Ze Tian

A Small Molecule Inhibitor of Ubiquitin-Specific Protease-7 Induces Apoptosis in Multiple Myeloma Cells and Overcomes Bortezomib Resistance

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.

In Vitro and In Vivo Selective Antitumor Activity of a Novel Orally Bioavailable Proteasome Inhibitor MLN9708 against Multiple Myeloma Cells

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 used 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 endoplasmic reticulum (ER) stress response proteins Bip, phospho-eIF2-α, and CHOP; and inhibition of nuclear factor kappa B. Finally, combining MLN2238 with lenalidomide, histone deacetylase inhibitor suberoylanilide hydroxamic acid, 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. Clin Cancer Res; 17(16); 5311–21. ©2011 AACR.

HDAC inhibition by LBH589 affects the phenotype and function of human myeloid dendritic cells

LBH589 is a novel pan-histone deacetylase (HDAC) inhibitor that has potent antitumor activity in multiple myeloma and other hematological malignancies. However, its impact on the 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, which was associated with DC maturation (CD83↓), antigen presentation (human leukocyte antigen-ABC↓) and T-cell co-stimulation (CD40↓ and CD86↑). LBH589 decreased both protein and polysaccharide antigen uptake capacities by DCs. Importantly, LBH589 impaired DC function to stimulate antigen-specific immune responses, resulting in the significant reduction of invariant natural killer T-cell (CD1d-restricted) and T-cell (major histocompatibility complex-restricted) activation in innate and adaptive immunity. LBH589 also significantly repressed the production of interleukin (IL)-6, IL-10, IL-12p70, IL-23 and tumor necrosis factor-α by Toll-like receptor (TLR)3 and TLR4-induced DC activation, indicating an important role of HDAC activity in immune regulation and inflammation. RelB, a component of the nuclear factor-κ B signaling pathway, was the key component regulated by HDAC inhibition in DCs. Together, our preclinical study demonstrates that LBH589 significantly impairs the 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.

Investigational agent MLN9708/2238 targets tumor-suppressor miR33b in MM cells

miRs play a critical role in tumor pathogenesis as either oncogenes or tumor-suppressor genes. However, the role of miRs and their regulation in response to proteasome inhibitors in multiple myeloma (MM) is unclear. In the current study, miR profiling in proteasome inhibitor MLN2238-treated MM.1S MM cells shows up-regulation of miR33b. Mechanistic studies indicate that the induction of miR33b is predominantly via transcriptional regulation. Examination of miR33b in patient MM cells showed a constitutively low expression. Overexpression of miR33b decreased MM cell viability, migration, colony formation, and increased apoptosis and sensitivity of MM cells to MLN2238 treatment. In addition, overexpression of miR33b or MLN2238 exposure negatively regulated oncogene PIM-1 and blocked PIM-1 wild-type, but not PIM-1 mutant, luciferase activity. Moreover, PIM-1 overexpression led to significant abrogation of miR33b- or MLN2238-induced cell death. SGI-1776, a biochemical inhibitor of PIM-1, triggered apoptosis in MM. Finally, overexpression of miR33b inhibited tumor growth and prolonged survival in both subcutaneous and disseminated human MM xenograft models. Our results show that miR33b is a tumor suppressor that plays a role during MLN2238-induced apoptotic signaling in MM cells, and these data provide the basis for novel therapeutic strategies targeting miR33b in MM.

Preclinical activity of the oral proteasome inhibitor MLN9708 in Myeloma bone disease.

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-κB ligand)-induced NF-κB activation, F-actin ring disruption, and diminished expression of αVβ3 integrin. A similar range of MLN2238 concentrations promoted in vitro osteoblastogenesis and osteoblast activity (even in osteoprogenitors from patients with myeloma), partly mediated by activation of TCF/β-catenin signaling and upregulation of the IRE1 component of the unfolded protein response. In a mouse model of bone marrow–disseminated human multiple myeloma, orally administered MLN2238 was equally effective as bortezomib to control tumor burden and also provided a marked benefit in associated bone disease (sustained by both bone anabolic and anticatabolic activities). Conclusion: Given favorable data on pharmacologic properties and emerging clinical safety profile of MLN9708, it is conceivable that this proteasome inhibitor may achieve bone beneficial effects in addition to its antimyeloma activity in patients with myeloma. Clin Cancer Res; 20(6); 1542–54. ©2014 AACR.

A novel TLR-9 agonist C792 inhibits plasmacytoid dendritic cell-induced myeloma cell growth and enhance cytotoxicity of bortezomib

Our prior study in multiple myeloma (MM) patients showed increased numbers of plasmacytoid dendritic cells (pDCs) in the bone marrow (BM), which both contribute to immune dysfunction as well as promote tumor cell growth, survival and drug resistance. Here we show that a novel Toll-like receptor (TLR-9) agonist C792 restores the ability of MM patient-pDCs to stimulate T-cell proliferation. Coculture of pDCs with MM cells induces MM cell growth; and importantly, C792 inhibits pDC-induced MM cell growth and triggers apoptosis. In contrast, treatment of either MM cells or pDCs alone with C792 does not affect the viability of either cell type. In agreement with our in vitro data, C792 inhibits pDC-induced MM cell growth in vivo in a murine xenograft model of human MM. Mechanistic studies show that C792 triggers maturation of pDCs, enhances interferon-α and interferon-λ secretion and activates TLR-9/MyD88 signaling axis. Finally, C792 enhances the anti-MM activity of bortezomib, lenalidomide, SAHA or melphalan. Collectively, our preclinical studies provide the basis for clinical trials of C792, either alone or in combination, to both improve immune function and overcome drug resistance in MM.

A novel hypoxia-selective epigenetic agent RRx-001 triggers apoptosis and overcomes drug resistance in multiple myeloma cells

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: (i) activation of caspases; (ii) release of ROS and nitrogen species; (iii) induction of DNA damage via ATM/γ-H2AX; and (iv) decrease in DNA methyltransferase (DNMT) and global methylation. RNA interference study shows a predominant role of DNMT1 in MM cell survival versus DNMT3a or DNMT3b. The 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.

Abstract C94: Deubiquitylases as novel anticancer targets: Discovery and development of novel USP7 inhibitors

The ubiquitin‐proteasome pathway is a complex system consisting of enzymes that conjugate or deconjugate ubiquitin to/from target proteins upstream of the 26S proteasome. In particular, ubiquitin deconjugation is performed by families of proteases known as deubiquitylating enzymes (DUBs). The therapeutic validation of the ubiquitin‐proteasome system was provided by the marketing approval of bortezomib (PS‐341) for the treatment of multiple myeloma (MM). In our search for alternative approaches for the treatment of MM, we investigated the therapeutic potential of inhibiting another component of the ubiquitin‐proteasome pathway, the DUB USP7. Our rationale for targeting USP7 is as follows: 1) Blockade of USP7 using siRNA or homologous recombination results in elevated levels of the tumor suppressor p53 and cell cycle arrest in cancer cells. In contrast to the situation in the majority of human tumors, mutations or deletions of p53 in MM are relatively rare and thus the activation of p53 is likely to offer a therapeutic benefit in MM. 2) Knockdown of USP7 destabilizes the adaptor protein Claspin, inhibiting activation of Chk1, an effector kinase in the DNA damage response, abrogating the G2M cell cycle checkpoint and providing a mechanism for inducing tumor cell death in a p53 independent manner. Thus inhibition of USP7 would be predicted to activate pro‐apoptotic and inactivate pro‐survival pathways in multiple myeloma, resulting in tumor suppression. Recently, using its proprietary screening technology, Progenra discovered and characterized a small molecule inhibitor of USP7, P5091. Specifically, P5091 selectively inhibits USP7 relative to other proteases and exhibits cytotoxic activity against both p53 wild type and p53 mutant tumor cell lines. Furthermore, P5091 destabilizes the pharmacodynamic markers HDM2 and Claspin in tumor cells and induces cell death synergistically in combination with genotoxic agents. Medicinal chemistry has identified a series of potent and selective novel chemical entities. Initial preclinical studies at the Dana‐Farber Cancer Institute demonstrate that P5091 induces apoptosis and is cytotoxic against human MM cells but not normal plasma cells. In conclusion, these data demonstrate that 1) USP7 is a viable target for the treatment of MM and other cancers; and 2) P5091 is an efficacious inhibitor of USP7. Preclinical development of the P5091 series continues, augmented by a comprehensive medicinal chemistry program designed to identify a clinical candidate from this series in 2010. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C94.