Oncolytic virotherapy counteracts the selection of IFN-unresponsive cancer cells post-immunotherapy but is limited by the emergence of dedifferentiated cancer cells.

Genetic mutations disrupting interferon (IFN) signaling have been described as an evolutionary route for tumor cells to evade immune checkpoint inhibitor (ICI) therapy. We hypothesized that loss of IFN signaling not only enables cancer cells to evade immune surveillance, but also renders them susceptible to oncolytic viruses. To experimentally address this hypothesis, we generated IFN-unresponsive mouse melanoma cell variants via CRISPR/Cas9 and investigated the impact of sequential immuno-virotherapy on their evolutionary dynamics in immunocompetent syngeneic hosts. In wild-type mice, Natural Killer (NK) cells eliminated IFN-unresponsive subclones in mixed tumor cell transplants due to their very low expression of major histocompatibility complex class I (MHC-I) molecules. Upon NK cell depletion, IFN-unresponsive subclones established and were preferentially selected by T cell-directed immunotherapy, consistent with observations in patients. Sequential oncolytic virotherapy was able to specifically target and counter-select these IFN-unresponsive clones. Unexpectedly, IFN-responsive tumor cells re-emerged with a dedifferentiated phenotype that resisted both immune and viral control. Together, these findings touch upon the paradoxical anti- and pro-tumoral roles of cancer cell-intrinsic IFN signaling and highlight dedifferentiation as a potential convergent resistance mechanism that ultimately limits the efficacy of both treatment approaches. Our findings may provide an explanation for why combination immunotherapy and oncolytic virotherapy did not meet clinical expectations.