The Role of Annexin A1 Expression in Glioblastoma-Associated Astrocytes

About This Grant

Astrocytes profoundly influence immunity, but they are negligibly characterized in glioblastoma (GBM). My preliminary data suggest that GBM-associated astrocytes expressing annexin A1 (ANXA1) contribute to immunosuppression in GBM: 1) ANXA1 expression in non-malignant astrocytes is correlated with early GBM recurrence and inversely correlated with survival; 2) The ANXA1 receptor formyl peptide receptor 1 (FPR1) is expressed in GBM cells and inversely correlated with survival; 3) In molecular barcoding RABID-seq studies, I detected increased ANXA1 in astrocytes interacting with GBM cells; 4) Fpr1 inactivation in mouse glioma cells leads to extended survival, increased tumor-reactive T-cell responses, increased NF-kB and inflammasome signaling in astrocytes, and increased necroptosis signaling in tumor cells; 5) Inactivation of Anxa1 in astrocytes extends survival of glioma-bearing mice; 6) Human GBM cells suppress inflammasome activation in cultured human astrocytes in an FPR1-dependent manner; and 7) Tumor associated macrophages (TAMs) interacting with ANXA1+ astrocytes express immunosuppressed gene pathways. Thus, I hypothesize that ANXA1+ astrocytes promote immunosuppression in GBM and represent a novel therapeutic target. This revised CDA-2 consists of 3 aims probing the immunosuppressive mechanisms of ANXA1+ astrocytes, and a 4-year training plan closing gaps in my skillset: (1) quantification of immunofluorescence microscopy; (2) multivariate survival analysis; and (3) analysis of single-cell RNA-seq data, including the technique, RABID-seq. SPECIFIC AIM 1: Establish the effects of ANXA1 on astrocytes and glioma-reactive T-cells. My data suggest that ANXA1 suppresses immunostimulatory NF-kB and inflammasome signaling in astrocytes, leading to glioma-specific T-cell dysfunction. Thus, I will inactivate Anxa1 in astrocytes in a GBM mouse model and: A) quantify astrocyte NF-kB and inflammasome activity; and B) measure T-cell dysfunction. SPECIFIC AIM 2: Do ANXA1+ astrocytes influence survival in murine and human high-grade glioma? Disruption of ANXA1-FPR1 signaling increases tumor-specific T-cell responses in mouse GBM models and increases necroptosis (immunogenic cell death) in glioma cells, suggesting that ANXA1 blockade could render GBM responsive to as-of-yet ineffective T-cell directed therapies. I will measure survival of glioma-bearing mice with astrocyte-specific Anxa1 inactivation: A) in combination with anti-PD1 immunotherapy; and B) in mice bearing gliomas with inactivation of the necroptosis executioner, Mlkl. Third, C) I will define the link between astrocyte ANXA1 expression and clinical outcomes in Veteran GBM patients. SPECIFIC AIM 3: Elucidate the role of ANXA1+ astrocyte interactions with GBM-associated macrophages. Astrocytes promote or inhibit T-cell responses, depending on the context, via interactions with macrophages. TAMs interacting with ANXA1+ astrocytes express immunosuppressive gene programs. Thus, here I will perform cell barcoding RABID-seq on GBM tissue from 4 surgeries at VA Boston and: A) detect mediators of interactions between ANXA1+ astrocytes and TAMs expressing oncostatin M, which reside in niches relatively abundant in T-cells in GBM. I will validate clinical relevance these mediators of ANXA1+ (astrocyte)—TAM crosstalk by B) multivariate survival analysis from The Cancer Genome Atlas; C) immunofluorescence of GBM autopsies from 2 VA sites; and D) survival analysis of glioma-bearing mice following astrocyte-specific gene inactivation. IN SUMMARY: I will investigate mechanisms of immunosuppression in ANXA1+ astrocytes with the long-term objective of identifying novel therapeutic targets to sensitize GBM to immunotherapies. I am a board-certified neurologist and neuro-oncologist with PhD training in brain tumor cell vaccines and neuroscience. The project will be completed at the VA Medical Center, Boston, complement my clinical practice in neuro-oncology, and enable me to establish an independent research program at VAMC focused on cell crosstalk in GBM.