Abstract B015: CDK12 pharmacological targeting promotes cell death by abrogating DNA damage response, impairs PAX3-FOXO1 expression and synergizes with BRD4 and PARP inhibitors in fusion positive rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is the most frequent soft tissue sarcoma of childhood. The fusion positive (FP) subtype expressing the chimeric transcriptional factor PAX3-FOXO1, the driver of malignancy, has limited treatment options due to resistance to treatments. As a pediatric cancer, RMS has a low mutational burden with epigenetic transcriptional deregulations playing key roles. Thus, targeting the basic transcriptional machinery including catalytic effectors of transcription is an emerging anti-cancer approach. The transcriptional cyclin-dependent kinase CDK12 regulates the transcription elongation phase, particularly of the DNA damage response (DDR) genes, by phosphorylating Ser2 (pSer) on RNA PolII. In Ewing sarcoma, CDK12 inhibition leads to a DNA repair deficient phenotype and high sensitivity to DNA damaging agents. PAX3-FOXO1 RMS cells display elevated levels of replication stress highly relying to DDR for survival. Therefore, in this study we evaluate the impact of CDK12 inhibition on PAX3-FOXO1 RMS tumorigenic features. We found that CDK12 mRNA levels were highly up-regulated in RMS primary samples compared to healthy control muscles, with one of the highest expression among the adult and pediatric tumors analyzed. Then, we treated PAX3-FOXO1 cell lines with THZ531, an inhibitor of CDK12 and its paralog CDK13. Six hours of THZ531 treatment at nanomolar concentrations decreased pSer2 of RNA PolII and reduced PAX3-FOXO1 transcripts and protein levels. The drug also reduced cell survival in 2D and 3D culture conditions. THZ531-treated cells accumulated in G2/M phase and underwent PARP- and Casp 3/7-dependent apoptosis, increased markers of DNA damage associated to DNA double-strand breaks (neutral comet assay), and reduced the levels of the DDR players ATM, ATR, RAD51, BRCA1 and BRCA2. Similar results were obtained after treatment with another CDK12/13i with Cyclin K degrader ability, SR-4835, and with a CDK12-specific degrader, BSJ-4-116, suggesting on-target effects. The three drugs showed similar low nanomolar concentrations effective on PAX3-FOXO1 RMS organoids. Preliminary RNAseq data in two PAX3-FOXO1 RMS cell lines showed that THZ531 downregulated only a low number of genes, common to the two cell lines. The most downregulated gene families were those of DDR pathways. Moreover, a negative correlation between gene length and gene expression levels was found. CDK12 KO in two RMS cell lines affected cell survival in 2D and 3D conditions compared to control cells suggesting on-target activity of the drugs. Phosphoproteomics analysis after THZ531 treatment showed a significant decrease of a number of serines and threonine phosphopeptides also involving a subset on DDR proteins. THZ531 treatment resulted synergic in vitro with Olaparib (PARPi) and with JQ1 (BRD4i). Interestingly, BRD4 is needed for PAX3-FOXO1 activity on oncogenic super-enhancers. Analysis of nascent RNAs and mapping of RNA PolII to identify direct targets of CDK12 is ongoing. Supported by Associazione Italiana per la Ricerca sul Cancro (AIRC) IG #27794 grant to RR Citation Format: Marika Attili, Lucrezia D'Archivio, Riccardo Mazzocchi, Francesca Antonella Aiello, Matteo Cassandri, Silvia Pomella, Adam D. Durbin, Francesco Marampon, Kim PJ Schellekens, Michael T Meister, Jarno Drost, Concetta Quintarelli, Simone Sidoli, Michele Ceribelli, Craig J Thomas, Franco Locatelli, Biagio De Angelis, Kristy R Stengel, Rossella Rota. CDK12 pharmacological targeting promotes cell death by abrogating DNA damage response, impairs PAX3-FOXO1 expression and synergizes with BRD4 and PARP inhibitors in fusion positive rhabdomyosarcoma abstract. In: Proceedings of the AACR Special Conference in Cancer Research: Discovery and Innovation in Pediatric Cancer— From Biology to Breakthrough Therapies; 2025 Sep 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85 (18Suppl₂): Abstract nr B015.