Doctor of Philosophy (PhD)
Dr. Amarnath Natarajan, Ph.D.
Cyclin-dependent kinases (CDKs) are a family of serine-threonine kinases involved in various cellular functions, such as regulating the cell cycle and gene transcription. CDK9, a transcriptional CDK, regulates highly expressed enhancer-associated oncogenic transcription factors, including the oncogene Myc. CDK9 is responsible for the transcription and stabilization of Myc; consequently, it was a validated target for pancreatic cancer treatment.
As such, we developed a panel of aminopyrazole based proteolysis targeting chimera where we identified PROTAC 2 as a selective degrader of CDK9 (DC50 = 158 ± 6 nM). PROTAC 2 was capable of cereblon mediated proteasomal degradation of CDK9 while sparing other CDK family members. However, PROTAC 2 binds to CDK2 and CDK5, inhibiting their kinase activity, but did not induce their degradation, likely due to differentially exposed lysine residues. In MiaPaCa2 and Suit-2, we observed reduced PROTAC 2 mediated degradation of CDK9. Mass spectrometry-based kinome profiling further validated PROTAC 2 selective degradation of CDK9 in HEK293 and MiaPaCa2 cells. PROTAC 2 sensitized MiaPaCa2 cells to Venetoclax mediated growth inhibition, likely due to concurrent inhibition of Mcl-1 and Bcl2.
Furthermore, this panel of PROTACs also included PROTAC 4, which we identified as a selective CDK12 degrader (DC50 = 1.1 ± 0.3 μM). CDK12 plays a significant role in transcriptional elongation/termination, mRNA splicing, and DNA damage response. CDK12 is upregulated in several cancers, including breast, ovarian, stomach, and colorectal. We observed colorectal cancer-specific cereblon mediated proteasomal degradation of CDK12. PROTAC 4 selective degradation of CDK12 in HCT116 sensitized them to Gemcitabine mediated growth inhibition.
Since PROTAC 2 and PROTAC 4 only vary in linker length and composition, our studies demonstrate the importance of chemical characteristics in developing selective degraders utilizing the PROTAC strategy. Moreover, PROTACs offer a new therapeutic approach.
King, Hannah, "Utilizing Proteolysis-Targeting Chimeras to Target the Transcriptional Cyclin-Dependent Kinases 9 and 12" (2021). Theses & Dissertations. 545.