(P 294) DNA-DAMAGE REPAIR DYSREGULATION AND CHEMOTHERAPY RESPONSE IN SARCOMA MODELS

Objective: Chemotherapy remains the most important systemic treatment option for most patients with sarcomas harboring complex karyotypes. However, patients do not respond uniformly well to therapy and biomarkers to predict response have not yet been identified. DNA damage repair (DDR) alterations have been shown to be predictive for some chemotherapies in colorectal cancer and brain tumors but its role in sarcomas is unknown. We studied the impact of mismatch repair deficiency (dMMR) and the role of MGMT on chemotherapy response in preclinical sarcoma models.

Methods: We used Cas9-based knockout (KO) to generate isogenic sarcoma cell line models (originated from myxofibrosarcoma (MFS) and leiomyosarcoma (LMS) cell lines) devoid of MSH2 and MGMT expression. Protein expression was assessed by Western Blot (WB). Microsatellite instability (MSI) was evaluated by Microsatellite replication assay (Promega). MGMT promoter methylation was assessed by methylation-specific PCR. MGMT was overexpressed in a MGMT-negative sarcoma model via lentiviral overexpression. The same MGMT-negative LMS model was continuously treated with temozolomide (TMZ). Cell lines were then characterized for sensitivity to various chemotherapeutic drugs using sulforhodamine B (SRB) cell viability assays.

Results: MSH2 KO caused microsatellite instability (MSI) in one myxofibrosarcoma model. Cell viability assays revealed MSI to confer resistance to nucleoside analoga gemcitabin [GR50: 22 nM vs. > 5 µM – GR50 not reached] and 5-fluorouracil [GR50: 6.6 µM vs. 37.0 µM]. Notably, no shift in sensitivity was observed for other chemotherapeutic durgs (doxorubicin, cisplatin and ifosfamide). MGMT overexpression in a MGMT-negative LMS cell line led to a 5- fold increased TMZ tolerance [GR50: 100µM vs 500µM]. Continuous TMZ treatment of MGMT negative cells resulted in TMZ resistance accompanied by strong endogenous re-expression of MGMT. Notably, MGMT re-expression was not caused by change in MGMT methylation status. MGMT KO in this model reversed TMZ-resistant phenotype. MSH2 KO in MGMT-negative LMS cell line conferred resistance to TMZ.

Conclusion: We here introduce novel, genetically modified in vitro models to study DNA damage repair alterations (dMMR / MSI and MGMT-status) in soft tissue sarcomas. Our data suggest a predictive value of MGMT expression for TMZ/dacarbazine response. Furthermore, MSI appears to be a valid biomarker for the efficacy of nucleoside analoga. We currently investigate the prevalence of these biomarkers in the context of pretreatment as well as correlation with chemotherapeutic response in a retrospective setting.