Controlling senescence by PML and PML nuclear bodies

Cellular senescence is a stress response wherein proliferating competent cells undergo a stable cell cycle arrest in response to a variety of intrinsic and extrinsic stimuli, including telomere shortening, oxidative stress, DNA damage or the constitutive activation of oncogenes among others. All these stimuli have in common the potential to initiate or promote neoplastic transformation that can degenerate in cancer. Senescence, particularly in human cells, is established and maintained by at least two major tumor suppressor pathways: the p53/p21CIP and p16INK4A/pRB pathways and is now accepted as a potent cell-autonomous mechanism for suppressing the development of cancer. Both pathways are able to activate and increase the expression of the tumor suppressor protein PML. As a tumor suppressor, PML is sufficient to induce senescence in normal cells; however, upon the same stimuli, cancer cells fail to engage a complete senescence response.  Given this, the first aim of this thesis is to investigate the resistance mechanisms of cancer cells to PML-induced senescence.   We found that overexpression of the CDK4 and CDK6 (which are often up-regulated in cancer) are sufficient to bypass PML-induced senescence in normal cells. In cancer cells the expression of these kinases impairs the PML-induced senescence. By inhibiting the expression and/or function of CDK4/6 we were able to restore the senescence program in cancer cells. Also, the specific CDK4/6 inhibitor palbociclib (currently used in clinical trials) increased the ability of PML to regulate a stronger and more permanent growth inhibition in cell culture and decreased tumor progression in mice.  This complete senescence response correlated with an increase in autophagy markers, repression of E2F target genes and a gene expression signature of blocked DNA methylation. Furthermore, CDK4/6 inhibition by palbociclib promotes autophagy-dependant degradation of the DNA methyltransferase DNMT1. More important, we were able to demonstrate that CDK4 directly interacts and phosphorylates DNMT1 in vitro. These results highlight the potential value of CDK4/6 inhibitors as epigenetic modulators to facilitate activation of cellular senescence in cancer cells. PML-induced senescence is tightly regulated by post-translational modifications (PTMs). Among these PTMs, SUMOylation plays an important role in the scaffold function of PML and the formation of the PML-NBs (PML-Nuclear Bodies). PML-NBs are dynamic structures triggered by stress such as oncogene-induced senescence, and its formation allows the sequestration of target proteins for their regulation and/or its post-translational modification. By protein recruitment, PML-NBs regulate several cellular functions such as senescence, apoptosis, antiviral response, DNA repair and gene regulation. Given this; the second aim of this thesis is to characterize the role of SUMOylation in oncogene mediated cellular senescence, specifically by the expression of the oncogene RAS. By a SUMO3 proteome analysis of senescent cells we were able to identify 25 SUMO sites in 23 proteins that were significantly regulated during senescence. Importantly, most of these proteins were PML-NB associated. Interestingly, UBC9 (the only SUMO E2 enzyme), was found more SUMOylated in senescence on its Lys-49. Functional studies of a UBC9 mutant in Lys-49 showed a decreased association to PML-NBs and the loss of UBC9’s ability to delay senescence. Moreover, forced localization of UBC9 into PML-NBs counteracted RAS and PML-induced senescence. These results allowed us to propose a pro- and an anti-senescence function of protein SUMOylation, specifically for UBC9. Keywords: Senescence, PML, CDK4/6, DNA methylation, palbociclib, PML-NBs, SUMOylation, UBC9

Keyphrases: Senescence, Pml, Palbociclib, Cancer, SUMOylation, UBC9, CDK4/6, DNA methylation, PML-NBs