Abstract

While PD-1 immune checkpoint blockade (ICB) has revolutionized cancer treatment, most patients are not cured underscoring the need to new approaches. Given that anti-PD-1 therapy depends on the co-receptor CD28, and CD28-PI 3K activates AKT, we asked whether direct small-molecule activation of AKT could bypass the need for biologics and overcome resistance to anti-PD-1 immunotherapy. This has never been assessed before. In this context, tumor cell interferon-γ (IFN-γ) signaling plays a crucial role in immune surveillance. Here, we show that direct AKT activation potently promoted tumor rejection due to a dominant phenotype, namely, the marked upregulation of IFN-γ on all CD4 and CD8+ TIL subsets. Further, surprisingly, AKT activation could inter-convert suppressor Treg TILs in tumors to CD4+Th1-like T-cells, accompanied by a reduced STAT5 phospho-activation. The pathway was also dependent on T-bet and regressed tumors resistant to anti-PD-1 therapy. Our study offers a novel therapeutic approach using small molecules to activate AKT for the regression of tumors.

Abstract

Introduction: Hodgkin’s Lymphoma (HL) is the most common cancer in young adults that is initially treated with chemotherapy. PD1 inhibitors are effective at relapse but associated with immune related adverse events (IRAE). By understanding the mechanisms PD1 inhibitors inhibit tumor growth and cause autoimmunity may lead to safer, more effective immunotherapy treatments.
Methods: We performed single-cell RNA sequencing (scRNA-seq) using 10xGenomics of six samples of peripheral blood mononuclear cells taken from one relapsed HL patient treated with the PD-1 inhibitor pembrolizumab. Each sample corresponds to a timepoint with distinct disease and IRAE statuses (pericarditis). 26,739 cells were clustered using Seurat in order to analyze cell identities across the 6 timepoints to further understand immune pathway activation.
Results: Using Seurat’s Azimuth tool for referenced-based single-cell analysis and canonical markers for T cell effector and exhausted states, we identified 20 cell clusters present at each time point. Two clusters demonstrated the most dynamic and significant changes of gene expression at all time points: CD8+ exhausted and CD8+ effector T cells. B cells also displayed significant changes. In both CD8 T cells and B cells, different clones (identified by the T and B cell receptor genes) were enriched at IRAE and disease state time points and corresponding to different gene expression patterns.
Conclusion: scRNA-seq may be useful to identify dynamic changes in T and B cell clones present in the peripheral blood of patients treated with PD1 inhibitors, providing insights into the mechanisms of anti-tumor and auto-immune responses in immunotherapy-treated patients.

Abstract

Background: Triple negative breast cancer (TNBC) accounts for 15% of all invasive breast cancers. TNBC is currently only treated with surgery and chemotherapy [1]. Compared to other breast cancer types, TNBC has higher rates of genetic mutations and contains more tumor infiltrating lymphocytes [2]. These characteristics provide a strong rationale to use novel immunotherapies such as immunogenic autologous tumor cell vaccines to therapeutically target TNBC.
Hypothesis and aims: We hypothesized that an infected cell vaccine (ICV) which is made from irradiated and infected TNBC cells with oncolytic virus, VSVd51, will induce beneficial tumor- targeted immune responses in translational cancer models. The specific aims are 1) To characterize vaccine induced immune responses in vivo using syngeneic TNBC mouse models and 2) evaluate the immunogenicity of TNBC patient resected tumors following ex-vivo infection with VSVd51.
Results: We detected an improvement in NK and T cell cytotoxicity and cytokine secretion, a reduction in lung tumor burden, and an increase in survival in ICV treated mice. Combination treatment with the checkpoint inhibitor anti-PD1 further extended survival in ICV treated mice compared to monotherapy ICV or anti-PD1. Ex vivo infection of patients’ resected TNBC tumors with VSVd51 demonstrated that ICV enhances immune signatures and biomarkers of immunogenic cell death in human TNBC tissues. Our immediate goal is to further improve ICV efficacy by applying a “prime-boost” cancer vaccination approach.
Conclusion: These promising preclinical results demonstrate the therapeutic potential of oncolytic virus based immunogenic tumor vaccines to treat TNBC.

Abstract

Melanomas commonly undergo a phenotype switch, from a proliferative to an invasive state. Melanoma plasticity exhibited as phenotype switching contributes to immunotherapy resistance, however the mechanisms are not completely understood and thus therapeutically unexploited. Here, using a transgenic melanoma mouse model, we demonstrated a critical role of the MNK1/2-eIF4E axis in melanoma plasticity and resistance to immunotherapy. We showed that phospho-eIF4E deficient murine melanomas express high levels of melanocytic antigens, with similar results verified in patient melanomas. Mechanistically, we identified that phospho-eIF4E controls the translation of NGFR, a critical effector of phenotype switching. In patients with melanoma, the expression of MKNK1, the kinase for eIF4E, positively correlated with markers of immune exhaustion. Genetic ablation of phospho-eIF4E reprogrammed the immunosuppressive microenvironment, exemplified by lowered production of inflammatory factors and increased CD8⁺ T cell infiltrates. Blocking phospho-eIF4E, using MNK1/2 inhibitors, offers a new strategy to inhibit melanoma plasticity and improve the survival response to anti-PD-1 immunotherapy.

Abstract

Lung cancer is the leading cause of cancer mortality, but recent advances in cancer immunotherapy, including immune checkpoint inhibitors (ICIs) have revolutionized patient care and prognosis. Recent retrospective clinical data have shown that obesity is associated with enhanced response to ICIs in lung cancer patients. Given the challenges in predicting which patients will benefit from immunotherapy, the enhanced efficacy in patients with obesity raises is an intriguing observation. The objective of this project is to identify the mechanism driving enhanced response to ICIs in patients with obesity. I hypothesize that tumor-intrinsic characteristics of lung tumors in combination with a subset of infiltrating immune cells, both unique to patients with obesity, create a microenvironment conducive to enhanced ICI efficacy. We have generated a subcutaneous syngeneic model of lung adenocarcinoma in diet-induced obese and lean mice to show that the degree of response to ICIs is greater in the obese mice. I will use flow cytometry to determine immune cell dynamics associated with improved ICI response in the setting of obesity. In parallel, I am using patient-derived lung tumor organoids to determine tumor- intrinsic and tumor-extrinsic (microenvironmental) factors that mediate cancer progression and response to therapy in lean versus obese hosts. I will identify transcriptional signatures in tumors that are characteristic of obesity and identify how tumor intrinsic versus extrinsic factors affect the anti-tumor immune response. Improving our understanding of the immunological landscape of cancer and obesity will provide insight to optimize ICI responses across a broader patient population.

Abstract

Liver is the most common site for colorectal cancer metastasis. Colorectal cancer liver metastasis (CRCLM) grow in two major histolopathological growth patterns (HGP): angiogenic and co-opting. The two HGPs do not only differ in their histopathology, but also their response to treatment and patient overall survival. The biological explanation for the different treatment responses and existence of the different HGPs are unknown and under current investigation. Our study focuses on the composition of the immune cells in each HGPs. Specifically, we looked at the difference in immune cell profiles between the two HGPs to delineate both the innate and adaptive cell density. Using Nanostring GeoMx® spatial protein profiler, using their 96 protein panel on FFPE slides, we established the immune microenvironment of the lesions and performed immunohistochemistry with CD4, CD8, CD68, and elastase markers to confirm Nanostring’s data and describe the immune topography. Interestingly, in chemonaive lesions there was a significantly higher population of CD4 and CD8 T cells in the angiogenic lesion and a significantly higher population of macrophages in co- opting lesions. Previous work from the lab had already confirmed a higher population of neutrophils in co-opting lesions. Therefore, there is a pattern of more adaptive immune cells being present in angiogenic lesions and more innate immune cells in co-opting chemonaive lesions. Therefore, we hypothesize that the different immune landscape of the two HGPs correlate with the different responses to cancer treatment.

Abstract

The receptors and signaling pathways that regulate T-cell activation are central to controlling responses against tumors. We have previously documented the involvement of proximal kinases such as p56^lck and downstream immune cell adaptors such as ADAP in the T-cell activation process. More recently, we identified a new signaling pathway in T-cells that involves the binding of immune adaptor SLP-76 directly to SUMO-RanGAP1 of the nuclear pore complex (NPC) (Mol Cell 2015 59(5):840-9). We approximate that this interaction accounts for 40-50% of transcription factor entry into the nucleus and is a second checkpoint for NFATc1 nuclear entry. RanGAP1 binds to a motif in SLP-76 involving the lysine at residue 56 where its mutation ablates complex formation. In this new study, we report the generation of a K56E SLP-76 knock-in (KI) mouse which are markedly impaired in the control of tumor growth. This loss of control was accompanied by a marked reduction in the presence CD8+ effector TILs and in the production of interferon-g1 and CD8 cytolytic effector molecules, perforin and granzyme B (GZMB). Further, K56E T-cells show an impairment in metabolism. Seahorse analysis showed a >70% decrease in glycolysis and a >40% decrease in oxidative in response to TCR ligation due to this single mutation in a single adaptor protein. Further, K56E T-cells show a decrease in the expression of Glut1, other metabolic regulators and in c-Myc translocation into the nucleus of T- cells. By contrast, the expression of other mediators such as PPARα and CPT-1A, involved in fatty acid oxidation were unaffected. Our findings document a novel signaling pathway in T-cells that involves the direct regulation of the NPC complex by SLP-76 in control of immunity against tumors.