Efficacy of CDK4/6 inhibitors in preclinical models of malignant pleural mesothelioma

Abstract

Background: There is no effective therapy for patients with malignant pleural mesothelioma (MPM) who progressed to platinum-based chemotherapy and immunotherapy.

Methods: We aimed to investigate the antitumor activity of CDK4/6 inhibitors using in vitro and in vivo preclinical models of MPM.

Results: Based on publicly available transcriptomic data of MPM, patients with CDK4 or CDK6 overexpression had shorter overall survival. Treatment with abemaciclib or palbociclib at 100 nM significantly decreased cell proliferation in all cell models evaluated. Both CDK4/6 inhibitors significantly induced G1 cell cycle arrest, thereby increasing cell senescence and increased the expression of interferon signalling pathway and tumour antigen presentation process in culture models of MPM. In vivo preclinical studies showed that palbociclib significantly reduced tumour growth and prolonged overall survival using distinct xenograft models of MPM implanted in athymic mice.

Conclusions: Treatment of MPM with CDK4/6 inhibitors decreased cell proliferation, mainly by promoting cell cycle arrest at G1 and by induction of cell senescence. Our preclinical studies provide evidence for evaluating CDK4/6 inhibitors in the clinic for the treatment of MPM.

Fig. 1. Quantification of the expression levels of key cell cycle regulators and response to treatment with CDK4/6 inhibitors in a panel of commercial MPM cell lines and primary patient-derived cultures.

a Baseline protein expression levels by Western blot of CDK4, CDK6, cyclin D1, Rb, phosphor-RB and p16. b Number of viable cells was determined in vitro by cell counting in the panel of cells after 3 days of treatment with increasing concentrations (0, 10, 100 and 500 nM) of abemaciclib or palbociclib. Bar plots represent the means ± SD of three measurements in three biological replicates. Adjusted p values were calculated with Wilcoxon’s signed-rank tests. In the graph, the p values are reported with respect to 0 nM (*p < 0.05; **p < 0.01). c Colony formation assay displaying treatment response to abemaciclib and palbociclib. A representative image from three biologically independent replicates is displayed.

Fig. 2. Effects of CDK4/6 inhibitors in cell cycle and cell senescence in MPM cell lines.

Effects of cell line treatments with CDK4/6 inhibitors abemaciclib or palbociclib at 0, 250 or 500 nM doses to induce (a, b) cell cycle arrest and (c) senescence. a MSTO-211H, H28 and ICO_MPM3 cells were untreated and treated with both inhibitors for 24 h and DNA content was analysed by flow cytometry. Cell cycle arrest at the G1 phase was induced by both CDK4/6 inhibitors in the cell lines. b Percentage of cells in each phase of the cell cycle phase in response to abemaciclib and palbociclib treatments at doses of 0, 250 or 500 nM for 24 h. Cell cycle phase distribution analysis was done using the FlowJo software. Each value represents the mean ± SD of three replicates. Adjusted p values were considered significant when the mean differed from control within each phase of the cell cycle (*p < 0.05; ***p < 0.001). c A significant increase in the number of SA-β-gal-positive cells was detected in MSTO-211H, H28 and ICO_MPM2 treated. Data are expressed as a percentage of senescent cells obtained from the mean value ± SD of three replicates. Adjusted p values <0.05 were considered significant.

Fig. 3. In vivo treatment with palbociclib in MSTO-211H subcutaneous xenografted MPM model.

A xenografted subcutaneous tumour model was established by inoculation of MSTO-211H cells into the flanks of athymic nude mice (n = 7 per group). Tumours’ volume (a) was monitored by calliper measure every 4 days (at each time point, a SD bar is shown). b Differences in tumour volume for each day were represented using boxplots and compared using Kruskal–Wallis test, adjusted by FDR and were significantly different at day 16. At the end of the experiment, mice were sacrificed and (c, d) the tumours were removed, weighted and photographed. Asterisks indicated the absence of apparent macroscopic tumour at sacrifice, while residual cells were identified by H&E analysis. e Summary of the body weight values among first and last day of treatment from all mice in in vivo subcutaneous tumour xenograft growth experiment. Palbociclib did not exert any substantial change in the mice body weight. Differences were evaluated by Mann–Whitney test and adjusted for FDR. f Representative IHC images for (F.A) mouse macrophages that express F4/80 (brown staining) and (F.B) mouse NK cells that express NCR1 (brown staining) infiltrated in the subcutaneous MPM tumours xenografted in athymic nude mice after 26 days of treatment with vehicle, platinum plus pemetrexed or palbociclib. Inset photos contain the digital whole slide image showing the infiltrated area of tumour. Scale bar = 50 µm. Dot plots showing (g) the mean F4/80⁺ intensity per pixel (x-axis) or (h) the percentage of NCR1⁺ cells over total cells infiltrated in the subcutaneous MPM tumour xenografted in athymic nude mice after 26 days of treatment with vehicle, platinum plus pemetrexed or palbocilib (n = 5–7 per group). Data are expressed as single data values (dots) + the mean. ANOVA test was used to detect statistical differences between treatments (*p < 0.05).

Fig. 4. In vivo treatment with CDK4/6 inhibitors in advanced orthotopic MPM models.

Two advanced orthotopic models were generated by implantation in the pleural space of mice small solid fragments (2–3 mm³) of previously generated (a–c) MSTO-211H subcutaneous cisplatin plus pemetrexed-resistant tumour xenograft or (d–f) chemoresistant patient-derived tumour xenograft. a Kaplan–Meier curves showing survival of MSTO-211H orthotopic tumour-bearing mice (n = 33). b Representative MSTO-211H images of orthotopic tumours dissected from each group of treatment and c histological characterisation on H&E sections (scale bars = 100 µm). d Kaplan–Meier curves showing survival of ICO_MPM3 orthotopic tumour-bearing mice (n = 29). e Representative images of patient-derived orthotopic tumours dissected from each group of treatment and f histological characterisation on H&E sections (scale bars = 100 µm). Both orthotopic models accurately reproduce human MPM disease characteristics as tumours grown from the site of implantation to all the pleural space. Tumour mass area is delimited by a white line.

Fig. 5. Overall survival analysis according to CDK4 and CDK6 expression in two cohorts of patients with MPM.

Kaplan–Meier plots of overall survival (OS) in MPM patients according to a CDK4 and b CDK6 gene expression levels based on data obtained from Bueno et al. (left column) and Hmeljak et al. (middle column) cohorts or the combination of both (right column). High levels of CDK4 or CDK6 (red line) were significantly associated with poor OS in patients with MPM. In each cohort, the high and low expression levels were defined based upon the median. P values and hazard ratios (HRs) were calculated by likelihood ratio test and multivariate Cox regression analysis, respectively.