Sbk2, a Newly Discovered Atrium-Enriched Regulator of Sarcomere Integrity.

BACKGROUND: Heart development relies on tight spatiotemporal control of cardiac gene expression. Genes involved in this intricate process have been identified using animals and pluripotent stem cell-based models of cardio(myo)genesis. Recently, the repertoire of cardiomyocyte differentiation models has been expanded with iAM-1, a monoclonal line of conditionally immortalized neonatal rat atrial myocytes (NRAMs), which allows toggling between proliferative and differentiated (ie, excitable and contractile) phenotypes in a synchronized and homogenous manner. METHODS: In this study, the unique properties of conditionally immortalized NRAMs (iAMs) were exploited to identify and characterize (lowly expressed) genes with an as-of-yet uncharacterized role in cardiomyocyte differentiation. RESULTS: Transcriptome analysis of iAM-1 cells at different stages during one cycle of differentiation and subsequent dedifferentiation identified ≈13 000 transcripts, of which the dynamic changes in expression upon cardiomyogenic differentiation mostly opposed those during dedifferentiation. Among the genes whose expression increased during differentiation and decreased during dedifferentiation were many with known (lineage-specific) functions in cardiac muscle formation. Filtering for cardiac-enriched low-abundance transcripts, identified multiple genes with an uncharacterized role during cardio(myo)genesis including Sbk2 (SH3 domain binding kinase family member 2). Sbk2 encodes an evolutionarily conserved putative serine/threonine protein kinase, whose expression is strongly up- and downregulated during iAM-1 cell differentiation and dedifferentiation, respectively. In neonatal and adult rats, the protein is muscle-specific, highly atrium-enriched, and localized around the A-band of cardiac sarcomeres. Knockdown of Sbk2 expression caused loss of sarcomeric organization in NRAMs, iAMs and their human counterparts, consistent with a decrease in sarcomeric gene expression as evinced by transcriptome and proteome analyses. Interestingly, co-immunoprecipitation using Sbk2 as bait identified possible interaction partners with diverse cellular functions (translation, intracellular trafficking, cytoskeletal organization, chromatin modification, sarcomere formation). CONCLUSIONS: iAM-1 cells are a relevant and suitable model to identify (lowly expressed) genes with a hitherto unidentified role in cardiomyocyte differentiation as exemplified by Sbk2: a regulator of atrial sarcomerogenesis.

The α-(1,3)-glucan synthase gene agsE impacts the secretome of Aspergillus niger.

Aspergillus niger is widely used as a cell factory for the industrial production of enzymes. Previously, it was shown that deletion of α-1-3 glucan synthase genes results in smaller micro-colonies in liquid cultures of Aspergillus nidulans. Also, it has been shown that small wild-type Aspergillus niger micro-colonies secrete more protein than large mirco-colonies. We here assessed whether deletion of the agsC or agsE α-1-3 glucan synthase genes results in smaller A. niger micro-colonies and whether this is accompanied by a change in protein secretion. Biomass formation was not affected in the deletion strains but pH of the culture medium had changed from 5.2 in the case of the wild-type to 4.6 and 6.4 for ΔagsC and ΔagsE, respectively. The diameter of the ΔagsC micro-colonies was not affected in liquid cultures. In contrast, diameter of the ΔagsE micro-colonies was reduced from 3304 ± 338 µm to 1229 ± 113 µm. Moreover, the ΔagsE secretome was affected with 54 and 36 unique proteins with a predicted signal peptide in the culture medium of MA234.1 and the ΔagsE, respectively. Results show that these strains have complementary cellulase activity and thus may have complementary activity on plant biomass degradation. Together, α-1-3 glucan synthesis (in)directly impacts protein secretion in A. niger.

Proteomics and Phosphoproteomics Profiling of Drug-Addicted BRAFi-Resistant Melanoma Cells.

Acquired resistance to MAPK inhibitors limits the clinical efficacy in melanoma treatment. We and others have recently shown that BRAF inhibitor (BRAFi)-resistant melanoma cells can develop a dependency on the therapeutic drugs to which they have acquired resistance, creating a vulnerability for these cells that can potentially be exploited in cancer treatment. In drug-addicted melanoma cells, it was shown that this induction of cell death was preceded by a specific ERK2-dependent phenotype switch; however, the underlying molecular mechanisms are largely lacking. To increase the molecular understanding of this drug dependency, we applied a mass spectrometry-based proteomic approach on BRAFi-resistant BRAFMUT 451Lu cells, in which ERK1, ERK2, and JUNB were silenced separately using CRISPR-Cas9. Inactivation of ERK2 and, to a lesser extent, JUNB prevents drug addiction in these melanoma cells, while, conversely, knockout of ERK1 fails to reverse this phenotype, showing a response similar to that of control cells. Our analysis reveals that ERK2 and JUNB share comparable proteome responses dominated by reactivation of cell division. Importantly, we find that EMT activation in drug-addicted melanoma cells upon drug withdrawal is affected by silencing ERK2 but not ERK1. Moreover, transcription factor (regulator) enrichment shows that PIR acts as an effector of ERK2 and phosphoproteome analysis reveals that silencing of ERK2 but not ERK1 leads to amplification of GSK3 kinase activity. Our results depict possible mechanisms of drug addiction in melanoma, which may provide a guide for therapeutic strategies in drug-resistant melanoma.

FlbA-Regulated Gene rpnR Is Involved in Stress Resistance and Impacts Protein Secretion when Aspergillus niger Is Grown on Xylose.

Proteins are secreted throughout the mycelium of Aspergillus niger except for the sporulating zone. A link between sporulation and repression of protein secretion was underlined by the finding that inactivation of the sporulation gene flbA results in mycelial colonies that secrete proteins throughout the colony. However, ΔflbA strain hyphae also lyse and have thinner cell walls. This pleiotropic phenotype is associated with differential expression of 36 predicted transcription factor genes, one of which, rpnR, was inactivated in this study. Sporulation, biomass, and secretome complexity were not affected in the ΔrpnR deletion strain of the fungus. In contrast, ribosomal subunit expression and protein secretion into the medium were reduced when A. niger was grown on xylose. Moreover, the ΔrpnR strain showed decreased resistance to H2O2 and the proteotoxic stress-inducing agent dithiothreitol. Taking the data together, RpnR is involved in proteotoxic stress resistance and impacts protein secretion when A. niger is grown on xylose.IMPORTANCEAspergillus niger secretes a large amount and diversity of industrially relevant enzymes into the culture medium. This makes the fungus a widely used industrial cell factory. For instance, carbohydrate-active enzymes of A. niger are used in biofuel production from lignocellulosic feedstock. These enzymes represent a major cost factor in this process. Higher production yields could substantially reduce these costs and therefore contribute to a more sustainable economy and less dependence on fossil fuels. Enzyme secretion is inhibited in A. niger by asexual reproduction. The sporulation protein FlbA is involved in this process by impacting the expression of 36 predicted transcription factor genes. Here, we show that one of these predicted transcriptional regulators, RpnR, regulates protein secretion and proteotoxic stress resistance. The gene is thus an interesting target to improve enzyme production in A. niger.

The physiology of Agaricus bisporus in semi-commercial compost cultivation appears to be highly conserved among unrelated isolates.

The white button mushroom Agaricus bisporus is one of the most widely produced edible fungus with a great economical value. Its commercial cultivation process is often performed on wheat straw and animal manure based compost that mainly contains lignocellulosic material as a source of carbon and nutrients for the mushroom production. As a large portion of compost carbohydrates are left unused in the current mushroom cultivation process, the aim of this work was to study wild-type A. bisporus strains for their potential to convert the components that are poorly utilized by the commercial strain A15. We therefore focused our analysis on the stages where the fungus is producing fruiting bodies. Growth profiling was used to identify A. bisporus strains with different abilities to use plant biomass derived polysaccharides, as well as to transport and metabolize the corresponding monomeric sugars. Six wild-type isolates with diverse growth profiles were compared for mushroom production to A15 strain in semi-commercial cultivation conditions. Transcriptome and proteome analyses of the three most interesting wild-type strains and A15 indicated that the unrelated A. bisporus strains degrade and convert plant biomass polymers in a highly similar manner. This was also supported by the chemical content of the compost during the mushroom production process. Our study therefore reveals a highly conserved physiology for unrelated strains of this species during growth in compost.

Proteome-wide profiling of protein assemblies by cross-linking mass spectrometry.

We describe an integrated workflow that robustly identifies cross-links from endogenous protein complexes in human cellular lysates. Our approach is based on the application of mass spectrometry (MS)-cleavable cross-linkers, sequential collision-induced dissociation (CID)-tandem MS (MS/MS) and electron-transfer dissociation (ETD)-MS/MS acquisitions, and a dedicated search engine, XlinkX, which allows rapid cross-link identification against a complete human proteome database. This approach allowed us to detect 2,179 unique cross-links (1,665 intraprotein cross-links at a 5% false discovery rate (FDR) and 514 interprotein cross-links at 1% FDR) in HeLa cell lysates. We validated the confidence of our cross-linking results by using a target-decoy strategy and mapping the observed cross-link distances onto existing high-resolution structures. Our data provided new structural information about many protein assemblies and captured dynamic interactions of the ribosome in contact with different elongation factors.

Robust, Sensitive, and Automated Phosphopeptide Enrichment Optimized for Low Sample Amounts Applied to Primary Hippocampal Neurons.

Because of the low stoichiometry of protein phosphorylation, targeted enrichment prior to LC-MS/MS analysis is still essential. The trend in phosphoproteome analysis is shifting toward an increasing number of biological replicates per experiment, ideally starting from very low sample amounts, placing new demands on enrichment protocols to make them less labor-intensive, more sensitive, and less prone to variability. Here we assessed an automated enrichment protocol using Fe(III)-IMAC cartridges on an AssayMAP Bravo platform to meet these demands. The automated Fe(III)-IMAC-based enrichment workflow proved to be more effective when compared to a TiO2-based enrichment using the same platform and a manual Ti(IV)-IMAC-based enrichment workflow. As initial samples, a dilution series of both human HeLa cell and primary rat hippocampal neuron lysates was used, going down to 0.1 µg of peptide starting material. The optimized workflow proved to be efficient, sensitive, and reproducible, identifying, localizing, and quantifying thousands of phosphosites from just micrograms of starting material. To further test the automated workflow in genuine biological applications, we monitored EGF-induced signaling in hippocampal neurons, starting with only 200 000 primary cells, resulting in ∼50 µg of protein material. This revealed a comprehensive phosphoproteome, showing regulation of multiple members of the MAPK pathway and reduced phosphorylation status of two glutamate receptors involved in synaptic plasticity.

Toward an Optimized Workflow for Middle-Down Proteomics.

Mass spectrometry (MS)-based proteomics workflows can crudely be classified into two distinct regimes, targeting either relatively small peptides (i.e., 0.7 kDa < Mw < 3.0 kDa) or small to medium sized intact proteins (i.e., 10 kDa < Mw < 30 kDa), respectively, termed bottom-up and top-down proteomics. Recently, a niche has started to be explored covering the analysis of middle-range peptides (i.e., 3.0 kDa < Mw < 10 kDa), aptly termed middle-down proteomics. Although middle-down proteomics can follow, in principle, a modular workflow similar to that of bottom-up proteomics, we hypothesized that each of these modules would benefit from targeted optimization to improve its overall performance in the analysis of middle-range sized peptides. Hence, to generate middle-range sized peptides from cellular lysates, we explored the use of the proteases Asp-N and Glu-C and a nonenzymatic acid induced cleavage. To increase the depth of the proteome, a strong cation exchange (SCX) separation, carefully tuned to improve the separation of longer peptides, combined with reversed phase-liquid chromatography (RP-LC) using columns packed with material possessing a larger pore size, was used. Finally, after evaluating the combination of potentially beneficial MS settings, we also assessed the peptide fragmentation techniques, including higher-energy collision dissociation (HCD), electron-transfer dissociation (ETD), and electron-transfer combined with higher-energy collision dissociation (EThcD), for characterization of middle-range sized peptides. These combined improvements clearly improve the detection and sequence coverage of middle-range peptides and should guide researchers to explore further how middle-down proteomics may lead to an improved proteome coverage, beneficial for, among other things, the enhanced analysis of (co-occurring) post-translational modifications.

Assessment of SRM, MRM(3) , and DIA for the targeted analysis of phosphorylation dynamics in non-small cell lung cancer.

Hypothesis-driven MS-based targeted proteomics has gained great popularity in a relatively short timespan. Next to the widely established selected reaction monitoring (SRM) workflow, data-independent acquisition (DIA), also referred to as sequential window acquisition of all theoretical spectra (SWATH) was introduced as a high-throughput targeted proteomics method. DIA facilitates increased proteome coverage, however, does not yet reach the sensitivity obtained with SRM. Therefore, a well-informed method selection is crucial for designing a successful targeted proteomics experiment. This is especially the case when targeting less conventional peptides such as those that contain PTMs, as these peptides do not always adhere to the optimal fragmentation considerations for targeted assays. Here, we provide insight into the performance of DIA, SRM, and MRM cubed (MRM(3) ) in the analysis of phosphorylation dynamics throughout the phosphoinositide 3-kinase mechanistic target of rapamycin (PI3K-mTOR) and mitogen-activated protein kinase (MAPK) signaling network. We observe indeed that DIA is less sensitive when compared to SRM, however demonstrates increased flexibility, by postanalysis selection of alternative phosphopeptide precursors. Additionally, we demonstrate the added benefit of MRM(3) , allowing the quantification of two poorly accessible phosphosites. In total, targeted proteomics enabled the quantification of 42 PI3K-mTOR and MAPK phosphosites, gaining a so far unachieved in-depth view mTOR signaling events linked to tyrosine kinase inhibitor resistance in non-small cell lung cancer.

Daily rhythms in the cyanobacterium synechococcus elongatus probed by high-resolution mass spectrometry-based proteomics reveals a small defined set of cyclic proteins.

Circadian rhythms are self-sustained and adjustable cycles, typically entrained with light/dark and/or temperature cycles. These rhythms are present in animals, plants, fungi, and several bacteria. The central mechanism behind these "pacemakers" and the connection to the circadian regulated pathways are still poorly understood. The circadian rhythm of the cyanobacterium Synechococcus elongatus PCC 7942 (S. elongatus) is highly robust and controlled by only three proteins, KaiA, KaiB, and KaiC. This central clock system has been extensively studied functionally and structurally and can be reconstituted in vitro. These characteristics, together with a relatively small genome (2.7 Mbp), make S. elongatus an ideal model system for the study of circadian rhythms. Different approaches have been used to reveal the influence of the central S. elongatus clock on rhythmic gene expression, rhythmic mRNA abundance, rhythmic DNA topology changes, and cell division. However, a global analysis of its proteome dynamics has not been reported yet. To uncover the variation in protein abundances during 48 h under light and dark cycles (12:12 h), we used quantitative proteomics, with TMT 6-plex isobaric labeling. We queried the S. elongatus proteome at 10 different time points spanning a single 24-h period, leading to 20 time points over the full 48-h period. Employing multidimensional separation and high-resolution mass spectrometry, we were able to find evidence for a total of 82% of the S. elongatus proteome. Of the 1537 proteins quantified over the time course of the experiment, only 77 underwent significant cyclic variations. Interestingly, our data provide evidence for in- and out-of-phase correlation between mRNA and protein levels for a set of specific genes and proteins. As a range of cyclic proteins are functionally not well annotated, this work provides a resource for further studies to explore the role of these proteins in the cyanobacterial circadian rhythm.

Uncovering the abilities of Agaricus bisporus to degrade plant biomass throughout its life cycle.

The economically important edible basidiomycete mushroom Agaricus bisporus thrives on decaying plant material in forests and grasslands of North America and Europe. It degrades forest litter and contributes to global carbon recycling, depolymerizing (hemi-)cellulose and lignin in plant biomass. Relatively little is known about how A. bisporus grows in the controlled environment in commercial production facilities and utilizes its substrate. Using transcriptomics and proteomics, we showed that changes in plant biomass degradation by A. bisporus occur throughout its life cycle. Ligninolytic genes were only highly expressed during the spawning stage day 16. In contrast, (hemi-)cellulolytic genes were highly expressed at the first flush, whereas low expression was observed at the second flush. The essential role for many highly expressed plant biomass degrading genes was supported by exo-proteome analysis. Our data also support a model of sequential lignocellulose degradation by wood-decaying fungi proposed in previous studies, concluding that lignin is degraded at the initial stage of growth in compost and is not modified after the spawning stage. The observed differences in gene expression involved in (hemi-)cellulose degradation between the first and second flushes could partially explain the reduction in the number of mushrooms during the second flush.

Protein acetylation affects acetate metabolism, motility and acid stress response in Escherichia coli.

Although protein acetylation is widely observed, it has been associated with few specific regulatory functions making it poorly understood. To interrogate its functionality, we analyzed the acetylome in Escherichia coli knockout mutants of cobB, the only known sirtuin-like deacetylase, and patZ, the best-known protein acetyltransferase. For four growth conditions, more than 2,000 unique acetylated peptides, belonging to 809 proteins, were identified and differentially quantified. Nearly 65% of these proteins are related to metabolism. The global activity of CobB contributes to the deacetylation of a large number of substrates and has a major impact on physiology. Apart from the regulation of acetyl-CoA synthetase, we found that CobB-controlled acetylation of isocitrate lyase contributes to the fine-tuning of the glyoxylate shunt. Acetylation of the transcription factor RcsB prevents DNA binding, activating flagella biosynthesis and motility, and increases acid stress susceptibility. Surprisingly, deletion of patZ increased acetylation in acetate cultures, which suggests that it regulates the levels of acetylating agents. The results presented offer new insights into functional roles of protein acetylation in metabolic fitness and global cell regulation.

Similar is not the same: differences in the function of the (hemi-)cellulolytic regulator XlnR (Xlr1/Xyr1) in filamentous fungi.

The transcriptional activator XlnR (Xlr1/Xyr1) is a major regulator in fungal xylan and cellulose degradation as well as in the utilization of d-xylose via the pentose catabolic pathway. XlnR homologs are commonly found in filamentous ascomycetes and often assumed to have the same function in different fungi. However, a comparison of the saprobe Aspergillus niger and the plant pathogen Magnaporthe oryzae showed different phenotypes for deletion strains of XlnR. In this study wild type and xlnR/xlr1/xyr1 mutants of five fungi were compared: Fusarium graminearum, M. oryzae, Trichoderma reesei, A. niger and Aspergillus nidulans. Growth profiling on relevant substrates and a detailed analysis of the secretome as well as extracellular enzyme activities demonstrated a common role of this regulator in activating genes encoding the main xylanolytic enzymes. However, large differences were found in the set of genes that is controlled by XlnR in the different species, resulting in the production of different extracellular enzyme spectra by these fungi. This comparison emphasizes the functional diversity of a fine-tuned (hemi-)cellulolytic regulatory system in filamentous fungi, which might be related to the adaptation of fungi to their specific biotopes. Data are available via ProteomeXchange with identifier PXD001190.

CNTN4 modulates neural elongation through interplay with APP.

The neuronal cell adhesion molecule contactin-4 (CNTN4) is genetically associated with autism spectrum disorder (ASD) and other psychiatric disorders. Cntn4-deficient mouse models have previously shown that CNTN4 plays important roles in axon guidance and synaptic plasticity in the hippocampus. However, the pathogenesis and functional role of CNTN4 in the cortex has not yet been investigated. Our study found a reduction in cortical thickness in the motor cortex of Cntn4 -/- mice, but cortical cell migration and differentiation were unaffected. Significant morphological changes were observed in neurons in the M1 region of the motor cortex, indicating that CNTN4 is also involved in the morphology and spine density of neurons in the motor cortex. Furthermore, mass spectrometry analysis identified an interaction partner for CNTN4, confirming an interaction between CNTN4 and amyloid-precursor protein (APP). Knockout human cells for CNTN4 and/or APP revealed a relationship between CNTN4 and APP. This study demonstrates that CNTN4 contributes to cortical development and that binding and interplay with APP controls neural elongation. This is an important finding for understanding the physiological function of APP, a key protein for Alzheimer's disease. The binding between CNTN4 and APP, which is involved in neurodevelopment, is essential for healthy nerve outgrowth.

Deletion of flbA results in increased secretome complexity and reduced secretion heterogeneity in colonies of Aspergillus niger.

Aspergillus niger is a cell factory for the production of enzymes. This fungus secretes proteins in the central part and at the periphery of the colony. The sporulating zone of the colony overlapped with the nonsecreting subperipheral zone, indicating that sporulation inhibits protein secretion. Indeed, strain ΔflbA that is affected early in the sporulation program secreted proteins throughout the colony. In contrast, the ΔbrlA strain that initiates but not completes sporulation did not show altered spatial secretion. The secretome of 5 concentric zones of xylose-grown ΔflbA colonies was assessed by quantitative proteomics. In total 138 proteins with a signal sequence for secretion were identified in the medium of ΔflbA colonies. Of these, 18 proteins had never been reported to be part of the secretome of A. niger, while 101 proteins had previously not been identified in the culture medium of xylose-grown wild type colonies. Taken together, inactivation of flbA results in spatial changes in secretion and in a more complex secretome. The latter may be explained by the fact that strain ΔflbA has a thinner cell wall compared to the wild type, enabling efficient release of proteins. These results are of interest to improve A. niger as a cell factory.

Heterogeneity in Spore Aggregation and Germination Results in Different Sized, Cooperative Microcolonies in an Aspergillus niger Culture.

The fungus Aspergillus niger is among the most abundant fungi in the world and is widely used as a cell factory for protein and metabolite production. This fungus forms asexual spores called conidia that are used for dispersal. Notably, part of the spores and germlings aggregate in an aqueous environment. The aggregated conidia/germlings give rise to large microcolonies, while the nonaggregated spores/germlings result in small microcolonies. Here, it is shown that small microcolonies release a larger variety and quantity of secreted proteins compared to large microcolonies. Yet, the secretome of large microcolonies has complementary cellulase activity with that of the small microcolonies. Also, large microcolonies are more resistant to heat and oxidative stress compared to small microcolonies, which is partly explained by the presence of nongerminated spores in the core of the large microcolonies. Together, it is proposed that heterogeneity in germination and aggregation has evolved to form a population of different sized A. niger microcolonies, thereby increasing stress survival and producing a meta-secretome more optimally suited to degrade complex substrates. IMPORTANCE Aspergillus niger can form microcolonies of different size due to partial aggregation of spores and germlings. So far, this heterogeneity was considered a negative trait by the industry. We here, however, show that heterogeneity in size within a population of microcolonies is beneficial for food degradation and stress survival. This functional heterogeneity is not only of interest for the industry to make blends of enzymes (e.g., for biofuel or bioplastic production) but could also play a role in nature for effective nutrient cycling and survival of the fungus.

Spatially resolving the secretome within the mycelium of the cell factory Aspergillus niger.

Aspergillus niger is an important cell factory for the industrial production of enzymes. These enzymes are released into the culture medium, from which they can be easily isolated. Here, we determined with stable isotope dimethyl labeling the secretome of five concentric zones of 7-day-old xylose-grown colonies of A. niger that had either or not been treated with cycloheximide. As expected, cycloheximide blocked secretion of proteins at the periphery of the colony. Unexpectedly, protein release was increased by cycloheximide in the intermediate and central zones of the mycelium when compared to nontreated colonies. Electron microscopy indicated that this is due to partial degradation of the cell wall. In total, 124 proteins were identified in cycloheximide-treated colonies, of which 19 secreted proteins had not been identified before. Within the pool of 124 proteins, 53 secreted proteins were absent in nontreated colonies, and additionally, 35 proteins were released ≥4-fold in the central and subperipheral zones of cycloheximide-treated colonies when compared to nontreated colonies. The composition of the secretome in each of the five concentric zones differed. This study thus describes spatial release of proteins in A. niger, which is instrumental in understanding how fungi degrade complex substrates in nature.

Multistep mechanism of G-quadruplex resolution during DNA replication.

G-quadruplex (or G4) structures form in guanine-rich DNA sequences and threaten genome stability when not properly resolved. G4 unwinding occurs during S phase via an unknown mechanism. Using Xenopus egg extracts, we define a three-step G4 unwinding mechanism that acts during DNA replication. First, the replicative helicase composed of Cdc45, MCM2-7 and GINS (CMG) stalls at a leading strand G4 structure. Second, the DEAH-box helicase 36 (DHX36) mediates bypass of the CMG past the intact G4 structure, allowing approach of the leading strand to the G4. Third, G4 structure unwinding by the Fanconi anemia complementation group J helicase (FANCJ) enables DNA polymerase to synthesize past the G4 motif. A G4 on the lagging strand template does not stall CMG but still requires DNA replication for unwinding. DHX36 and FANCJ have partially redundant roles, conferring pathway robustness. This previously unknown genome maintenance pathway promotes faithful G4 replication, thereby avoiding genome instability.

Ribociclib Induces Broad Chemotherapy Resistance and EGFR Dependency in ESR1 Wildtype and Mutant Breast Cancer.

While endocrine therapy is highly effective for the treatment of oestrogen receptor-α (ERα)-positive breast cancer, a significant number of patients will eventually experience disease progression and develop treatment-resistant, metastatic cancer. The majority of resistant tumours remain dependent on ERα-action, with activating ESR1 gene mutations occurring in 15-40% of advanced cancers. Therefore, there is an urgent need to discover novel effective therapies that can eradicate cancer cells with aberrant ERα and to understand the cellular response underlying their action. Here, we evaluate the response of MCF7-derived, CRISPR-Cas9-generated cell lines expressing mutant ERα (Y537S) to a large number of drugs. We report sensitivity to numerous clinically approved inhibitors, including CDK4/6 inhibitor ribociclib, which is a standard-of-care therapy in the treatment of metastatic ERα-positive breast cancer and currently under evaluation in the neoadjuvant setting. Ribociclib treatment induces senescence in both wildtype and mutant ERα breast cancer models and leads to a broad-range drug tolerance. Strikingly, viability of cells undergoing ribociclib-induced cellular senescence is maintained via engagement of EGFR signalling, which may be therapeutically exploited in both wildtype and mutant ERα-positive breast cancer. Our study highlights a wide-spread reduction in sensitivity to anti-cancer drugs accompanied with an acquired vulnerability to EGFR inhibitors following CDK4/6 inhibitor treatment.

Dynamic remodelling of the human host cell proteome and phosphoproteome upon enterovirus infection.

The group of enteroviruses contains many important pathogens for humans, including poliovirus, coxsackievirus, rhinovirus, as well as newly emerging global health threats such as EV-A71 and EV-D68. Here, we describe an unbiased, system-wide and time-resolved analysis of the proteome and phosphoproteome of human cells infected with coxsackievirus B3. Of the ~3,200 proteins quantified throughout the time course, a large amount (~25%) shows a significant change, with the majority being downregulated. We find ~85% of the detected phosphosites to be significantly regulated, implying that most changes occur at the post-translational level. Kinase-motif analysis reveals temporal activation patterns of certain protein kinases, with several CDKs/MAPKs immediately active upon the infection, and basophilic kinases, ATM, and ATR engaging later. Through bioinformatics analysis and dedicated experiments, we identify mTORC1 signalling as a major regulation network during enterovirus infection. We demonstrate that inhibition of mTORC1 activates TFEB, which increases expression of lysosomal and autophagosomal genes, and that TFEB activation facilitates the release of virions in extracellular vesicles via secretory autophagy. Our study provides a rich framework for a system-level understanding of enterovirus-induced perturbations at the protein and signalling pathway levels, forming a base for the development of pharmacological inhibitors to treat enterovirus infections.