Abstract PO5-05-02: Discovery of BTX-9341, a bifunctional degrader of CDK4 and CDK6 for HR+/HER2- breast cancer

Abstract

Abstract CDK4/6 inhibitors (CDK4/6i) such as palbociclib, abemaciclib and ribociclib are used to treat HR+/HER2- breast cancer, but patients can develop resistance via many mechanisms, several of which converge on the upregulation of cyclin D-CDK4/6 signaling node. 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). To address acquired resistance, we sought a degrader approach. We utilized our PRODEGY platform of Cereblon (CRBN) binders to synthesize CRBN mediated CDK4/6 bifunctional degraders and identified BTX-9341 as a development candidate due to its potency in vitro, in CDK4/6i resistant models and its effectiveness at inhibiting tumor growth in vivo. Degradation of CDK4/6 by immunoblot analysis of the triple negative breast cancer (TNBC) cell line, MDA-MB-231, treated with BTX-9341 for 6 hours showed up to 85% degradation of CDK4 and CDK6 with DC50s of < 1nM. Similar degradation depth and DC50 values were seen in HR+ breast cancer cells including T47D, MCF7 and ZR-751. CDK4/6 phosphorylates the protein RB which releases the transcription factor E2F, inducing the expression of genes which promote cell cycle progression. To determine the effect of CDK4/6 degradation on downstream signaling, we examined RB phosphorylation by in-cell western. Twenty-four hours treatment with BTX-9341 blocked RB phosphorylation robustly, with phospho-RB IC50s at < 30nM in HR+ breast cancer cells as well as MDA-MB-231 cells. Cell cycle analysis by staining with propidium iodide after 24 hours of treatment with BTX-9341 caused 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. BTX-9341 showed potent inhibition of cell proliferation with CFA IC50s of < 50nM in TNBC cell lines and < 20nM in HR+ cell lines. We demonstrated that BTX-9341 was significantly more potent in vitro than the CDK4/6i, which had CFA values between 50 and 700nM in HR+ cell lines and between 100 and 1000nM in TNBC. This increased activity was due to CRBN mediated target degradation, as demonstrated by a shift in CFA IC50 values in a CRBN knockout cell line towards the values seen with the inhibitors. In palbociclib-resistant HR+/HER2- cell lines models which exhibit high CDK6 expression, BTX-9341 maintained a low CFA IC50 (< 150nM) while other CDK4/6 inhibitors displayed micromolar CFA IC50s. BTX-9341 displays excellent pharmacokinetic properties and oral bioavailability, which allowed for oral dosing in xenograft studies. MCF7 xenograft results with BTX-9341 showed dose-dependent tumor growth inhibition, tumor regression at higher doses and overall greater potency compared to the clinical CDK4/6i. BTX-9341 shows good exposure in the brain and exhibits tumor growth inhibition in an intracranial MCF-7 xenograft model, indicating that BTX-9341 could inhibit the growth of brain metastases. These results show that BTX-9341 displays excellent single agent activity in vitro and in vivo particularly in comparison to clinically approved CDK4/6i and that this activity is maintained in CDK4/6i resistant models. This indicates that a degrader approach to targeting this pathway may be more effective than current therapies, and that using this modality in a post CDK4/6i setting may be more effective than switching CDK4/6 inhibitors. Reference: Scheidemann, Erin R, and Ayesha N Shajahan-Haq. “Resistance to CDK4/6 Inhibitors in Estrogen Receptor-Positive Breast Cancer.” International journal of molecular sciences vol. 22,22 12292. 14 Nov. 2021, doi:10.3390/ijms222212292 Citation Format: Hannah Majeski, Akinori Okano, Kirti Chahal, Angela Pasis, Casey Carlson, Arvind Shakya, Qiao Liu, Shenlin Huang, Aparajita Hoskote Chourasia, Leah Fung. Discovery of BTX-9341, a bifunctional degrader of CDK4 and CDK6 for HR+/HER2- breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-05-02.