Gasdermin pores permeabilize mitochondria to augment caspase-3 activation during apoptosis and inflammasome activation.

Gasdermin E (GSDME/DFNA5) cleavage by caspase-3 liberates the GSDME-N domain, which mediates pyroptosis by forming pores in the plasma membrane. Here we show that GSDME-N also permeabilizes the mitochondrial membrane, releasing cytochrome c and activating the apoptosome. Cytochrome c release and caspase-3 activation in response to intrinsic and extrinsic apoptotic stimuli are significantly reduced in GSDME-deficient cells comparing with wild type cells. GSDME deficiency also accelerates cell growth in culture and in a mouse model of melanoma. Phosphomimetic mutation of the highly conserved phosphorylatable Thr6 residue of GSDME, inhibits its pore-forming activity, thus uncovering a potential mechanism by which GSDME might be regulated. Like GSDME-N, inflammasome-generated gasdermin D-N (GSDMD-N), can also permeabilize the mitochondria linking inflammasome activation to downstream activation of the apoptosome. Collectively, our results point to a role of gasdermin proteins in targeting the mitochondria to promote cytochrome c release to augment the mitochondrial apoptotic pathway

FOXD3 Regulates VISTA Expression in Melanoma.

Immune checkpoint inhibitors have improved patient survival in melanoma, but the innate resistance of many patients necessitates the investigation of alternative immune targets. Many immune checkpoint proteins lack proper characterization, including V-domain Ig suppressor of T cell activation (VISTA). VISTA expression on immune cells can suppress T cell activity; however, few studies have investigated its expression and regulation in cancer cells. In this study, we observe that VISTA is expressed in melanoma patient samples and cell lines. Tumor cell-specific expression of VISTA promotes tumor onset in vivo, associated with increased intratumoral T regulatory cells, and enhanced PDL-1 expression on tumor-infiltrating macrophages. VISTA transcript levels are regulated by the stemness factor Forkhead box D3 (FOXD3). BRAF inhibition upregulates FOXD3 and reduces VISTA expression. Overall, this study demonstrates melanoma cell expression of VISTA and its regulation by FOXD3, contributing to the rationale for therapeutic strategies that combine targeted inhibitors with immune checkpoint blockade

Exploiting Murine Cytomegalovirus as an Anti-Tumor Therapy and Model for Virus-Specific Tumor Infiltrating Lymphocytes

The immune system is tightly intertwined with tumor development, i.e. tumor immune surveillance, and can be manipulated to induce anti-tumor responses by targeting tumor immune escape pathways. Recently, it has become clear that several different escape mechanisms need to be targeted simultaneously for robust effects. This dissertation will outline the various intricacies of tumor immune surveillance and tumor immune therapies, offering new perspectives, therapies, and questions that need addressing. In this body of work, murine cytomegalovirus (MCMV) was used as an anti-tumor therapy and to model virus-specific tumor infiltrating lymphocytes (TIL). MCMV is a promising tumor vaccine vector as it induces accumulation of massive numbers of antigen-specific CD8+ T cells (i.e. memory inflation), which enter most tissues and remain functional. To explore this, MCMV expressing a modified gp100 melanoma antigen was used. Systemic vaccination was able to induce gp100-specific memory inflation, but minimal therapeutic effect. However, intratumoral delivery of MCMV significantly delayed tumor growth and prolonged survival in a CD8+ T cell-dependent, gp100-independent manner. Despite MCMV infecting and killing B16F0s in vitro, infection was restricted to tumor- associated macrophages in vivo, suggesting it was not inducing direct oncolysis. Importantly, the therapy synergized with anti-PD-L1 therapy, resulting in tumor clearance and protection in over half of the treated mice. These data demonstrate that multiple immune escape mechanisms must be targeted in order to induce tumor clearance. CD8+ TIL are correlated with positive prognosis in cancer patients. Broad phenotypic markers are used to determine TIL correlations, mostly assuming that TIL are tumor-specific. It is unknown if CD8+ T cells without tumor specificity can become TIL at steady state or in response to pathogens. To address this, tumor- specific and virus-specific CD8+ TIL were compared using MCMV-gp100 and vaccinia virus. Virus-specific CD8+ T cells migrated to tumors and became TIL in a CXCR3-dependent manner and independent of antigen during acute, cleared or latent infections. Surprisingly, virus-specific CD8+ TIL were fully functional compared to tumor-specific TIL, independent of antigen-dependent PD-1 expression. These results suggest that CD8+ TIL can reflect an individual’s immune status and may not always be tumor-specific, potentially skewing prognostic correlations

Intratumoral infection by CMV may change the tumor environment by directly interacting with tumor-associated macrophages to promote cancer immunity

Cytomegalovirus (CMV) is a herpesvirus that induces an extremely robust and sustained immune response. For this reason, CMV has been proposed as a vaccine vector to promote immunity to both pathogens and cancer. However, exploration of CMV as a vaccine vector is at an early stage and there are many questions. Using a mouse melanoma model, we recently found that a CMV-based vaccine induced large populations of melanoma-specific T cells, but was not effective at slowing tumor growth unless it was injected directly into the tumor. These surprising results have led us to hypothesize that CMV may be adept at modulating the tumor micro-environment through its infection of macrophages. Importantly, injection of CMV into the growing tumor synergized with blockade of the PD-1 checkpoint to clear well-established tumors. Here, we discuss our results in the context of CMV-based vaccines for pathogens and cancer

Virus-Specific CD8(+) T Cells Infiltrate Melanoma Lesions and Retain Function Independently of PD-1 Expression

It is well known that CD8(+) tumor-infiltrating lymphocytes (TILs) are correlated with positive prognoses in cancer patients and are used to determine the efficacy of immune therapies. Although it is generally assumed that CD8(+) TILs will be tumor-associated Ag (TAA) specific, it is unknown whether CD8(+) T cells with specificity for common pathogens also infiltrate tumors. If so, the presence of these T cells could alter the interpretation of prognostic and diagnostic TIL assays. We compared TAA-specific and virusspecific CD8(+) T cells in the same tumors using murine CMV, a herpesvirus that causes a persistent/latent infection, and vaccinia virus, a poxvirus that is cleared by the host. Virus-specific CD8+ TILs migrated into cutaneous melanoma lesions during acute infection with either virus, after a cleared vaccinia virus infection, and during a persistent/latent murine CMV infection. Virusspecific TILs developed independently of viral Ag in the tumor and, interestingly, expressed low or intermediate levels of fulllength PD-1 in the tumor environment. Importantly, PD-1 expression could be markedly induced by Ag but did not correlate with dysfunction for virus-specific TILs, in sharp contrast to TAA-specific TILs in the same tumors. These data suggest that CD8(+) TILs can reflect an individual's immune status, rather than exclusively representing TAA-specific T cells, and that PD-1 expression on CD8(+) TILs is not always associated with repeated Ag encounter or dysfunction. Thus, functional virus-specific CD8(+) TILs could skew the results of prognostic or diagnostic TIL assays.National Institutes of Health Grants R03 CA174979 and R01 AI106810 and by American Cancer Society Grants ACS-IRG-08-060-04 and RSG-15-184-01)info:eu-repo/semantics/publishedVersio

CADM1 is a TWIST1-regulated suppressor of invasion and survival

Abstract Metastatic cancer remains a clinical challenge; however, patients diagnosed prior to metastatic dissemination have a good prognosis. The transcription factor, TWIST1 has been implicated in enhancing the migration and invasion steps within the metastatic cascade, but the range of TWIST1-regulated targets is poorly described. In this study, we performed expression profiling to identify the TWIST1-regulated transcriptome of melanoma cells. Gene ontology pathway analysis revealed that TWIST1 and epithelial to mesenchymal transition (EMT) were inversely correlated with levels of cell adhesion molecule 1 (CADM1). Chromatin immunoprecipitation (ChIP) studies and promoter assays demonstrated that TWIST1 physically interacts with the CADM1 promoter, suggesting TWIST1 directly represses CADM1 levels. Increased expression of CADM1 resulted in significant inhibition of motility and invasiveness of melanoma cells. In addition, elevated CADM1 elicited caspase-independent cell death in non-adherent conditions. Expression array analysis suggests that CADM1 directed non-adherent cell death is associated with loss of mitochondrial membrane potential and subsequent failure of oxidative phosphorylation pathways. Importantly, tissue microarray analysis and clinical data from TCGA indicate that CADM1 expression is inversely associated with melanoma progression and positively correlated with better overall survival in patients. Together, these data suggest that CADM1 exerts tumor suppressive functions in melanoma by reducing invasive potential and may be considered a biomarker for favorable prognosis