Induction of p16Ink4a-dependent senescence through exogenous signals

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Data from our laboratory have shown that simultaneous activation of the interferon (IFN) and of the tumor necrosis factor (TNF)-signaling pathways results in a stable cell cycle arrest in a broad range of tumor cells. This cytokine-induced cell cycle arrest strictly depends on the Cdk4/6 inhibitor p16INK4a and shows all characteristic features of cellular senescence (CIS). As TNF is too toxic for systemic applications, here we asked whether molecules of the TNF-signaling pathway could be targeted. Combing such molecules with IFN may allow establishing an efficient cancer therapy. Based on the data gained during the first funding period, this application has three related projects: (I) Searching for molecules that induce senescence in cancer cells when combined with IFN, we identified JunB as a key messenger important for the regulation of senescence and synthetic lethality in response to IFN. We have constructed tumor cells that overexpress JunB. Surprisingly, CIS can be induced in JunB-overexpressing cells with IFN alone. Thus JunB overexpression can replace the TNF required for CIS. This opens not only the approach for a druggable molecule. More interestingly, our data strongly suggest that JunB is a tunable molecule that ultimately determines whether an IFN signal leads to survival, senescence or apoptosis. (II) Searching for druggable molecules that can induce apoptosis in senescent cancer cells (senolysis) we found that combining IFN with HDAC inhibitors can induce senolysis. A key molecule that seems to be critical for the induction of senolysis was DNA-methyl transferase 1 (DNMT1). We will now exactly analyze the role of DNMT1 in CIS and senolysis by establishing knock-out cells (DNMT10/0) and especially also tumor cells with constitutive DNMT1-expression (DNMT1CONST). Intraperitoneal application of (DNMT10/0) or of (DNMT1CONST) tumor cells, followed by treatment with IFN with HDAC inhibitors will unravel whether DNMT1 is the postulated key messenger required for cancer immune control by HDAC inhibitors, when combined with IFN. (III) A key question concerns the natural clearance mechanisms of senescent cancer cells, and the biological meaning of this clearance. Here we identified TRAIL and iNOS as natural signals capable of inducing senolysis in vitro; moreover, we found that senolysis is needed to clear senescent cancer cells by macrophages or dendritic cells. It will now be important to identify the role of TRAIL and iNOS in inducing senolysis in vivo, and the need of these molecules for the in vivo-clearance of senescent cancers.Together, the data will explain how the immune system promotes synthetic lethality in cancers and how this can be exploited to clear cancers, when combined with either senescence-inducing molecules or drugs targeting cancer drivers, when oncogene-induced senescence fails.