Discovery of CDK4/6 bifunctional degraders for ER+/HER2- breast cancer.

person Hannah Majeski San Diego, CA info_outline Hannah Majeski, Akinori Okano, Angela Pasis, Casey Carlson, Arvind Shakya, Shenlin Huang, Aparajita Hoskote Chourasia, Leah M. Fung, Qiao Liu

Authors person Hannah Majeski San Diego, CA info_outline Hannah Majeski, Akinori Okano, Angela Pasis, Casey Carlson, Arvind Shakya, Shenlin Huang, Aparajita Hoskote Chourasia, Leah M. Fung, Qiao Liu Organizations San Diego, CA, Biotheryx, Inc, San Diego, CA, Biotheryx, Inc., San Diego, CA, Biotheryx, San Diego, CA Abstract Disclosures Research Funding Pharmaceutical/Biotech Company Biotheryx, Inc Background: CDK4/6 inhibitors (CDK4/6i) such as palbociclib and ribociclib are used to treat ER+/HER2- breast cancer, but patients can develop resistance via many mechanisms, several of which converge on the upregulation of CDK6. This has been shown to limit the effectiveness of CDK4/6i in ER+ breast cancer with up to 20% patients exhibiting innate resistance and up to 70% patients developing acquired resistance after 3 years on therapy (Scheidemann, 2021, doi:10.3390/ijms222212292). Methods: To address this limitation, we utilized our PRODEGY platform of Cereblon (CRBN) binders to synthesize CRBN mediated CDK4/6 bifunctional degraders to potently inhibit tumor growth for treatment of naïve ER+/HER2- breast cancer and CDK4/6i resistant tumors. Results: Target degradation by immunoblot analysis of the triple negative breast cancer (TNBC) cell line, MDA-MB-231, treated with our CDK4/6 bifunctional degraders for 6 hours showed up to 85% degradation of CDK4 and CDK6 with DC 50 s of 1-100nM. CDK4/6 phosphorylates the protein RB which releases the transcription factor E2F, inducing the expression of genes which promote cell cycle progression. Analysis of RB phosphorylation by in-cell western upon 24 hours of CDK4/6 degrader treatment showed phospho-RB IC 50 s at <30nM. Cell cycle analysis by staining with propidium iodide after 24 hours of treatment with CDK4/6 degraders induced G0/G1 cell cycle arrest at concentrations as low as 10nM. We used a 2D colony formation assay (CFA) as a readout for inhibition of proliferation by cell cycle arrest. Our CDK4/6 degraders showed potent inhibition of cell proliferation with CFA IC 50 s of <100nM in TNBC cell lines and <25nM in ER+ cell lines, including MCF7, T47D and ZR751 compared to CDK4/6i which ranged from 200nM to 500nM in MCF7 cells. We demonstrated that our CDK4/6 bifunctional degraders were significantly more potent in vitro than the CDK4/6i ribociclib and palbociclib, and the increased activity was due to CRBN mediated target degradation. Our CDK4/6 bifunctional degraders display excellent pharmacokinetic properties in mice with half-lives between 2-10 hours, oral bioavailability between 50-96%. MCF7 xenograft results with our proof-of-concept CDK4/6 degrader showed dose-dependent tumor growth inhibition and greater potency compared to the clinical CDK4/6i ribociclib. We saw tumor regression with our degrader at higher doses which we did not see at any dose of CDK4/6i. Conclusions: Our CDK4/6 bifunctional degraders display excellent single agent activity in vitro and in vivo particularly in comparison to clinically approved CDK4/6i, indicating that using a degrader approach to targeting this pathway may be more effective than current inhibitor therapies.

Pre-clinical