Abstract LB-323:Atrxdeletion delays tumor formation and increases radiosensitivity of a primary mouse model of soft tissue sarcoma

Abstract

Soft tissue sarcomas are tumors of the connective tissue that account for an estimated 12,000 new cancer cases annually and carry a poor prognosis with a five year survival rate of 50% despite treatment. An important research objective in improving therapy for soft tissue sarcoma patients is to understand how genetic mutations affect soft tissue sarcoma development and radiation response. Intriguingly, next-generation sequencing data from The Cancer Genome Atlas and other massive cancer sequencing efforts have identified Alpha Thalassemia and Mental Retardation X-linked, or ATRX, as the second most frequently mutated gene in soft tissue sarcoma. The vast majority of these ATRX mutations are frameshift mutations. To dissect the role of ATRX in soft tissue sarcoma development and therapeutic response, we generated a primary mouse model of soft tissue sarcoma. This model utilizes Cre-LoxP technology to conditionally activate oncogenic KrasG12D, delete p53, and delete both alleles of Atrx (KPA) in mouse hindlimb Pax7+ muscle satellite cells. When compared to mice with activation of oncogenic KrasG12D, deletion of p53 and at least one wild-type Atrx allele (KP), KPA mice showed a significant delay in time to tumor formation (35 vs. 58 days median), but no significant difference in tumor growth rate as measured by time to tumor quintupling. Next, we generated primary soft tissue sarcoma cell lines from our KP mouse model, and performed CRISPR-Cas9 mediated knockout of Atrx to generate isogenic cell lines. Testing of these isogenic cell lines revealed that Atrx deletion in soft tissue sarcoma cells increased sensitivity to double strand break inducing chemotherapeutics and ionizing radiation, but not to single strand break inducing chemotherapeutics in vitro. In clonogenic assays, Atrx deletion increased radiosensitivity of soft tissue sarcoma cell lines more than fourfold across all radiation doses tested. We next used our KP and KPA models to test the effect of Atrx deletion on radiosensitivity in vivo. Results showed that, relative to the KP model, Atrx deletion improved the response of soft tissue sarcomas to radiation therapy in vivo, increasing the time to tumor quintupling after radiation by 20%. Further analysis demonstrated that Atrx deletion significantly increases mitotic segregation defects after ionizing radiation, suggesting a potential mechanism for the radiosensitivity observed in our studies. Our in vivo study provides the first insight into how ATRX alters the development and therapeutic response of soft tissue sarcoma. Citation Format: Robert W. Floyd, Lixia Luo, David Kirsch. Atrx deletion delays tumor formation and increases radiosensitivity of a primary mouse model of soft tissue sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-323.