Abstract A14: Exploiting DNA damage repair defects to enhance PD-L1 expression in Ewing sarcoma

Abstract

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.

Organoids as a new approach for improving pediatric cancer research

“…Maurer et al developed ES organoids and monolayers from a metastatic pulmonary lesion from a patient with an inherited BRCA1 Associated RING Domain 1 (BARD1) mutation. The organoids demonstrated high sensitivity to poly (ADP-ribose) polymerase (PARP) inhibitors (70). The same study group published the results of their second study, stating that the loss of BARD1 increases ES sensitivity to DNA damage, and that Guanylate-binding protein 1 (GBP1) expression contributes to DNA damage response in ES organoids (74).…”

Organoids: A New Chapter in Sarcoma Diagnosis and Treatment

“…Maurer et al developed Ewing sarcoma organoids and monolayers from a metastatic pulmonary lesion from a patient with an inherited BRCA1 Associated RING Domain 1 ( BARD1 ) mutation. The organoids surprisingly demonstrated high sensitivity to poly (ADP-ribose) polymerase (PARP) inhibitors [ 38 ]. Two years after their initial publication, the same study group published the results of their second study, stating that the loss of BARD1 increases Ewing sarcoma sensitivity to DNA damage, and that Guanylate-binding protein 1 (GBP1) expression contributes to DNA damage response in Ewing sarcoma organoids [ 39 ].…”