Abstract 6941: Preclinical efficacy of the CK1 alpha/CDK7/CDK9 inhibitor BTX-A51 in <i>CIC</i>-rearranged sarcoma

Abstract CIC-rearranged sarcoma is an aggressive mesenchymal neoplasm characterized by high metastatic potential and poor clinical outcomes with median overall survival of only 12 months. The lack of effective systemic therapies highlights the urgent need to identify biologic vulnerabilities for therapeutic targeting. CIC-rearranged sarcoma shows molecular dependence on MYC, which can be targeted indirectly through cyclin-dependent kinase (CDK) 9 inhibition. In leukemia, casein kinase 1 alpha (CK1α) inhibition has been shown to have anti-tumor activity through activation of p53. We here investigated the anti-tumor effect of the CK1α/CDK7/CDK9 inhibitor BTX-A51 in preclinical models of CIC-rearranged sarcoma and determined the underlying mechanism of response. To evaluate the anti-tumor effect of BTX-A51 in vitro, we treated the CIC01, CIC02, NCC-CDS1-X1-C1 (CDS1), and NCC-CDS2-C1 (CDS2) cell lines with BTX-A51 (dose range 0.49-1000 nM) and performed BrdU cell proliferation, CellTiter-Glo cell viability, and Caspase-Glo 3/7 apoptosis assays. Daudi (MYC-driven Burkitt lymphoma) cells were used as control. IC50 values were calculated using an AAT Bioquest IC50 Calculator. We also assessed the effect of BTX-A51 on cell cycle progression using flow cytometry analysis and measured cell counts. The expression of key signaling proteins after 3, 6, 12, or 24 hours of treatment with BTX-A51 was determined through immunoblotting studies. In vivo studies assessing the anti-tumor effect of BTX-A51 in the CIC01 and CIC02 patient-derived xenografts are currently underway, and results will be reported. The CIC01, CIC02, CDS1, and CDS2 cell lines were highly sensitive to BTX-A51 with proliferation IC50 values ranging from 13-50 nM. In all 4 cell lines, BTX-A51 substantially inhibited cell viability and induced caspase 3/7-mediated apoptosis. At the same time, BTX-A51 treatment substantially inhibited expression of MYC, MCL-1, MDM2, phospho-RPB1 CTD Ser2 (a substrate of CDK9), phospho-RPB1 CTD Ser5 (a substrate of CDK7), phospho-RB1 Ser795, and RB1, and induced expression of phospho-histone H2A.X Ser13, p53, cleaved PARP (Asp214), and cleaved caspase 3. BTX-A51 treatment for 12 hours reduced the fraction of cells in S phase in cell cycle phase analysis consistent with G1 arrest and significantly reduced cell counts in all 4 cell lines (P = 0.0381 each, Wilcoxon rank-sum test). These findings demonstrate that BTX-A51 has anti-tumor activity in preclinical models of CIC-rearranged sarcoma. Treatment with BTX-A51 induces caspase 3-mediated apoptotic cell death through combined stabilization of p53 and inactivation of oncogenic drivers. Together with ongoing in vivo studies, our data will provide a compelling preclinical rationale for the evaluation of BTX-A51 in patients with CIC-rearranged sarcoma. Citation Format: Quentin Odom-Lewis, Samantha L. Davis, Stephan Pieper, Chen Chu, Emanuele Mazzola, Renyan Liu, Nicole Solimini, Prafulla C. Gokhale, Geoffrey I. Shapiro, George D. Demetri, Inga-Marie Schaefer. Preclinical efficacy of the CK1 alpha/CDK7/CDK9 inhibitor BTX-A51 in CIC-rearranged sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6941.