Therapeutic Targeting of a Major Immune Resistance Pathway in Triple-negative Breast Cancer Abstract Checkpoint blockade results in moderate survival benefit in a subset of patients with triple-negative breast cancer (TNBC) but most patients currently fail to benefit from immunotherapy. We recently discovered that the genes encoding the integrin αV and SOX4 proteins (ITGAV and SOX4 genes) render tumor cells resistant to killing by cytotoxic T cells. Integrin αVβ6 and SOX4 form a resistance pathway that is particularly relevant to TNBC: Integrin αVβ6 releases TGFβ from an inactive latent complex by a force-dependent mechanism, and active TGFβ induces expression of the SOX4 transcription factor that inhibits T cell-mediated tumor immunity. The integrin αVβ6 heterodimer is expressed at a low level by healthy epithelial cells, but its expression is highly upregulated in many epithelial cancers, including TNBC. TGFβ is an important immunosuppressive cytokine in human tumors, but has been difficult to target due to its pleiotropic biology in different cell types and tissues. This approach provides an opportunity for more selective targeting of TGFβ for cancer immunotherapy. Our preliminary data demonstrate that an integrin αVβ6 blocking mAb inhibits SOX4 expression and sensitizes TNBC cells to cytotoxic T cells. This antibody confers a substantial survival benefit in two aggressive mouse models of TNBC that are resistant to checkpoint blockade. In Aim 1, we will study the integrin αVβ6 – SOX4 resistance pathway as an immunotherapy target in aggressive and highly metastatic mouse models of TNBC. In particular, we will examine how inhibition of integrin αVβ6 induces a substantial influx of CD8 T cells into TNBCs that are poorly infiltrated by T cells. In Aim 2, we will investigate the molecular mechanisms by which the integrin αVβ6 – SOX4 pathway inhibits T cell-mediated tumor immunity. Preliminary data demonstrate that the SOX4 transcription factor inhibits expression of genes from multiple innate immune pathways in tumor cells, including the cytosolic dsRNA and dsDNA sensing pathways as well as the type 1 interferon response pathway. We will define the direct transcriptional targets of SOX4 and study how SOX4 cooperates with other transcription factors to render tumor cells resistant to T cell-mediated tumor immunity. In Aim 3, we will study the significance of the integrin αVβ6 – SOX4 pathway in human TNBC. Our hypothesis is that this resistance pathway inhibits T cell infiltration in human TNBC, and we will therefore examine the spatial relationship between integrin αVβ6/SOX4 expression and T cell infiltration in human TNBC specimens. High-affinity integrin αVβ6 antibodies and a small molecule inhibitor are already being tested in clinical trials for fibrosis indications. The studies described here could thus provide the scientific rationale for testing of such inhibitors in TNBC and other human cancers of epithelial origin.