Degradation of AMPK by a cancer-specific ubiquitin ligase.

AMP-activated protein kinase (AMPK) is a master sensor and regulator of cellular energy status. Upon metabolic stress, AMPK suppresses anabolic and promotes catabolic processes to regain energy homeostasis. Cancer cells can occasionally suppress the growth-restrictive AMPK pathway by mutation of an upstream regulatory kinase. Here, we describe a widespread mechanism to suppress AMPK through its ubiquitination and degradation by the cancer-specific MAGE-A3/6-TRIM28 ubiquitin ligase. MAGE-A3 and MAGE-A6 are highly similar proteins normally expressed only in the male germline but frequently re-activated in human cancers. MAGE-A3/6 are necessary for cancer cell viability and are sufficient to drive tumorigenic properties of non-cancerous cells. Screening for targets of MAGE-A3/6-TRIM28 revealed that it ubiquitinates and degrades AMPKα1. This leads to inhibition of autophagy, activation of mTOR signaling, and hypersensitization to AMPK agonists, such as metformin. These findings elucidate a germline mechanism commonly hijacked in cancer to suppress AMPK.

Regulation of WASH-dependent actin polymerization and protein trafficking by ubiquitination.

Endosomal protein trafficking is an essential cellular process that is deregulated in several diseases and targeted by pathogens. Here, we describe a role for ubiquitination in this process. We find that the E3 RING ubiquitin ligase, MAGE-L2-TRIM27, localizes to endosomes through interactions with the retromer complex. Knockdown of MAGE-L2-TRIM27 or the Ube2O E2 ubiquitin-conjugating enzyme significantly impaired retromer-mediated transport. We further demonstrate that MAGE-L2-TRIM27 ubiquitin ligase activity is required for nucleation of endosomal F-actin by the WASH regulatory complex, a known regulator of retromer-mediated transport. Mechanistic studies showed that MAGE-L2-TRIM27 facilitates K63-linked ubiquitination of WASH K220. Significantly, disruption of WASH ubiquitination impaired endosomal F-actin nucleation and retromer-dependent transport. These findings provide a cellular and molecular function for MAGE-L2-TRIM27 in retrograde transport, including an unappreciated role of K63-linked ubiquitination and identification of an activating signal of the WASH regulatory complex.

A Cancer-Specific Ubiquitin Ligase Drives mRNA Alternative Polyadenylation by Ubiquitinating the mRNA 3' End Processing Complex.

Alternative polyadenylation (APA) contributes to transcriptome complexity by generating mRNA isoforms with varying 3' UTR lengths. APA leading to 3' UTR shortening (3' US) is a common feature of most cancer cells; however, the molecular mechanisms are not understood. Here, we describe a widespread mechanism promoting 3' US in cancer through ubiquitination of the mRNA 3' end processing complex protein, PCF11, by the cancer-specific MAGE-A11-HUWE1 ubiquitin ligase. MAGE-A11 is normally expressed only in the male germline but is frequently re-activated in cancers. MAGE-A11 is necessary for cancer cell viability and is sufficient to drive tumorigenesis. Screening for targets of MAGE-A11 revealed that it ubiquitinates PCF11, resulting in loss of CFIm25 from the mRNA 3' end processing complex. This leads to APA of many transcripts affecting core oncogenic and tumor suppressors, including cyclin D2 and PTEN. These findings provide insights into the molecular mechanisms driving APA in cancer and suggest therapeutic strategies.

Translational Repression of G3BP in Cancer and Germ Cells Suppresses Stress Granules and Enhances Stress Tolerance.

Stress granules (SGs) are membrane-less ribonucleoprotein condensates that form in response to various stress stimuli via phase separation. SGs act as a protective mechanism to cope with acute stress, but persistent SGs have cytotoxic effects that are associated with several age-related diseases. Here, we demonstrate that the testis-specific protein, MAGE-B2, increases cellular stress tolerance by suppressing SG formation through translational inhibition of the key SG nucleator G3BP. MAGE-B2 reduces G3BP protein levels below the critical concentration for phase separation and suppresses SG initiation. Knockout of the MAGE-B2 mouse ortholog or overexpression of G3BP1 confers hypersensitivity of the male germline to heat stress in vivo. Thus, MAGE-B2 provides cytoprotection to maintain mammalian spermatogenesis, a highly thermosensitive process that must be preserved throughout reproductive life. These results demonstrate a mechanism that allows for tissue-specific resistance against stress and could aid in the development of male fertility therapies.

Cytosolic Iron-Sulfur Assembly Is Evolutionarily Tuned by a Cancer-Amplified Ubiquitin Ligase.

The cytosolic iron-sulfur (Fe-S) cluster assembly (CIA) pathway functions to incorporate inorganic Fe-S cofactors into a variety of proteins, including several DNA repair enzymes. However, the mechanisms regulating the CIA pathway are unknown. We describe here that the MAGE-F1-NSE1 E3 ubiquitin ligase regulates the CIA pathway through ubiquitination and degradation of the CIA-targeting protein MMS19. Overexpression or knockout of MAGE-F1 altered Fe-S incorporation into MMS19-dependent DNA repair enzymes, DNA repair capacity, sensitivity to DNA-damaging agents, and iron homeostasis. Intriguingly, MAGE-F1 has undergone adaptive pseudogenization in select mammalian lineages. In contrast, MAGE-F1 is highly amplified in multiple human cancer types and amplified tumors have increased mutational burden. Thus, flux through the CIA pathway can be regulated by degradation of the substrate-specifying MMS19 protein and its downregulation is a common feature in cancer and is evolutionarily controlled.

TAC-tics for Leveraging Proximity Biology in Drug Discovery.

Chemically induced proximity (CIP) refers to co-opting naturally occurring biological pathways using synthetic molecules to recruit neosubstrates that are not normally encountered or to enhance the affinity of naturally occurring interactions. Leveraging proximity biology through CIPs has become a rapidly evolving field and has garnered considerable interest in basic research and drug discovery. PROteolysis TArgeting Chimera (PROTAC) is a well-established CIP modality that induces the proximity between a target protein and an E3 ubiquitin ligase, causing target protein degradation via the ubiquitin-proteasome system. Inspired by PROTACs, several other induced proximity modalities have emerged to modulate both proteins and RNA over recent years. In this review, we summarize the critical advances and opportunities in the field, focusing on protein degraders, RNA degraders and non-degrader modalities such as post-translational modification (PTM) and protein-protein interaction (PPI) modulators. We envision that these emerging proximity-based drug modalities will be valuable resources for both biological research and therapeutic discovery in the future.

An Acetyldegron Triggers CRBN to Take Down the "Q".

In this issue of Molecular Cell, Nguyen et al. (2016) show that p300/CBP-mediated acetylation of glutamine synthetase (GS) triggers recognition by the CRL4(CRBN) E3 ubiquitin ligase, resulting in its ubiquitylation and degradation in response to high glutamine concentrations.

USP7 Acts as a Molecular Rheostat to Promote WASH-Dependent Endosomal Protein Recycling and Is Mutated in a Human Neurodevelopmental Disorder.

Endosomal protein recycling is a fundamental cellular process important for cellular homeostasis, signaling, and fate determination that is implicated in several diseases. WASH is an actin-nucleating protein essential for this process, and its activity is controlled through K63-linked ubiquitination by the MAGE-L2-TRIM27 ubiquitin ligase. Here, we show that the USP7 deubiquitinating enzyme is an integral component of the MAGE-L2-TRIM27 ligase and is essential for WASH-mediated endosomal actin assembly and protein recycling. Mechanistically, USP7 acts as a molecular rheostat to precisely fine-tune endosomal F-actin levels by counteracting TRIM27 auto-ubiquitination/degradation and preventing overactivation of WASH through directly deubiquitinating it. Importantly, we identify de novo heterozygous loss-of-function mutations of USP7 in individuals with a neurodevelopmental disorder, featuring intellectual disability and autism spectrum disorder. These results provide unanticipated insights into endosomal trafficking, illuminate the cooperativity between an ubiquitin ligase and a deubiquitinating enzyme, and establish a role for USP7 in human neurodevelopmental disease.

Target and tissue selectivity of PROTAC degraders.

Targeted protein degradation (TPD) strategies have revolutionized how scientists tackle challenging protein targets deemed undruggable with traditional small molecule inhibitors. Many promising campaigns to inhibit proteins have failed due to factors surrounding inhibition selectivity and targeting of compounds to specific tissues and cell types. One of the major improvements that PROTAC (proteolysis targeting chimera) and molecular glue technology can exert is highly selective control of target inhibition. Multiple studies have shown that PROTACs can gain selectivity for their protein targets beyond that of their parent ligands via optimization of linker length and stabilization of ternary complexes. Due to the bifunctional nature of PROTACs, the tissue selective nature of E3 ligases can be exploited to uncover novel targeting mechanisms. In this review, we provide critical analysis of the recent progress towards making selective PROTAC molecules and new PROTAC technologies that will continue to push the boundaries of achieving selectivity. These efforts have wide implications in the future of treating disease as they will broaden the possible targets that can be addressed by small molecules, like undruggable proteins or broadly active targets that would benefit from degradation in specific tissue types.

CSAG2 is a cancer-specific activator of SIRT1.

SIRT1 is a NAD+ -dependent deacetylase that controls key metabolic and signaling pathways, including inactivating the p53 tumor suppressor. However, the mechanisms controlling SIRT1 enzymatic activity in the context of cancer are unclear. Here, we show that the previously undescribed CSAG2 protein is a direct activator of SIRT1. CSAG2 is normally restricted to expression in the male germline but is frequently re-activated in cancers. CSAG2 is necessary for cancer cell proliferation and promotes tumorigenesis in vivo. Biochemical studies revealed that CSAG2 directly binds to and stimulates SIRT1 activity toward multiple substrates. Importantly, CSAG2 enhances SIRT1-mediated deacetylation of p53, inhibits p53 transcriptional activity, and improves cell survival in response to genotoxic stress. Mechanistically, CSAG2 binds SIRT1 catalytic domain and promotes activity independent of altering substrate affinity. Together, our results identify a previously undescribed mechanism for SIRT1 activation in cancer cells and highlight unanticipated approaches to therapeutically modulate SIRT1.

Emerging roles of the MAGE protein family in stress response pathways.

The melanoma antigen (MAGE) proteins all contain a MAGE homology domain. MAGE genes are conserved in all eukaryotes and have expanded from a single gene in lower eukaryotes to ∼40 genes in humans and mice. Whereas some MAGEs are ubiquitously expressed in tissues, others are expressed in only germ cells with aberrant reactivation in multiple cancers. Much of the initial research on MAGEs focused on exploiting their antigenicity and restricted expression pattern to target them with cancer immunotherapy. Beyond their potential clinical application and role in tumorigenesis, recent studies have shown that MAGE proteins regulate diverse cellular and developmental pathways, implicating them in many diseases besides cancer, including lung, renal, and neurodevelopmental disorders. At the molecular level, many MAGEs bind to E3 RING ubiquitin ligases and, thus, regulate their substrate specificity, ligase activity, and subcellular localization. On a broader scale, the MAGE genes likely expanded in eutherian mammals to protect the germline from environmental stress and aid in stress adaptation, and this stress tolerance may explain why many cancers aberrantly express MAGEs Here, we present an updated, comprehensive review on the MAGE family that highlights general characteristics, emphasizes recent comparative studies in mice, and describes the diverse functions exerted by individual MAGEs.

Regulation of MAGE-A3/6 by the CRL4-DCAF12 ubiquitin ligase and nutrient availability.

Melanoma antigen genes (MAGEs) are emerging as important oncogenic drivers that are normally restricted to expression in male germ cells but are aberrantly expressed in cancers and promote tumorigenesis. Mechanistically, MAGEs function as substrate specifying subunits of E3 ubiquitin ligases. Thus, the activation of germline-specific genes in cancer can drive metabolic and signaling pathways through altered ubiquitination to promote tumorigenesis. However, the mechanisms regulating MAGE expression and activity are unclear. Here, we describe how the MAGE-A3/6 proteins that function as repressors of autophagy are downregulated in response to nutrient deprivation. Short-term cellular starvation promotes rapid MAGE-A3/6 degradation in a proteasome-dependent manner. Proteomic analysis reveals that degradation of MAGE-A3/6 is controlled by the CRL4-DCAF12 E3 ubiquitin ligase. Importantly, the degradation of MAGE-A3/6 by CRL4-DCAF12 is required for starvation-induced autophagy. These findings suggest that oncogenic MAGEs can be dynamically controlled in response to stress to allow cellular adaptation, autophagy regulation, and tumor growth and that CRL4-DCAF12 activity is responsive to nutrient status.

Phenyl-Glutarimides: Alternative Cereblon Binders for the Design of PROTACs.

Targeting cereblon (CRBN) is currently one of the most frequently reported proteolysis-targeting chimera (PROTAC) approaches, owing to favorable drug-like properties of CRBN ligands, immunomodulatory imide drugs (IMiDs). However, IMiDs are known to be inherently unstable, readily undergoing hydrolysis in body fluids. Here we show that IMiDs and IMiD-based PROTACs rapidly hydrolyze in commonly utilized cell media, which significantly affects their cell efficacy. We designed novel CRBN binders, phenyl glutarimide (PG) analogues, and showed that they retained affinity for CRBN with high ligand efficiency (LE >0.48) and displayed improved chemical stability. Our efforts led to the discovery of PG PROTAC 4 c (SJ995973), a uniquely potent degrader of bromodomain and extra-terminal (BET) proteins that inhibited the viability of human acute myeloid leukemia MV4-11 cells at low picomolar concentrations (IC50 =3 pM; BRD4 DC50 =0.87 nM). These findings strongly support the utility of PG derivatives in the design of CRBN-directed PROTACs.

Scc1 sumoylation by Mms21 promotes sister chromatid recombination through counteracting Wapl.

DNA double-strand breaks (DSBs) fuel cancer-driving chromosome translocations. Two related structural maintenance of chromosomes (Smc) complexes, cohesin and Smc5/6, promote DSB repair through sister chromatid homologous recombination (SCR). Here we show that the Smc5/6 subunit Mms21 sumoylates multiple lysines of the cohesin subunit Scc1. Mms21 promotes cohesin-dependent small ubiquitin-like modifier (SUMO) accumulation at laser-induced DNA damage sites in S/G2 human cells. Cells expressing the nonsumoylatable Scc1 mutant (15KR) maintain sister chromatid cohesion during mitosis but are defective in SCR and sensitive to ionizing radiation (IR). Scc1 15KR is recruited to DNA damage sites. Depletion of Wapl, a negative cohesin regulator, rescues SCR defects of Mms21-deficient or Scc1 15KR-expressing cells. Expression of the acetylation-mimicking Smc3 mutant does not bypass the requirement for Mms21 in SCR. We propose that Scc1 sumoylation by Mms21 promotes SCR by antagonizing Wapl at a step after cohesin loading at DSBs and in a way not solely dependent on Smc3 acetylation.

DNA-damage-induced degradation of EXO1 exonuclease limits DNA end resection to ensure accurate DNA repair.

End resection of DNA double-strand breaks (DSBs) to generate 3'-single-stranded DNA facilitates DSB repair via error-free homologous recombination (HR) while stymieing repair by the error-prone non-homologous end joining (NHEJ) pathway. Activation of DNA end resection involves phosphorylation of the 5' to 3' exonuclease EXO1 by the phosphoinositide 3-kinase-like kinases ATM (ataxia telangiectasia-mutated) and ATR (ATM and Rad3-related) and by the cyclin-dependent kinases 1 and 2. After activation, EXO1 must also be restrained to prevent over-resection that is known to hamper optimal HR and trigger global genomic instability. However, mechanisms by which EXO1 is restrained are still unclear. Here, we report that EXO1 is rapidly degraded by the ubiquitin-proteasome system soon after DSB induction in human cells. ATR inhibition attenuated DNA-damage-induced EXO1 degradation, indicating that ATR-mediated phosphorylation of EXO1 targets it for degradation. In accord with these results, EXO1 became resistant to degradation when its SQ motifs required for ATR-mediated phosphorylation were mutated. We show that upon the induction of DNA damage, EXO1 is ubiquitinated by a member of the Skp1-Cullin1-F-box (SCF) family of ubiquitin ligases in a phosphorylation-dependent manner. Importantly, expression of degradation-resistant EXO1 resulted in hyper-resection, which attenuated both NHEJ and HR and severely compromised DSB repair resulting in chromosomal instability. These findings indicate that the coupling of EXO1 activation with its eventual degradation is a timing mechanism that limits the extent of DNA end resection for accurate DNA repair.

MAGE-RING protein complexes comprise a family of E3 ubiquitin ligases.

The melanoma antigen (MAGE) family consists of more than 60 genes, many of which are cancer-testis antigens that are highly expressed in cancer and play a critical role in tumorigenesis. However, the biochemical and cellular functions of this enigmatic family of proteins have remained elusive. Here, we identify really interesting new gene (RING) domain proteins as binding partners for MAGE family proteins. Multiple MAGE family proteins bind E3 RING ubiquitin ligases with specificity. The crystal structure of one of these MAGE-RING complexes, MAGE-G1-NSE1, reveals structural insights into MAGE family proteins and their interaction with E3 RING ubiquitin ligases. Biochemical and cellular assays demonstrate that MAGE proteins enhance the ubiquitin ligase activity of RING domain proteins. For example, MAGE-C2-TRIM28 is shown to target p53 for degradation in a proteasome-dependent manner, consistent with its tumorigenic functions. These findings define a biochemical and cellular function for the MAGE protein family.

Cellular and disease functions of the Prader-Willi Syndrome gene MAGEL2.

Melanoma antigen L2 (MAGEL2 or MAGE-L2) is a member of the MAGE family of ubiquitin ligase regulators. It is maternally imprinted and often paternally deleted or mutated in the related neurodevelopmental syndromes, Prader-Willi Syndrome (PWS) and Schaaf-Yang Syndrome (SHFYNG). MAGEL2 is highly expressed in the hypothalamus and plays an important role in a fundamental cellular process that recycles membrane proteins from endosomes through the retromer sorting pathway. MAGEL2 is part of a multi-subunit protein complex consisting of MAGEL2, the TRIM27 E3 ubiquitin ligase, and the USP7 deubiquitinating enzyme. The MAGEL2-USP7-TRIM27 (or MUST) complex facilitates the retromer recycling pathway through ubiquitination and activation of the WASH actin nucleation promoting factor. This review provides an overview of the MAGE protein family of ubiquitin ligases regulators and details the molecular and cellular role of MAGEL2 in ubiquitination, actin regulation and endosomal sorting processes, as well as MAGEL2 implications in PWS and SHFYNG disorders. The physiological functions of MAGEL2, elucidated through the study of Magel2 knockout mouse models, are also discussed.

Feel the breeze: Opening the therapeutic window with RIPTACs and induced proximity.

In this issue of Cell Chemical Biology, Raina et al.1 demonstrate proof of concept of a new chemical induced proximity strategy for targeted cancer therapeutics. Building on a recent surge in induced proximity modalities, RIPTACs represent a novel approach that offers promise in treating cancers with improved safety profiles.

RNATACs: Multispecific small molecules targeting RNA by induced proximity.

RNA-targeting small molecules (rSMs) have become an attractive modality to tackle traditionally undruggable proteins and expand the druggable space. Among many innovative concepts, RNA-targeting chimeras (RNATACs) represent a new class of multispecific, induced proximity small molecules that act by chemically bringing RNA targets into proximity with an endogenous RNA effector, such as a ribonuclease (RNase). Depending on the RNA effector, RNATACs can alter the stability, localization, translation, or splicing of the target RNA. Although still in its infancy, this new modality has the potential for broad applications in the future to treat diseases with high unmet need. In this review, we discuss potential advantages of RNATACs, recent progress in the field, and challenges to this cutting-edge technology.

Melanoma antigens in pediatric medulloblastoma contribute to tumor heterogeneity and species-specificity of group 3 tumors.

Background: Medulloblastoma (MB) is the most malignant childhood brain cancer. Group 3 MB subtype accounts for about 25% of MB diagnoses and is associated with the most unfavorable outcomes. Herein, we report that more than half of group 3 MB tumors express melanoma antigens (MAGEs), which are potential prognostic and therapeutic markers. MAGEs are tumor antigens, expressed in several types of adult cancers and associated with poorer prognosis and therapy resistance; however, their expression in pediatric cancers is mostly unknown. The aim of this study was to determine whether MAGEs are activated in pediatric MB. Methods: To determine MAGE frequency in pediatric MB, we obtained formalin-fixed paraffin-embedded tissue (FFPE) samples of 34 patients, collected between 2008 - 2015, from the Children's Medical Center Dallas pathology archives and applied our validated reverse transcription quantitative PCR (RT-qPCR) assay to measure the relative expression of 23 MAGE cancer-testis antigen genes. To validate our data, we analyzed several published datasets from pediatric MB patients and patient-derived orthotopic xenografts, totaling 860 patients. We then examined how MAGE expression affects the growth and oncogenic potential of medulloblastoma cells by CRISPR-Cas9- and siRNA-mediated gene depletion. Results: Our RT-qPCR analysis suggested that MAGEs were expressed in group 3/4 medulloblastoma. Further mining of bulk and single-cell RNA-sequencing datasets confirmed that 50-75% of group 3 tumors activate a subset of MAGE genes. Depletion of MAGEAs, B2, and Cs alter MB cell survival, viability, and clonogenic growth due to decreased proliferation and increased apoptosis. Conclusions: These results indicate that targeting MAGEs in medulloblastoma may be a potential therapeutic option for group 3 medulloblastomas. Key Points: Several Type I MAGE CTAs are expressed in >60% of group 3 MBs. Type I MAGEs affect MB cell proliferation and apoptosis. MAGEs are potential biomarkers and therapeutic targets for group 3 MBs. Importance of the Study: This study is the first comprehensive analysis of all Type I MAGE CTAs ( MAGEA , -B , and -C subfamily members) in pediatric MBs. Our results show that more than 60% of group 3 MBs express MAGE genes, which are required for the viability and growth of cells in which they are expressed. Collectively, these data provide novel insights into the antigen landscape of pediatric MBs. The activation of MAGE genes in group 3 MBs presents potential stratifying and therapeutic options.