SUMOylation inhibitor TAK-981 (subasumstat) synergizes with 5-azacytidine in preclinical models of acute myeloid leukemia.

Acute myeloid leukemias (AML) are severe hematomalignancies with dismal prognosis. The post-translational modification SUMOylation plays key roles in leukemogenesis and AML response to therapies. Here, we show that TAK-981 (subasumstat), a first-in-class SUMOylation inhibitor, is endowed with potent anti-leukemic activity in various preclinical models of AML. TAK-981 targets AML cell lines and patient blast cells in vitro and in vivo in xenografted mice with minimal toxicity on normal hematopoietic cells. Moreover, it synergizes with 5-azacytidine (AZA), a DNA-hypomethylating agent now used in combination with the BCL-2 inhibitor venetoclax to treat AML patients unfit for standard chemotherapies. Interestingly, TAK-981+AZA combination shows higher anti-leukemic activity than AZA+venetoclax combination both in vitro and in vivo, at least in the models tested. Mechanistically, TAK-981 potentiates the transcriptional reprogramming induced by AZA, promoting apoptosis, alteration of the cell cycle and differentiation of the leukemic cells. In addition, TAK-981+AZA treatment induces many genes linked to inflammation and immune response pathways. In particular, this leads to the secretion of type-I interferon by AML cells. Finally, TAK-981+AZA induces the expression of natural killer-activating ligands (MICA/B) and adhesion proteins (ICAM-1) at the surface of AML cells. Consistently, TAK-981+AZA-treated AML cells activate natural killer cells and increase their cytotoxic activity. Targeting SUMOylation with TAK-981 may thus be a promising strategy to both sensitize AML cells to AZA and reduce their immune-escape capacities.

DeSUMOylation of chromatin-bound proteins limits the rapid transcriptional reprogramming induced by daunorubicin in acute myeloid leukemias.

Genotoxicants have been used for decades as front-line therapies against cancer on the basis of their DNA-damaging actions. However, some of their non-DNA-damaging effects are also instrumental for killing dividing cells. We report here that the anthracycline Daunorubicin (DNR), one of the main drugs used to treat Acute Myeloid Leukemia (AML), induces rapid (3 h) and broad transcriptional changes in AML cells. The regulated genes are particularly enriched in genes controlling cell proliferation and death, as well as inflammation and immunity. These transcriptional changes are preceded by DNR-dependent deSUMOylation of chromatin proteins, in particular at active promoters and enhancers. Surprisingly, inhibition of SUMOylation with ML-792 (SUMO E1 inhibitor), dampens DNR-induced transcriptional reprogramming. Quantitative proteomics shows that the proteins deSUMOylated in response to DNR are mostly transcription factors, transcriptional co-regulators and chromatin organizers. Among them, the CCCTC-binding factor CTCF is highly enriched at SUMO-binding sites found in cis-regulatory regions. This is notably the case at the promoter of the DNR-induced NFKB2 gene. DNR leads to a reconfiguration of chromatin loops engaging CTCF- and SUMO-bound NFKB2 promoter with a distal cis-regulatory region and inhibition of SUMOylation with ML-792 prevents these changes.

The NADPH oxidase NOX2 is a marker of adverse prognosis involved in chemoresistance of acute myeloid leukemias.

Resistance to chemotherapeutic drugs is a major cause of treatment failure in acute myeloid leukemias (AML). To better characterize the mechanisms of chemoresistance, we first identified genes whose expression is dysregulated in AML cells resistant to daunorubicin or cytarabine, the main drugs used for induction therapy. The genes found to be activated are mostly linked to immune signaling and inflammation. Among them, we identified a strong upregulation of the NOX2 NAPDH oxidase subunit genes (CYBB, CYBA, NCF1, NCF2, NCF4 and RAC2). The ensuing increase in NADPH oxidase expression and production of reactive oxygen species, which is particularly strong in daunorubicin-resistant cells, participates in the acquisition and/or maintenance of resistance to daunorubicin. Gp91phox (CYBB-encoded Nox2 catalytic subunit), was found to be more expressed and active in leukemic cells from patients with the French-American-British (FAB) M4/M5 subtypes of AML than in those from patients with the FAB M0-M2 ones. Moreover, its expression was increased at the surface of patients' chemotherapy-resistant AML cells. Finally, using a gene expression based score we demonstrated that high expression of NOX2 subunit genes is a marker of adverse prognosis in AML patients. The prognostic NOX score we defined is independent of the cytogenetic-based risk classification, FAB subtype, FLT3/NPM1 mutational status and age.

SUMO and Transcriptional Regulation: The Lessons of Large-Scale Proteomic, Modifomic and Genomic Studies.

One major role of the eukaryotic peptidic post-translational modifier SUMO in the cell is transcriptional control. This occurs via modification of virtually all classes of transcriptional actors, which include transcription factors, transcriptional coregulators, diverse chromatin components, as well as Pol I-, Pol II- and Pol III transcriptional machineries and their regulators. For many years, the role of SUMOylation has essentially been studied on individual proteins, or small groups of proteins, principally dealing with Pol II-mediated transcription. This provided only a fragmentary view of how SUMOylation controls transcription. The recent advent of large-scale proteomic, modifomic and genomic studies has however considerably refined our perception of the part played by SUMO in gene expression control. We review here these developments and the new concepts they are at the origin of, together with the limitations of our knowledge. How they illuminate the SUMO-dependent transcriptional mechanisms that have been characterized thus far and how they impact our view of SUMO-dependent chromatin organization are also considered.

The NF-κB member p65 controls glutamine metabolism through miR-23a.

Cancer cells have elevated aerobic glycolysis that is termed the Warburg effect. But several tumor cells, including leukemic cells, also increase glutamine metabolism, which is initiated by glutaminase (GLS). The microRNA (miRNA) miR-23 targets GLS mRNA and inhibits expression of GLS protein. Here we show that in human leukemic Jurkat cells the NF-κB p65 subunit binds to miR-23a promoter and inhibits miR-23a expression. Histone deacetylase (HDAC) inhibitors release p65-induced inhibition. Jurkat cells growing in glutamine decrease proliferation due to cell accumulation in G0/G1 phase. Nevertheless, cells get used to this new source of energy by increasing GLS expression, which correlates with an increase in p65 expression and its translocation to the nucleus, leading to a higher basal NF-κB activity. Jurkat cells overexpressing p65 show increase basal GLS expression and proliferate faster than control cells in glutamine medium. Overexpressing miR-23a in leukemic cells impaired glutamine use and induces mitochondrial dysfunction leading to cell death. Therefore, p65 activation decreases miR-23a expression, which facilitates glutamine consumption allowing leukemic cells to use this alternative source of carbon and favoring their adaptation to the metabolic environment.

An NF-kappaB-dependent role for JunB in the induction of proinflammatory cytokines in LPS-activated bone marrow-derived dendritic cells.

BACKGROUND: Dendritic cells (DCs) play a key role in the induction of adaptive and memory immune responses. Upon encounter with pathogens, they undergo a complex maturation process and migrate toward lymphoid organs where they stimulate immune effector cells. This process is associated with dramatic transcriptome changes, pointing to a paramount role for transcription factors in DC activation and function. The regulation and the role of these transcription factors are however ill-defined and require characterization. Among those, AP-1 is a family of dimeric transcription complexes with an acknowledged role in the control of immunity. However, it has not been studied in detail in DCs yet. METHODOLOGY/PRINCIPAL FINDINGS: Here, we have investigated the regulation and function of one of its essential components, JunB, in primary bone marrow-derived DCs induced to maturate upon stimulation by Escherichia coli lipopolysaccharide (LPS). Our data show fast and transient NF-kappaB-dependent transcriptional induction of the junb gene correlating with the induction of the TNFalpha, IL-6, and IL-12 proinflammatory cytokines. Inhibition of JunB protein induction by RNA interference hampered the transcriptional activation of the TNF-alpha, IL-6, and IL-12p40 genes. Consistently, chromatin immunoprecipitation experiments showed LPS-inducible binding of JunB at AP-1-responsive sites found in promoter regions of these genes. Concomitant LPS-inducible NF-kappaB/p65 binding to these promoters was also observed. CONCLUSIONS/SIGNIFICANCE: We identified a novel role for JunB--that is, induction of proinflammatory cytokines in LPS-activated primary DCs with NF-kappaB acting not only as an inducer of JunB, but also as its transcriptional partner.

SUMO under stress.

During the last decade, SUMOylation has emerged as a central regulatory post-translational modification in the control of the fate and function of proteins. However, how SUMOylation is regulated itself has just started to be delineated. It appears now that SUMO (small ubiquitin-related modifier) conjugation/deconjugation equilibrium is affected by various environmental stresses, including osmotic, hypoxic, heat, oxidative and genotoxic stresses. This regulation occurs either at the level of individual targets, through an interplay between stress-induced phosphorylation and SUMOylation, or via modulation of the conjugation/deconjugation machinery abundance or activity. The present review gives an overview of the connections between stress and SUMOylation, the underlying molecular mechanisms and their effects on cellular functions.

The alpha helix of ubiquitin interacts with yeast cyclin-dependent kinase subunit CKS1.

Ubiquitin serves as a molecular zipcode to direct and sort ubiquitinylated proteins into distinct biological pathways. Although novel modes of ubiquitin interaction have recently been characterized, conventional ubiquitin-binding domains (UBDs) recognize ubiquitin through a hydrophobic pocket centered around isoleucine 44 and lined by residues in beta sheets 3 and 4. In this study, we report a novel mode of interaction between ubiquitin and the cyclin-dependent kinase subunit of Saccharomyces cerevisiae, Cks1p, an adaptor protein involved in transcriptional regulation through recruitment of proteasomal subunits to gene promoters. Cks1p interacts specifically with monoubiquitin and tetraubiquitin with an affinity several orders of magnitude greater than that of other ubiquitin-binding domains and in an unconventional fashion, which differs from interactions documented so far between ubiquitin and conventional UBDs. The loop between helices alpha 1 and alpha 2, and to a minor extent the N-terminal alpha-helix of Cks1p, are involved in the interaction with the alpha-helix of ubiquitin, instead of its I44-centered hydrophobic pocket. Not only is this the first time the alpha-helix of ubiquitin is implicated in a protein/protein interaction, thereby shedding new light on the mechanisms of ubiquitin recognition, but also the first report of a direct physical interaction between ubiquitin and Cks1p, inferring a role for ubiquitin binding in the transcriptional function of Cks1p.

Multisite protein kinase A and glycogen synthase kinase 3beta phosphorylation leads to Gli3 ubiquitination by SCFbetaTrCP.

Gli3 is a zinc finger transcription factor proteolytically processed into a truncated repressor lacking C-terminal activation domains. Gli3 processing is stimulated by protein kinase A (PKA) and inhibited by Hedgehog signaling, a major signaling pathway in vertebrate development and disease. We show here that multisite glycogen synthase kinase 3beta (GSK3beta) phosphorylation and ubiquitination by SCFbetaTrCP are required for Gli3 processing. We identified multiple betaTrCP-binding sites related to the DSGX2-4S motif in Gli3, which are intertwined with PKA and GSK3beta sites, and SCFbetaTrCP target lysines that are essential for processing. Our results support a simple model whereby PKA triggers a cascade of Gli3 phosphorylation by GSK3beta and CK1 that leads to direct betaTrCP binding and ubiquitination by SCFbetaTrCP. Binding of betaTrCP to Gli3 N- and C-terminal domains lacking DSGX2-4S-related motifs was also observed, which could reflect indirect interaction via other components of Hedgehog signaling, such as the tumor suppressor Sufu. Gli3 therefore joins a small set of transcription factors whose processing is regulated by the ubiquitin-proteasome pathway. Our study sheds light on the role of PKA phosphorylation in Gli3 processing and will help to analyze how dose-dependent tuning of Gli3 processing is achieved by Hedgehog signaling.

Stable transmission of targeted gene modification using single-stranded oligonucleotides with flanking LNAs.

Targeted mutagenesis directed by oligonucleotides (ONs) is a promising method for manipulating the genome in higher eukaryotes. In this study, we have compared gene editing by different ONs on two new target sequences, the eBFP and the rd1 mutant photoreceptor betaPDE cDNAs, which were integrated as single copy transgenes at the same genomic site in 293T cells. Interestingly, antisense ONs were superior to sense ONs for one target only, showing that target sequence can by itself impart strand-bias in gene editing. The most efficient ONs were short 25 nt ONs with flanking locked nucleic acids (LNAs), a chemistry that had only been tested for targeted nucleotide mutagenesis in yeast, and 25 nt ONs with phosphorothioate linkages. We showed that LNA-modified ONs mediate dose-dependent target modification and analyzed the importance of LNA position and content. Importantly, when using ONs with flanking LNAs, targeted gene modification was stably transmitted during cell division, which allowed reliable cloning of modified cells, a feature essential for further applications in functional genomics and gene therapy. Finally, we showed that ONs with flanking LNAs aimed at correcting the rd1 stop mutation could promote survival of photoreceptors in retinas of rd1 mutant mice, suggesting that they are also active in vivo.

Integrase mutants defective for interaction with LEDGF/p75 are impaired in chromosome tethering and HIV-1 replication.

The insertion of a DNA copy of its RNA genome into a chromosome of the host cell is mediated by the viral integrase with the help of mostly uncharacterized cellular cofactors. We have recently described that the transcriptional co-activator LEDGF/p75 strongly interacts with HIV-1 integrase. Here we show that interaction of HIV-1 integrase with LEDGF/p75 is important for viral replication. Using multiple approaches including two-hybrid interaction studies, random and directed mutagenesis, we could demonstrate that HIV-1 virus harboring a single mutation that disrupts integrase-LEDGF/p75 interaction, resulted in defective HIV-1 replication. Furthermore, we found that LEDGF/p75 tethers HIV-1 integrase to chromosomes and that this interaction may be important for the integration process and the replication of HIV-1.