CDK12 mediated transcriptional regulation in U2OS cells

While activation of canonical NF-?B signaling through the IKK complex is well studied, few regulators of NIK-dependent non-canonical p52 nuclear translocation have been identified. We discovered a novel role for cyclin dependent kinase 12 (CDK12) in transcriptionally regulating the non-canonical NF-?B pathway. High-content phenotypic screening identified a novel compound, 919278, which inhibits lymphotoxin ß receptor (LTßR)- and FN14-dependent p52 nuclear translocation, but not TNFa receptor (TNFR)-mediated, canonical NF-?B p65 nuclear translocation. Chemoproteomics identified cyclin dependent kinase 12 (CDK12) as the target of 919278. CDK12 inhibition by 919278, THZ1, or siRNA knock down all affect similar global transcriptional changes and prevent LTßR and FN14-dependent MAP3K14 (NIK) mRNA induction and subsequent protein accumulation. In addition, 919278 and THZ1 treatment reduce RNA Pol II CTD phosphorylation. This powerful approach of coupling a phenotypic screen with chemoproteomics revealed a novel regulatory pathway of the non-canonical NF-?B pathway that could serve as a therapeutic target in autoimmunity and cancer. Overall design: There are TWEAK stimulated and unstimulated conditions, 4hr and 24hr time points. 7 treatments (DMSO, BIO0702697, BIO0919278, BIO032202, NTsiRNA, siRNAs523626, siRNAs523629) in duplicates. In total, 56 sample were sequenced and analyzed.

56

Henry KL, Kellner D, Bajrami B, Anderson JE, Beyna M, Bhisetti G, Cameron T, Capacci AG, Bertolotti-Ciarlet A, Feng J, Gao B, Hopkins B, Jenkins T, Li K, May-Dracka T, Murugan P, Wei R, Zeng W, Allaire N, Buckler A, Loh C, Juhasz P, Lucas B, Ennis KA, Vollman E, Cahir-McFarland E, Hett EC, Ols ML