Summary Bortezomib therapy has proven successful for the treatment of relapsed/refractory, relapsed and newly diagnosed multiple myeloma (MM); however, dose-limiting toxicities and the development of resistance limit its long-term utility. Here we show that P5091 is an inhibitor of deubiquitylating enzyme USP7, which induces apoptosis in MM cells resistant to conventional and bortezomib therapies. Biochemical and genetic studies show that blockade of HDM2 and p21 abrogates P5091-induced cytotoxicity. In animal tumor model studies, P5091 is well tolerated, inhibits tumor growth, and prolongs survival. Combining P5091 with lenalidomide, HDAC inhibitor SAHA, or dexamethasone triggers synergistic anti-MM activity. Our preclinical study therefore supports clinical evaluation of USP7 inhibitor, alone or in combination, as a potential MM therapy.

Key Points• Deubiquitylating enzymes USP14 and UCHL5 are involved in the tumorigenesis of MM.• b-AP15 is a specific USP14 and UCHL5 inhibitor, which blocks growth and induces apoptosis in MM cells.Proteasome inhibitors have demonstrated that targeting protein degradation is effective therapy in multiple myeloma (MM). Here we show that deubiquitylating enzymes (DUBs) USP14 and UCHL5 are more highly expressed in MM cells than in normal plasma cells. USP14 and UCHL5 short interfering RNA knockdown decreases MM cell viability. A novel 19S regulatory particle inhibitor b-AP15 selectively blocks deubiquitylating activity of USP14 and UCHL5 without inhibiting proteasome activity. b-AP15 decreases viability in MM cell lines and patient MM cells, inhibits proliferation of MM cells even in the presence of bone marrow stroma cells, and overcomes bortezomib resistance. Anti-MM activity of b-AP15 is associated with growth arrest via downregulation of CDC25C, CDC2, and cyclin B1 as well as induction of caspase-dependent apoptosis and activation of unfolded protein response. In vivo studies using distinct human MM xenograft models show that b-AP15 is well tolerated, inhibits tumor growth, and prolongs survival. Combining b-AP15 with suberoylanilide hydroxamic acid, lenalidomide, or dexamethasone induces synergistic anti-MM activity. Our preclinical data showing efficacy of b-AP15 in MM disease models validates targeting DUBs in the ubiquitin proteasomal cascade to overcome proteasome inhibitor resistance and provides the framework for clinical evaluation of USP14/UCHL5 inhibitors to improve patient outcome in MM. (Blood. 2014;123(5):706-716)

Purpose The success of bortezomib therapy for treatment of multiple myeloma (MM) led to the development of structurally and pharmacologically distinct novel proteasome inhibitors. In the present study, we evaluated the efficacy of one such novel orally bioactive proteasome inhibitor MLN9708/MLN2238 in MM using well-established in vitro and in vivo models. Experimental Design MM cell lines, primary patient cells, and the human MM xenograft animal model were utilized to study the antitumor activity of MN2238. Results Treatment of MM cells with MLN2238 predominantly inhibits chymotrypsin-like activity of the proteasome and induces accumulation of ubiquitinated proteins. MLN2238 inhibits growth and induces apoptosis in MM cells resistant to conventional and bortezomib therapies without affecting the viability of normal cells. In animal tumor model studies, MLN2238 is well tolerated and inhibits tumor growth with significantly reduced tumor recurrence. A head-to-head analysis of MLN2238 versus bortezomib showed a significantly longer survival time in mice treated with MLN2238 than mice receiving bortezomib. Immununostaining of MM tumors from MLN2238-treated mice showed growth inhibition, apoptosis, and a decrease in associated angiogenesis. Mechanistic studies showed that MLN2238-triggered apoptosis is associated with activation of caspase-3, caspase-8, and caspase-9; increase in p53, p21, Noxa, PUMA, and E2F; induction of ER stress response proteins Bip, phospho-eIF2-α, and CHOP; and inhibition of NF-κB. Finally, combining MLN2238 with lenalidomide, HDAC inhibitor SAHA or dexamethasone triggers synergistic anti-MM activity. Conclusion Our preclinical study supports clinical evaluation of MLN9708, alone or in combination, as a potential MM therapy.

OBJECTIVES-To investigate a B-cell-depleting strategy to reverse diabetes in naïve NOD mice. RESEARCH DESIGN AND METHODS-We targeted the CD22 receptor on B-cells of naïve NOD mice to deplete and reprogram B-cells to effectively reverse autoimmune diabetes.RESULTS-Anti-CD22/cal monoclonal antibody (mAb) therapy resulted in early and prolonged B-cell depletion and delayed disease in pre-diabetic mice. Importantly, when new-onset hyperglycemic mice were treated with the anti-CD22/cal mAb, 100% of B-celldepleted mice became normoglycemic by 2 days, and 70% of them maintained a state of long-term normoglycemia. Early therapy after onset of hyperglycemia and complete B-cell depletion are essential for optimal efficacy. Treated mice showed an increase in percentage of regulatory T-cells in islets and pancreatic lymph nodes and a diminished immune response to islet peptides in vitro. Transcriptome analysis of reemerging B-cells showed significant changes of a set of proinflammatory genes. Functionally, reemerging B-cells failed to present autoantigen and prevented diabetes when cotransferred with autoreactive CD4 ϩ T-cells into NOD.SCID hosts. Most individuals affected by type 1 diabetes exhibit multiple features associated with impaired B-cell function, including autoantibodies against a variety of islet cell antigens (6,7). Data from different groups using NOD mice, the best animal model for the study of type 1 diabetes, have confirmed the importance of B-cells in the onset of diabetes (2-4,8,9). NOD mice that are deficient in B-cells have been shown to be protected from autoimmune diabetes (3,10,11) and are deficient in the development of a T-cell response to major autoantigens (such as 65-kDa glutamate decarboxylase) (3,10,11). In humans, the production of autoantibodies to islet antigens is well documented as an early indicator of disease onset (12). These observations render B-cell targeting a particularly attractive and novel strategy for the treatment of type 1 diabetes (13-15). Unfortunately, this strategy has not been fully described in naïve NOD mice. Only recently did a publication show the positive effects of an anti-CD20 -based B-cell-depleting strategy in transgenic NOD mice expressing the humanized CD20 receptor on B-cells (8). Interestingly, use of B-cell depletion as a therapy for human autoimmune disease (16 -20), including in patients with new-onset type 1 diabetes, is ongoing (21,22). CONCLUSIONS-TargetingWe made use of a newly developed reagent (anti-CD22 calicheamicin-conjugated monoclonal antibody [anti-CD22/cal mAb]) that efficiently depletes mature B-cells in mice (13) to establish a therapeutic approach for type 1 diabetes. Our main hypothesis was that depleting B-cells by targeting CD22 should prevent diabetes onset and restore normoglycemia in newly hyperglycemic NOD mice. Furthermore, we hypothesize that our approach will generate a pool of reemerging B-cells that may function to regulate the autoimmune response in vivo, establishing a state of long-term tolerance toward autoantigens. RESEA...

Despite the growing understanding of PDGF signaling, studies of PDGF function have encountered two major obstacles: the functional redundancy of PDGFRα and PDGFRβ in vitro and their distinct roles in vivo. Here we used wild-type mouse embryonic fibroblasts (MEF), MEF null for either PDGFRα, β, or both to dissect PDGF-PDGFR signaling pathways. These four PDGFR genetically defined cells provided us a platform to study the relative contributions of the pathways triggered by the two PDGF receptors. They were treated with PDGF-BB and analyzed for differential gene expression, in vitro proliferation and differential response to pharmacological effects. No genes were differentially expressed in the double null cells, suggesting minimal receptor-independent signaling. Protean differentiation and proliferation pathways are commonly regulated by PDGFRα, PDGFRβ and PDGFRα/β while each receptor is also responsible for regulating unique signaling pathways. Furthermore, some signaling is solely modulated through heterodimeric PDGFRα/β.

LBH589 is a novel pan-HDAC inhibitor which has potent antitumor activity in multiple myeloma and other hematologic malignancies. However, its impact on immune system has not been defined. We here evaluated the effects of LBH589 on human myeloid dendritic cells (DCs) at clinically relevant concentrations. Exposure to LBH589 affected the surface molecule expression on immature and mature DCs, associated with DC maturation (CD83↓), antigen presentation (HLA-ABC↓), and T cell co-stimulation (CD40↓ and CD86↑). LBH589 decreased both protein and polysaccharide antigen uptake capacities by DCs. Importantly, LBH589 impaired DCs function to stimulate antigen-specific immune responses, resulting in the significant reduction of invariant NKT cell (CD1d-restricted) and T cell (MHC-restricted) activation in innate and adaptive immunity. LBH589 also significantly repressed the production of IL-6, IL-10, IL-12p70, IL-23 and TNF-α by TLR3 and TLR4-induced DCs activation, indicating an important role of HDAC activity in immune regulation and inflammation. RelB, a component of NF-κB signaling pathway, was the key component regulated by HDAC inhibition in DCs. Together, our preclinical study demonstrates that LBH589 significantly impairs phenotype and function of DCs, indicating a need for monitoring the immune status in patients receiving HDAC inhibitor therapy. It also provides a rationale to evaluate LBH589 activity for the treatment of inflammation.

Purpose: MLN9708 (ixazomib citrate), which hydrolyzes to pharmacologically active MLN2238 (ixazomib), is a next-generation proteasome inhibitor with demonstrated preclinical and clinical antimyeloma activity, but yet with an unknown effect on myeloma bone disease. Here, we investigated its bone anabolic and antiresorptive effects in the myeloma setting and in comparison with bortezomib in preclinical models.Experimental Design: The in vitro effect of MLN2238 was tested on osteoclasts and osteoclast precursors from healthy donors and patients with myeloma, and on osteoprogenitors derived from bone marrow mesenchymal stem cells also from both origins. We used an in vivo model of bone marrow-disseminated human myeloma to evaluate MLN2238 antimyeloma and bone activities.Results: Clinically achievable concentrations of MLN2238 markedly inhibited in vitro osteoclastogenesis and osteoclast resorption; these effects involved blockade of RANKL (receptor activator of NF-kB ligand)-induced NF-kB activation, F-actin ring disruption, and diminished expression of aVb3 integrin. A similar range of MLN2238 concentrations promoted in vitro osteoblastogenesis and osteoblast activity (even in osteoprogenitors from patients with myeloma

The hypoxic bone-marrow (BM) microenvironment confers growth/survival and drug-resistance in multiple myeloma (MM) cells. Novel therapies targeting the MM cell in its hypoxic-BM milieu may overcome drug resistance. Recent studies led to the development of a novel molecule RRx-001 with hypoxia-selective epigenetic and Nitric Oxide-donating properties. Here we demonstrate that RRx-001 decreases the viability of MM cell lines and primary patient cells, as well as overcomes drug-resistance. RRx-001 inhibits MM cell growth in the presence of BM stromal cells. RRx-001 induced apoptosis is associated with: 1) activation of caspases; 2) release of ROS and nitrogen-species; 3) induction of DNA damage via ATM/γ-H2AX; and 4) decrease in DNA methytransferase (DNMT) and global methylation. RNA interference study shows a predominant role of DNMT1 in MM cell survival versus DNMT3a or DNMT3b. Deubiquitylating enzyme USP7 stimulates DNMT1 activity; and conversely, USP7-siRNA reduced DNMT1 activity and decreased MM cell viability. RRx-001 plus USP7 inhibitor P5091 triggered synergistic anti-MM activity. MM xenograft studies show that RRx-001 is well tolerated, inhibits tumor growth, and enhances survival. Combining RRx-001 with pomalidomide, bortezomib or SAHA induces synergistic anti-MM activity. Our results provide the rationale for translation of RRx-001, either alone or in combination, to clinical evaluation in MM.