Smaug1 membrane-less organelles respond to AMPK/mTOR and affect mitochondrial function‡

Smaug is a conserved translational regulator that binds numerous mRNAs, including nuclear transcripts that encode mitochondrial enzymes. Smaug orthologs form cytosolic membrane-less organelles (MLOs) in several organisms and cell types. We have performed single-molecule FISH assays that revealed that SDHB and UQCRC1 mRNAs associate with Smaug1 bodies in U2OS cells. Loss of function of Smaug1 and Smaug2 affected both mitochondrial respiration and morphology of the mitochondrial network. Phenotype rescue by Smaug1 transfection depends on the presence of its RNA binding domain. Moreover, we identified specific Smaug1 domains involved in MLO formation, and found that impaired Smaug1 MLO condensation correlates with mitochondrial defects. Mitochondrial Complex I inhibition by rotenone –but not strong mitochondrial uncoupling by CCCP– rapidly induced Smaug1 MLOs dissolution. Metformin and rapamycin elicited similar effects, which were blocked by pharmacological inhibition of AMPK. Finally, we found that Smaug1 MLO dissolution weakens the interaction with target mRNAs, thus enabling their release. We propose that mitochondrial respiration and the AMPK/mTOR balance controls the condensation and dissolution of Smaug1 MLOs, thus regulating nuclear mRNAs that encode key mitochondrial proteins.

Study on the effect of fengycin on the respiration and metabolic mechanism of Penicillium expansum

This study investigate the inhibiting effect of fengycin on respiration and nutrient utilization of Penicillium expansum. The respiratory inhibition rate of the P. expansum was determined by the test of dissolved oxygen fengycin, The effect of fengycin treatment on the activity of P. expansum mitochondrial complex enzyme was detected by mitochondrial enzyme activity assay. The ability of fengycin treatment to P. expansum the utilization of total sugar and total protein was determined by DNS colorimetric method and biuret method. After fengycin treatments, the TCA pathway of respiratory metabolism in P. expansum was inhibited. Besides, fengycin could block the gene expression in P. expansum by binding P. expansum mitochondrial complex enzyme II and III related genes. Therefore, the activity of mitochondrial enzymes was affected. With the increasement of fengycin concentration, the absorption and utilization capacity of P. expansum to total sugar and total protein decreased significantly. Fengycin could inhibit the respiratory metabolism and reduce the biochemical metabolism level in P. expansum and finally caused the growth inhibition.

Identification of ncRNA-mediated prognostic Value and Immune Infiltration of MTHFD2 in Bladder Cancer

Background: Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2), one of mitochondrial enzymes, is involved in folate and nucleic acid metabolism and maintains the cellular redox balance. However, the function of MTHFD2 in bladder cancer is still poorly characterized. This study was designed to elucidate the effect and regulatory mechanism of MTHFD2 on bladder cancer cells and explore the relationships between MTHFD2 and immune cell infiltration in tumor microenvironment (TME). Methods: The data from Oncomine, TIMER, The Cancer Genome Atlas (TCGA) and The Human Protein Atlas (HPA) database were extracted to evaluate the expression of MTHFD2 and its prognostic role in pan-cancer, especially in bladder cancer. Enrichment analyses, were utilized to explore the underlying cellular mechanisms. The ncRNA regulatory axis was established by Starbase database, and the PPI network was constructed by Cytoscape software. Ultimately, the relations between the expression of MTHFD2 and immune cell infiltration in bladder cancer was indicated by TCGA and TIMER databases.Results: Our results demonstrated that MTHFD2 expression was generally up-regulated in pan-cancers and its high expression was correlated with poor prognosis of patients with bladder cancer. Specifically, our study revealed that MTHFD2 was a powerful risk factor and involved in the tumor development of bladder cancer. Furthermore, hsa_circ_0046140 and hsa_circ_0006769/miR-383-5p/MTHFD2 axis could also play a significant role in tumorigenesis. Ultimately, a strong correlation was observed between MTHFD2 expression and various immune cell infiltration, which implied that MTHFD2 might serve as an agent in tumor immunotherapy. Conclusion: MTHFD2 is widely overexpressed in pan-cancers, and its expression is related with the prognosis of patients and the multiple immune cell infiltrates in TME. Besides, hsa_circ_0046140 and hsa_circ_0006769/miR-383-5p/MTHFD2 axis are implicated with the proliferation and invasion of tumors.

Investigation of Carboxylic Acid Isosteres for Inhibition of the Human SIRT5 Lysine Deacylase Enzyme

Sirtuin 5 (SIRT5) is a protein lysine deacylase enzyme that regulates diverse biology by hydrolyzing -N-carboxyacyllysine posttranslational modifications in the cell. Inhibition of SIRT5 has been linked to potential treatment of several cancers but potent compounds with activity in cells have been lacking. Here we developed mechanism-based inhibitors that incorporate isosteres of a carboxylic acid residue that is important for high-affinity binding to the enzyme active site. By masking of the tetrazole moiety of the most potent candidate from our initial SAR study, we achieved potent and cytoselective growth inhibition for the treatment of SIRT5-dependent leukemic cancer cell lines in culture. Thus, we provide an efficient, cellularly active small molecule that targets SIRT5, which can help elucidate its function and potential as a future drug target. This work shows that masked biosisosteres of carboxylic acids are viable chemical motifs for the development of inhibitors that target mitochondrial enzymes, which may have applications beyond the sirtuin field.

Complete Remission with Devimistat (CPI-613) in Refractory Burkitt Lymphoma

Introduction: Patients (pts) with primary refractory or relapsed high-grade lymphoma (HGL) including Burkitt lymphoma (BL) and high-grade B-cell lymphoma with rearrangements of MYC and BCL2 and/or BCL6 (double-hit lymphoma, DHL) have a dismal prognosis with patients almost never achieving a meaningful remission to second line therapy. No standard second line therapeutic approach exists, particularly for BL. The characteristic hallmark of these diseases is a dysregulated MYC oncogene with both downstream effects on proliferation and a high metabolic fluxes which use tricarboxylic acid (TCA) cycle intermediates as biosynthetic precursors. CPI-613 (devimistat) is a non-redox active analogue of lipoic acid, a required cofactor for two key mitochondrial enzymes of the TCA cycle, pyruvate dehydrogenase and alpha ketoglutarate dehydrogenase. Disruption of mitochondrial function by CPI-613 results in a shutdown of ATP and biosynthetic-intermediate production, leading to cancer cell death by apoptosis or necrosis. In the initial phase I trial (n=26) one patient with multiply refractory BL had a partial remission sustained for over one year and then consolidated by surgical resection. She remains alive 7 years later. As of July 2021, 20 clinical studies for various cancers have been conducted (ongoing/completed) with devimistat with over 700 patients having received study drug. We initiated a phase II trial to further explore efficacy in HGL. Devimistat has FDA orphan status for BL and 4 other cancers. Methods: NCT03793140 is a multicenter study aiming to enroll 17 patients on each of two cohorts, BL and DHL, with a Simon's 2-stage design for each cohort, requiring one response among the first 9 treated patients to expand to 17. Patients must have had at least one prior line of therapy or are refusing standard of care and must be more than 3 months after a prior stem cell transplant. Active central nervous system (CNS) parenchymal disease is excluded, but prior leptomeningeal disease is allowed if the CSF is negative for more than 4 weeks at enrollment and maintenance intrathecal therapy is ongoing. Devimistat is given by central line over 2 hours daily x 5 days for two 14-day cycles and then as maintenance x5 days every 21 days. Pts were evaluable for response if they received at least 4 infusions over 5 days of the first cycle. Results: 9 pts were enrolled in the DHL/THL arm. Mediannumber of prior therapies were 3 (range, 1-6). No responses were seen, with only 1 patient achieving stable disease as best response, resulting in cohort closure. Thus far, 8 BL pts were enrolled. Median number of prior therapies was 3 (range, 2-4). Two patients were inevaluable for response. 1/6 patients had stable disease through cycle 7 and one had a complete response (CR). This CR patient (HIV+) with 4 prior therapies entered the study with only a biopsy proven thigh mass. He was not a transplant candidate for social reasons. He had a near complete metabolic remission after 4 cycles of devimistat and a CR after cycle 7. (Table and Figure) As of July 2021, he is in cycle 11, having had a 4-week treatment delay of cycle 5 due to CoVID 19 infection. ECOG improved from 3 to 0. Adverse events (AE): As of July30, 2021, no patient experienced a serious adverse event related to study drug. Four patients had grade 3 events at least possibly related: 2 neutropenia, 1 thrombocytopenia and 1 elevated bilirubin. 1 patient had a dose reduction for grade 2 alanine aminotransferase increase. Conclusions: Although our results are preliminary, the complete remission in this patient is promising in a disease where no viable treatment options exist in the relapsed, refractory BL. Enrollment to the BL cohort is ongoing. Figure 1 Figure 1. Disclosures Nikolaenko: Pfizer: Research Funding; Rafael Pharmaceuticals: Research Funding. Pardee: Celgene/BMS: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Pharmacyclics: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; AbbVie: Membership on an entity's Board of Directors or advisory committees; CBM Biopharma: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Research Funding; Rafael Pharmaceuticals: Research Funding. Abramson: Genentech: Consultancy; Kymera: Consultancy; Karyopharm: Consultancy; AbbVie: Consultancy; Seagen Inc.: Research Funding; Allogene Therapeutics: Consultancy; Astra-Zeneca: Consultancy; Incyte Corporation: Consultancy; BeiGene: Consultancy; Bluebird Bio: Consultancy; Genmab: Consultancy; EMD Serono: Consultancy; Bristol-Myers Squibb Company: Consultancy, Research Funding; C4 Therapeutics: Consultancy; Morphosys: Consultancy; Kite Pharma: Consultancy; Novartis: Consultancy. Horwitz: Vividion Therapeutics: Consultancy; Shoreline Biosciences, Inc.: Consultancy; Tubulis: Consultancy; Verastem: Research Funding; ONO Pharmaceuticals: Consultancy; Myeloid Therapeutics: Consultancy; SecuraBio: Consultancy, Research Funding; Trillium Therapeutics: Consultancy, Research Funding; Seattle Genetics: Consultancy, Research Funding; Millennium /Takeda: Consultancy, Research Funding; Kura Oncology: Consultancy; Janssen: Consultancy; Kyowa Hakko Kirin: Consultancy, Research Funding; Forty Seven, Inc.: Research Funding; Daiichi Sankyo: Research Funding; C4 Therapeutics: Consultancy; Celgene: Research Funding; Aileron: Research Funding; Affimed: Research Funding; Acrotech Biopharma: Consultancy; ADC Therapeutics: Consultancy, Research Funding. Matasar: GlaxoSmithKline: Honoraria, Research Funding; Teva: Consultancy; Janssen: Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Genentech, Inc.: Consultancy, Honoraria, Research Funding; Merck Sharp & Dohme: Current holder of individual stocks in a privately-held company; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; IGM Biosciences: Research Funding; Merck: Consultancy; Juno Therapeutics: Consultancy; TG Therapeutics: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria, Research Funding; Memorial Sloan Kettering Cancer Center: Current Employment; Pharmacyclics: Honoraria, Research Funding; Daiichi Sankyo: Consultancy; ImmunoVaccine Technologies: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Rocket Medical: Consultancy, Research Funding. Noy: Rafael Parhma: Research Funding; Morphosys: Consultancy; Targeted Oncology: Consultancy; Medscape: Consultancy; Pharmacyclics: Consultancy, Research Funding; Janssen: Consultancy, Honoraria; Epizyme: Consultancy. OffLabel Disclosure: CPI-613 (devimistat) is a non-redox active analogue of lipoic acid, a required cofactor for two key mitochondrial enzymes of the TCA cycle, pyruvate dehydrogenase and alpha ketoglutarate dehydrogenase. Disruption of mitochondrial function by CPI-613 results in a shutdown of ATP and biosynthetic-intermediate production, leading to cancer cell death by apoptosis or necrosis

Distribution and Functions of Monodehydroascorbate Reductases in Plants: Comprehensive Reverse Genetic Analysis of Arabidopsis thaliana Enzymes

Monodehydroascorbate reductase (MDAR) is an enzyme involved in ascorbate recycling. Arabidopsis thaliana has five MDAR genes that encode two cytosolic, one cytosolic/peroxisomal, one peroxisomal membrane-attached, and one chloroplastic/mitochondrial isoform. In contrast, tomato plants possess only three enzymes, lacking the cytosol-specific enzymes. Thus, the number and distribution of MDAR isoforms differ according to plant species. Moreover, the physiological significance of MDARs remains poorly understood. In this study, we classify plant MDARs into three classes: class I, chloroplastic/mitochondrial enzymes; class II, peroxisomal membrane-attached enzymes; and class III, cytosolic/peroxisomal enzymes. The cytosol-specific isoforms form a subclass of class III and are conserved only in Brassicaceae plants. With some exceptions, all land plants and a charophyte algae, Klebsormidium flaccidum, contain all three classes. Using reverse genetic analysis of Arabidopsis thaliana mutants lacking one or more isoforms, we provide new insight into the roles of MDARs; for example, (1) the lack of two isoforms in a specific combination results in lethality, and (2) the role of MDARs in ascorbate redox regulation in leaves can be largely compensated by other systems. Based on these findings, we discuss the distribution and function of MDAR isoforms in land plants and their cooperation with other recycling systems.

Higher expression of proline dehydrogenase altered mitochondrial function and increased Trypanosoma cruzi differentiation in vitro and in the insect vector

The pathogenic protist Trypanosoma cruzi uses kissing bugs as intermediate hosts that vectorize the infection among mammals. This parasite oxidizes proline to glutamate through two enzymatic steps and one nonenzymatic step. In insect vectors, T. cruzi differentiates from a noninfective replicating form to nonproliferative infective forms. Proline sustains this differentiation, but to date, a link between proline metabolism and differentiation has not been established. In T. cruzi, the enzymatic steps of the proline-glutamate oxidation pathway are catalysed exclusively by the mitochondrial enzymes proline dehydrogenase [TcPRODH, EC: 1.5.5.2] and D1-pyrroline-5-carboxylate dehydrogenase [TcP5CDH, EC: 1.2.1.88]. Both enzymatic steps produce reducing equivalents that are able to directly feed the mitochondrial electron transport chain (ETC) and thus produce ATP. In this study, we demonstrate the contribution of each enzyme of the proline-glutamate pathway to ATP production. In addition, we show that parasites overexpressing these enzymes produce increased levels of H2O2, but only those overexpressing TcP5CDH produce increased levels of superoxide anion. We show that parasites overexpressing TcPRODH, but not parasites overexpressing TcP5CDH, exhibit a higher rate of differentiation into metacyclic trypomastigotes in vitro. Finally, insect hosts infected with parasites overexpressing TcPRODH showed a diminished parasitic load but a higher percent of metacyclic trypomastigotes, when compared with controls. Our data show that parasites overexpressing both, PRODH and P5CDH had increased mitochondrial functions that orchestrated different oxygen signalling, resulting in different outcomes in relation to the efficiency of parasitic differentiation in the invertebrate host.

The Combined Effects of Wood Vinegar and Perfluorooctanoic Acid on Enzymatic Activities, DNA Integrity and Gene Transcription in Dugesia Japonica

Perfluorooctanoic acid (PFOA) has been identified widely in aquatic environments, while there are few reports detailing the environmental risk of wood vinegar (WV) on freshwater ecosystems. We used freshwater planarians, Dugesia japonica, to evaluate the combined effects of PFOA and WV by examining the adverse influence of enzymatic activities, DNA damage and gene transcription on planarians. Compared with control and PFOA groups, the malonaldehyde content was lower in planarians treated with WV. In addition, WV enhanced the activities of antioxidant enzymes and mitochondrial enzymes, relieving the toxicity effects of PFOA. We also studied the genotoxicity of PFOA and WV on planarians using the comet assay. WV decreased the proportion of DNA in the tail and the olive tail moment. Furthermore, expression of gpx, gst and gr genes in planarians was significantly altered following exposure to both PFOA and WV, compared with individual exposure. Our results suggest that WV could alleviate the adverse effects of PFOA on the aquatic environment, but ecological risk assessments and toxicological safety evaluations of WV are necessary.