels in OCLs/OBs versus WT and MVNP/Igf1-cKO, with none detectable in OCLs/OBs of Igf1-cKO mice. Mechanistically, IL-6 induced the increased OCL-IGF1 in MVNP mice. These results suggest that high OCL-IGF1 levels increase bone formation and PDLs in PD by enhancing EphB2/EphB4 expression in vivo and suggest OCL-IGF1 may contribute to normal bone remodeling.

Persistent Activation of Calcium-Sensing Receptor Suppresses Bone Turnover, Increases Microcracks, and Decreases Bone Strength.

Activating mutations of calcium-sensing receptor (CaSR) cause autosomal dominant hypocalcemia type 1 (ADH1). Patients with ADH1 exhibit similar features to patients with hypoparathyroidism, including reduced serum parathyroid hormone (PTH) and Ca with low bone turnover. Although persistent suppression of bone turnover may increase bone fragility, bone strength in ADH1 patients has been unclear. We created knock-in mice harboring the A843E activating mutation of CaSR, mimicking severe features of ADH1 patients. The severe form of ADH1 model mice showed smaller body and bone size with lower bone mineral density (BMD) and cortical area of the femur compared with age-matched wild-type (WT) mice. Bone strength in the femur was lower in ADH1 mice even after correction by bone geometry and/or BMD. Microcracks were markedly increased in ADH1 mice, but were rarely detected in WT mice. There was a negative correlation between bone strength corrected by bone geometry and/or BMD and microcrack number or density in ADH1 and WT mice. Among ADH1 mice, negative correlation was still observed between bone strength and microcrack number or density. Microcracks increased with age in ADH1 mice, and were negatively correlated with bone strength. Treatment with PTH(1-34) or a calcilytic, JTT-305, increased bone turnover, reduced microcracks, and increased bone strength to similar levels to those in WT mice. The increase in microcracks was associated with a reduction in bone strength in ADH1 mice, and aging aggravates these changes. These results demonstrate that activating mutation of CaSR causes reduction in PTH secretion with suppressed bone turnover, that reduced bone turnover is associated with an age-dependent increase in microcracks with a reduction in bone strength, and that both PTH(1-34) and calcilytic ameliorate all these changes in bone turnover and strength. It is suggested that fracture susceptibility may be increased in severe types of ADH1 patients especially in the elderly. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

PD-L1 upregulation in myeloma cells by panobinostat in combination with interferon-γ.

Immunotherapy is revolutionizing the treatment paradigm for multiple myeloma (MM). Interferon (IFN)-γ is essential for immune responses, whereas immune checkpoint molecules, such as programmed cell death-1 ligand-1 (PD-L1), mitigate the beneficial anti-tumor immune responses. As HDAC inhibitors alter the immunogenicity and anti-tumor immune responses, we here explored the regulation of PD-L1 expression in MM cells by the clinically available HDAC inhibitor panobinostat in the presence of IFN-γ. IFN-γ activated the STAT1-IRF1 pathway to upregulate PD-L1 expression in MM cells, and panobinostat was able to upregulate their PD-L1 expression without activating the STAT1-IRF1 pathway. Of note, panobinostat enhanced IFN-γR1 expression, which substantially increased the total and phosphorylated levels of STAT1 protein but reduced IRF1 protein levels through proteasomal degradation in the presence of IFN-γ. Panobinostat further enhanced the IFN-γ-mediated durable STAT1 activation in MM cells; STAT1 gene silencing abolished the PD-L1 upregulation by panobinostat and IFN-γ in combination, indicating a critical role for STAT1. These results suggest that panobinostat enhances PD-L1 expression by facilitating the IFN-γ-STAT1 pathway in a ligand-dependent manner in MM cells with ambient IFN-γ. PD-L1 upregulation should be taken into account when combining immunotherapies with panobinostat.

Effective impairment of myeloma cells and their progenitors by hyperthermia.

Multiple myeloma (MM) remains incurable, and MM-initiating cells or MM progenitors are considered to contribute to disease relapse through their drug-resistant nature. In order to improve the therapeutic efficacy for MM, we recently developed novel superparamagnetic nanoparticles which selectively accumulate in MM tumors and extirpate them by heat generated with magnetic resonance. We here aimed to clarify the therapeutic effects on MM cells and their progenitors by hyperthermia. Heat treatment at 43°C time-dependently induced MM cell death. The treatment upregulated endoplasmic reticulum (ER) stress mediators, ATF4 and CHOP, while reducing the protein levels of Pim-2, IRF4, c-Myc and Mcl-1. Combination with the proteasome inhibitor bortezomib further enhanced ER stress to potentiate MM cell death. The Pim inhibitor SMI-16a also enhanced the reduction of the Pim-2-driven survival factors, IRF4 and c-Myc, in combination with the heat treatment. The heat treatment almost completely eradicated "side population" fractions in RPMI8226 and KMS-11 cells and suppressed their clonogenic capacity as determined by in vitro colony formation and tumorigenic capacity in SCID mice. These results collectively demonstrated that hyperthermia is able to impair clonogenic drug-resistant fractions of MM cells and enhance their susceptibility to chemotherapeutic drugs.

Unique anti-myeloma activity by thiazolidine-2,4-dione compounds with Pim inhibiting activity.

Proviral Integrations of Moloney virus 2 (PIM2) is overexpressed in multiple myeloma (MM) cells, and regarded as an important therapeutic target. Here, we aimed to validate the therapeutic efficacy of different types of PIM inhibitors against MM cells for their possible clinical application. Intriguingly, the thiazolidine-2,4-dione-family compounds SMI-16a and SMI-4a reduced PIM2 protein levels and impaired MM cell survival preferentially in acidic conditions, in contrast to other types of PIM inhibitors, including AZD1208, CX-6258 and PIM447. SMI-16a also suppressed the drug efflux function of breast cancer resistance protein, minimized the sizes of side populations and reduced in vitro colony-forming capacity and in vivo tumourigenic activity in MM cells, suggesting impairment of their clonogenic capacity. PIM2 is known to be subject to ubiquitination-independent proteasomal degradation. Consistent with this, the proteasome inhibitors bortezomib and carfilzomib increased PIM2 protein levels in MM cells without affecting its mRNA levels. However, SMI-16a mitigated the PIM2 protein increase and cooperatively enhanced anti-MM effects in combination with carfilzomib. Collectively, the thiazolidine-2,4-dione-family compounds SMI-16a and SMI-4a uniquely reduce PIM2 protein in MM cells, which may contribute to their profound efficacy in addition to their immediate kinase inhibition. Their combination with proteasome inhibitors is envisioned.