Improved tools to evaluate the sensitivity of cancer cells to CDK4 inhibitors

- Introduction :The complexes between D-type cyclins (CCND1-3) and cyclin-dependent kinases CDK4/6 are key regulators of the cell cycle entry, by phosphorylating the onco-suppressor retinoblastoma protein (pRb). Their deregulated activity is a major oncogenic event in most types of tumours. Our group identified the T172-phosphorylation of CDK4 as the determining step for its activation and have described critical regulations of this event in various cell systems (1,2,3). CDK4/6 inhibitory compounds have been tested in a growing number of phase II/III clinical trials. Due to unprecedented improvement of the progression-free survival (4), FDA approved Palbociclib (PD0332991), the first specific inhibitor of CDK4/6, for the treatment of advanced estrogen receptor positive breast tumours in combination with letrozole. Breast cancers are mostly classified in five major groups that are clinically relevant as they influence the sensitivity to therapy. In order to predict tumour’s response to Palbociclib and develop associated biomarkers, cancer cell line panels have been used to evaluate the impact of the drug on cell growth (5,6,7). However, divergent IC50s have been described probably due to the different assays used to quantify the drug effects.- Aims :To develop and validate a drug response assay that more directly assesses the cell cycle responsiveness to CDK4/6 inhibition, and compare this to the expression of cell cycle effectors.- Methods :We have studied a subset of 20 breast cancer cell lines representative of different Palbociclib sensitivities and molecular subtypes. We adapted the 5-Bromo-2’-deoxyuridine (BrDU) proliferation method, which labels and detects neo-synthesized DNA, to a 96-wells plate format. Drug effects were also determined in parallel with the sulforhodamine B (SRB) (which measures protein content) and the MTT assays (which reflects a mitochondrial enzymatic activity) to compare our results to the previous ones. The protein levels of total and phosphorylated pRb, CDK4, Cyclin D1, Cyclin D3, Cyclin E and p16 were also evaluated.- Results and discussion :The BrDU proliferation assay quantifies the proportion of cells actively synthesizing DNA, a readout directly associated with cell cycle progression. The proportion of labelled cells after a 1h BrDU pulse in control asynchronous growth varied from 20 to 50%. In 13 out of the 20 cell lines examined, Palbociclib reduced this proportion in a dose-dependent way. In all except one, the cell lines responses to the drug were comparable to Finn’s data (5), although with lower and narrower IC50 ranges than with the ones reported. Furthermore, our IC50 values are closer to the concentration required to block the CDK4 activity in vitro (15 nM). However, sensitivities measured by the three techniques were distinct. The impact of CDK4/6 inhibition was less pronounced and more in line with the Finn’s results when Palbociclib action was assayed by sulforhodamine or MTT assays. Using the BrDU assay, all luminal and most ERBB2-amplified cell lines were sensitive to the drug while, interestingly, basal-like cells were either sensitive or insensitive. Phosphorylated pRb was absent in four insensitive cell lines but detected in three other totally or partially resistant ones. p16 was either present or absent in sensitive cell lines while it was highly expressed in completely resistant cells. Overall, none of these G1 phase regulators, used alone, could serve as a qualitative biomarker of CDK4 inhibitor sensitivity.- Conclusions :The BrDU labeling is the best method to evaluate CDK4 inhibitors sensitivity. The breast cancer cell line panel is a useful tool to identify molecular determinants of CDK4 inhibition and proteomic/genomic characterization is on going to define biomarkers of CDK4 inhibitor sensitivity.- References :1. Bockstaele, L. et al. (2006). Mol.Cell Biol. 26, 5070-5085.2. Paternot, S. et al. (2010). Cell Cycle 9, 689-699.3. Bisteau, X. et al. (2013) PLoS Genet. 9(5):e1003546. doi: 10.1371/journal.pgen.1003546.4. Finn, R.S. et al. (2015). Lancet Oncol.16, 25-35.5. Finn, R.S. et al. (2009). Breast Cancer Res. 11, R77.6. Garnett, M.J. et al. (2012). Nature. 483, 570–575.7. Barretina J. et al. (2012) Nature. 483: 603–607.