Abstract P5-05-14: Cyclin-dependent kinases inhibitors improve antimetabolite drug potency depending on SAMHD1 expression
Authors: Felip Eudald , Badia Roger , Margelí Mireia , Castellví Marc , Quiroga Vanesa , Teruel Iris , Cirauqui Beatriz , Romeo Margarita , Ezeonwumelu Ifeanyi Jude , Riveira-Muñoz Eva , Esté José , Ballana Ester 
Published: March 2020

Abstract
Introduction Cyclin-dependent kinase (CDK) 4/6 inhibitors represent a new paradigm in the treatment of hormone receptor (HR) positive and HER-2 negative metastatic breast cancer (MBC). However, despite their evident clinical benefit, the cellular and molecular mechanisms underlying treatment efficacy and drug failure are still largely unknown. CDK4/6 inhibitors block the cyclin D1-CDK4/6 interaction, leading to inhibition of cell cycle progression through the G1/S phase and inhibiting cell proliferation. SAMHD1 is a dNTPase that maintains the intracellular dNTP pool at levels adequate for DNA replication and repair. SAMHD1 function is regulated by phosphorylation, a process controlled by CDKs. Moreover, SAMHD1 has also been shown to modify the efficacy of different nucleotide analogues. Here, we aim to evaluate the capacity of selective CDK4/6 inhibitors to modify the efficacy of different antimetabolites currently used for cancer treatment. Material and methods: Cytotoxic activity of 5-fluorouracil, its prodrug capecitabine and the anti-folate pemetrexed was evaluated alone or in combination with the CDK4/6 inhibitor palbociclib in breast cancer cell lines (T47D and MDA-MB-468) and primary cells expressing or not SAMHD1 through HIV-2 Vpx mediated degradation. Cytotoxicity was measured either by colorimetric tetrazolium dye MTT method or flow cytometry. Drug combinations were evaluated by calculating the combination index (CI) through the ixobologram equation, considering a synergic drug combination a CI &lt;1. Intracellular dNTP content was determined using a polymerase- based method. Expression of relevant genes was measured by qPCR mRNA detection. SAMHD1 expression and phosphorylation were measured by Western blot. Results Analysis of cell cycle profile and protein expression of drug-treated cells confirmed that CDK4/6 control SAMHD1 phosphorylation and decrease intracellular dNTPs. CDK4/6 inhibitors palbociclib, ribociclib and abemaciclib blocked SAMHD1 phosphorylation, whereas SAMHD1 protein expression was not affected. In addition, we observed a concomitant dephosphorylation and decreased expression of Rb, the natural substrate of CDK6, and a known marker of CDK4/6 inhibitor activity. In combination, palbociclib enhanced the efficacy of pemetrexed or 5-fluorouracil up to 6-fold, indicating strong synergy (CI=0,0049-0,404). Interestingly, the synergistic effect was lost in cells not expressing SAMHD1. Palbociclib did not change antimetabolite efficacy in the breast cancer MDA-MB-468 cell line. However, Rb and pRb were not detected in MDA-MB-468 cells by mRNA qPCR or Western blot. Conclusion CDK4/6 inhibitors induce the activation of SAMHD1. Taken together, our results suggest that CDK4/6 inhibitor-mediated enhancement of antimetabolite activity is dependent on the regulation of SAMHD1 function. CDK4/6 inhibitors restrict cells in G1 but also prevent SAMHD1 phosphorylation that sensitizes tumoral cells to antimetabolite drugs. Thus, pharmacological modulation of SAMHD1 activity has the potential to improve the efficacy of cancer therapies and paves the way to the identification of malignancies that may be treated with new drug combinations. Combinations with selective CDK4/6 inhibitors plus pemetrexed or capecitabine could be effective in the treatment of cancer after first line drug-failure. SAMHD1 could be a useful biomarker to predict treatment response.
Citation Format: Eudald Felip, Roger Badia, Mireia Margelí, Marc Castellví, Vanesa Quiroga, Iris Teruel, Beatriz Cirauqui, Margarita Romeo, Ifeanyi Jude Ezeonwumelu, Eva Riveira-Muñoz, José Esté, Ester Ballana. Cyclin-dependent kinases inhibitors improve antimetabolite drug potency depending on SAMHD1 expression [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-05-14.