Reovirus-Induced Cell-Mediated Immunity for the Treatment of Multiple Myeloma Within the Resistant Bone Marrow Niche
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Open access Original research J Immunother Cancer: first published as 10.1136/jitc-2020-001803 on 19 March 2021. Downloaded from Reovirus- induced cell- mediated immunity for the treatment of multiple myeloma within the resistant bone marrow niche Louise M E Müller,1 Gemma Migneco,1 Gina B Scott,1 Jenny Down,2 Sancha King,2 1 3 4 1 Basem Askar, Victoria Jennings, Babatunde Oyajobi, Karen Scott , Emma West,1 Christy Ralph,1 Adel Samson,1 Elizabeth J Ilett,1 Munitta Muthana,2 Matt Coffey,5 Alan Melcher,3 Christopher Parrish,6 Gordon Cook,7 2 1 Michelle Lawson, Fiona Errington- Mais To cite: Müller LME, Migneco G, ABSTRACT BACKGROUND Scott GB, et al. Reovirus- Background Multiple myeloma (MM) remains an Multiple myeloma (MM) is a tumor of induced cell- mediated immunity incurable disease and oncolytic viruses offer a well- terminally differentiated plasma cells which for the treatment of multiple tolerated addition to the therapeutic arsenal. Oncolytic myeloma within the resistant expand in the bone marrow (BM). MM reovirus has progressed to phase I clinical trials and bone marrow niche. Journal results in the onset of myeloma- induced bone its direct lytic potential has been extensively studied. for ImmunoTherapy of Cancer disease (MBD) due to increased bone resorp- However, to date, the role for reovirus- induced 2021;9:e001803. doi:10.1136/ tion by osteoclasts and loss of osteoblast func- jitc-2020-001803 immunotherapy against MM, and the impact of the bone marrow (BM) niche, have not been reported. tion. MBD is one of the many debilitating Methods This study used human peripheral blood features of MM which results in bone pain ► Additional material is 1 published online only. To view, mononuclear cells from healthy donors and in vitro co- and frequent fractures. The worldwide inci- please visit the journal online culture of MM cells and BM stromal cells to recapitulate dence of MM is ~114 000 cases per year and (http:// dx. doi. org/ 10. 1136/ jitc- the resistant BM niche. Additionally, the 5TGM1- Kalw/ numbers are expected to increase with an 2020- 001803). RijHSD immunocompetent in vivo model was used to aging population.2 Currently, MM remains an examine reovirus efficacy and characterize reovirus- incurable disease in need of novel treatment Accepted 17 February 2021 induced immune responses in the BM and spleen following strategies that are safe and well- tolerated.2 intravenous administration. Collectively, these in vitro and The BM microenvironment has a diverse http://jitc.bmj.com/ in vivo models were used to characterize the development cellular composition, including haemato- of innate and adaptive antimyeloma immunity following reovirus treatment. poietic stem cells, mesenchymal stem cells, Results Using the 5TGM1- Kalw/RijHSD BM stromal cells (BMSCs; fibroblasts and immunocompetent in vivo model we have demonstrated epithelial cells) and immune cells. Moreover, that reovirus reduces both MM tumor burden and the BM niche provides a range of cytokines, on September 25, 2021 by guest. Protected copyright. myeloma- induced bone disease. Furthermore, detailed chemokines and growth factors which all immune characterization revealed that reovirus: (i) contribute to MM progression and therapy + increased natural killer (NK) cell and CD8 T cell resistance.3 4 Oncolytic viruses (OVs) prefer- + numbers; (ii) activated NK cells and CD8 T cells and (iii) entially infect and kill malignant cells, and upregulated effector- memory CD8+ T cells. Moreover, + use multiple mechanisms to eradicate tumor increased effector- memory CD8 T cells correlated with cells, including engagement of both innate decreased tumor burden. Next, we explored the potential and adaptive antitumor immune responses.5 6 for reovirus-induced immunotherapy using human co- culture models to mimic the myeloma- supportive Oncolytic virotherapy has gained increasing BM niche. MM cells co- cultured with BM stromal cells attention over recent years following the displayed resistance to reovirus-induced oncolysis and Food and Drug Administration approval of © Author(s) (or their employer(s)) 2021. Re- use bystander cytokine- killing but remained susceptible to Talimogene laherparepvec (T-V ec), a modi- permitted under CC BY. killing by reovirus-activa ted NK cells and MM-specific fied herpes simplex virus-1 (HSV-1), which Published by BMJ. cytotoxic T lymphocytes. encodes granulocyte-macrophage colony- For numbered affiliations see Conclusion These data highlight the importance of stimulating factor (GM-CSF), for the treat- end of article. reovirus-induced immunotherapy for targeting MM cells ment of advanced melanoma. Unfortunately, within the BM niche and suggest that combination with in comparison to solid malignancies, OVs are Correspondence to agents which boost antitumor immune responses should relatively under investigated in the context Dr Fiona Errington- Mais; be a priority. F. Errington@ leeds. ac. uk of hematological malignancies (HM) and, as Müller LME, et al. J Immunother Cancer 2021;9:e001803. doi:10.1136/jitc-2020-001803 1 Open access J Immunother Cancer: first published as 10.1136/jitc-2020-001803 on 19 March 2021. Downloaded from such, their progression to clinical trials has been limited; Reovirus therapy experiment to date, phase I clinical trials in MM have been reported Both female and male mice aged 5–8 weeks were used for reovirus, measles virus (MV) and vesicular stomatitis and all groups were randomized based on age and sex. virus.7 Mouse group numbers were ascertained using the power Reovirus is a double-stranded RNA virus that uses calculation formula (2(SD)2×f(α,β)/Δ2). The α (signifi- junctional adhesion molecule A (JAM- A) for viral entry,8 cance level) was 0.05, β (power level) was 90% and both kills neoplastic cells through both apoptotic and non- Δ (least practicable difference between groups) and SD apoptotic mechanisms9 10 and can use activated RAS were taken from a pilot study to determine reovirus effi- signaling for replication and oncolysis9; significantly, cacy. This gave rise to the following power calculation JAM- A is overexpressed in MM11 and mutated Ras is (2(8.33)2×10.5/152=6.47) and seven mice/group were associated with MM progression.12 In accordance with used for all subsequent in vivo experiments. this, the direct lytic potential of reovirus against MM On day 0, mice were injected with 2×106 bone- homing has been reported13 and early phase clinical trials have 5TGM1 cells (a kind gift from Prof Oyajobi)19 in 100 µL been carried out using reovirus type 3 Dearing strain phosphate- buffered saline (PBS) via the lateral tail vein. (T3D; pelareorep).14 Pelareorep was well-tolerated in On day 7–9, reovirus or control (PBS) treatment was patients with MM, and only low- grade adverse effects initiated with 3 weekly injections (Monday/Wednesday/ were reported.14 15 Friday) of 2×107 plaque- forming units (PFU)/mL Innate and adaptive immunity are important for reovirus in 100 µL PBS or 100 µL PBS alone, respectively. reovirus efficacy and the secretion of type I inter- Treatment continued until the development of HLP in feron (IFN) is a key component of the innate immune PBS- treated mice (20–27 days) when all mice were sacri- response.16 IFN-α is important for natural killer (NK) cell ficed and tissues were harvested for assessment of tumor activation in response to reovirus,16 although a range of burden, bone analysis and immunophenotyping (see other pro- inflammatory cytokines mediate the recruit- online supplemental tables S1 and S2 for details of the ment of NK cells and dendritic cells (DC) to the tumor.17 flow cytometry antibodies used). Reovirus also enhances the ability of DC to present tumor- associated antigens (TAA) for priming of tumor-specific Assessment of MBD by micro-CT cytotoxic T lymphocytes (CTLs).18 Harnessing reovirus- To assess bone disease, tibiae were analyzed by micro-CT induced antitumor immunity has the potential to induce (µCT) using a SkyScan 1272 ex vivo µCT scanner at 50 kV immunological memory and long-term cancer remission and 200 µA, using an aluminum filter of 0.5 mm and pixel in patients with MM. size of 4.3 µm2, as described previously.20 Bone volume Unfortunately, despite promising preclinical data, (BV)/total volume, trabecular number, trabecular thick- single- agent reovirus treatment was ineffective at treating ness, cortical thickness, lesion area and lesion number relapsed/refractory MM. This dichotomy highlights the parameters were then assessed as described previously20 21 need for us to better understand reovirus–host interac- and according to standard guidelines. http://jitc.bmj.com/ tions, in particular, which reovirus effector mechanisms are induced within the BM niche and which have the SYBR Green reovirus RT-qPCR capacity to eradicate MM cells that are supported by the One- step quantitative PCR (qPCR) reactions were tumor microenvironment. Once identified, key effector prepared in triplicate in 96-well MicroAmp Optical Reac- mechanisms can be prioritized for the development tion plates (Applied Biosystems) using the Power SYBR Green RNA- to- C 1- Step Kit (ThermoFisher Scientific); of effective combination strategies designed to boost T on September 25, 2021 by guest. Protected copyright. reovirus efficacy in clinical trials. Therefore, this study 20 µL reaction mixes included: 0.3 µg RNA, 1× Power aimed to characterize the immune response to reovirus in SYBR Green RT- PCR Mix, 1× RT Enzyme Mix, 0.5 µM of a BM niche and identify key effector mechanisms under- σ3 forward and reverse primers and RNase- free water. A pinning reovirus efficacy. 10fold serial dilution of reovirus RNA, isolated from stock reovirus, was included for quantification of reovirus RNA. Thermal cycling was performed on the QuantStudio 5 Real- Time PCR System (Applied Biosystems). METHODS 5TGM1 in vivo model Cell culture C57BL/KaLw/RijHSD mice were purchased at age 5–10 NCI-H929, U266B, JIM3 and OPM2 human MM cell lines, weeks from the St James’s Biomedical Services, Univer- KG-1 (an acute myeloid leukemia cell line) and HS-5 sity of Leeds.