A cytosolic surveillance mechanism activates the mitochondrial UPR.

The mitochondrial unfolded protein response (UPRmt) is essential to safeguard mitochondria from proteotoxic damage by activating a dedicated transcriptional response in the nucleus to restore proteostasis1,2. Yet, it remains unclear how the information on mitochondria misfolding stress (MMS) is signalled to the nucleus as part of the human UPRmt (refs. 3,4). Here, we show that UPRmt signalling is driven by the release of two individual signals in the cytosol-mitochondrial reactive oxygen species (mtROS) and accumulation of mitochondrial protein precursors in the cytosol (c-mtProt). Combining proteomics and genetic approaches, we identified that MMS causes the release of mtROS into the cytosol. In parallel, MMS leads to mitochondrial protein import defects causing c-mtProt accumulation. Both signals integrate to activate the UPRmt; released mtROS oxidize the cytosolic HSP40 protein DNAJA1, which leads to enhanced recruitment of cytosolic HSP70 to c-mtProt. Consequently, HSP70 releases HSF1, which translocates to the nucleus and activates transcription of UPRmt genes. Together, we identify a highly controlled cytosolic surveillance mechanism that integrates independent mitochondrial stress signals to initiate the UPRmt. These observations reveal a link between mitochondrial and cytosolic proteostasis and provide molecular insight into UPRmt signalling in human cells.

Functional Translatome Proteomics Reveal Converging and Dose-Dependent Regulation by mTORC1 and eIF2α.

Regulation of translation is essential during stress. However, the precise sets of proteins regulated by the key translational stress responses-the integrated stress response (ISR) and mTORC1-remain elusive. We developed multiplexed enhanced protein dynamics (mePROD) proteomics, adding signal amplification to dynamic-SILAC and multiplexing, to enable measuring acute changes in protein synthesis. Treating cells with ISR/mTORC1-modulating stressors, we showed extensive translatome modulation with ∼20% of proteins synthesized at highly reduced rates. Comparing translation-deficient sub-proteomes revealed an extensive overlap demonstrating that target specificity is achieved on protein level and not by pathway activation. Titrating cap-dependent translation inhibition confirmed that synthesis of individual proteins is controlled by intrinsic properties responding to global translation attenuation. This study reports a highly sensitive method to measure relative translation at the nascent chain level and provides insight into how the ISR and mTORC1, two key cellular pathways, regulate the translatome to guide cellular survival upon stress.

Growth Factor Receptor Signaling Inhibition Prevents SARS-CoV-2 Replication.

SARS-CoV-2 infections are rapidly spreading around the globe. The rapid development of therapies is of major importance. However, our lack of understanding of the molecular processes and host cell signaling events underlying SARS-CoV-2 infection hinders therapy development. We use a SARS-CoV-2 infection system in permissible human cells to study signaling changes by phosphoproteomics. We identify viral protein phosphorylation and define phosphorylation-driven host cell signaling changes upon infection. Growth factor receptor (GFR) signaling and downstream pathways are activated. Drug-protein network analyses revealed GFR signaling as key pathways targetable by approved drugs. The inhibition of GFR downstream signaling by five compounds prevents SARS-CoV-2 replication in cells, assessed by cytopathic effect, viral dsRNA production, and viral RNA release into the supernatant. This study describes host cell signaling events upon SARS-CoV-2 infection and reveals GFR signaling as a central pathway essential for SARS-CoV-2 replication. It provides novel strategies for COVID-19 treatment.

Papain-like protease regulates SARS-CoV-2 viral spread and innate immunity.

The papain-like protease PLpro is an essential coronavirus enzyme that is required for processing viral polyproteins to generate a functional replicase complex and enable viral spread1,2. PLpro is also implicated in cleaving proteinaceous post-translational modifications on host proteins as an evasion mechanism against host antiviral immune responses3-5. Here we perform biochemical, structural and functional characterization of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) PLpro (SCoV2-PLpro) and outline differences with SARS-CoV PLpro (SCoV-PLpro) in regulation of host interferon and NF-κB pathways. SCoV2-PLpro and SCoV-PLpro share 83% sequence identity but exhibit different host substrate preferences; SCoV2-PLpro preferentially cleaves the ubiquitin-like interferon-stimulated gene 15 protein (ISG15), whereas SCoV-PLpro predominantly targets ubiquitin chains. The crystal structure of SCoV2-PLpro in complex with ISG15 reveals distinctive interactions with the amino-terminal ubiquitin-like domain of ISG15, highlighting the high affinity and specificity of these interactions. Furthermore, upon infection, SCoV2-PLpro contributes to the cleavage of ISG15 from interferon responsive factor 3 (IRF3) and attenuates type I interferon responses. Notably, inhibition of SCoV2-PLpro with GRL-0617 impairs the virus-induced cytopathogenic effect, maintains the antiviral interferon pathway and reduces viral replication in infected cells. These results highlight a potential dual therapeutic strategy in which targeting of SCoV2-PLpro can suppress SARS-CoV-2 infection and promote antiviral immunity.

ULK1-mediated phosphorylation regulates the conserved role of YKT6 in autophagy.

Autophagy is a catabolic process during which cytosolic material is enwrapped in a newly formed double-membrane structure called the autophagosome, and subsequently targeted for degradation in the lytic compartment of the cell. The fusion of autophagosomes with the lytic compartment is a tightly regulated step and involves membrane-bound SNARE proteins. These play a crucial role as they promote lipid mixing and fusion of the opposing membranes. Among the SNARE proteins implicated in autophagy, the essential SNARE protein YKT6 is the only SNARE protein that is evolutionarily conserved from yeast to humans. Here, we show that alterations in YKT6 function, in both mammalian cells and nematodes, produce early and late autophagy defects that result in reduced survival. Moreover, mammalian autophagosomal YKT6 is phospho-regulated by the ULK1 kinase, preventing premature bundling with the lysosomal SNARE proteins and thereby inhibiting autophagosome-lysosome fusion. Together, our findings reveal that timely regulation of the YKT6 phosphorylation status is crucial throughout autophagy progression and cell survival.

SARS-CoV-2 Variants Show Different Host Cell Proteome Profiles With Delayed Immune Response Activation in Omicron-Infected Cells.

The ancestral SARS-CoV-2 strain that initiated the Covid-19 pandemic at the end of 2019 has rapidly mutated into multiple variants of concern with variable pathogenicity and increasing immune escape strategies. However, differences in host cellular antiviral responses upon infection with SARS-CoV-2 variants remain elusive. Leveraging whole-cell proteomics, we determined host signaling pathways that are differentially modulated upon infection with the clinical isolates of the ancestral SARS-CoV-2 B.1 and the variants of concern Delta and Omicron BA.1. Our findings illustrate alterations in the global host proteome landscape upon infection with SARS-CoV-2 variants and the resulting host immune responses. Additionally, viral proteome kinetics reveal declining levels of viral protein expression during Omicron BA.1 infection when compared to ancestral B.1 and Delta variants, consistent with its reduced replication rates. Moreover, molecular assays reveal deferral activation of specific host antiviral signaling upon Omicron BA.1 and BA.2 infections. Our study provides an overview of host proteome profile of multiple SARS-CoV-2 variants and brings forth a better understanding of the instigation of key immune signaling pathways causative for the differential pathogenicity of SARS-CoV-2 variants.

Lipid and protein content profiling of isolated native autophagic vesicles.

Autophagy is responsible for clearance of an extensive portfolio of cargoes, which are sequestered into vesicles, called autophagosomes, and are delivered to lysosomes for degradation. The pathway is highly dynamic and responsive to several stress conditions. However, the phospholipid composition and protein contents of human autophagosomes under changing autophagy rates are elusive so far. Here, we introduce an antibody-based FACS-mediated approach for the isolation of native autophagic vesicles and ensured the quality of the preparations. Employing quantitative lipidomics, we analyze phospholipids present within human autophagic vesicles purified upon basal autophagy, starvation, and proteasome inhibition. Importantly, besides phosphoglycerides, we identify sphingomyelin within autophagic vesicles and show that the phospholipid composition is unaffected by the different conditions. Employing quantitative proteomics, we obtain cargo profiles of autophagic vesicles isolated upon the different treatment paradigms. Interestingly, starvation shows only subtle effects, while proteasome inhibition results in the enhanced presence of ubiquitin-proteasome pathway factors within autophagic vesicles. Thus, here we present a powerful method for the isolation of native autophagic vesicles, which enabled profound phospholipid and cargo analyses.

A20 binding and inhibitor of nuclear factor kappa B (NF-κB)-1 (ABIN-1): a novel modulator of mitochondrial autophagy.

A20 binding inhibitor of nuclear factor kappa B (NF-κB)-1 (ABIN-1), a polyubiquitin-binding protein, is a signal-induced autophagy receptor that attenuates NF-κB-mediated inflammation and cell death. The present study aimed to elucidate the potential role of ABIN-1 in mitophagy, a biological process whose outcome is decisive in diverse physiological and pathological settings. Microtubule-associated proteins 1A/1B light chain 3B-II (LC3B-II) was found to be in complex with ectopically expressed hemagglutinin (HA)-tagged-full length (FL)-ABIN-1. Bacterial expression of ABIN-1 and LC3A and LC3B showed direct binding of ABIN-1 to LC3 proteins, whereas mutations in the LC3-interacting region (LIR) 1 and 2 motifs of ABIN-1 abrogated ABIN-1/LC3B-II complex formation. Importantly, induction of autophagy in HeLa cells resulted in colocalization of ABIN-1 with LC3B-II in autophagosomes and with lysosomal-associated membrane protein 1 (LAMP-1) in autophagolysosomes, leading to degradation of ABIN-1 with p62. Interestingly, ABIN-1 was found to translocate to damaged mitochondria in HeLa-mCherry-Parkin transfected cells. In line with this observation, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated deletion of ABIN-1 significantly inhibited the degradation of the mitochondrial outer membrane proteins voltage-dependent anion-selective channel 1 (VDAC-1), mitofusin-2 (MFN2), and translocase of outer mitochondrial membrane (TOM)20. In addition, short interfering RNA (siRNA)-mediated knockdown of ABIN-1 significantly decreased lysosomal uptake of mitochondria in HeLa cells expressing mCherry-Parkin and the fluorescence reporter mt-mKEIMA. Collectively, our results identify ABIN-1 as a novel and selective mitochondrial autophagy regulator that promotes mitophagy, thereby adding a new player to the complex cellular machinery regulating mitochondrial homeostasis.

The iron chelator and OXPHOS inhibitor VLX600 induces mitophagy and an autophagy-dependent type of cell death in glioblastoma cells.

Induction of alternative, non-apoptotic cell death programs such as cell-lethal autophagy and mitophagy represent possible strategies to combat glioblastoma (GBM). Here we report that VLX600, a novel iron chelator and oxidative phosphorylation (OXPHOS) inhibitor, induces a caspase-independent type of cell death that is partially rescued in adherent U251 ATG5/7 (autophagy related 5/7) knockout (KO) GBM cells and NCH644 ATG5/7 knockdown (KD) glioma stem-like cells (GSCs), suggesting that VLX600 induces an autophagy-dependent cell death (ADCD) in GBM. This ADCD is accompanied by decreased oxygen consumption, increased expression/mitochondrial localization of BNIP3 (BCL2 interacting protein 3) and BNIP3L (BCL2 interacting protein 3 like), the induction of mitophagy as demonstrated by diminished levels of mitochondrial marker proteins [e.g., COX4I1 (cytochrome c oxidase subunit 4I1)] and the mitoKeima assay as well as increased histone H3 and H4 lysine tri-methylation. Furthermore, the extracellular addition of iron is able to significantly rescue VLX600-induced cell death and mitophagy, pointing out an important role of iron metabolism for GBM cell homeostasis. Interestingly, VLX600 is also able to completely eliminate NCH644 GSC tumors in an organotypic brain slice transplantation model. Our data support the therapeutic concept of ADCD induction in GBM and suggest that VLX600 may be an interesting novel drug candidate for the treatment of this tumor.NEW & NOTEWORTHY Induction of cell-lethal autophagy represents a possible strategy to combat glioblastoma (GBM). Here, we demonstrate that the novel iron chelator and OXPHOS inhibitor VLX600 exerts pronounced tumor cell-killing effects in adherently cultured GBM cells and glioblastoma stem-like cell (GSC) spheroid cultures that depend on the iron-chelating function of VLX600 and on autophagy activation, underscoring the context-dependent role of autophagy in therapy responses. VLX600 represents an interesting novel drug candidate for the treatment of this tumor.

SpinTip: A Simple, Robust, and Versatile Preanalytical Method for Microscale Suspension Cell Proteomics.

Sample loss and contamination are critical preanalytical pitfalls in microscale proteomic applications of nonadhering cells. Common assays and workflows are not easily adoptable to microscale sample sizes of suspension cells due to inadvertent sample loss. This impedes preanalytical experimental manipulation of limited suspension cell samples for microscale proteomics applications, such as encountered for primary human materials. Here, we describe and test a simple manual batch technique for single-step 100-fold concentration of scarce numbers of diluted suspension cells (down to 5000 cells) by volume reduction, facilitating microscale experiments with suspension cells. Pipette tips with heat-sealed orifices (SpinTips) are manufactured within 1 min and serve as versatile microcentrifugation vessels from which supernatant can be aspirated with minimal cell loss. A residual volume of approximately 3 µL can be achieved without visualization of the cell pellet. The results show that SpinTips enable the concentration, medium exchange, washing, and culture of highly limited amounts of suspension cells for functional manipulation and microscale proteomics and are readily incorporated into standard workflows. The application is illustrated by profiling ex vivo responses of primary acute myeloid leukemia (AML) cells from one AML patient to daunorubicin (DNR) to a depth of 3462 quantified proteins with excellent repeatability.

Effectiveness of Resistive Vibration Exercise and Whey Protein Supplementation Plus Alkaline Salt on the Skeletal Muscle Proteome Following 21 Days of Bed Rest in Healthy Males.

Muscle atrophy is a deleterious consequence of physical inactivity and is associated with increased morbidity and mortality. The aim of this study was to decipher the mechanisms involved in disuse muscle atrophy in eight healthy men using a 21 day bed rest with a cross-over design (control, with resistive vibration exercise (RVE), or RVE combined with whey protein supplementation and an alkaline salt (NEX)). The main physiological findings show a significant reduction in whole-body fat-free mass (CON -4.1%, RVE -4.3%, NEX -2.7%, p < 0.05), maximal oxygen consumption (CON -20.5%, RVE -6.46%, NEX -7.9%, p < 0.05), and maximal voluntary contraction (CON -15%, RVE -12%, and NEX -9.5%, p < 0.05) and a reduction in mitochondrial enzyme activity (CON -30.7%, RVE -31.3%, NEX -17%, p < 0.05). The benefits of nutrition and exercise countermeasure were evident with an increase in leg lean mass (CON -1.7%, RVE +8.9%, NEX +15%, p < 0.05). Changes to the vastus lateralis muscle proteome were characterized using mass spectrometry-based label-free quantitative proteomics, the findings of which suggest alterations to cell metabolism, mitochondrial metabolism, protein synthesis, and degradation pathways during bed rest. The observed changes were partially mitigated during RVE, but there were no significant pathway changes during the NEX trial. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD006882. In conclusion, resistive vibration exercise, when combined with whey/alkalizing salt supplementation, could be an effective strategy to prevent skeletal muscle protein changes, muscle atrophy, and insulin sensitivity during medium duration bed rest.

Analysis of femurs from mice embarked on board BION-M1 biosatellite reveals a decrease in immune cell development, including B cells, after 1 wk of recovery on Earth.

Bone loss and immune dysregulation are among the main adverse outcomes of spaceflight challenging astronauts' health and safety. However, consequences on B-cell development and responses are still under-investigated. To fill this gap, we used advanced proteomics analysis of femur bone and marrow to compare mice flown for 1 mo on board the BION-M1 biosatellite, followed or not by 1 wk of recovery on Earth, to control mice kept on Earth. Our data revealed an adverse effect on B lymphopoiesis 1 wk after landing. This phenomenon was associated with a 41% reduction of B cells in the spleen. These reductions may contribute to explain increased susceptibility to infection even if our data suggest that flown animals can mount a humoral immune response. Future studies should investigate the quality/efficiency of produced antibodies and whether longer missions worsen these immune alterations.-Tascher, G., Gerbaix, M., Maes, P., Chazarin, B., Ghislin, S., Antropova, E., Vassilieva, G., Ouzren-Zarhloul, N., Gauquelin-Koch, G., Vico, L., Frippiat, J.-P., Bertile, F. Analysis of femurs from mice embarked on board BION-M1 biosatellite reveals a decrease in immune cell development, including B cells, after 1 wk of recovery on Earth.

Profiling the Murine SUMO Proteome in Response to Cardiac Ischemia and Reperfusion Injury.

SUMOylation is a reversible posttranslational modification pathway catalyzing the conjugation of small ubiquitin-related modifier (SUMO) proteins to lysine residues of distinct target proteins. SUMOylation modifies a wide variety of cellular regulators thereby affecting a multitude of key processes in a highly dynamic manner. The SUMOylation pathway displays a hallmark in cellular stress-adaption, such as heat or redox stress. It has been proposed that enhanced cellular SUMOylation protects the brain during ischemia, however, little is known about the specific regulation of the SUMO system and the potential target proteins during cardiac ischemia and reperfusion injury (I/R). By applying left anterior descending (LAD) coronary artery ligation and reperfusion in mice, we detect dynamic changes in the overall cellular SUMOylation pattern correlating with decreased SUMO deconjugase activity during I/R injury. Further, unbiased system-wide quantitative SUMO-proteomics identified a sub-group of SUMO targets exhibiting significant alterations in response to cardiac I/R. Notably, transcription factors that control hypoxia- and angiogenesis-related gene expression programs, exhibit altered SUMOylation during ischemic stress adaptation. Moreover, several components of the ubiquitin proteasome system undergo dynamic changes in SUMO conjugation during cardiac I/R suggesting an involvement of SUMO signaling in protein quality control and proteostasis in the ischemic heart. Altogether, our study reveals regulated candidate SUMO target proteins in the mouse heart, which might be important in coping with hypoxic/proteotoxic stress during cardiac I/R injury.

Seasonal changes in eicosanoid metabolism in the brown bear.

Polyunsaturated fatty acids (PUFAs) exert several important functions across organ systems. During winter, hibernators divert PUFAs from oxidation, retaining them in their tissues and membranes, to ensure proper body functions at low body temperature. PUFAs are also precursors of eicosanoids with pro- and anti-inflammatory properties. This study investigated seasonal changes in eicosanoid metabolism of free-ranging brown bears (Ursus arctos). By using a lipidomic approach, we assessed (1) levels of specific omega-3 and omega-6 fatty acids involved in the eicosanoid cascade and (2) concentrations of eicosanoids in skeletal muscle and blood plasma of winter hibernating and summer active bears. We observed significant seasonal changes in the specific omega-3 and omega-6 precursors. We also found significant seasonal alterations of eicosanoid levels in both tissues. Concentrations of pro-inflammatory eicosanoids, such as thromboxane B2, 5-hydroxyeicosatetraenoic acid (HETE), and 15-HETE and 18-HETE, were significantly lower in muscle and/or plasma of hibernating bears compared to summer-active animals. Further, plasma and muscle levels of 5,6-epoxyeicosatrienoic acid (EET), as well as muscle concentration of 8,9-EET, tended to be lower in bears during winter hibernation vs. summer. We also found lower plasma levels of anti-inflammatory eicosanoids, such as 15dPGJ2 and PGE3, in bears during winter hibernation. Despite of the limited changes in omega-3 and omega-6 precursors, plasma and muscle concentrations of the products of all pathways decreased significantly, or remained unchanged, independent of their pro- or anti-inflammatory properties. These findings suggest that hibernation in bears is associated with a depressed state of the eicosanoid cascade.

Proteome-wide Adaptations of Mouse Skeletal Muscles during a Full Month in Space.

The safety of space flight is challenged by a severe loss of skeletal muscle mass, strength, and endurance that may compromise the health and performance of astronauts. The molecular mechanisms underpinning muscle atrophy and decreased performance have been studied mostly after short duration flights and are still not fully elucidated. By deciphering the muscle proteome changes elicited in mice after a full month aboard the BION-M1 biosatellite, we observed that the antigravity soleus incurred the greatest changes compared with locomotor muscles. Proteomics data notably suggested mitochondrial dysfunction, metabolic and fiber type switching toward glycolytic type II fibers, structural alterations, and calcium signaling-related defects to be the main causes for decreased muscle performance in flown mice. Alterations of the protein balance, mTOR pathway, myogenesis, and apoptosis were expected to contribute to muscle atrophy. Moreover, several signs reflecting alteration of telomere maintenance, oxidative stress, and insulin resistance were found as possible additional deleterious effects. Finally, 8 days of recovery post flight were not sufficient to restore completely flight-induced changes. Thus in-depth proteomics analysis unraveled the complex and multifactorial remodeling of skeletal muscle structure and function during long-term space flight, which should help define combined sets of countermeasures before, during, and after the flight.

GPCR Function in Autophagy Control: A Systematic Approach of Chemical Intervention.

Autophagy facilitates the degradation of cellular content via the lysosome and is involved in cellular homeostasis and stress response pathways. As such, malfunction of autophagy is linked to a variety of diseases ranging from organ-specific illnesses like cardiomyopathy to systemic illnesses such as cancer or metabolic syndromes. Given the variety of autophagic functions within a cell and tissue, regulation of autophagy is complex and contains numerous positive and negative feedback loops. While our knowledge of mechanisms for cargo selectivity has significantly improved over the last decade, our understanding of signaling routes activating individual autophagy pathways remains rather sparse. In this resource study, we report on a well-characterized chemical library containing 77 GPCR-targeting ligands that was used to systematically analyze LC3B-based autophagy as well as ER-phagy flux upon compound treatment. Upon others, compounds TC-G 1004, BAY 60-6583, PSNCBAM-1, TC-G 1008, LPA2 Antagonist 1, ML-154, JTC-801 and ML-290 targeting adenosine receptor A2a (ADORA2A), adenosine receptor A2b (ADORA2B), cannabinoid receptor 1 (CNR1), G-protein coupled receptor 39 (GPR39), lysophosphatidic acid receptor 2 (LPAR2), neuropeptide S receptor 1 (NPSR1), opioid related nociceptin receptor 1 (OPRL1), and relaxin receptor 1 (RXFP1), respectively, were hit compounds for general autophagy flux. From these compounds, only JTC-801 markly increased ER-phagy flux. In addition, the global impact of these selected hit compounds were analyzed by TMT-based mass spectrometry and demonstrated the differential impact of targeting GPCRs on autophagy-associated proteins. This chemical screening exercise indicates to a significant cross-talk between GPCR signaling and regulation of autophagy pathways.

FACS-mediated isolation of native autophagic vesicles.

Autophagosome isolation enables the thorough investigation of structural components and engulfed materials. Recently, we introduced a novel antibody-based FACS-mediated method for isolation of native macroautophagic/autophagic vesicles and confirmed the quality of the preparations. We performed phospholipidomic and proteomic analyses to characterize autophagic vesicle-associated phospholipids and protein cargoes under different autophagy conditions. Lipidomic analyses identified phosphoglycerides and sphingomyelins within autophagic vesicles and revealed that the lipid composition was unaffected by different rates of autophagosome formation. Proteomic analyses identified more than 4500 potential autophagy substrates and showed that in comparison to autophagic vesicles isolated under basal autophagy conditions, starvation only marginally affected the cargo profile. Proteasome inhibition, however, resulted in the enhanced degradation of ubiquitin-proteasome system components. Taken together, the novel isolation method enriched large quantities of autophagic vesicles and enabled detailed analyses of their lipid and cargo composition.

Characterization of nucleolar SUMO isopeptidases unveils a general p53-independent checkpoint of impaired ribosome biogenesis.

Ribosome biogenesis is a multi-step process, in which a network of trans-acting factors ensures the coordinated assembly of pre-ribosomal particles in order to generate functional ribosomes. Ribosome biogenesis is tightly coordinated with cell proliferation and its perturbation activates a p53-dependent cell-cycle checkpoint. How p53-independent signalling networks connect impaired ribosome biogenesis to the cell-cycle machinery has remained largely enigmatic. We demonstrate that inactivation of the nucleolar SUMO isopeptidases SENP3 and SENP5 disturbs distinct steps of 40S and 60S ribosomal subunit assembly pathways, thereby triggering the canonical p53-dependent impaired ribosome biogenesis checkpoint. However, inactivation of SENP3 or SENP5 also induces a p53-independent checkpoint that converges on the specific downregulation of the key cell-cycle regulator CDK6. We further reveal that impaired ribosome biogenesis generally triggers the downregulation of CDK6, independent of the cellular p53 status. Altogether, these data define the role of SUMO signalling in ribosome biogenesis and unveil a p53-independent checkpoint of impaired ribosome biogenesis.

Impact of mesenchymal stromal cell-derived vesicular cargo on B-cell acute lymphoblastic leukemia progression.

Leukemia cells reciprocally interact with their surrounding bone marrow microenvironment (BMM), rendering it hospitable to leukemia cell survival, for instance through the release of small extracellular vesicles (sEVs). In contrast, we show here that BMM deficiency of pleckstrin homology domain family M member 1 (PLEKHM1), which serves as a hub between fusion and secretion of intracellular vesicles and is important for vesicular secretion in osteoclasts, accelerates murine BCR-ABL1+ B-cell acute lymphoblastic leukemia (B-ALL) via regulation of the cargo of sEVs released by BMM-derived mesenchymal stromal cells (MSCs). PLEKHM1-deficient MSCs and their sEVs carry increased amounts of syntenin and syndecan-1, resulting in a more immature B-cell phenotype and an increased number/function of leukemia-initiating cells (LICs) via focal adhesion kinase and AKT signaling in B-ALL cells. Ex vivo pretreatment of LICs with sEVs derived from PLEKHM1-deficient MSCs led to a strong trend toward acceleration of murine and human BCR-ABL1+ B-ALL. In turn, inflammatory mediators such as recombinant or B-ALL cell-derived tumor necrosis factor α or interleukin-1ß condition murine and human MSCs in vitro, decreasing PLEKHM1, while increasing syntenin and syndecan-1 in MSCs, thereby perpetuating the sEV-associated circuit. Consistently, human trephine biopsies of patients with B-ALL showed a reduced percentage of PLEKHM1+ MSCs. In summary, our data reveal an important role of BMM-derived sEVs for driving specifically BCR-ABL1+ B-ALL, possibly contributing to its worse prognosis compared with BCR-ABL1- B-ALL, and suggest that secretion of inflammatory cytokines by cancer cells in general may similarly modulate the tumor microenvironment.

An atypical GABARAP binding module drives the pro-autophagic potential of the AML-associated NPM1c variant.

The nucleolar scaffold protein NPM1 is a multifunctional regulator of cellular homeostasis, genome integrity, and stress response. NPM1 mutations, known as NPM1c variants promoting its aberrant cytoplasmic localization, are the most frequent genetic alterations in acute myeloid leukemia (AML). A hallmark of AML cells is their dependency on elevated autophagic flux. Here, we show that NPM1 and NPM1c induce the autophagy-lysosome pathway by activating the master transcription factor TFEB, thereby coordinating the expression of lysosomal proteins and autophagy regulators. Importantly, both NPM1 and NPM1c bind to autophagy modifiers of the GABARAP subfamily through an atypical binding module preserved within its N terminus. The propensity of NPM1c to induce autophagy depends on this module, likely indicating that NPM1c exerts its pro-autophagic activity by direct engagement with GABARAPL1. Our data report a non-canonical binding mode of GABARAP family members that drives the pro-autophagic potential of NPM1c, potentially enabling therapeutic options.