Systematic discovery of mutation-directed neo-protein-protein interactions in cancer.

Comprehensive sequencing of patient tumors reveals genomic mutations across tumor types that enable tumorigenesis and progression. A subset of oncogenic driver mutations results in neomorphic activity where the mutant protein mediates functions not engaged by the parental molecule. Here, we identify prevalent variant-enabled neomorph-protein-protein interactions (neoPPI) with a quantitative high-throughput differential screening (qHT-dS) platform. The coupling of highly sensitive BRET biosensors with miniaturized coexpression in an ultra-HTS format allows large-scale monitoring of the interactions of wild-type and mutant variant counterparts with a library of cancer-associated proteins in live cells. The screening of 17,792 interactions with 2,172,864 data points revealed a landscape of gain of interactions encompassing both oncogenic and tumor suppressor mutations. For example, the recurrent BRAF V600E lesion mediates KEAP1 neoPPI, rewiring a BRAFV600E/KEAP1 signaling axis and creating collateral vulnerability to NQO1 substrates, offering a combination therapeutic strategy. Thus, cancer genomic alterations can create neo-interactions, informing variant-directed therapeutic approaches for precision medicine.

A Cellular Mechanism to Detect and Alleviate Reductive Stress.

Metazoan organisms rely on conserved stress response pathways to alleviate adverse conditions and preserve cellular integrity. Stress responses are particularly important in stem cells that provide lifetime support for tissue formation and repair, but how these protective systems are integrated into developmental programs is poorly understood. Here we used myoblast differentiation to identify the E3 ligase CUL2FEM1B and its substrate FNIP1 as core components of the reductive stress response. Reductive stress, as caused by prolonged antioxidant signaling or mitochondrial inactivity, reverts the oxidation of invariant Cys residues in FNIP1 and allows CUL2FEM1B to recognize its target. The ensuing proteasomal degradation of FNIP1 restores mitochondrial activity to preserve redox homeostasis and stem cell integrity. The reductive stress response is therefore built around a ubiquitin-dependent rheostat that tunes mitochondrial activity to redox needs and implicates metabolic control in coordination of stress and developmental signaling.

The Oncogenic Action of NRF2 Depends on De-glycation by Fructosamine-3-Kinase.

The NRF2 transcription factor controls a cell stress program that is implicated in cancer and there is great interest in targeting NRF2 for therapy. We show that NRF2 activity depends on Fructosamine-3-kinase (FN3K)-a kinase that triggers protein de-glycation. In its absence, NRF2 is extensively glycated, unstable, and defective at binding to small MAF proteins and transcriptional activation. Moreover, the development of hepatocellular carcinoma triggered by MYC and Keap1 inactivation depends on FN3K in vivo. N-acetyl cysteine treatment partially rescues the effects of FN3K loss on NRF2 driven tumor phenotypes indicating a key role for NRF2-mediated redox balance. Mass spectrometry reveals that other proteins undergo FN3K-sensitive glycation, including translation factors, heat shock proteins, and histones. How glycation affects their functions remains to be defined. In summary, our study reveals a surprising role for the glycation of cellular proteins and implicates FN3K as targetable modulator of NRF2 activity in cancer.

Hypoxia Rescues Frataxin Loss by Restoring Iron Sulfur Cluster Biogenesis.

Friedreich's ataxia (FRDA) is a devastating, multisystemic disorder caused by recessive mutations in the mitochondrial protein frataxin (FXN). FXN participates in the biosynthesis of Fe-S clusters and is considered to be essential for viability. Here we report that when grown in 1% ambient O2, FXN null yeast, human cells, and nematodes are fully viable. In human cells, hypoxia restores steady-state levels of Fe-S clusters and normalizes ATF4, NRF2, and IRP2 signaling events associated with FRDA. Cellular studies and in vitro reconstitution indicate that hypoxia acts through HIF-independent mechanisms that increase bioavailable iron as well as directly activate Fe-S synthesis. In a mouse model of FRDA, breathing 11% O2 attenuates the progression of ataxia, whereas breathing 55% O2 hastens it. Our work identifies oxygen as a key environmental variable in the pathogenesis associated with FXN depletion, with important mechanistic and therapeutic implications.

BACH1 Stabilization by Antioxidants Stimulates Lung Cancer Metastasis.

For tumors to progress efficiently, cancer cells must overcome barriers of oxidative stress. Although dietary antioxidant supplementation or activation of endogenous antioxidants by NRF2 reduces oxidative stress and promotes early lung tumor progression, little is known about its effect on lung cancer metastasis. Here, we show that long-term supplementation with the antioxidants N-acetylcysteine and vitamin E promotes KRAS-driven lung cancer metastasis. The antioxidants stimulate metastasis by reducing levels of free heme and stabilizing the transcription factor BACH1. BACH1 activates transcription of Hexokinase 2 and Gapdh and increases glucose uptake, glycolysis rates, and lactate secretion, thereby stimulating glycolysis-dependent metastasis of mouse and human lung cancer cells. Targeting BACH1 normalized glycolysis and prevented antioxidant-induced metastasis, while increasing endogenous BACH1 expression stimulated glycolysis and promoted metastasis, also in the absence of antioxidants. We conclude that BACH1 stimulates glycolysis-dependent lung cancer metastasis and that BACH1 is activated under conditions of reduced oxidative stress.

Nrf2 Activation Promotes Lung Cancer Metastasis by Inhibiting the Degradation of Bach1.

Approximately 30% of human lung cancers acquire mutations in either Keap1 or Nfe2l2, resulting in the stabilization of Nrf2, the Nfe2l2 gene product, which controls oxidative homeostasis. Here, we show that heme triggers the degradation of Bach1, a pro-metastatic transcription factor, by promoting its interaction with the ubiquitin ligase Fbxo22. Nrf2 accumulation in lung cancers causes the stabilization of Bach1 by inducing Ho1, the enzyme catabolizing heme. In mouse models of lung cancers, loss of Keap1 or Fbxo22 induces metastasis in a Bach1-dependent manner. Pharmacological inhibition of Ho1 suppresses metastasis in a Fbxo22-dependent manner. Human metastatic lung cancer display high levels of Ho1 and Bach1. Bach1 transcriptional signature is associated with poor survival and metastasis in lung cancer patients. We propose that Nrf2 activates a metastatic program by inhibiting the heme- and Fbxo22-mediated degradation of Bach1, and that Ho1 inhibitors represent an effective therapeutic strategy to prevent lung cancer metastasis.

Chemistry-First Approach for Nomination of Personalized Treatment in Lung Cancer.

Diversity in the genetic lesions that cause cancer is extreme. In consequence, a pressing challenge is the development of drugs that target patient-specific disease mechanisms. To address this challenge, we employed a chemistry-first discovery paradigm for de novo identification of druggable targets linked to robust patient selection hypotheses. In particular, a 200,000 compound diversity-oriented chemical library was profiled across a heavily annotated test-bed of >100 cellular models representative of the diverse and characteristic somatic lesions for lung cancer. This approach led to the delineation of 171 chemical-genetic associations, shedding light on the targetability of mechanistic vulnerabilities corresponding to a range of oncogenotypes present in patient populations lacking effective therapy. Chemically addressable addictions to ciliogenesis in TTC21B mutants and GLUT8-dependent serine biosynthesis in KRAS/KEAP1 double mutants are prominent examples. These observations indicate a wealth of actionable opportunities within the complex molecular etiology of cancer.

Oxidative Stress.

Oxidative stress is two sided: Whereas excessive oxidant challenge causes damage to biomolecules, maintenance of a physiological level of oxidant challenge, termed oxidative eustress, is essential for governing life processes through redox signaling. Recent interest has focused on the intricate ways by which redox signaling integrates these converse properties. Redox balance is maintained by prevention, interception, and repair, and concomitantly the regulatory potential of molecular thiol-driven master switches such as Nrf2/Keap1 or NF-κB/IκB is used for system-wide oxidative stress response. Nonradical species such as hydrogen peroxide (H2O2) or singlet molecular oxygen, rather than free-radical species, perform major second messenger functions. Chemokine-controlled NADPH oxidases and metabolically controlled mitochondrial sources of H2O2 as well as glutathione- and thioredoxin-related pathways, with powerful enzymatic back-up systems, are responsible for fine-tuning physiological redox signaling. This makes for a rich research field spanning from biochemistry and cell biology into nutritional sciences, environmental medicine, and molecular knowledge-based redox medicine.

Proteasomal and Autophagic Degradation Systems.

Autophagy and the ubiquitin-proteasome system are the two major quality control pathways responsible for cellular homeostasis. As such, they provide protection against age-associated changes and a plethora of human diseases. Ubiquitination is utilized as a degradation signal by both systems, albeit in different ways, to mark cargoes for proteasomal and lysosomal degradation. Both systems intersect and communicate at multiple points to coordinate their actions in proteostasis and organelle homeostasis. This review summarizes molecular details of how proteasome and autophagy pathways are functionally interconnected in cells and indicates common principles and nodes of communication that can be therapeutically exploited.

Environmental pollutants and the immune response.

Environmental pollution is one of the most serious challenges to health in the modern world. Pollutants alter immune responses and can provoke immunotoxicity. In this Review, we summarize the major environmental pollutants that are attracting wide-ranging concern and the molecular basis underlying their effects on the immune system. Xenobiotic receptors, including the aryl hydrocarbon receptor (AHR), sense and respond to a subset of environmental pollutants by activating the expression of detoxification enzymes to protect the body. However, chronic activation of the AHR leads to immunotoxicity. KEAP1-NRF2 is another important system that protects the body against environmental pollutants. KEAP1 is a sensor protein that detects environmental pollutants, leading to activation of the transcription factor NRF2. NRF2 protects the body from immunotoxicity by inducing the expression of genes involved in detoxification, antioxidant and anti-inflammatory activities. Intervening in these sensor-response systems could protect the body from the devastating immunotoxicity that can be induced by environmental pollutants.

SDHA gain-of-function engages inflammatory mitochondrial retrograde signaling via KEAP1-Nrf2.

Whether screening the metabolic activity of immune cells facilitates discovery of molecular pathology remains unknown. Here we prospectively screened the extracellular acidification rate as a measure of glycolysis and the oxygen consumption rate as a measure of mitochondrial respiration in B cells from patients with primary antibody deficiency. The highest oxygen consumption rate values were detected in three study participants with persistent polyclonal B cell lymphocytosis (PPBL). Exome sequencing identified germline mutations in SDHA, which encodes succinate dehydrogenase subunit A, in all three patients with PPBL. SDHA gain-of-function led to an accumulation of fumarate in PPBL B cells, which engaged the KEAP1-Nrf2 system to drive the transcription of genes encoding inflammatory cytokines. In a single patient trial, blocking the activity of the cytokine interleukin-6 in vivo prevented systemic inflammation and ameliorated clinical disease. Overall, our study has identified pathological mitochondrial retrograde signaling as a disease modifier in primary antibody deficiency.

Primary Cilium-Autophagy-Nrf2 (PAN) Axis Activation Commits Human Embryonic Stem Cells to a Neuroectoderm Fate.

Under defined differentiation conditions, human embryonic stem cells (hESCs) can be directed toward a mesendoderm (ME) or neuroectoderm (NE) fate, the first decision during hESC differentiation. Coupled with lineage-specific G1 lengthening, a divergent ciliation pattern emerged within the first 24 hr of induced lineage specification, and these changes heralded a neuroectoderm decision before any neural precursor markers were expressed. By day 2, increased ciliation in NE precursors induced autophagy that resulted in the inactivation of Nrf2 and thereby relieved transcriptional activation of OCT4 and NANOG. Nrf2 binds directly to upstream regions of these pluripotency genes to promote their expression and repress NE derivation. Nrf2 suppression was sufficient to rescue poorly neurogenic iPSC lines. Only after these events had been initiated did neural precursor markers get expressed at day 4. Thus, we have identified a primary cilium-autophagy-Nrf2 (PAN) control axis coupled to cell-cycle progression that directs hESCs toward NE.

Repression of the Antioxidant NRF2 Pathway in Premature Aging.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare, invariably fatal premature aging disorder. The disease is caused by constitutive production of progerin, a mutant form of the nuclear architectural protein lamin A, leading, through unknown mechanisms, to diverse morphological, epigenetic, and genomic damage and to mesenchymal stem cell (MSC) attrition in vivo. Using a high-throughput siRNA screen, we identify the NRF2 antioxidant pathway as a driver mechanism in HGPS. Progerin sequesters NRF2 and thereby causes its subnuclear mislocalization, resulting in impaired NRF2 transcriptional activity and consequently increased chronic oxidative stress. Suppressed NRF2 activity or increased oxidative stress is sufficient to recapitulate HGPS aging defects, whereas reactivation of NRF2 activity in HGPS patient cells reverses progerin-associated nuclear aging defects and restores in vivo viability of MSCs in an animal model. These findings identify repression of the NRF2-mediated antioxidative response as a key contributor to the premature aging phenotype.

NRF2 Promotes Tumor Maintenance by Modulating mRNA Translation in Pancreatic Cancer.

Pancreatic cancer is a deadly malignancy that lacks effective therapeutics. We previously reported that oncogenic Kras induced the redox master regulator Nfe2l2/Nrf2 to stimulate pancreatic and lung cancer initiation. Here, we show that NRF2 is necessary to maintain pancreatic cancer proliferation by regulating mRNA translation. Specifically, loss of NRF2 led to defects in autocrine epidermal growth factor receptor (EGFR) signaling and oxidation of specific translational regulatory proteins, resulting in impaired cap-dependent and cap-independent mRNA translation in pancreatic cancer cells. Combined targeting of the EGFR effector AKT and the glutathione antioxidant pathway mimicked Nrf2 ablation to potently inhibit pancreatic cancer ex vivo and in vivo, representing a promising synthetic lethal strategy for treating the disease.

Endogenous hydrogen sulfide production is essential for dietary restriction benefits.

Dietary restriction (DR) without malnutrition encompasses numerous regimens with overlapping benefits including longevity and stress resistance, but unifying nutritional and molecular mechanisms remain elusive. In a mouse model of DR-mediated stress resistance, we found that sulfur amino acid (SAA) restriction increased expression of the transsulfuration pathway (TSP) enzyme cystathionine γ-lyase (CGL), resulting in increased hydrogen sulfide (H2S) production and protection from hepatic ischemia reperfusion injury. SAA supplementation, mTORC1 activation, or chemical/genetic CGL inhibition reduced H2S production and blocked DR-mediated stress resistance. In vitro, the mitochondrial protein SQR was required for H2S-mediated protection during nutrient/oxygen deprivation. Finally, TSP-dependent H2S production was observed in yeast, worm, fruit fly, and rodent models of DR-mediated longevity. Together, these data are consistent with evolutionary conservation of TSP-mediated H2S as a mediator of DR benefits with broad implications for clinical translation. PAPERFLICK:

AIRE relies on Z-DNA to flag gene targets for thymic T cell tolerization.

AIRE is an unconventional transcription factor that enhances the expression of thousands of genes in medullary thymic epithelial cells and promotes clonal deletion or phenotypic diversion of self-reactive T cells1-4. The biological logic of AIRE's target specificity remains largely unclear as, in contrast to many transcription factors, it does not bind to a particular DNA sequence motif. Here we implemented two orthogonal approaches to investigate AIRE's cis-regulatory mechanisms: construction of a convolutional neural network and leveraging natural genetic variation through analysis of F1 hybrid mice5. Both approaches nominated Z-DNA and NFE2-MAF as putative positive influences on AIRE's target choices. Genome-wide mapping studies revealed that Z-DNA-forming and NFE2L2-binding motifs were positively associated with the inherent ability of a gene's promoter to generate DNA double-stranded breaks, and promoters showing strong double-stranded break generation were more likely to enter a poised state with accessible chromatin and already-assembled transcriptional machinery. Consequently, AIRE preferentially targets genes with poised promoters. We propose a model in which Z-DNA anchors the AIRE-mediated transcriptional program by enhancing double-stranded break generation and promoter poising. Beyond resolving a long-standing mechanistic conundrum, these findings suggest routes for manipulating T cell tolerance.

Oxidative stress controls regulatory T cell apoptosis and suppressor activity and PD-L1-blockade resistance in tumor.

Live regulatory T cells (Treg cells) suppress antitumor immunity, but how Treg cells behave in the metabolically abnormal tumor microenvironment remains unknown. Here we show that tumor Treg cells undergo apoptosis, and such apoptotic Treg cells abolish spontaneous and PD-L1-blockade-mediated antitumor T cell immunity. Biochemical and functional analyses show that adenosine, but not typical suppressive factors such as PD-L1, CTLA-4, TGF-ß, IL-35, and IL-10, contributes to apoptotic Treg-cell-mediated immunosuppression. Mechanistically, apoptotic Treg cells release and convert a large amount of ATP to adenosine via CD39 and CD73, and mediate immunosuppression via the adenosine and A2A pathways. Apoptosis in Treg cells is attributed to their weak NRF2-associated antioxidant system and high vulnerability to free oxygen species in the tumor microenvironment. Thus, the data support a model wherein tumor Treg cells sustain and amplify their suppressor capacity through inadvertent death via oxidative stress. This work highlights the oxidative pathway as a metabolic checkpoint that controls Treg cell behavior and affects the efficacy of therapeutics targeting cancer checkpoints.

ATDC binds to KEAP1 to drive NRF2-mediated tumorigenesis and chemoresistance in pancreatic cancer.

Pancreatic ductal adenocarcinoma is a lethal disease characterized by late diagnosis, propensity for early metastasis and resistance to chemotherapy. Little is known about the mechanisms that drive innate therapeutic resistance in pancreatic cancer. The ataxia-telangiectasia group D-associated gene (ATDC) is overexpressed in pancreatic cancer and promotes tumor growth and metastasis. Our study reveals that increased ATDC levels protect cancer cells from reactive oxygen species (ROS) via stabilization of nuclear factor erythroid 2-related factor 2 (NRF2). Mechanistically, ATDC binds to Kelch-like ECH-associated protein 1 (KEAP1), the principal regulator of NRF2 degradation, and thereby prevents degradation of NRF2 resulting in activation of a NRF2-dependent transcriptional program, reduced intracellular ROS and enhanced chemoresistance. Our findings define a novel role of ATDC in regulating redox balance and chemotherapeutic resistance by modulating NRF2 activity.

Collagenolysis-dependent DDR1 signalling dictates pancreatic cancer outcome.

Pancreatic ductal adenocarcinoma (PDAC) is a highly desmoplastic, aggressive cancer that frequently progresses and spreads by metastasis to the liver1. Cancer-associated fibroblasts, the extracellular matrix and type I collagen (Col I) support2,3 or restrain the progression of PDAC and may impede blood supply and nutrient availability4. The dichotomous role of the stroma in PDAC, and the mechanisms through which it influences patient survival and enables desmoplastic cancers to escape nutrient limitation, remain poorly understood. Here we show that matrix-metalloprotease-cleaved Col I (cCol I) and intact Col I (iCol I) exert opposing effects on PDAC bioenergetics, macropinocytosis, tumour growth and metastasis. Whereas cCol I activates discoidin domain receptor 1 (DDR1)-NF-κB-p62-NRF2 signalling to promote the growth of PDAC, iCol I triggers the degradation of DDR1 and restrains the growth of PDAC. Patients whose tumours are enriched for iCol I and express low levels of DDR1 and NRF2 have improved median survival compared to those whose tumours have high levels of cCol I, DDR1 and NRF2. Inhibition of the DDR1-stimulated expression of NF-κB or mitochondrial biogenesis blocks tumorigenesis in wild-type mice, but not in mice that express MMP-resistant Col I. The diverse effects of the tumour stroma on the growth and metastasis of PDAC and on the survival of patients are mediated through the Col I-DDR1-NF-κB-NRF2 mitochondrial biogenesis pathway, and targeting components of this pathway could provide therapeutic opportunities.

The Center Cannot Hold: NRF2 Battles Ferroptosis in the 3rd Dimension.

Takahashi et al. (2020) conduct a focused CRISPR/Cas9 screen against NRF2 target and other redox regulatory genes in both 2D- and 3D-culture systems, uncovering a vulnerability of spheroid cancer cells deprived of extracellular matrix to undergo ferroptosis.