PR-924, a Selective Inhibitor of the Immunoproteasome Subunit LMP-7 Blocks Multiple Myeloma Cell Growth Both in Vitro and In Vivo.

Therapeutic targeting of Ubiquitin-Proteasome pathway is exemplified by the recent FDA approval of dipeptidyl boronic acid bortezomib first-in-class proteasome inhibitor for the treatment of multiple myeloma (MM). As with other agents, dose-limiting toxicities and the development of drug resistance limit its long-term utility. The β5, β1 and β2 catalytic subunits within the 26S proteasome mediate chymotrypsin-like, caspase-like, and trypsin-like activities, respectively. Importantly, these catalytic subunits have corresponding immunoproteasome components LMP-7, LMP-2 and multicatalytic endopeptidase complex subunit-1 (MECL-1), which regulate immune cell function and cytokine production; however, the role of the immunoproteasome in MM cells is still unclear. Recent studies have therefore focused on the discovery and development of small molecule inhibitors of the immunoproteasomes, which will both delineate the function of immunproteasomes and allow for specific therapeutic targeting of the UPS in order to reduce off-target activities and associated toxicities. Here, we examined PR-924, an LMP-7-selective peptide-ketoepoxide proteasome inhibitor related to carfilzomib. PR-924, like carfilzomib, contains a ketopoxide pharmacophore that covalently modifies proteasomal N-terminal threonine active sites. We examined the effects of PR-924 in MM cell lines and primary patient cells in vitro. To determine the in vivo efficacy of PR-924, we utilized two xenograft models of human MM in SCID mice, a subcutaneous tumor plasmacytoma model and the SCID-hu model, which best reflects the human MM-BM microenvironment in vivo.

We utilized MM.1S, MM.1R, RPMI-8226, U266, DOX40, KMS12, LR-5, OPM1, OPM2 and INA-6 (an IL-6 dependent) human MM cell lines, as well as purified tumor cells from patients with MM relapsing after prior therapies including lenalidomide or bortezomib. Cell viability and apoptosis assays were performed using Trypan blue, MTT and Annexin V staining. Immunoblot analysis was performed using antibodies to caspase-8, caspase-9, caspase-3, caspase-7, PARP, Bcl-2, BID, or GAPDH. For tumor xenograft studies, CB-17 SCID male mice (n = 10; 5 mice/each group) were subcutaneously inoculated with 5.0 × 106 MM.1S cells in 100 microliters of serum-free RPMI-1640 medium. When tumors were measurable (∼150 mm3) 2-3 weeks after MM cell injection, mice were injected IV with either PR-924 (6 mg/kg BW) or vehicle twice weekly. Mice were sacrificed when their tumors reached >2 cm3. In the SCID-hu model, 2 × 106 INA-6 cells were injected directly into human bone chips implanted subcutaneously in SCID mice (n=10: 5 mice/EA group), and MM cell growth was assessed by serial measurements of circulating levels of soluble human interleukin-6 receptor (shulIL6R) in mouse serum. Statistical significance of differences observed in PR-924 vs. vehicle treated mice was determined using a Student t test.

PR-924 significantly inhibits growth of all the MM cell lines in a time- and dose-dependent manner (IC50 range: 3-5 μM; P <0.005 for all cell lineIt alsos. reduced the viability of primary patient cells (P < 0.05; n=5), without significant effects on normal peripheral mononuclear cells. The PR-924-triggered decrease in MM cell viability is due to apoptosis, as evidenced by Annexin V/PI staining. Moreover, PR-924-induced apoptosis in MM.1S and MM.1R MM cells is associated with activation of caspase-3, caspase-8, caspase-9, caspase-7, BID and PARP. In vivo PR-924 triggered significant tumor growth inhibition in tumor plasmacytoma xenografts (2.3 fold decrease in tumor volume in mice receiving PR-924 versus mice injected with vehicle alone; P value = 0.01). Similarly, a significant reduction in the shuIL6R levels (3.4 fold decrease; P value = 0.02) was observed in mice treated with PR-924 versus vehicle-control. PR-924 treatment was well tolerated, as evidenced by the lack of weight loss even after three weeks of treatment. Importantly, treatment of tumor bearing mice with PR-924, but not vehicle alone, significantly prolonged survival (P < 0.005).

Our preclinical findings establish immunoproteasome LMP-7 as a novel therapeutic target in MM.

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