Abstract 2340: Thymidine kinase activity as a response marker for CDK 4/6 inhibition
Authors: Shapiro Geoffrey , Neumuller Magnus , Lööf Sara , Bacanu Smaranda , Seki Takahiro , Hilton John , Do Khan , Chau Nicole , Gandhi Leena , Gibson Joseph W. , Distel Robert , Niekrasz Pawel , Suh Edward M. , Bergqvist Mattias 
Published: July 2017

Abstract
Selective CDK4/6 inhibition is now part of standard treatment for HR+ breast cancer. There is a pressing need for a practical biomarker that can provide early indication of the biologic activity of these agents and correlate with clinical outcome. Expression of thymidine kinase (TK) occurs in actively proliferating cells, is E2F-dependent and is downregulated after CDK4/6 inhibitor-mediated G1 arrest. Here, we have investigated TK activity (TKA) as a pharmacodynamic marker for CDK4/6 inhibition in both preclinical and clinical contexts and demonstrate the potential of this assay as CDK4/6 inhibitors are further developed. TKA in response to the CDK 4/6 inhibitor palbociclib (palbo) was studied in cell culture, mouse models and samples from clinical studies. TKA was determined by the DiviTum Assay (Biovica, Sweden). In culture, the intracellular TKA and TK release in response to palbo were determined by analysis of cellular extracts and tissue culture media. In vivo, mice bearing human xenografts were treated with vehicle or palbo to determine effects on TKA in serum and in tumor extracts. Finally, serum TKA (s-TKA) was assayed in patient samples procured before and after palbo treatment in a Phase 1 study of palbo and the MEK inhibitor PD0325901 (NCT02022982). Palbo was administered on 3/4 (21 of every 28 days) or 4/4-week (continuous) schedules. In K562 cells, intracellular TKA levels exhibited a clear dose response to palbo. Reductions in TKA were seen at lower drug concentrations than those affecting cell viability. Similar results were observed in MCF-7 xenografts, where lower TKA in serum and tumor was observed after palbo treatment, whereas in E0771 xenografts, no change in TKA was observed. Clinically, serum samples were obtained before and at various time points after palbo exposure in 20 cancer patients. One patient, who discontinued study treatment at the end of cycle 1, demonstrated an increase in s-TKA. In contrast, for the other 19 patients, there was a marked decrease in s-TKA at day 21. Pre-treatment s-TKA values were higher than C1D21 (paired t-test, p&lt;.0001). In 11 patients there was a reduction in s-TKA to below the assay detection limit. Five patients exhibited a reduction in s-TKA within the working range, with mean residual activity at day 21 of 32%. In the 6 patients receiving continuous palbo, s-TKA remained low after day 21. Of the 14 patients on the 3/4-week schedule, 9 exhibited an increasing level of s-TKA between day 21 and day 1 of cycle 2, consistent with reversible effects of palbo on cell cycle progression after cessation of exposure. These data suggest that s-TKA reflects MOA response to the selective CDK4/6 inhibitor palbo. The DiviTum assay may provide a practical, non-invasive tool for monitoring the effects of CDK4/6 inhibitors in both preclinical models and patients. We plan inclusion of the assay into additional studies to determine whether the assay can predict clinical outcome or portend development of resistance.
Citation Format: Geoffrey Shapiro, Magnus Neumuller, Sara Lööf, Smaranda Bacanu, Takahiro Seki, John Hilton, Khan Do, Nicole Chau, Leena Gandhi, Joseph W. Gibson, Robert Distel, Pawel Niekrasz, Edward M. Suh, Mattias Bergqvist. Thymidine kinase activity as a response marker for CDK 4/6 inhibition [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 2340. doi:10.1158/1538-7445.AM2017-2340