The Hippo pathway component WW domain-containing transcription regulator 1 (TAZ) is a transcriptional coactivator and an oncogene in breast and lung cancer. Transcriptional targets of TAZ that modulate immune cell function in the tumor microenvironment are poorly understood. Here, we perform a comprehensive screen for immune-related genes regulated by TAZ and its paralog YAP using NanoString gene expression profiling. We identify the immune checkpoint molecule as a target of Hippo signaling. The upstream kinases of the Hippo pathway, mammalian STE20-like kinase 1 and 2 (MST1/2), and large tumor suppressor 1 and 2 (LATS1/2), suppress PD-L1 expression while TAZ and YAP enhance PD-L1 levels in breast and lung cancer cell lines. PD-L1 expression in cancer cell lines is determined by TAZ activity and TAZ/YAP/TEAD increase promoter activity. Critically, TAZ-induced PD-L1 upregulation in human cancer cells is sufficient to inhibit T-cell function. The relationship between TAZ and PD-L1 is not conserved in multiple mouse cell lines, likely due to differences between the human and mouse promoters. To explore the extent of divergence in TAZ immune-related targets between human and mouse cells, we performed a second NanoString screen using mouse cell lines. We show that many targets of TAZ may be differentially regulated between these species. These findings highlight the role of Hippo signaling in modifying human/murine physiologic/pathologic immune responses and provide evidence implicating TAZ in human cancer immune evasion. Human-specific activation of PD-L1 by a novel Hippo signaling pathway in cancer immune evasion may have a significant impact on research in immunotherapy. .

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Platforms enabling targeted delivery of proteins into cells are needed to fully realize the potential of protein-based therapeutics with intracellular sites-of-action. Bacterial toxins are attractive systems to consider as templates for designing protein transduction systems as they naturally bind and enter specific cells with high efficiency. Here we investigated the capacity of diphtheria toxin to function as an intracellular protein delivery vector. We report that diphtheria toxin delivers an impressive array of passenger proteins spanning a range of sizes, structures, and stabilities into cells in a manner that indicates that they are "invisible" to the translocation machinery. Further, we show that α-amylase delivered into cells by a detoxified diphtheria toxin chimera digests intracellular glycogen in live cells, providing evidence that delivered cargo is folded, active, and abundant. The efficiency and versatility of diphtheria toxin over existing systems open numerous possibilities for intracellular delivery of bioactive proteins.

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The interaction between the Programmed Death Ligand 1 (PD-L1) immune checkpoint on the tumor cell surface with the Programmed Death-1 (PD-1) receptor on cytotoxic T lymphocytes (CTLs) leads to CTL inactivation, thereby promoting tumor cell escape from adaptive immunity. We previously demonstrated that signaling by PD-L1/PD-1 is bidirectional and leads to activation of oncogenic pathways as well as drug resistance in tumor cells. We also have preliminary evidence that Immunity Related GTPase M, an important mediator of autophagy, is up-regulated by PD-1/PD-L1 reverse signaling. Autophagy is a well-established mechanism of drug resistance in cancer cells. This led us to hypothesize that PD-1/PD-L1 signaling induces drug resistance in tumor cells by up-regulating autophagy. The MEK/ERK and the PI3K/Akt signaling pathways are known to increase and decrease autophagy, respectively. Breast cancer cells exposed to rPD-1 showed a time dependent increase in extracellular signal–regulated kinase (ERK) activation and a decrease in protein kinase B (Akt) activation. Conversion of microtubule-associated protein light chain 3 (LC3)-I to LC3-II is a requirement for autophagosome formation and is a robust marker of autophagy. Exposure of human breast cancer cells to recombinant PD-1 (rPD-1) showed a time-dependent increase in LC-3 II. We are currently conducting additional studies to confirm that the activation of PD-L1 signaling in tumor cells up-regulates autophagy. These results provide evidence that PD-1/PD-L1 reverse signaling activates autophagy as a potential mechanism of cancer cell chemoresistance. (Supported by a grant from the Canadian Institutes of Health Research.) Citation Format: Lori M. Minassian, Shannyn K. MacDonald-Goodfellow, Peter Truesdell, Daniel Sanwalka, Andrew W. Craig, Madhuri Koti, D Robert Siemens, Charles H. Graham. Tumor cell drug resistance induced by the programmed death ligand 1 (PD-L1) immune checkpoint is associated with autophagy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3960. doi:10.1158/1538-7445.AM2017-3960