Immunotherapy has revolutionized outcomes for cancer patients, but the mechanisms of resistance remain poorly defined. We used a series of whole-genome clustered regularly interspaced short palindromic repeat (CRISPR)-based screens performed in vitro and in vivo to identify mechanisms of tumor immune evasion from cytotoxic lymphocytes [CD8 T cells and natural killer (NK) cells]. Deletion of key genes within the tumor necrosis factor (TNF) signaling, interferon-γ (IFN-γ) signaling, and antigen presentation pathways provided protection of tumor cells from CD8 T cell-mediated killing and blunted antitumor immune responses in vivo. Deletion of a number of genes in the TNF pathway also emerged as the key mechanism of immune evasion from primary NK cells. Our screens also identified that the metabolic protein 2-aminoethanethiol dioxygenase (Ado) modulates sensitivity to TNF-mediated killing by cytotoxic lymphocytes and is required for optimal control of tumors in vivo. Remarkably, we found that tumors delete the same genes when exposed to perforin-deficient CD8 T cells, demonstrating that the dominant immune evasion strategy used by tumor cells is acquired resistance to T cell-derived cytokine-mediated antitumor effects. We demonstrate that TNF-mediated bystander killing is a potent T cell effector mechanism capable of killing antigen-negative tumor cells. In addition to highlighting the importance of TNF in CD8 T cell- and NK cell-mediated killing of tumor cells, our study also provides a comprehensive picture of the roles of the TNF, IFN, and antigen presentation pathways in immune-mediated tumor surveillance.

The BRAF mutant, BRAF V600E , is expressed in nearly half of melanomas, and oral BRAF inhibitors induce substantial tumor regression in patients with BRAF V600E metastatic melanoma. The inhibitors are believed to work primarily by inhibiting BRAF V600E -induced oncogenic MAPK signaling; however, some patients treated with BRAF inhibitors exhibit increased tumor immune infiltration, suggesting that a combination of BRAF inhibitors and immunotherapy may be beneficial. We used two relatively resistant variants of Braf V600E -driven mouse melanoma (SM1 and SM1WT1) and melanoma-prone mice to determine the role of host immunity in type I BRAF inhibitor PLX4720 antitumor activity. We found that PLX4720 treatment downregulated tumor Ccl2 gene expression and decreased tumor CCL2 expression in both Braf V600E mouse melanoma transplants and in de novo melanomas in a manner that was coincident with reduced tumor growth. While PLX4720 did not directly increase tumor immunogenicity, analysis of SM1 tumor-infiltrating leukocytes in PLX4720-treated mice demonstrated a robust increase in CD8 + T/FoxP3 + CD4 + T cell ratio and NK cells. Combination therapy with PLX4720 and anti-CCL2 or agonistic anti-CD137 antibodies demonstrated significant antitumor activity in mouse transplant and de novo tumorigenesis models. These data elucidate a role for host CCR2 in the mechanism of action of type I BRAF inhibitors and support the therapeutic potential of combining BRAF inhibitors with immunotherapy.

The nuclear receptor peroxisome proliferator-activated receptor ␦ [PPAR␦/␤ (NR1C2)] has been implicated in colorectal carcinogenesis by various molecular genetic observations. These observations have recently been supported by studies of activation of PPAR␦ by pharmacological agents. Here we present the first report of the stimulation of breast and prostate cancer cell growth using PPAR␦ selective agonists. Activation of PPAR␦ with compound F stimulated proliferation in breast (T47D, MCF7) and prostate (LNCaP, PNT1A) cell lines, which are responsive to sex hormones. Conversely, we have found that several steroid-independent cell lines, including colon lines, were unresponsive to compound F. These findings were confirmed with an additional high-affinity PPAR␦ agonist, GW501516. Conditional expression of PPAR␦ in MCF7 Tet-On cells resulted in a doxycycline-enhanced response to GW501516, thus providing direct genetic evidence for the role of PPAR␦ in the proliferative response to this drug. Activation of PPAR␦ in T47D cells resulted in increased expression of the proliferation marker Cdk2 and also vascular endothelial growth factor ␣ (VEGF␣) and its receptor, FLT-1, thus, suggesting that PPAR␦ may initiate an autocrine loop for cellular proliferation and possibly angiogenesis. Consistent with this hypothesis, we demonstrated a pro-proliferative effect of GW501516 on human umbilical vein endothelial cell cultures and found that GW501516 also regulated the expression of VEGF␣ and FLT-1 in these cells. Our observations provide the first evidence that activation of PPAR␦ can result in increased growth in breast and prostate cancer cell lines and primary endothelial cells and supports the possibility that PPAR␦ antagonists may be of therapeutic value in the treatment of breast and prostate cancer.