TFEB Translocation Research Articles

Lactate Contributes to Remote Ischemic Preconditioning–Mediated Protection Against Myocardial Ischemia Reperfusion Injury by Facilitating Autophagy via the AMP-Activated Protein Kinase–Mammalian Target of Rapamycin–Transcription Factor EB–Connexin 43 Axis

Remote ischemic preconditioning (RIPC) exerts a protective role on myocardial ischemia reperfusion (I/R) injury by the release of various humoral factors. Lactate is a common metabolite in ischemic tissues. Nevertheless, little is known about the role lactate plays in myocardial I/R injury and its underlying mechanism. This investigation revealed that RIPC elevated the level of lactate in blood and myocardium. Furthermore, AZD3965, a selective monocarboxylate transporter 1 (MCT1) inhibitor and 2-Deoxy-D-glucose (2-DG), a glycolysis inhibitor, mitigated the effects of RIPC-induced elevated lactate in the myocardium and prevented RIPC against myocardial I/R injury. In an in vitro hypoxia reoxygenation (H/R) model, lactate markedly mitigated H/R-induced cell damage in H9c2 cells. Meanwhile, further studies suggested that lactate contributed to RIPC rescuing I/R-induced autophagy deficiency by promoting TFEB translocation to the nucleus through activating the AMPK-mTOR pathway without influencing the PI3K-Akt pathway, thus reducing cardiomyocytes damage. Interestingly, we also found that lactate upregulated the mRNA and protein expression of CX43 by facilitating the binding of TFEB to CX43 promoter in the myocardium. Functionally, silencing of TFEB attenuated the protective effect of lactate on cell damage, which was reversed by overexpression of CX43. Further mechanistic studies suggested lactate facilitated CX43-regulated autophagy via AMPK-mTOR-TFEB signaling pathway. Collectively, our research demonstrates that RIPC protects against myocardial I/R injury through lactate-mediated myocardial autophagy via AMPK-mTOR-TFEB-CX43 axis.

A potential early-atheroprotective target: Irgm1 mediates lymphangiogenesis through LEC autophagy by Tfeb translocation

Lymphatic dysfunction is a pivotal pathological mechanism underlying the development of early atherosclerotic plaques. Potential targets of lymphatic function must be identified to realize the early prevention and treatment of atherosclerosis (AS). The immunity-related GTPase Irgm1 is involved in orchestrating cellular autophagy and apoptosis. However, the effect of Irgm1 on early AS progression, particularly through alterations in lymphatic function, remains unclear. In this study, we confirmed the protective effect of lymphangiogenesis on early-AS in vivo. Subsequently, an in vivo model of early AS mice with Irgm1 knockdown shows that Irgm1 reduces early atherosclerotic plaque burden by promoting lymphangiogenesis. Given that lymphatic endothelial cell (LEC) autophagy significantly contributes to lymphangiogenesis, Irgm1 may enhance lymphatic circulation by promoting LEC autophagy. Moreover, Irgm1 orchestrates autophagy in LECs by inhibiting mTOR and facilitating nuclear translocation of Tfeb. Collectively, these processes lead to lymphangiogenesis. Thus, this study establishes a link between Irgm1 and early AS, thus revealing a novel mechanism by which Irgm1 exerts an early protective influence on AS within the context of lymphatic circulation. The insights gained from this study have the potential to revolutionize the approach and management of AS onset.

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.

Fluvoxamine Exerts Sigma-1R to Rescue Autophagy via Pom121-Mediated Nucleocytoplasmic Transport of TFEB

Expansion of the GGGGCC-RNA repeat is a known cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), which currently have no cure. Recent studies have indicated the activation of Sigma-1 receptor plays an important role in providing neuroprotection, especially in ALS and Alzheimer’s disease. Nevertheless, the mechanisms underlying Sigma-1R activation and its effect on (G4C2)n-RNA-induced cell death remain unclear. In this study, we demonstrated that fluvoxamine is a Sigma-1R agonist that can increase chaperone activity and stabilize the protein expression of Pom121 in (G4C2)31-RNA-expressing NSC34 cells, leading to increased colocalization at the nuclear envelope. Interestingly, fluvoxamine treatment increased Pom121 protein expression without affecting transcription. In C9orf72-ALS, the nuclear translocation of TFEB autophagy factor decreased owing to nucleocytoplasmic transport defects. Our results showed that pretreatment of NSC34 cells with fluvoxamine promoted the shuttling of TFEB into the nucleus and elevated the expression of LC3-II compared to the overexpression of (G4C2)31-RNA alone. Additionally, even when used alone, fluvoxamine increases Pom121 expression and TFEB translocation. To summarize, fluvoxamine may act as a promising repurposed medicine for patients with C9orf72-ALS, as it stabilizes the nucleoporin Pom121 and promotes the translocation of TFEB in (G4C2)31-RNA-expressing NSC34 cells.

Increased DNMT1 Involvement in the Activation of LO2 Cell Death Induced by Silver Nanoparticles via Promoting TFEB-Dependent Autophagy.

The accumulation of exogenous silver nanoparticles (AgNPs) will terminally bring about liver injury, including cell death, where DNA methylation tends to be a crucial epigenetic modulator. The change in the cell autophagy level verified to be closely associated with hepatocyte death has been followed with wide interest. But the molecular toxicological mechanisms of AgNPs in relation to DNA methylation, autophagy, and cell death remain inconclusive. To address the issue above, in LO2 cells treated with increasing concentrations of AgNPs (0, 5, 10, and 20 μg/mL), a cell cytotoxicity assay was performed to analyze the level of cell death, which also helped to choose an optimal concentration for next experiments. An immunofluorescence assay was used to determine the autophagic flux as well as TFEB translocation, with qRT-PCR and western blot being used to analyze the expression level of autophagy-related genes and proteins. According to our findings, in the determination of cell viability, 20 μg/mL (AgNPs) was adopted as the best working concentration. LO2 cell death, autophagy, and TFEB nuclear translocation were induced by AgNPs, which could be inhibited by lysosome inhibitor chloroquine (CQ) or siRNA specific for TFEB. Moreover, AgNP exposure led to DNA hypermethylation, with DNMT1 taking part mainly, which could be obviously prevented by 5-Aza-2'-deoxycytidine (5-AzaC) or trichostatin A (TSA) treatment or DNMT1 knockout in LO2 cells. Our studies suggest that through TFEB-dependent cell autophagy, increased DNMT1 may facilitate cell death induced by AgNPs.

Functional TFEB activation characterizes multiple models of renal cystic disease and loss of polycystin-1.

Polycystic kidney disease is a disorder of renal epithelial growth and differentiation. Transcription factor EB (TFEB), a master regulator of lysosome biogenesis and function, was studied for a potential role in this disorder. Nuclear translocation and functional responses to TFEB activation were studied in three murine models of renal cystic disease, including knockouts of folliculin, folliculin interacting proteins 1 and 2, and polycystin-1 (Pkd1) as well as in mouse embryonic fibroblasts lacking Pkd1 and three-dimensional cultures of Madin-Darby canine kidney cells. Nuclear translocation of Tfeb characterized cystic but not noncystic renal tubular epithelia in all three murine models as both an early and sustained response to cyst formation. Epithelia expressed elevated levels of Tfeb-dependent gene products, including cathepsin B and glycoprotein nonmetastatic melanoma protein B. Nuclear Tfeb translocation was observed in mouse embryonic fibroblasts lacking Pkd1 but not wild-type fibroblasts. Pkd1 knockout fibroblasts were characterized by increased Tfeb-dependent transcripts, lysosomal biogenesis and repositioning, and increased autophagy. The growth of Madin-Darby canine kidney cell cysts was markedly increased following exposure to the TFEB agonist compound C1, and nuclear Tfeb translocation was observed in response to both forskolin and compound C1 treatment. Nuclear TFEB also characterized cystic epithelia but not noncystic tubular epithelia in human patients with autosomal dominant polycystic kidney disease. Noncanonical activation of TFEB is characteristic of cystic epithelia in multiple models of renal cystic disease including those associated with loss of Pkd1. Nuclear TFEB translocation is functionally active in these models and may be a component of a general pathway contributing to cystogenesis and growth.NEW & NOTEWORTHY Changes in epithelial cell metabolism are important in renal cyst development. The role of TFEB, a transcriptional regulator of lysosomal function, was explored in several models of renal cystic disease and human ADPKD tissue sections. Nuclear TFEB translocation was uniformly observed in cystic epithelia in each model of renal cystic disease examined. TFEB translocation was functionally active and associated with lysosomal biogenesis and perinuclear repositioning, increased TFEB-associated protein expression, and activation of autophagic flux. Compound C1, a TFEB agonist, promoted cyst growth in 3-D cultures of MDCK cells. Nuclear TFEB translocation is an underappreciated signaling pathway for cystogenesis that may represent a new paradigm for cystic kidney disease.

Comprehensive study of nine novel cases of TFEB-amplified renal cell carcinoma: an aggressive tumour with frequent PDL1 expression.

First described in 2014, renal cell carcinoma (RCC) with TFEB amplification (6p21) is a rare molecular subgroup whose diagnosis is challenging. The prognosis and therapeutic implications remain unclear. We report here the clinical, histological, immunohistochemical, and genetic features of nine novel cases. The pathological and immunohistochemical features were centrally reviewed by expert uropathologists. Fluorescence in situ hybridisation (FISH) confirmed the diagnosis and comparative genomic hybridisation (CGH) was performed to determine quantitative genomic alterations. We also performed an exhaustive review of the literature and compiled our data. TFEB-amplified RCC were locally advanced, with initial lymph node involvement in one case and liver metastasis in another case. They were high-grade eosinophilic tumours with papillary/pseudopapillary architecture, frequent positivity for melanocytic markers, and frequent PDL1 expression. FISH demonstrated high-level TFEB amplification in six cases. One case showed concomitant TFEB translocation. CGH analysis identified complex alterations with frequent losses of 1p, 2q, 3p, 6p, and frequent 6p and 8q gains. VEGFA coamplification was identified in all cases with a lower level than TFEB. The prognosis was poor, with five patients having lymph node or distant metastases. TFEB-amplified RCC is a rare molecular subgroup with variable morphology whose diagnosis is confirmed by FISH analysis. The complex alterations identified by CGH are consistent with an aggressive clinical behaviour. The coamplification of VEGFA and the expression of PDL1 could suggest a potential benefit from antiangiogenics and targeted immunotherapy in combination for these aggressive tumours.

Immuno-oncology (IO) combinations +/- VEGF targeted therapy (VEGF TT) in patients (pts) with advanced mit family translocation renal cell carcinomas (tRCC): Results from an international multicenter study.

343 Background: IO combinations are standard of care for first-line therapy of clear-cell RCC; however, non-clear cell histologies including tRCC were not included in the registrational trials. We previously reported a modest efficacy (objective response rate [ORR] <20%) with IO monotherapy (PD-1 blockade) in tRCC (Boilève et al, JITC. 2018). The efficacy of IO combinations +/- VEGF TT has not been reported. Methods: This is a retrospective, international, multicenter study of pts with tRCC treated with IO-IO or IO+VEGF TT at 11 centers in the US, France, and Belgium. Only pts with confirmed TFE3 or TFEB translocation by fluorescent-in-situ hybridization (FISH) were included. ORR and progression-free survival (PFS) were assessed by RECIST. Overall survival (OS) was assessed by Kaplan-Meier methods. OS was measured from initiation of therapy till death or last follow up. We also assessed the association between OS and baseline prognostic variables. Results: 29 patients with metastatic tRCC were included in this analysis. Median age at starting therapy was 38 (IQR 27, 53) years. Female:Male ratio was 0.9:1. FISH revealed a translocation involving TFE3 and TFEB in 22 and 7 patients, respectively. Most frequent metastatic sites at diagnosis were lungs (76%), liver (52%), retroperitoneal adenopathy (48%), and bone (38%). IMDC risk at diagnosis was favorable (31%), intermediate (45%) and poor (24%). Combinations of IO+VEGF TT and anti-PD1 (L1) + anti-CTLA-4 (IO+IO) were used in 11 and 18 pts, respectively. 17 (59%) pts received IO combinations as 1L, 7 (24%) pts as 2L and 5 (17%) pts as ≥3L. ORR in the IO+IO group was 1/18 (5.5%), while in IO+VEGF TT group was 4/11 (36%). For 20 (69%) pts, progressive disease was the best overall response. At a median follow-up of 12.9 months (mo), median PFS was 3.2 mo and median OS was 13.5 mo for all 29 pts (Table). Median PFS in the IO+IO group was 2.8 mo, and 5.4 mo in IO+VEGF TT group (HR=0.81, 95% CI: 0.35-1.84). Conclusions: In this small retrospective study of tRCC, IO+IO therapy produced modest activity based on low ORR and short PFS while IO+VEGF TT produced numerically higher ORR and longer PFS. Insights into the biological basis of tRCC are necessary to develop more effective therapies for this rare and aggressive RCC variant.[Table: see text]

Wild-type GBA1 increases the α-synuclein tetramer–monomer ratio, reduces lipid-rich aggregates, and attenuates motor and cognitive deficits in mice

Loss-of-function mutations in acid beta-glucosidase 1 (GBA1) are among the strongest genetic risk factors for Lewy body disorders such as Parkinson's disease (PD) and Lewy body dementia (DLB). Altered lipid metabolism in PD patient-derived neurons, carrying either GBA1 or PD αS mutations, can shift the physiological α-synuclein (αS) tetramer-monomer (T:M) equilibrium toward aggregation-prone monomers. A resultant increase in pSer129+ αS monomers provides a likely building block for αS aggregates. 3K αS mice, representing a neuropathological amplification of the E46K PD-causing mutation, have decreased αS T:M ratios and vesicle-rich αS+ aggregates in neurons, accompanied by a striking PD-like motor syndrome. We asked whether enhancing glucocerebrosidase (GCase) expression could benefit αS dyshomeostasis by delivering an adeno-associated virus (AAV)-human wild-type (wt) GBA1 vector into the brains of 3K neonates. Intracerebroventricular AAV-wtGBA1 at postnatal day 1 resulted in prominent forebrain neuronal GCase expression, sustained through 6 mo. GBA1 attenuated behavioral deficits both in working memory and fine motor performance tasks. Furthermore, wtGBA1 increased αS solubility and the T:M ratio in both 3K-GBA mice and control littermates and reduced pS129+ and lipid-rich aggregates in 3K-GBA. We observed GCase distribution in more finely dispersed lysosomes, in which there was increased GCase activity, lysosomal cathepsin D and B maturation, decreased perilipin-stabilized lipid droplets, and a normalized TFEB translocation to the nucleus, all indicative of improved lysosomal function and lipid turnover. Therefore, a prolonged increase of the αS T:M ratio by elevating GCase activity reduced the lipid- and vesicle-rich aggregates and ameliorated PD-like phenotypes in mice, further supporting lipid modulating therapies in PD.

A cellular protection racket: How lysosomal Ca2+ fluxes prevent kidney injury

LC3-lipidation is activated by lysosomal damage by mechanisms that are unknown and divergent from canonical autophagy. In this study, Nakamura et al, show that lysosomal damage induced by lysosomotropic agents or oxalate in renal proximal tubule cells causes lipidated LC3 to insert into the lysosomal membrane to activate TRPML1 channels and release Ca2+ from lysosomes. This leads to TFEB dephosphorylation and translocation into the nucleus which results in clearance of damaged lysosomes and their contents which may reduce the deleterious effects of crystal nephropathy.

Abstract 3525: Epidemiology and clinicopathology prognostic factors of TFEB translocation renal cell carcinoma (TBRCC): Analysis of updated pooled database

Abstract Renal cell carcinomas harboring the t(6;11) (p21;q12) translocation with a specific Alpha(MALAT1)-TFEB gene fusion is a recognized rare subtype of the Microphthalmia Transcription factor (MiT) family of translocation RCC. TBRCC is an indolent disease that occurs at a younger age. Though it may take on a wide spectrum of morphology, TBRCC has been classically characterized by distinctive biphasic morphology with larger epithelioid cells and smaller cells clustered around eosinophilic spheres formed by basement membrane material. This analysis was conducted to update and expand our existing knowledge of this rare disease. In order to study the demographic characteristics, cytogenetic and molecular signatures, survival, and prognostic factors, we compiled a pooled database of 140 cases. Kaplan-Meier survival curves were constructed. Cox proportional-hazards model and Log-rank tests were used to assess the influence of demographic and clinicopathologic factors on survival. The median age was 32 (3-78) years with a peak incidence between ages 17 and 30 years. Males and females were afflicted equally. The median overall survival (OS) of the whole group was not reached with more than 78% alive at 10 years. The group's median disease-free survival (DFS) was 96 months. Six percent of this cohort recurred. Among patients who recurred, the median time to recurrence was 24(6-75) months. Sex did not impact OS or DFS. Similarly, the molecular partner of the TFEB gene fusion, Alpha (MALAT1) versus others, did not seem to impact OS or DFS. The presence of necrosis seemed to adversely impact OS (HR:14.3, p<0.0001) and DFS (HR:14.4, p<0.0001). Nuclear grade negatively impacted DFS, the higher the grade the worse the DFS. The presence of eosinophilia, psammoma bodies, and pigmentation did not affect OS or DFS. Similarly, the histologic morphology, biphasic vs others, did not affect OS or DFS. Multivariate analysis revealed older age (HR:1.08, p=0.01) and larger tumor size (HR:10.12, p=0.002) adversely impacted OS. This study presents an updated epidemiologic and clinicopathologic data from a pooled cohort of patients with TBRCC. Age, size, and necrosis were found to adversely impact the OS. However, higher nuclear grade only adversely impacted the cohort's DFS. Citation Format: Philip A. Haddad, Dalia Hammoud, Kevin Gallagher. Epidemiology and clinicopathology prognostic factors of TFEB translocation renal cell carcinoma (TBRCC): Analysis of updated pooled database [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3525.

Clinicopathologic and Molecular Analysis of the TFEB Fusion Variant Reveals New Members of TFEB Translocation Renal Cell Carcinomas (RCCs): Expanding the Genomic Spectrum.

Xp11 renal cell carcinoma (RCC) with different gene fusions may have different clinicopathologic features. We sought to identify variant fusions in TFEB translocation RCC. A total of 31 cases of TFEB RCCs were selected for the current study; MALAT1-TFEB fusion was identified in 25 cases (81%, 25/31) using fusion probes. The remaining 6 cases (19%, 6/31) were further analyzed by RNA sequencing and 5 of them were detected with TFEB-associated gene fusions, including 2 ACTB-TFEB, 1 EWSR1-TFEB, 1 CLTC-TFEB, and 1 potential PPP1R10-TFEB (a paracentric inversion of the TFEB gene, consistent with "negative" TFEB split FISH result, and advising a potential diagnostic pitfall in detecting TFEB gene rearrangement). Four of the 5 fusion transcripts were successfully validated by reverse transcription-polymerase chain reaction and Sanger sequencing. Morphologically, approximately one third (29%, 9/31) of TFEB RCCs showed typical biphasic morphology. The remaining two thirds of the cases (71%, 22/31) exhibited nonspecific morphology, with nested, sheet-like, or papillary architecture, resembling other types of renal neoplasms, such as clear cell RCC, Xp11 RCC, perivascular epithelioid cell tumor (PEComa), or papillary RCC. Although cases bearing a MALAT1-TFEB fusion demonstrated variable morphologies, all 9 cases featuring typical biphasic morphology were associated with MALAT1-TFEB genotype. Accordingly, typical biphasic morphology suggests MALAT1-TFEB fusion, whereas atypical morphology did not suggest the specific type of fusion. Isolated or clustered eosinophilic cells were a common feature in TFEB RCCs, which may be a useful morphology diagnostic clue for TFEB RCCs. Clinicopathologic variables assessment showed that necrosis was the only morphologic feature that correlated with the aggressive behavior of TFEB RCC (P=0.004). In summary, our study expands the genomic spectrum and the clinicopathologic features of TFEB RCCs, and highlights the challenges of diagnosis and the importance of subtyping of this tumor by combining morphology and multiple molecular techniques.

Diagnosis of uncommon renal epithelial neoplasms: performances of fluorescence in situ hybridization

Renal cell carcinomas (RCC) are divided in several subtypes, characterized by morphological and histological features, protein expression patterns and genetics criteria. The main subtypes include Clear cell renal cell carcinoma (CCRCC), Papillary RCC (PRCC), Chromophobe RCC (ChRCC), oncocytoma, TFE3 and TFEB Translocation renal cell carcinoma (TRCC). In most cases, RCC can be easily classified according to histological criteria and immunohistochemistry. Nevertheless, the subtyping process can be more complex in some cases: differential diagnosis (CCRCC or TFE3 TRCC, PRCC or TFE3 TRCC, oncocytic tumors corresponding to ChRCC or oncocytoma), molecular confirmation (TFEB TRCC) and unclassified RCC. Complementary analyses are required such as fluorescence in situ hybridization (FISH) for the detection of chromosomal abnormalities associated to each subtype. In this aim, this study assessed the performance of FISH analysis in the histological classification of 359 RCC exhibiting unusual histological characteristics and/or occurring in young people. FISH probes were selected according to the histological features of each tumor. FISH analysis contributed to the histological classification in 73% of the RCC (261/359). Conversely, FISH did not contribute to the diagnosis in 19% of the cases (69/359) and a hybridization failure was observed for the remaining tumors (8%; 29/359). Considering the different RCC subtypes, FISH analysis was highly efficient to confirm the histological diagnosis of CCRCC, PRCC, and TFE3 TRCC and to identify abnormalities of the TFEB gene. However, this strategy showed some limitations for the diagnosis of oncocytic tumors and unclassified RCC, suggesting that additional molecular assays should be evaluated in these cases.

Abstract 985: Development and validation of cell-based TFEB translocation assay in a high-content and high-throughput screening format

Abstract Transcription factor EB (TFEB) is a master regulator of lysosomal gene expression and mitochondrial biogenesis. Autophagy is known to act as a double edged sword in cancer. Autophagy acts as a tumor suppressor during the tumorigenesis process but it also helps to sustain the survival of already transformed cancer cells. Lysosomes sit at the end of the autophagy pathway, which is critical to meet the needs of autophagy. Therefore, small molecules that inhibit TFEB-mediated lysosomal biogenesis and autophagic degradation may be helpful for inhibiting cancer cell growth and limiting cancer progression. On the other hand, small molecules that enhance TFEB-mediated lysosomal degradation may be used to prevent tumorigenesis. In order to identify compounds that modulate TFEB translocation, an image-based TFEB translocation assay was developed in a 1536-well plate format with a GFP-TFEB stable AML12 cell line, which is an immortalized mouse hepatocyte cell line. Torin 1, a known TFEB translocation inducer, was used as positive control in the study. Torin 1 increased TFEB nuclear translocation in a concentration dependent manner with an EC50 of 150nM. To optimize the assay condition, various cell densities per well and time courses were tested. To validate this assay, a group of 384 known anticancer compounds were screened at 11 concentrations ranging from 0.8 nM to 46 μM. From this screening, we identified 25 compounds that enhanced TFEB nuclear translocation with EC50s ranging from 2 nM to 12 μM. Several known and novel TFEB inducers were among these compounds. These results demonstrate that this high-content and high-throughput assay can be used to screen large numbers of chemicals and provide a robust in vitro system to identify TFEB agonists and antagonists, which may be used to either prevent (agonists) or treat (antagonist) cancer. Citation Format: Li Zhang, Ruili Huang, Wen Xing Ding, Menghang Xia. Development and validation of cell-based TFEB translocation assay in a high-content and high-throughput screening format [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 985.

NEO212 induces mitochondrial apoptosis and impairs autophagy flux in ovarian cancer

BackgroundTemozolomide-perillyl alcohol conjugate (NEO212), a novel temozolomide (TMZ) analog, was previously reported to exert its anti-cancer effect in non-small cell lung cancer (NSCLC), and human nasopharyngeal carcinoma (NPC), etc.. In the current study, we intend to illuminate the potential anticancer property and the underly mechanisms of NEO212 in ovarian cancer cells.MethodsThe cytotoxicity of NEO212 was detected by MTT, colony formation analysis and xenograft model. The proteins involved in cell proliferation, DNA damage, autophagy and lysosomal function were detected by western blots; mitochondria, lysosome and autophagosome were visualized by TEM and/or immunofluorescence; Apoptosis, cell cycle analysis and mitochondrial transmembrane potential were detected by flow cytometry. TFEB translocation was detected by immunofluorescence and western blot.ResultsNEO212 has the potential anticancer property in ovarian cancer cells, as evidence from cell proliferation inhibition, G2/M arrest, DNA damage, xenograft, mitochondrial dysfunction and apoptosis. Importantly, we observed that although it induced significant accumulation of autophagosomes, NEO212 quenched GFP-LC3 degradation, down-regulated a series of lysosome related gene expression and blocked the autophagic flux, which significantly facilitated it induced apoptosis and was largely because it inhibited the nuclear translocation of transcription factor EB (EB).ConclusionsNEO212 inhibited TFEB translocation, and impaired the lysosomal function, implying NEO212 might avoid from autophagy mediated chemo-resistance, thus proposing NEO212 as a potential therapeutic candidate for ovarian cancer.

Clinical Utility of Chromosome Genomic Array Testing for Unclassified and Advanced-Stage Renal Cell Carcinomas.

Cytogenomic analysis provides a useful adjunct to traditional pathology in the categorization of renal cell carcinomas (RCCs), particularly in morphologically ambiguous cases, but it has disadvantages, including cost. To define the clinical scenarios in which this technology has direct clinical applications. DNA was isolated from paraffin-embedded tissue from 40 selected cases of RCC. Chromosome genomic array testing was performed using the OncoScan. Of 23 cases of unclassified renal tumors, 19 (83%) were reclassified with incorporation of cytogenetic and histologic features, including 10 as clear cell RCC, 2 as collecting duct carcinoma, 2 as papillary RCC, and 1 as novel TFEB-amplified tumor lacking TFEB translocation. Of 5 tumors with "hybrid" oncocytic features, 3 were reclassified as an eosinophilic variant of chromophobe RCC and 1 as oncocytoma. Appropriate staging in 2 patients was determined by identifying distinct, nonshared cytogenetic profiles. Of 11 cases of metastatic clear cell RCC, 7 (63%) had cytogenetic features associated with a poor prognosis. We identified 5 scenarios in which chromosome genomic array testing has direct clinical utility: (1) to investigate unclassified RCCs, (2) to understand tumors with "hybrid" features and "collision" tumors, (3) to determine appropriate staging in questions of bilateral tumors and/or metastases, (4) to identify chromosomal aberrations in metastatic clear cell RCCs associated with a worse prognosis, and (5) to identify new entities. This has practical value in our institution, where a molecular profile diagnostically separating morphologically difficult to classify clear cell, papillary, chromophobe, and unclassified RCC influences treatment recommendations and clinical trial eligibility.

Peripheral Melanocortin 3 Receptor (MC3R) Regulates Hepatic Autophagy in Obesity

We have previously reported homozygosity for two MC3R SNPs (C17A+G241A) that partially inactive MC3R increases adiposity in humans and mouse models. However, the precise mechanisms by which hypoactive MC3R alters metabolic homeostasis remain unclear. Given the essential role of hepatic autophagy in cellular mechanisms of obesity, we hypothesized that diminished peripheral MC3R activity leads to hepatic lipid accumulation by interfering with autophagy, leading to steatosis. We used knock-in mouse models that carry either homozygous human “wild type” (MC3RhWT/hWT) or “double mutant” MC3R (C17A+G241A, MC3RhDM/hDM). We studied hepatic autophagic pathways with biochemical/imaging analysis in wild type Mc3r (C57/BL6) and MC3RhWT/hWT as controls (WT mice), and in MC3RhDM/hDM, and mice lacking Mc3r (Mc3r-/-) which each have greater fat mass and hepatic triglycerides. We first demonstrated the MC3R specific agonist D-trp-γ-MSH induced LC3II (a marker of autophagosome formation) in hepatocytes from WT, but not Mc3r-/- or MC3RhDM/hDM mice. Interestingly D-trp-γ-MSH also induced TFEB (a master regulator of lysosomal biogenesis) translocation from cytosol to nucleus in TFEB-overexpressed NIH-3T3 cells, indicating MC3R may regulate autophagy by affecting TFEB signaling. Overnight fasting induced liver LC3II expression in WT mice, but not in MC3RhDM/hDM or Mc3r-/-; rather, basal LC3II was upregulated even in the fed state for the latter two genotypes. Hepatocytes from MC3RhDM/hDM or Mc3r-/- failed to increase autophagic flux with starvation or with the presence of chloroquine, suggesting MC3R may play a role in the regulation of autolysosomal degradation under the control of TFEB. Electron microscopy analysis of liver sections further demonstrated MC3RhDM/hDM mice exhibited fewer lipid droplet-associated autophagosomes compared to MC3RhWT/hWT mice. These data suggest MC3R plays an important peripheral role for the regulation of hepatic autophagy that is targeted to lipids. Disclosure J. Jun: None. A.Y. Seo: None. T.P. Patel: None. A.J. Uhlman: None. N.J. Levi: None. R. Roberson: None. J.A. Yanovski: Research Support; Self; Rhythm Pharmaceuticals Inc., Zafgen.

Detection of 6 TFEB-amplified renal cell carcinomas and 25 renal cell carcinomas with MITF translocations: systematic morphologic analysis of 85 cases evaluated by clinical TFE3 and TFEB FISH assays

Renal cell carcinomas with MITF aberrations demonstrate a wide morphologic spectrum, highlighting the need to consider these entities within the differential diagnosis of renal tumors encountered in clinical practice. Herein, we describe our experience with application of clinical fluorescence in situ hybridization (FISH) assays for detection of TFE3 and TFEB gene aberrations from 85 consecutive renal cell carcinoma cases submitted to our genitourinary FISH service. Results from 170 FISH assays performed on these tumors were correlated with available clinicopathologic findings. Ninety-eight percent of renal tumors submitted for FISH evaluation were from adult patients. Thirty-one (37%) tumors were confirmed to demonstrate MITF aberrations (21 TFE3 translocation, 4 TFEB translocation, and 6 TFEB amplification cases). Overall, renal cell carcinomas with MITF aberrations demonstrated morphologic features overlapping with clear cell, papillary, or clear cell papillary renal cell carcinomas. Renal cell carcinomas with MITF aberrations were significantly more likely to demonstrate dual (eosinophilic and clear) cytoplasmic tones (P=0.030), biphasic TFEB translocation renal cell carcinoma-like morphology (P=0.002), psammomatous calcifications (P=0.002), and nuclear pseudoinclusions (P=0.001) than renal cell carcinomas without MITF aberrations. Notably, 7/9 (78%) renal cell carcinomas exhibiting subnuclear clearing and linear nuclear array (6 of which showed high World Health Organization/International Society of Urological Pathology nucleolar grade) demonstrated TFE3 translocation, an association that was statistically significant when compared with renal cell carcinomas without MITF aberrations (P=0.009). In this cohort comprising consecutive cases, TFEB-amplified renal cell carcinomas were more commonly identified than renal cell carcinomas with TFEB translocations, and four (67%) of these previously unreported TFEB-amplified renal cell carcinomas demonstrated oncocytic and papillary features with a high World Health Organization/International Society of Urological Pathology nucleolar grade. In summary, TFE3 and TFEB FISH evaluation aids in identification and accurate classification of renal cell carcinomas with MITF aberrations, including TFEB-amplified renal cell carcinoma, which may demonstrate aggressive behavior.

Melanotic MiT family translocation neoplasms: Expanding the clinical and molecular spectrum of this unique entity of tumors

MiT family translocation tumors are a group of neoplasms characterized by translocations involving MiT family transcription factors. The translocation renal cell carcinomas, TFE3 (Xp11.2) and TFEB (t6;11) are known members of this family. Melanotic Xp11 translocation renal cancer is a more recently described entity. To date only 14 cases have been described. It is characterized by a distinct set of features including a nested epithelioid morphology, melanin pigmentation, labeling for markers of melanocytic differentiation, lack of labeling for markers of renal tubular differentiation, predominance in a younger age population and association with aggressive clinical behavior. There are noted similarities between that entity and TFE3 associated PEComas. There are no cases reported of equivalent melanotic TFEB translocation renal cancer. We report 2 rare cases of melanotic translocation renal neoplasms. The first is a melanotic TFE3 translocation renal cancer with an indolent clinical course, occurring in a patient more than 3-decades older than the usual average age in which such tumors have been described. The other case is, to our knowledge, the first reported melanotic TFEB translocation cancer of the kidney. Both cases exhibit the same H&E morphology as previously reported in melanotic translocation renal cancers and label accordingly with HMB45 and Melan-A. While the TFE3 melanotic tumor lacked any evidence of renal tubular differentiation, the TFEB melanotic cancer exhibited some staining for renal tubular markers. Based on the unique features noted above, these two cases expand the clinical and molecular spectrum of the melanotic translocation renal cancers.

Autophagy is required for PDAC glutamine metabolism.

Macroautophagy (autophagy) is believed to maintain energy homeostasis by degrading unnecessary cellular components and molecules. Its implication in regulating cancer metabolism recently started to be uncovered. However, the precise roles of autophagy in cancer metabolism are still unclear. Here, we show that autophagy plays a critical role in glutamine metabolism, which is required for tumor survival. Pancreatic ductal adenocarcinoma (PDAC) cells require both autophagy and typical glutamine transporters to maintain intracellular glutamine levels. Glutamine deprivation, but not that of glucose, led to the activation of macropinocytosis-associated autophagy through TFEB induction and translocation into the nucleus. In contrast, glutamine uptake increased as a compensatory response to decreased intracellular glutamine levels upon autophagy inhibition. Moreover, autophagy inhibition and glutamine deprivation did not induce cell death, while glutamine deprivation dramatically activated apoptotic cell death upon autophagy inhibition. Interestingly, the addition of α-ketoglutarate significantly rescued the apoptotic cell death caused by the combination of the inhibition of autophagy with glutamine deprivation. Our data suggest that macropinocytosis-associated autophagy is a critical process providing glutamine for anaplerosis of the TCA cycle in PDAC. Thus, targeting both autophagy and glutamine metabolism to completely block glutamine supply may provide new therapeutic approaches to treat refractory tumors.