Characterization of BTX-9341, a bifunctional degrader of CDK4 and CDK6 for HR+/HER2- breast cancer and glioblastoma multiforme.
Authors: Majeski Hannah , Chahal Kirti , Pasis Angela , Carlson Casey , Liu Qiao , Shakya Arvind , Okano Akinori , Huang Shenlin , Chourasia Aparajita Hoskote , Fung Leah M. 
Published: June 2024

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Background: CDK4/6 inhibitors (CDK4/6i) are used to treat HR+/HER2- breast cancer, but patients develop resistance via many mechanisms, many of which involve on the upregulation of the cyclin D-CDK4/6 or cyclin E-CDK2 signaling nodes. This has been shown to limit the effectiveness of CDK4/6i in HR+ breast cancer with up to 20% patients exhibiting innate resistance and up to 70% patients developing acquired resistance after 3 years on therapy (doi:10.3390/ijms222212292). Glioblastoma multiforme (GBM) is an aggressive form of brain cancer with a high frequency of dysregulated CDKN2A-cyclin D-CDK4/CDK6 signaling, however CDK4/6i have shown limited efficacy in GBM due to poor brain exposure. Methods: 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. We examined retinoblastoma (RB) phosphorylation by in-cell western, E2F target gene expression by western and qPCR, cell cycle progression by flow cytometry, and cell proliferation by colony formation assay (CFA). Results: Breast cancer cell lines treated with BTX-9341 showed up to 85% degradation of CDK4 and CDK6 with DC50s <1nM. CDK4/6 phosphorylates RB which releases the transcription factor E2F, inducing the expression of genes including CDK2 and Cyclin E which promote cell cycle progression. BTX-9341 displayed phospho-RB IC50s <30nM, E2F target gene downregulation and G0/G1 cell cycle arrest at concentrations as low as 10nM. These downstream effects including inhibition of CDK2 and Cyclin E transcription were sustained up to 72 hours with BTX-9341 treatment but recovered at 24 hours with palbociclib treatment. BTX-9341 inhibited cell proliferation with CFA IC50s of 20-50nM while CDK4/6i had CFA IC50s of 50-1000nM. In a palbociclib-resistant HR+/HER2- cell line model BTX-9341 maintained a low CFA IC50 (<150nM). In HR+ breast cancer cells, BTX-9341 in combination with fulvestrant had a synergistic anti-proliferation effect. BTX-9341 has good oral bioavailability and high brain to plasma ratios which allowed for oral dosing and the assessment of BTX-9341 in both breast cancer and GBM in vivo models. 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. BTX-9341 inhibited tumor growth and promoted survival in both intracranial and subcutaneous GBM efficacy models. Conclusions: BTX-9341, a degrader of CDK4 and CDK6 and inhibitor of CDK2 and Cyclin E transcription, displayed enhanced activity compared to CDK4/6i in breast cancer and GBM in vitro and in vivo. This indicates that a degrader approach to targeting this pathway in breast cancer may be more effective than current therapies, and that BTX-9341 may also be a promising candidate for brain metastases and GBM.