Study of the combined effects of CDK1 inhibitors and senolytic drugs for the clearance of aneuploid-senescent cells

Despite the progresses in discovering new therapeutic drugs and treatments, cancer is still one of the main causes of death. The biggest part of available treatments, moreover, is not always effective against tumour spread and it also has negative effects on the healthy tissues of the individual. For this reason, it is extremely relevant to find new strategies to avoid side effects during the anti-cancer therapies. Aneuploidy, an aberrant number of chromosomes in the cell, is a typical condition of cancer cells caused mainly by segregation errors and chromosomal instability (CIN). CIN is a process by which higher rate of chromosome segregation defects occurs by different mechanisms (chromosome mis-alignments, spindle alterations, mitotic defects cytokinesis failure…) resulting in aneuploidy that, by inducing proteotoxic stress, energy stress and DNA damages, affects proliferation of normal cells. On the other hand, CIN and aneuploidy allow cancer cells to escape pathways leading to cell death (apoptosis), cell cycle arrest and cellular senescence. The reactivation of pathways leading to apoptosis or cellular senescence is a powerful strategy to halt proliferation of cancer cells. In particular, cellular senescence, an irreversible cell cycle arrest, presents itself as an effective mean to stop proliferation of cancer cells that could be then killed specifically, for example by senolytic drugs. Generally, cellular senescence originates from a G1 arrest of the cell cycle caused by DNA damage or other cellular stresses/alterations (such as defects in chromosome segregations). The cyclin-dependent kinase CDK1 is the most overexpressed kinase in malignant tumours compared to other CDKs. Its function is important for the correct cell cycle progression and its action is highly regulated, in order to preserve the right progression of mitosis. Once CDK1 is degraded, cell can successfully ultimate mitosis, proceeding from anaphase to telophase. Evidence showed how CDK1 and not its partner cyclin B1 is the main responsible of the right progression of mitosis1. Aneuploidy has been suggested also as a trigger for cellular senescence and aging. Thus, I hypothesized that inducing a cell cycle arrest in the G2/M phase by inhibiting CDK1 could trigger mitotic errors leading to a subsequent G1 arrest and likely senescence of cancer cells. To this aim I have inhibited CDK1 by RNAi and the selective inhibitor RO-3306, an ATP-competitor which interferes with CDK1 activation, in cultured cancer cells and not transformed aneuploid human cells. Moreover, as a positive control of senescence, the flavonoid Curcumin was used due to its known senescence inducer activity in cancer cells. After senescence induction, cells have been treated with the senolytic drugs Fisetin and Quercetin in order to specifically eliminate senescent cells. Globally, my work shows that CDK1 depletion, by RNAi and pharmacological inhibition, leads to G2/M arrest followed by p21waf1/cip1 rise and triggers cellular senescence in cancer cells as shown by SA-βGal positive cells. Accordingly, even though with different mechanisms, Curcumin has induced cell cycle arrest in the G2/M phase, as well as senescence, in tumor cells. Interestingly, the percentage of senescent cells reduces following treatment with the drugs Quercetin and Fisetin, confirming the senolytic action of these compounds. Thus, combining CDK1 inhibition with senolytic drugs can be a powerful strategy for the clearance of aneuploid – senescent cancer cells.