Abstract
Introduction The bone marrow (BM) microenvironment confers growth, survival, and drug resistance in multiple myeloma (MM) cells (Chauhan et al, Cancer Cell 2009, 16:309-323) BM hypoxia (low oxygenation) plays a role in promoting MM cell survival, drug resistance, migration, and metastasis. Novel therapies that selectively target the MM cell in its hypoxic BM milieu may therefore overcome conventional drug resistance. Recent studies led to the development of a novel aerospace industry-derived Phase 2 molecule RRx-001 with hypoxia-selective epigenetic and NO-donating properties. A Phase I clinical trial demonstrated promising evidence of anti-tumor activity in a heavily pretreated population with no dose-limiting toxicities (Reid et al. J Clin Oncol 32:5s, 2014 suppl; abstr 2578, Reid et al, Lancet Oncology, in press). RRx-001 is currently under investigation in multiple Phase II clinical trials. Here we examined both the mechanism of action and anti-MM activity of RRx-001 using in vitro and in vivo models of MM.
Methods Cell viability, apoptosis, and migration assays were performed using MTT, Annexin V staining, and transwell Inserts, respectively. ROS and NO generation was measured as previously described (Chauhan et al., Blood, 2004, 104:2458). Synergistic anti-MM activity was assessed by isobologram analysisusing "CalcuSyn" software program. In vitro angiogenesis was assessed using matrigel capillary-like tube formation assays. DNMT1 activity was measured using DNMT1 assay kit. USP7 siRNA was purchased from Dharmacon. CB-17 SCID-mice were subcutaneously inoculated with MM.1S cells as previously described (Chauhan et al., Cancer Cell 2012, 11:345-358). Statistical significance of data was determined using a Student's t test. RRx-001 was obtained from EpicentRx, CA, USA; USP7 inhibitor P5091, bortezomib, SAHA, and pomalidomide were purchased from Selleck chemicals, USA.
Results Treatment of MM cell lines (MM.1S, MM.1R, RPMI-8226, OPM2, H929, Dox-40 ARP-1, KMS-11, ANBL6.WT, ANBL6.BR, and LR5) and primary patient cells for 48h significantly decreased their viability (IC50 range 1.25nM to 2.5nM) (p < 0.05; n=3) without markedly affecting PBMCs from normal healthy donors, suggesting specific anti-MM activity and a favorable therapeutic index for RRx-001. Tumor cells from 3 of 5 patients were obtained from patients whose disease was progressing while on bortezomib, dexamethasone, and lenalidomide therapies. Moreover, RRx-001 inhibits proliferation of MM cells, even in the presence of BM stromal cells. Washout experiments showed that a short time (3h) exposure of MM cells to RRx-001 triggered irreversible cell death. RRx-001-triggered apoptosis is associated with: 1) induction of DNA damage response signaling via ATM/p53/gH2AX axis; 2) activation of caspases mediating both intrinsic and extrinsic apoptotic pathways; 3) increase in oxidative stress through release of ROS and generation of NO; and 4) decrease in DNMT1 activity and global methylation levels. Furthermore, RRx-001 blocked migration of MM cells and angiogenesis. Deubiqyitylating enzyme USP7 stimulates DNMT1 enzymatic activity. USP7-siRNA reduced DNMT1 activity and decreased MM cell viability. Importantly, the combination of USP7 inhibitor P5091 and RRx-001 triggered synergistic anti-MM activity associated with a robust decrease in DNMT1 activity, as well as increased degradation of USP7 substrate MDM2 and induction of downstream p21/p53 signaling axis. In vivo studies using a subcutaneous human MM xenograft model shows that RRx-001 is well tolerated, inhibits tumor growth, and enhances survival. Finally, combining RRx-001 with pomalidomide, bortezomib or SAHA induces synergistic anti-MM activity in p53-WT and p53-null MM cells, and overcomes drug resistance.
Conclusion Our preclinical studies demonstrate that RRx-001, a ROS-mediated epigenetic inhibitor with anti-angiogenic properties selectively targets MM cells in vivo and synergizes with existing anti-MM agents to overcome therapeutic resistance. Our data also suggest a potential mechanism of action for RRx-001-induced epigenetic changes via USP7-DNMT1 complex and downstream p53/p21 signaling cascade. Collectively, these results provide a rationale for rapid translation of RRx-001, either alone or in combination, in a clinical trial of relapsed refractory MM.
Disclosures
Oronsky: epicentrx: Employment. Scicinski:epicentrx: Employment. Chauhan:Stemline Therapeutics: Consultancy.
Cathepsin L inhibition suppresses drug resistance in vitro and in vivo: a putative mechanism
