Targeting The P27kip1/Cdk4/Cdk2/Rb Axis In Breast Cancer Using A Peptidomimetic Of Brk'S Sh3 Domain

Purpose: Cyclin D-cdk4 (DK4) has been a highly sought after therapeutic target because it drives cancer proliferation in a majority of human tumors. We have explored the clinical utility of a recently discovered mechanism of cell cycle control exerted on DK4 by p27Kip1 and its activator, the Breast tumor Related Kinase (Brk), in predicting responsiveness to therapy and as a new target for treatment. Although known as a DK4 assembly factor and cdk2 inhibitor, p27 also acts as a DK4 ON/OFF “switch.” Tyrosine (Y) phosphorylation of p27 (pY) by Brk gatekeeps both ATP binding and CAK phosphorylation of cdk4’s T loop, essential for DK4 activation. This function is restricted to cdk4: p27’s association with cdk2, whether Y phosphorylated or not, appears to be inhibitory. However, in vivo Y phosphorylated p27 is a target for cdk2-dependent ubiquitin-mediated degradation, reducing p27’s association with cdk2, indirectly activating this complex. We showed that blocking p27 pY inactivates cdk4 directly AND cdk2 indirectly, and thus represents a novel way to block cancer cell proliferation. pY also serves as a predictive biomarker of cdk4 activity and tumor response. Methods: We used a small peptide, ALT, which contains a portion of Brk’s SH3 domain. ALT binds to p27, blocks Brk’s association and ability to phosphorylate p27, inhibiting cdk4 and increasing p27’s ability to inhibit cdk2. We engineered a lipid-based nanoparticle delivery vehicle (NP-ALT), permitting us to test ALT as a first generation therapeutic in breast cancer cell lines that were both responsive and non-responsive to cdk4i therapy. ALT was also used with Palbociclib to determine if combination therapy reduced drug resistance. We developed a dual IHC assay for p27 and pY, which we used to analyze paraffin-embedded, archival human tumor samples, to determine whether we could pinpoint patients who would have responded to cdk4 inhibition therapy. Results: NP-ALT blocks pY, cdk4 and cdk2 activity, and proliferation in both Palbociclib sensitive and resistant cell lines. As a dual therapy, ALT treatment synergized with Palbociclib to arrest cells for u003e30 days, increased senescence, and in animal models caused tumor regression instead of just slowing tumor growth as seen with Palbociclib alone. Analysis of human cancer, obtained from archival sources, demonstrated that pY is never detected in quiescent benign mammary tissue, but is detected in about half of the advanced ER/PR+/Her2- tumors analyzed, and using explant culture techniques, we were able to stratify pY with Palbociclib response. Conclusion: Use of an Brk SH3 based peptide (NP-ALT) has proven effective in blocking p27 pY, inhibiting both cdk2 and cdk4, inducing senescence and increased durability. pY levels correlate with Palbociclib sensitivity in low, moderate and non-responders, suggesting that this may be a biomarker highlighting responsiveness to cdk4i therapy. Citation Format: Stacy W. Blain, Jason Quinones, Priyank Patel, Vladislav Tsiperson, Susan Gottesman, Jonathan Somma, Yun Wu. Targeting the p27kip1/cdk4/cdk2/Rb axis in breast cancer using a peptidomimetic of Brk’s SH3 domain [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2348. doi:10.1158/1538-7445.AM2017-2348