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Regeneration of mesenchymal tissues depends on a resident stem cell population, that in most cases remains elusive in terms of cellular identity and differentiation signals. We here show that primary cell cultures derived from adipose tissue or skeletal muscle differentiate into adipocytes when cultured in high glucose. High glucose induces ROS production and PKCβ activation. These two events appear crucial steps in this differentiation process that can be directly induced by oxidizing agents and inhibited by PKCβ siRNA silencing. The differentiated adipocytes, when implanted in vivo, form viable and vascularized adipose tissue. Overall, the data highlight a previously uncharacterized differentiation route triggered by high glucose that drives not only resident stem cells of the adipose tissue but also uncommitted precursors present in muscle cells to form adipose depots. This process may represent a feed-forward cycle between the regional increase in adiposity and insulin resistance that plays a key role in the pathogenesis of diabetes mellitus.

Activating mutations of Src homology-2 domain-containing protein tyrosine phosphatase-2 (Shp2) cause multiple childhood conditions for which there is an unmet therapeutic need, including juvenile myelomonocytic leukemia (JMML) and Noonan syndrome. SFX-01, an α-cyclodextrin-stabilized sulforaphane complex currently in clinical development, covalently adducts cysteine residues. Using unbiased proteomics, its protein targets were identified, including Shp2. SFX-01 induced an inhibitory dithiolethione modification at the Shp2 active site cysteine. Importantly, in a transgenic mouse model of human Noonan syndrome with hyperactive D61G Shp2, SFX-01 concomitantly normalized their phosphatase activity and myeloid cell count. Furthermore, SFX-01 also attenuated JMML human patient-derived hematopoietic stem cell proliferation that was linked to STAT1 signaling and decreased cyclin D1 expression, resulting in cell-cycle arrest. We conclude that SFX-01 is an activating mutant Shp2 inhibitor and may offer beneficial effects in patients with JMML or Noonan syndrome. Synopsis The sulforaphane complex SFX-01 targets hyperactive Shp2 by modifying its active site. In models of Noonan syndrome and JMML, SFX-01 normalises Shp2 activity, reduces myeloid cells number, inhibits stem cell proliferation and induces cell-cycle arrest, demonstrating the drug’s therapeutic potential. Activating mutations of Shp2 cause conditions such as Noonan syndrome and juvenile myelomonocytic leukemia (JMML), with unmet therapeutic needs. SFX-01, a sulforaphane complex, modifies cysteine residues and targets proteins including Shp2, where it induces an inhibitory modification at the active site of this protein phosphatase. In a transgenic mouse model of Noonan syndrome and human JMML stem cells, SFX-01 normalized Shp2 activity, reduced myeloid cell counts, and induced cell-cycle arrest, highlighting its therapeutic potential. The sulforaphane complex SFX-01 targets hyperactive Shp2 by modifying its active site. In models of Noonan syndrome and JMML, SFX-01 normalises Shp2 activity, reduces myeloid cells number, inhibits stem cell proliferation and induces cell-cycle arrest, demonstrating the drug’s therapeutic potential.

Fibrosis is a chronic disease characterized by excessive extracellular matrix production, which leads to disruption of organ function. Fibroblasts are key effector cells of this process, responding chiefly to the pleiotropic cytokine transforming growth factor-β1 (TGF-β1), which promotes fibroblast to myofibroblast differentiation. We found that extracellular nutrient availability profoundly influenced the TGF-β1 transcriptome of primary human lung fibroblasts and that biosynthesis of amino acids emerged as a top enriched TGF-β1 transcriptional module. We subsequently uncovered a key role for pyruvate in influencing glutaminase (GLS1) inhibition during TGF-β1-induced fibrogenesis. In pyruvate-replete conditions, GLS1 inhibition was ineffective in blocking TGF-β1-induced fibrogenesis, as pyruvate can be used as the substrate for glutamate and alanine production via glutamate dehydrogenase (GDH) and glutamic-pyruvic transaminase 2 (GPT2), respectively. We further show that dual targeting of either GPT2 or GDH in combination with GLS1 inhibition was required to fully block TGF-β1-induced collagen synthesis. These findings embolden a therapeutic strategy aimed at additional targeting of mitochondrial pyruvate metabolism in the presence of a glutaminolysis inhibitor to interfere with the pathological deposition of collagen in the setting of pulmonary fibrosis and potentially other fibrotic conditions.

During obesity, macrophages infiltrate the breast tissue leading to low‐grade chronic inflammation, a factor considered responsible for the higher risk of breast cancer associated with obesity. Here, we formally demonstrate that breast epithelial cells acquire malignant properties when exposed to medium conditioned by macrophages derived from human healthy donors. These effects were mediated by the breast cancer oncogene IKKε and its downstream target—the serine biosynthesis pathway as demonstrated by genetic or pharmacological tools. Furthermore, amlexanox, an FDA‐approved drug targeting IKKε and its homologue TBK1, delayed in vivo tumour formation in a combined genetic mouse model of breast cancer and high‐fat diet‐induced obesity/inflammation. Finally, in human breast cancer tissues, we validated the link between inflammation–IKKε and alteration of cellular metabolism. Altogether, we identified a pathway connecting obesity‐driven inflammation to breast cancer and a potential therapeutic strategy to reduce the risk of breast cancer associated with obesity. Synopsis In this article a new pathway has been identified that links macrophage‐driven inflammation and cellular metabolism to the acquisition of malignant traits in breast epithelium providing new insight into the well‐established association between breast cancer and obesity and offering a new preventive strategy. Medium conditioned by macrophages derived from healthy donors induces acquisition of malignant properties in breast epithelial cells. Inhibition of the kinase IKKε and the serine biosynthesis pathway prevents the acquisition of the malignant phenotypes induced by macrophage‐conditioned medium. Amlexanox – an FDA approved drug targeting IKKε and its homologue TBK1 – defers tumour appearance in vivo in a combined genetic mouse model of breast cancer and high fat diet‐induced obesity/inflammation. Graphical Abstract In this article a new pathway has been identified that links macrophage‐driven inflammation and cellular metabolism to the acquisition of malignant traits in breast epithelium providing new insight into the well‐established association between breast cancer and obesity and offering a new preventive strategy.

Caspases provide vital links in non-apoptotic regulatory networks controlling inflammation, compensatory proliferation, morphology and cell migration. How caspases are activated under non-apoptotic conditions and process a selective set of substrates without killing the cell remain enigmatic. Here we find that the Drosophila unconventional myosin CRINKLED (CK) selectively interacts with the initiator caspase DRONC and regulates some of its non-apoptotic functions. Loss of CK in the arista, border cells or proneural clusters of the wing imaginal discs affects DRONC-dependent patterning. Our data indicate that CK acts as substrate adaptor, recruiting SHAGGY46/GSK3-β to DRONC, thereby facilitating caspase-mediated cleavage and localized modulation of kinase activity. Similarly, the mammalian CK counterpart, MYO7A, binds to and impinges on CASPASE-8, revealing a new regulatory axis affecting receptor interacting protein kinase-1 (RIPK1)>CASPASE-8 signalling. Together, our results expose a conserved role for unconventional myosins in transducing caspase-dependent regulation of kinases, allowing them to take part in specific signalling events. In addition to their role in apoptosis, caspases are also involved in mediating non-apoptotic events. Here the authors show that the Drosophila myosin family member CRINKLED and its mammalian counterpart act as substrate adaptor that facilitate caspase-mediated cleavage and localised kinase activity.

Voltage-gated hydrogen channel 1 (Hvcn1) is a voltage-gated proton channel, which reduces cytosol acidification and facilitates the production of ROS. The increased expression of this channel in some cancers has led to proposing Hvcn1 antagonists as potential therapeutics. While its role in most leukocytes has been studied in depth, the function of Hvcn1 in T cells remains poorly defined. We show that Hvcn1 plays a nonredundant role in protecting naive T cells from intracellular acidification during priming. Despite sharing overall functional impairment in vivo and in vitro, Hvcn1-deficient CD4+ and CD8+ T cells display profound differences during the transition from naive to primed T cells, including in the preservation of T cell receptor (TCR) signaling, cellular division, and death. These selective features result, at least in part, from a substantially different metabolic response to intracellular acidification associated with priming. While Hvcn1-deficient naive CD4+ T cells reprogram to rescue the glycolytic pathway, naive CD8+ T cells, which express high levels of this channel in the mitochondria, respond by metabolically compensating mitochondrial dysfunction, at least in part via AMPK activation. These observations imply heterogeneity between adaptation of naive CD4+ and CD8+ T cells to intracellular acidification during activation.

Mitochondrial dysfunction is implicated in the pathogenesis of the neurological condition autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS), yet precisely how the mitochondrial metabolism is affected is unknown. Thus, to better understand changes in the mitochondrial metabolism caused by loss of the sacsin protein (encoded by the SACS gene, which is mutated in ARSACS), we performed mass spectrometry-based tracer analysis, with both glucose- and glutamine-traced carbon. Comparing the metabolite profiles between wild-type and sacsin-knockout cell lines revealed increased reliance on aerobic glycolysis in sacsin-deficient cells, as evidenced by the increase in lactate and reduction of glucose. Moreover, sacsin knockout cells differentiated towards a neuronal phenotype had increased levels of tricarboxylic acid cycle metabolites relative to the controls. We also observed disruption in the glutaminolysis pathway in differentiated and undifferentiated cells in the absence of sacsin. In conclusion, this work demonstrates consequences for cellular metabolism associated with a loss of sacsin, which may be relevant to ARSACS.

Cardiomyocytes rely on metabolic substrates, not only to fuel cardiac output, but also for growth and remodelling during stress. Here we show that mitochondrial pyruvate carrier (MPC) abundance mediates pathological cardiac hypertrophy. MPC abundance was reduced in failing hypertrophic human hearts, as well as in the myocardium of mice induced to fail by angiotensin II or through transverse aortic constriction. Constitutive knockout of cardiomyocyte MPC1/2 in mice resulted in cardiac hypertrophy and reduced survival, while tamoxifen-induced cardiomyocyte-specific reduction of MPC1/2 to the attenuated levels observed during pressure overload was sufficient to induce hypertrophy with impaired cardiac function. Failing hearts from cardiomyocyte-restricted knockout mice displayed increased abundance of anabolic metabolites, including amino acids and pentose phosphate pathway intermediates and reducing cofactors. These hearts showed a concomitant decrease in carbon flux into mitochondrial tricarboxylic acid cycle intermediates, as corroborated by complementary 1,2-[ 13 C 2 ]glucose tracer studies. In contrast, inducible cardiomyocyte overexpression of MPC1/2 resulted in increased tricarboxylic acid cycle intermediates, and sustained carrier expression during transverse aortic constriction protected against cardiac hypertrophy and failure. Collectively, our findings demonstrate that loss of the MPC1/2 causally mediates adverse cardiac remodelling. Mitochondrial metabolism is critical for cardiac function and growth. Fernandez-Caggiano et al. show that maintenance of mitochondrial pyruvate transporter expression is a critical mediator of cardiac hypertrophy and heart failure.

The inhibitors of apoptosis (IAP) proteins cIAP1 and cIAP2 have recently emerged as key ubiquitin-E3 ligases regulating innate immunity and cell survival. Much of our knowledge of these IAPs stems from studies using pharmacological inhibitors of IAPs, dubbed Smac mimetics (SMs). Although SMs stimulate auto-ubiquitylation and degradation of cIAPs, little is known about the molecular determinants through which SMs activate the E3 activities of cIAPs. In this study, we find that SM-induced rapid degradation of cIAPs requires binding to tumour necrosis factor (TNF) receptor-associated factor 2 (TRAF2). Moreover, our data reveal an unexpected difference between cIAP1 and cIAP2. Although SM-induced degradation of cIAP1 does not require cIAP2, degradation of cIAP2 critically depends on the presence of cIAP1. In addition, degradation of cIAP2 also requires the ability of the cIAP2 RING finger to dimerise and to bind to E2s. This has important implications because SM-mediated degradation of cIAP1 causes non-canonical activation of NF- κ B, which results in the induction of cIAP2 gene expression. In the absence of cIAP1, de novo synthesised cIAP2 is resistant to the SM and suppresses TNF α killing. Furthermore, the cIAP2-MALT1 oncogene, which lacks cIAP2's RING, is resistant to SM treatment. The identification of mechanisms through which cancer cells resist SM treatment will help to improve combination therapies aimed at enhancing treatment response.