Abstract

Introduction Proteasome inhibitor bortezomib is an effective therapy for the treatment of relapsed and refractory multiple myeloma (RRMM); however, prolonged treatment can be associated with toxicity and drug resistance. A novel proteasome inhibitor marizomib is distinct from bortezomib in its chemical structure, mechanisms of action, and effects on proteasomal activities (Chauhan et al., Cancer Cell 2005, 8:407-419). Pomalidomide is an analogue of thalidomide with potent immunomodulatory activity. Based on increased progression-free survival, pomalidomide has been approved by the FDA for the treatment of patients with RRMM who have received at least two prior therapies, including lenalidomide and bortezomib, and who showed disease progression on or within 60 days of completion of the most recent therapy. Here we utilized in vitro and in vivo models of MM to examine the anti-MM activity of combined marizomib and pomalidomide. Animal model studies examined the efficacy of marizomib (PO) using both single weekly and twice weekly schedule either alone or together with pomalidomide (PO).

Methods Cell viability, and apoptosis were performed using WST/MTT, and Annexin V, respectively. Synergistic anti-MM activity was determined with CalcuSyn software program. Proteasome activity was measured, as in prior study (Chauhan et al., Cancer Cell 2005). MM.1S-tumor-bearing mice were treated with vehicle control, marizomib (PO) pomalidomide (PO), or marizomib plus pomalidomide at the indicated doses for 21 days on a twice-weekly or once weekly schedule for marizomib and 4 consecutive days weekly for pomalidomide. Statistical significance was determined using a Student's t test. Pomalidomide was purchased from Selleck chemicals; and marizomib was obtained from Triphase Accelerator, USA.

Results MM cell lines (MM.1R, MM.1S, INA-6, RPMI-8226, LR5, Dox-40, bortezomib-sensitive ANBL6.WT, and bortezomib-resistant ANBL6.BR) and primary patient MM cells were pretreated with pomalidomide for 24h; marizomib was then added for an additional 24h, followed by measurement of cell viability. A significant decrease in viability of all cell lines was observed in response to treatment with combined low doses of marizomib and pomalidomide vs. either agent alone. Isobologram analysis confirmed the synergistic anti-MM activity of these agents (CI < 1.0). The cytotoxicity of combination therapy was observed in MM cell lines sensitive and resistant to novel therapies, and in p53-null ARP-1 MM cells. A significant decrease in cell viability of all patient MM cells was noted after combination therapy as compared to either compound alone (p < 0.05 for all patients). In contrast, combined low doses of marizomib plus pomalidomide did not significantly affect the viability of normal PBMCs, suggesting a favorable therapeutic index for this combination regimen. Tumor cells from 5 of 7 patients were obtained from patients whose disease was progressing while on bortezomib, dex, and lenalidomide therapies. Marizomib plus pomalidomide-induced apoptosis was associated with: 1) activation of caspase-8, caspase-9, caspase-3, and PARP cleavage; 2) downregulation of cereblon (CRBN), IRF4, c-Myc, and Mcl-1; and 3) suppression of CT-L, C-L, and T-L proteasome activities. CRBN-siRNA attenuated marizomib plus pomalidomide-induced MM cells death. Furthermore, marizomib plus pomalidomide inhibited the migration of MM cells and tumor-associated angiogenesis, and overcame cytoprotective effects of BM milieu. Human MM xenograft model study showed that combined low doses of marizomib (twice weekly; PO)) and pomalidomide (4 consecutive days weekly; PO) for 3 weeks were well tolerated, inhibited tumor growth, and prolonged survival. Importantly, combination of marizomib on once weekly (PO) schedule with pomalidomide (PO) was active and led to prolongation of survival. Finally, inhibition of CT-L (63%), T-L (40%) and C-L (29%) proteasome activity was observed in tumors from marizomib plus pomalidomide-treated mice vs. untreated mice.

Conclusion Our preclinical data from in vitro studies and in vivo MM xenograftmodels demonstrate that oral marizomib plus pomalidomide trigger synergistic anti-MM activity, enhance proteasome inhibition, and overcome drug resistance. These studies support the continuation of clinical trials of combined marizomib and pomalidomide to improve outcome in patients with RRMM.

Disclosures

Trikha:Triphase Accelerator Corporation: Employment. Chauhan:Stemline Therapeutics: Consultancy.

Author notes

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Asterisk with author names denotes non-ASH members.