A Murine Cell Line Based Model of Chronic CDK9 Inhibition to Study Widespread Non-Genetic Transcriptional Elongation Defects (TE^deff) in Cancers

We have previously reported that a subset of cancers is defined by global transcriptional deregulations with widespread deficiencies in mRNA transcription elongation (TE)—we call such cancers as TE^deff. Notably, TE^deff cancers are characterized by spurious transcription and faulty mRNA processing in a large set of genes, such as interferon/JAK/STAT and TNF/NF-κB pathways, leading to their suppression. The TE^deff subtype of tumors in renal cell carcinoma and metastatic melanoma patients significantly correlate with poor response and outcome in immunotherapy_. Given the importance of investigating TE^deff cancers—as it portends a significant roadblock against immunotherapy—the goal of this protocol is to establish an in vitro TE^deff mouse model to study these widespread, non-genetic transcriptional abnormalities in cancers and gain new insights, novel uses for existing drugs, or find new strategies against such cancers. We detail the use of chronic flavopiridol mediated CDK9 inhibition to abrogate phosphorylation of serine 2 residue on the C-terminal repeat domain (CTD) of RNA polymerase II (RNA Pol II), suppressing the release of RNA Pol II into productive transcription elongation. Given that TE^deff cancers are not classified under any specific somatic mutation, a pharmacological model is advantageous, and best mimics the widespread transcriptional and epigenetic defects observed in them. The use of an optimized sublethal dose of flavopiridol is the only efficacious strategy in creating a generalizable model of non-genetic widespread disruption in transcription elongation and mRNA processing defects, closely mimicking the clinically observed TE^deff characteristics. Therefore, this model of TE^deff can be leveraged to dissect, cell-autonomous factors enabling them in resisting immune-mediated cell attack.