SUCLA2-coupled regulation of GLS succinylation and activity counteracts oxidative stress in tumor cells.

Glutaminase regulates glutaminolysis to promote cancer cell proliferation. However, the mechanism underlying glutaminase activity regulation is largely unknown. Here, we demonstrate that kidney-type glutaminase (GLS) is highly expressed in human pancreatic ductal adenocarcinoma (PDAC) specimens with correspondingly upregulated glutamine dependence for PDAC cell proliferation. Upon oxidative stress, the succinyl-coenzyme A (CoA) synthetase ADP-forming subunit ß (SUCLA2) phosphorylated by p38 mitogen-activated protein kinase (MAPK) at S79 dissociates from GLS, resulting in enhanced GLS K311 succinylation, oligomerization, and activity. Activated GLS increases glutaminolysis and the production of nicotinamide adenine dinucleotide phosphate (NADPH) and glutathione, thereby counteracting oxidative stress and promoting tumor cell survival and tumor growth in mice. In addition, the levels of SUCLA2 pS79 and GLS K311 succinylation, which were mutually correlated, were positively associated with advanced stages of PDAC and poor prognosis for patients. Our findings reveal critical regulation of GLS by SUCLA2-coupled GLS succinylation regulation and underscore the regulatory role of metabolites in glutaminolysis and PDAC development.

Identification of a Novel Homozygous GLS Gene Variant Associated with Developmental and Epileptic Encephalopathy (DEE) Type 71.

Developmental and epileptic encephalopathy (DEEs) (OMIM#618,328) is characterized by seizures, hypotonia, and brain abnormalities, often arising from mutations in genes crucial for brain function. Among these genes, GLS stands out due to its vital role in the central nervous system (CNS), with homozygous variants potentially causing DEE type 71. Using Whole Exome Sequencing (WES) on a patient exhibiting symptoms of epileptic encephalopathy, we identified a novel homozygous variant, NM_014905.5:c.1849G > T; p.(Asp617Tyr), in the GLS gene. The 5-year-old patient, born to consanguineous parents, presented with developmental delay, encephalopathy, frequent seizures, and hypotonia. Sanger sequencing further validated the GLS gene variant in both the patient and his family. Furthermore, our bioinformatics analysis indicated that this missense variant could lead to alteration of splicing, resulting in the activation of a cryptic donor site and potentially causing loss of protein function. Our finding highlights the pathogenic significance of the GLS gene, particularly in the context of brain disorders, specifically DEE71.

Senescence and senolysis in cancer: The latest findings.

Aging is a life phenomenon that occurs in most living organisms and is a major risk factor for many diseases, including cancer. Cellular senescence is a cellular trait induced by various genomic and epigenetic stresses. Senescent cells are characterized by irreversible cell growth arrest and excessive secretion of inflammatory cytokines (senescence-associated secretory phenotypes, SASP). Chronic tissue microinflammation induced by SASP contributes to the pathogenesis of a variety of age-related diseases, including cancer. Senolysis is a promising new strategy to selectively eliminate senescent cells in order to suppress chronic inflammation, suggesting its potential use as an anticancer therapy. This review summarizes recent findings on the molecular basis of senescence in cancer cells and senolysis.

IGF2BP3 promotes glutamine metabolism of endometriosis by interacting with UCA1 to enhances the mRNA stability of GLS1.

BACKGROUND: Insulin like growth factor II mRNA binding protein 3 (IGF2BP3) has been implicated in numerous inflammatory and cancerous conditions. However, its precise molecular mechanisms in endometriosis (EMs) remains unclear. The aim of this study is to examine the influence of IGF2BP3 on the occurrence and progression of EMs and to elucidate its underlying molecular mechanism. METHODS: Efects of IGF2BP3 on endometriosis were confrmed in vitro and in vivo. Based on bioinformatics analysis, RNA immunoprecipitation (RIP), RNA pull-down assays and Fluorescent in situ hybridization (FISH) were used to show the association between IGF2BP3 and UCA1. Single-cell spatial transcriptomics analysis shows the expression distribution of glutaminase 1 (GLS1) mRNA in EMs. Study the effect on glutamine metabolism after ectopic endometriotic stromal cells (eESCs) were transfected with Sh-IGF2BP3 and Sh-UCA1 lentivirus. RESULTS: Immunohistochemical staining have revealed that IGF2BP3 was upregulated in ectopic endometriotic lesions (EC) compared to normal endometrial tissues (EN). The proliferation and migration ability of eESCs were greatly reduced by downregulating IGF2BP3. Additionally, IGF2BP3 has been observed to interact with urothelial carcinoma associated 1 (UCA1), leading to increased stability of GLS1 mRNA and subsequently enhancing glutamine metabolism. Results also demonstrated that IGF2BP3 directly interacts with the 3' UTR region of GLS1 mRNA, influencing its expression and stability. Furthermore, UCA1 was able to bind with c-MYC protein, stabilizing c-MYC mRNA and consequently enhancing GLS1 expression through transcriptional promotion. CONCLUSION: These discoveries underscored the critical involvement of IGF2BP3 in the elevation and stability of GLS1 mRNA in the context of glutamine metabolism by interacting with UCA1 in EMs. The implications of our study extended to the identification of possible therapeutic targets for individuals with EMs.

Left ventricular global longitudinal strain is worse in BRCA mutation positive breast cancer patients prior to cancer treatment and premature menopause.

PURPOSE: Breast cancer patients with mutations in human tumor suppressor genes BRCA1 and BRCA2 are at higher risk of cardiovascular disease (CVD) than the general population, as they are frequently exposed to cardiotoxic chemotherapy, anti-estrogen therapy, radiation, and/or oophorectomy for cancer-related treatment and prophylaxis. Animal and cell culture models suggest that BRCA mutations may play an independent role in heart failure. We sought to evaluate cardiac structure and function in female BRCA1 and BRCA2 mutation carriers with breast cancer compared to BRCA wildtype women with breast cancer. METHODS: We performed a 1:2 age- and hypertension-matched retrospective cohort study comparing BRCA1 and BRCA2 mutation carriers (n = 38) versus BRCA wildtype controls (n = 76) with a new diagnosis of breast cancer. Echocardiographic data were obtained within 6 months of breast cancer diagnosis and prior to chemotherapy, anti-estrogen therapy, radiation, or oophorectomy. Left ventricular global longitudinal strain (LV-GLS), a highly sensitive marker of LV function, was measured using QLab 15 (Philips Healthcare). RESULTS: In the total cohort of 114 patients with a new diagnosis of breast cancer, the median age was 45 ± 11 years and the prevalence of hypertension was 8%. There were no differences in traditional cardiovascular disease risk factors between cases and controls. BRCA carriers had lower LV-GLS (- 18.1% ± 4.7% vs. - 20.1% ± 3.8%, p = 0.02) and greater right atrial area (12.9 cm2 ± 2.7 cm2 vs. 11.8 cm2 ± 2.0 cm2, p = 0.04) compared to controls; however, both LV-GLS and right atrial area were within the normal range. Compared to controls, BRCA carriers had a trend toward worse LV posterior wall thickness (0.89 cm ± 0.15 cm vs. 0.83 cm ± 0.16 cm, p = 0.06) although not statistically significant. CONCLUSION: In women with newly diagnosed breast cancer and prior to treatment, LV-GLS was worse in BRCA1 and BRCA2 mutation carriers compared to those with BRCA wildtype. These findings suggest that BRCA mutations may be associated with subtle changes in cardiac function. Whether differences in GLS translate to increased cardiovascular risk in women with BRCA mutations needs to be further characterized.

HOXC10 promotes hypertrophic scar fibroblast fibrosis through the regulation of STMN2 and the TGF-ß/Smad signaling pathway.

The pathophysiology of hypertrophic scar (HS) shares similarities with cancer. HOXC10, a gene significantly involved in cancer development, exhibits higher expression levels in HS than in normal skin (NS), suggesting its potential role in HS regulation. And the precise functions and mechanisms by which HOXC10 influences HS require further clarification. Gene and protein expressions were analyzed using raeal-time quantitative polymerase chain reaction (RT-qPCR) and western blot techniques. Cell proliferation and migration were evaluated using EdU proliferation assays, CCK-8 assays, scratch assays, and Transwell assays. Chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays were conducted to investigate the interactions between HOXC10 and STMN2. HOXC10 and STMN2 expression levels were significantly higher in HS tissues compared with NS tissues. Silencing HOXC10 led to decreased activation, proliferation, migration, and fibrosis in hypertrophic scar fibroblasts (HSFs). Our findings also indicate that HOXC10 directly targets STMN2. The promotional effects of HOXC10 knockdown on HSF activation, proliferation, migration, and fibrosis were reversed by STMN2 overexpression. We further demonstrated that HOXC10 regulates HSF activity through the TGF-ß/Smad signaling pathway. HOXC10 induces the activation and fibrosis of HSFs by promoting the transcriptional activation of STMN2 and engaging the TGF-ß/Smad signaling pathway. This study suggests that HOXC10 could be a promising target for developing treatments for HS.

TIM-3/Galectin-9 interaction and glutamine metabolism in AML cell lines, HL-60 and THP-1.

BACKGROUND: T cell immunoglobulin and mucin-domain containing-3 (TIM-3) is a cell surface molecule that was first discovered on T cells. However, recent studies revealed that it is also highly expressed in acute myeloid leukemia (AML) cells and it is related to AML progression. As, Glutamine appears to play a prominent role in malignant tumor progression, especially in their myeloid group, therefore, in this study we aimed to evaluate the relation between TIM-3/Galectin-9 axis and glutamine metabolism in two types of AML cell lines, HL-60 and THP-1. METHODS: Cell lines were cultured in RPMI 1640 which supplemented with 10% FBS and 1% antibiotics. 24, 48, and 72 h after addition of recombinant Galectin-9 (Gal-9), RT-qPCR analysis, RP-HPLC and gas chromatography techniques were performed to evaluate the expression of glutaminase (GLS), glutamate dehydrogenase (GDH) enzymes, concentration of metabolites; Glutamate (Glu) and alpha-ketoglutarate (α-KG) in glutaminolysis pathway, respectively. Western blotting and MTT assay were used to detect expression of mammalian target of rapamycin complex (mTORC) as signaling factor, GLS protein and cell proliferation rate, respectively. RESULTS: The most mRNA expression of GLS and GDH in HL-60 cells was seen at 72 h after Gal-9 treatment (p = 0.001, p = 0.0001) and in THP-1 cell line was observed at 24 h after Gal-9 addition (p = 0.001, p = 0.0001). The most mTORC and GLS protein expression in HL-60 and THP-1 cells was observed at 72 and 24 h after Gal-9 treatment (p = 0.0001), respectively. MTT assay revealed that Gal-9 could promote cell proliferation rate in both cell lines (p = 0.001). Glu concentration in HL-60 and α-KG concentration in both HL-60 (p = 0.03) and THP-1 (p = 0.0001) cell lines had a decreasing trend. But, Glu concentration had an increasing trend in THP-1 cell line (p = 0.0001). CONCLUSION: Taken together, this study suggests TIM-3/Gal-9 interaction could promote glutamine metabolism in HL-60 and THP-1 cells and resulting in AML development.

Cyclopeptide RA-V from Rubia yunnanensis restores activity of Adagrasib against colorectal cancer by reducing the expression of Nrf2.

Adagrasib (MRTX849), an approved and promising KRAS G12C inhibitor, has shown the promising results for treating patients with advanced non-small cell lung cancer (NSCLC) or colorectal cancer (CRC) harboring KRAS-activating mutations. However, emergence of the acquired resistance limits its long-term efficacy and clinical application. Further understanding of the mechanism of the acquired resistance is crucial for developing more new effective therapeutic strategies. Herein, we firstly found a new connection between the acquired resistance to MRTX849 and nuclear factor erythroid 2-related factor 2 (Nrf2). The expression levels of Nrf2 and GLS1 proteins were substantially elevated in different CRC cell lines with the acquired resistance to MRTX849 in comparison with their corresponding parental cell lines. Next, we discovered that RA-V, one of natural cyclopeptides isolated from the roots of Rubia yunnanensis, could restore the response of resistant CRC cells to MRTX849. The results of molecular mechanisms showed that RA-V suppressed Nrf2 protein through the ubiquitin-proteasome-dependent degradation, leading to the induction of oxidative and ER stress, and DNA damage in CRC cell lines. Consequently, RA-V reverses the resistance to MRTX849 by inhibiting the Nrf2/GLS1 axis, which shows the potential for further developing into one of novel adjuvant therapies of MRTX849.

Analysis of anti-tumor effect and mechanism of GLS1 inhibitor CB-839 in colorectal cancer using a stroma-abundant tumor model.

BACKGROUND: Glutaminase 1 (GLS1), a key enzyme in glutamine metabolism in cancer cells, acts as a tumor promoter and could be a potential therapeutic target. CB-839, a GLS1-specific inhibitor, was developed recently. Herein, we aimed to elucidate the anti-tumor effects and mechanism of action of CB-839 in colorectal cancer (CRC). METHODS: Using the UCSC Xena public database, we evaluated GLS1 expression in various cancers. Immunostaining for GLS1 was performed on 154 surgically resected human CRC specimens. Subsequently, we examined the GLS1 mRNA expression levels in eight CRC cell lines and evaluated the association between GLS1 expression and CB-839 efficacy. To create a reproducible CRC model with abundant stroma and an allogeneic immune response, we co-transplanted CT26 and stem cells into BALB/c mice and treated them with CB-839. Finally, RNA sequencing of mouse tumors was performed. RESULTS: Database analysis showed higher GLS1 expression in CRC tissues than in normal colon tissues. Clinical samples from 114 of the 154 patients with CRC showed positive GLS1 expression. GLS1 expression in clinical CRC tissues correlated with vascular invasion. CB-839 treatment inhibited cancer cell proliferation depending on GLS1 expression in vitro and inhibited tumor growth and metastasis in the CRC mouse model. RNA sequencing revealed that CB-839 treatment inhibited stromal activation, tumor growth, migration, and angiogenesis. These findings were validated through in vitro and in vivo experiments and clinical specimen analysis. CONCLUSIONS: GLS1 expression in CRC plays important roles in tumor progression. CB-839 has inhibitory effects on cancer proliferation and the tumor microenvironment.

Effect of residual myocardial ischemia on recovery of left ventricular function after primary percutaneous coronary intervention.

BACKGROUND: It is unknown whether the existence of severe bystander damage will affect left ventricular (LV) healing following primary percutaneous coronary intervention (PPCI). The aim of the present analysis was to follow LV recovery using 2D speckle tracking echocardiography (2-D STE) in cases with single versus multiple vessel disease with acute myocardial infarction (AMI) who underwent PPCI and to assess major adverse cardiovascular events (MACEs) within 3 months. PATIENTS AND METHODS: This work was conducted at Assiut University Heart Hospital. Of 1026 screened subjects with AMI needing PPCI and assessed for eligibility, only 89 cases fulfilled the inclusion criteria. They were classified into Group A: single vessel and Group B: multiple vessel (≥ 2 vessels) disease. Their data were obtained on admittance and after 90 days. RESULTS: In group A compared to group B, there was a statistically preferable value at baseline in the global longitudinal strain- Apical 2 chamber (GLS-A2C) (-12.05 ± 3.57 vs. -10.38 ± 3.92, P = 0.039). At follow-up, the improvement was in all 2-D STE variables, including GLS-long axis (GLS-LAX) (-13.09 ± 3.84 vs.-10.75 ± 3.96, P = 0.006), GLS- apical 4 chamber (GLS-A4C) (-13.23 ± 3.51 vs.-10.62 ± 4.08, P = 0.002), GLS-A2C (-13.85 ± 3.41 vs-10.93 ± 3.97, P < 0.001) and GLS- average (GLS-AVG, P = 0.001). There was a considerable negative correlation between the recovery of LV performance and the existence of multi-vessel lesions (P = 0.009). There was no variance between the groups regarding MACEs. CONCLUSIONS: Patients with single vessel lesions who underwent PPCI to the culprit lesion had better recovery of LV function than those with multi-vessel (≥ 2 vessels) lesions who underwent PPCI to the culprit lesion only. The presence of multivessel involvement was an independent risk factor for deterioration in GLS. TRIAL REGISTRATION: Registered in clinical trial, clinicalTrial.gov ID NCT04103008 (25/09/2019). IRB registration: 17,100,834 (05/11/2019).

Echocardiographic parameters of cardiac structure and function in the diagnosis of acute myocarditis in adult patients: A systematic review and meta-analysis.

BACKGROUND: Transthoracic echocardiography (TTE) plays a key role in the initial work-up of myocarditis where the identification of pathologic structural and functional changes may assist in its diagnosis and management. The aim of this systematic review was to appraise the evidence for the utility of echocardiographic parameters of cardiac structure and function in the diagnosis of myocarditis in adult populations. METHODS: A systematic literature search of medical databases was performed using PRISMA principles to identify all relevant studies assessing TTE parameters in adult patients with myocarditis (1995-2020; English only; PROSPERO registration CRD42021243598). Data for a range of structural and functional TTE parameters were individually extracted and those with low heterogeneity were then meta-analyzed using a random-effects model for effect size, and assessed through standardized mean difference (SMD). RESULTS: Available data from six studies (with a pooled total of 269 myocarditis patients and 240 controls) revealed that myocarditis can be reliably differentiated from healthy controls using echocardiographic measures of left ventricular (LV) size and systolic function, in particular LV end-diastolic diameter, LV ejection fraction (LVEF) and LV global longitudinal strain (LV-GLS) (p ≤ .01 for all). LV-GLS demonstrated the highest overall effect size, followed by LVEF and LVEDD (SMD: |0.46-1.98|). Two studies also demonstrated that impairment in LV-GLS was associated with adverse cardiovascular outcomes in this population, irrespective of LVEF. CONCLUSIONS: LV-GLS demonstrated the greatest overall effect size and therefore ability to differentiate myocarditis populations from healthy controls. GLS was also shown to be a predictor of adverse cardiovascular outcomes, in this population. HIGHTLIGHTS: What is already known on this subject? Myocarditis is a disease process that is often a diagnosis of exclusion, as it frequently mimics other acute cardiac pathologies. Transthoracic echocardiography is traditionally the initial imaging modality used for noninvasive structural assessment in populations with myocarditis. What might this study add? This study demonstrates that left ventricular (LV) global longitudinal strain, LV ejection fraction and LV end-diastolic diameter can differentiate between myocarditis patients and healthy controls. LV-GLS demonstrated the greatest overall effect size when comparing these two populations, in comparison to the other measures. How might this impact on clinical practice? This study demonstrates that assessment of myocardial deformation indices allows for sensitive discrimination between myocarditis patients from healthy controls. Routine assessment of LV-GLS may serve as an important diagnostic tool in the acute care setting.

Clinical study of two-dimensional speckle tracking to evaluate abnormal myocardial motion due to coronary lesions.

OBJECTIVE: To investigate the predictive ability of global longitudinal strain (GLS) and mechanical dispersion for coronary stenosis and provide a more reliable noninvasive method for diagnosis of obstructive coronary artery disease(OCAD). METHODS: Sixty-seven patients diagnosed with suspected CAD were included in the study. Patients with coronary stenosis greater than 50% were assigned as OCAD, while the others were assigned as non obstructive coronary artery disease(NOCAD). General information was collected and patients underwent speckle tracking echocardiogram(STE). RESULTS: Spearman's correlation analysis showed that GLS and mechanical dispersion were positively correlated with the degree of coronary stenosis (r = 0.383, 0.342, p < 0.05), and there was also a positive correlation between GLS and mechanical dispersion (r = 0.327, p < 0.05). GLS, longitudinal strain (LS) of each chamber, and mechanical dispersion were higher in the OCAD group than in the NOCAD group (p < 0.05). Univariate regression analysis showed that GLS, each lumen LS and mechanical dispersion were statistically significant (p < 0.05). Multifactorial regression analysis showed that elevated GLS (p = 0.007) and elevated mechanical dispersion (p = 0.030) were independent risk factors for OCAD. The ROC curves showed that GLS predicted OCAD (AUC area 0.745, 95% CI 0.624 to 0.865) versus mechanical discrete prediction of OCAD (AUC area 0.702, 95% CI 0.569 to 0.834) were more diagnostic than conventional cardiac ultrasound observations of ventricular wall motion abnormalities (AUC area 0.566, 95% CI 0.463 to 0.669). CONCLUSIONS: Combining GLS with mechanical dispersion can rapidly assess OCAD in a very short period, which has strong promotion value and in-depth research value.

Incremental prognostic value of speckle tracking echocardiography and early follow-up echo assessment in predicting left ventricular recovery after reperfusion for ST-segment elevation myocardial infarction (STEMI).

PURPOSE: Up to 50% of patients do not achieve significant left ventricular ejection fraction (LVEF) recovery after primary percutaneous intervention (PPCI) for STEMI. We aimed to identify the echocardiographic predictors for LVEF recovery and assess the value of early follow-up echocardiography (Echo) in risk assessment of post-myocardial infarction (MI) patients. METHODS: One hundred one STEMI patients undergoing PPCI were enrolled provided EF below 50%. Baseline echocardiography assessed LVEF, volumes, wall motion score index (WMSI), global longitudinal strain (GLS), global circumferential strain (GCS), and E/e'. Follow-up echocardiography after 6 weeks reassessed left ventricular volumes, LVEF and GLS.GCS was not assessed at follow up. Patients were classified into recovery and non-recovery groups. Predictors of LVEF recovery and major adverse cardiovascular events (MACE) at 6 months were analysed. RESULTS: The mean change of EF was 8.04 ± 3.32% in group I versus -.39 ± 5.09 % in group II (p < .001). Recovered patients had better baseline GLS, baseline GCS, E/e', and follow-up GLS. Multivariate regression analysis revealed E/e', GCS, and follow-up GLS after 6 weeks to be strong independent predictors for LVEF recovery. Composite MACE was considerably higher in group II (32.7% vs. 4.1%, p < .001) mainly driven by higher heart failure hospitalisation Multivariate regression analysis revealed baseline GLS, E/e', and ejection fraction (EF) percentage recovery as strong independent predictors for MACE. CONCLUSIONS: Multiparametric echocardiographic approach incorporating LVEF, strain parameters, and diastolic function could allow early optimal risk stratification after STEMI treated with PPCI. Follow-up GLS and LVEF percentage change are the strongest predictors for early LV recovery and long term clinical outcome, respectively.

Assessment of cancer therapy-related cardiac dysfunction in breast cancer women using a new speckle tracking echocardiography index: The GAVS.

BACKGROUND: Recently, peak atrial longitudinal strain (PALS) has emerged as a possible predictor of Cancer therapy-related cardiac dysfunction (CTRCD) in cancer patients (CP), in addition to left ventricular global longitudinal strain (GLS). Thus, considering the link between left atrium and left ventricle, the aim of this study was to assess the global atrio-ventricular strain (GAVS) in CP, to detect early cardiotoxicity. METHODS: A prospective study was carried out enrolling 131 breast cancer women (mean age 51.4 ± 10.4 years) receiving anti-cancer treatment. Clinical and echocardiographic evaluation was performed at baseline (T0), 3 (T1), 6 (T2) and 12 months (T3) after starting treatment. CTRCD was defined according to the 2022 ESC Cardio-Oncology guidelines. RESULTS: Forty-four patients developed CTRCD (3 moderate and 41 mild CTRCD group A) and 87 patients did not (group B). In group A, significant changes in GLS, PALS, GAVS, LASi (left atrial stiffness index) and LVEF/GLS occurred earlier than LVEF, that reduced significantly only at T3 (p-value < .05). Significant changes in LASi, PALS and GAVS occurred even in group B but reduction in GAVS (-21% vs. -5%) and PALS (-24% vs. -12%) was significantly greater in group A compared to group B (p-value = .04). CONCLUSIONS: Our study confirms high sensitivity of speckle tracking echocardiography in detecting subclinical myocardial damage in CP and the usefulness of a multiparametric echocardiographic evaluation including PALS and GLS (GAVS) for having a global evaluation of the phenomenon cardiotoxicity.

Glutaminase inhibition as potential cancer therapeutics: current status and future applications.

Alterations in normal metabolic processes are defining features of cancer. Glutamine, an abundant amino acid in the human blood, plays a critical role in regulating several biosynthetic and bioenergetic pathways that support tumour growth. Glutaminolysis is a metabolic pathway that converts glutamine into various metabolites involved in the tricarboxylic acid (TCA) cycle and generates antioxidants that are vital for tumour cell survival. As glutaminase catalyses the initial step of this metabolic pathway, it is of great significance in cancer metabolism and tumour progression. Inhibition of glutaminase and targeting of glutaminolysis have emerged as promising strategies for cancer therapy. This review explores the role of glutaminases in cancer metabolism and discusses various glutaminase inhibitors developed as potential therapies for tumour regression.

ERK5 Interacts with Mitochondrial Glutaminase and Regulates Its Expression.

Many of the biological processes of the cell, from its structure to signal transduction, involve protein-protein interactions. On this basis, our aim was to identify cellular proteins that interact with ERK5, a serine/threonine protein kinase with a key role in tumor genesis and progression and a promising therapeutic target in many tumor types. Using affinity chromatography, immunoprecipitation, and mass spectrometry techniques, we unveiled an interaction between ERK5 and the mitochondrial glutaminase GLS in pancreatic tumor cells. Subsequent co-immunoprecipitation and immunofluorescence studies supported this interaction in breast and lung tumor cells as well. Genetic approaches using RNA interference techniques and CRISPR/Cas9 technology demonstrated that the loss of ERK5 function led to increased protein levels of GLS isoforms (KGA/GAC) and a concomitant increase in their activity in tumor cells. It is well known that the tumor cell reprograms its intermediary metabolism to meet its increased metabolic needs. In this sense, mitochondrial GLS is involved in the first step of glutamine catabolism, one of the main energy sources in the context of cancer. Our data suggest that ERK5 contributes to the regulation of tumor cell energy metabolism via glutaminolysis.

Mesothelioma cancer cells are glutamine addicted and glutamine restriction reduces YAP1 signaling to attenuate tumor formation.

Glutamine addiction is an important phenotype displayed in some types of cancer. In these cells, glutamine depletion results in a marked reduction in the aggressive cancer phenotype. Mesothelioma is an extremely aggressive disease that lacks effective therapy. In this study, we show that mesothelioma tumors are glutamine addicted suggesting that glutamine depletion may be a potential therapeutic strategy. We show that glutamine restriction, by removing glutamine from the medium or treatment with inhibitors that attenuate glutamine uptake (V-9302) or conversion to glutamate (CB-839), markedly reduces mesothelioma cell proliferation, spheroid formation, invasion, and migration. Inhibition of the SLC1A5 glutamine importer, by knockout or treatment with V-9302, an SLC1A5 inhibitor, also markedly reduces mesothelioma cell tumor growth. A relationship between glutamine utilization and YAP1/TEAD signaling has been demonstrated in other tumor types, and the YAP1/TEAD signaling cascade is active in mesothelioma cells and drives cell survival and proliferation. We therefore assessed the impact of glutamine depletion on YAP1/TEAD signaling. We show that glutamine restriction, SLC1A5 knockdown/knockout, or treatment with V-9302 or CB-839, reduces YAP1 level, YAP1/TEAD-dependent transcription, and YAP1/TEAD target protein (e.g., CTGF, cyclin D1, COL1A2, COL3A1, etc.) levels. These changes are observed in both cells and tumors. These findings indicate that mesothelioma is a glutamine addicted cancer, show that glutamine depletion attenuates YAP1/TEAD signaling and tumor growth, and suggest that glutamine restriction may be useful as a mesothelioma treatment strategy.

Design and structural optimization of thiadiazole derivatives with potent GLS1 inhibitory activity.

GLS1 is an attractive target not only as anticancer agents but also as candidates for various potential pharmaceutical applications such as anti-aging and anti-obesity treatments. We performed docking simulations based on the complex crystal structure of GLS1 and its inhibitor CB-839 and found that compound A bearing a thiadiazole skeleton exhibits GLS1 inhibition. Furthermore, we synthesized 27 thiadiazole derivatives in an effort to obtain a more potent GLS1 inhibitor. Among the synthesized derivatives, 4d showed more potent GLS1 inhibitory activity (IC50 of 46.7 µM) than known GLS1 inhibitor DON and A. Therefore, 4d is a very promising novel GLS1 inhibitor.

Design, synthesis, structure-activity relationship studies, and evaluation of novel GLS1 inhibitors.

Glutaminase converts glutamine into glutamic acid and has two isoforms: glutaminase 1 (GLS1) and glutaminase 2 (GLS2). GLS1 is overexpressed in several tumors, and research to develop glutaminase inhibitors as antitumor drugs is currently underway. The present study examined candidate GLS1 inhibitors using in silico screening and attempted to synthesize novel GLS1 inhibitors and assess their GLS1 inhibitory activities in a mouse kidney extract and against recombinant mouse and human GLS1. Novel compounds were synthesized using compound C as the lead compound, and their GLS1 inhibitory activities were evaluated using the mouse kidney extract. Among the derivatives tested, the trans-4-hydroxycyclohexylamide derivative 2j exhibited the strongest inhibitory activity. We also assessed the GLS1 inhibitory activities of the derivatives 2j, 5i, and 8a against recombinant mouse and human GLS1. The derivatives 5i and 8a significantly decreased the production of glutamic acid at 10 mM. In conclusion, we herein identified two compounds that exhibited GLS1 inhibitory activities with equal potencies as known GLS1 inhibitors. These results will contribute to the development of effective novel GLS1 inhibitors with more potent inhibitory activity.

Coronary microvascular dysfunction affects left ventricular global longitudinal strain response to dipyridamole stress echocardiography: a pilot study.

The aim is to investigate, by means of speckle tracking echocardiography, left ventricular (LV) contractile function at rest and during dipyridamole stress in patients with coronary microvascular dysfunction (CMD). 59 patients (39% women, mean age 65.6 ± 6.1 years) with history of chest pain and without obstructive coronary artery disease (CAD) underwent dipyridamole stress echocardiography. Coronary flow was assessed in the left anterior descending coronary artery. Coronary flow reserve (CFR) was determined as the ratio of hyperaemic to baseline diastolic coronary flow velocity. CMD was defined as CFR < 2. Global longitudinal strain (GLS) was measured at rest and at peak dose. Nineteen patients (32%) among the overall population showed CMD. Baseline GLS was significantly lower in patients with CMD (- 16.8 ± 2.7 vs. - 19.1 ± 3.1, p < 0.01). A different contractile response to dipyridamole infusion was observed between the two groups: GLS significantly increased up to peak dose in patients without CMD (from - 19.1 ± 3.1 to - 20.2 ± 3.1, p < 0.01), and significantly decreased in patients with CMD (from - 16.8 ± 2.7 to - 15.8 ± 2.7, p < 0.01). There was a significant inverse correlation between CFR and ∆GLS (r = - 0.82, p < 0.01). Rest GLS and GLS response to dipyridamole stress are markedly impaired among patients with chest pain syndrome, non-obstructive CAD and CMD, reflecting subclinical LV systolic dysfunction and lack of LV contractile reserve due to underlying myocardial ischemia.