Abstract SY19-01: Synthetic lethality opportunities for tumors with MMEJ deficiency

Abstract Normal human cells have several DNA repair pathways which maintain genomic stability. Tumor cells are often deficient in one of these pathways. Loss of a pathway provides the tumor cell with enhanced mutagenesis and chromosome instability, thereby giving the tumor cell a selective advantage as well as a means for developing drug resistance. Interestingly, tumor cells deficient in one DNA repair pathway are hyperdependent on another pathway. This dependency provides an opportunity for the treatment of these tumors through the mechanism of synthetic lethality. For instance, if a tumor is deficient in homologous recombination repair, such as a BRCA1/2 deficient breast or ovarian cancer, it may be hyperdependent on MMEJ (Microhomology Mediated End Joining) repair. An MMEJ inhibitor, such as a POLQ inhibitor, provides a synthetic lethal strategy for killing this tumor. In contrast, no tumors have been identified to date which have an underlying defect in MMEJ. Ewing sarcoma (EwS) is a group of bone and soft tissue cancers that can occur in children and young adults. The cancer is driven by a chromosomal translocation at 11;22, resulting in expression of the EWS-FLI1 fusion oncoprotein, formed by the N-terminus of EWSR1 and the C-terminus of FLI1. EwS cells have pronounced sensitivity to chemotherapy and radiation-induced DNA damage, suggesting that there may be defects in DNA damage repair (DDR) mechanisms. Recently, it has been reported that the EWS-FLI1 oncoprotein promotes R-loop formation, impairing BRCA1 recruitment to the chromatin and resulting in a HRD phenotype. However, despite this preclinical data, clinical trials did not demonstrate PARP inhibitor response in EwS patients. Therefore, there could be deficits in another DSB repair pathway, distinct from HR, which have yet to be identified. We have recently determined that the loss of wildtype EWSR1, or expression of the EWS-FLI1 oncoprotein, causes a defect in microhomology-mediated end-joining (MMEJ). EWSR1 loss phenocopies MMEJ-deficient cells, resulting in hypersensitivity to irradiation, topoisomerase inhibitors, and ATR inhibitors. In addition, EWSR1 loss exhibited synthetic lethality with BRCA1/2 loss. We also confirmed that depletion of EWSR1 significantly decreased MMEJ activity, as measured by multiple MMEJ reporters. To investigate the effects of EWSR1 loss on the MMEJ pathway, we characterized the expression of individual MMEJ-related proteins and found that the expression of DNA polymerase theta (POLQ), the primary DNA polymerase in the MMEJ pathway encoded by POLQ gene, is exclusively and markedly reduced among other MMEJ-related molecules, including LIG3 and APEX2. In addition, depletion of EWSR1 also decreased endogenous POLQ foci at DSB loci. To investigate the potential mechanism underlying the decreased POLQ expression following EWSR1 loss, we performed a deep RNA-seq analysis. Loss of EWSR1 leads to the skipping of several exons of the POLQ pre-mRNA transcript, including exon25. Formation of EWSR1 condensates, generated by its prion-like domain, plays a pivotal role in maintaining faithful splicing of the POLQ pre-mRNA. A mass spec analysis revealed that wildtype EWSR1 interacts with RNA binding proteins including spliceosomes via its prion-like domain. Depletion of several splicing factors that can bind to wildtype EWSR1, but not to an EWSR1 37YS mutant, results in decreased condensate formation, decreased POLQ expression, and increased exon skipping of the POLQ transcript. Importantly, we found that POLQ exon25 skipping occurs in primary EWS tumor samples but not in other pediatric cancers. Furthermore, ectopic expression of the EWS-FLI1 oncoprotein also inhibits the recruitment of endogenous POLQ to the DSB loci, resulting in a severe impairment of MMEJ. Depletion of the EWS-FLI1 oncoprotein in EwS cells restores MMEJ activity and endogenous POLQ formation. Interestingly, an EWS-FLI1 37YS mutant, which fails to form condensates with wildtype EWSR1, failed to inhibit the MMEJ activity, indicating that the EWS-FLI1 oncoprotein disrupts wildtype EWSR1 condensates and prevents the faithful splicing of the POLQ transcript. Dual inhibition of MMEJ and non-homologous end joining (NHEJ) results in synthetic lethality. We next treated EwS cells with Peposertib, a DNA-PKc inhibitor. Compared to an osteosarcoma cell line, U2OS, EwS cells exhibited hypersensitivity to Peposertib. In addition, the combination of Peposertib and the topoisomerase inhibitor etoposide synergistically eliminated EwS cells in vitro and in vivo. Collectively, our data indicate that EwS cells have a unique DDR defect-namely, impaired MMEJ-activity. Therefore, this unique DDR defect could be exploited for therapeutic benefit by inhibiting another DNA repair pathway, such as NHEJ or HR. Based on these data, we have reached the following conclusions. First, Ewing sarcoma tumors have a deficiency in POLQ expression and a corresponding loss of MMEJ activity. Second, the EWSR1 complex, consisting of the EWSR1, FUBP1, and KHSRP splicing factors, binds to a cis-acting Exonic Splicing Enhancer (ESE) in POLQ exon 25 of the POLQ pre-mRNA, and prevents exon 25 skipping. Disruption of this complex, or mutation of the six nucleotide ESE in exon 25, causes exon skipping and loss of POLQ protein expression. Third, the EWS-FLI1 fusion oncoprotein binds and inactivates EWSR1 and causes an exon 25 splicing defect, leading to exon skipping and loss of POLQ protein expression. Fourth, the MMEJ deficiency of EwS cells results in enhanced cellular sensitivity to inhibitors of other DNA Repair pathways, providing a strategy for synthetic lethality therapy. Citation Format: Alan D. D'Andrea. Synthetic lethality opportunities for tumors with MMEJ deficiency abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts) ; 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86 (8Suppl): Abstract nr SY19-01.