Targeting the ubiquitin ligase Siah2, central in cell autonomous proliferation and activity of Tregs, may thus offer the rationale for an innovative therapeutic approach. Methods Animals and tumor models All Eptifibatide experimental animal procedures were approved by the Institutional Animal Care and Use Committee of Sanford Burnham Prebys Medical Discovery Institute (approval # 16C070, 17C043) and complied with all relevant ethical regulations for animal testing and research. mice subjected to PD-1 blockade, indicating synergy between PD-1 blockade and loss. Low and expression is identified in immune responsive human melanoma tumors. Overall, Siah2 regulation of Treg recruitment and cell cycle progression effectively controls melanoma development and Siah2 loss in the host sensitizes melanoma to anti-PD-1 therapy. mice To evaluate Siah2 function in the tumor environment, we injected cells of the BRAF-mutant melanoma line YUMMER1.7 into syngeneic wild-type (WT) or mice. The YUMMER1.7 line carries a high somatic mutation burden and is more immunogenic than the parental YUMM1.7 line31,32. Growth of YUMMER1.7 cells was largely attenuated in relative to WT mice, (Fig.?1a), with no obvious changes in gross tumor morphology or melanoma marker expression (Supplementary Fig.?1a, b). Notably, 6 of 14 tumors (42%) grown in Eptifibatide mice exhibited complete regression as compared with 2/14 (14%) tumors in WT mice (Fig.?1a). While melanoma development in the first few days following tumor cell inoculation was similar in both the WT and mice, within 10C14 days tumors began to regress in the mice, while they continued growing in the WT genotype. Increasing the number of tumor cells inoculated (from 4??105 to 1 1??106) abrogated the tumor rejection phenotype in mice (Supplementary Fig.?1c), suggesting that tumor burden is a critical determinant of effective Siah2-dependent immune cell function. Open in a separate window Fig. 1 Siah2-deficient mice limits melanoma growth.a YUMMER1.7 melanoma cells (400,000) were injected s.c. into the flank of 5C7-weeks-old WT or male mice, and mean (lower panel) and individual (upper panel) tumor growth (volume) was measured over time (mice?(= 5 for both genotypes). Heat map shows the most upregulated and downregulated pathways in mice based on Eptifibatide comparisons Rabbit Polyclonal to Collagen V alpha1 of YUMMER1.7 tumors (tumors. Analysis was performed 10 days after tumor injection. Cutoff applied: versus WT tumors. Cutoff is color coded: green?=?mice that may contribute to tumor growth inhibition, we performed RNA sequencing (RNAseq) on both WT or tumors. An enhanced inflammatory gene signature was identified in tumors harvested from relative to WT mice, a signature characterized by upregulation of genes implicated in the Th1 pathway and NOS2 signaling (Supplementary Fig.?1d). To further map the effect of Siah2 on immune signaling, we performed PanCancer Immune Profiling using the NanoString technology. Common to both RNAseq and NanoString analyses were increased expression of genes that function in immune cell inflammatory and effector phenotypes (among them, and mice. was among the most upregulated genes in mice, while levels is consistent with improved anti-tumor immunity and attenuated tumor growth. Accordingly, both RNAseq and NanoString analyses revealed significantly reduced expression of mice, a decrease confirmed by quantitative PCR (qPCR) analysis (Fig.?1d). Overall, these findings reveal an increased inflammatory and activated immune phenotype in the tumor immune environment, concomitant with reduced Treg infiltration. Increased T effector cells and fewer Tregs in mice Eptifibatide grown tumors We next compared the type and quantity of infiltrating immune cells in tumors grown in and WT littermates. Flow cytometry analysis performed on tumors collected 11 days after melanoma cell inoculation, a time point when tumors begin to shrink in mice (Supplementary Fig.?2a) revealed a comparable number (Fig.?2a) or proportion (Fig.?2b) of CD45.2+, CD4+, CD8+, CD11b+ F4/80+, CD11c+, and CD11b+GR1+ cells in both genotypes (Fig.?2a, b, Supplementary Fig.?2b). However, a 3-fold increase in the T-bet+ cell population and a 2-fold decrease in FOXP3+CD25+ cells within the CD4+ population was seen in tumors grown in mice as compared Eptifibatide to WT mice (Fig.?2c, d), while WT and tumors showed comparable expression of FOXP3 within the Treg cell population (Supplementary Fig.?2c). These findings suggest that reduced infiltration of Treg cells is accompanied by.