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Wnt/β-catenin signaling is active in small subpopulations of Ewing sarcoma cells, and these cells display a more metastatic phenotype, in part due to antagonism of EWS-FLI1-dependent transcriptional activity. Importantly, these β-catenin-activated Ewing sarcoma cells also alter secretion of extracellular matrix (ECM) proteins. We thus hypothesized that, in addition to cell-autonomous mechanisms, Wnt/β-catenin-active tumor cells might contribute to disease progression by altering the tumor microenvironment (TME). Analysis of transcriptomic data from primary patient biopsies and from β-catenin-active versus -nonactive tumor cells identified angiogenic switch genes as being highly and reproducibly upregulated in the context of β-catenin activation. In addition, in silico and in vitro analyses, along with chorioallantoic membrane assays, demonstrated that β-catenin-activated Ewing cells secreted factors that promote angiogenesis. In particular, activation of canonical Wnt signaling leads Ewing sarcoma cells to upregulate expression and secretion of proangiogenic ECM proteins, collectively termed the angiomatrix. Significantly, our data show that induction of the angiomatrix by Wnt-responsive tumor cells is indirect and is mediated by TGF-β. Mechanistically, Wnt/β-catenin signaling antagonizes EWS-FLI1-dependent repression of TGF-β receptor type 2, thereby sensitizing tumor cells to TGF-β ligands. Together, these findings suggest that Wnt/β-catenin-active tumor cells can contribute to Ewing sarcoma progression by promoting angiogenesis in the local TME.

Developmental transcription programs are epigenetically regulated by multi-protein complexes, including the menin- and MLL-containing trithorax (TrxG) complexes, which promote gene transcription by depositing the H3K4me3 activating mark at target gene promoters. We recently reported that in Ewing sarcoma, MLL1 (lysine methyltransferase 2A, KMT2A) and menin are overexpressed and function as oncogenes. Small molecule inhibition of the menin-MLL interaction leads to loss of menin and MLL1 protein expression, and to inhibition of growth and tumorigenicity. Here, we have investigated the mechanistic basis of menin-MLL-mediated oncogenic activity in Ewing sarcoma. Bromouridine sequencing (Bru-seq) was performed to identify changes in nascent gene transcription in Ewing sarcoma cells, following exposure to the menin-MLL interaction inhibitor MI-503. Menin-MLL inhibition resulted in early and widespread reprogramming of metabolic processes. In particular, the serine biosynthetic pathway (SSP) was the pathway most significantly affected by MI-503 treatment. Baseline expression of SSP genes and proteins (PHGDH, PSAT1, and PSPH), and metabolic flux through the SSP were confirmed to be high in Ewing sarcoma. In addition, inhibition of PHGDH resulted in reduced cell proliferation, viability, and tumor growth in vivo, revealing a key dependency of Ewing sarcoma on the SSP. Loss of function studies validated a mechanistic link between menin and the SSP. Specifically, inhibition of menin resulted in diminished expression of SSP genes, reduced H3K4me3 enrichment at the PHGDH promoter, and complete abrogation of de novo serine and glycine biosynthesis, as demonstrated by metabolic tracing studies with 13 C-labeled glucose. These data demonstrate that the SSP is highly active in Ewing sarcoma and that its oncogenic activation is maintained, at least in part, by menin-dependent epigenetic mechanisms involving trithorax complexes. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Background: Ewing sarcoma is the second most common pediatric bone cancer. Metastatic disease is almost always fatal, and there is currently no method to predict which patients are at risk for metastasis. Better therapies are needed to prevent and treat metastatic disease, which means the mechanisms that drive Ewing sarcoma metastasis must be better elucidated. It is becoming increasingly clear that interactions between tumor cells and the tumor microenvironment (TME) play an essential role in metastasis, but the specific mechanisms through which the TME contributes to Ewing sarcoma progression remain largely unknown. Our previous work has demonstrated that activation of canonical Wnt and TGF-β pathways in a subset of Ewing sarcoma cells induces changes in gene expression and protein secretion that are associated with enhanced metastatic engraftment, changes in the extracellular matrix (ECM), and increased angiogenesis. As bone, the primary site for Ewing sarcoma, is an excellent source of ligands for both pathways, we hypothesize that crosstalk between Wnt/TGF-β-activated tumor cells and the local bone microenvironment contributes to osteolysis and metastatic progression. Methods: Ewing sarcoma cells were treated with control or Wnt3a media +/- recombinant TGF-β1, and then the mRNA levels of target genes were measured by Q-RT-PCR. ELISA and Luminex assays were also performed to determine the levels of secreted proteins. Conditioned media collected from stimulated cells were used to treat osteoblast precursor cells, which were then assayed for effects on differentiation and function. Subcutaneous, femur, and vossicle transplant xenograft models were established in mice and are being validated. Results: We have demonstrated that in response to Wnt3a and TGF-β1, Ewing sarcoma cells upregulate expression of canonical Wnt targets (e.g., LEF1), ECM-associated genes (e.g., TNC, COL1A1, and MMP2), and the pro-osteolytic factor PTHrP. We have also shown that osteoblast precursor cells can be induced to differentiate in the presence of conditioned media from Ewing sarcoma cell lines. Immunohistochemical staining is being used to evaluate Wnt and TGF-β pathway activation, angiogenesis, osteolysis, and the ECM in our bone tumor xenograft models. Conclusions: Wnt3a/TGF-β1-stimulated Ewing sarcoma cells upregulate expression of genes associated with the ECM and osteolysis. We are in the process of demonstrating the functional consequences of those changes using in vitro conditioned media experiments and in vivo transplant models. Understanding the mechanisms by which these pathways contribute to Ewing sarcoma phenotypes is important for the development of new therapeutics for patients with metastatic disease. Citation Format: Kelsey Temprine, Sydney Treichel, Allegra Hawkins, Tahra Suhan, Wei Jiang, Parker Acevedo, Amy Koh, Kurt Hankenson, Laurie K. McCauley, Elizabeth R. Lawlor. Investigating the role of tumor:bone microenvironment crosstalk in Ewing sarcoma progression [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B74.

Developmental transcription programs are epigenetically regulated by multi-protein complexes, including the menin- and MLL-containing trithorax (TrxG) complexes, which promote gene transcription by depositing the H3K4me3 activating mark at target gene promoters. We recently reported that in Ewing sarcoma, MLL1 (lysine methyltransferase 2A, KMT2A) and menin are overexpressed and function as oncogenes. Small molecule inhibition of the menin-MLL interaction leads to loss of menin and MLL1 protein expression and to inhibition of growth and tumorigenicity. Here, we have investigated the mechanistic basis of menin-MLL-mediated oncogenic activity in Ewing sarcoma. Bromouridine sequencing (Bru-seq) was performed to identify changes in nascent gene transcription in Ewing sarcoma cells, following exposure to the menin-MLL interaction inhibitor MI-503. Menin-MLL inhibition resulted in early and widespread reprogramming of metabolic processes. In particular, the serine biosynthetic pathway (SSP) was the pathway most significantly affected by MI-503 treatment. Baseline expression of SSP genes and proteins (PHGDH, PSAT1, and PSPH), and metabolic flux through the SSP were confirmed to be high in Ewing sarcoma. In addition, inhibition of PHGDH resulted in reduced cell proliferation, viability and tumor growth in vivo, revealing a key dependency of Ewing sarcoma on the SSP. Loss of function studies validated a mechanistic link between menin and the SSP. Specifically, inhibition of menin resulted in diminished expression of SSP genes, reduced H3K4me3 enrichment at the PHGDH promoter, and complete abrogation of de novo serine and glycine biosynthesis, as demonstrated by metabolic tracing studies with (13)C-labeled glucose. These data demonstrate that the SSP is highly active in Ewing sarcoma and that its oncogenic activation is maintained, at least in part, by menin-dependent epigenetic mechanisms involving trithorax complexes.

Local and metastatic progression of solid tumors depends on crosstalk between tumor cells and the tumor microenvironment (TME), including both stromal cells and the extracellular matrix (ECM). We recently showed that high Wnt/beta-catenin activity in Ewing sarcoma correlates with diminished patient survival and that canonical Wnt signaling alters the tumor secretome, influencing ECM protein composition. In light of this, we investigated the hypothesis that Wnt/beta-catenin supports tumor progression by modulating tumor: TME crosstalk. Our results reveal that, in discrete tumor cell sub-populations, beta-catenin activation sensitizes cells to TGF-beta ligands through derepression of the TGF-beta receptor, TGFBR2, resulting in canonical Wnt-induced, TGF-beta-dependent upregulation of TGF-beta targets. Significantly, these Wnt/TGF-beta responsive targets include multiple AngioMatrix genes that are known to alter the TME to promote angiogenesis, including tenascin-C and collagens. Studies of Ewing sarcoma models, in vitro and in vivo, as well as in two independent patient cohorts, confirm that a direct relationship exists between beta-catenin and TGF-beta activation in tumor cells and angiogenesis in the local TME. Mechanistically, this is due, in part, to tenascin C-mediated promotion of endothelial cell proliferation. Thus, functional cooperation between canonical Wnt and TGF-beta signaling in Ewing cells induces secretion of pro-angiogenic factors. This study reveals a novel link between Wnt and TGF-beta signaling in Ewing sarcoma and illustrates the critical contribution of tumor cell heterogeneity and tumor: TME crosstalk to sarcoma progression. Citation Format: Allegra G. Hawkins, Elisabeth A. Pedersen, Wei Jiang, Sydney Treichel, Colin Sperring, Jay Read, Brian Magnuson, Rajiv M. Patel, Dafydd Thomas, Rashmi Chugh, Elizabeth R. Lawlor. Cooperation between canonical Wnt and TGF-beta pathways promotes sarcoma angiogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3645.