Selective Targeting of Protein Kinase C (PKC)-θ Nuclear Translocation Reduces Mesenchymal Gene Signatures and Reinvigorates Dysfunctional CD8(+) T Cells in Immunotherapy-Resistant and Metastatic Cancers

SIMPLE SUMMARY: Some important signaling proteins that control how cells grow and behave not only act in the cytoplasm but also in the nucleus, where they tether to chromatin. This is especially true for protein kinase C (PKC)-θ, which acts in the nucleus to mediate cancer hallmarks that drive metastasis and in normal T cells. However, current PKC-θ inhibitors are either non-specific or target only its cytoplasmic function. In a bid to develop a novel class of PKC-θ inhibitor that maintains cytoplasmic signaling but inhibits its nuclear function, here we present a novel PKC-θ inhibitor (nPKC-θi2) that specifically inhibits nuclear translocation of PKC-θ without interrupting normal signaling in healthy T cells. We show for the first time that nPKC-θ mediates immunotherapy resistance via its activity in circulating tumor cells and dysfunctional CD8(+) T cells. Our novel inhibitor provides a means to target this process by simultaneously overcoming T-cell exhaustion and cancer stem cell burden. As part of a sequential approach with other therapies, this work paves the way for improving outcomes in cancer patients with immunotherapy-resistant relapse and metastasis. ABSTRACT: Protein kinase C (PKC)-θ is a serine/threonine kinase with both cytoplasmic and nuclear functions. Nuclear chromatin-associated PKC-θ (nPKC-θ) is increasingly recognized to be pathogenic in cancer, whereas its cytoplasmic signaling is restricted to normal T-cell function. Here we show that nPKC-θ is enriched in circulating tumor cells (CTCs) in patients with triple-negative breast cancer (TNBC) brain metastases and immunotherapy-resistant metastatic melanoma and is associated with poor survival in immunotherapy-resistant disease. To target nPKC-θ, we designed a novel PKC-θ peptide inhibitor (nPKC-θi2) that selectively inhibits nPKC-θ nuclear translocation but not PKC-θ signaling in healthy T cells. Targeting nPKC-θ reduced mesenchymal cancer stem cell signatures in immunotherapy-resistant CTCs and TNBC xenografts. PKC-θ was also enriched in the nuclei of CD8(+) T cells isolated from stage IV immunotherapy-resistant metastatic cancer patients. We show for the first time that nPKC-θ complexes with ZEB1, a key repressive transcription factor in epithelial-to-mesenchymal transition (EMT), in immunotherapy-resistant dysfunctional PD1(+)/CD8(+) T cells. nPKC-θi2 inhibited the ZEB1/PKC-θ repressive complex to induce cytokine production in CD8(+) T cells isolated from patients with immunotherapy-resistant disease. These data establish for the first time that nPKC-θ mediates immunotherapy resistance via its activity in CTCs and dysfunctional CD8(+) T cells. Disrupting nPKC-θ but retaining its cytoplasmic function may offer a means to target metastases in combination with chemotherapy or immunotherapy.