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.

Harnessing immunomodulation during DNA damage in Ewing sarcoma.

Ewing sarcoma is a fusion-oncoprotein-driven primary bone tumor most commonly diagnosed in adolescents. Given the continued poor outcomes for patients with metastatic and relapsed Ewing sarcoma, testing innovative therapeutic approaches is essential. Ewing sarcoma has been categorized as a 'BRCAness' tumor with emerging data characterizing a spectrum of DNA damage repair defects within individual Ewing tumors, including the presence of EWSR1::FLI1 itself, recurrent somatic mutations, and rare germline-based defects. It is critical to understand the cumulative impact of various DNA damage repair defects on an individual Ewing tumor's response to therapy. Further, in addition to DNA-damage-directed therapies, subsets of Ewing tumors may be more susceptible to DNA-damage/immunotherapy combinations given the significant cross-talk between DNA damage and inflammatory pathways in the tumor microenvironment. Here we review potential approaches utilizing DNA-damaging agents as modulators of the Ewing tumor immune microenvironment, with a focus on radiation and opportunities during disease metastasis and relapse.

BRCA1-associated RING domain-1 (BARD1) loss and GBP1 expression enhance sensitivity to DNA damage in Ewing sarcoma.

Ewing sarcoma is a fusion oncoprotein-driven primary bone tumor. A subset of patients (~10%) with Ewing sarcoma are known to harbor germline variants in a growing number of genes involved in DNA damage repair. We recently reported our discovery of a germline mutation in the DNA damage repair protein BARD1 (BRCA1-associated RING domain-1) in a patient with Ewing sarcoma. BARD1 is recruited to the site of DNA double stranded breaks via the poly(ADP-ribose) polymerase (PARP) protein and plays a critical role in DNA damage response pathways including homologous recombination. We thus questioned the impact of BARD1 loss on Ewing cell sensitivity to DNA damage and the Ewing sarcoma transcriptome. We demonstrate that PSaRC318 cells, a novel patient-derived cell line harboring a pathogenic BARD1 variant, are sensitive to PARP inhibition and by testing the effect of BARD1 depletion in additional Ewing sarcoma cell lines, we confirm that BARD1 loss enhances cell sensitivity to PARP inhibition plus radiation. Additionally, RNA-seq analysis revealed that loss of BARD1 results in the upregulation of GBP1 (guanylate-binding protein 1), a protein whose expression is associated with variable response to therapy depending on the adult carcinoma subtype examined. Here, we demonstrate that GBP1 contributes to the enhanced sensitivity of BARD1 deficient Ewing cells to DNA damage. Together, our findings demonstrate the impact of loss-of function mutations in DNA damage repair genes, such as BARD1, on Ewing sarcoma treatment response.

Systemic and Nodular Hyperinflammation in a Patient with Refractory Familial Hemophagocytic Lymphohistiocytosis 2.

Familial hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory syndrome resulting from defective cytotoxicity. A previously healthy 3-month-old female presented with fever, irritability, abdominal distention, and tachypnea. She ultimately met all eight HLH-2004 diagnostic criteria, accompanied by elevated CXCL9. Initial empiric anti-inflammatory treatment included anakinra and IVIg, which stabilized ferritin and cytopenias. She had molecular and genetic confirmation of perforin deficiency and was started on dexamethasone and etoposide per HLH-94. She clinically improved, though CXCL9 and sIL-2Ra remained elevated. She was readmitted at week 8 for relapsed HLH without clear trigger and HLH-94 induction therapy was reinitiated. Her systemic HLH symptoms failed to respond and she soon developed symptomatic CNS HLH. She was incidentally found to have multifocal lung and kidney nodules, which were sterile and consisted largely of histiocytes and activated, oligoclonal CD8 T cells. The patient had a laboratory response to salvage therapy with alemtuzumab and emapalumab, but progressive neurologic decline led to withdrawal of care. This report highlights HLH foci manifest as pulmonary/renal nodules, demonstrates the utility of monitoring an array of HLH biomarkers, and suggests possible benefit of earlier salvage therapy.