Human forebrain endothelial cell therapy for psychiatric disorders.

Abnormalities of or reductions in GABAergic interneurons are implicated in the pathology of severe neuropsychiatric disorders, for which effective treatments are still elusive. Transplantation of human stem cell-derived interneurons is a promising cell-based therapy for treatment of these disorders. In mouse xenograft studies, human stem cell-derived-interneuron precursors could differentiate in vivo, but required a prolonged time of four to seven months to migrate from the graft site and integrate with the host tissue. This poses a serious roadblock for clinical translation of this approach. For transplantation to be effective, grafted neurons should migrate to affected areas at a faster rate. We have previously shown that endothelial cells of the periventricular vascular network are the natural substrates for GABAergic interneurons in the developing mouse forebrain, and provide valuable guidance cues for their long-distance migration. In addition, periventricular endothelial cells house a GABA signaling pathway with direct implications for psychiatric disease origin. In this study we translated this discovery into human, with significant therapeutic implications. We generated human periventricular endothelial cells, using human pluripotent stem cell technology, and extensively characterized its molecular, cellular, and functional properties. Co-culture of human periventricular endothelial cells with human interneurons significantly accelerated interneuron migration in vitro and led to faster migration and wider distribution of grafted interneurons in vivo, compared to neuron-only transplants. Furthermore, the co-transplantation strategy was able to rescue abnormal behavioral symptoms in a pre-clinical model of psychiatric disorder, within 1 month after transplantation. We anticipate this strategy to open new doors and facilitate exciting advances in angiogenesis-mediated treatment of psychiatric disorders.

Aurora kinase inhibition sensitizes melanoma cells to T-cell-mediated cytotoxicity.

Although immunotherapy has achieved impressive durable clinical responses, many cancers respond only temporarily or not at all to immunotherapy. To find novel, targetable mechanisms of resistance to immunotherapy, patient-derived melanoma cell lines were transduced with 576 open reading frames, or exposed to arrayed libraries of 850 bioactive compounds, prior to co-culture with autologous tumor-infiltrating lymphocytes (TILs). The synergy between the targets and TILs to induce apoptosis, and the mechanisms of inhibiting resistance to TILs were interrogated. Gene expression analyses were performed on tumor samples from patients undergoing immunotherapy for metastatic melanoma. Finally, the effect of inhibiting the top targets on the efficacy of immunotherapy was investigated in multiple preclinical models. Aurora kinase was identified as a mediator of melanoma cell resistance to T-cell-mediated cytotoxicity in both complementary screens. Aurora kinase inhibitors were validated to synergize with T-cell-mediated cytotoxicity in vitro. The Aurora kinase inhibition-mediated sensitivity to T-cell cytotoxicity was shown to be partially driven by p21-mediated induction of cellular senescence. The expression levels of Aurora kinase and related proteins were inversely correlated with immune infiltration, response to immunotherapy and survival in melanoma patients. Aurora kinase inhibition showed variable responses in combination with immunotherapy in vivo, suggesting its activity is modified by other factors in the tumor microenvironment. These data suggest that Aurora kinase inhibition enhances T-cell cytotoxicity in vitro and can potentiate antitumor immunity in vivo in some but not all settings. Further studies are required to determine the mechanism of primary resistance to this therapeutic intervention.

Novel coronavirus treatment with ribavirin: Groundwork for an evaluation concerning COVID-19.

Confronting the challenge of the outbreak of COVID-19 should sharpen our focus on global drug access as a key issue in antiviral therapy testing. The testing and adoption of effective therapies for novel coronaviruses are hampered by the challenge of conducting controlled studies during a state of emergency. The access to direct antiviral drugs, such as ribavirin, that have an existing inventory and reliable supply chain may be a priority consideration for therapies developed for the 2019-nCoV infection outbreaks and any strain variants that may emerge. On the basis of the direct antiviral activity of ribavirin against 2019-nCoV in vitro and evidence for potency enhancement strategies developed during the prior SARS and MERS outbreaks, ribavirin may significantly impact our ability to end the lingering outbreaks in China and slow outbreaks in other countries. The apparent COVID-19 pandemic provides an opportunity to follow dosage guidelines for treatment with ribavirin, test new therapeutic concepts, and conduct controlled testing to apply the scientific rigor required to address the controversy around this mainstay of antiviral therapy.

B-Raf regulation of integrin α4ß1-mediated resistance to shear stress through changes in cell spreading and cytoskeletal association in T cells.

The regulation of integrin-mediated adhesion is of vital importance to adaptive and innate immunity. Integrins are versatile proteins and mediate T cell migration and trafficking by binding to extracellular matrix or other cells as well as initiating intracellular signaling cascades promoting survival or activation. The MAPK pathway is known to be downstream from integrins and to regulate survival, differentiation, and motility. However, secondary roles for canonical MAPK pathway members are being discovered. We show that chemical inhibition of RAF by sorafenib or shRNA-mediated knockdown of B-Raf reduces T cell resistance to shear stress to α4ß1 integrin ligands vascular cell adhesion molecule 1 (VCAM-1) and fibronectin, whereas inhibition of MEK/ERK by U0126 had no effect. Microscopy showed that RAF inhibition leads to significant inhibition of T cell spreading on VCAM-1. The association of α4ß1 integrin with the actin cytoskeleton was shown to be dependent on B-Raf activity or expression, whereas α4ß1 integrin affinity for soluble VCAM-1 was not. These effects were shown to be specific for α4ß1 integrin and not other integrins, such as α5ß1 or LFA-1, or a variety of membrane proteins. We demonstrate a novel role for B-Raf in the selective regulation of α4ß1 integrin-mediated adhesion.

ACE2-Fc fusion protein overcomes viral escape by potently neutralizing SARS-CoV-2 variants of concern.

COVID-19, an infectious disease caused by the SARS-CoV-2 virus, emerged globally in early 2020 and has remained a serious public health issue. To date, although several preventative vaccines have been approved by FDA and EMA, vaccinated individuals increasingly suffer from breakthrough infections. Therapeutic antibodies may provide an alternative strategy to neutralize viral infection and treat serious cases; however, the clinical data and our experiments show that some FDA-approved monoclonal antibodies lose function against COVID-19 variants such as Omicron. Therefore, in this study, we present a novel therapeutic agent, SI-F019, an ACE2-Fc fusion protein whose neutralization efficiency is not compromised, but actually strengthened, by the mutations of dominant variants including Omicron. Comprehensive biophysical analyses revealed the mechanism of increased inhibition to be enhanced interaction of SI-F019 with all the tested spike variants, in contrast to monoclonal antibodies which tended to show weaker binding to some variants. The results imply that SI-F019 may be a broadly useful agent for treatment of COVID-19.

Endothelial cell-derived GABA signaling modulates neuronal migration and postnatal behavior.

The cerebral cortex is essential for integration and processing of information that is required for most behaviors. The exquisitely precise laminar organization of the cerebral cortex arises during embryonic development when neurons migrate successively from ventricular zones to coalesce into specific cortical layers. While radial glia act as guide rails for projection neuron migration, pre-formed vascular networks provide support and guidance cues for GABAergic interneuron migration. This study provides novel conceptual and mechanistic insights into this paradigm of vascular-neuronal interactions, revealing new mechanisms of GABA and its receptor-mediated signaling via embryonic forebrain endothelial cells. With the use of two new endothelial cell specific conditional mouse models of the GABA pathway (Gabrb3ΔTie2-Cre and VgatΔTie2-Cre), we show that partial or complete loss of GABA release from endothelial cells during embryogenesis results in vascular defects and impairs long-distance migration and positioning of cortical interneurons. The downstream effects of perturbed endothelial cell-derived GABA signaling are critical, leading to lasting changes to cortical circuits and persistent behavioral deficits. Furthermore, we illustrate new mechanisms of activation of GABA signaling in forebrain endothelial cells that promotes their migration, angiogenesis and acquisition of blood-brain barrier properties. Our findings uncover and elucidate a novel endothelial GABA signaling pathway in the CNS that is distinct from the classical neuronal GABA signaling pathway and shed new light on the etiology and pathophysiology of neuropsychiatric diseases, such as autism spectrum disorders, epilepsy, anxiety, depression and schizophrenia.

SLC45A2: A Melanoma Antigen with High Tumor Selectivity and Reduced Potential for Autoimmune Toxicity.

Cytotoxic T lymphocyte (CTL)-based immunotherapies have had remarkable success at generating objective clinical responses in patients with advanced metastatic melanoma. Although the melanocyte differentiation antigens (MDA) MART-1, PMEL, and tyrosinase were among the first melanoma tumor-associated antigens identified and targeted with immunotherapy, expression within normal melanocytes of the eye and inner ear can elicit serious autoimmune side effects, thus limiting their clinical potential as CTL targets. Using a tandem mass spectrometry (MS) approach to analyze the immunopeptidomes of 55 melanoma patient-derived cell lines, we identified a number of shared HLA class I-bound peptides derived from the melanocyte-specific transporter protein SLC45A2. Antigen-specific CTLs generated against HLA-A*0201- and HLA-A*2402-restricted SLC45A2 peptides effectively killed a majority of HLA-matched cutaneous, uveal, and mucosal melanoma cell lines tested (18/25). CTLs specific for SLC45A2 showed significantly reduced recognition of HLA-matched primary melanocytes that were, conversely, robustly killed by MART1- and PMEL-specific T cells. Transcriptome analysis revealed that SLC45A2 mRNA expression in normal melanocytes was less than 2% that of other MDAs, therefore providing a more favorable melanoma-to-melanocyte expression ratio. Expression of SLC45A2 and CTL sensitivity could be further upregulated in BRAF(V600E)-mutant melanoma cells upon treatment with BRAF or MEK inhibitors, similarly to other MDAs. Taken together, our study demonstrates the feasibility of using tandem MS as a means of discovering shared immunogenic tumor-associated epitopes and identifies SLC45A2 as a promising immunotherapeutic target for melanoma with high tumor selectivity and reduced potential for autoimmune toxicity. Cancer Immunol Res; 5(8); 618-29. ©2017 AACR.

BRAFV600E Co-opts a Conserved MHC Class I Internalization Pathway to Diminish Antigen Presentation and CD8+ T-cell Recognition of Melanoma.

Oncogene activation in tumor cells induces broad and complex cellular changes that contribute significantly to disease initiation and progression. In melanoma, oncogenic BRAF(V600E) has been shown to drive the transcription of a specific gene signature that can promote multiple mechanisms of immune suppression within the tumor microenvironment. We show here that BRAF(V600E) also induces rapid internalization of MHC class I (MHC-I) from the melanoma cell surface and its intracellular sequestration within endolysosomal compartments. Importantly, MAPK inhibitor treatment quickly restored MHC-I surface expression in tumor cells, thereby enhancing melanoma antigen-specific T-cell recognition and effector function. MAPK pathway-driven relocalization of HLA-A*0201 required a highly conserved cytoplasmic serine phosphorylation site previously implicated in rapid MHC-I internalization and recycling by activated immune cells. Collectively, these data suggest that oncogenic activation of BRAF allows tumor cells to co-opt an evolutionarily conserved MHC-I trafficking pathway as a strategy to facilitate immune evasion. This link between MAPK pathway activation and the MHC-I cytoplasmic tail has direct implications for immunologic recognition of tumor cells and provides further evidence to support testing therapeutic strategies combining MAPK pathway inhibition with immunotherapies in the clinical setting.

Forging a link between oncogenic signaling and immunosuppression in melanoma.

Immunosuppressive tumor microenvironments limit the efficacy of T cell-based immunotherapy. We have recently demonstrated that the inhibition of BRAFV600E with vemurafenib relieves interleukin-1 (IL-1)-induced T-cell suppression as mediated by melanoma tumor associated fibroblasts (TAFs). These results suggest that inhibitors of the MAPK pathway in combination with T cell-based immunotherapies may induce long-lasting and durable responses.

In silico prediction of tumor antigens derived from functional missense mutations of the cancer gene census.

Antigen-specific immune responses against peptides derived from missense gene mutations have been identified in multiple cancers. The application of personalized peptide vaccines based on the tumor mutation repertoire of each cancer patient is a near-term clinical reality. These peptides can be identified for pre-validation by leveraging the results of massive gene sequencing efforts in cancer. In this study, we utilized NetMHC 3.2 to predict nanomolar peptide binding affinity to 57 human HLA-A and B alleles. All peptides were derived from 5,685 missense mutations in 312 genes annotated as functionally relevant in the Cancer Genome Project. Of the 26,672,189 potential 8-11 mer peptide-HLA pairs evaluated, 0.4% (127,800) display binding affinities < 50 nM, predicting high affinity interactions. These peptides can be segregated into two groups based on the binding affinity to HLA proteins relative to germline-encoded sequences: peptides for which both the mutant and wild-type forms are high affinity binders, and peptides for which only the mutant form is a high affinity binder. Current evidence directs the attention to mutations that increase HLA binding affinity, as compared with cognate wild-type peptide sequences, as these potentially are more relevant for vaccine development from a clinical perspective. Our analysis generated a database including all predicted HLA binding peptides and the corresponding change in binding affinity as a result of point mutations. Our study constitutes a broad foundation for the development of personalized peptide vaccines that hone-in on functionally relevant targets in multiple cancers in individuals with diverse HLA haplotypes.

Combination small molecule MEK and PI3K inhibition enhances uveal melanoma cell death in a mutant GNAQ- and GNA11-dependent manner.

PURPOSE: Activating Q209L/P mutations in GNAQ or GNA11 (GNAQ/11) are present in approximately 80% of uveal melanomas. Mutant GNAQ/11 are not currently therapeutically targetable. Inhibiting key down-stream effectors of GNAQ/11 represents a rational therapeutic approach for uveal melanomas that harbor these mutations. The mitogen-activated protein/extracellular signal-regulated kinase/mitogen-activated protein kinase (MEK/MAPK) and PI3K/AKT pathways are activated in uveal melanoma. In this study, we test the effect of the clinically relevant small molecule inhibitors GSK1120212 (MEK inhibitor) and GSK2126458 (pan class I PI3K inhibitor) on uveal melanoma cells with different GNAQ/11 mutation backgrounds. EXPERIMENTAL DESIGN: We use the largest set of genetically annotated uveal melanoma cell lines to date to carry out in vitro cellular signaling, cell-cycle regulation, growth, and apoptosis analyses. RNA interference and small molecule MEK and/or PI3K inhibitor treatment were used to determine the dependency of uveal melanoma cells with different GNAQ/11 mutation backgrounds on MEK/MAPK and/or PI3K/AKT signaling. Proteomic network analysis was done to unveil signaling alterations in response to MEK and/or PI3K small molecule inhibition. RESULTS: GNAQ/11 mutation status was not a determinant of whether cells would undergo cell-cycle arrest or growth inhibition to MEK and/or phosphoinositide 3-kinase (PI3K) inhibition. A reverse correlation was observed between MAPK and AKT phosphorylation after MEK or PI3K inhibition, respectively. Neither MEK nor PI3K inhibition alone was sufficient to induce apoptosis in the majority of cell lines; however, the combination of MEK + PI3K inhibitor treatment resulted in the marked induction of apoptosis in a GNAQ/11 mutant-dependent manner. CONCLUSIONS: MEK + PI3K inhibition may be an effective combination therapy in uveal melanoma, given the inherent reciprocal activation of these pathways within these cells.

Oncogenic BRAF(V600E) promotes stromal cell-mediated immunosuppression via induction of interleukin-1 in melanoma.

PURPOSE: In this study, we assessed the specific role of BRAF(V600E) signaling in modulating the expression of immune regulatory genes in melanoma, in addition to analyzing downstream induction of immune suppression by primary human melanoma tumor-associated fibroblasts (TAF). EXPERIMENTAL DESIGN: Primary human melanocytes and melanoma cell lines were transduced to express WT or V600E forms of BRAF, followed by gene expression analysis. The BRAF(V600E) inhibitor vemurafenib was used to confirm targets in BRAF(V600E)-positive melanoma cell lines and in tumors from melanoma patients undergoing inhibitor treatment. TAF lines generated from melanoma patient biopsies were tested for their ability to inhibit the function of tumor antigen-specific T cells, before and following treatment with BRAF(V600E)-upregulated immune modulators. Transcriptional analysis of treated TAFs was conducted to identify potential mediators of T-cell suppression. RESULTS: Expression of BRAF(V600E) induced transcription of interleukin 1 alpha (IL-1α) and IL-1ß in melanocytes and melanoma cell lines. Further, vemurafenib reduced the expression of IL-1 protein in melanoma cell lines and most notably in human tumor biopsies from 11 of 12 melanoma patients undergoing inhibitor treatment. Treatment of melanoma-patient-derived TAFs with IL-1α/ß significantly enhanced their ability to suppress the proliferation and function of melanoma-specific cytotoxic T cells, and this inhibition was partially attributable to upregulation by IL-1 of COX-2 and the PD-1 ligands PD-L1 and PD-L2 in TAFs. CONCLUSIONS: This study reveals a novel mechanism of immune suppression sensitive to BRAF(V600E) inhibition, and indicates that clinical blockade of IL-1 may benefit patients with BRAF wild-type tumors and potentially synergize with immunotherapeutic interventions.

Natural splice variant of MHC class I cytoplasmic tail enhances dendritic cell-induced CD8+ T-cell responses and boosts anti-tumor immunity.

Dendritic cell (DC)-mediated presentation of MHC class I (MHC-I)/peptide complexes is a crucial first step in the priming of CTL responses, and the cytoplasmic tail of MHC-I plays an important role in modulating this process. Several species express a splice variant of the MHC-I tail that deletes exon 7-encoding amino acids (Δ7), including a conserved serine phosphorylation site. Previously, it has been shown that Δ7 MHC-I molecules demonstrate extended DC surface half-lives, and that mice expressing Δ7-K(b) generate significantly augmented CTL responses to viral challenge. Herein, we show that Δ7-D(b)-expressing DCs stimulated significantly more proliferation and much higher cytokine secretion by melanoma antigen-specific (Pmel-1) T cells. Moreover, in combination with adoptive Pmel-1 T-cell transfer, Δ7-D(b) DCs were superior to WT-D(b) DCs at stimulating anti-tumor responses against established B16 melanoma tumors, significantly extending mouse survival. Human DCs engineered to express Δ7-HLA-A*0201 showed similarly enhanced CTL stimulatory capacity. Further studies demonstrated impaired lateral membrane movement and clustering of human Δ7-MHC-I/peptide complexes, resulting in significantly increased bioavailability of MHC-I/peptide complexes for specific CD8+ T cells. Collectively, these data suggest that targeting exon 7-encoded MHC-I cytoplasmic determinants in DC vaccines has the potential to increase CD8+ T-cell stimulatory capacity and substantially improve their clinical efficacy.