Altered TFEB subcellular localization in nigral neurons of subjects with incidental, sporadic and GBA-related Lewy body diseases.

Transcription factor EB (TFEB) is a master regulator of genes involved in the maintenance of autophagic and lysosomal homeostasis, processes which have been implicated in the pathogenesis of GBA-related and sporadic Parkinson's disease (PD), and dementia with Lewy bodies (DLB). TFEB activation results in its translocation from the cytosol to the nucleus. Here, we investigated TFEB subcellular localization and its relation to intracellular alpha-synuclein (aSyn) accumulation in post-mortem human brain of individuals with either incidental Lewy body disease (iLBD), GBA-related PD/DLB (GBA-PD/DLB) or sporadic PD/DLB (sPD/DLB), compared to control subjects. We analyzed nigral dopaminergic neurons using high-resolution confocal and stimulated emission depletion (STED) microscopy and semi-quantitatively scored the TFEB subcellular localization patterns. We observed reduced nuclear TFEB immunoreactivity in PD/DLB patients compared to controls, both in sporadic and GBA-related cases, as well as in iLBD cases. Nuclear depletion of TFEB was more pronounced in neurons with Ser129-phosphorylated (pSer129) aSyn accumulation in all groups. Importantly, we observed previously-unidentified TFEB-immunopositive perinuclear clusters in human dopaminergic neurons, which localized at the Golgi apparatus. These TFEB clusters were more frequently observed and more severe in iLBD, sPD/DLB and GBA-PD/DLB compared to controls, particularly in pSer129 aSyn-positive neurons, but also in neurons lacking detectable aSyn accumulation. In aSyn-negative cells, cytoplasmic TFEB clusters were more frequently observed in GBA-PD/DLB and iLBD patients, and correlated with reduced GBA enzymatic activity as well as increased Braak LB stage. Altered TFEB distribution was accompanied by a reduction in overall mRNA expression levels of selected TFEB-regulated genes, indicating a possible early dysfunction of lysosomal regulation. Overall, we observed cytoplasmic TFEB retention and accumulation at the Golgi in cells without apparent pSer129 aSyn accumulation in iLBD and PD/DLB patients. This suggests potential TFEB impairment at the early stages of cellular disease and underscores TFEB as a promising therapeutic target for synucleinopathies.

Dimorphic effect of TFE3 in determining mitochondrial and lysosomal content in muscle following denervation.

BACKGROUND: Muscle atrophy is a common consequence of the loss of innervation and is accompanied by mitochondrial dysfunction. Mitophagy is the adaptive process through which damaged mitochondria are removed via the lysosomes, which are regulated in part by the transcription factor TFE3. The role of lysosomes and TFE3 are poorly understood in muscle atrophy, and the effect of biological sex is widely underreported. METHODS: Wild-type (WT) mice, along with mice lacking TFE3 (KO), a transcriptional regulator of lysosomal and autophagy-related genes, were subjected to unilateral sciatic nerve denervation for up to 7 days, while the contralateral limb was sham-operated and served as an internal control. A subset of animals was treated with colchicine to capture mitophagy flux. RESULTS: WT females exhibited elevated oxygen consumption rates during active respiratory states compared to males, however this was blunted in the absence of TFE3. Females exhibited higher mitophagy flux rates and greater lysosomal content basally compared to males that was independent of TFE3 expression. Following denervation, female mice exhibited less muscle atrophy compared to male counterparts. Intriguingly, this sex-dependent muscle sparing was lost in the absence of TFE3. Denervation resulted in 45% and 27% losses of mitochondrial content in WT and KO males respectively, however females were completely protected against this decline. Decreases in mitochondrial function were more severe in WT females compared to males following denervation, as ROS emission was 2.4-fold higher. In response to denervation, LC3-II mitophagy flux was reduced by 44% in females, likely contributing to the maintenance of mitochondrial content and elevated ROS emission, however this response was dysregulated in the absence of TFE3. While both males and females exhibited increased lysosomal content following denervation, this response was augmented in females in a TFE3-dependent manner. CONCLUSIONS: Females have higher lysosomal content and mitophagy flux basally compared to males, likely contributing to the improved mitochondrial phenotype. Denervation-induced mitochondrial adaptations were sexually dimorphic, as females preferentially preserve content at the expense of function, while males display a tendency to maintain mitochondrial function. Our data illustrate that TFE3 is vital for the sex-dependent differences in mitochondrial function, and in determining the denervation-induced atrophy phenotype.

Genome-wide identification of ZmMYC2 binding sites and target genes in maize.

BACKGROUND: Jasmonate (JA) is the important phytohormone to regulate plant growth and adaption to stress signals. MYC2, an bHLH transcription factor, is the master regulator of JA signaling. Although MYC2 in maize has been identified, its function remains to be clarified. RESULTS: To understand the function and regulatory mechanism of MYC2 in maize, the joint analysis of DAP-seq and RNA-seq is conducted to identify the binding sites and target genes of ZmMYC2. A total of 3183 genes are detected both in DAP-seq and RNA-seq data, potentially as the directly regulating genes of ZmMYC2. These genes are involved in various biological processes including plant growth and stress response. Besides the classic cis-elements like the G-box and E-box that are bound by MYC2, some new motifs are also revealed to be recognized by ZmMYC2, such as nGCATGCAnn, AAAAAAAA, CACGTGCGTGCG. The binding sites of many ZmMYC2 regulating genes are identified by IGV-sRNA. CONCLUSIONS: All together, abundant target genes of ZmMYC2 are characterized with their binding sites, providing the basis to construct the regulatory network of ZmMYC2 and better understanding for JA signaling in maize.

Liver ChREBP deficiency inhibits fructose-induced insulin resistance in pregnant mice and female offspring.

High fructose intake during pregnancy increases insulin resistance (IR) and gestational diabetes mellitus (GDM) risk. IR during pregnancy primarily results from elevated hormone levels. We aim to determine the role of liver carbohydrate response element binding protein (ChREBP) in insulin sensitivity and lipid metabolism in pregnant mice and their offspring. Pregnant C57BL/6J wild-type mice and hepatocyte-specific ChREBP-deficient mice were fed with a high-fructose diet (HFrD) or normal chow diet (NC) pre-delivery. We found that the combination of HFrD with pregnancy excessively activates hepatic ChREBP, stimulating progesterone synthesis by increasing MTTP expression, which exacerbates IR. Increased progesterone levels upregulated hepatic ChREBP via the progesterone-PPARγ axis. Placental progesterone activated the progesterone-ChREBP loop in female offspring, contributing to IR and lipid accumulation. In normal dietary conditions, hepatic ChREBP modestly affected progesterone production and influenced IR during pregnancy. Our findings reveal the role of hepatic ChREBP in regulating insulin sensitivity and lipid homeostasis in both pregnant mice consuming an HFrD and female offspring, and suggest it as a potential target for managing gestational metabolic disorders, including GDM.

LncRNA XXYLT1-AS2 promotes tumor progression via autophagy inhibition through ubiquitinated degradation of TFEB in hepatocellular carcinoma

Purpose There is compelling evidence that long-stranded non-coding RNAs (lncRNAs) play an important role in the progression of hepatocellular carcinoma (HCC). The aim of this study was to investigate the role of lncRNA XXYLT1 antisense-2 (XXYLT1-AS2) in HCC progression. Methods Real-time PCR was used to assess the levels of XXYLT1-AS2 in plasma from HCC and normal patients. Cell proliferation, apoptosis, migration, and invasion were monitored, and tumor xenografts were established to investigate the biological functions of XXYLT1-AS2 by gain-of-function and loss-of-function studies in vitro and in vivo, the expression of autophagy biomarkers and transcriptional factor EB (TFEB) was examined by immunoprecipitation, ubiquitination assays, and western blotting. Autophagy inhibitor, 3-methyladenine (3MA), and proteasome inhibitor, MG132, were used to verify the role of autophagy in HCC progression and the effect of XXYLT1-AS2 on TFEB ubiquitination, respectively. Results In this study, we identified that lncRNA XXYLT1-AS2 is highly expressed in HCC plasma and promotes tumor growth in vivo. In functional studies, it was found that silent expression of XXYLT1-AS2 inhibited HCC proliferation, migration, invasion, and activated autophagy of HCC cells, which were attenuated by autophagy inhibitor, 3MA. Mechanistically, XXYLT1-AS2 decreased the protein level of TFEB through promoting its degradation by ubiquitin proteasome pathway. Conclusion XXYLT1-AS2 plays an oncogenic role in HCC progression through inhibition of autophagy via promoting the degradation of TFEB, and thus could be a novel target for HCC treatment (AU)

Impaired TFEB-mediated autophagy-lysosome fusion promotes tubular cell cycle G2/M arrest and renal fibrosis by suppressing ATP6V0C expression and interacting with SNAREs.

Increasing evidence suggests that autophagy plays a major role during renal fibrosis. Transcription factor EB (TFEB) is a critical regulator of autophagy- and lysosome-related gene transcription. However, the pathophysiological roles of TFEB in renal fibrosis and fine-tuned mechanisms by which TFEB regulates fibrosis remain largely unknown. Here, we found that TFEB was downregulated in unilateral ureteral obstruction (UUO)-induced human and mouse fibrotic kidneys, and kidney-specific TFEB overexpression using recombinant AAV serotype 9 (rAAV9)-TFEB in UUO mice alleviated renal fibrosis pathogenesis. Mechanically, we found that TFEB's prevention of extracellular matrix (ECM) deposition depended on autophagic flux integrity and its subsequent blockade of G2/M arrest in tubular cells, rather than the autophagosome synthesis. In addition, we together RNA-seq with CUT&Tag analysis to determine the TFEB targeted gene ATP6V0C, and revealed that TFEB was directly bound to the ATP6V0C promoter only at specific site to promote its expression through CUT&Run-qPCR and luciferase reporter assay. Interestingly, TFEB induced autophagic flux integrity, mainly dependent on scaffold protein ATP6V0C-mediated autophagosome-lysosome fusion by bridging with STX17 and VAMP8 (major SNARE complex) by co-immunoprecipitation analysis, rather than its mediated lysosomal acidification and degradation function. Moreover, we further investigated the underlying mechanism behind the low expression of TEFB in UUO-induced renal fibrosis, and clearly revealed that TFEB suppression in fibrotic kidney was due to DNMT3a-associated TFEB promoter hypermethylation by utilizing methylation specific PCR (MSP) and bisulfite-sequencing PCR (BSP), which could be effectively recovered by 5-Aza-2'-deoxycytidine (5A-za) to alleviate renal fibrosis pathogenesis. These findings reveal for the first time that impaired TFEB-mediated autophagosome-lysosome fusion disorder, tubular cell G2/M arrest and renal fibrosis appear to be sequentially linked in UUO-induced renal fibrosis and suggest that DNMT3a/TFEB/ATP6V0C may serve as potential therapeutic targets to prevent renal fibrosis.

Comparative genomics incorporating translocation renal cell carcinoma mouse model reveals molecular mechanisms of tumorigenesis.

Translocation renal cell carcinoma (tRCC) most commonly involves an ASPSCR1-TFE3 fusion, but molecular mechanisms remain elusive and animal models are lacking. Here, we show that human ASPSCR1-TFE3 driven by Pax8-Cre (a credentialed clear cell RCC driver) disrupted nephrogenesis and glomerular development, causing neonatal death, while the clear cell RCC failed driver, Sglt2-Cre, induced aggressive tRCC (as well as alveolar soft part sarcoma) with complete penetrance and short latency. However, in both contexts, ASPSCR1-TFE3 led to characteristic morphological cellular changes, loss of epithelial markers, and an epithelial-mesenchymal transition. Electron microscopy of tRCC tumors showed lysosome expansion, and functional studies revealed simultaneous activation of autophagy and mTORC1 pathways. Comparative genomic analyses encompassing an institutional human tRCC cohort (including a hitherto unreported SFPQ-TFEB fusion) and a variety of tumorgraft models (ASPSCR1-TFE3, PRCC-TFE3, SFPQ-TFE3, RBM10-TFE3, and MALAT1-TFEB) disclosed significant convergence in canonical pathways (cell cycle, lysosome, and mTORC1) and less established pathways such as Myc, E2F, and inflammation (IL-6/JAK/STAT3, interferon-γ, TLR signaling, systemic lupus, etc.). Therapeutic trials (adjusted for human drug exposures) showed antitumor activity of cabozantinib. Overall, this study provides insight into MiT/TFE-driven tumorigenesis, including the cell of origin, and characterizes diverse mouse models available for research.

Evaluation of TRIM63 RNA in situ hybridization (RNA-ISH) as a potential biomarker for alveolar soft-part sarcoma (ASPS).

Alveolar soft-part sarcoma (ASPS) is a rare soft tissue tumor with a broad morphologic differential diagnosis. While histology and immunohistochemistry can be suggestive, diagnosis often requires exclusion of other entities followed by confirmatory molecular analysis for its characteristic ASPSCR1-TFE3 fusion. Current stain-based biomarkers (such as immunohistochemistry for cathepsin K and TFE3) show relatively high sensitivity but may lack specificity, often showing staining in multiple other entities under diagnostic consideration. Given the discovery of RNA in situ hybridization (RNA-ISH) for TRIM63 as a sensitive and specific marker of MiTF-family aberration renal cell carcinomas, we sought to evaluate its utility in the workup of ASPS. TRIM63 RNA-ISH demonstrated high levels (H-score greater than 200) of expression in 19/20 (95%) cases of ASPS (average H-score 330) and was weak or negative in cases of paraganglioma, clear cell sarcoma, rhabdomyosarcoma, malignant epithelioid hemangioendothelioma, as well as hepatocellular and adrenal cortical carcinomas. Staining was also identified in tumors with known subsets characterized by TFE3 alterations such as perivascular epithelioid cell neoplasm (PEComa, average H-score 228), while tumors known to exhibit overexpression of TFE3 protein without cytogenetic alterations, such as melanoma and granular cell tumor, generally showed less TRIM63 ISH staining (average H-scores 147 and 96, respectively). Quantitative assessment of TRIM63 staining by RNA-ISH is potentially a helpful biomarker for tumors with molecular TFE3 alterations such as ASPS.

Cathelicidin LL-37 promotes wound healing in diabetic mice by regulating TFEB-dependent autophagy.

Diabetic patients often experience impaired wound healing. Human cathelicidin LL-37 possesses various biological functions, such as anti-microbial, anti-inflammatory, and pro-wound healing activities. Autophagy has important effects on skin wound healing. However, little is known about whether LL-37 accelerates diabetic wound healing by regulating autophagy. In the study, we aimed to investigate the role of autophagy in LL-37-induced wound healing and uncover the underlying mechanisms involved. A full-thickness wound closure model was established in diabetic mice to evaluate the effects of LL-37 and an autophagy inhibitor (3-MA) on wound healing. The roles of LL-37 and 3-MA in regulating keratinocyte migration were assessed using transwell migration and wound healing assays. The activation of transcription factor EB (TFEB) was measured using western blotting and immunofluorescence (IF) assays of its nuclear translocation. The results showed that LL-37 treatment improved wound healing in diabetic mice, whereas these effects were reversed by 3-MA. In vitro, 3-MA decreased the effects of LL-37 on promoting HaCat keratinocyte migration in the presence of high glucose (HG). Mechanistically, LL-37 promoted TFEB activation and resulted in subsequent activation of autophagy, as evidenced by increased nuclear translocation of TFEB and increased expression of ATG5, ATG7, and beclin 1 (BECN1), whereas these changes were blocked by TFEB knockdown. As expected, TFEB knockdown damaged the effects of LL-37 on promoting keratinocyte migration. Collectively, these results suggest that LL-37 accelerates wound healing in diabetic mice by activating TFEB-dependent autophagy, providing new insights into the mechanism by which LL-37 promotes diabetic wound healing.

The MYC2-PUB22-JAZ4 module plays a crucial role in jasmonate signaling in tomato.

Jasmonates (JAs), a class of lipid-derived stress hormones, play a crucial role across an array of plant physiological processes and stress responses. Although JA signaling is thought to rely predominantly on the degradation of specific JAZ proteins by SCFCOI1, it remains unclear whether other pathways are involved in the regulation of JAZ protein stability. Here, we report that PUB22, a plant U-box type E3 ubiquitin ligase, plays a critical role in the regulation of plant resistance against Helicoverpa armigera and other JA responses in tomato. Whereas COI1 physically interacts with JAZ1/2/5/7, PUB22 physically interacts with JAZ1/3/4/6. PUB22 ubiquitinates JAZ4 to promote its degradation via the 26S proteasome pathway. Importantly, we observed that pub22 mutants showreduced resistance to H. armigera, whereas jaz4 single mutants and jaz1 jaz3 jaz4 jaz6 quadruple mutants have enhanced resistance. The hypersensitivity of pub22 mutants to herbivores could be partially rescued by JAZ4 mutation. Moreover, we found that expression of PUB22 can be transcriptionally activated by MYC2, thus forming a positive feedback circuit in JA signaling. We noticed that the PUB22-JAZ4 module also regulates other JA responses, including defense against B. cinerea, inhibition of root elongation, and anthocyanin accumulation. Taken together, these results indicate that PUB22 plays a crucial role in plant growth and defense responses, together with COI1-regulated JA signaling, by targeting specific JAZs.

A SPLICS reporter reveals [Formula: see text]-synuclein regulation of lysosome-mitochondria contacts which affects TFEB nuclear translocation.

Mitochondrial and lysosomal activities are crucial to maintain cellular homeostasis: optimal coordination is achieved at their membrane contact sites where distinct protein machineries regulate organelle network dynamics, ions and metabolites exchange. Here we describe a genetically encoded SPLICS reporter for short- and long- juxtapositions between mitochondria and lysosomes. We report the existence of narrow and wide lysosome-mitochondria contacts differently modulated by mitophagy, autophagy and genetic manipulation of tethering factors. The overexpression of α-synuclein (α-syn) reduces the apposition of mitochondria/lysosomes membranes and affects their privileged Ca2+ transfer, impinging on TFEB nuclear translocation. We observe enhanced TFEB nuclear translocation in α-syn-overexpressing cells. We propose that α-syn, by interfering with mitochondria/lysosomes tethering impacts on local Ca2+ regulated pathways, among which TFEB mediated signaling, and in turn mitochondrial and lysosomal function. Defects in mitochondria and lysosome represent a common hallmark of neurodegenerative diseases: targeting their communication could open therapeutic avenues.

The GATOR2 complex maintains lysosomal-autophagic function by inhibiting the protein degradation of MiT/TFEs.

Lysosomes are central to metabolic homeostasis. The microphthalmia bHLH-LZ transcription factors (MiT/TFEs) family members MITF, TFEB, and TFE3 promote the transcription of lysosomal and autophagic genes and are often deregulated in cancer. Here, we show that the GATOR2 complex, an activator of the metabolic regulator TORC1, maintains lysosomal function by protecting MiT/TFEs from proteasomal degradation independent of TORC1, GATOR1, and the RAG GTPase. We determine that in GATOR2 knockout HeLa cells, members of the MiT/TFEs family are ubiquitylated by a trio of E3 ligases and are degraded, resulting in lysosome dysfunction. Additionally, we demonstrate that GATOR2 protects MiT/TFE proteins in pancreatic ductal adenocarcinoma and Xp11 translocation renal cell carcinoma, two cancers that are driven by MiT/TFE hyperactivation. In summary, we find that the GATOR2 complex has independent roles in TORC1 regulation and MiT/TFE protein protection and thus is central to coordinating cellular metabolism with control of the lysosomal-autophagic system.

ASPSCR1-TFE3 reprograms transcription by organizing enhancer loops around hexameric VCP/p97.

The t(X,17) chromosomal translocation, generating the ASPSCR1::TFE3 fusion oncoprotein, is the singular genetic driver of alveolar soft part sarcoma (ASPS) and some Xp11-rearranged renal cell carcinomas (RCCs), frustrating efforts to identify therapeutic targets for these rare cancers. Here, proteomic analysis identifies VCP/p97, an AAA+ ATPase with known segregase function, as strongly enriched in co-immunoprecipitated nuclear complexes with ASPSCR1::TFE3. We demonstrate that VCP is a likely obligate co-factor of ASPSCR1::TFE3, one of the only such fusion oncoprotein co-factors identified in cancer biology. Specifically, VCP co-distributes with ASPSCR1::TFE3 across chromatin in association with enhancers genome-wide. VCP presence, its hexameric assembly, and its enzymatic function orchestrate the oncogenic transcriptional signature of ASPSCR1::TFE3, by facilitating assembly of higher-order chromatin conformation structures demonstrated by HiChIP. Finally, ASPSCR1::TFE3 and VCP demonstrate co-dependence for cancer cell proliferation and tumorigenesis in vitro and in ASPS and RCC mouse models, underscoring VCP's potential as a novel therapeutic target.

Berberine Alleviates Ischemic Brain Injury by Enhancing Autophagic Flux via Facilitation of TFEB Nuclear Translocation.

Berberine has been demonstrated to alleviate cerebral ischemia/reperfusion injury, but its neuroprotective mechanism has yet to be understood. Studies have indicated that ischemic neuronal damage was frequently driven by autophagic/lysosomal dysfunction, which could be restored by boosting transcription factor EB (TFEB) nuclear translocation. Therefore, this study investigated the pharmacological effects of berberine on TFEB-regulated autophagic/lysosomal signaling in neurons after cerebral stroke. A rat model of ischemic stroke and a neuronal ischemia model in HT22 cells were prepared using middle cerebral artery occlusion (MCAO) and oxygen-glucose deprivation (OGD), respectively. Berberine was pre-administered at a dose of 100[Formula: see text]mg/kg/d for three days in rats and 90[Formula: see text][Formula: see text]M in HT22 neurons for 12[Formula: see text]h. 24[Formula: see text]h after MCAO and 2[Formula: see text]h after OGD, the penumbral tissues and OGD neurons were obtained to detect nuclear and cytoplasmic TFEB, and the key proteins in the autophagic/lysosomal pathway were examined using western blot and immunofluorescence, respectively. Meanwhile, neuron survival, infarct volume, and neurological deficits were assessed to evaluate the therapeutic efficacy. The results showed that berberine prominently facilitated TFEB nuclear translocation, as indicated by increased nuclear expression in penumbral neurons as well as in OGD HT22 cells. Consequently, both autophagic activity and lysosomal capacity were simultaneously augmented to alleviate the ischemic injury. However, berberine-conferred neuroprotection could be greatly counteracted by lysosomal inhibitor Bafilomycin A1 (Baf-A1). Meanwhile, autophagy inhibitor 3-Methyladenine (3-MA) also slightly neutralized the pharmacological effect of berberine on ameliorating autophagic/lysosomal dysfunction. Our study suggests that berberine-induced neuroprotection against ischemic stroke is elicited by enhancing autophagic flux via facilitation of TFEB nuclear translocation in neurons.

Advanced oxidation protein products attenuate the autophagy-lysosome pathway in ovarian granulosa cells by modulating the ROS-dependent mTOR-TFEB pathway.

Oxidative stress dysfunction has recently been found to be involved in the pathogenesis of premature ovarian insufficiency (POI). Previously, we found that advanced oxidation protein products (AOPPs) in plasma were elevated in women with POI and had an adverse effect on granulosa cell proliferation. However, the mechanism underlying the effects of AOPPs on autophagy-lysosome pathway regulation in granulosa cells remains unclear. In this study, the effect of AOPPs on autophagy and lysosomal biogenesis and the underlying mechanisms were explored by a series of in vitro experiments in KGN and COV434 cell lines. AOPP-treated rat models were employed to determine the negative effect of AOPPs on the autophagy-lysosome systems in vivo. We found that increased AOPP levels activated the mammalian target of rapamycin (mTOR) pathway, and inhibited the autophagic response and lysosomal biogenesis in KGN and COV434 cells. Furthermore, scavenging of reactive oxygen species (ROS) with N-acetylcysteine and blockade of the mTOR pathway with rapamycin or via starvation alleviated the AOPP-induced inhibitory effects on autophagy and lysosomal biogenesis, suggesting that these effects of AOPPs are ROS-mTOR dependent. The protein expression and nuclear translocation of transcription factor EB (TFEB), the key regulator of lysosomal and autophagic function, were also impaired by the AOPP-activated ROS-mTOR pathway. In addition, TFEB overexpression attenuated the AOPP-induced impairment of autophagic flux and lysosomal biogenesis in KGN and COV434 cells. Chronic AOPP stimulation in vivo also impaired autophagy and lysosomal biogenesis in granulosa cells of rat ovaries. The results highlight that AOPPs lead to impairment of autophagic flux and lysosomal biogenesis via ROS-mTOR-TFEB signaling in granulosa cells and participate in the pathogenesis of POI.

Epithelioid hemangioendothelioma (EHE) with WWTR1::TFE3 gene fusion, a novel fusion variant.

Epithelioid hemangioendothelioma (EHE) is a rare endothelial sarcoma associated with a high incidence of metastases and for which there are no standard treatment options. Based on disease-defining mutations, most EHEs are classified into two subtypes: WWTR1::CAMTA1-fused EHE or YAP1::TFE3-fused EHE. However, rare non-canonical fusions have been identified in clinical samples of EHE cases and are challenging to classify. In this study, we report the identification of a novel WWTR1::TFE3 fusion variant in an EHE patient using targeted RNA sequencing. Histologically, the tumor exhibited hybrid morphological characteristics between WWTR1::CAMTA1-fused EHE and YAP1::TFE3-fused EHE. In addition to the driver fusion, there were six additional secondary mutations identified, including a loss-of-function FANCA mutation. Furthermore, in vitro studies were conducted to investigate the tumorigenic function of the WWTR1::TFE3 fusion protein in NIH3T3 cells and demonstrated that WWTR1::TFE3 promotes colony formation in soft agar. Finally, as the wild-type WWTR1 protein relies on binding the TEAD family of transcription factors to affect gene transcription, mutation of the WWTR1 domain of the fusion protein to inhibit such binding abrogates the transformative effect of WWTR1::TFE3. Overall, we describe a novel gene fusion in EHE with a hybrid histological appearance between the two major genetic subtypes of EHE. Further cases of this very rare subtype of EHE will need to be identified to fully elucidate the clinical and pathological characteristics of this unusual subtype of EHE.

MYC the oncogene from hell: Novel opportunities for cancer therapy.

Cancer comprises a heterogeneous disease, characterized by diverse features such as constitutive expression of oncogenes and/or downregulation of tumor suppressor genes. MYC constitutes a master transcriptional regulator, involved in many cellular functions and is aberrantly expressed in more than 70 % of human cancers. The Myc protein belongs to a family of transcription factors whose structural pattern is referred to as basic helix-loop-helix-leucine zipper. Myc binds to its partner, a smaller protein called Max, forming an Myc:Max heterodimeric complex that interacts with specific DNA recognition sequences (E-boxes) and regulates the expression of downstream target genes. Myc protein plays a fundamental role for the life of a cell, as it is involved in many physiological functions such as proliferation, growth and development since it controls the expression of a very large percentage of genes (∼15 %). However, despite the strict control of MYC expression in normal cells, MYC is often deregulated in cancer, exhibiting a key role in stimulating oncogenic process affecting features such as aberrant proliferation, differentiation, angiogenesis, genomic instability and oncogenic transformation. In this review we aim to meticulously describe the fundamental role of MYC in tumorigenesis and highlight its importance as an anticancer drug target. We focus mainly on the different categories of novel small molecules that act as inhibitors of Myc function in diverse ways hence offering great opportunities for an efficient cancer therapy. This knowledge will provide significant information for the development of novel Myc inhibitors and assist to the design of treatments that would effectively act against Myc-dependent cancers.

ABCG2 is an itaconate exporter that limits antibacterial innate immunity by alleviating TFEB-dependent lysosomal biogenesis.

Itaconate is a metabolite that synthesized from cis-aconitate in mitochondria and transported into the cytosol to exert multiple regulatory effects in macrophages. However, the mechanism by which itaconate exits from macrophages remains unknown. Using a genetic screen, we reveal that itaconate is exported from cytosol to extracellular space by ATP-binding cassette transporter G2 (ABCG2) in an ATPase-dependent manner in human and mouse macrophages. Elevation of transcription factor TFEB-dependent lysosomal biogenesis and antibacterial innate immunity are observed in inflammatory macrophages with deficiency of ABCG2-mediated itaconate export. Furthermore, deficiency of ABCG2-mediated itaconate export in macrophages promotes antibacterial innate immune defense in a mouse model of S. typhimurium infection. Thus, our findings identify ABCG2-mediated itaconate export as a key regulatory mechanism that limits TFEB-dependent lysosomal biogenesis and antibacterial innate immunity in inflammatory macrophages, implying the potential therapeutic utility of blocking itaconate export in treating human bacterial infections.

Glycoprotein Nonmetastatic Melanoma Protein B (GPNMB) Immunohistochemistry Can Be a Useful Ancillary Tool to Identify Perivascular Epithelioid Cell Tumor.

Perivascular epithelioid cell tumors (PEComas) are rare mesenchymal tumors that express smooth muscle and melanocytic makers. Diagnosis of PEComas can be challenging due to focal or lost expression of traditional immunohistochemical markers, limited availability of molecular testing, and morphological overlap with much more common smooth muscle tumors. This study evaluates the use of glycoprotein nonmetastatic melanoma protein B (GPNMB) immunohistochemical staining as a surrogate marker for TSC1/2/MTOR alteration or TFE3 rearrangement to differentiate PEComas from other mesenchymal tumors. Cathepsin K was also assessed for comparison. A total of 399 tumors, including PEComas, alveolar soft part sarcomas, and other histologic PEComa mimics, were analyzed using GPNMB and cathepsin K immunohistochemistry. GPNMB expression was seen in all PEComas and alveolar soft part sarcomas with the majority showing diffuse and moderate-to-strong labeling, whereas other sarcomas were negative or showed focal labeling. When a cutoff of diffuse and at least moderate staining was used, GPNMB demonstrated 95% sensitivity and 97% specificity in distinguishing PEComas from leiomyosarcoma, well-differentiated/dedifferentiated liposarcomas, and undifferentiated pleomorphic sarcomas. Cathepsin K with a cutoff of any labeling had lower sensitivity (78%) and similar specificity (94%) to GPNMB. This study highlights GPNMB as a highly sensitive marker for PEComas and suggests its potential use as an ancillary tool within a panel of markers for accurate classification of these tumors.

Mitochondrial translocation of TFEB regulates complex I and inflammation.

TFEB is a master regulator of autophagy, lysosome biogenesis, mitochondrial metabolism, and immunity that works primarily through transcription controlled by cytosol-to-nuclear translocation. Emerging data indicate additional regulatory interactions at the surface of organelles such as lysosomes. Here we show that TFEB has a non-transcriptional role in mitochondria, regulating the electron transport chain complex I to down-modulate inflammation. Proteomics analysis reveals extensive TFEB co-immunoprecipitation with several mitochondrial proteins, whose interactions are disrupted upon infection with S. Typhimurium. High resolution confocal microscopy and biochemistry confirms TFEB localization in the mitochondrial matrix. TFEB translocation depends on a conserved N-terminal TOMM20-binding motif and is enhanced by mTOR inhibition. Within the mitochondria, TFEB and protease LONP1 antagonistically co-regulate complex I, reactive oxygen species and the inflammatory response. Consequently, during infection, lack of TFEB specifically in the mitochondria exacerbates the expression of pro-inflammatory cytokines, contributing to innate immune pathogenesis.