Molecular basis for substrate recruitment to the PRMT5 methylosome.

PRMT5 is an essential arginine methyltransferase and a therapeutic target in MTAP-null cancers. PRMT5 uses adaptor proteins for substrate recruitment through a previously undefined mechanism. Here, we identify an evolutionarily conserved peptide sequence shared among the three known substrate adaptors (CLNS1A, RIOK1, and COPR5) and show that it is necessary and sufficient for interaction with PRMT5. We demonstrate that PRMT5 uses modular adaptor proteins containing a common binding motif for substrate recruitment, comparable with other enzyme classes such as kinases and E3 ligases. We structurally resolve the interface with PRMT5 and show via genetic perturbation that it is required for methylation of adaptor-recruited substrates including the spliceosome, histones, and ribosomal complexes. Furthermore, disruption of this site affects Sm spliceosome activity, leading to intron retention. Genetic disruption of the PRMT5-substrate adaptor interface impairs growth of MTAP-null tumor cells and is thus a site for development of therapeutic inhibitors of PRMT5.

A conditional mouse expressing an activating mutation in NRF2 displays hyperplasia of the upper gastrointestinal tract and decreased white adipose tissue.

Activation of the nuclear factor (erythroid-derived 2)-like 2 (NFE2L2 or NRF2) transcription factor is a critical and evolutionarily conserved cellular response to oxidative stress, metabolic stress, and xenobiotic insult. Deficiency of NRF2 results in hypersensitivity to a variety of stressors, whereas its aberrant activation contributes to several cancer types, most commonly squamous cell carcinomas of the esophagus, oral cavity, bladder, and lung. Between 10% and 35% of patients with squamous cell carcinomas display hyperactive NRF2 signaling, harboring activating mutations and copy number amplifications of the NFE2L2 oncogene or inactivating mutations or deletions of KEAP1 or CUL3, the proteins of which co-complex to ubiquitylate and degrade NRF2 protein. To better understand the role of NRF2 in tumorigenesis and more broadly in development, we engineered the endogenous Nfe2l2 genomic locus to create a conditional mutant LSL-Nrf2E79Q mouse model. The E79Q mutation, one of the most commonly observed NRF2-activating mutations in human squamous cancers, codes for a mutant protein that does not undergo KEAP1/CUL3-dependent degradation, resulting in its constitutive activity. Expression of NRF2 E79Q protein in keratin 14 (KRT14)-positive murine tissues resulted in hyperplasia of squamous cell tissues of the tongue, forestomach, and esophagus, a stunted body axis, decreased weight, and decreased visceral adipose depots. RNA-seq profiling and follow-up validation studies of cultured NRF2E79Q murine esophageal epithelial cells revealed known and novel NRF2-regulated transcriptional programs, including genes associated with squamous cell carcinoma (e.g. Myc), lipid and cellular metabolism (Hk2, Ppard), and growth factors (Areg, Bmp6, Vegfa). These data suggest that in addition to decreasing adipogenesis, KRT14-restricted NRF2 activation drives hyperplasia of the esophagus, forestomach, and tongue, but not formation of squamous cell carcinoma. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Mercury Benefits of Climate Policy in China: Addressing the Paris Agreement and the Minamata Convention Simultaneously.

National commitments under the Paris Agreement on climate change interact with other global environmental objectives, such as those of the Minamata Convention on Mercury. We assess how mercury emissions and deposition reductions from national climate policy in China under the Paris Agreement could contribute to the country's commitments under the Minamata Convention. We examine emissions under climate policy scenarios developed using a computable general equilibrium model of China's economy, end-of-pipe control scenarios that meet China's commitments under the Minamata Convention, and these policies in combination, and evaluate deposition using a global atmospheric transport model. We find climate policy in China can provide mercury benefits when implemented with Minamata policy, achieving in the year 2030 approximately 5% additional reduction in mercury emissions and deposition in China when climate policy achieves a 5% reduction per year in carbon intensity (CO2 emissions 9.7 Gt in 2030). This corresponds to 63 Mg additional mercury emissions reductions in 2030 when implemented with Minamata Convention policy, compared to Minamata policy implemented alone. Climate policy provides emissions reductions in sectors not considered under the Minamata Convention, such as residential combustion. This changes the combination of sectors that contribute to emissions reductions.

Identification and Characterization of MCM3 as a Kelch-like ECH-associated Protein 1 (KEAP1) Substrate.

KEAP1 is a substrate adaptor protein for a CUL3-based E3 ubiquitin ligase. Ubiquitylation and degradation of the antioxidant transcription factor NRF2 is considered the primary function of KEAP1; however, few other KEAP1 substrates have been identified. Because KEAP1 is altered in a number of human pathologies and has been proposed as a potential therapeutic target therein, we sought to better understand KEAP1 through systematic identification of its substrates. Toward this goal, we combined parallel affinity capture proteomics and candidate-based approaches. Substrate-trapping proteomics yielded NRF2 and the related transcription factor NRF1 as KEAP1 substrates. Our targeted investigation of KEAP1-interacting proteins revealed MCM3, an essential subunit of the replicative DNA helicase, as a new substrate. We show that MCM3 is ubiquitylated by the KEAP1-CUL3-RBX1 complex in cells and in vitro Using ubiquitin remnant profiling, we identify the sites of KEAP1-dependent ubiquitylation in MCM3, and these sites are on predicted exposed surfaces of the MCM2-7 complex. Unexpectedly, we determined that KEAP1 does not regulate total MCM3 protein stability or subcellular localization. Our analysis of a KEAP1 targeting motif in MCM3 suggests that MCM3 is a point of direct contact between KEAP1 and the MCM hexamer. Moreover, KEAP1 associates with chromatin in a cell cycle-dependent fashion with kinetics similar to the MCM2-7 complex. KEAP1 is thus poised to affect MCM2-7 dynamics or function rather than MCM3 abundance. Together, these data establish new functions for KEAP1 within the nucleus and identify MCM3 as a novel substrate of the KEAP1-CUL3-RBX1 E3 ligase.

Histone deacetylase inhibitors activate CIITA and MHC class II antigen expression in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL), the most common form of non-Hodgkin's lymphoma (NHL) diagnosed in the USA, consists of at least two distinct subtypes: germinal centre B (GCB) and activated B-cell (ABC). Decreased MHC class II (MHCII) expression on the tumours in both DLBCL subtypes directly correlates with significant decreases in patient survival. One common mechanism accounting for MHCII down-regulation in DLBCL is reduced expression of the MHC class II transactivator (CIITA), the master regulator of MHCII transcription. Furthermore, reduced CIITA expression in ABC DLBCL correlates with the presence of the transcriptional repressor positive regulatory domain-I-binding factor-1 (PRDI-BF1). However, the mechanisms underlying down-regulation of CIITA in GCB DLBCL are currently unclear. In this study, we demonstrate that neither PRDI-BF1 nor CpG hypermethylation at the CIITA promoters are responsible for decreased CIITA in GCB DLBCL. In contrast, histone modifications associated with an open chromatin conformation and active transcription were significantly lower at the CIITA promoters in CIITA(-) GCB cells compared with CIITA(+) B cells, which suggests that epigenetic mechanisms contribute to repression of CIITA transcription. Treatment of CIITA(-) or CIITA(low) GCB cells with several different histone deacetylase inhibitors (HDACi) activated modest CIITA and MHCII expression. However, CIITA and MHCII levels were significantly higher in these cells after exposure to the HDAC-1-specific inhibitor MS-275. These results suggest that CIITA transcription is repressed in GCB DLBCL cells through epigenetic mechanisms involving HDACs, and that HDACi treatment can alleviate repression. These observations may have important implications for patient therapy.

Early Clinical Success of MTA-Cooperative PRMT5 Inhibitors for the Treatment of CDKN2A/MTAP-Deleted Cancers.

SUMMARY: CDKN2A encodes the tumor suppressors p16 and p14ARF and is the most common homozygously deleted gene in all human cancers; tumors frequently codelete the nearby gene MTAP, creating a dependency on PRMT5. In this issue of Cancer Discovery, Engstrom and colleagues report an MTA-cooperative PRMT5 methyltransferase inhibitor MRTX1719 that selectively kills CDKN2A/MTAP-codeleted cancers and demonstrates early efficacy in clinical trials for solid tumors harboring the CDKN2A/MTAP codeletion. See related article by Engstrom et al., p. 2412 (1).

Discovery of a First-in-Class Inhibitor of the PRMT5-Substrate Adaptor Interaction.

PRMT5 and its substrate adaptor proteins (SAPs), pICln and Riok1, are synthetic lethal dependencies in MTAP-deleted cancer cells. SAPs share a conserved PRMT5 binding motif (PBM) which mediates binding to a surface of PRMT5 distal to the catalytic site. This interaction is required for methylation of several PRMT5 substrates, including histone and spliceosome complexes. We screened for small molecule inhibitors of the PRMT5-PBM interaction and validated a compound series which binds to the PRMT5-PBM interface and directly inhibits binding of SAPs. Mode of action studies revealed the formation of a covalent bond between a halogenated pyridazinone group and cysteine 278 of PRMT5. Optimization of the starting hit produced a lead compound, BRD0639, which engages the target in cells, disrupts PRMT5-RIOK1 complexes, and reduces substrate methylation. BRD0639 is a first-in-class PBM-competitive inhibitor that can support studies of PBM-dependent PRMT5 activities and the development of novel PRMT5 inhibitors that selectively target these functions.

Coordinate loss of MHC class II expression in the diffuse large B cell lymphoma cell line OCI-Ly2 is due to a novel mutation in RFX-AP.

Loss of major histocompatibility complex class II (MHCII) antigen expression on diffuse large B cell lymphoma (DLBCL) corresponds closely with significant decreases in patient survival. However, the mechanisms accounting for MHCII loss in DLBCL have not been thoroughly characterized to date. In this report, we demonstrate that coordinate loss of MHCII expression in OCI-Ly2 DLBCL cells is associated with an 11-base deletion in the cDNA encoding RFX-AP, one of the subunits of the heterotrimeric regulatory factor X (RFX) that is required for activating MHCII transcription. This deletion results in a frameshift in the RFX-AP protein beginning at amino acid 234 and, therefore, in the loss of C-terminal amino acids that are required for function. Stable transfection of OCI-Ly2 DLBCL cells with an expression vector for wild-type RFX-AP restores MHCII expression, which strongly suggests that the defect in RFX-AP accounts for MHCII loss in these cells.

Health co-benefits and mitigation costs as per the Paris Agreement under different technological pathways for energy supply.

This study assesses the reductions in air pollution emissions and subsequent beneficial health effects from different global mitigation pathways consistent with the 2 °C stabilization objective of the Paris Agreement. We use an integrated modelling framework, demonstrating the need for models with an appropriate level of technology detail for an accurate co-benefit assessment. The framework combines an integrated assessment model (GCAM) with an air quality model (TM5-FASST) to obtain estimates of premature mortality and then assesses their economic cost. The results show that significant co-benefits can be found for a range of technological options, such as introducing a limitation on bioenergy, carbon capture and storage (CCS) or nuclear power. Cumulative premature mortality may be reduced by 17-23% by 2020-2050 compared to the baseline, depending on the scenarios. However, the ratio of health co-benefits to mitigation costs varies substantially, ranging from 1.45 when a bioenergy limitation is set to 2.19 when all technologies are available. As for regional disaggregation, some regions, such as India and China, obtain far greater co-benefits than others.

Loss of SWI/SNF Chromatin Remodeling Alters NRF2 Signaling in Non-Small Cell Lung Carcinoma.

The NF-E2-related factor 2 (referred to as NRF2) transcription factor binds antioxidant responsive elements within the promoters of cytoprotective genes to induce their expression. Next-generation sequencing studies in lung cancer have shown a significant number of activating mutations within the NRF2 signaling pathway. Mutations in components of the SWI/SNF chromatin-remodeling complex, a general regulator of transcription using either BRG1 or BRM as the catalytic subunit, also frequently occur in lung cancers. Importantly, low BRG1 expression levels in primary human NSCLC correlated with increased NRF2-target gene expression. Here, we show that loss of SWI/SNF complex function activated a subset of NRF2-mediated transcriptional targets. Using a series of isogenic NSCLC lines with reduced or depleted BRG1 and/or BRM expression, we observed significantly increased expression of the NRF2-target genes HMOX1 and GSTM4. In contrast, expression of the NRF2 target genes NQO1 and GCLM modestly increased following BRM reduction. Chromatin immunoprecipitation showed that BRG1 knockdown led to increased NRF2 binding at its respective ARE sites in the HMOX1 promoter but not in NQO1 and GCLM. Our data demonstrate that loss of BRG1 or BRM in lung cancer results in activation of the NRF2/KEAP1 pathway and HMOX1 expression. Therefore, we provide an additional molecular explanation for why patients harboring BRG1 or BRM mutations show poor prognoses. A better understanding of this mechanism may yield novel insights into the design of targeted treatment modalities. IMPLICATIONS: Our study identifies a novel mechanism for how mutations in the SMARCA4 gene may drive progression of human lung adenocarcinomas.

Engineering a genetically encoded competitive inhibitor of the KEAP1-NRF2 interaction via structure-based design and phage display.

In its basal state, KEAP1 binds the transcription factor NRF2 (Kd = 5 nM) and promotes its degradation by ubiquitylation. Changes in the redox environment lead to modification of key cysteines within KEAP1, resulting in NRF2 protein accumulation and the transcription of genes important for restoring the cellular redox state. Using phage display and a computational loop grafting protocol, we engineered a monobody (R1) that is a potent competitive inhibitor of the KEAP1-NRF2 interaction. R1 bound to KEAP1 with a Kd of 300 pM and in human cells freed NRF2 from KEAP1 resulting in activation of the NRF2 promoter. Unlike cysteine-reactive small molecules that lack protein specificity, R1 is a genetically encoded, reversible inhibitor designed specifically for KEAP1. R1 should prove useful for studying the role of the KEAP1-NRF2 interaction in several disease states. The structure-based phage display strategy employed here is a general approach for engineering high-affinity binders that compete with naturally occurring interactions.

Proteomic analysis of ubiquitin ligase KEAP1 reveals associated proteins that inhibit NRF2 ubiquitination.

Somatic mutations in the KEAP1 ubiquitin ligase or its substrate NRF2 (NFE2L2) commonly occur in human cancer, resulting in constitutive NRF2-mediated transcription of cytoprotective genes. However, many tumors display high NRF2 activity in the absence of mutation, supporting the hypothesis that alternative mechanisms of pathway activation exist. Previously, we and others discovered that via a competitive binding mechanism, the proteins WTX (AMER1), PALB2, and SQSTM1 bind KEAP1 to activate NRF2. Proteomic analysis of the KEAP1 protein interaction network revealed a significant enrichment of associated proteins containing an ETGE amino acid motif, which matches the KEAP1 interaction motif found in NRF2. Like WTX, PALB2, and SQSTM1, we found that the dipeptidyl peptidase 3 (DPP3) protein binds KEAP1 via an "ETGE" motif to displace NRF2, thus inhibiting NRF2 ubiquitination and driving NRF2-dependent transcription. Comparing the spectrum of KEAP1-interacting proteins with the genomic profile of 178 squamous cell lung carcinomas characterized by The Cancer Genome Atlas revealed amplification and mRNA overexpression of the DPP3 gene in tumors with high NRF2 activity but lacking NRF2 stabilizing mutations. We further show that tumor-derived mutations in KEAP1 are hypomorphic with respect to NRF2 inhibition and that DPP3 overexpression in the presence of these mutants further promotes NRF2 activation. Collectively, our findings further support the competition model of NRF2 activation and suggest that "ETGE"-containing proteins such as DPP3 contribute to NRF2 activity in cancer.