Abstract LB404: ATR inhibitors synergize with DHFR inhibitors in rhabdomyosarcoma cells by disrupting DNA damage checkpoint

Abstract

Abstract Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and adolescents. RMS tumors are classified into four subtypes: embryonal, alveolar, spindle cell/sclerosing, and pleomorphic. Treatment options for RMS include a combination of surgery, chemotherapy, and radiation therapy and regimens for patients with relapsed and metastatic RMS remain limited. However, a recent study has reported that pharmacological inhibitors targeting ataxia telangiectasia and Rad3-related protein (ATR) are currently undergoing phase I/II trials and may be utilized for the treatment of alveolar RMS. Interestingly, we identified that the gene signature of replication stress positively correlated with dihydrofolate reductase (DHFR) expression in the cancer cell line database (CellMinerCDB, discover.nci.nih.gov/cellminercdb; Pearson correlation (r)=0.66). DHFR is an important therapeutic target for cancer and autoimmune diseases, and its inhibitors are routinely used and clinically approved. DHFR inhibitors are known to exhibit their anticancer effects by inhibiting nucleotide synthesis and DNA replication. In this study, we aimed to explore a potential synergistic interaction using a combination treatment of ATR inhibitors (elimusertib and ceralasertib) and DHFR inhibitors (pralatrexate, methotrexate, and raltitrexated) in multiple RMS cell lines. We used a drug matrix reduction format and found that combined treatment of ATR inhibitors and DHFR inhibitors induced a synergistically decreased RMS cell viability, showing higher synergy in fusion-positive RMS cells than in fusion-negative RMS cells. Mechanistically, ATR inhibitors abrogated DNA damage response checkpoint CHK1 activation caused by DHFR inhibitor, leading to increased γH2AX and decreased DNA replication activity. Accordingly, the accumulation of DNA damage in combination with ATR inhibitor and DHFR inhibitor triggered higher cell cycle arrest and apoptotic cell death than single agent treatment of DHFR inhibitor. Our findings warrant further research into identifying genomic regions where the DNA damage is enriched in combination treatment by using END-sequencing and synthesis-associated with repair sequencing (SAR-seq). In conclusion, this study provides promising rationale for the combination treatment of ATR and DHFR inhibitors to treat RMS patients, including those with advanced, relapsed, and metastatic states. Citation Format: Ukhyun Jo, George K Annor, Jack F. Shern. ATR inhibitors synergize with DHFR inhibitors in rhabdomyosarcoma cells by disrupting DNA damage checkpoint [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB404.