Mammalian target of rapamycin inhibition protects glioma cells from temozolomide-induced cell death.

Glioblastoma is an incurable brain tumor with a median survival below two years. Trials investigating targeted therapy with inhibitors of the kinase mTOR have produced ambiguous results. Especially combination of mTOR inhibition with standard temozolomide radiochemotherapy has resulted in reduced survival in a phase II clinical trial. To date, this phenomenon is only poorly understood. To recreate the therapeutic setting in vitro, we exposed glioblastoma cell lines to co-treatment with rapamycin and temozolomide and assessed cell viability, DNA damage and reactive oxygen species. Additionally, we employed a novel translatomic based mass spectrometry approach ("mePROD") to analyze acute changes in translated proteins. mTOR inhibition with rapamycin protected glioblastoma cells from temozolomide toxicity. Following co-treatment of temozolomide with rapamycin, an increased translation of reactive oxygen species (ROS)-detoxifying proteins was detected by mass spectrometry. This was accompanied by improved ROS-homeostasis and reduced DNA damage. Additionally, rapamycin induced the expression of the DNA repair enzyme O-6-methylguanine-DNA methyltransferase (MGMT) in glioblastoma cells with an unmethylated MGMT gene promotor. Inhibition of mTOR antagonized the cytotoxic effects of temozolomide in vitro. The induction of antioxidant defences and MGMT are two underlying candidate mechanisms. Further functional experiments in vitro and in vivo are warranted to characterize this effect that appears relevant for combinatorial therapeutic strategies.

Mmp12 Is Translationally Regulated in Macrophages during the Course of Inflammation.

Despite the importance of rapid adaptive responses in the course of inflammation and the notion that post-transcriptional regulation plays an important role herein, relevant translational alterations, especially during the resolution phase, remain largely elusive. In the present study, we analyzed translational changes in inflammatory bone marrow-derived macrophages upon resolution-promoting efferocytosis. Total RNA-sequencing confirmed that apoptotic cell phagocytosis induced a pro-resolution signature in LPS/IFNγ-stimulated macrophages (Mϕ). While inflammation-dependent transcriptional changes were relatively small between efferocytic and non-efferocytic Mϕ; considerable differences were observed at the level of de novo synthesized proteins. Interestingly, translationally regulated targets in response to inflammatory stimuli were mostly downregulated, with only minimal impact of efferocytosis. Amongst these targets, pro-resolving matrix metallopeptidase 12 (Mmp12) was identified as a translationally repressed candidate during early inflammation that recovered during the resolution phase. Functionally, reduced MMP12 production enhanced matrix-dependent migration of Mϕ. Conclusively, translational control of MMP12 emerged as an efficient strategy to alter the migratory properties of Mϕ throughout the inflammatory response, enabling Mϕ migration within the early inflammatory phase while restricting migration during the resolution phase.

USP28 controls SREBP2 and the mevalonate pathway to drive tumour growth in squamous cancer.

SREBP2 is a master regulator of the mevalonate pathway (MVP), a biosynthetic process that drives the synthesis of dolichol, heme A, ubiquinone and cholesterol and also provides substrates for protein prenylation. Here, we identify SREBP2 as a novel substrate for USP28, a deubiquitinating enzyme that is frequently upregulated in squamous cancers. Our results show that silencing of USP28 reduces expression of MVP enzymes and lowers metabolic flux into this pathway. We also show that USP28 binds to mature SREBP2, leading to its deubiquitination and stabilisation. USP28 depletion rendered cancer cells highly sensitive to MVP inhibition by statins, which was rescued by the addition of geranyl-geranyl pyrophosphate. Analysis of human tissue microarrays revealed elevated expression of USP28, SREBP2 and MVP enzymes in lung squamous cell carcinoma (LSCC) compared to lung adenocarcinoma (LADC). Moreover, CRISPR/Cas-mediated deletion of SREBP2 selectively attenuated tumour growth in a KRas/p53/LKB1 mutant mouse model of lung cancer. Finally, we demonstrate that statins synergise with a dual USP28/25 inhibitor to reduce viability of SCC cells. Our findings suggest that combinatorial targeting of MVP and USP28 could be a therapeutic strategy for the treatment of squamous cell carcinomas.

Inhibition of mTOR signaling protects human glioma cells from hypoxia-induced cell death in an autophagy-independent manner.

Although malignant gliomas frequently show aberrant activation of the mammalian target of rapamycin (mTOR), mTOR inhibitors have performed poorly in clinical trials. Besides regulating cell growth and translation, mTOR controls the initiation of autophagy. By recycling cellular components, autophagy can mobilize energy resources, and has thus been attributed cancer-promoting effects. Here, we asked whether the activation of autophagy represents an escape mechanism to pharmacological mTOR inhibition in glioma cells, and explored co-treatment with mTOR and autophagy inhibitors as a therapeutic strategy. Mimicking conditions of the glioma microenvironment, glioma cells were exposed to nutrient starvation and hypoxia. We analyzed autophagic activity, cell growth, viability and oxygen consumption following (co-)treatment with the mTOR inhibitors torin2 or rapamycin, and autophagy inhibitors bafilomycin A1 or MRT68921. Changes in global proteome were quantified by mass spectrometry. In the context of hypoxia and starvation, autophagy was strongly induced in glioma cells and further increased by mTOR inhibition. While torin2 enhanced glioma cell survival, co-treatment with torin2 and bafilomycin A1 failed to promote cell death. Importantly, treatment with bafilomycin A1 alone also protected glioma cells from cell death. Mechanistically, both compounds significantly reduced cell growth and oxygen consumption. Quantitative proteomics analysis showed that bafilomycin A1 induced broad changes in the cellular proteome. More specifically, proteins downregulated by bafilomycin A1 were associated with the mitochondrial respiratory chain and ATP synthesis. Taken together, our results show that activation of autophagy does not account for the cytoprotective effects of mTOR inhibition in our in vitro model of the glioma microenvironment. Our proteomic findings suggest that the pharmacological inhibition of autophagy induces extensive changes in the cellular proteome that can support glioma cell survival under nutrient-deplete and hypoxic conditions. These findings provide a novel perspective on the complex role of autophagy in gliomas.

Translatome proteomics identifies autophagy as a resistance mechanism to on-target FLT3 inhibitors in acute myeloid leukemia.

Internal tandem duplications (ITD) in the receptor tyrosine kinase FLT3 occur in 25 % of acute myeloid leukemia (AML) patients, drive leukemia progression and confer a poor prognosis. Primary resistance to FLT3 kinase inhibitors (FLT3i) quizartinib, crenolanib and gilteritinib is a frequent clinical challenge and occurs in the absence of identifiable genetic causes. This suggests that adaptive cellular mechanisms mediate primary resistance to on-target FLT3i therapy. Here, we systematically investigated acute cellular responses to on-target therapy with multiple FLT3i in FLT3-ITD + AML using recently developed functional translatome proteomics (measuring changes in the nascent proteome) with phosphoproteomics. This pinpointed AKT-mTORC1-ULK1-dependent autophagy as a dominant resistance mechanism to on-target FLT3i therapy. FLT3i induced autophagy in a concentration- and time-dependent manner specifically in FLT3-ITD + cells in vitro and in primary human AML cells ex vivo. Pharmacological or genetic inhibition of autophagy increased the sensitivity to FLT3-targeted therapy in cell lines, patient-derived xenografts and primary AML cells ex vivo. In mice xenografted with FLT3-ITD + AML cells, co-treatment with oral FLT3 and autophagy inhibitors synergistically impaired leukemia progression and extended overall survival. Our findings identify a molecular mechanism responsible for primary FLT3i treatment resistance and demonstrate the pre-clinical efficacy of a rational combination treatment strategy targeting both FLT3 and autophagy induction.

Mitochondrial RNA modifications shape metabolic plasticity in metastasis.

Aggressive and metastatic cancers show enhanced metabolic plasticity1, but the precise underlying mechanisms of this remain unclear. Here we show how two NOP2/Sun RNA methyltransferase 3 (NSUN3)-dependent RNA modifications-5-methylcytosine (m5C) and its derivative 5-formylcytosine (f5C) (refs.2-4)-drive the translation of mitochondrial mRNA to power metastasis. Translation of mitochondrially encoded subunits of the oxidative phosphorylation complex depends on the formation of m5C at position 34 in mitochondrial tRNAMet. m5C-deficient human oral cancer cells exhibit increased levels of glycolysis and changes in their mitochondrial function that do not affect cell viability or primary tumour growth in vivo; however, metabolic plasticity is severely impaired as mitochondrial m5C-deficient tumours do not metastasize efficiently. We discovered that CD36-dependent non-dividing, metastasis-initiating tumour cells require mitochondrial m5C to activate invasion and dissemination. Moreover, a mitochondria-driven gene signature in patients with head and neck cancer is predictive for metastasis and disease progression. Finally, we confirm that this metabolic switch that allows the metastasis of tumour cells can be pharmacologically targeted through the inhibition of mitochondrial mRNA translation in vivo. Together, our results reveal that site-specific mitochondrial RNA modifications could be therapeutic targets to combat metastasis.

PTEN mutant non-small cell lung cancer require ATM to suppress pro-apoptotic signalling and evade radiotherapy.

BACKGROUND: Despite advances in treatment of patients with non-small cell lung cancer, carriers of certain genetic alterations are prone to failure. One such factor frequently mutated, is the tumor suppressor PTEN. These tumors are supposed to be more resistant to radiation, chemo- and immunotherapy. RESULTS: We demonstrate that loss of PTEN led to altered expression of transcriptional programs which directly regulate therapy resistance, resulting in establishment of radiation resistance. While PTEN-deficient tumor cells were not dependent on DNA-PK for IR resistance nor activated ATR during IR, they showed a significant dependence for the DNA damage kinase ATM. Pharmacologic inhibition of ATM, via KU-60019 and AZD1390 at non-toxic doses, restored and even synergized with IR in PTEN-deficient human and murine NSCLC cells as well in a multicellular organotypic ex vivo tumor model. CONCLUSION: PTEN tumors are addicted to ATM to detect and repair radiation induced DNA damage. This creates an exploitable bottleneck. At least in cellulo and ex vivo we show that low concentration of ATM inhibitor is able to synergise with IR to treat PTEN-deficient tumors in genetically well-defined IR resistant lung cancer models.

Proteomic landscape of SARS-CoV-2- and MERS-CoV-infected primary human renal epithelial cells.

Acute kidney injury is associated with mortality in COVID-19 patients. However, host cell changes underlying infection of renal cells with SARS-CoV-2 remain unknown and prevent understanding of the molecular mechanisms that may contribute to renal pathology. Here, we carried out quantitative translatome and whole-cell proteomics analyses of primary renal proximal and distal tubular epithelial cells derived from human donors infected with SARS-CoV-2 or MERS-CoV to disseminate virus and cell type-specific changes over time. Our findings revealed shared pathways modified upon infection with both viruses, as well as SARS-CoV-2-specific host cell modulation driving key changes in innate immune activation and cellular protein quality control. Notably, MERS-CoV infection-induced specific changes in mitochondrial biology that were not observed in response to SARS-CoV-2 infection. Furthermore, we identified extensive modulation in pathways associated with kidney failure that changed in a virus- and cell type-specific manner. In summary, we provide an overview of the effects of SARS-CoV-2 or MERS-CoV infection on primary renal epithelial cells revealing key pathways that may be essential for viral replication.

PBLMM: Peptide-based linear mixed models for differential expression analysis of shotgun proteomics data.

Here, we present a peptide-based linear mixed models tool-PBLMM, a standalone desktop application for differential expression analysis of proteomics data. We also provide a Python package that allows streamlined data analysis workflows implementing the PBLMM algorithm. PBLMM is easy to use without scripting experience and calculates differential expression by peptide-based linear mixed regression models. We show that peptide-based models outperform classical methods of statistical inference of differentially expressed proteins. In addition, PBLMM exhibits superior statistical power in situations of low effect size and/or low sample size. Taken together our tool provides an easy-to-use, high-statistical-power method to infer differentially expressed proteins from proteomics data.

Quantitative Translation Proteomics Using mePROD.

Multiplexed enhanced protein dynamic mass spectrometry (mePROD MS) enables robust quantification of translation in cell culture. Tandem mass tags (TMT) are combined with pulsed stable isotope labeling in cell culture (pSILAC) to monitor newly synthesized proteins on a proteome wide scale. While approaches combining pSILAC and TMT typically require long labeling times to reach sufficient intensity of the newly synthesized peptides in the mass spectrometer, mePROD uses a carrier signal that boosts the survey scan intensity and strongly increases identification rates. Hence, this protocol provides an easy and cost-efficient method to profile proteome-wide translatome changes at a temporal resolution of minutes.

Inhibition of USP28 overcomes Cisplatin-resistance of squamous tumors by suppression of the Fanconi anemia pathway.

Squamous cell carcinomas (SCC) frequently have an exceptionally high mutational burden. As consequence, they rapidly develop resistance to platinum-based chemotherapy and overall survival is limited. Novel therapeutic strategies are therefore urgently required. SCC express ∆Np63, which regulates the Fanconi Anemia (FA) DNA-damage response in cancer cells, thereby contributing to chemotherapy-resistance. Here we report that the deubiquitylase USP28 is recruited to sites of DNA damage in cisplatin-treated cells. ATR phosphorylates USP28 and increases its enzymatic activity. This phosphorylation event is required to positively regulate the DNA damage repair in SCC by stabilizing ∆Np63. Knock-down or inhibition of USP28 by a specific inhibitor weakens the ability of SCC to cope with DNA damage during platin-based chemotherapy. Hence, our study presents a novel mechanism by which ∆Np63 expressing SCC can be targeted to overcome chemotherapy resistance. Limited treatment options and low response rates to chemotherapy are particularly common in patients with squamous cancer. The SCC specific transcription factor ∆Np63 enhances the expression of Fanconi Anemia genes, thereby contributing to recombinational DNA repair and Cisplatin resistance. Targeting the USP28-∆Np63 axis in SCC tones down this DNA damage response pathways, thereby sensitizing SCC cells to cisplatin treatment.

SIK2 orchestrates actin-dependent host response upon Salmonella infection.

Salmonella is an intracellular pathogen of a substantial global health concern. In order to identify key players involved in Salmonella infection, we performed a global host phosphoproteome analysis subsequent to bacterial infection. Thereby, we identified the kinase SIK2 as a central component of the host defense machinery upon Salmonella infection. SIK2 depletion favors the escape of bacteria from the Salmonella-containing vacuole (SCV) and impairs Xenophagy, resulting in a hyperproliferative phenotype. Mechanistically, SIK2 associates with actin filaments under basal conditions; however, during bacterial infection, SIK2 is recruited to the SCV together with the elements of the actin polymerization machinery (Arp2/3 complex and Formins). Notably, SIK2 depletion results in a severe pathological cellular actin nucleation and polymerization defect upon Salmonella infection. We propose that SIK2 controls the formation of a protective SCV actin shield shortly after invasion and orchestrates the actin cytoskeleton architecture in its entirety to control an acute Salmonella infection after bacterial invasion.

Characterization of Extracellular Vesicles from Preconditioned Human Adipose-Derived Stromal/Stem Cells.

Cell-free therapy using extracellular vesicles (EVs) from adipose-derived mesenchymal stromal/stem cells (ASCs) seems to be a safe and effective therapeutic option to support tissue and organ regeneration. The application of EVs requires particles with a maximum regenerative capability and hypoxic culture conditions as an in vitro preconditioning regimen has been shown to alter the molecular composition of released EVs. Nevertheless, the EV cargo after hypoxic preconditioning has not yet been comprehensively examined. The aim of the present study was the characterization of EVs from hypoxic preconditioned ASCs. We investigated the EV proteome and their effects on renal tubular epithelial cells in vitro. While no effect of hypoxia was observed on the number of released EVs and their protein content, the cargo of the proteins was altered. Proteomic analysis showed 41 increased or decreased proteins, 11 in a statistically significant manner. Furthermore, the uptake of EVs in epithelial cells and a positive effect on oxidative stress in vitro were observed. In conclusion, culture of ASCs under hypoxic conditions was demonstrated to be a promising in vitro preconditioning regimen, which alters the protein cargo and increases the anti-oxidative potential of EVs. These properties may provide new potential therapeutic options for regenerative medicine.

Aprotinin Inhibits SARS-CoV-2 Replication.

Severe acute respiratory syndrome virus 2 (SARS-CoV-2) is the cause of the current coronavirus disease 19 (COVID-19) pandemic. Protease inhibitors are under consideration as virus entry inhibitors that prevent the cleavage of the coronavirus spike (S) protein by cellular proteases. Herein, we showed that the protease inhibitor aprotinin (but not the protease inhibitor SERPINA1/alpha-1 antitrypsin) inhibited SARS-CoV-2 replication in therapeutically achievable concentrations. An analysis of proteomics and translatome data indicated that SARS-CoV-2 replication is associated with a downregulation of host cell protease inhibitors. Hence, aprotinin may compensate for downregulated host cell proteases during later virus replication cycles. Aprotinin displayed anti-SARS-CoV-2 activity in different cell types (Caco2, Calu-3, and primary bronchial epithelial cell air-liquid interface cultures) and against four virus isolates. In conclusion, therapeutic aprotinin concentrations exert anti-SARS-CoV-2 activity. An approved aprotinin aerosol may have potential for the early local control of SARS-CoV-2 replication and the prevention of COVID-19 progression to a severe, systemic disease.

Unbiased translation proteomics upon cell stress.

The mammalian target of rapamycin and the integrated stress response are central cellular hubs regulating translation upon stress. The precise proteins and pathway specificity of translation targets of these pathways remained largely unclear. We recently described a new method for quantitative translation proteomics and found that both pathways control translation of the same sets of proteins.

The Lipid Receptor G2A (GPR132) Mediates Macrophage Migration in Nerve Injury-Induced Neuropathic Pain.

Nerve injury-induced neuropathic pain is difficult to treat and mechanistically characterized by strong neuroimmune interactions, involving signaling lipids that act via specific G-protein coupled receptors. Here, we investigated the role of the signaling lipid receptor G2A (GPR132) in nerve injury-induced neuropathic pain using the robust spared nerve injury (SNI) mouse model. We found that the concentrations of the G2A agonist 9-HODE (9-Hydroxyoctadecadienoic acid) are strongly increased at the site of nerve injury during neuropathic pain. Moreover, G2A-deficient mice show a strong reduction of mechanical hypersensitivity after nerve injury. This phenotype is accompanied by a massive reduction of invading macrophages and neutrophils in G2A-deficient mice and a strongly reduced release of the proalgesic mediators TNFα, IL-6 and VEGF at the site of injury. Using a global proteome analysis to identify the underlying signaling pathways, we found that G2A activation in macrophages initiates MyD88-PI3K-AKT signaling and transient MMP9 release to trigger cytoskeleton remodeling and migration. We conclude that G2A-deficiency reduces inflammatory responses by decreasing the number of immune cells and the release of proinflammatory cytokines and growth factors at the site of nerve injury. Inhibiting the G2A receptor after nerve injury may reduce immune cell-mediated peripheral sensitization and may thus ameliorate neuropathic pain.

Instrument Logic Increases Identifications during Mutliplexed Translatome Measurements.

Pulsed Stable Isotope Labeling in Cell culture (SILAC) approaches allow measurement of protein dynamics, including protein translation and degradation. However, its use for quantifying acute changes has been limited due to low labeled peptide stoichiometry. Here, we describe the use of instrument logic to select peaks of interest via targeted mass differences (TMD) for overcoming this limitation. Comparing peptides artificially mixed at low heavy-to-light stoichiometry measured using standard data dependent acquisition with or without TMD revealed 2-3-fold increases in identification without significant loss in quantification precision for both MS2 and MS3 methods. Our benchmarked method approach increased throughput by reducing the necessary machine time. We anticipate that all pulsed SILAC measurements, combined with tandem mass tagging (TMT) or not, would greatly benefit from instrument logic based approaches.

Serine-dependent redox homeostasis regulates glioblastoma cell survival.

BACKGROUND: The amino acid serine is an important substrate for biosynthesis and redox homeostasis. We investigated whether glioblastoma (GBM) cells are dependent on serine for survival under conditions of the tumour microenvironment. METHODS: Serine availability in GBM cells was modulated pharmacologically, genetically and by adjusting serine and glycine concentrations in the culture medium. Cells were investigated for regulation of serine metabolism, proliferation, sensitivity to hypoxia-induced cell death and redox homeostasis. RESULTS: Hypoxia-induced expression of phosphoglycerate dehydrogenase (PHGDH) and the mitochondrial serine hydroxymethyltransferase (SHMT2) was observed in three of five tested glioma cell lines. Nuclear factor erythroid 2-related factor (Nrf) 2 activation also induced PHGDH and SHMT2 expression in GBM cells. Low levels of endogenous PHGDH as well as PHGDH gene suppression resulted in serine dependency for cell growth. Pharmacological inhibition of PHGDH with CBR-5884 reduced proliferation and sensitised cells profoundly to hypoxia-induced cell death. This effect was accompanied by an increase in reactive oxygen species and a decrease in the NADPH/NADP+ ratio. Similarly, hypoxia-induced cell death was enhanced by PHGDH gene suppression and reduced by PHGDH overexpression. CONCLUSIONS: Serine facilitates adaptation of GBM cells to conditions of the tumour microenvironment and its metabolism could be a plausible therapeutic target.