Placental co-transcriptional activator Vestigial-like 1 (VGLL1) drives tumorigenesis via increasing transcription of proliferation and invasion genes.

Introduction: Vestigial-like 1 (VGLL1) is a co-transcriptional activator that binds to TEA domain-containing transcription factors (TEADs). Its expression is upregulated in a variety of aggressive cancer types, including pancreatic and basal-like breast cancer, and increased transcription of VGLL1 is strongly correlated with poor prognosis and decreased overall patient survival. In normal tissues, VGLL1 is most highly expressed within placental trophoblast cells, which share the common attributes of rapid cellular proliferation and invasion with tumor cells. The impact of VGLL1 in cancer has not been fully elucidated and no VGLL1-targeted therapy currently exists. Methods: The aim of this study was to evaluate the cellular function and downstream genomic targets of VGLL1 in placental, pancreatic, and breast cancer cells. Functional assays were employed to assess the role of VGLL1 in cellular invasion and proliferation, and ChIP-seq and RNAseq assays were performed to identify VGLL1 target genes and potential impact using pathway analysis. Results: ChIP-seq analysis identified eight transcription factors with a VGLL1-binding motif that were common between all three cell types, including TEAD1-4, AP-1, and GATA6, and revealed ~3,000 shared genes with which VGLL1 interacts. Furthermore, increased VGLL1 expression led to an enhancement of cell invasion and proliferation, which was supported by RNAseq analysis showing transcriptional changes in several genes known to be involved in these processes. Discussion: This work expands our mechanistic understanding of VGLL1 function in tumor cells and provides a strong rationale for developing VGLL1-targeted therapies for treating cancer patients.

Vestigial-like 1 (VGLL1): An ancient co-transcriptional activator linking wing, placenta, and tumor development.

Vestigial-like 1 (VGLL1) is a recently discovered driver of proliferation and invasion that is expressed in many aggressive human malignancies and is strongly associated with poor prognosis. The VGLL1 gene encodes for a co-transcriptional activator that shows intriguing structural similarity to key activators in the hippo pathway, providing important clues to its functional role. VGLL1 binds to TEAD transcription factors in an analogous fashion to YAP1 but appears to activate a distinct set of downstream gene targets. In mammals, VGLL1 expression is found almost exclusively in placental trophoblasts, cells that share many hallmarks of cancer. Due to its role as a driver of tumor progression, VGLL1 has become a target of interest for potential anticancer therapies. In this review, we discuss VGLL1 from an evolutionary perspective, contrast its role in placental and tumor development, summarize the current knowledge of how signaling pathways can modulate VGLL1 function, and discuss potential approaches for targeting VGLL1 therapeutically.

Charge-based interactions through peptide position 4 drive diversity of antigen presentation by human leukocyte antigen class I molecules.

Human leukocyte antigen class I (HLA-I) molecules bind and present peptides at the cell surface to facilitate the induction of appropriate CD8+ T cell-mediated immune responses to pathogen- and self-derived proteins. The HLA-I peptide-binding cleft contains dominant anchor sites in the B and F pockets that interact primarily with amino acids at peptide position 2 and the C-terminus, respectively. Nonpocket peptide-HLA interactions also contribute to peptide binding and stability, but these secondary interactions are thought to be unique to individual HLA allotypes or to specific peptide antigens. Here, we show that two positively charged residues located near the top of peptide-binding cleft facilitate interactions with negatively charged residues at position 4 of presented peptides, which occur at elevated frequencies across most HLA-I allotypes. Loss of these interactions was shown to impair HLA-I/peptide binding and complex stability, as demonstrated by both in vitro and in silico experiments. Furthermore, mutation of these Arginine-65 (R65) and/or Lysine-66 (K66) residues in HLA-A*02:01 and A*24:02 significantly reduced HLA-I cell surface expression while also reducing the diversity of the presented peptide repertoire by up to 5-fold. The impact of the R65 mutation demonstrates that nonpocket HLA-I/peptide interactions can constitute anchor motifs that exert an unexpectedly broad influence on HLA-I-mediated antigen presentation. These findings provide fundamental insights into peptide antigen binding that could broadly inform epitope discovery in the context of viral vaccine development and cancer immunotherapy.

Evolution of CD8+ T Cell Receptor (TCR) Engineered Therapies for the Treatment of Cancer.

Engineered T cell receptor T (TCR-T) cell therapy has facilitated the generation of increasingly reliable tumor antigen-specific adaptable cellular products for the treatment of human cancer. TCR-T cell therapies were initially focused on targeting shared tumor-associated peptide targets, including melanoma differentiation and cancer-testis antigens. With recent technological developments, it has become feasible to target neoantigens derived from tumor somatic mutations, which represents a highly personalized therapy, since most neoantigens are patient-specific and are rarely shared between patients. TCR-T therapies have been tested for clinical efficacy in treating solid tumors in many preclinical studies and clinical trials all over the world. However, the efficacy of TCR-T therapy for the treatment of solid tumors has been limited by a number of factors, including low TCR avidity, off-target toxicities, and target antigen loss leading to tumor escape. In this review, we discuss the process of deriving tumor antigen-specific TCRs, including the identification of appropriate tumor antigen targets, expansion of antigen-specific T cells, and TCR cloning and validation, including techniques and tools for TCR-T cell vector construction and expression. We highlight the achievements of recent clinical trials of engineered TCR-T cell therapies and discuss the current challenges and potential solutions for improving their safety and efficacy, insights that may help guide future TCR-T studies in cancer.

Reversing Epigenetic Gene Silencing to Overcome Immune Evasion in CNS Malignancies.

Glioblastoma (GBM) is an aggressive brain malignancy with a dismal prognosis. With emerging evidence to disprove brain-immune privilege, there has been much interest in examining immunotherapy strategies to treat central nervous system (CNS) cancers. Unfortunately, the limited success of clinical studies investigating immunotherapy regimens, has led to questions about the suitability of immunotherapy for these cancers. Inadequate inherent populations of tumor infiltrating lymphocytes (TILs) and limited trafficking of systemic, circulating T cells into the CNS likely contribute to the poor response to immunotherapy. This paucity of TILs is in concert with the finding of epigenetic silencing of genes that promote immune cell movement (chemotaxis) to the tumor. In this study we evaluated the ability of GSK126, a blood-brain barrier (BBB) permeable small molecule inhibitor of EZH2, to reverse GBM immune evasion by epigenetic suppression of T cell chemotaxis. We also evaluated the in vivo efficacy of this drug in combination with anti-PD-1 treatment on tumor growth, survival and T cell infiltration in syngeneic mouse models. GSK126 reversed H3K27me3 in murine and human GBM cell lines. When combined with anti-PD-1 treatment, a significant increase in activated T cell infiltration into the tumor was observed. This resulted in decreased tumor growth and enhanced survival both in sub-cutaneous and intracranial tumors of immunocompetent, syngeneic murine models of GBM. Additionally, a significant increase in CXCR3+ T cells was also seen in the draining lymph nodes, suggesting their readiness to migrate to the tumor. Closer examination of the mechanism of action of GSK126 revealed its ability to promote the expression of IFN-γ driven chemokines CXCL9 and CXCL10 from the tumor cells, that work to traffic T cells without directly affecting T maturation and/or proliferation. The loss of survival benefit either with single agent or combination in immunocompromised SCID mice, suggest that the therapeutic efficacy of GSK126 in GBM is primarily driven by lymphocytes. Taken together, our data suggests that in glioblastoma, epigenetic modulation using GSK126 could improve current immunotherapy strategies by reversing the epigenetic changes that enable immune cell evasion leading to enhanced immune cell trafficking to the tumor.

Neoantigen vaccination induces clinical and immunologic responses in non-small cell lung cancer patients harboring EGFR mutations.

BACKGROUND: Neoantigen (NeoAg) peptides displayed at the tumor cell surface by human leukocyte antigen molecules show exquisite tumor specificity and can elicit T cell mediated tumor rejection. However, few NeoAgs are predicted to be shared between patients, and none to date have demonstrated therapeutic value in the context of vaccination. METHODS: We report here a phase I trial of personalized NeoAg peptide vaccination (PPV) of 24 stage III/IV non-small cell lung cancer (NSCLC) patients who had previously progressed following multiple conventional therapies, including surgery, radiation, chemotherapy, and tyrosine kinase inhibitors (TKIs). Primary endpoints of the trial evaluated feasibility, tolerability, and safety of the personalized vaccination approach, and secondary trial endpoints assessed tumor-specific immune reactivity and clinical responses. Of the 16 patients with epidermal growth factor receptor (EGFR) mutations, nine continued TKI therapy concurrent with PPV and seven patients received PPV alone. RESULTS: Out of 29 patients enrolled in the trial, 24 were immunized with personalized NeoAg peptides. Aside from transient rash, fatigue and/or fever observed in three patients, no other treatment-related adverse events were observed. Median progression-free survival and overall survival of the 24 vaccinated patients were 6.0 and 8.9 months, respectively. Within 3-4 months following initiation of PPV, seven RECIST-based objective clinical responses including one complete response were observed. Notably, all seven clinical responders had EGFR-mutated tumors, including four patients that had continued TKI therapy concurrently with PPV. Immune monitoring showed that five of the seven responding patients demonstrated vaccine-induced T cell responses against EGFR NeoAg peptides. Furthermore, two highly shared EGFR mutations (L858R and T790M) were shown to be immunogenic in four of the responding patients, all of whom demonstrated increases in peripheral blood neoantigen-specific CD8+ T cell frequencies during the course of PPV. CONCLUSIONS: These results show that personalized NeoAg vaccination is feasible and safe for advanced-stage NSCLC patients. The clinical and immune responses observed following PPV suggest that EGFR mutations constitute shared, immunogenic neoantigens with promising immunotherapeutic potential for large subsets of NSCLC patients. Furthermore, PPV with concurrent EGFR inhibitor therapy was well tolerated and may have contributed to the induction of PPV-induced T cell responses.

Vestigial-like 1 is a shared targetable cancer-placenta antigen expressed by pancreatic and basal-like breast cancers.

Cytotoxic T lymphocyte (CTL)-based cancer immunotherapies have shown great promise for inducing clinical regressions by targeting tumor-associated antigens (TAA). To expand the TAA landscape of pancreatic ductal adenocarcinoma (PDAC), we performed tandem mass spectrometry analysis of HLA class I-bound peptides from 35 PDAC patient tumors. This identified a shared HLA-A*0101 restricted peptide derived from co-transcriptional activator Vestigial-like 1 (VGLL1) as a putative TAA demonstrating overexpression in multiple tumor types and low or absent expression in essential normal tissues. Here we show that VGLL1-specific CTLs expanded from the blood of a PDAC patient could recognize and kill in an antigen-specific manner a majority of HLA-A*0101 allogeneic tumor cell lines derived not only from PDAC, but also bladder, ovarian, gastric, lung, and basal-like breast cancers. Gene expression profiling reveals VGLL1 as a member of a unique group of cancer-placenta antigens (CPA) that may constitute immunotherapeutic targets for patients with multiple cancer types.

Dexamethasone-induced immunosuppression: mechanisms and implications for immunotherapy.

BACKGROUND: Corticosteroids are routinely utilized to alleviate edema in patients with intracranial lesions and are first-line agents to combat immune-related adverse events (irAEs) that arise with immune checkpoint blockade treatment. However, it is not known if or when corticosteroids can be administered without abrogating the efforts of immunotherapy. The purpose of this study was to evaluate the impact of dexamethasone on lymphocyte activation and proliferation during checkpoint blockade to provide guidance for corticosteroid use while immunotherapy is being implemented as a cancer treatment. METHODS: Lymphocyte proliferation, differentiation, and cytokine production were evaluated during dexamethasone exposure. Human T cells were stimulated through CD3 ligation and co-stimulated either directly by CD28 ligation or by providing CD80, a shared ligand for CD28 and CTLA-4. CTLA-4 signaling was inhibited by antibody blockade using ipilimumab which has been approved for the treatment of several solid tumors. The in vivo effects of dexamethasone during checkpoint blockade were evaluated using the GL261 syngeneic mouse intracranial model, and immune populations were profiled by flow cytometry. RESULTS: Dexamethasone upregulated CTLA-4 mRNA and protein in CD4 and CD8 T cells and blocked CD28-mediated cell cycle entry and differentiation. Naïve T cells were most sensitive, leading to a decrease of the development of more differentiated subsets. Resistance to dexamethasone was conferred by blocking CTLA-4 or providing strong CD28 co-stimulation prior to dexamethasone exposure. CTLA-4 blockade increased IFNγ expression, but not IL-2, in stimulated human peripheral blood T cells exposed to dexamethasone. Finally, we found that CTLA-4 blockade partially rescued T cell numbers in mice bearing intracranial gliomas. CTLA-4 blockade was associated with increased IFNγ-producing tumor-infiltrating T cells and extended survival of dexamethasone-treated mice. CONCLUSIONS: Dexamethasone-mediated T cell suppression diminishes naïve T cell proliferation and differentiation by attenuating the CD28 co-stimulatory pathway. However, CTLA-4, but not PD-1 blockade can partially prevent some of the inhibitory effects of dexamethasone on the immune response.