Ajita V. Singh

A novel orally active proteasome inhibitor ONX 0912 triggers in vitro and in vivo cytotoxicity in multiple myeloma

Bortezomib therapy has proven successful for the treatment of relapsed, relapsed/refractory, and newly diagnosed multiple myeloma (MM). At present, bortezomib is available as an intravenous injection, and its prolonged treatment is associated with toxicity and development of drug resistance. Here we show that the novel proteasome inhibitor ONX 0912, a tripeptide epoxyketone, inhibits growth and induces apoptosis in MM cells resistant to conventional and bortezomib therapies. The anti-MM activity of ONX-0912 is associated with activation of caspase-8, caspase-9, caspase-3, and poly(ADP) ribose polymerase, as well as inhibition of migration of MM cells and angiogenesis. ONX 0912, like bortezomib, predominantly inhibits chymotrypsin-like activity of the proteasome and is distinct from bortezomib in its chemical structure. Importantly, ONX 0912 is orally bioactive. In animal tumor model studies, ONX 0912 significantly reduced tumor progression and prolonged survival. Immununostaining of MM tumors from ONX 0912-treated mice showed growth inhibition, apoptosis, and a decrease in associated angiogenesis. Finally, ONX 0912 enhances anti-MM activity of bortezomib, lenalidomide dexamethasone, or pan-histone deacetylase inhibitor. Taken together, our study provides the rationale for clinical protocols evaluating ONX 0912, either alone or in combination, to improve patient outcome in MM.

Functional interaction of plasmacytoid dendritic cells with multiple myeloma cells: a therapeutic target.

Multiple myeloma (MM) remains incurable despite novel therapies, suggesting the need for further identification of factors mediating tumorigenesis and drug resistance. Using both in vitro and in vivo MM xenograft models, we show that plasmacytoid dendritic cells (pDCs) in the bone marrow (BM) microenvironment both mediate immune deficiency characteristic of MM and promote MM cell growth, survival, and drug resistance. Microarray, cell signaling, cytokine profile, and immunohistochemical analysis delineate the mechanisms mediating these sequelae. Although pDCs are resistant to novel therapies, targeting toll-like receptors with CpG oligodeoxynucleotides both restores pDC immune function and abrogates pDC-induced MM cell growth. Our study therefore validates targeting pDC-MM interactions as a therapeutic strategy to overcome drug resistance in MM.

Combination of novel proteasome inhibitor NPI-0052 and lenalidomide trigger in vitro and in vivo synergistic cytotoxicity in multiple myeloma

Our recent study demonstrated that a novel proteasome inhibitor NPI-0052 is distinct from bortezomib (Velcade) and, importantly, triggers apoptosis in multiple myeloma (MM) cells resistant to bortezomib. Here we demonstrate that combining NPI-0052 and lenalidomide (Revlimid) induces synergistic anti-MM activity in vitro using MM-cell lines or patient MM cells. NPI-0052 plus lenalidomide-induced apoptosis is associated with (1) activation of caspase-8, caspase-9, caspase-12, caspase-3, and poly(ADP) ribose polymerase; (2) activation of BH-3 protein BIM; (3) translocation of BIM to endoplasmic reticulum; (4) inhibition of migration of MM cells and angiogenesis; and (5) suppression of chymotrypsin-like, caspase-like, and trypsin-like proteasome activities. Importantly, blockade of BIM using siRNA significantly abrogates NPI-0052 plus lenalidomide-induced apoptosis. Furthermore, studies using biochemical inhibitors of caspase-8 versus caspase-9 demonstrate that NPI-0052 plus lenalidomide-triggered apoptosis is primarily dependent on caspase-8 signaling. In animal tumor model studies, low-dose combination of NPI-0052 and lenalidomide is well tolerated, significantly inhibits tumor growth, and prolongs survival. Taken together, our study provides the preclinical rationale for clinical protocols evaluating lenalidomide together with NPI-0052 to improve patient outcome in MM.

PR‐924, a selective inhibitor of the immunoproteasome subunit LMP‐7, blocks multiple myeloma cell growth both in vitro and in vivo

PR‐924 is an LMP‐7‐selective tripeptide epoxyketone proteasome inhibitor that covalently modifies proteasomal N‐terminal threonine active sites. In the present study, we show that PR‐924 inhibits growth and triggers apoptosis in multiple myeloma (MM) cell lines and primary patient MM cells, without significantly affecting normal peripheral blood mononuclear cells. PR‐924‐induced apoptosis in MM cells is associated with activation of caspase‐3, caspase‐8, caspase‐9, BID, PARP and cytochrome–c release. In vivo administration of PR‐924 inhibits tumour growth in human plasmacytoma xenografts. Results from SCID‐hu model show a significant reduction in the shIL‐6R levels in mice treated with PR‐924 versus vehicle‐control. PR‐924 treatment was well tolerated as evidenced by the lack of weight loss. Importantly, treatment of tumour‐bearing mice with PR‐924, but not vehicle alone, prolonged survival. Our preclinical findings therefore validate immunoproteasome LMP‐7 subunit as a novel therapeutic target in MM.

Pharmacodynamic and Efficacy Studies of the Novel Proteasome Inhibitor NPI-0052 (marizomib) in a Human Plasmacytoma Xenograft Murine Model

Our previous study showed that the novel proteasome inhibitor NPI‐0052 induces apoptosis in multiple myeloma (MM) cells resistant to conventional and bortezomib (Velcade™, Takeda, Boston, MA, USA) therapies. In vivo studies using human MM‐xenografts demonstrated that NPI‐0052 is well tolerated, prolongs survival, and reduces tumour recurrence. These preclinical studies provided the basis for an ongoing phase‐1 clinical trial of NPI‐0052 in relapsed/refractory MM patients. Here we performed pharmacodynamic (PD) studies of NPI‐0052 using human MM xenograft murine model. Our results showed that NPI‐0052: (i) rapidly left the vascular compartment in an active form after intravenous (i.v.) administration, (ii) inhibited 20S proteasome chymotrypsin‐like (CT‐L, β5), trypsin‐like (T‐L, β2), and caspase‐like (C‐L, β1) activities in extra‐vascular tumours, packed whole blood (PWB), lung, liver, spleen, and kidney, but not brain and (iii) triggered a more sustained (>24 h) proteasome inhibition in tumours and PWB than in other organs (<24 h). Tissue distribution analysis of radiolabeled compound (3H‐NPI‐0052) in mice demonstrated that NPI‐0052 left the vascular space and entered organs as the parent compound. Importantly, treatment of MM.1S‐bearing mice with NPI‐0052 showed reduced tumour growth without significant toxicity, which was associated with prolonged inhibition of proteasome activity in tumours and PWB but not normal tissues.

A novel vascular disrupting agent plinabulin triggers JNK-mediated apoptosis and inhibits angiogenesis in multiple myeloma cells

Previous studies have established a role of vascular-disrupting agents as anti- cancer agents. Plinabulin is a novel vascular-disrupting agent that exhibits potent interruption of tumor blood flow because of the disruption of tumor vascular endothelial cells, resulting in tumor necrosis. In addition, plinabulin exerts a direct action on tumor cells, resulting in apoptosis. In the present study, we examined the anti-multiple myeloma (MM) activity of plinabulin. We show that low concentrations of plinabulin exhibit a potent antiangiogenic action on vascular endothelial cells. Importantly, plinabulin also induces apoptotic cell death in MM cell lines and tumor cells from patients with MM, associated with mitotic growth arrest. Plinabulin-induced apoptosis is mediated through activation of caspase-3, caspase-8, caspase-9, and poly(ADP-ribose) polymerase cleavage. Moreover, plinabulin triggered phosphorylation of stress response protein JNK, as a primary target, whereas blockade of JNK with a biochemical inhibitor or small interfering RNA strategy abrogated plinabulin-induced mitotic block or MM cell death. Finally, in vivo studies show that plinabulin was well tolerated and significantly inhibited tumor growth and prolonged survival in a human MM.1S plasmacytoma murine xenograft model. Our study therefore provides the rationale for clinical evaluation of plinabulin to improve patient outcome in MM.

Preclinical evaluation of a novel SIRT1 modulator SRT1720 in multiple myeloma cells.

SIRT1 belongs to the silent information regulator 2 (Sir2) protein family of enzymes and functions as a NAD+‐dependent class III histone deacetylase. Here, we examined the anti‐multiple myeloma (MM) activity of a novel oral agent, SRT1720, which targets SIRT1. Treatment of MM cells with SRT1720 inhibited growth and induced apoptosis in MM cells resistant to conventional and bortezomib therapies without significantly affecting the viability of normal cells. Mechanistic studies showed that anti‐MM activity of SRT1720 is associated with: (i) activation of caspase‐8, caspase‐9, caspase‐3, poly(ADP) ribose polymerase; (ii) increase in reactive oxygen species; (iii) induction of phosphorylated ataxia telangiectasia mutated/checkpoint kinase 2 signalling; (iv) decrease in vascular endothelial growth factor‐induced migration of MM cells and associated angiogenesis; and (v) inhibition of nuclear factor‐κB. Blockade of ATM attenuated SRT1720‐induced MM cell death. In animal tumour model studies, SRT1720 inhibited MM tumour growth. Finally, SRT1720 enhanced the cytotoxic activity of bortezomib or dexamethasone. Our preclinical studies provide the rationale for novel therapeutics targeting SIRT1 in MM.