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Loss of GBP1 expression does not impact PSaRC318 cell migration.

Prevalence of somatic DNA damage repair gene variants in Ewing cell lines included in original studies of PARP inhibition.

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 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. In addition, RNA-sequencing 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.Significance:This work provides preclinical support for the inclusion of pediatric patients with advanced Ewing sarcoma and pathogenic germline variants in BARD1 in future clinical trials testing novel agents inducing DNA damage/targeting DNA damage repair.

Ewing sarcoma is a bone tumor most commonly diagnosed in adolescents and young adults. Survival for patients with recurrent or metastatic Ewing sarcoma is dismal and there is a dire need to better understand the mechanisms of cell metastasis specific to this disease. Our recent work demonstrated that microenvironmental stress leads to increased Ewing sarcoma cell invasion through Src activation. Additionally, we have shown that the matricellular protein tenascin C (TNC) promotes metastasis in Ewing sarcoma. A major role of both TNC and Src is mediation of cell–cell and cell-matrix interactions resulting in changes in cell motility, invasion, and adhesion. However, it remains largely unknown, if and how, TNC and Src are linked in these processes. We hypothesized that TNC is a positive regulator of invadopodia formation in Ewing sarcoma through its ability to activate Src. We demonstrate here that both tumor cell endogenous and exogenous TNC can enhance Src activation and invadopodia formation in Ewing sarcoma. We found that microenvironmental stress upregulates TNC expression and this is dampened with application of the Src inhibitor dasatinib, suggesting that TNC expression and Src activation cooperate to promote the invasive phenotype. This work reports the impact of stress-induced TNC expression on enhancing cell invadopodia formation, provides evidence for a feed forward loop between TNC and Src to promote cell metastatic behavior, and highlights a pathway by which microenvironment-driven TNC expression could be therapeutically targeted in Ewing sarcoma.

Metastatic and relapsed Ewing sarcoma typically afflicts the adolescent population and is largely fatal. These bone tumors are most commonly driven by the fusion oncoprotein EWS-FLI1. Ewing tumors demonstrate significant intra-tumoral heterogeneity, and individual tumor cells can express highly variable and dynamic levels of EWS-FLI1. Recent studies revealed that the EWS-FLI1 oncoprotein level (high versus low expression) greatly influences the behavior of Ewing tumor cells. As compared to cells with high EWS-FLI1, Ewing cells in the EWS-FLI1 low state demonstrate an increased propensity for metastasis. In light of these observations, we sought to determine how tumor cell EWS-FLI1 level influences the anti-tumor cell immune response. Since ICAM-1, which can promote tumor cell/T-cell interaction and T-cell activation, is highly expressed on EWS-FLI1 low cells, we hypothesized that EWS-FLI1 low cells would be more susceptible to T-cell mediated tumor cell apoptosis as compared to cells with high EWS-FLI1. Unexpectedly, we found that EWS-FLI1 low cells are more resistant to T-cell mediated apoptosis than EWS-FLI1 high cells. We investigated the potential mechanisms by which EWS-FLI1 level might influence the T-cell anti-tumor response, and discovered that low EWS-FLI1 expression results in upregulation of PD-L1 and PD-L2, both important ligands for the PD-1 immune checkpoint receptor on T-cells. We demonstrated that blocking PD-1 results in a greater increase of T-cell mediated killing of EWS-FLI1 low tumor cells as compared to cells with higher EWS-FLI1 expression. Our studies suggest that Ewing cells in the EWS-FLI1 low expression state may serve as a niche of tumor immune-evasion.

Ewing sarcoma, an oncofusion-driven primary bone tumor, can occur in the setting of various germline mutations in DNA damage repair pathway genes. 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. PARP inhibitors (PARPi) are effective against Ewing sarcoma cells in vitro, though have demonstrated limited success in clinical trials to date. In order to assess the impact of BARD1 loss on Ewing sarcoma sensitivity to PARP inhibitor therapy, we generated the novel PSaRC318 patient-derived Ewing tumor cell from our patient with a germline BARD1 mutation and then analyzed the response of these cells to PARPi. We demonstrate that PSaRC318 cells are sensitive to PARP inhibition and by testing the effect of BARD1 depletion in additional Ewing sarcoma cell lines, we confirm that loss of BARD1 enhances PARPi sensitivity. In certain malignancies, DNA damage can activate the IRF1 (interferon response factor 1) immunoregulatory pathway, and the activation of this pathway can drive immunosuppression through upregulation of the immune checkpoint protein PD-L1. In order to determine the ability of PARPi to alter Ewing tumor immunoregulation, we evaluated whether PARPi results in upregulation of the IRF1-PDL1 pathway. Indeed, we now demonstrate that PARPi leads to increased PD-L1 expression in Ewing sarcoma. Together, these data thus far suggest that while Ewing tumors harboring germline mutations in DNA damage repair proteins may in respond to PARPi in vitro, in vivo benefit of PARPi may only be demonstrated when counteracting the immunosuppressive effects of DNA damage by concurrently targeting immune checkpoint proteins.

Objective: The purpose of this work is to determine whether PD-L1 expression can be upregulated by PARP inhibitor treatment in Ewing tumor cells harboring DNA damage repair defects. Further, we aimed to determine whether enhanced PD-L1 expression renders Ewing cells more susceptible to T-cell mediated apoptosis following PD-1 inhibition. Experimental Procedures: Patient-derived tumor organoids and monolayers (named PSaRC-318) were established from a viably cryofrozen metastatic lung lesion from a patient with Ewing sarcoma and a paternally inherited germline BARD1 mutation. A673, CHLA9, and CHLA10 Ewing tumor cell lines were also employed. Sensitivity of PSaRC-318 to the PARP inhibitors olaparib and talazoparib was established by performing IncuCyte apoptosis assays. PD-L1 expression was examined using RT-PCR, Western blot, and flow cytometry. BARD1, BRCA, and RAD51D siRNA were also utilized to test the role of specific DNA damage repair proteins on this effect. T-cell/tumor cell cocultures and tumor-primed T-cells were established. T cell-induced tumor cell apoptosis was monitored in real-time using IncuCyte caspase and annexin assays. Results: PSaRC-318 organoids demonstrate exquisite sensitivity to PARP inhibitors, with an IC50 for talazoparib of 3.54nM. Treatment of PARP inhibitor-resistant Ewing cell lines, such as CHLA10, with BARD1 siRNA rendered cells sensitive to PARP inhibition. Ewing tumor cells were found to upregulate tumor cell surface expression of the checkpoint protein PD-L1 in response to treatment with PARP inhibitors. The impact of BARD1, BRCA1, and RAD51D on PD-L1 expression in Ewing tumor cells was also determined. Next, Ewing tumor cells were pretreated with PARP inhibitors and then cocultured with tumor-primed T-cells. T cell-mediated tumor cell apoptosis was monitored in real time using caspase IncuCyte assays. PD-1 blocking antibody added to T-cell/tumor cell cocultures resulted in an increase in early tumor cell death compared to IgG controls. Conclusion: Single-agent PARP inhibitors have not demonstrated significant clinical benefit in relapsed Ewing sarcoma to date, suggesting that combinatorial therapy with PARP inhibitors may be a more effective approach. Here, we have shown that Ewing tumor cells with germline mutations in DNA-damage repair proteins, such as BARD1, can upregulate PD-L1 expression following treatment with PARP inhibitors. Thus, PARP inhibitor/anti-PD-1 therapy may be a logical combination for the treatment of relapsed Ewing sarcoma. Ongoing preclinical studies are continuing to examine this combination. Citation Format: Lisa M. Maurer, Rose M. Venier, Claire Julian, Kelly M. Bailey. Exploiting DNA damage repair defects to enhance PD-L1 expression in Ewing sarcoma [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 A14.

BACKGROUND The authors investigated the acceptability to women of the preventive strategies available for dealing with hereditary breast/ovarian carcinoma in France, the United Kingdom, and Canada, countries selected because of their cultural differences. The authors aimed to discover the existence of specific factors that may affect acceptability of these preventative measures. METHODS A cross-sectional, multicenter survey was conducted in Marseilles, France (n = 141), in Manchester, England (n = 130), and in Montreal, Quebec (n = 84). All of the women attending cancer genetic clinics for the first time because of a family history of breast-ovarian carcinoma completed a self-administered questionnaire before their clinic consultation. RESULTS Variations in responses to different preventative options presented on the questionnaire were seen within the sample of patients considered as a whole. The highest levels of acceptability were obtained for mammographic screening (87%) and chemoprevention (58%). In contrast, prophylactic oophorectomy and prophylactic mastectomy were thought to be acceptable at an early age (before 35 years), an age associated with the highest prophylactic efficacy, by only 19% and 16% of the respondents, respectively. After multivariate adjustment, the statistical data showed that the British respondents were more in favor of oophorectomy (P < 0.0001) and more in favor of chemoprevention than the French (P < 0.001) and the Canadian respondents (P < 0.001). The British (overall adjusted response [ORadj] = 3.9; P < 0.001) and Canadian respondents (ORadj = 3; P < 0.01) were more in favor of prophylactic mastectomy than the French. The cumulated acceptability of mammography before the age of 40 years was found to be greater in the French (ORadj = 2.8; P < 0.01) and Canadian (ORadj = 3.1; P < 0.05) samples than in the British sample. CONCLUSION These results demonstrated the existence of international variations in the acceptability of the preventive strategies available for women at risk for hereditary breast/ovarian carcinoma. Therefore, these results suggested that when establishing medical recommendations or planning public health interventions, physicians must integrate the population's perception of advantages and drawbacks with the patient's individual decision making. Cancer 2001;92:959–68. © 2001 American Cancer Society.