Abstract B018: Triple CDK2/4/6 inhibition via p27 modulation: A novel approach to targeting cell cycle dysregulation
Authors: Chen Grace , Zisman Natalia , Allamneni Krishna , Blain Stacy W 
Published: February 2024

The dysregulation of cell cycle control is a hallmark of cancer and targeting cyclin-dependent kinases (CDKs) has emerged as a promising strategy in advanced breast cancer. The G1 CDKs are the recipients of the oncogenic signals that travel from the cell surface to the nucleus, and as such, selective inhibition of CDK2, CDK4 and CDK6 has been a goal in oncology for several decades. We present a novel approach involving modulation of the CDK interacting protein p27Kip1, to simultaneously inhibit CDK2/4/6. p27 is responsible for turning the G1 CDKs ON and OFF during periods of proliferation and quiescence, respectively, and this transition is mediated by a specific modification to p27 itself, by the tyrosine kinase BRK (Breast tumor Related Kinase). A naturally occurring ALTernatively spliced form of BRK, named ALT, can bind to p27, blocking BRK's association with and phosphorylation of p27. The binding of ALT to p27 locks both CDK4/6 and CDK2 into the OFF conformation, inhibiting activity of both kinases. p27’s association with CDK2 also prevents p27’s degradation. In in vitro kinase assays, inducible ALT expression in ER+ breast cancer cells, inhibited CDK4/6 and CDK2 activities simultaneously, resulting in durable growth arrest. As p27 associates preferentially with the G1 CDKs, the specificity of ALT for these kinases is achieved by p27 itself, thus avoiding off-target kinase interactions with the rest of the CDK kinome. We engineered IpY.20, a smaller, more stable variant of ALT, formulated within a liposome to increase its serum residence time and facilitate delivery into tumor cells. IpY.20 inhibits cell proliferation of HR+ and triple negative (TN) breast cancer, ovarian, uterine and NSCLC, preferentially over non-tumor cells, supporting its clinical utility. Unlike CDK4/6i and CDK2i which only cause cytostasis, IpY.20 arrests proliferation and induces cell death, demonstrating that p27 inhibition is a viable therapeutic approach to combat CDK4/6i-dependent drug resistance. In vivo, IpY.20 reduces tumor volume in palbociclib-resistant ER+ xenografts, TN xenografts and the MMTV-ErbB2 spontaneous breast cancer model. IpY.20 does not cause the adverse effects seen in the presence of CDK4/6i or CDK2i: IpY.20 does not cause neutropenia, and in combination with palbociclib, IpY.20 rescues the CDK4/6i-dependent decrease in neutrophils, suggesting better tolerability in patients. IpY.20 is detected in tumors within 15 min. of single dose I.V. administration and continues to be detectable for 24 h. IpY.20 is currently completing CMC process optimization, batch-to-batch consistency, and stability as it moves into the IND program. These findings underscore the significance of targeting CDK2, CDK4, and CDK6 simultaneously via p27 modulation as a potential therapy. Given the difficulties in inhibiting the CDK2/4/6 hub with small molecule CDKi (off-target kinase selectivity, adverse effects, lack of tumor cell killing), targeting p27 may provide advantages to the post-CDK4/6i setting.

Citation Format: Grace Chen, Natalia Zisman, Krishna Allamneni, Stacy W Blain. Triple CDK2/4/6 inhibition via p27 modulation: A novel approach to targeting cell cycle dysregulation [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B018.