Research: Deciphering the role of transcriptional CDKs in Rhabdomyosarcoma, a pediatric soft tissue sarcoma
Title of the project: “Deciphering the role of transcriptional CDKs in Rhabdomyosarcoma, a pediatric soft tissue sarcoma”
Background: Pediatric rhabdomyosarcoma (RMS) is an aggressive soft tissue sarcoma of myogenic origin representing ~8% of all pediatric tumors. Despite heavy multi-modal therapy for patients with metastatic and recurrent RMS, 5-years overall survival remains <30% due to emergency of resistance to conventional treatments. Differently from adult cancers, pediatric malignancies have low mutational burdens with epigenetic deregulations playing key roles. Therefore, our project is focused on the dissection of a subgroup of transcription-associated Cyclin-dependent kinases (CDKs) that supports the transcription of DNA damage response (DDR) genes through Serine2 RNA Pol II phosphorylation.
Objectives: the main aims of this project are (i) to clarify whether these CDKs can represent an exploitable vulnerability in RMS and (ii) whether their inhibition sensitizes RMS to DNA-damaging agents.
Experimental plan: Task1) Define the role of the identified CDKs in regulating DNA damage response and cell survival in RMS. To this end, CDKs pharmacologic and genetic inhibition will be done in vitro on high risk-representative RMS cell lines and functional assays (cell growth, apoptosis, differentiation, stemness) and global gene expression profiling (RNA-seq) and chromatin enhancer landscape (H3K27ac ChIP-seq) will be analyzed. Phosphoproteomics (RPPA) will identify proteins phosphorylated by CDK12. Targets modulated by CDK12 and involved in RMS tumorigenicity will be confirmed through genetic approaches such as gene silencing or overexpression. In vivo validation will be done measuring the growth of CDKs-depleted or inhibited RMS cells inoculated in immunodeficient mice.
Task2) Investigate if the inhibition of transcriptional CDKs sensitizes RMS cells to radiotherapy. The effects of irradiation in combination with CDKs inhibition or depletion will be studied in vitro by clonogenic assay, apoptosis and DNA damage detection together with the measure of expression of DDR genes and key genes in RMS. We will also assay patient-derived xenografts (PDXs) testing the effects of CDKs inhibition or depletion in combination with irradiation. Results will be validated in preclinical models of immunocompromised mice inoculated with RMS cells and treated with CDKs inhibitors and/or irradiation to measure tumor growth.
Expected Results: Results from this proposal will contribute to shed light on the role of transcriptional CDKs on the survival, growth, differentiation and stemness of RMS cells. They could also identify potential future combinatorial therapies that promote the response to irradiation for high risk RMS. Moreover, dissecting transcriptional CDKs functions will provide novel insights into oncogenic transcriptional mechanisms governing RMS.