Ewing's sarcoma (EwS) is a pediatric solid tumor entity with low somatic mutational burden and a low rate of tumor-infiltrating T cells, indicating a low extent of immunogenicity. In EwS, immunogenicity may furthermore be significantly diminished by a predominantly M2 macrophage driven pro-tumorigenic tumor microenvironment. In the past, we demonstrated that CHM1319-specific TCR-transgenic T cells are able to control EwS growth in a preclinical mouse model as well as in a patient with metastatic disease. However, new adjuvant techniques to induce long lasting and curative CHM1319-specific TCR-transgenic T cell-mediated anti-tumor responses are needed. In this work, we sought to identify a technique to improve the cytotoxic effect of CHM1319-specific TCR-transgenic T cell by altering the immunogenic cell surface marker expression on EwS cell lines using different cytokines. We demonstrate that TNF, IL-6, IL-1β and PGE2 cause pro-immunogenic CD83, MHC class I and II as well as ICAM-1 upregulation in EwS cell lines. This observation was associated with significantly improved recognition and killing of the tumor cells by EwS-specific CHM1319/HLA-A*02:01-restricted TCR-transgenic T cells. Conclusively, we demonstrate that the induction of an inflammatory signature renders EwS more susceptible to adoptive T cell therapy. TNF, which is upregulated during inflammatory processes, is of particular translational interest as its secretion may be induced in the patients e.g., by irradiation and hyperthermia in the clinical setting. In future clinical protocols, this finding may be important to identify appropriate conditioning regimens as well as point of time for adoptive T cell-based immunotherapy in EwS patients.
Previously, we used inhibitors blocking BET bromodomain binding proteins (BRDs) in Ewing sarcoma (EwS) and observed that long term treatment resulted in the development of resistance. Here, we analyze the possible interaction of BRD4 with cyclin-dependent kinase (CDK) 9.Co-immunoprecipitation experiments (CoIP) to characterize BRD4 interaction and functional consequences of inhibiting transcriptional elongation were assessed using drugs targeting of BRD4 or CDK9, either alone or in combination.CoIP revealed an interaction of BRD4 with EWS-FLI1 and CDK9 in EwS. Treatment of EwS cells with CDKI-73, a specific CDK9 inhibitor (CDK9i), induced a rapid downregulation of EWS-FLI1 expression and block of contact-dependent growth. CDKI-73 induced apoptosis in EwS, as depicted by cleavage of Caspase 7 (CASP7), PARP and increased CASP3 activity, similar to JQ1. Microarray analysis following CDKI-73 treatment uncovered a transcriptional program that was only partially comparable to BRD inhibition. Strikingly, combined treatment of EwS with BRD- and CDK9-inhibitors re-sensitized cells, and was overall more effective than individual drugs not only in vitro but also in a preclinical mouse model in vivo.Treatment with BRD inhibitors in combination with CDK9i offers a new treatment option that significantly blocks the pathognomonic EWS-ETS transcriptional program and malignant phenotype of EwS.
Ewing sarcoma (EwS) is an aggressive pediatric cancer of bone and soft tissues characterized by scant T cell infiltration and predominance of immunosuppressive myeloid cells. Given the important roles of extracellular vesicles (EVs) in cancer-host crosstalk, we hypothesized that EVs secreted by EwS tumors target myeloid cells and promote immunosuppressive phenotypes. Here, EVs were purified from EwS and fibroblast cell lines and exhibited characteristics of small EVs, including size (100-170 nm) and exosome markers CD63, CD81, and TSG101. Treatment of healthy donor-derived CD33+ and CD14+ myeloid cells with EwS EVs but not with fibroblast EVs induced pro-inflammatory cytokine release, including IL-6, IL-8, and TNF. Furthermore, EwS EVs impaired differentiation of these cells towards monocytic-derived dendritic cells (moDCs), as evidenced by reduced expression of co-stimulatory molecules CD80, CD86 and HLA-DR. Whole transcriptome analysis revealed activation of gene expression programs associated with immunosuppressive phenotypes and pro-inflammatory responses. Functionally, moDCs differentiated in the presence of EwS EVs inhibited CD4+ and CD8+ T cell proliferation as well as IFNγ release, while inducing secretion of IL-10 and IL-6. Therefore, EwS EVs may promote a local and systemic pro-inflammatory environment and weaken adaptive immunity by impairing the differentiation and function of antigen-presenting cells.
<p>Supplementary Figures S1-6. Supplementary Fig S1: Combined patient characteristics and subjected methods Supplementary Fig. S2 A: ROC curves were constructed by using the somatic scores from the sequenced genotypes. Supplementary Fig. S3: Circos plots of representative Ewing sarcomas. Supplementary Fig. S4: Sanger sequencing results confirmed (A) somatic inactivating mutations of the STAG2 gene (positions according to RefSeq NG 033796.2) and (B) the activating N546 mutation of FGFR1. Supplementary Fig. S5: FGFR-1 expression in Ewing Sarcomas by means of real time RT PCR (A, normalized to FGFR-1 expression in VH-64). (B) Array-based analysis of FGFR1 expression in ES compared to normal tissue (NBA; SI Materials and Methods). (C) Knock-down by means of lentiviral transduced FGFR1.shRNA effectively reduced FGFR1 mRNA expression by 70 % or more in Ewing sarcoma cell lines as compared to scrambled shRNA. (D) Knockdown was confirmed on the protein level by means of western blotting. Supplementary Fig. S6: Amplification of the FGFR-1 gene (A) as well as gene expression (B) are associated with a trend towards inferior survival in patients with ES. (C) Treatment with Ponatinib inhibited growth of ES cell lines significantly. (D) FGFR1 tyrosine kinase inhibitor therapy in a patient with relapsed Ewing sarcoma.</p>
Introduction Pediatric sarcomas, including osteosarcoma (OS), Ewing sarcoma (EwS) and rhabdomyosarcoma (RMS) carry low somatic mutational burden and low MHC-I expression, posing a challenge for T cell therapies. Our previous study showed that mediators of monocyte maturation sensitized the EwS cell line A673 to lysis by HLA-A*02:01/CHM1 319 -specific allorestricted T cell receptor (TCR) transgenic CD8 + T cells (CHM1 319 CD8 + T cells). Methods In this study, we tested a panel of monocyte maturation cytokines for their ability to upregulate immunogenic cell surface markers on OS, EwS and RMS cell lines, using flow cytometry. xCELLigence, SRB and ELISpot assays were used to assess whether TNF pretreatment increases CD8 + T cell cytotoxicity. Results We observed that TNF and IL-1β upregulated MHC class I, ICAM-1 as well as CD83 and PD-L1 on the surface of pediatric sarcoma cell lines, while IL-4, GM-CSF, IL-6 and PGE 2 failed to induce respective effects. Although pretreatment of pediatric sarcoma cell lines with TNF did not improve unspecific peripheral blood mononuclear cells (PBMCs) cytotoxicity, TNF enhanced specific lysis of 1/3 HLA-A2 + EwS cell lines by CHM1 319 CD8 + T cells depending on MHC-I expression and ICAM-1 upregulation. Discussion Our study supports utilization of TNF or TNF-inducing regimens for upregulation of MHC-I and costimulatory surface molecules on pediatric sarcoma cells and for enhancing recognition of responsive HLA-A2 + EwS tumor cells by antigen-specific CD8 + T cells.
Abstract Ewing sarcomas (ES) are highly malignant, osteolytic bone or soft tissue tumors, which are characterized by ews/ets translocations and early metastasis into lung and bone. In this study, we analyzed the role of the BRICHOS domain-containing genes CHM1 and ITM2A in ES pathogenesis, especially in bone-associated tumor growth and metastasis. Both genes are overexpressed in ES and among others appear to be important for chondrogenic differentiation. CHM1 in addition seems directly up-regulated by EWS/FLI1. Using RNA interference, we demonstrate that CHM1 and ITM2A slightly reduced bone invasiveness and osteolysis in vivo that is likely mediated by the suppression of the osteolytic genes HIF1α, IL6, JAG1 and VEGF. Furthermore, CHM1 and ITM2A suppressed the chondrogenic and to a lesser extend the osteogenic differentiation status of ES cells indicating that osteomimicry might play a role in homing, colonizing and invasion into bone tissues. Additionally, we show that both BRICHOS domain-containing genes significantly increased the expression of the stem cell genes, ABCG2 and PROM1. Together with the observed undifferentiated phenotype of ES cells, we demonstrate that CHM1 and ITM2A clearly enhanced lung metastasis in a xenograft mouse model in vivo. Our results suggest that CHM1 and ITM2A are essential players with respect to the undifferentiated phenotype and the metastatic behavior of ES cells. Citation Format: Kristina von Heyking, Annette Fasan, Stefan Burdach, Günther H. Richter. BRICHOS genes CHM1 and ITM2A maintain an undifferentiated, invasive phenotype in Ewing sarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3973. doi:10.1158/1538-7445.AM2014-3973
