France

More than two decades of basic research is finally beginning to bear fruit, as the first early phase trials are evaluating the safety and efficacy of SUMO pathway inhibition in cancer patients. In cancer, SUMO machinery components are often dysregulated at the genomic level and by conditions typically present in cancer, such as hypoxia. Many molecular mechanisms relevant to the pathogenesis of specific cancers involve SUMO, highlighting the relevance of SUMO machinery components as potential prognostic markers and therapeutic targets. Our research focuses on characterizing mechanisms involving SUMO that drive cancer growth, ultimately helping find subgroups of patients putatively benefitting of drugs targeting SUMOylation. Our results of SUMOylation in breast cancer, head and neck cancer and neuroblastoma will be discussed.

Sumo Modification in Cell Signalling

Targeting SUMO machinery in cancer – any promise for patients?

SUMO (Small Ubiquitin-like Modifier) modification has recently emerged as a critical class of post translational modification (PTM), playing a major role in diverse cellular pathways in all eukaryotes. A key function of SUMO is to act as a vital counterpoise to Ubiquitination, adding a layer of control above ubiquitination including competition for targets and prevention of ubiquitin-dependent protein degradation. Phosphorylation is also regulated by SUMOylation of kinases and phosphatases. Crucially, unlike ubiquitination and phosphorylation which are controlled by thousands of genes, only 33-50 genes regulate the SUMO modification pathway in eukaryotes making SUMO an ideal experimental system to decipher the language and rules of PTMs.


Discovering how SUMO transduces environmental signals into specific physiological responses will be key to understanding this new set of fundamental ‘rules of life’ that regulate processes ranging from development to disease resistance in an ever-changing environment. This 2- day satellite meeting will highlight the established cellular role of SUMO in a range of eukaryotes in development to disease along with identifying knowledge gaps and horizon scanning. Particular emphasis will be paid to emerging technologies such as single cell techniques that will be crucial to decipher the SUMO code across eukaryotes. The meeting will consist of a series of talks from established to early career researchers who are using a variety of techniques to understand SUMO mediated cellular signalling in a range of systems (including yeast, plants and animals). Each session will end in a panel discussion with the audience hosted by the chair of the session.

This 2- day satellite meeting will highlight the established cellular role of SUMO in a range of eukaryotes in development to disease along with identifying knowledge gaps and horizon scanning. Particular emphasis will be paid to emerging technologies such as single cell techniques that will be crucial to decipher the SUMO code across eukaryotes.

keynote

TAK-981, a first in class inhibitor of SUMOylation, exhibits strong synergy with genotoxics and epidrugs used for acute myeloid leukemia treatments, against AML cell lines and primary patient blast cells in cellulo and in vivo. TAK-981 and epidrugs combination increases transcription of genes involved in apoptosis, cell cycle control but also triggers an anti-tumoral immunity mediated by Natural Killer cells.

TAK-981, a first in class inhibitor of SUMOylation, synergizes with Acute Myeloid Leukemias treatments and induces anti-tumor immunity in preclinical models

SUMO conjugation is emerging as an important mechanism to transduce environmental cues into cellular signaling. We present evidence to show that SUMOylation of key transcriptional regulators such as DELLA and BZR1, provides a conduit for environmental influence of root growth and development during salt stress. SUMOylation stabilizes BZR1 in the nucleus by inhibiting its interaction with BIN2 kinase. During salt stress, Arabidopsis plants arrest growth through deSUMOylation of BZR1 in the cytoplasm by promoting the accumulation of the BZR1 targeting SUMO protease, ULP1a. ULP1a mutants are salt tolerant and insensitive to the BR inhibitor, brassinazole with regards to root growth. BR treatment stimulates ULP1a degradation, allowing SUMOylated BZR1 to accumulate and promote growth. This study uncovers a mechanism for integrating environmental cues into BR signaling to shape root growth. 
Intriguingly an interacting partner of BZR1, the growth repressing DELLA proteins are also SUMOylated. We demonstrate that a proportion of DELLAs are conjugated to SUMO, the extent of conjugation increases during salt stress. We identify a SUMO interacting motif (SIM) in the GA receptor GID1 and demonstrate that SUMO-conjugated DELLA binds to this motif in a GA-independent manner. The consequent sequestration of GID1 by SUMO-conjugated DELLAs leads to an accumulation of non-SUMOylated DELLAs resulting in repression of root growth in salinity. We present a model depicting how SUMO decodes environmental cues to shape root growth and development.

Deciphering the SUMO code for adaptive responses in roots

Post-translational modifications by SUMO (Small Ubiquitin-like MOdifier) modulate protein activity through regulation of subcellular localization, protein activity and stability, and protein-protein interactions. In plants, SUMO is essential during seed development, modulates hormone signaling, and biotic/abiotic stress responses. In the cell, SUMO conjugates are highly enriched in the nucleus, consistently with a preferential nuclear localization of the SUMOylation machinery components. In this scenario, a major unsolved question is to uncover the mechanism that facilitates the conjugation of the cytosolic SUMO targets.
We will present our findings pointing to a major role of the large subunit of the SUMO activating enzyme, SAE2, controlling nucleocytoplasmic partitioning of SUMOylation. We have found that the C-terminus of SAE2 is processed, and this modification facilitates partial localization of SAE2 to the cytosol. Accumulation of SAE2 in the cytosol promotes cytosolic SUMOylation and behave as a signal for growth arrest. We will also discuss the potential implications of this mechanism in seed appearance during plant evolution.

Seed proteolytic proteoforms of SUMO-activating enzyme 2 (SAE2) mediate nucleocytoplasmic partitioning of SUMOylation.

In the model plant Arabidopsis, two closely related members of the plant specific SYP12 clade of secretory syntaxins (PEN1 and SYP122) are indispensable for the formation of evolutionarily conserved defense structures that provide general immunity to filamentous pathogens. However, in addition to the conserved SYP12 clade function shared with SYP122, PEN1 also provides resistance to attack by powdery mildew fungi. To elucidate the molecular properties that enables PEN1, but not SYP122, to mediate powdery mildew resistance, we made a series of chimeric constructs in which domains and individual amino acids from the two syntaxins were interchanged. In plants lacking native PEN1 and SYP122, we analyzed these chimeric syntaxins for their ability to restore the conserved SYP12 function and the specific function mediated by PEN1 alone. Here we have uncovered SUMOylation to be a critical modifier of PEN1 functionality and its localization in response to powdery mildew attack. To our knowledge, this is the first example in which SUMOylation positively regulates the function of a secretory syntaxin. Accordingly, PEN1 SUMOylation could represent a new mechanistic function for this post-translational modification.

SUMOylation regulates plant syntaxin function in innate immunity

During in vitro tissue culture, plants form callus and regenerate organs by reprograming developmental pathways. In addition to hormonal signals, stress-induced cues, such as those from wounding, can trigger and promote cellular reprogramming. Conjugation of the small ubiquitin-related modifier (SUMO) to target transcriptional regulators is thought to rapidly and precisely modulate their activity in response to stress. We previously investigated if SUMOylation is involved in wound-induced reprograming and showed that loss of the E3 ligase SIZ1 causes a hyper-active wound response and enhances shoot regeneration (Coleman et al, 2020). Based on these observations, we hypothesized that SUMOylation suppresses wounding-induced regulator(s) and searched for early acting players underlying the stress-induced cellular reprograming. By analyzing the transcriptome of wounded plants, we found that HEAT SHOCK FACTOR A1s (HSFA1s) are activated after wounding. Mutant and overexpression analysis showed that HSFA1s, also known to be activated by heat stress, promote shoot regeneration in Arabidopsis. Furthermore, the regulation of HSFA1s appeared independent of transcription and biochemical analysis revealed that they are subject to SUMOylation in response to stress. Here, I will discuss how key stress-induced transcription factors regulate cellular reprograming and how SUMOylation modulates this regenerative response in plants.

The role of SUMOylation during shoot regeneration in Arabidopsis

Seed germination is the crucial physiological process regulated by both environmental and endogenous phytohormones. ABA negatively regulates seed germination, post-germination growth and floral transition, however, the cross talk between multiple regulatory pathways are still unclear. Here, we show that ABA activates two MAP kinases, AtMPK3/AtMPK6 and selectively regulates the transcript of AtMPK3 through ABI5, a master regulator of ABA signaling. As a feedback loop, AtMPK3 interacts and phosphorylates ABI5 at the serine-314 position. ABI5 phosphorylation by MAP kinases positively regulates ABI5 nuclear localization and negatively regulates its dimerization. Subcellular localization of ABI5 phospho-null protein further suggests the role of phosphorylation in regulation of its cytoplasmic stability and its nuclear dimerization. Overexpression of phosphor-null ABI5 in abi5-8 mutant restored the ABA sensitivity during seed germination and delayed the floral transition as compared to phospho-mimic ABI5. Additionally, overexpression of constitutive phosphorylated ABI5 in abi5-8 mutants suggest that phosphorylation makes ABI5 partially inactive. Furthermore, phospho-null ABI5 plants showed drought sensitive phenotype whereas, mpk3, mkk4, mkk5, abi5-8 and phosphor-mimic plants showed drought tolerant phenotype. Further, ABI5 has SUMO interacting motifs and the possibility of ABI5 under the regulation SUMOlyation will also be discussed. Our findings present a new insight between MAP kinase cascade and ABA signaling which collectively regulates the ABA response through ABI5 phosphorylation.

A novel positive feedback mechanism of ABI5 phosphorylation by mitogen activated protein kinase-3 regulates ABA signaling in Arabidopsis

Brassinosteroids (BRs) are a class of steroid molecules perceived at the cell surface that act as plant hormones. The BR LRR-RLK receptor BRI1 offers a model to understand receptor-mediated signaling in plants and the role of post-translational modifications. We previously reported that undergoes Ub-mediated endocytosis following its massive decoration with K63-linked polyUb chains. A non-ubiquitinatable BRI1 variant lacking 25 intracellular lysine residues indeed shows slower internalization kinetics and inability to reach the lytic vacuole. Interestingly, a slight increase in ambient temperature triggers BRI1 destabilization, presumably through Ub-mediated endocytosis since the BRI125KR variant fails to respond. Here we identify SUMOylation as a new modification, targeting BRI1 to regulate its activity. We show that BRI1 is SUMOylated in planta on two lysine residues, which are also Ub targets, and that the levels of BRI1-SUMO conjugates are controlled by the Desi3a SUMO protease. We demonstrate that BRI1 is deSUMOylated at elevated temperature by Desi3a, leading to increased BRI1 interaction with the negative regulator of BR signaling BIK1 and enhanced BRI1 endocytosis. Loss of Desi3a or BIK1 results in increased response to temperature elevation, indicating that BRI1 deSUMOylation acts as a safety mechanism necessary to keep temperature responses in check. Altogether, our work establishes BRI1 deSUMOylation as a molecular crosstalk mechanism between temperature and BR signaling, allowing plants to translate environmental inputs into growth response. Besides, it sheds light on the possible interplay between SUMO and Ub at the same target lysine residues to control BRI1 stability.

Integration of plant steroid hormone signaling and temperature responses : interplay between SUMO and Ub

SUMOylation, the covalent attachment of the small ubiquitin-like modifier (SUMO) to target proteins, is emerging as a key modulator of eukaryotic immune function. In plants, a SUMO1/2-dependent process has been proposed to control the deployment of host defense responses. Our findings suggest that increasing nitric oxide levels following pathogen recognition promote S-nitrosylation of the Arabidopsis SUMO E2 enzyme, SCE1, at Cys139. The SUMO-conjugating activities of both SCE1 and its human homolog, UBC9, were inhibited following this modification. Accordingly, mutation of Cys139 resulted in increased levels of SUMO1/2 conjugates, disabled immune responses, and enhanced pathogen susceptibility. Our findings imply that S-nitrosylation of SCE1 at Cys139 enables NO bioactivity to drive immune activation by relieving SUMO1/2-mediated suppression. The control of global SUMOylation is thought to occur predominantly at the level of each substrate via complex local machineries. Our findings uncover a parallel and complementary mechanism by suggesting that total SUMO conjugation may also be regulated directly by SNO formation at SCE1 Cys139. This Cys is evolutionary conserved and specifically S-nitrosylated in UBC9, implying that this immune-related regulatory process might be conserved across phylogenetic kingdoms.

A role for S-nitrosylation of the SUMO-conjugating enzyme SCE1 in plant immunity

Membrane-less organelles are condensates formed by phase separation whose functions often remain enigmatic. Upon oxidative stress, PML scaffolds Nuclear Bodies (NBs) to regulate senescence or stress adaptation (1). PML was the first identified protein of the SUMO-targeted Ubiquitin-mediated RNF4 ligase (2). We previously showed that PML NBs concentrates UBC9 together with PML partner proteins and thus proposed that PML NBs might favor partner sumoylation and subsequent poly-ubiquitination by NB-associated RNF4 (3). To establish whether PML controls partner sumoylation upon physiological stress response, we recently explored different models with distinct NB-status (4). SUMO2-conjugation is increased upon arsenic-induced PML NB re-organization in Acute Promyelocytic Leukemia (APL) mice, as well as in mouse Embryonic Stem Cells (mESCs) compared to Pml deficient mESCs. Differential in vivo SUMO2 proteomics identified as PML NB-dependent SUMO2-targets several members of the repressive KAP1 complex, as well as the DPPA2 transcription factor. Their sumoylation drives functional activation of this key epigenetic repressor and inhibition of DPPA2. Accordingly, Pml deficient mESCs re-express transposable elements and display features of 2-cell-like cells, a state of the mESCs reassembly to the 2-cell stage of embryogenesis. Finally, PML is required for adaptive stress responses in mESCs. Collectively, PML regulates mESC fate by orchestrating SUMO2-conjugation of key transcriptional or epigenetic regulators.

Exploration of PML in stress response and embryonic stem cell fate

Adrenal Cortex; SENP2; cellular differentiation; WNT/Beta-catenin

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TAK-981, a first in class inhibitor of SUMOylation, synergizes with Acute Myeloid Leukemias treatments and induces anti-tumor immunity in preclinical models

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A novel positive feedback mechanism of ABI5 phosphorylation by mitogen activated protein kinase-3 regulates ABA signaling in Arabidopsis

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