CDK Inhibition as a new Potential Target Approach in Pleural Mesothelioma Models

CDK Inhibition as a new Potential Target Approach in Pleural Mesothelioma Models Assoc. Prof. Mara Bonelli1, Dr. Maricla Galetti2, Prof. Silvia La Monica1, Dr. Emanuela Falcone1, Prof. Luca Ampollini1, Dr. Claudia Fumarola1, Prof. Roberta Alfieri1, Dr. Davide De Forni3, Dr. Barbara Poddesu3, Dr. Franco Lori3, Prof. Pier Giorgio Petronini1 1University of Parma, Parma, Italy, 2Department of Occupational and Environmental Medicine, Epidemiology and Hygiene, INAIL-Italian Workers' Compensation Authority, Rome, Italy, 3ViroStatics srl, Sassari, Italy Objectives: Loss of CDKN2A/ARF gene is the most common genetic alteration in pleural mesothelioma (PM), with an incidence rate ranging from 50% to 100%, depending on tumor histotype, suggesting a possible target approach for the therapy of this untreatable tumor. We previously demonstrated the efficacy of the treatment with the CDK4/6 inhibitors in inhibiting tumor cell proliferation, impairing tumor cell metabolism and preventing the acquisition of chemoresistance. Based on these findings, this study aimed to investigate the efficacy of CDK4/6 inhibitors in chemo-resistant PM cells. As metabolic reprogramming frequently occurs during the acquisition of chemo-resistance, we also evaluated the metabolic features of chemo-resistant cells. Furthermore, we evaluated the efficacy of the new molecule myrtleciclib, which is a dual CDK4/6 and CDK9 inhibitor, in PM cells and assessed its ability to overcome chemo-resistance. Methods: Experiments were performed on a panel of PM cell lines of different histotypes. In vivo studies were performed in an immune-competent orthotopic rat recurrence model of PM. Results: PM clones resistant to cisplatin and pemetrexed were obtained by exposing the H2452 epithelioid cell line to increasing concentrations of the drug combination over a period of more than six months. The chemo-resistant clones showed increased activity of the MAPK and AKT/mTOR pathways, as well as the pRb/E2F/Myc axis, as compared to the parental cell line. As both E2F and c-Myc are involved in regulating energy cell metabolism, the metabolic profile of chemo-resistant clones was studied. Most of the clones analyzed exhibited an 'energetic phenotype', characterized by increased glycolysis and mitochondrial respiration compared to the parental cell line. Given the genetic characteristics of the cells (CDKN2A/ARF loss) and the hyperactivation of Rb signalling, we evaluated the efficacy of the CDK4/6 inhibitor abemaciclib in chemo-resistant cells. Interestingly, all chemo-resistant clones were sensitive to the drug, which inhibited cell proliferation with IC₅₀ values similar to or lower than those of the parental cell line. Taking advantage of the new molecule myrtleciclib, we evaluated its efficacy in PM models as well as its potential to overcome chemo-resistance. Myrtleciclib exhibited greater anti-proliferative efficacy than commercially available CDK4/6 inhibitors, with approximately 50-fold lower IC50 values. It also induced apoptosis, by decreasing the expression of the anti-apoptotic molecule Mcl-1, a CDK9 target. The efficacy of myrtleciclib was also demonstrated in vivo in an orthotopic rat recurrence model of PM: the treatment with myrtleciclib strongly prevented tumor relapses compared to control animals. Furthermore, myrtleciclib demonstrated high anti-proliferative activity in chemo-resistant clones, inducing cell death and proving its efficacy in overcoming chemo-resistance. Conclusion: The mechanisms underlying chemo-resistance in PM patients are poorly understood, and no effective treatment has yet been developed to treat these patients and ameliorate their survival. Overall, our results demonstrated the therapeutic potential of CDK inhibitors in chemo-resistant PM cells, which is consistent with the recent positive results of the MiST2 arm in abemaciclib-treated patients.