Junctional adhesion molecule-A is overexpressed in advanced multiple myeloma and determines response to oncolytic reovirus

Abstract

Despite the development of several new agents for multiple myeloma (MM) therapy over the last decade, drug resistance continues to be a significant problem. Patients with relapsed/refractory disease have high mortality rates and desperately need new precision approaches that directly target specific molecular features that are prevalent in the refractory setting. Reolysin is a proprietary formulation of reovirus for cancer therapy that has demonstrated efficacy in multiple clinical trials. Its selective effects against solid tumors have been largely attributed to RAS-mediated control of reovirus replication. However, the mechanisms regulating its preferential anti-neoplastic effects in MM and other hematological malignancies have not been rigorously studied. Here we report that the reovirus receptor, junctional adhesion molecule-A (JAM-A) is highly expressed in primary cells from patients with MM and the majority of MM cell lines compared to normal controls. A series of experiments demonstrated that JAM-A expression, rather than RAS, was required for Reolysin-induced cell death in MM models. Notably, analysis of paired primary MM specimens revealed that JAM-A expression was significantly increased at relapse compared to diagnosis. Two different models of acquired resistance to bortezomib also displayed both higher JAM-A expression and elevated sensitivity to Reolysin compared to parental cells, suggesting that Reolysin may be an effective agent for patients with relapsed/refractory disease due to their high JAM-A levels. Taken together, these findings support further investigation of Reolysin for the treatment of patients with relapsed/refractory MM and of JAM-A as a predictive biomarker for sensitivity to Reolysin-induced cell death.

Keywords: JAM-A; NOXA; bortezomib; myeloma; reovirus.

Figure 1. Reovirus replication in MM cells induces apoptosis independently of RAS activity status

A. PBMCs from healthy donors and 7 MM cell lines were treated with 30 PFU/Cell Reolysin for 48 h. Reovirus replication was determined by transmission electron microscopy. Arrows denote reovirus accumulation. Bar represents 2 microns or 500 nm as indicated on each image. B. Reolysin decreases cell viability in MM cell lines while showing little activity against PBMCs or OPM-2 cells. PBMCs and MM cell lines were treated with the indicated amounts of Reolysin for 72 h and cell viability was measured by MTT assay. Mean ± SD, n = 3. C. Reolysin induces caspase-3 processing in all MM cell lines except OPM-2. MM cells were treated for 48 hours with the indicated concentrations of Reolysin. Active caspase-3 was measured using fluorescent antibody staining and flow cytometry. Mean ± SD, n = 3. D. Cells susceptible to Reolysin-mediated apoptosis induce NOXA expression. Cells were treated for 48 h with 30 PFU/Cell Reolysin. NOXA expression was determined by immunoblotting. E. Reolysin stimulates apoptosis in all MM cell lines except OPM-2. Cells were treated with the indicated concentrations of Reolysin and apoptosis was measured by PI-FACS analysis. Mean ± SD, n = 3. F. Determination of RAS activity in MM cell lines. Constitutively active RAS levels were determined in MM cell lines using an active RAS pull-down kit. GTPγS and GDP treated cells served as positive and negative controls, respectively.

Figure 2. JAM-A is overexpressed in MM cells

A. JAM-A was measured by immunoblotting in 7 MM cell lines and normal PBMCs. Tubulin served as a loading control. B. Cell surface expression of JAM-A on MM cell lines and PBMCs. JAM-A was measured by fluorescent staining and flow cytometry. Mean ± SD, n = 3. C. Measurement of JAM-A (F11R) transcript levels. F11R expression was determined in MM cell lines by qRT-PCR analysis. Mean ± SD, n = 3. D. F11R expression in normal plasma cells (NPC) from 22 healthy subjects, 44 subjects with MGUS, and 351 patients with newly diagnosed MM. E. Kaplan-Meier analyses of PFS revealed inferior outcomes among patients with high JAM-A expression compared with patients with low JAM-A expression. Groupings are based on quartiles with p values calculated for the top half compared to the bottom half (50/50) of JAM-A expression *Indicates a significant difference from low JAM-A expression group, p = 0.04.

Figure 3. JAM-A regulates reovirus sensitivity in MM

A. shRNA-mediated knockdown of JAM-A in RPMI-8226 cells. Cells were infected with JAM-A lentiviral shRNA and knockdown was determined by immunoblotting. B. Cells with silenced JAM-A are significantly less sensitive to Reolysin. Cells infected with control or JAM-A shRNA were treated with Reolysin for 72 h. Cell viability was measured by MTT assay. Mean ± SD, n = 3, *p < 0.05 compared to shRNA control treated cells. C. Cells with decreased JAM-A levels are resistant to Reolysin-mediated apoptosis. RPMI-8226 cells were treated with 30 PFU/Cell Reolysin for 48 h. Active caspase-3 levels were measured by fluorescent staining and flow cytometry. Mean ± SD, n = 3, *p < 0.05 compared to shRNA control treated cells. D. Overexpression of JAM-A in OPM-2 cells. JAM-A was transfected into OPM-2 cells and immunoblotting confirmed increased expression at 72 h following transfection. E. Forced expression of JAM-A sensitizes OPM-2 cells to Reolysin. Vector control and JAM-A transfected cells were treated with the indicated concentrations of Reolysin for 72 h. Cell viability was measured by MTT assay. Mean ± SD, n = 3, *p < 0.05 compared to Vector control treated cells. F. JAM-A overexpression sensitizes OPM-2 cells to Reolysin-mediated apoptosis. OPM-2 cells were treated with 30 PFU/Cell Reolysin for 48 h and apoptosis was measured by active caspase-3 staining and flow cytometry. Mean ± SD, n = 3, *p < 0.05 compared to Vector control treated cells.

Figure 4. JAM-A (F11R) expression is significantly upregulated in MM patients at relapse

A. F11R levels are increased in MM patients at relapse. A total of 51 paired CD138+ specimens from MM patients were measured for F11R expression (P = 0.0002). B. F11R levels are increased in freshly isolated CD138+ cells from refractory/relapsed MM patients (n = 4) and one refractory Waldenstrom's macroglobulinemia patient compared to those with newly diagnosed MM (n = 6). F11R was determined by qRT-PCR. Mean ± SD, *p < 0.05 compared to newly diagnosed MM. C. Relapsed/refractory MM/WM patients (n = 5) display increased sensitivity to Reolysin compared to newly diagnosed MM patients (n = 6). CD138+ cells from patients were treated with varying concentrations of Reolysin and cell viability was determined by MTT assay. IC₅₀ values to Reolysin were calculated and plotted. Mean ± SD, *p < 0.05 compared to newly diagnosed MM cells treated with Reolysin.

Figure 5. BZ-resistant MM cells display elevated JAM-A expression and enhanced reovirus replication

A-B. BZ resistance in MM cell lines. Parental and BZ-resistant cells were treated with the indicated concentrations of BZ for 72 h. Cell viability was determined by MTT assay. Mean ± SD, n = 3. *p < 0.05 compared to parental cells treated with Reolysin. C. BZ-resistant cells display increased reovirus levels by electron microscopy. Cells were treated with 30 PFU/Cell Reolysin for 48 h and harvested for electron microscopy. Arrows denote reovirus accumulation. Bar represents 2 microns. D-E. BZ-resistant cells exhibit increased JAM-A expression. F11R/JAM-A levels were measured by qRT-PCR and immunoblotting. Mean ± SD, n = 3, *Significant increase compared to parental cells, p < 0.05.

Figure 6. BZ-resistant MM cells exhibit increased sensitivity to Reolysin-induced NOXA upregulation and apoptosis

A. BZ-resistant cells display increased sensitivity to Reolysin. Cells were treated with the indicated concentrations of Reolysin for 72 h. Cell viability was determined by MTT assay. Mean ± SD, n = 3, *Significant difference compared to Reolysin treated parental cells, p < 0.05. B. BZ-resistant cells are more sensitive to Reolysin-mediated apoptosis. Cells were treated with 30 PFU/Cell Reolysin for 48 h. Apoptosis was measured by active caspase-3 assay (Left) and PI-FACS analysis (Right). Mean ± SD, n = 3, *Significant increase compared to Reolysin treated parental cells, p < 0.05. C-D. NOXA (PMAIP1) expression following Reolysin treatment. Parental and BZ-resistant variants were treated with 30 PFU/Cell Reolysin for 48 h. PMAIP1/NOXA expression was measured by qRT-PCR and immunoblotting. Mean ± SD, n = 3, *Significant increase compared to Reolysin treated parental cells, p < 0.05. Immunoblot band intensity was quantified by densitometry.