Immunocompetent murine model of Ewing sarcoma reveals role for TGFß inhibition to enhance immune infiltrates in Ewing tumors during radiation.

Ewing sarcoma (ES) is an aggressive cancer diagnosed in adolescents and young adults. The fusion oncoprotein (EWSR1::FLI1) that drives Ewing sarcoma is known to downregulate TGFBR2 expression (part of the TGFß receptor). Because TGFBR2 is downregulated, it was thought that TGFß likely plays an inconsequential role in Ewing biology. However, the expression of TGFß in the Ewing tumor immune microenvironment (TIME) and functional impact of TGFß in the TIME remains largely unknown given the historical lack of immunocompetent preclinical models. Here, we use single-cell RNAseq analysis of human Ewing tumors to show that immune cells, such as NK cells, are the largest source of TGFß production in human Ewing tumors. We develop a humanized (immunocompetent) mouse model of ES and demonstrate distinct TME signatures and metastatic potential in these models as compared to tumors developed in immunodeficient mice. Using this humanized model, we study the effect of TGFß inhibition on the Ewing TME during radiation therapy, a treatment that both enhances TGFß activation and is used to treat aggressive ES. Utilizing a trivalent ligand TGFß TRAP to inhibit TGFß, we demonstrate that in combination with radiation, TGFß inhibition both increases ES immune cell infiltration and decreases lung metastatic burden in vivo . The culmination of these data demonstrates the value of humanized models to address immunobiologic preclinical questions in Ewing sarcoma and suggests TGFß inhibition as a promising intervention during radiation therapy to promote metastatic tumor control.

Grainyhead-like-2, an epithelial master programmer, promotes interferon induction and suppresses breast cancer recurrence.

Type-I and -III interferons play a central role in immune rejection of pathogens and tumors, thus promoting immunogenicity and suppressing tumor recurrence. Double strand RNA is an important ligand that stimulates tumor immunity via interferon responses. Differentiation of embryonic stem cells to pluripotent epithelial cells activates the interferon response during development, raising the question of whether epithelial vs. mesenchymal gene signatures in cancer potentially regulate the interferon pathway as well. Here, using genomics and signaling approaches, we show that Grainyhead-like-2 (GRHL2), a master programmer of epithelial cell identity, promotes type-I and -III interferon responses to double-strand RNA. GRHL2 enhanced the activation of IRF3 and relA/NF-kB and the expression of IRF1; a functional GRHL2 binding site in the IFNL1 promoter was also identified. Moreover, time to recurrence in breast cancer correlated positively with GRHL2 protein expression, indicating that GRHL2 is a tumor recurrence suppressor, consistent with its enhancement of interferon responses. These observations demonstrate that epithelial cell identity supports interferon responses in the context of cancer.

Expression of lymphoid structure-associated cytokine/chemokine gene transcripts in tumor and protein in serum are prognostic of melanoma patient outcomes.

Background: Proinflammatory chemokines/cytokines support development and maturation of tertiary lymphoid structures (TLS) within the tumor microenvironment (TME). In the current study, we sought to investigate the prognostic value of TLS-associated chemokines/cytokines (TLS-kines) expression levels in melanoma patients by performing serum protein and tissue transcriptomic analyses, and to then correlate these data with patients clinicopathological and TME characteristics. Methods: Levels of TLS-kines in patients' sera were quantitated using a custom Luminex Multiplex Assay. The Cancer Genomic Atlas melanoma cohort (TCGA-SKCM) and a Moffitt Melanoma cohort were used for tissue transcriptomic analyses. Associations between target analytes and survival outcomes, clinicopathological variables, and correlations between TLS-kines were statistically analyzed. Results: Serum of 95 patients with melanoma were evaluated; 48 (50%) female, median age of 63, IQR 51-70 years. Serum levels of APRIL/TNFSF13 were positively correlated with levels of both CXCL10 and CXCL13. In multivariate analyses, high levels of serum APRIL/TNFSF13 were associated with improved event-free survival after adjusting for age and stage (HR = 0.64, 95% CI 0.43-0.95; p = 0.03). High expression of APRIL/TNFSF13 tumor transcripts was significantly associated with improved OS in TCGA-SKCM (HR = 0.69, 95% CI 0.52-0.93; p = 0.01) and in Moffitt Melanoma patients (HR = 0.51, 95% CI: 0.32-0.82; p = 0.006). Further incorporation of CXCL13 and CXCL10 tumor transcript levels in a 3-gene index revealed that high APRIL/CXCL10/CXCL13 expression was associated with improved OS in the TCGA SKCM cohort (HR = 0.42, 95% CI 0.19-0.94; p = 0.035). Melanoma differentially expressed genes positively associated with high APRIL/CXCL10/CXCL13 tumor expression were linked to tumor infiltration by a diverse array of proinflammatory immune cell types. Conclusion: Serum protein and tumor transcript levels of APRIL/TNFSF13 are associated with improved survival outcomes. Patients exhibiting high coordinate expression of APRIL/CXCL10/CXCL13 transcripts in their tumors displayed superior OS. Further investigation of TLS-kine expression profiles related to clinical outcomes in larger cohort studies is warranted.

Immune checkpoint blockade in triple negative breast cancer influenced by B cells through myeloid-derived suppressor cells.

Triple negative breast cancer holds a dismal clinical outcome and as such, patients routinely undergo aggressive, highly toxic treatment regimens. Clinical trials for TNBC employing immune checkpoint blockade in combination with chemotherapy show modest prognostic benefit, but the percentage of patients that respond to treatment is low, and patients often succumb to relapsed disease. Here, we show that a combination immunotherapy platform utilizing low dose chemotherapy (FEC) combined with oncolytic virotherapy (oHSV-1) increases tumor-infiltrating lymphocytes, in otherwise immune-bare tumors, allowing 60% of mice to achieve durable tumor regression when treated with immune checkpoint blockade. Whole-tumor RNA sequencing of mice treated with FEC + oHSV-1 shows an upregulation of B cell receptor signaling pathways and depletion of B cells prior to the start of treatment in mice results in complete loss of therapeutic efficacy and expansion of myeloid-derived suppressor cells. Additionally, RNA sequencing data shows that FEC + oHSV-1 suppresses genes associated with myeloid-derived suppressor cells, a key population of cells that drive immune escape and mediate therapeutic resistance. These findings highlight the importance of tumor-infiltrating B cells as drivers of antitumor immunity and their potential role in the regulation of myeloid-derived suppressor cells.

Type I interferons and related pathways in cell senescence.

This review article addresses the largely unanticipated convergence of two landmark discoveries. The first is the discovery of interferons, critical signaling molecules for all aspects of both innate and adaptive immunity, discovered originally by Isaacs and Lindenmann at the National Institute for Medical Research, London, in 1957 (Proceedings of the Royal Society of London. Series B: Biological Sciences, 1957, 147, 258). The second, formerly unrelated discovery, by Leonard Hayflick and Paul Moorhead (Wistar Institute, Philadelphia) is that cultured cells undergo an irreversible but viable growth arrest, termed senescence, after a finite and predictable number of cell divisions (Experimental Cell Research, 1961, 25, 585). This phenomenon was suspected to relate to organismal aging, which was confirmed subsequently (Nature, 2011, 479, 232). Cell senescence has broad-ranging implications for normal homeostasis, including immunity, and for diverse disease states, including cancer progression and response to therapy (Nature Medicine, 2015, 21, 1424; Cell, 2019, 179, 813; Cell, 2017, 169, 1000; Trends in Cell Biology, 2018, 28, 436; Journal of Cell Biology, 2018, 217, 65). Here, we critically address the bidirectional interplay between interferons (focusing on type I) and cell senescence, with important implications for health and healthspan.

Grainyhead-like-2 confers NK-sensitivity through interactions with epigenetic modifiers.

Natural Killer (NK) cells suppress tumor initiation and metastasis. Most carcinomas are heterogeneous mixtures of epithelial, mesenchymal and hybrid tumor cells, but the relationships of these phenotypes to NK susceptibility are understood incompletely. Grainyhead-like-2 (GRHL2) is a master programmer of the epithelial phenotype, that is obligatorily down-regulated during experimentally induced Epithelial-Mesenchymal Transition (EMT). Here, we utilize GRHL2 re-expression to discover unifying molecular mechanisms that link the epithelial phenotype with NK-sensitivity. GRHL2 enhanced the expression of ICAM-1, augmenting NK-target cell synaptogenesis and NK killing of target cells. The expression of multiple interferon response genes, including ICAM1, anti-correlated with EMT. We identified two novel GRHL2-interacting proteins, the histone methyltransferases KMT2C and KMT2D. Mesenchymal-epithelial transition, NK-sensitization and ICAM-1 expression were promoted by GRHL2-KMT2C/D interactions and by GRHL2 inhibition of p300, revealing novel and potentially targetable epigenetic mechanisms connecting the epithelial phenotype with target cell susceptibility to NK killing.