Cdk12/13 Inhibition Cooperates With The Ewing Sarcoma Oncoprotein Ews/Fli To Attenuate Homologous Recombination Repair In Ewing Sarcoma Cells

A therapeutic challenge in pediatric oncology is the paucity of readily “druggable” genetic events in many of the childhood malignancies. These tumors are frequently defined by sentinel abnormalities involving transcription factors in an otherwise quiet genomic landscape. One approach to treating these tumors would involve direct targeting of the aberrant transcription factor; however, this is a drug discovery challenge. A second approach would be to identify synthetic lethal relationships in the context of the aberrant transcription factor. THZ1, a covalent and potent inhibitor of CDK7, CDK12, and CDK13, kinases involved in transcriptional regulation, recently emerged as a targeted strategy to impair aberrant transcription. Extensive profiling of THZ1 against a diverse panel of u003e1,000 cancer cell lines revealed that the pediatric solid tumor, Ewing sarcoma, was exceptionally sensitive to this compound. We found that the anti-proliferative effects of THZ1 in Ewing sarcoma can be attributed primarily to CDK12/13 inhibition. Treatment of Ewing sarcoma cells with THZ531, a covalent and selective CDK12/13 inhibitor, decreased the phosphorylation of the C-terminal domain of RNA polymerase II, induced apoptosis, and markedly decreased colony formation capacity of Ewing sarcoma cell lines. In contrast, treatment with a selective CDK7 inhibitor had minimal effect. EWS/FLI is the transcription factor fusion protein that typically drives tumor establishment and maintenance in Ewing sarcoma tumors. Based on prior reports of this compound class inhibiting a small subset of highly expressed genes critical to tumor maintenance, we expected that these inhibitors would disrupt oncogenic EWS/FLI-driven transcription as the mechanism of inducing cell death. Surprisingly, however, global gene expression profiling revealed that THZ531 did not selectively repress EWS/FLI or EWS/FLI target genes. Rather, we observed that THZ531 preferentially repressed genes involved in DNA damage repair. Consistent with this finding, we found that THZ531 induced defects in DNA damage repair and highly synergized with DNA damaging agents that induce lesions repaired by homologous recombination (HR). Furthermore, we found that suppression of EWS/FLI attenuated sensitivity to THZ531 and the PARP inhibitor olaparib and abrogated synergy observed with this drug combination. Thus, we conclude that EWS/FLI establishes tumor cell synthetic lethality to CDK12/13 inhibitors by imparting sensitivity to DNA repair defects. This work establishes a novel mechanism of action of CDK12/13 inhibitors and gives further credence to the role of EWS/FLI in DNA damage response. Ongoing work is dedicated to the in vivo testing of THZ1 alone, and in combination with olaparib, as a novel targeted therapy for the treatment of Ewing sarcoma. Citation Format: Amanda Balboni, Bjorn Stolte, Peter Kalev, Nicholas Kwiatkowski, Tinghu Zhang, Brian Abraham, Gabriela Alexe, Dipanjan Chowdhury, Richard A. Young, Nathanael S. Gray, Kimberly Stegmaier. CDK12/13 inhibition cooperates with the Ewing sarcoma oncoprotein EWS/FLI to attenuate homologous recombination repair in Ewing sarcoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2441.