Abstract P4-11-02: Characterization of BTX-9341, a bifunctional degrader of CDK4 and CDK6 for HR+/HER2- breast cancer

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 upregulation of the 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% of patients exhibiting innate resistance and up to 70% of patients developing acquired resistance after 3 years on therapy (Scheidemann, 2021). To address acquired resistance, we sought to apply 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. Breast cancer cell lines treated with BTX-9341 for 6 hours demonstrated up to 85% degradation of CDK4 and CDK6 with DC50s <1nM. BTX-9341 showed specificity for CDK4/6, with minimal off-target binding or degradation. CDK4/6 phosphorylates retinoblastoma protein (Rb) which releases the transcription factor E2F, inducing the expression of genes including CDK2 and Cyclin E which promote cell cycle progression. To determine the effect of BTX-9341 on downstream signaling, we examined Rb phosphorylation by in-cell western, and E2F target gene expression by qPCR and western. BTX-9341 was potent in all assays, with phospho-Rb IC50s <30nM, and E2F target gene downregulation at concentrations as low as 10nM. These downstream effects were sustained for 72 hours with BTX-9341 treatment, whereas rapid recovery was seen with CDK4/6i treatment. We used a 2D colony formation assay (CFA) to assess inhibition of proliferation. BTX-9341 potently inhibited cell proliferation with CFA IC50s of 20-50nM while CDK4/6i had CFA IC50s of 50-1000nM. 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. Combining BTX-9341 with selective estrogen receptor degraders (SERDs) resulted in a synergistic anti-proliferative effect in HR+ breast cancer cells. In a palbociclib-resistant HR+/HER2- cell line model BTX-9341 maintained a low CFA IC50 (<150nM) while CDK4/6i displayed IC50s > 1µM. [SG1] BTX-9341 showed enhanced inhibition of Rb phosphorylation and a stronger synergistic effect with SERDs in CFA assays than CDK4/6i in this palbociclib resistant model. BTX-9341 displays excellent pharmacokinetic properties which allowed for oral dosing in xenograft studies. In several breast cancer xenograft models, BTX-9341 showed dose-dependent tumor growth inhibition, tumor regression at higher dose levels, and was effective with multiple alternate dosing regimens. These results demonstrate the excellent activity of BTX-9341 as a single agent and in combination with SERDs, particularly in comparison to CDK4/6i. BTX-9341 mediated downregulation of CDK2 and cyclin E1, which are known to drive resistance to CDK4/6i, was more pronounced and more sustained than that mediated by CDK4/6 inhibitors including approved inhibitors such as palbociclib and inhibitors under clinical development such as PF-07220060. BTX-9341 maintained single agent activity and synergistic activity with SERDs in a CDK4/6i resistant model. These results indicate that utilizing a degrader such as BTX-9341 may be more effective in a post CDK4/6i setting than switching to a new CDK4/6 inhibitor. Based on this data, we have initiated a Phase 1 clinical trial with BTX-9341 as a monotherapy and in combination with fulvestrant, for HR+/HER2- breast cancer patients who have progressed on CDK4/6i therapy. 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, Kirti Kandhwal Chahal, Angela Pasis, Casey Carlson, Qiao Liu, Arvind Shakya, Akinori Okano, Shenlin Huang, Aparajita Chourasia, Leah Fung. Characterization of BTX-9341, a bifunctional degrader of CDK4 and CDK6 for HR+/HER2- breast cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P4-11-02.