Grant: CIHR - ICR / Cancer Research Society Partnership - Research Grants

Oncolytic viruses (OVs) are viruses that specifically kill cancer cells while leaving normal tissues unharmed. Our recent research has focused on breast cancer: we developed several OV-therapies that greatly improved outcomes and were able to uncover the crucial role of anti-tumor immunity induced by OV-therapy, but the mechanisms by which OVs trigger cancer-specific immunity remain to be fully understood. The recognition and elimination of cancer cells by T cells require the presentation of tumor antigens at the cell surface. The first step of this process is the generation of antigenic peptides, a process that requires the optimal function of the immunoproteasome (ImP): a modified form of the proteasome for which 3 alternative subunits are incorporated. The processing of proteins by the ImP generates peptides that have higher affinity for MHC molecules compared to the ones produced by the constitutive proteasome. The ImP-generated peptides are therefore optimal for antigen presentation. The ImP is induced by pro-inflammatory stimuli such as interferons, which are produced in substantial amounts in response to OVs such as the Vesicular stomatitis virus (VSV), our model OV for this study. We therefore hypothesize that the mechanism by which OVs like VSV trigger anti-tumor immunity is by inducing the ImP and modifying the peptidic repertoire of infected cancer cells. Focusing on breast cancer, our specific objectives are to: 1) Measure ImP levels and function in breast cancer cells in response to VSV infection 2) Determine the importance of the ImP for the efficacy of oncolytic VSV using murine breast cancer models 3) Characterize the peptide repertoire of VSV-infected vs non-infected breast cancer cells Scientific approach: The E0771, 4T1 and EMT6 murine breast cancer cells lines will be used for both in vitro and in vivo studies. We have extensive experience using these tumor models. ImP-/- variants of the cell lines will be generated using the CRISPR-Cas9 system. The MCF7, T-47D, MDA-MB-231, BT-549, ZR-75 and Hs 578T human breast cancer cell lines will be used in key in vitro validation experiments to confirm the relevance of our findings to the human disease. The constitutive proteasome and ImP expression from infected and non-infected tumor lysates will be quantified by western blot analysis. The incorporation of the different ImP subunits into the proteasome (assembly of the ImP) and ImP function will be measured after proteasome isolation followed by western blot analysis. In addition, commercially available colorimetric assays will be used to characterize the cleavage activity of the isolated proteasomes (chymotryspine-like, caspase-like and trypsine-like cleavage). Finally, the peptide repertoires with and without VSV treatment will be compared by mass spectrometry. For immune response analysis, spleens, blood and lymph nodes will be collected at different time points for IFN-gamma ELISPOT assays and flow cytometry analysis. For efficacy studies, tumor growth and Kaplan-Meier survival curves will be generated. Expected outcome: Overall, this proposal will not only elucidate a mechanism by which VSV induces anti-tumor immunity, but it will also point to improvements in this treatment platform. The understanding of the role of the ImP for OV-therapy and the generation of new improved OV variants could provide a new therapy and dramatically improve the long-term protection breast cancer patients.