Signalling involving MET and FAK supports cell division independent of the activity of the cell cycle-regulating CDK4/6 kinases

Deregulation of cyclin-dependent kinases 4 and 6 (CDK4/6) is highly prevalent in cancer; yet, inhibitors against these kinases are currently used only in restricted tumour contexts. The extent to which cancers depend on CDK4/6 and the mechanisms that may undermine such dependency are poorly understood. Here, we report that signalling engaging the MET proto-oncogene receptor tyrosine kinase/focal adhesion kinase (FAK) axis leads to CDK4/6-independent CDK2 activation, involving as critical mechanistic events loss of the CDKI p21CIP1 and gain of its regulator, the ubiquitin ligase subunit SKP2. Combined inhibition of MET/FAK and CDK4/6 eliminates the proliferation capacity of cancer cells in culture, and enhances tumour growth inhibition in vivo. Activation of the MET/FAK axis is known to arise through cancer extrinsic and intrinsic cues. Our work predicts that such cues support cell division independent of the activity of the cell cycle-regulating CDK4/6 kinases and identifies MET/FAK as a tractable route to broaden the utility of CDK4/6 inhibitor-based therapies in the clinic.

were obtained from Qiagen.
RNAi screen: Screens used the kinase-covering component of the Dharmacon siGENOME SMARTpool TM library. Library pools were mixed at equimolar ratio with SMARTpool TM oligonucleotides targeting TP53 or non-targeting oligonucleotide, then reverse transfected at a combined concentration of 20nM into HCT116-PSLD, seeded into triplicate wells of opaque, tissue culture treated, 96-well plates with transparent base (Packard View plates, Perkin Elmer). Transfected cells were incubated for 24 h prior to treatment with CDK4/6 inhibitor palbociclib (450nM) or vehicle (DMSO) for 24 h. Plates were fixed in 4% formaldehyde for 10 minutes at room temperature, Prior to imaging the fixed cells were permeablized in TBS buffer (25 mM Tris, 140 mM NaCl, pH 7.5) containing 0.1% Triton X-100 supplemented with 1g/ ml Hoechst 33342 DNA dye and imaged using an INCell Analyzer 3000 (GE Healthcare) or an Opera (Perkin-Elmer) high-content imager platform.
Data were processed using CellProfiler open-source image analysis software [3] as described in [4]. A custom perl script, described in [4] was used to compute the percentage of imaged cells with nuc/cyto fluorescence ratio of > 1.5 in each well.
Protein concentrations were measured by BCA protein assay (Pierce). Samples were separated on SDS polyacrylamide gels and transferred to an Immobilon-FL membrane (Millipore), before incubation with primary antibodies overnight at 4 °C. Membranes were washed with TBST (25 mM Tris, 140 mM NaCl, 0.1% Tween-20, pH 7.5) and probed with IRDye 680-or IRDye 800CW-conjugated secondary antibodies (LI-COR) for 1 h at room temperature. The imaging and quantification of protein bands was carried out using an Odyssey CL (LI-COR) infrared imaging system. Immunoprecipitation and in vitro kinase assay. Cells were lysed in ice-cold immunoprecipitation (IP) buffer (50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 50 mM NaF, 20 mM β-glycerophosphate, 4 mM Na 3 VO 4 , 1 mM DTT and 0.1% Tween-20, pH 8), lysates were cleared by centrifugations at 10,000 x g at 4 °C. Cleared supernatants were mixed with 50 µl of Dynabead coupled protein A (Life Technologies) bound with 2 µg of IgG.
Beads were incubated with rotation for 20 min at 4 °C, then washed three times in IP buffer.
Precipitates were analysed using SDS polyacrylamide gel electrophoresis followed by immunoblotting or were used in vitro kinase activity assays. Kinase activity assays were performed as previously described [5], using as a substrate GST-RB1 763-928 recombinant protein (500 ng/reaction), purified as described [6], in 20 µl kinase reaction buffer (25 mM HEPES, 0.1% β-mercaptoethanol, 0.1 mM EGTA, 25 mM MgCl 2 and 400 µM ATP, pH 7.9) added directly to the beads. The mixture was incubated for 10 min at 30 °C, reaction products were separated on SDS polyacrylamide gels and subjected to immunoblot analysis using antibody directed against the proline-directed phosphorylation site Ser807 of human RB1 (anti-pRB1 S807 ) to quantify substrate phosphorylation and rabbit anti-GST antiserum, to visualise substrate present in each reaction.
Cell-based immunostaining assay: Antibody staining was performed in 96 well plates as in [1]. Cells grown in 96-well plates were fixed with 4% formalin for 10 minutes, permeablized in TBS (25 mM Tris, 140 mM NaCl, pH 7.5) supplemented with 0.1% Triton X-100 and then washed in TBS containing 0.1% Tween 20 (TBST) and 5% skimmed milk. Primary antibodies were diluted to 1:250 in TBST. Cells were incubated with diluted antibodies overnight at 4 ºC, prior to washing in TBST buffer. Cells were subsequently probed with Alexafluor-647®-coupled secondary antibodies containing 1 g/ mL Hoechst 33342. Cells were imaged as described preciously [4]. A custom script, described in [4], was used to calculate the percentage of cells with a nuclear fluorescence intensity above threshold.
Detection of senescence-associated β-Galactosidase activity: Detection of SA-β-Gal activity using with C 12 FDG staining (Life Technologies) was carried out as previously described [7]. Fluorescence intensity data within the perinuclear region of individual cells was quantified using CellProfiler free open-source image analysis software (http://cellprofiler.org/). Images of Hoechst 33342 fluorescence were used to generate a nuclear mask with which each imaged cell was located and subsequently measured for perinuclear staining in corresponding C 12 FDG fluorescence images. Thresholds to gate for cells exhibiting SA-β-Gal positivity above background were determined using histogram plots generated using the 'R' free statistical software (https://www.r-project.org). A custom perl script, described in [4], was used to calculate the percentage of cells with a perinulcear C 12 FDG intensity above threshold.
Time-lapse microscopy. Time-lapse observations were carried out in cells expressing H2B-GFP using a IncuCyte TM live cell analysis system (Essen Bioscience). Cells were seeded into 96-well plates 24 h prior to treatment, and followed by fluorescence imaging every two 4 hours at 37 °C with 5% CO 2 . Data were collected from a minimum of three parallel wells per condition.
Clonogenic assay. Cells seeded into 6-well plates were treated with drug(s) for 5 days, then detached using trypsin. Cells were counted and reseeded at fixed cell numbers into duplicate wells of 6-well plates into drug-free medium. After 14 days cells were fixed with 4% formaldehyde, and stained with 0.5% crystal violet dye dissolved in 25% methanol. Excess dye was thoroughly removed by repeat washing with tap water. Digital images were generated using a PC desktop scanner, then bound crystal violet dye was eluted using 30% v/v acetic acid, 30% v/v DMSO, 1% SDS and photometrically quantified at 590 nm absorbance.
In vivo human xenograft studies: Compounds were dissolved in sodium lactate pH 4.0 (palbociclib) or sterile saline (crizotinib) and administered at 100 mg/ kg once daily p.o.. Six to seven week-old female NCr-Foxn1 nu mice were inoculated s.c. into the right flank with 3 x 10 6 HCT116 TP53 -/cells. Treatment was initiated when tumour grafts reached 5 to 6 mm in diameter when mice were randomly assigned to each treatment group. Tumour size was measured twice per week using callipers and volumes were calculated using the formula 1/2 (length (mm)) × (width (mm)). Pharmacokinetic studies used a single dose of 100 mg/ kg palbociclib and/or crizotinib, administered p.o. Plasma and tumour samples were collected at 4, 16 and 24 h post-administration, and analysed as described previously [8]. For pharmacodynamic studies, tumour samples were collected at 24 h post-administration.
Laboratory staff providing animal care, including treatment and assessment of tumour growth were blinded to the group allocation of mice. Animal experiment reporting follows guidelines provided in [9]. Post-hoc analysis revealed power of the study to detect a difference in tumour volume between any two of the treatment groups of > 95.9% based on data from the study end (day 13), and > 68.9 based on data for the time at which drug administration was ended (day 8).
Statistical Analyses. Statistical assessments used two-tailed unpaired Student's t-test, 1way ANOVA and 2-way ANOVA. Calculations were performed using GraphPad 7.0.
Calculation of p-values for pathway enrichment analysis, measuring the likelihood that the intersection between the input genes and a particular network was obtained by chance, were carried out using MetaCore TM as described in the user manual. All data are expressed as normalized means and standard deviation (SD) or standard error (SE) from multiple independent measurements, as stated. Z-scores, describing the distance from the target mean to the population mean in units of standard error, were computed using Z-test statistics in EXCEL. Gene clustering was performed using the heatmap function within 'R' 5 open source software (https://www.r-project.org). Tests for interaction between target knockdown and treatment were performed as previously described [1]. The degree (index) of interaction (SI) was calculated by subtracting the observed combined effect of treatments from the product of the observed individual effects. https://clincalc.com/stats/Power.aspx. was used to conduct post hoc power analysis relating to the in vivo xenograft studies.  c Graph depicting the fraction of cells with GFP-PSLD nuc/cyto ratio > 1.5 (low CDK2). Data represent mean ±SD for n = 3 independent repeats. Cells treated as in a; **p ≤0.01, ***p ≤0.001, ns p> 0.05, 2-tailed Student's t-test.

Supplementary
d Population scatter plot, depicting the relationship between CDK2 activity, assessed using the GFP-PSLD reporter, and CDK4/6 activity, assessed using immunostaining for pRB1 S780 . Representative images of HCT116 treated with 500 nM inhibitors for 120 h and detection of SA-β-gal activity by incubation with C 12 FDG at acidic pH. Scale bar = 20 µm.
b Distribution of perinuclear C 12 FDG-fluorescence intensity in cells treated as in a. Dotted lines indicated threshold used to identify cells with high SA-β-gal activity. Results for TP53 WT and TP53 -/-HCT116 are shown.
c CI value plots for data shown in Fig. 4d, evaluating the interaction of MET and CDK4/6 inhibition on effecting SA-β-gal activity in the cells population. Data presenting mean ± range for 2 independent repeats, expressed as fractions relative to vehicle. 2-way ANOVA comparing in (a) the effect size of the combination in control and RBdepleted cells (**p≤ 0.01, ***p≤ 0.001) or the effect size of single agent with that of their combination in RB-depleted cells ( ns p > 0.05), in (b) the effect size of single agent palbociclib with that of the combination ( ns p > 0.05). 1-way ANOVA assessing in (b) the effect of treatment compared to vehicle (*p≤ 0.05, **p≤ 0.01, ***p≤ 0.001), in (c) the effect of vehicle vs. that of the combination. a TP53 WT , b TP53 -/-, c MCF7, d A549, d. Effect of SKIP2 loss on CDK2 regulation by Palbociclib. Charts show the percentage of cells with a GFP-PSLD nuc/cyto ratio of > 1.5, indicating low CDK2 activity. Cells were transfected with siRNA combinations as indicated 24 h followed by treatment with 500 nM palbociclib or vehicle for a further 24 h. Results for HCT116 and MCF7 cells are shown. Data 10 are mean values ±SD for n = 3 independent repeats. *p≤ 0.05, **p≤ 0.01, 2-way ANOVA comparing the effect size in TP53 compromised versus T53 normal cells and comparing the effect size of palbociclib vs vehicle in unperturbed (siNT) and SKP2 ablated (siSKP2) cells, SI values for palbociclib + siSKP2 are indicated.
(Related to Figure 7)