The Role of Egfl6 in Tumor Immunity - Ovarian cancer (OvCa) has an extremely high mortality rate, indicating a clear need for new therapeutic approaches. One such approach is immune checkpoint inhibitor (ICI) therapy. Unfortunately, despite the presence of anti-tumor effector cells in many OvCa patients, ICI therapies have poor response rates in OvCa. One reason for this may large numbers of immunosuppressive myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) present in the OvCa. Both MDSC and TAM negatively regulate immunity in cancer and therapeutic approaches which can disrupt the recruitment/function of these cells can enhance ICI response. A potential therapeutic target to disrupt MDSC/TAM is Egfl6. Egfl6 is a developmental growth factor which is known to regulate cellular migration, proliferation, and differentiation and is highly expressed in OvCa. We found that mice which overexpress Egfl6 have increased numbers of granulocytes and monocytes in both the bone marrow (BM) and spleen. Egfl6 treatment of BM myeloid cells was associated with an integrin dependent increase in p-Syk, and differentiation to IL-10+ and S100A9+ granulocytic MDSC. Murine tumors which overexpress Egfl6, compared to controls, (i) have a significant increase in MDSC accumulation and M2- like TAM, (iii) express CXCL2 and CLEC5a, factors associated with neutrophil extracellular traps (NETs) formation, and (iii) are refractory to ICI therapy. We thus hypothesize that Egfl6, via Syk activation, promotes an immunosuppressive TME by increasing numbers of MDSC/TAM and their production of NETs in tumors. As an extension of this, we hypothesize that inhibition of Egfl6, will enhance the efficacy of ICI therapy. To test our hypotheses, we propose: SA1. To evaluate the requirement for Syk in Egfl6 signaling on myeloid cells. As Syk is required for integrin signaling in granulocytes, we hypothesize that targeted disruption of Syk will prevent Egfl6- dependent granulocyte accumulation. Using specific Syk inhibitor compounds and Syk-KO granulocytes/monocytes, we will dissect the Egfl6-Syk regulatory axis. SA2. To analyze the role of Egfl6 in NETs formation and resistance to ICI therapy. We found Egfl6 expressing tumors have increased levels and NETosis and that Egfl6 induces CXCL2 and CLEC5a, factors associated with NETosis. This occurs both in the presence and absence of ICI therapy. We hypothesize that Egfl6, by induction of NETs, limits the anti-tumor immunity promoting resistance to ICI therapy. We will use CXCL2 and CLEC5a mediated inhibition/KO approaches to define the mechanisms by which Egfl6 induces NETosis and the impact of Egfl6 induced NETosis on ICI cytotoxic immunity. SA 3. Evaluate the impact of Egfl6 neutralizing antibody on the efficacy of ICI therapy and anti-tumor immunity. We will use an immune competent ICI responsive murine OvCa model to evaluate the ability of an Egfl6 neutralizing antibody to overcome Egfl6 mediated immune suppression. Combined these studies will characterize a novel mechanism of OvCa mediated tumor immune suppression and validate EGFL6 antibody as a novel therapeutic approach to enhance ICI therapy in OvCa patients.
