Final Report Summary - SUMOSTRESS (Integrating the activities of SUMO in gene expression, inflammation and cancer) Unravelling the mechanisms that maintain and stabilize cells in the face of external and internal stresses that threaten cellular function, viability or identity is crucial to understanding normal physiological processes and how these are corrupted in disease. One such mechanism is given by the post-translational modification of proteins by the Small Ubiquitin-related MOdifier (SUMO; 'sumoylation'). Since its discovery some twenty years ago, sumoylation has emerged as an essential regulatory mechanism of protein function involved in virtually every normal or pathological process, including cancer, inflammatory and neurological disorders, and, more recently, in pathways governing cell fate decisions. Like ubiquitinylation and other ubiquitin-like (Ubl-) modification pathways, sumoylation is unusual in that cellular proteins are modified not by the addition of simple chemical groups (methyl-, acetyl-, phospho-, etc.), but by the covalent attachment of other proteins (SUMO1; -2, 3) by a unique and therefore druggable pathway. Our studies in the framework of the ERC-SUMOSTRESS project have firmly established sumoylation as a critical chromatin-associated process in gene expression regulation and as a barrier to both a pathogenic innate immune response and to induced cell fate conversion. The aims of our work were to explore novel mechanisms regulating global cellular sumoylation,to characterize the dynamic SUMO chromatin landscape, to dissect SUMO-dependent gene regulatory mechanisms and to investigate specific roles of sumoylation in inflammation and tumor initiation and development.Specifically, we found significant accumulations of SUMO-modified proteins on the regulatory regions (promoters and enhancers) of actively expressed genes in growing, but not in senescent (i.e. permanently growth-arrested) cells. The characterization of this 'SUMO chromatin landscape' thus firmly established sumoylation as a key pathway dynamically regulating active transcription on a genome-wide scale. Our subsequent work showed that histone gene expression, repressed in the senescent state, is critically regulated by cell cycle-dependent waves of (repressive) sumoylation and de-sumoylation.Further studies on the gene expression consequences of deregulated sumoylation also revealed that sumoylation regulates the stability of the RNAi protein Ago2 as well as the transcriptional properties and molecular dynamics of BRD4, a regulator of transcriptional elongation and high-profile target for cancer therapy using 'iBET' drugs.Next, we discovered that reducing global sumoylation in immune cells leads to a strikingly monomorphic phenotype: the activation of the whole inflammatory program. Upon toll-like receptor (TLR) engagement (i.e. mimicking bacterial infection) in myeloid cells, deficient sumoylation leads to strong, acute innate immune response: the secretion of inflammatory cytokines and a massive type I interferon (IFN-I) anti-viral gene signature, the latter mediated by unscheduled de-repression of the Infb1 gene. Consistent with this, reduced sumoylation in animal models was found to exacerbate both inflammatory (endotoxic shock) and antiviral responses. Studies on the effect of infection by the enteropathogen Shigella flexneri further supported the important role of sumoylation in restraining inflammation and, in addition, uncovered a novel mechanism regulating global cellular sumoylation by induced, calpain-dependent degradation of the SUMO pathway E1 enzyme. Given the evidence that the phenotypic effects of altered sumoylation are mediated principally by processes operating at the chromatin level (e.g. at the histone or IFN loci), we surmised key functions for sumoylation also in mechanisms underlying cell fate transitions. We showed that SUMO modification acts as an epigenetic barrier, acting at specific chromatin loci, to the reprogramming of differentiated (fibroblast) to pluripotent (iPSC) cells and to the spontaneous reversion of pluripotent (ESC) to totipotent (2-cell embryo, "2C-like") cells, as well as in several differentiation and trans-differentiation paradigms.Our - still ongoing - studies of in vitro and in vivo models of intestinal cancer have provided strong evidence that sumoylation plays apparently paradoxical, i.e. dose- and context-dependent roles in tumor initiation and development, thus acting in both tumor-suppressive (in MEF transformation and in genetic intestinal tumorigenesis provoked by inducible APC tumor suppressor inactivation) and tumor-promoting (in inflammation-driven colon cancer) mechanisms.Finally, altogether, this work with important therapeutic implications for understanding the mechanisms that underlie inflammation, tumorigenesis and the maintenance of cell identity, has also informed an ongoing phase I clinical trial using a sumoylation inhibitor (Takeda Pharmaceutical's TAK-981) in refractory cancer patients.
