The coordinate regulation of HLA class II (HLA-II) is controlled by the class II transactivator, CIITA, and is crucial for the development of anti-tumor immunity. HLA-II in breast carcinoma is associated with increased IFN-γ levels, reduced expression of the estrogen receptor (ER) and reduced age at diagnosis. Here, we tested the hypothesis that estradiol (E₂) and ERα signaling contribute to the regulation of IFN-γ inducible HLA-II in breast cancer cells. Using a panel of established ER⁻ and ER⁺ breast cancer cell lines, we showed that E₂ attenuated HLA-DR in two ER⁺ lines (MCF-7 and BT-474), but not in T47D, while it augmented expression in ER⁻ lines, SK-BR-3 and MDA-MB-231. To further study the mechanism(s), we used paired transfectants: ERα⁺ MC2 (MDA-MB-231 c10A transfected with the wild type ERα gene) and ERα⁻ VC5 (MDA-MB-231 c10A transfected with the empty vector), treated or not with E₂ and IFN-γ. HLA-II and CIITA were severely reduced in MC2 compared to VC5 and were further exacerbated by E₂ treatment. Reduced expression occurred at the level of the IFN-γ inducible CIITA promoter IV. The anti-estrogen ICI 182,780 and gene silencing with ESR1 siRNA reversed the E₂ inhibitory effects, signifying an antagonistic role for activated ERα on CIITA pIV activity. Moreover, STAT1 signaling, necessary for CIITA pIV activation, and selected STAT1 regulated genes were variably downregulated by E₂ in transfected and endogenous ERα positive breast cancer cells, whereas STAT1 signaling was noticeably augmented in ERα⁻ breast cancer cells. Collectively, these results imply immune escape mechanisms in ERα⁺ breast cancer may be facilitated through an ERα suppressive mechanism on IFN-γ signaling.

As the current efficacy of oncolytic viruses (OVs) as monotherapy is limited, exploration of OVs as part of a broader immunotherapeutic treatment strategy for cancer is necessary. Here, we investigated the ability for immune checkpoint blockade to enhance the efficacy of oncolytic reovirus (RV) for the treatment of breast cancer (BrCa). In vitro, oncolysis and cytokine production were assessed in human and murine BrCa cell lines following RV exposure. Furthermore, RV-induced upregulation of tumor cell PD-L1 was evaluated. In vivo, the immunocompetent, syngeneic EMT6 murine model of BrCa was employed to determine therapeutic and tumor-specific immune responses following treatment with RV, anti-PD-1 antibodies or in combination. RV-mediated oncolysis and cytokine production were observed following BrCa cell infection and RV upregulated tumor cell expression of PD-L1. In vivo, RV monotherapy significantly reduced disease burden and enhanced survival in treated mice, and was further enhanced by PD-1 blockade. RV therapy increased the number of intratumoral regulatory T cells, which was reversed by the addition of PD-1 blockade. Finally, dual treatment led to the generation of a systemic adaptive anti-tumor immune response evidenced by an increase in tumor-specific IFN-γ producing CD8⁺ T cells, and immunity from tumor re-challenge. The combination of PD-1 blockade and RV appears to be an efficacious immunotherapeutic strategy for the treatment of BrCa, and warrants further investigation in early-phase clinical trials.

Purpose: In addition to their direct cytopathic effects, oncolytic viruses are capable of priming antitumor immune responses. However, strategies to enhance the immunotherapeutic potential of these agents are lacking. Here, we investigated the ability of the multi-tyrosine kinase inhibitor and first-line metastatic renal cell carcinoma (RCC) agent, sunitinib, to augment the antitumor immune response generated by oncolytic reovirus. Experimental Design: In vitro, oncolysis and chemokine production were assessed in a panel of human and murine RCC cell lines after exposure to reovirus, sunitinib, or their combination. In vivo, the RENCA syngeneic murine model of RCC was employed to determine therapeutic and tumor-specific immune responses after treatment with reovirus (intratumoral), sunitinib, or their combination. Parallel investigations employing the KLN205 syngeneic murine model of lung squamous cell carcinoma (NSCLC) were conducted for further validation. Results: Reovirus-mediated oncolysis and chemokine production was observed following RCC infection. Reovirus monotherapy reduced tumor burden and was capable of generating a systemic adaptive antitumor immune response evidenced by increased numbers of tumor-specific CD8+ IFNγ-producing cells. Coadministration of sunitinib with reovirus further reduced tumor burden resulting in improved survival, decreased accumulation of immune suppressor cells, and the establishment of protective immunity upon tumor rechallenge. Similar results were observed for KLN205 tumor–bearing mice, highlighting the potential broad applicability of this approach. Conclusions: The ability to repurpose sunitinib for augmentation of reovirus' immunotherapeutic efficacy positions this novel combination therapy as an attractive strategy ready for clinical testing against a range of histologies, including RCC and NSCLC. Clin Cancer Res; 22(23); 5839–50. ©2016 AACR.

The possible link between infection/inflammation/immune activation and a cancer patient’s outcome from both a causative and outcome point of view has long been postulated. Substantial progress in the understanding of tumour associated antigens/epitopes, immune cellular subpopulations, cytokine pathways/expression, the tumour microenvironment, and the balance between tumour immune suppression and stimulation have been made over the past decade. This knowledge has heralded a new era of tumor immunotherapy utilizing vaccines, immune checkpoint inhibition and oncolytic viruses. Despite significant progress in the molecular era now with targeted therapeutics such as EGFR tyrosine kinase inhibitors and ALK fusion protein inhibitors that have significantly improved the outcome of these specific lung cancer subpopulations, the overall 5 year survival for all non-small cell lung carcinoma (NSCLC) is still less than 20%. Unlike malignancies such as malignant melanoma, renal cell carcinoma and neuroblastoma given their documented spontaneous remission rates lung cancer historically has been felt to be resistant to immune approaches likely related to an immunosuppressive tumour microenvironment and/or lack of immune recognition. Defining responding populations, understanding the mechanism(s) underlying durable immune responses and the role of chemotherapy, radiation, oncolytic viruses and other tumour disrupting agents in augmenting immune responses have led to improved optimization of immune therapeutic strategies. The purpose of this mini review is to focus on the recent advances in lung immunotherapy with an emphasis on recent clinical trials in the last 5 years in NSCLC.

The oncolytic reovirus (RV) has demonstrated clinical efficacy and minimal toxicity in a variety of cancers, including multiple myeloma (MM). MM is a malignancy of plasma cells that is considered treatable but incurable because of the 90% relapse rate that is primarily from drug resistance. The systemic nature of MM and the antitumor immunosuppression by its tumor microenvironment presents an ongoing therapeutic challenge. In the present study, we demonstrate that RV synergizes with the standard-of-care MM drug bortezomib (BTZ) and, importantly, enhances its therapeutic potential in therapy-resistant human MM cell lines in vitro. Using the syngeneic Vk*MYC BTZ-resistant immunocompetent transplantable MM murine model, we also demonstrate that mice harboring BTZ-insensitive MM tumors respond to the RV/BTZ combination treatment in terms of decreased tumor burden and improved overall survival (P < .00001). We demonstrate that BTZ augments RV replication in tumor-associated endothelial cells and myeloma cells, leading to enhanced viral delivery and thereby stimulating cytokine release, immune activity, apoptosis, and reduction of the MM-associated immune suppression. We conclude that combined RV/BTZ is an attractive therapeutic strategy with no safety signals for the treatment of MM. Key Points RV and BTZ synergize to reduce immune suppression and drug resistance of MM. BTZ triggers viral replication in the tumor microenvironment enhancing anticancer cytotoxic and immune modulatory signals. Visual Abstract