UNIVERSITY OF CALGARY
Investigating the Administration of Sunitinib with Oncolytic Reovirus as a Novel Treatment Strategy for Multiple Myeloma
By
Winston Gee Kong Yuen
A THESIS SUBMITTED TO THE CUMMING SCHOOL OF MEDICINE IN PARTIAL FULFILMENT OF THE REQUREMENTS FOR THE DEGREE OF BACHELOR OF HEALTH SCIENCES HONOURS
Bachelor of Health Sciences Cumming School of Medicine University of Calgary Calgary, AB
© Winston Gee Kong Yuen 2015
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I. Abstract Purpose: While oncolytic reovirus (RV) is an attractive bio-therapeutic for multiple myeloma (MM) with low side effects, treatment resistance in some phenotypes remains problematic. In this study, we sought to combine the immunomodulatory effects of sunitinib with RV to overcome treatment resistance, and provide evidence for this combination as a novel therapeutic to treat MM. Experimental Design: RV and sunitinib effective dose 50 (ED50) values were established in MM cell lines OPM2, KMS11, and RPMI8226, which have characteristically diverse sensitivities to RV. RV and sunitinib combinative effects were measured using the WST-1 cell viability assay, and combination indexes (CI) were calculated via the Chao-Talalay method in OPM2 and RPMI8226 cells. Apoptosis, measured through flow cytometry, and viral replication, measured through plaque titration assay, were assessed as mechanisms of combinative effects. Results: The Chao-Talalay method revealed that RV and sunitinib combination were either additive or synergistic in all concentrations of RV and sunitinib tested in OPM2, while this was antagonistic in RPMI8226. Preliminary results indicate that apoptosis is a major mediator of combinative RV and sunitinib treatment in both OPM2 and RPMI8226 MM cell lines. Additionally, pilot data suggests that RV replication is increased in the presence of sunitinib compared to RV alone in OPM2 cells. Conclusion: In this study, we have shown that combining sunitinib with RV can overcome RV resistance in MM cells, as exemplified by OPM2 cells. Moving forward, this provides important preliminary data to extrapolate into an in vivo murine models, ex vivo MM patient specimen testing for these combinations, and phase I MM clinical trials. This evidence can lead to the future
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application of RV and sunitinib combination therapy as a novel treatment strategy for MM. Further research should also exploit immune modulatory effects of RV and sunitinib in MM.
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II. Acknowledgments I would like to acknowledge my supervisors Dr. Don Morris and Dr. Chandini Thirukkumaran. Both have provided me with superb feedback in my writing and presentations, and kept me on track. I would also like to acknowledge the staff of the Morris Laboratory. Qiao Shi has been a significant help in teaching me the various assays, including providing me with all the necessary tools to succeed. Kathy Gratton and Ahmed Mostafa have been a tremendous help with the analysis of my flow cytometry data. Lastly, I would like to acknowledge the rest of the members of the Tom Baker Translational Lab including Cay Egan, Adrijana D’Silva, Peter Liu, Dr. Gwyn Bebb, Lars Peterson, Allison Childers, and Emeka Enwere. They have been of tremendous help both inside and outside of the laboratory.
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III. Dedication This thesis is dedicated to my grandmother, Ah Ching Poon, who passed away at the Tom Baker Cancer Centre on July 18, 2013. This thesis is also dedicated to the staff of TBCC Unit 36 whom have been the most wonderful of care support to Ah Ching and my family.
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Table of Contents I. Abstract ………………..……………………………………………………………………… ii II. Acknowledgments ….………………………………………………………………………... iv III. Dedication ….…………………………………………….…………………………………. v IV. List of Tables ….……………………………..………….…………………………………. vii V. List of Figures ….…………………………………………….…..…………………………. vii
1. Review of Literature ………………………………………………………………………....... 1 2. Methods ………..……………………………………………………………………………… 5 3. Results …………..…………………………………………...…………………………..……. 7 4. Discussion ………………………………………………………………………….………. 10 5. Conclusion …....…………….……………….……………….……………………………… 12 6. References ………………….………………………………………………………………. 14 7. Appendix A: Figures and Tables ……………………………………………..………………18 8. Appendix B: Ethics Approval …………………………………………………………….…. 24
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IV. List of Tables Table 1. Sunitinib and reovirus ED50 doses for MM cell lines calculated using linear regression analysis. Table 2. Combination indexes (CI) generated by the Chou-Talalay method for OPM2 and RPMI8226 treated with reovirus and sunitinib at constant ratios of ED50 values.
V. List of Figures Figure 1. Effect of reovirus on multiple myeloma cell lines. Figure 2. Dose response of (A) OPM2, (B) KMS11, and (C) RPMI8226 cells treated with serial dilutions of sunitinib from 0.47 to 30 ÂľM in vitro. Figure 3. Synergistic and additive effects between reovirus and sunitinib in OPM2 and antagonistic effects in RPMI8226 cells. Figure 4. Apoptosis is a major mediator of cytotoxicity in RV and sunitinib treated MM cell lines. Figure 5. Viral progeny production in OPM2 cells following infection with RV or RV and sunitinib combination.
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1. Review of Literature 1.1. Multiple myeloma Multiple myeloma (MM) is a clonal neoplasm of plasma cell malignancy derived from Blymphocytes and is characterized most commonly by the accumulation of immunoglobulin G secreting plasma cells in the bone marrow microenvironment [1]. Patients who suffer from MM experience hypercalcemia, recurring bacterial infection, renal insufficiency, anemia and bone lesions [2]. MM is the most common bone cancer, affecting over 70,000 people in North America and 2,300 Canadians annually, with the incidences doubling over the last fifty years [3]. MM accounts for approximately 10% of all hematological malignancies and approximately 1-2% of all cancer-related deaths [1]. Treatment for MM have rapidly evolved over the past decade. Stem cell rescue following a high-dose chemotherapy with autologous transplantation is a standard treatment for over 50% of MM patients [3-5]. More recently, novel therapeutics including the proteasome inhibitors, bortezomib, carfilzomib, and the immunomodulatory agents, thalidomide, lenalidomide, have become frontline therapies for MM patients [3-5]. With the advent of these novel therapeutics, stem cell rescue, and other treatments such as dexamethasone in the MM treatment arsenal, the median survival of MM has increased from a previously reported 3-5 years, to 8-10 years [3-5]. Despite these advances, MM remains incurable and all patients succumb to this disease. Doses of treatment necessary to achieve total tumour remission are often toxic to patients [6]. In addition, treatment resistance can arise, further limiting their pharmaceutical efficacy. Thus, it is imperative that more effective treatment strategies are explored. 1.2. Reovirus
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Oncolytic viruses offer a biological alternative to pharmaceutical therapeutics with the advantage that they can infect, propagate and lyse cancer cells while sparing non-cancerous cells [7]. As such, reovirus (RV), a ubiquitous, non-enveloped double-stranded RNA virus, has been shown to have extensive efficacy and negligible pathogenicity in humans in breast, prostate, brain tumors, renal carcinoma, and hematological malignancies in vitro, in vivo, ex vivo and in clinical trials [7,8]. With the over 1000 patients treated with RV to date, the maximum tolerated dose has not been reached, further exhibiting the safety and tolerance of the treatment. Furthermore, RV exemplifies genomic stability, and ease of manufacturing, the ideal characteristics an oncolytic virus should possess [8]. The wide range of histologies that are treatable with RV may be mediated through activated aberrant oncogenic pathways that these cancers possess [9-10]. The aberrant signaling pathways that allow cancer cells to proliferate also create a permissive environment for the RV RNA translation and the propagation of RV. Our laboratory has previously shown that treatment of MM with RV downregulates phosphorylation of Akt, a potent mediator of cancerous proliferation, promotes rapid translocation of NF-ÎşB, and increases terminal caspase activity [11-12]. Interestingly, in experimental murine models, RV treatment is also capable of priming innate and adaptive anti-tumour immune responses [13-15] as well as build long-term tumour immunosurveillance [16-18]. In a phase I clinical trial, when prostate cancer patients who had exhausted cytotoxic chemotherapy and radiotherapy were treated with intratumoural RV, an increase of CD8+ cells were noted in the RV treated tumour and not in adjacent non-cancerous tissues [19]. Thus the effects of RV are not limited to the direct oncolytic effects only. In addition, the release of MM mediated immune suppression by immunomodulatory drugs may also facilitate RV mediated anti-tumour immune responses in MM patients.
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1.3. Mechanisms of RV mediated cell death With lytic cycles as a key step of viral replication, many viruses have shown capabilities of both inducing and inhibiting apoptosis following infection. Evidence in our lab, and others, have shown that RV oncolysis is mediated primarily via apoptotic mechanisms [11,20-24]. Transcription factor NF-ÎşB (nuclear factor-kappa B) has been shown to be an instrumental molecule in apoptosis induction in the HeLa cervical carcinoma cell line [20]. In addition, presensitization of ovarian cancer cell lines (OVCAR3, PA-1 and SKOV-3), breast (ZR75-1) and lung (H157), with TNF-related apoptosis-inducing ligand (TRIAL), which activates caspase-8 dependent apoptosis, was shown to synergistically enhance apoptosis [20]. In contrast, this interaction in some colorectal cancer cell lines (C26 and HCT116) was not observed [23]. RVmediated oncolysis in these colorectal cancer cells were found to be dependent of ras mutations rather than RV replicative abilities [23]. In our laboratory, treatment of breast cancer cell lines MCF7 and HTB 133 resulted in the upregulation of genes such as tumour necrosis factor alpha induced protein (TNFÎąI-P), TRAIL receptor 2, TNF receptor 6, TNF member 1, and TNF receptor superfamily member 6 associated factor, as well as, 2-27 fold increases NF-kB, signal transducer and activator of transcription- 5 (STAT 5), and p53 upregulated modulator of apoptosis (PUMA) [11]. More recently, we have shown in our laboratory that inhibition of apoptotic pathways via Z-VAD-FML-001 in MM cancer cell lines RPMI8226, NCI-H929 and U266, lead to decreased, but incomplete inhibition of cell death. Further tests resulted in the novel finding that RV-mediated oncolysis is also attributed to autophagic pathways [25].
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1.4. Sunitinib Sunitinib is a multi-targeted tyrosine-kinase inhibitor (TKI) with anti-angiogenic effects, with activity against the stem cell-factor receptor (Kit) and platelet-derived growth-factor receptor (PDGFR), vascular endothelial growth-factor receptor (VEGFR), colony-stimulating factor-1 receptor (CSF1R), and Fms-like tyrosine kinase-3 receptor (FLT3) [26]. However, more recent studies have highlighted sunitinib’s enhancement of cell-mediated immunity against tumoural cells, independent of its antitumoural activity [27]. This group of renal cell carcinoma patients showed that undergoing sunitinib therapy that were administrated two cycles of 50 mg daily doses for 4 weeks every 6 weeks showed an increased percentage of interferon (IFN)-γ producing Tcells compared to no treatment [27]. As such, sunitinib has undergone phase II clinical trial in patients with relapsed MM [28]. More recent studies with sunitinib have also been shown to reduce tumour burden and improve overall survival in murine models of melanoma, hepatocellular carcinoma, and colorectal metastasis, following immunotherapeutic administration [29-33]. However, the aforementioned clinical trial has shown that sunitinib monotherapy was shown to prolong event free survival of MM patients only for 2.86 months [28], thus suggesting the need for its evaluation in conjunction with other agents. 1.5. Multiple myeloma and the immune system MM has a unique ability to evade immunosurveillance through mechanisms such as over expression of myeloid derived suppressor cells (MDSCs), expansion of regulatory T cells (Treg), reduced T-cell cytotoxic activity and responsiveness to IL-2 defects in B-cell immunity, and induction of dendritic cell (DC) dysfunction [30]. These immune evading tactics may, contribute
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significantly to the unsustainable responses to treatment in MM. Work conducted in our laboratory has shown that sunitinib can augment RV mediated immune modulation through the suppression of tumour infiltrating MDSCs and altering cytokine profiles in renal cell carcinoma, breast, and lung cancer models [29, 31]. However, this has not been studied in MM. Thus, in the current study, we sought to evaluate the synergy of effects between RV and sunitinib.
2. Methods 2.1. Cell Lines and Virus OPM2, KMS11, RPMI8226, and L-929 cell lines were obtained from the American Type Tissue Collection (Manassas, VA). OPM2, KMS11 and RPMI8226 were cultured in RPMI medium supplemented with 10% heat-inactivated fetal bovine serum (FBS; Invitrogen, Grand Island, NY). OPM2 medium was additionally supplemented with 1% HEPES, 1% sodium pyruvate, and 1 mg/ml D-glucose. Cultures were free of antibiotics. RV serotype 3 (Dearing strain) was grown in L-929 cells then purified and tittered as described [19]. RV was inactivated via a short wavelength ultraviolet (UV) and checked for activity and sterility as previously described [19]. 2.2. Cell Viability Assay To evaluate the effective dose 50 (ED50) of cells to sunitinib, OPM2, KMS11 and RPMI8226 cells were plated in 96 well plates at 6.0 x 104 cells/well, 3.0 x 104 cells/well and 3.5 x 104 cells/well for OPM2, KMS11 and 8226 respectively. Sunitinib was serially diluted at concentrations of 0.47 µM to 30 µM in a total concentration of 200 µl. After an incubation period of 48 hours at 37ºC 5% CO2, 100 µl WST-1 (Roche, Basel) was added to each well and incubated
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for 2 hours. Absorbance was quantified using a Bio-rad® plate reader (Hercules, CA). Percent viability was calculated as the absorbance ratio of treated/untreated cells multiplied by 100. ED50 values were calculated using linear regression. To evaluate the combination of RV and sunitinib, cells were seeded at a density of 6.0 x 104 cells/well, 2.0 x 104 cells/well and 2.5 x 104 cells/well for OPM2, KMS11, and RPMI8226 respectively into 96 well micro-titre plates in serum free media. RV, sunitinib, or their combination were then added to each well and incubated for 48 hours in 10% serum containing media in a total volume of 200µl. Following this, 150µl of WST-1 was added to each well and incubated for 2 hours and absorbance was measured as recorded above. 2.3. Viral Progeny Assay OPM2 and RPMI8226 cells were treated with RV or sunitinib and RV combination in 12well plates in their respective media for 0, 12, 24, 36, 48, 60 and 72 hours and frozen at -80ºC. These frozen culture plates were then subjected to three freeze-thaw cycles and supernatants were harvested. To quantify progeny production, harvested supernatants were plaque titrated on monolayers of L-929 cells in semi-solid medium, consisting of DMEM, 10% FBS and 1% Bactoagar (BD Biosciences, Franklin Lakes, NJ). After a 72 hour incubation, a medium-agar mixture consisting of DMEM, 10% FBS, 1% Bacto-agar, and 0.02% Neutral Red Solution (Sigma-Aldrich, St. Louis, MO), and plaques counted after an additional 24-hour incubation. 2.4. In Vitro Synergy Assay Dose response curves for all cell lines to RV and sunitinib were generated. Calcusyn software (Biosoft, Great Shelford, Cambridge, UK) was utilized to generate effective dose for 50% cytotoxicity (ED50) values for each cell line to RV and sunitinib from the dose response data. Cell
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lines were then treated with escalating doses of RV and sunitinib concurrently at a fixed ratio of ED50:ED50. Cell viability was quantified as per the cell viability assay and combination index values were generated using calcusyn software with a CI value < 1 denoting an synergistic response, > 1 denoting an antagonistic response and = 1 denoting additive response. 2.5. Flow Cytometry 1 x 106 OPM2 or RPMI8226 cells treated with, dead virus (DV) control, RV, sunitinib or a combination of RV and sunitinib at fixed ratio of ED50:ED50. Cells were then washed twice with ice cold phosphate buffered saline (PBS) and BD Perm/Washâ&#x201E;˘, permeabilized using the BD Cytofix/Cytopermâ&#x201E;˘ Fixation/Permeabilization solution, and stained with V450 rabbit AntiActive Caspase-3 antibody (BD Horizon, San Jose, CA; Clone: C92-605, Cat: 560627) at 1:5 dilutions, and fixed in 1% formalin. FACS analysis was performed by the Flow Cytometry Core Facility at the University of Calgary using BD LSR II Cytometer (San Jose, CA).
3. Results 3.1. Sunitinib is cytotoxic towards multiple myeloma cell lines OPM2, KMS11 and RPMI8226 in vitro. Previous data in our laboratory (Figure. 1) suggests that RV has oncolytic activity in a wide array of MM cell lines. As such, these cells presented varying sensitivities and resistance to RV treatment, ranging from ED50 values of 2.32 MOI to 63.06 MOI (Table. 1). As such, three cell lines of varying resistance to RV, OPM2, KMS11, and RPMI8226, were chosen as candidate cell lines to emulate the varying phenotype of MM in response to RV treatment and to move forward
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with sunitinib experiments. OPM2, KMS11, and RPMI8226 had RV ED50 values of 63.01 MOI, 13.25 MOI, and 2.32 MOI respectively as previously testing in our laboratory has shown. Treatment of OPM2, KMS11 and RPMI8226 with sunitinib in vitro resulted in a dose dependent decrease in cell viability at 48 hours (Figure. 2A-C) as determined by WST-1 assay. ED50 values for each cell line in response to sunitinib was calculated from dose response curves using a linear regression analysis (Figure. 2A-C). These values were 1.95µM ± 0.10, 3.43µM ± 0.63 and 2.70µM ± 0.49 for OPM2, KMS11, and RPMI8226 respectively (Table. 1). 3.2. Combination therapy with reovirus and sunitinib mediates synergistic cytotoxicity in OPM2 and antagonism in RPMI8226 in vitro. To evaluate in vitro synergy between RV and sunitinib, the most RV sensitive and most RV resistant cell lines OPM2 and RPMI8226 were selected. The combination index (CI) values using the Chou and Talalay method was determined for each cell line. Cells were then treated simultaneously with sunitinib and RV for 48 hours at increasing doses of these agents at fixed ratios of ED50:ED50, from ED50/4 to 2x ED50 values as referred to in Table 1. Cell viability was then determined via the WST-1 assay (Figure. 3A and 3B). CI values less than 1 indicate an in vitro synergistic effect, whereas equal to 1 and greater than 1 indicate additive and antagonistic effects respectively. Using calcusyn software, it was revealed that RV and sunitinib combination therapy in OPM2 creates an additive effect in the ED50/4 dose of treatment and synergistic effects in the higher doses (Table. 2). Antagonistic effects were seen in RV and sunitinib treated RPMI8226 cells in all concentrations, with CI values trending towards synergistic or additive effects with higher concentrations (Table. 2). 3.3. Apoptosis is a major mediator of cytotoxicity in reovirus and sunitinib combinatory killing.
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Next, we wanted to determine the molecular mechanism of RV and sunitinib combination mediated cytotoxicity. Apoptosis was evaluated by measuring activated (cleaved) caspase-3 using flow cytometry. OPM2 and RMPI8226 cells were treated with RV and sunitinib at fixed ED50 concentrations ratios and harvested at 24, 48 and 72 hours. OPM2 cells treated with RV, sunitinib and a combination of RV and sunitinib at ED50:ED50 and 2x ED50 concentrations all exemplified increases of activated caspase-3 levels from 24 to 72 hours (Figure. 4A). For ED50:ED50 concentrations, activated caspase-3 levels in the total population of live cells increased by 2.17-fold compared to 1x RV treatment at 72 hours and by 2.27-fold as compared to 1x sunitinib alone 72 hours. In 2x ED50 concentrations, these levels were increased by 1.2-fold and 2.9-fold as compared to 72 hour 2x RV and 2x Sunitinib treatments respectively. As depicted in Figure. 4B, the percent total cell population that showed activated caspase3 positive was heightened when RV and drug combination therapy. Treatment of RPMI8226 cells with ED50:ED50 and 2x ED concentrations of combination treatment showed higher activated caspase-3 activity as compared to RV or sunitinib alone at their respective ED50 values. Interestingly, caspase-3 levels increased at ED50:ED50 concentrations from 24 hour to 72 hour. However, this is decreased at the 2x ED50 level by a 1.59-fold decrease across time points. This may be reflective of increased cell death, hence less measurable activated caspase-3 levels. Interestingly, these two cell lines show differing responses to 2x ED50 concentrations over the course of 72 hours. 2x ED50 treated OPM2 and RPMI8226 cells showed opposite trends in activated caspase-3 levels, indicating that there may be differing drug kinetics between MM cell lines. Overall, with increases in activated caspase-3 levels in RV and sunitinib combination
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treatment cells, this data demonstrates that apoptosis is a major mediator of combination treatment cytotoxicity in both OPM2 and RPMI8226 cells in vitro. 3.4. Preliminary data suggests that RV replication is increased in sunitinib treated OPM2 cells. Since certain drugs could stimulate viral replication within cells, we next asked the question whether sunitinib would stimulate/enhance viral progeny production. As RV oncolysis is determined by viral replication, a plaque titration was performed to determine viral progeny in RV or RV and sunitinib treated cells. OPM2 cells were stimulated with either RV at ED50 or RV and sunitinib at ED50:ED50 and subjected to freeze-thaw cycles, and supernatants collected as described in the methods. Preliminary data of OPM2 cells at 12, 24 and 48 hours, indicate that combination treatment enhances viral progeny production in comparison to treatment with RV alone (Figure. 5). At 24 hours, an 11.5-fold increase in viral progeny was seen with combination treatment in comparison to RV alone. This difference, however, diminished at 48 hours, leading only to a 1.46fold increase in RV treated versus combination treatment. Although this result is interesting, in order to form definitive conclusions, this experiment should be repeated and statistically analyzed. Also, it is noteworthy to point out that this may be a cell line specific event.
4. Discussion In the present study, we sought to determine the viability of combining oncolytic RV with the anti-angiogenic and immunomodulatory drug sunitinib as a novel treatment strategy for MM. This was determined by studying the cytotoxicity of sunitinib in MM cell lines OPM2, KMS11 and RPMI8226 in vitro, and assessing the synergism between RV sunitinib in OPM2 and RPMI8226 via the Chou-Talalay algorithms. Lastly, the mechanisms combination cytotoxicity in
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cell lines were commenced, using flow cytometry to assess apoptosis and plaque titration assays to determine altered viral replication patterns. With this preliminary investigation, we have shown that using combination therapy with RV and sunitinib, which is currently not a front line treatment strategy for MM, one can overcome therapeutic resistance of MM. This is well exemplified OPM2 data, which displayed synergistic effects between RV and sunitinib (Table. 2). While the in vitro data is promising, it is unknown whether these results can be extrapolated into clinical applicability. As such, our lab is currently beginning in vivo investigations using the syngeneic Vk*myc murine model in collaboration with the Mayo Clinic. Particularly advantageous about using a syngeneic model rather than an athymic murine model is the ability to evaluate immunological-based effects of RV and sunitinib in the tumour microenvironment and assess how immune cells are altered [32]. Results of these studies would provide valuable data moving forward into a phase I clinical trial here at the Tom Baker Cancer Centre. In addition, ex vivo analysis with patient MM cells can be used to assess RV and sunitinib combination treatment efficacy. Previous studies in our laboratory have also shown that RV alone is successful in ex vivo purging of cancerous cells in patients undergoing autologous stem cell rescue [36]. With data suggesting that RV and sunitinib combination treatment can overcome RV resistance in vitro (Figure. 3A), combination treatment to purge resistant MM cells in hematopoietic stem cells populations is another potential application of these results. Computational analysis of RV and sunitinib combination treatment using the Chou-Talalay method in OPM2 revealed synergistic effect (Table. 2). Conversely, antagonistic effect was seen in RPMI8226, with the higher combined RV and sunitinib doses trending towards additive/synergy. Though the Chou-Talalay is widely used in literature, this mathematical formula may not be the most ideal method of assessing synergistic or antagonistic drug interactions. With
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experience in our laboratory, we have seen that when cell lines are resistant to one or more of these agents, they usually respond synergistically, whereas cell lines sensitive to these agents respond antagonistically [29]. These discrepancies can be ameliorated by assessing tumour burden reduction and survival analyses in in vivo animal models. In assessing the mechanistic action of RV and sunitinib combination treatment, it was shown that apoptosis is a major mechanism of cytotoxicity in both OPM2 and RPMI8226 (Figure. 4A-B). Particularly interesting is that the drug and virus kinetics differ between the RV resistant (OPM2) and the RV sensitive (RPMI8226) MM cell lines, and these need to be repeated with more cell lines. Previous studies in our laboratory have also shown that RV-mediated cell death in MM is preceded by phosphatidyl serine flipping and PARP cleavage [25]. However, modifications of these mechanisms when RV is paired with sunitinib has not yet been tested. Other mechanisms of cytotoxicity including autophagy and necrosis have yet to be exploredâ&#x20AC;&#x201D;autophagy of which has been previously shown in our laboratory as a mechanism of RV oncolysis in MM [25]. Consequently, these mechanisms may be altered in the milieu of the tumour microenvironment when examined in vivo. In light of the preliminary results obtained by, it is interesting that sunitinib is able to enhance viral production in OPM2 (Figure. 5). To confirm these results, further replicates are needed, with testing in multiple cell lines. An overall understanding of the mechanisms of RV and sunitinib combination therapy are necessary if this treatment is to be optimized for future patients.
5. Conclusion
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In conclusion, we have shown that RV and sunitinib combination therapy is able to overcome RV resistance in MM cells, as exemplified by the OPM2 cell line. Though there is an overall synergistic combinative effect in OPM2, RV and sunitinib combination in RPMI8226 produces an antagonistic effect as per the Chou-Talalay method. Preliminary data also suggests that apoptosis and increased viral replication are mediators of RV and sunitinib combinative effects. However, more replicates must be performed, in addition to exploring necrosis, necroptosis and autophagy as potential mechanisms of cytotoxicity. Moving forward, this provides important preliminary data to extrapolate into an in vivo murine models, an ex vivo models, and phase I clinical trial. This evidence can hopefully lead to the future application of RV and sunitinib combination therapy as a novel treatment strategy for MM.
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12. Thirukkumaran CM, Shi ZQ, Luider J, et al. Multiple myeloma oncolysis by reovirus is mediated through apoptosis via downregulation of Akt signalling and simultaneous activation of Caspase-3. AACR. 2007;48. 13. Errington F, Steele L, Prestwich R, et al. Reovirus activates human dendritic cells to promote innate antitumor immunity. J Immunol. 2008;180(9):6018-26. 14. Prestwich RJ, Errington F, IIett EJ, et al. Tumor infection by oncolytic reovirus primes adaptive antitumor immunity. Clin Cancer Res. 2008;14(22):7358-66. 15. Steele L, Errington F, Prestwich R, et al. Proinflammatory cytokine/chemokine production by reovirus treated melanoma cells is PKR/NF-ÎşB mediated and supports innate and adaptive anti-tumour immune priming. Mol Cancer. 2011 Feb;10:20. 16. Prestwich RJ, IIett EJ, Errington F, et al. Immune-mediated antitumor activity of reovirus is required for therapy and is independent of direct viral oncolysis and replication. Clin Cancer Res. 2009;15(13):4374-81. 17. Gujar SA, Marcato P, Pan D, et al. Reovirus virotherapy overrides tumor antigen presentation evasion and promotes protective antitumor immunity. Mol Cancer Ther. 2010;9(11):2924-33. 18. Gujar SA, Pan DA, Marcato, et al. Oncolytic virus-initiated protective immunity against prostate cancer. Mol Ther. 2011;19(4):797-804. 19. Thirukkumaran CM, Nodwell MJ, Hirasawa K, et al. Oncolytic viral therapy for prostate cancer: efficacy of reovirus as a biological therapeutic. Cancer Res 2010;70(6):2435-44. 20. Connolly JL, Rodgers SE, Clarke P, et al. Reovirus-induced apoptosis requires activation of transcription factor NF-kappaB. J Virol. 2000; 74(7):2981-2989. 21. Clarke P, Meintzer SM, Spalding AC, et al. Caspase 8-dependent sensitization of cancer cells to TRAIL-induced apoptosis following reovirus-infection. Oncogene. 2001;20(47):6910-6919. 22. Clarke P, Tyler KL. Down-regulation of cFLIP following reovirus infection sensitizes human ovarian cancer cells to TRAIL-induced apoptosis. Apoptosis. 2007;12(1):211-223.
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23. Smakman N, van den Wollenberg DJ, Elias SG et al. KRAS(D13) Promotes apoptosis of human colorectal tumor cells by ReovirusT3D and oxaliplatin but not by tumor necrosis factor-related apoptosis-inducing ligand. Cancer Res. 2006;66(10):5403-5408. 24. Thirukkumaran C, Thirukkumaran P, Morris D. Gene expression profiling of reovirus oncolysis in breast cancer [abstract]. In: Proceedings of the 96th Annual Meeting of the American Association for Cancer Research. AACR; 2005;46. Abstract nr. 24. 25. Thirukkumaran CM, Shi ZQ, Luider J, et al. Reovirus modulates autophagy during oncolysis of multiple myeloma. Autophagy. 2013 Mar;9(30)413-4. 26. Sun M, Lughezzani G, Perrotte P, et al. Treatment of metastatic renal cell carcinoma. Nat Rev Urol 2010;7(6):327-38. 27. Finke JH, Rini B, Ireland J, et al. Sunitinib reverses type-1 immune suppression and decreases T-regulatory cells in renal cell carcinoma patients. Clin Cancer Res 2008;14(20):6674-82. 28. National Cancer Institute [Internet]. Sunitinib in Treating Patients with Relapsed Multiple Myeloma; 2014 [cited 2015 Mar 19]. Available from: http://clinicaltrials.gov/ct2/show/study/NCT00514137 29. Lawson K, Shi ZQ, Spurrell J, et al. Repurposing sunitinib with oncolytic reovirus as a novel treatment strategy for renal cell carcinoma. Mol Can Therpeu. 2015 (Submitted) 30. Rutella S, Locatelli F. Targeting multiple-myeloma-induced immune dysfunction to improve immunotherapy outcomes. Clin Dev Immunol. 2012;2012:196063. 31. Mostafa AA, Gratton K, Lawson K, et al. PDL-1 blockade and sunitinib enhance the efficiency of oncolytic viral therapy. 2014. AACR Special Conferences titled Tumor Immunology and Immunotherapy: A New Chapter, being held from Monday, December 1, to Thursday, December 4, 2014, at Disneyâ&#x20AC;&#x2122;s contemporary Resort in Orlando Florida. 32. Chesi M, Robbiani DF, Sebag M, et al. AID-dependent activation of MYC transgene induces multiple myeloma in a conditional mouse model of post-germinal center malignancies. Cancer Cell. 2008 Feb;13(2)167-80. 33. Thirukkumaran CM, Shi ZQ, Luider J, et al. Reovirus as a successful ex vivo purging modality for multiple myeloma. Bone Marrow Transplant. 2014 Jan;49(1):80-6.
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7. Appendix A: Figures and Tables
Figure 1. Effect of reovirus on multiple myeloma (MM) cell lines. MM cells were cultured in the presence of live reovirus (LV) or dead reovirus (DV) at 40 MOI. After 48 and 72 hours post virus exposure cell viability was assessed using the WST assay. This data was generated by another lab member, Qiao Shi, prior to the start of thesis data collection.
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A - Sunitnib Dose Response in OPM2
OPM2 Sunitinib Dose Response Linear Regression 3
100
2.5 80
2
Log Effect
Percent Viable Cells
120
60 40 20
-0.5
0 0
0.46875 0.9375
1.875
3.75
7.5
15
y = 2.0521x - 0.584 R² = 0.9296
1 0.5 0 -0.5 0
0.5
1
1.5
2
-1
30
-1.5
Sunitinib Concentration (µM)
B - Sunitinib Dose Response in KMS11
Log Dose
KMS11 Sunitnib Dose Response Linear Regression
120
2.5
100
2 80
1.5
Log Effect
Percent Viable Cells
1.5
60 40 20
-0.5
0
1
0.5
y = 1.2647x - 0.5812 R² = 0.737
0 -0.5
0
0.5
1
-1 0
0.46875 0.9375
1.875
3.75
7.5
Sunitinib Concentration (µM)
15
30
-1.5
Log Dose
1.5
2
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C - Sunitnib Dose Response in RPMI8226
RPMI8226 Sunitinib Dose Response Linear Regression 2
100
1.5
80
Log Effect
Percent Viable Cells
120
60 40 20
y = 1.0764x - 0.4773 R² = 0.8091
1 0.5 0
-0.5
0
0.5
1
1.5
2
-0.5
0 0
0.46875 0.9375 1.875
3.75
7.5
Sunitinib Concentration (µM)
15
30
-1
Log Dose
Figure 2. Dose response of (A) OPM2, (B) KMS11, and (C) RPMI8226 cells treated with serial dilutions of sunitinib from 0.47 to 30 µM in vitro. Cell viability was assessed using the WST assay at 48 hours. (N=3 ±SE). Linear regression analysis used to calculate ED50 values for each respective cell line is also shown.
Table 1. Sunitinib and reovirus ED50 doses for MM cell lines calculated using linear regression analysis. Reovirus ED50 data was generated by another lab member, Qiao Shi, and presented here for completeness. Cell Line Reovirus (MOI) Sunitinib (µM) OPM2 63.06 1.95 ± 0.10 KMS11 13.25 3.43 ± 0.63 RPMI8226 2.32 2.70 ± 0.49
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Figure 3. Synergistic and additive effects between reovirus and sunitinib in OPM2 and antagonistic effects in RPMI8226 cells. (A) OPM2 cells were infected with constant ratios of ED50 values generated for RV (63.06 MOI) and sunitinib (1.95µM) and (B) RPMI8226 cells were infected with a constant ratio of RV (2.32 MOI) and sunitinib (2.70µM). Cell viability was assessed at 48 hours via the WST assay. UV-irradiated virus is denoted by DV. (N=3, ±SE) Table 2. Combination indexes (CI) generated by the Chou-Talalay method for OPM2 and RPMI8226 treated with reovirus and sunitinib at constant ratios of ED50 values. Cell Line ED50/4 ED50/2 ED50:ED50 2x ED50 OPM2 0.91 ± 0.28 0.59 ± 0.15 0.84 ± 0.11 0.59 ± 0.10 RPMI8226 24.2 ± 4.67 23.9 ± 9.58 7.13 ± 1.62 1.12 ± 0.42 Combination indices: = 1 Additive; >1 Antagonistic; <1 Synergistic
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70 60 50 40 30 20 10 0
B - Percent Positive Activated Caspase-3 in RPMI8226 % Positive Active Capsase-3
% Positive Active Caspase-3
A - Percent Positive Activated Caspase-3 in OPM2
35.0 30.0 25.0 20.0 15.0 10.0 5.0 0.0
Treatment Conditions 24 Hour
48 Hour
72 Hour
Treament Conditions 24 Hour
48 Hour
72 Hour
Figure 4. Apoptosis is a major mediator of cytotoxicity in RV and sunitinib treated MM cell lines. Percent positive population of activated (cleaved) terminator caspase-3 in response to RV, sunitinib, and combined at fixed ratios of ED50 values for (A) OPM2 and (B) RPMI8226 quantified using flow cytometry. UV-irradiated virus is denoted by DV. (N=1)
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Log 10 Viral Titre (PFU/ml)
OPM2 - Viral Progeny Assay 7.60 7.40 7.20 7.00 6.80 6.60 6.40 6.20 0
10
20
30
40
50
60
Time (Hours) 1x RV
ED50/ED50
Figure 5. Viral progeny production in OPM2 cells following infection with RV or RV and sunitinib combination. RV titre was measured in plaque-forming units (PFU). (N=1)
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8. Appendix B: Ethics Approval
Conjoint Health Research Ethics Board Research Services Office rd 3 Floor Mackimmie Library Tower (MLT 300) 2500 University Drive, NW Calgary AB T2N 1N4 Telephone: (403) 220-7990 Fax: (403) 289-0693 chreb@ucalgary.ca
CERTIFICATION OF INSTITUTIONAL ETHICS REVIEW This is to certify that the Conjoint Health Research Ethics Board at the University of Calgary has examined the following research proposal and found the proposed research involving human participants to be in accordance with University of Calgary Guidelines and the TriCouncil Policy Statement: Ethical Conduct for Research Involving Humans 2010 (TCPS 2). This form and accompanying letter constitute the Certification of Institutional Ethics Review. Ethics ID:
REB14-0079
Principal Investigator: Donald Morris Co-Investigator(s):
Chandini Thirukkumaran
Student CoInvestigator(s):
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Study Title:
Basic Studies Involving Oncolytic Virotherapy using Patient's Blood and/or Tumour Specimens to Target Therapy Resistance of Multiple Myeloma
Sponsor (if applicable):
1025525 / Cancer Research Society Inc.
Effective: October 29, 2014
Expires: October 29, 2015
Restrictions: This Certification is subject to the following conditions: 1. Approval is granted only for the project and purposes described in the application.
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2. Any modification to the authorized study must be submitted to the Chair, Conjoint Health Research Ethics Board for approval. 3. An annual report must be submitted within 45 days prior to expiry date of this Certification, and should provide the expected completion date for the study. 4. A final report must be sent to the Board when the project is complete or terminated. Date: Stacey A. Page, PhD, Chair , CHREB
October 29, 2014