Bone marrow fibrosis (BMF) develops in various hematological and non-hematological conditions and is a central pathological feature of myelofibrosis. Effective cell-targeted therapeutics are needed, but the cellular origin of BMF remains elusive. Here, we show using genetic fate tracing in two murine models of BMF that Gli1+ mesenchymal stromal cells (MSCs) are recruited from the endosteal and perivascular niche to become fibrosis-driving myofibroblasts in the bone marrow. Genetic ablation of Gli1+ cells abolished BMF and rescued bone marrow failure. Pharmacological targeting of Gli proteins with GANT61 inhibited Gli1+ cell expansion and myofibroblast differentiation and attenuated fibrosis severity. The same pathway is also active in human BMF, and Gli1 expression in BMF significantly correlates with the severity of the disease. In addition, GANT61 treatment reduced the myofibroblastic phenotype of human MSCs isolated from patients with BMF, suggesting that targeting of Gli proteins could be a relevant therapeutic strategy.

The causative agent of the coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected millions and killed hundreds of thousands of people worldwide, highlighting an urgent need to develop antiviral therapies. Here we present a quantitative mass spectrometry-based phosphoproteomics survey of SARS-CoV-2 infection in Vero E6 cells, revealing dramatic rewiring of phosphorylation on host and viral proteins. SARS-CoV-2 infection promoted casein kinase II (CK2) and p38 MAPK activation, production of diverse cytokines, and shutdown of mitotic kinases, resulting in cell cycle arrest. Infection also stimulated a marked induction of CK2-containing filopodial protrusions possessing budding viral particles. Eighty-seven drugs and compounds were identified by mapping global phosphorylation profiles to dysregulated kinases and pathways. We found pharmacologic inhibition of the p38, CK2, CDK, AXL, and PIKFYVE kinases to possess antiviral efficacy, representing potential COVID-19 therapies.

Biological processes are regulated by intermolecular interactions and chemical modifications that do not affect protein levels, thus escaping detection in classical proteomic screens. We demonstrate here that a global protein structural readout based on limited proteolysis-mass spectrometry (LiP-MS) detects many such functional alterations, simultaneously and in situ, in bacteria undergoing nutrient adaptation and in yeast responding to acute stress. The structural readout, visualized as structural barcodes, captured enzyme activity changes, phosphorylation, protein aggregation, and complex formation, with the resolution of individual regulated functional sites such as binding and active sites. Comparison with prior knowledge, including other 'omics data, showed that LiP-MS detects many known functional alterations within well-studied pathways. It suggested distinct metabolite-protein interactions and enabled identification of a fructose-1,6-bisphosphate-based regulatory mechanism of glucose uptake in E. coli. The structural readout dramatically increases classical proteomics coverage, generates mechanistic hypotheses, and paves the way for in situ structural systems biology.

Metabolic reprogramming is critical for tumor initiation and progression. However, the exact impact of specific metabolic changes on cancer progression is poorly understood. Here, we integrate multimodal analyses of primary and metastatic clonally-related clear cell renal cancer cells (ccRCC) grown in physiological media to identify key stage-specific metabolic vulnerabilities. We show that a VHL loss-dependent reprogramming of branched-chain amino acid catabolism sustains the de novo biosynthesis of aspartate and arginine enabling tumor cells with the flexibility of partitioning the nitrogen of the amino acids depending on their needs. Importantly, we identify the epigenetic reactivation of argininosuccinate synthase (ASS1), a urea cycle enzyme suppressed in primary ccRCC, as a crucial event for metastatic renal cancer cells to acquire the capability to generate arginine, invade in vitro and metastasize in vivo. Overall, our study uncovers a mechanism of metabolic flexibility occurring during ccRCC progression, paving the way for the development of novel stage-specific therapies.

Tissue-resident macrophages are required for homeostasis, but also contribute to tissue dysfunction in pathophysiological states. The sympathetic neurotransmitter norepinephrine (NE) induces an anti-inflammatory and tissue-reparative phenotype in macrophages. As NE has a well-established role in promoting triglyceride lipolysis in adipocytes, and macrophages accumulate triglyceride droplets in various physiological and disease states, we investigated the effect of NE on primary mouse bone marrow-derived macrophage triglyceride metabolism. Surprisingly, our data show that in contrast to the canonical role of NE in stimulating lipolysis, NE acting via beta2-adrenergic receptors (B2ARs) in macrophages promotes extracellular fatty acid uptake and their storage as triglycerides and reduces free fatty acid release from triglyceride-laden macrophages. We demonstrate that these responses are mediated by a B2AR activation-dependent increase in Hilpda and Dgat1 gene expression and activity. We further show that B2AR activation favors the storage of extracellular polyunsaturated fatty acids. Finally, we present evidence that macrophages isolated from hearts after myocardial injury, for which survival critically depends on leukocyte B2ARs, have a transcriptional signature indicative of a transient triglyceride accumulation. Overall, we describe a novel and unexpected role of NE in promoting triglyceride storage in macrophages that could have potential implications in multiple diseases.

Multi-omics datasets can provide molecular insights beyond the sum of individual omics. Various tools have been recently developed to integrate such datasets, but there are limited strategies to systematically extract mechanistic hypotheses from them. Here, we present COSMOS (Causal Oriented Search of Multi-Omics Space), a method that integrates phosphoproteomics, transcriptomics, and metabolomics datasets. COSMOS combines extensive prior knowledge of signaling, metabolic, and gene regulatory networks with computational methods to estimate activities of transcription factors and kinases as well as network-level causal reasoning. COSMOS provides mechanistic hypotheses for experimental observations across multi-omics datasets. We applied COSMOS to a dataset comprising transcriptomics, phosphoproteomics, and metabolomics data from healthy and cancerous tissue from eleven clear cell renal cell carcinoma (ccRCC) patients. COSMOS was able to capture relevant crosstalks within and between multiple omics layers, such as known ccRCC drug targets. We expect that our freely available method will be broadly useful to extract mechanistic insights from multi-omics studies.

The bioenergetics and molecular determinants of the metabolic response to mitochondrial dysfunction are incompletely understood, in part due to a lack of appropriate isogenic cellular models of primary mitochondrial defects. Here, we capitalize on a recently developed cell model with defined levels of m.8993T>G mutation heteroplasmy, mTUNE, to investigate the metabolic underpinnings of mitochondrial dysfunction. We found that impaired utilization of reduced nicotinamide adenine dinucleotide (NADH) by the mitochondrial respiratory chain leads to cytosolic reductive carboxylation of glutamine as a new mechanism for cytosol-confined NADH recycling supported by malate dehydrogenase 1 (MDH1). We also observed that increased glycolysis in cells with mitochondrial dysfunction is associated with increased cell migration in an MDH1-dependent fashion. Our results describe a novel link between glycolysis and mitochondrial dysfunction mediated by reductive carboxylation of glutamine.

Omic technologies allow us to generate extensive data, including transcriptomic, proteomic, phosphoproteomic and metabolomic. These data can be used to study signal transduction, gene regulation and metabolism. In this review, we summarise resources and methods to analysis these types of data. We focus on methods developed to recover functional insights using footprints. Footprints are signatures defined by the effect of molecules or processes of interest. They integrate information from multiple measurements whose abundances are under the influence of a common regulator. For example, transcripts controlled by a transcription factor or peptides phosphorylated by a kinase. Footprints can also be generalised across multiple types of omic data. Thus, we also present methods to integrate multiple types of omic data and features (such as the ones derived from footprints) together. We highlight some examples of studies that leverage such approaches to discover new biological mechanisms.

In diabetic patients, dyslipidemia frequently contributes to organ damage such as diabetic kidney disease (DKD). Dyslipidemia is associated with both excessive deposition of triacylglycerol (TAG) in lipid droplets (LDs) and lipotoxicity. Yet, it is unclear how these two effects correlate with each other in the kidney and how they are influenced by dietary patterns. By using a diabetes mouse model, we find here that high-fat diet enriched in the monounsaturated oleic acid (OA) caused more lipid storage in LDs in renal proximal tubular cells (PTCs) but less tubular damage than a corresponding butter diet with the saturated palmitic acid (PA). This effect was particularly evident in S2/S3 but not S1 segments of the proximal tubule. Combining transcriptomics, lipidomics, and functional studies, we identify endoplasmic reticulum (ER) stress as the main cause of PA-induced PTC injury. Mechanistically, ER stress is caused by elevated levels of saturated TAG precursors, reduced LD formation, and, consequently, higher membrane order in the ER. Simultaneous addition of OA rescues the cytotoxic effects by normalizing membrane order and increasing both TAG and LD formation. Our study thus emphasizes the importance of monounsaturated fatty acids for the dietary management of DKD by preventing lipid bilayer stress in the ER and promoting TAG and LD formation in PTCs.

Neuroimmune interactions between the sympathetic nervous system (SNS) and macrophages are required for the homeostasis of multiple tissues, including the adipose tissue. It has been proposed that the SNS maintains adipose tissue macrophages (ATMs) in an anti-inflammatory state via direct norepinephrine (NE) signaling to macrophages. This study aimed to investigate the physiological importance of this paradigm by utilizing a mouse model in which the adrenergic signaling from the SNS to macrophages, but not to other adipose tissue cells, was disrupted.

The biguanide drug metformin is a safe and widely prescribed drug for type 2 diabetes. Interestingly, hundreds of clinical trials have been set to evaluate the potential role of metformin in the prevention and treatment of cancer including colorectal cancer (CRC). However, the "metformin signaling" remains controversial.

Genetic alterations in cancer cells trigger oncogenic transformation, a process largely mediated by the dysregulation of kinase and transcription factor (TF) activities. While the mutational profiles of thousands of tumours have been extensively characterised, the measurements of protein activities have been technically limited until recently. We compiled public data of matched genomics and (phospho)proteomics measurements for 1,110 tumours and 77 cell lines that we used to estimate activity changes in 218 kinases and 292 TFs. Co-regulation of kinase and TF activities reflects previously known regulatory relationships and allows us to dissect genetic drivers of signalling changes in cancer. We find that loss-of-function mutations are not often associated with the dysregulation of downstream targets, suggesting frequent compensatory mechanisms. Finally, we identified the activities most differentially regulated in cancer subtypes and showed how these can be linked to differences in patient survival. Our results provide broad insights into the dysregulation of protein activities in cancer and their contribution to disease severity.

The COVID-19 Disease Map project is a large-scale community effort uniting 277 scientists from 130 Institutions around the globe. We use high-quality, mechanistic content describing SARS-CoV-2-host interactions and develop interoperable bioinformatic pipelines for novel target identification and drug repurposing.