Nucleolar stress caused by arginine-rich peptides triggers a ribosomopathy and accelerates aging in mice.

Nucleolar stress (NS) has been associated with age-related diseases such as cancer or neurodegeneration. To investigate how NS triggers toxicity, we used (PR)n arginine-rich peptides present in some neurodegenerative diseases as inducers of this perturbation. We here reveal that whereas (PR)n expression leads to a decrease in translation, this occurs concomitant with an accumulation of free ribosomal (r) proteins. Conversely, (PR)n-resistant cells have lower rates of r-protein synthesis, and targeting ribosome biogenesis by mTOR inhibition or MYC depletion alleviates (PR)n toxicity in vitro. In mice, systemic expression of (PR)97 drives widespread NS and accelerated aging, which is alleviated by rapamycin. Notably, the generalized accumulation of orphan r-proteins is a common outcome of chemical or genetic perturbations that induce NS. Together, our study presents a general model to explain how NS induces cellular toxicity and provides in vivo evidence supporting a role for NS as a driver of aging in mammals.

Decoding protein methylation function with thermal stability analysis.

Protein methylation is an important modification beyond epigenetics. However, systems analyses of protein methylation lag behind compared to other modifications. Recently, thermal stability analyses have been developed which provide a proxy of a protein functional status. Here, we show that molecular and functional events closely linked to protein methylation can be revealed by the analysis of thermal stability. Using mouse embryonic stem cells as a model, we show that Prmt5 regulates mRNA binding proteins that are enriched in intrinsically disordered regions and involved in liquid-liquid phase separation mechanisms, including the formation of stress granules. Moreover, we reveal a non-canonical function of Ezh2 in mitotic chromosomes and the perichromosomal layer, and identify Mki67 as a putative Ezh2 substrate. Our approach provides an opportunity to systematically explore protein methylation function and represents a rich resource for understanding its role in pluripotency.

Targeting the nucleolus as a therapeutic strategy in human disease.

The nucleolus is the site of ribosome biogenesis, one of the most resource-intensive processes in eukaryotic cells. Accordingly, nucleolar morphology and activity are highly responsive to growth signaling and nucleolar insults which are collectively included in the actively evolving concept of nucleolar stress. Importantly, nucleolar alterations are a prominent feature of multiple human pathologies, including cancer and neurodegeneration, as well as being associated with aging. The past decades have seen numerous attempts to isolate compounds targeting different facets of nucleolar activity. We provide an overview of therapeutic opportunities for targeting nucleoli in different pathologies and currently available therapies.

USP7 and VCPFAF1 define the SUMO/Ubiquitin landscape at the DNA replication fork.

The AAA+ ATPase VCP regulates the extraction of SUMO and ubiquitin-modified DNA replication factors from chromatin. We have previously described that active DNA synthesis is associated with a SUMO-high/ubiquitin-low environment governed by the deubiquitylase USP7. Here, we unveil a functional cooperation between USP7 and VCP in DNA replication, which is conserved from Caenorhabditis elegans to mammals. The role of VCP in chromatin is defined by its cofactor FAF1, which facilitates the extraction of SUMOylated and ubiquitylated proteins that accumulate after the block of DNA replication in the absence of USP7. The inactivation of USP7 and FAF1 is synthetically lethal both in C. elegans and mammalian cells. In addition, USP7 and VCP inhibitors display synergistic toxicity supporting a functional link between deubiquitylation and extraction of chromatin-bound proteins. Our results suggest that USP7 and VCPFAF1 facilitate DNA replication by controlling the balance of SUMO/Ubiquitin-modified DNA replication factors on chromatin.

Widespread displacement of DNA- and RNA-binding factors underlies toxicity of arginine-rich cell-penetrating peptides.

Due to their capability to transport chemicals or proteins into target cells, cell-penetrating peptides (CPPs) are being developed as therapy delivery tools. However, and despite their interesting properties, arginine-rich CPPs often show toxicity for reasons that remain poorly understood. Using a (PR)n dipeptide repeat that has been linked to amyotrophic lateral sclerosis (ALS) as a model of an arginine-rich CPP, we here show that the presence of (PR)n leads to a generalized displacement of RNA- and DNA-binding proteins from chromatin and mRNA. Accordingly, any reaction involving nucleic acids, such as RNA transcription, translation, splicing and degradation, or DNA replication and repair, is impaired by the presence of the CPPs. Interestingly, the effects of (PR)n are fully mimicked by protamine, a small arginine-rich protein that displaces histones from chromatin during spermatogenesis. We propose that widespread coating of nucleic acids and consequent displacement of RNA- and DNA-binding factors from chromatin and mRNA accounts for the toxicity of arginine-rich CPPs, including those that have been recently associated with the onset of ALS.

A chemical screen for modulators of mRNA translation identifies a distinct mechanism of toxicity for sphingosine kinase inhibitors.

We here conducted an image-based chemical screen to evaluate how medically approved drugs, as well as drugs that are currently under development, influence overall translation levels. None of the compounds up-regulated translation, which could be due to the screen being performed in cancer cells grown in full media where translation is already present at very high levels. Regarding translation down-regulators, and consistent with current knowledge, inhibitors of the mechanistic target of rapamycin (mTOR) signaling pathway were the most represented class. In addition, we identified that inhibitors of sphingosine kinases (SPHKs) also reduce mRNA translation levels independently of mTOR. Mechanistically, this is explained by an effect of the compounds on the membranes of the endoplasmic reticulum (ER), which activates the integrated stress response (ISR) and contributes to the toxicity of SPHK inhibitors. Surprisingly, the toxicity and activation of the ISR triggered by 2 independent SPHK inhibitors, SKI-II and ABC294640, the latter in clinical trials, are also observed in cells lacking SPHK1 and SPHK2. In summary, our study provides a useful resource on the effects of medically used drugs on translation, identified compounds capable of reducing translation independently of mTOR and has revealed that the cytotoxic properties of SPHK inhibitors being developed as anticancer agents are independent of SPHKs.

USP7 limits CDK1 activity throughout the cell cycle.

Chemical inhibitors of the deubiquitinase USP7 are currently being developed as anticancer agents based on their capacity to stabilize P53. Regardless of this activity, USP7 inhibitors also generate DNA damage in a p53-independent manner. However, the mechanism of this genotoxicity and its contribution to the anticancer effects of USP7 inhibitors are still under debate. Here we show that, surprisingly, even if USP7 inhibitors stop DNA replication, they also induce a widespread activation of CDK1 throughout the cell cycle, which leads to DNA damage and is toxic for mammalian cells. In addition, USP7 interacts with the phosphatase PP2A and supports its active localization in the cytoplasm. Accordingly, inhibition of USP7 or PP2A triggers very similar changes of the phosphoproteome, including a widespread increase in the phosphorylation of CDK1 targets. Importantly, the toxicity of USP7 inhibitors is alleviated by lowering CDK1 activity or by chemical activation of PP2A. Our work reveals that USP7 limits CDK1 activity at all cell cycle stages, providing a novel mechanism that explains the toxicity of USP7 inhibitors through untimely activation of CDK1.

Mislocalization of SMN from the I-band and M-band in human skeletal myofibers in spinal muscular atrophy associates with primary structural alterations of the sarcomere.

Spinal muscular atrophy (SMA) is caused by a deletion or mutation of the survival motor neuron 1 (SMN1) gene. Reduced SMN levels lead to motor neuron degeneration and muscular atrophy. SMN protein localizes to the cytoplasm and Cajal bodies. Moreover, in myofibrils from Drosophila and mice, SMN is a sarcomeric protein localized to the Z-disc. Although SMN participates in multiple functions, including the biogenesis of spliceosomal small nuclear ribonucleoproteins, its role in the sarcomere is unclear. Here, we analyzed the sarcomeric organization of SMN in human control and type I SMA skeletal myofibers. In control sarcomeres, we demonstrate that human SMN is localized to the titin-positive M-band and actin-positive I-band, and to SMN-positive granules that flanked the Z-discs. Co-immunoprecipitation assays revealed that SMN interacts with the sarcomeric protein actin, α-actinin, titin, and profilin2. In the type I SMA muscle, SMN levels were reduced, and atrophic (denervated) and hypertrophic (nondenervated) myofibers coexisted. The hypertrophied myofibers, which are potential primary targets of SMN deficiency, exhibited sites of focal or segmental alterations of the actin cytoskeleton, where the SMN immunostaining pattern was altered. Moreover, SMN was relocalized to the Z-disc in overcontracted minisarcomeres from hypertrophic myofibers. We propose that SMN could have an integrating role in the molecular components of the sarcomere. Consequently, low SMN levels might impact the normal sarcomeric architecture, resulting in the disruption of myofibrils found in SMA muscle. This primary effect might be independent of the neurogenic myopathy produced by denervation and contribute to pathophysiology of the SMA myopathy.

GRK2-Dependent HuR Phosphorylation Regulates HIF1α Activation under Hypoxia or Adrenergic Stress.

Adaptation to hypoxia is a common feature in solid tumors orchestrated by oxygen-dependent and independent upregulation of the hypoxia-inducible factor-1α (HIF-1α). We unveiled that G protein-coupled receptor kinase (GRK2), known to be overexpressed in certain tumors, fosters this hypoxic pathway via phosphorylation of the mRNA-binding protein HuR, a central HIF-1α modulator. GRK2-mediated HuR phosphorylation increases the total levels and cytoplasmic shuttling of HuR in response to hypoxia, and GRK2-phosphodefective HuR mutants show defective cytosolic accumulation and lower binding to HIF-1α mRNA in hypoxic Hela cells. Interestingly, enhanced GRK2 and HuR expression correlate in luminal breast cancer patients. GRK2 also promotes the HuR/HIF-1α axis and VEGF-C accumulation in normoxic MCF7 breast luminal cancer cells and is required for the induction of HuR/HIF1-α in response to adrenergic stress. Our results point to a relevant role of the GRK2/HuR/HIF-1α module in the adaptation of malignant cells to tumor microenvironment-related stresses.

PDS5 proteins are required for proper cohesin dynamics and participate in replication fork protection.

Cohesin is a chromatin-bound complex that mediates sister chromatid cohesion and facilitates long-range interactions through DNA looping. How the transcription and replication machineries deal with the presence of cohesin on chromatin remains unclear. The dynamic association of cohesin with chromatin depends on WAPL cohesin release factor (WAPL) and on PDS5 cohesin-associated factor (PDS5), which exists in two versions in vertebrate cells, PDS5A and PDS5B. Using genetic deletion in mouse embryo fibroblasts and a combination of CRISPR-mediated gene editing and RNAi-mediated gene silencing in human cells, here we analyzed the consequences of PDS5 depletion for DNA replication. We found that either PDS5A or PDS5B is sufficient for proper cohesin dynamics and that their simultaneous removal increases cohesin's residence time on chromatin and slows down DNA replication. A similar phenotype was observed in WAPL-depleted cells. Cohesin down-regulation restored normal replication fork rates in PDS5-deficient cells, suggesting that chromatin-bound cohesin hinders the advance of the replisome. We further show that PDS5 proteins are required to recruit WRN helicase-interacting protein 1 (WRNIP1), RAD51 recombinase (RAD51), and BRCA2 DNA repair associated (BRCA2) to stalled forks and that in their absence, nascent DNA strands at unprotected forks are degraded by MRE11 homolog double-strand break repair nuclease (MRE11). These findings indicate that PDS5 proteins participate in replication fork protection and also provide insights into how cohesin and its regulators contribute to the response to replication stress, a common feature of cancer cells.

Nucleolin reorganization and nucleolar stress in Purkinje cells of mutant PCD mice.

The Purkinje cell (PC) degeneration (pcd) mouse harbors a mutation in Agtpbp1 gene that encodes for the cytosolic carboxypeptidase, CCP1. The mutation causes degeneration and death of PCs during the postnatal life, resulting in clinical and pathological manifestation of cerebellar ataxia. Monogenic biallelic damaging variants in the Agtpbp1 gene cause infantile-onset neurodegeneration and cerebellar atrophy, linking loss of functional CCP1 with human neurodegeneration. Although CCP1 plays a key role in the regulation of tubulin stabilization, its loss of function in PCs leads to a severe nuclear phenotype with heterochromatinization and accumulation of DNA damage. Therefore, the pcd mice provides a useful neuronal model to investigate nuclear mechanisms involved in neurodegeneration, particularly the nucleolar stress. In this study, we demonstrated that the Agtpbp1 gene mutation induces a p53-dependent nucleolar stress response in PCs, which is characterized by nucleolar fragmentation, nucleoplasmic and cytoplasmic mislocalization of nucleolin, and dysfunction of both pre-rRNA processing and mRNA translation. RT-qPCR analysis revealed reduction of mature 18S rRNA, with a parallel increase of its intermediate 18S-5'-ETS precursor, that correlates with a reduced expression of Fbl mRNA, which encodes an essential factor for rRNA processing. Moreover, nucleolar alterations were accompanied by a reduction of PTEN mRNA and protein levels, which appears to be related to the chromosome instability and accumulation of DNA damage in degenerating PCs. Our results highlight the essential contribution of nucleolar stress to PC degeneration and also underscore the nucleoplasmic mislocalization of nucleolin as a potential indicator of neurodegenerative processes.

A Chemical Screen Identifies Compounds Limiting the Toxicity of C9ORF72 Dipeptide Repeats.

The expansion of GGGGCC repeats within the first intron of C9ORF72 constitutes the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Through repeat-associated non-ATG translation, these expansions are translated into dipeptide repeats (DPRs), some of which accumulate at nucleoli and lead to cell death. We here performed a chemical screen to identify compounds reducing the toxicity of ALS-related poly(PR) peptides. Our screening identified sodium phenylbutyrate, currently in clinical trials, and BET Bromodomain inhibitors as modifiers of poly(PR) toxicity in cell lines and developing zebrafish embryos. Mechanistically, we show that BET Bromodomain inhibitors rescue the nucleolar stress induced by poly(PR) or actinomycin D, alleviating the effects of the DPR in nucleolus-related functions such as mRNA splicing or translation. Our work suggests that BET Bromodomain inhibitors might have beneficial effects in diseases linked to nucleolar stress such as ALS/FTD.

TIAR marks nuclear G2/M transition granules and restricts CDK1 activity under replication stress.

The G2/M checkpoint coordinates DNA replication with mitosis and thereby prevents chromosome segregation in the presence of unreplicated or damaged DNA Here, we show that the RNA-binding protein TIAR is essential for the G2/M checkpoint and that TIAR accumulates in nuclear foci in late G2 and prophase in cells suffering from replication stress. These foci, which we named G2/M transition granules (GMGs), occur at low levels in normally cycling cells and are strongly induced by replication stress. In addition to replication stress response proteins, GMGs contain factors involved in RNA metabolism as well as CDK1. Depletion of TIAR accelerates mitotic entry and leads to chromosomal instability in response to replication stress, in a manner that can be alleviated by the concomitant depletion of Cdc25B or inhibition of CDK1. Since TIAR retains CDK1 in GMGs and attenuates CDK1 activity, we propose that the assembly of GMGs may represent a so far unrecognized mechanism that contributes to the activation of the G2/M checkpoint in mammalian cells.

CBP-mediated SMN acetylation modulates Cajal body biogenesis and the cytoplasmic targeting of SMN.

The survival of motor neuron (SMN) protein plays an essential role in the biogenesis of spliceosomal snRNPs and the molecular assembly of Cajal bodies (CBs). Deletion of or mutations in the SMN1 gene cause spinal muscular atrophy (SMA) with degeneration and loss of motor neurons. Reduced SMN levels in SMA lead to deficient snRNP biogenesis with consequent splicing pathology. Here, we demonstrate that SMN is a novel and specific target of the acetyltransferase CBP (CREB-binding protein). Furthermore, we identify lysine (K) 119 as the main acetylation site in SMN. Importantly, SMN acetylation enhances its cytoplasmic localization, causes depletion of CBs, and reduces the accumulation of snRNPs in nuclear speckles. In contrast, the acetylation-deficient SMNK119R mutant promotes formation of CBs and a novel category of promyelocytic leukemia (PML) bodies enriched in this protein. Acetylation increases the half-life of SMN protein, reduces its cytoplasmic diffusion rate and modifies its interactome. Hence, SMN acetylation leads to its dysfunction, which explains the ineffectiveness of HDAC (histone deacetylases) inhibitors in SMA therapy despite their potential to increase SMN levels.

G Protein-coupled Receptor Kinase 2 (GRK2) Promotes Breast Tumorigenesis Through a HDAC6-Pin1 Axis.

In addition to oncogenic drivers, signaling nodes can critically modulate cancer-related cellular networks to strength tumor hallmarks. We identify G-protein-coupled receptor kinase 2 (GRK2) as a relevant player in breast cancer. GRK2 is up-regulated in breast cancer cell lines, in spontaneous tumors in mice, and in a proportion of invasive ductal carcinoma patients. Increased GRK2 functionality promotes the phosphorylation and activation of the Histone Deacetylase 6 (HDAC6) leading to de-acetylation of the Prolyl Isomerase Pin1, a central modulator of tumor progression, thereby enhancing its stability and functional interaction with key mitotic regulators. Interestingly, a correlation between GRK2 expression and Pin1 levels and de-acetylation status is detected in breast cancer patients. Activation of the HDAC6-Pin1 axis underlies the positive effects of GRK2 on promoting growth factor signaling, cellular proliferation and anchorage-independent growth in both luminal and basal breast cancer cells. Enhanced GRK2 levels promote tumor growth in mice, whereas GRK2 down-modulation sensitizes cells to therapeutic drugs and abrogates tumor formation. Our data suggest that GRK2 acts as an important onco-modulator by strengthening the functionality of key players in breast tumorigenesis such as HDAC6 and Pin1.

Targeting the kinase activities of ATR and ATM exhibits antitumoral activity in mouse models of MLL-rearranged AML.

Among the various subtypes of acute myeloid leukemia (AML), those with chromosomal rearrangements of the MLL oncogene (AML-MLL) have a poor prognosis. AML-MLL tumor cells are resistant to current genotoxic therapies because of an attenuated response by p53, a protein that induces cell cycle arrest and apoptosis in response to DNA damage. In addition to chemicals that damage DNA, efforts have focused on targeting DNA repair enzymes as a general chemotherapeutic approach to cancer treatment. Here, we found that inhibition of the kinase ATR, which is the primary sensor of DNA replication stress, induced chromosomal breakage and death of mouse AML(MLL) cells (with an MLL-ENL fusion and a constitutively active N-RAS independently of p53. Moreover, ATR inhibition as a single agent exhibited antitumoral activity, both reducing tumor burden after establishment and preventing tumors from growing, in an immunocompetent allograft mouse model of AML(MLL) and in xenografts of a human AML-MLL cell line. We also found that inhibition of ATM, a kinase that senses DNA double-strand breaks, also promoted the survival of the AML(MLL) mice. Collectively, these data indicated that ATR or ATM inhibition represent potential therapeutic strategies for the treatment of AML, especially MLL-driven leukemias.

Efficacy of ATR inhibitors as single agents in Ewing sarcoma.

Ewing sarcomas (ES) are pediatric bone tumors that arise from a driver translocation, most frequently EWS/FLI1. Current ES treatment involves DNA damaging agents, yet the basis for the sensitivity to these therapies remains unknown. Oncogene-induced replication stress (RS) is a known source of endogenous DNA damage in cancer, which is suppressed by ATR and CHK1 kinases. We here show that ES suffer from high endogenous levels of RS, rendering them particularly dependent on the ATR pathway. Accordingly, two independent ATR inhibitors show in vitro toxicity in ES cell lines as well as in vivo efficacy in ES xenografts as single agents. Expression of EWS/FLI1 or EWS/ERG oncogenic translocations sensitizes non-ES cells to ATR inhibitors. Our data shed light onto the sensitivity of ES to genotoxic agents, and identify ATR inhibitors as a potential therapy for Ewing Sarcomas.

A Genome-wide CRISPR Screen Identifies CDC25A as a Determinant of Sensitivity to ATR Inhibitors.

One recurring theme in drug development is to exploit synthetic lethal properties as means to preferentially damage the DNA of cancer cells. We and others have previously developed inhibitors of the ATR kinase, shown to be particularly genotoxic for cells expressing certain oncogenes. In contrast, the mechanisms of resistance to ATR inhibitors remain unexplored. We report here on a genome-wide CRISPR-Cas9 screen that identified CDC25A as a major determinant of sensitivity to ATR inhibition. CDC25A-deficient cells resist high doses of ATR inhibitors, which we show is due to their failure to prematurely enter mitosis in response to the drugs. Forcing mitotic entry with WEE1 inhibitors restores the toxicity of ATR inhibitors in CDC25A-deficient cells. With ATR inhibitors now entering the clinic, our work provides a better understanding of the mechanisms by which these compounds kill cells and reveals genetic interactions that could be used for their rational use.

Dynamic Behavior of the RNA Polymerase II and the Ubiquitin Proteasome System During the Neuronal DNA Damage Response to Ionizing Radiation.

Neurons are highly vulnerable to genotoxic agents. To restore genome integrity upon DNA lesions, neurons trigger a DNA damage response (DDR) that requires chromatin modifications and transcriptional silencing at DNA damage sites. To study the reorganization of the active RNA polymerase II (Pol II), which transcribes all mRNA-encoding genes, and the participation of the ubiquitin-proteasome system (UPS) in the neuronal DDR, we have used rat sensory ganglion neurons exposed to X-rays (4 Gy) ionizing radiation (IR). In control neurons, Pol II appears concentrated in numerous chromatin microfoci identified as transcription factories by the incorporation of 5'-fluorouridine into nascent RNA. Upon IR treatment, numerous IR-induced foci (IRIF), which were immunoreactive for γH2AX and 53BP1, were observed as early as 30 min post-IR; their number progressively reduced at 3 h, 1 day, and 3 days post-IR. The formation of IRIF was associated with a decrease in Pol II levels by both immunofluorescence and Western blotting. Treatment with the proteasome inhibitor bortezomib strongly increased Pol II levels in both control and irradiated neurons, suggesting that proteasome plays a proteolytic role by clearing stalled Pol II complexes at DNA damage sites, as a prelude to DNA repair. Neuronal IRIF recruited ubiquitylated proteins, including ubiquitylated histone H2A (Ub-H2A), and the catalytic proteasome 20S. Ub-H2A has been associated with transcriptional silencing at DNA damage sites. On the other hand, the participation of UPS in neuronal DDR may be essential for the ubiquitylation of Pol II and other proteasome substrates of the DNA repair machinery and their subsequent proteasome-mediated degradation.

The SMN Tudor SIM-like domain is key to SmD1 and coilin interactions and to Cajal body biogenesis.

Cajal bodies (CBs) are nuclear organelles involved in the maturation of spliceosomal small nuclear ribonucleoproteins (snRNPs). They concentrate coilin, snRNPs and the survival motor neuron protein (SMN). Dysfunction of CB assembly occurs in spinal muscular atrophy (SMA). Here, we demonstrate that SMN is a SUMO1 target that has a small ubiquitin-related modifier (SUMO)-interacting motif (SIM)-like motif in the Tudor domain. The expression of SIM-like mutant constructs abolishes the interaction of SMN with the spliceosomal SmD1 (also known as SNRPD1), severely decreases SMN-coilin interaction and prevents CB assembly. Accordingly, the SMN SIM-like-mediated interactions are important for CB biogenesis and their dysfunction can be involved in SMA pathophysiology.