Nrf2 depletion in the context of loss-of-function Keap1 leads to mitolysosome accumulation.

Transcription factor nuclear factor erythroid 2 p45-related factor 2 (Nrf2) is the principal determinant of the cellular redox homeostasis, contributing to mitochondrial function, integrity and bioenergetics. The main negative regulator of Nrf2 is Kelch-like ECH associated protein 1 (Keap1), a substrate adaptor for Cul3/Rbx1 ubiquitin ligase, which continuously targets Nrf2 for ubiquitination and proteasomal degradation. Loss-of-function mutations in Keap1 occur frequently in lung cancer, leading to constitutive Nrf2 activation.

Dual inhibition of HSF1 and DYRK2 impedes cancer progression.

Preserving proteostasis is a major survival mechanism for cancer. Dual specificity tyrosine phosphorylation-regulated kinase 2 (DYRK2) is a key oncogenic kinase that directly activates the transcription factor heat-shock factor 1 (HSF1) and the 26S proteasome. Targeting DYRK2 has proven to be a tractable strategy to target cancers sensitive to proteotoxic stress; however, the development of HSF1 inhibitors remains in its infancy.

Biotinylation of an acetylenic tricyclic bis(cyanoenone) lowers its potency as an NRF2 activator while creating a novel activity against BACH1.

The transcription factor BACH1 regulates the expression of a variety of genes including genes involved in oxidative stress responses, inflammation, cell motility, cancer cell invasion and cancer metabolism. Based on this, BACH1 has become a promising therapeutic target in cancer (as anti-metastatic target) and also in chronic conditions linked to oxidative stress and inflammation, where BACH1 inhibitors share a therapeutic space with activators of transcription factor NRF2. However, while there is a growing number of NRF2 activators, there are only a few described BACH1 inhibitors/degraders.

The synthetic triterpenoids CDDO-TFEA and CDDO-Me, but not CDDO, promote nuclear exclusion of BACH1 impairing its activity.

The transcription factor BACH1 is a potential therapeutic target for a variety of chronic conditions linked to oxidative stress and inflammation, as well as cancer metastasis. However, only a few BACH1 degraders/inhibitors have been described. BACH1 is a transcriptional repressor of heme oxygenase 1 (HMOX1), which is positively regulated by transcription factor NRF2 and is highly inducible by derivatives of the synthetic oleanane triterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO).

Pirin, an Nrf2-Regulated Protein, Is Overexpressed in Human Colorectal Tumors.

The evolutionary conserved non-heme Fe-containing protein pirin has been implicated as an important factor in cell proliferation, migration, invasion, and tumour progression of melanoma, breast, lung, cervical, prostate, and oral cancers. Here we found that pirin is overexpressed in human colorectal cancer in comparison with matched normal tissue. The overexpression of pirin correlates with activation of transcription factor nuclear factor erythroid 2 p45-related factor 2 (Nrf2) and increased expression of the classical Nrf2 target NAD(P)H:quinone oxidoreductase 1 (NQO1), but interestingly and unexpectedly, not with expression of the aldo-keto reductase (AKR) family members AKR1B10 and AKR1C1, which are considered to be the most overexpressed genes in response to Nrf2 activation in humans.

A novel CDC25A/DYRK2 regulatory switch modulates cell cycle and survival.

The cell division cycle 25A (CDC25A) phosphatase is a key regulator of cell cycle progression that acts on the phosphorylation status of Cyclin-Cyclin-dependent kinase complexes, with an emergent role in the DNA damage response and cell survival control. The regulation of CDC25A activity and its protein level is essential to control the cell cycle and maintain genomic integrity. Here we describe a novel ubiquitin/proteasome-mediated pathway negatively regulating CDC25A stability, dependent on its phosphorylation by the serine/threonine kinase DYRK2.

TRAF6 Phosphorylation Prevents Its Autophagic Degradation and Re-Shapes LPS-Triggered Signaling Networks.

The ubiquitin E3 ligase TNF Receptor Associated Factor 6 (TRAF6) participates in a large number of different biological processes including innate immunity, differentiation and cell survival, raising the need to specify and shape the signaling output. Here, we identify a lipopolysaccharide (LPS)-dependent increase in TRAF6 association with the kinase IKKε (inhibitor of NF-κB kinase subunit ε) and IKKε-mediated TRAF6 phosphorylation at five residues. The reconstitution of TRAF6-deficient cells, with TRAF6 mutants representing phosphorylation-defective or phospho-mimetic TRAF6 variants, showed that the phospho-mimetic TRAF6 variant was largely protected from basal ubiquitin/proteasome-mediated degradation, and also from autophagy-mediated decay in autolysosomes induced by metabolic perturbation.

Emerging roles of DYRK2 in cancer.

Over the last decade, the CMGC kinase DYRK2 has been reported as a tumor suppressor across various cancers triggering major antitumor and proapoptotic signals in breast, colon, liver, ovary, brain, and lung cancers, with lower DYRK2 expression correlated with poorer prognosis in patients. Contrary to this, various medicinal chemistry studies reported robust antiproliferative properties of DYRK2 inhibitors, whereas unbiased 'omics' and genome-wide association study-based studies identified DYRK2 as a highly overexpressed kinase in various patient tumor samples. A major paradigm shift occurred in the last 4 years when DYRK2 was found to regulate proteostasis in cancer via a two-pronged mechanism.

The stress-responsive kinase DYRK2 activates heat shock factor 1 promoting resistance to proteotoxic stress.

To survive proteotoxic stress, cancer cells activate the proteotoxic-stress response pathway, which is controlled by the transcription factor heat shock factor 1 (HSF1). This pathway supports cancer initiation, cancer progression and chemoresistance and thus is an attractive therapeutic target. As developing inhibitors against transcriptional regulators, such as HSF1 is challenging, the identification and targeting of upstream regulators of HSF1 present a tractable alternative strategy.

High NRF2 Levels Correlate with Poor Prognosis in Colorectal Cancer Patients and with Sensitivity to the Kinase Inhibitor AT9283 In Vitro.

Aberrant hyperactivation of nuclear factor erythroid 2 (NF-E2) p45-related factor 2 (NRF2) is a common event in many tumour types and associates with resistance to therapy and poor patient prognosis; however, its relevance in colorectal tumours is not well-established. Measuring the expression of surrogate genes for NRF2 activity in silico, in combination with validation in patients' samples, we show that the NRF2 pathway is upregulated in colorectal tumours and that high levels of nuclear NRF2 correlate with a poor patient prognosis. These results highlight the need to overcome the protection provided by NRF2 and present an opportunity to selectively kill cancer cells with hyperactive NRF2.

Isomeric O-methyl cannabidiolquinones with dual BACH1/NRF2 activity.

Oxidative stress and inflammation in the brain are two key hallmarks of neurodegenerative diseases (NDs) such as Alzheimer's, Parkinson's, Huntington's and multiple sclerosis. The axis NRF2-BACH1 has anti-inflammatory and anti-oxidant properties that could be exploited pharmacologically to obtain neuroprotective effects. Activation of NRF2 or inhibition of BACH1 are, individually, promising therapeutic approaches for NDs.

Inhibition of dual-specificity tyrosine phosphorylation-regulated kinase 2 perturbs 26S proteasome-addicted neoplastic progression.

Dependence on the 26S proteasome is an Achilles' heel for triple-negative breast cancer (TNBC) and multiple myeloma (MM). The therapeutic proteasome inhibitor, bortezomib, successfully targets MM but often leads to drug-resistant disease relapse and fails in breast cancer. Here we show that a 26S proteasome-regulating kinase, DYRK2, is a therapeutic target for both MM and TNBC.

Induction of the Antioxidant Response by the Transcription Factor NRF2 Increases Bioactivation of the Mutagenic Air Pollutant 3-Nitrobenzanthrone in Human Lung Cells.

3-Nitrobenzanthrone (3-NBA) is a suspected human carcinogen present in diesel exhaust. It requires metabolic activation via nitroreduction in order to form DNA adducts and promote mutagenesis. We have determined that human aldo-keto reductases (AKR1C1-1C3) and NAD(P)H:quinone oxidoreductase 1 (NQO1) contribute equally to the nitroreduction of 3-NBA in lung epithelial cell lines and collectively represent 50% of the nitroreductase activity.

Phosphorylation-dependent regulation of the NOTCH1 intracellular domain by dual-specificity tyrosine-regulated kinase 2.

NOTCH proteins constitute a receptor family with a widely conserved role in cell cycle, growing and development regulation. NOTCH1, the best characterised member of this family, regulates the expression of key genes in cell growth and angiogenesis, playing an essential role in cancer development. These observations provide a relevant rationale to propose the inhibition of the intracellular domain of NOTCH1 (Notch1-IC) as a strategy for treating various types of cancer.

Cannabidiol induces antioxidant pathways in keratinocytes by targeting BACH1.

Cannabidiol (CBD) is a major non-psychotropic phytocannabinoid that attracted a great attention for its therapeutic potential against different pathologies including skin diseases. However, although the efficacy in preclinical models and the clinical benefits of CBD in humans have been extensively demonstrated, the molecular mechanism(s) and targets responsible for these effects are as yet unknown. Herein we characterized at the molecular level the effects of CBD on primary human keratinocytes using a combination of RNA sequencing (RNA-Seq) and sequential window acquisition of all theoretical mass spectrometry (SWATH-MS).

The isothiocyanate sulforaphane inhibits mTOR in an NRF2-independent manner.

Background: The isothiocyanate sulforaphane (SFN) has multiple protein targets in mammalian cells, affecting processes of fundamental importance for the maintenance of cellular homeostasis, among which are those regulated by the stress response transcription factor nuclear factor erythroid 2 p45-related factor 2 (NRF2) and the serine/threonine protein kinase mechanistic target of rapamycin (mTOR). Whereas the way by which SFN activates NRF2 is well established, the molecular mechanism(s) of how SFN inhibits mTOR is not understood.

Hypothesis/purpose: The aim of this study was to investigate the mechanism(s) by which SFN inhibits mTOR STUDY DESIGN AND METHODS: We used the human osteosarcoma cell line U2OS and its CRISPR/Cas9-generated NRF2-knockout counterpart to test the requirement for NRF2 and the involvement of mTOR regulators in the SFN-mediated inhibition of mTOR.

Heat Shock Factor 1 Is a Substrate for p38 Mitogen-Activated Protein Kinases.

Heat shock factor 1 (HSF1) monitors the structural integrity of the proteome. Phosphorylation at S326 is a hallmark for HSF1 activation, but the identity of the kinase(s) phosphorylating this site has remained elusive. We show here that the dietary agent phenethyl isothiocyanate (PEITC) inhibits heat shock protein 90 (Hsp90), the main negative regulator of HSF1; activates p38 mitogen-activated protein kinase (MAPK); and increases S326 phosphorylation, trimerization, and nuclear translocation of HSF1, and the transcription of a luciferase reporter, as well as the endogenous prototypic HSF1 target Hsp70.

New Insights into the Role of Histone Deacetylases as Coactivators of Inflammatory Gene Expression.

Significance: The expression and/or activity of histone deacetylases (HDACs) can be regulated by a variety of environmental conditions, including inflammation and oxidative stress. These events result in diminished or exaggerated protein acetylation, both of which can be causative for many ailments. While the anti-inflammatory activity of HDAC inhibitors (HDACis) is well known, recent studies started unraveling details of the molecular mechanisms underlying the pro-inflammatory function of HDACs.

Homeodomain-interacting protein kinase 2-dependent repression of myogenic differentiation is relieved by its caspase-mediated cleavage.

Differentiation of skeletal muscle cells is accompanied by drastic changes in gene expression programs that depend on activation and repression of genes at defined time points. Here we identify the serine/threonine kinase homeodomain-interacting protein kinase 2 (HIPK2) as a corepressor that inhibits myocyte enhancer factor 2 (MEF2)-dependent gene expression in undifferentiated myoblasts. Downregulation of HIPK2 expression by shRNAs results in elevated expression of muscle-specific genes, whereas overexpression of the kinase dampens transcription of these genes.

HIPK2 kinase activity depends on cis-autophosphorylation of its activation loop.

The multitude of mechanisms regulating the activity of protein kinases includes phosphorylation of amino acids contained in the activation loop. Here we show that the serine/threonine kinase HIPK2 (homeodomain-interacting protein kinase 2) is heavily modified by autophosphorylation, which occurs by cis-autophosphorylation at the activation loop and by trans-autophosphorylation at other phosphorylation sites. Cis-autophosphorylation of HIPK2 at Y354 and S357 in the activation loop is essential for its kinase function and the binding to substrates and the interaction partner Pin1.

A redox-regulated SUMO/acetylation switch of HIPK2 controls the survival threshold to oxidative stress.

Moderate concentrations of reactive oxygen species (ROS) serve as coregulatory signaling molecules, whereas exceedingly high concentrations trigger cell death. Here, we identify ROS-induced acetylation of the proapoptotic kinase HIPK2 as a molecular mechanism that controls the threshold discerning sensitivity from resistance toward ROS-mediated cell death. SUMOylation of HIPK2 at permissive ROS concentrations allows the constitutive association of HDAC3 and keeps HIPK2 in the nonacetylated state.