Metformin reduces the clonal fitness of Dnmt3aR878H hematopoietic stem and progenitor cells by reversing their aberrant metabolic and epigenetic state.

Clonal hematopoiesis (CH) arises when a hematopoietic stem cell (HSC) acquires a mutation that confers a competitive advantage over wild-type (WT) HSCs, resulting in its clonal expansion. Individuals with CH are at an increased risk of developing hematologic neoplasms and a range of age-related inflammatory illnesses1-3. Therapeutic interventions that suppress the expansion of mutant HSCs have the potential to prevent these CH-related illnesses; however, such interventions have not yet been identified. The most common CH driver mutations are in the DNA methyltransferase 3 alpha (DNMT3A) gene with arginine 882 (R882) being a mutation hotspot. Here we show that murine hematopoietic stem and progenitor cells (HSPCs) carrying the Dnmt3aR878H/+ mutation, which is equivalent to human DNMT3AR882H/+, have increased mitochondrial respiration compared with WT cells and are dependent on this metabolic reprogramming for their competitive advantage. Importantly, treatment with metformin, an oral anti-diabetic drug with inhibitory activity against complex I in the electron transport chain (ETC), reduced the fitness of Dnmt3aR878H/+ HSCs. Through a multi-omics approach, we discovered that metformin acts by enhancing the methylation potential in Dnmt3aR878H/+ HSPCs and reversing their aberrant DNA CpG methylation and histone H3K27 trimethylation (H3K27me3) profiles. Metformin also reduced the fitness of human DNMT3AR882H HSPCs generated by prime editing. Our findings provide preclinical rationale for investigating metformin as a preventive intervention against illnesses associated with DNMT3AR882 mutation-driven CH in humans.

Targeting STAT5 Signaling Overcomes Resistance to IDH Inhibitors in Acute Myeloid Leukemia through Suppression of Stemness.

Mutant isocitrate dehydrogenase 1 (IDH1) and IDH2 block the differentiation of acute myeloid leukemia (AML) cells through production of R-2-hydroxyglutarate (R-2-HG). IDH inhibitors can induce differentiation of AML cells by lowering R-2-HG but have limited clinical efficacy as single agents. Here, we performed a genome-wide CRISPR knockout screen in an Idh1-mutated hematopoietic progenitor cell line to identify genes that increased the differentiation response to ivosidenib, an IDH1 inhibitor. The screen identified C-type lectin member 5a (Clec5a), which encodes a spleen tyrosine kinase (SYK)-coupled surface receptor, as one of the top hits. Knockout of Clec5a and Syk rendered cells more sensitive to ivosidenib-induced differentiation through a reduction in STAT5-dependent expression of stemness-related genes, including genes in the homeobox (HOX) family. Importantly, direct inhibition of STAT5 activity was sufficient to increase the differentiation response to IDH inhibitors in primary human IDH1- and IDH2-mutated AML cells, including those harboring mutations in receptor tyrosine kinase (RTK) and MAPK genes that have been linked to drug resistance. In patient-derived xenograft models of IDH1-mutated AML, combination treatment with ivosidenib and the STAT5 inhibitor pimozide was superior to each agent alone in inducing differentiation in leukemic cells without compromising normal hematopoiesis. These findings demonstrate that STAT5 is a critical mediator of resistance to IDH inhibitors and provide the rationale for combining STAT5 and IDH inhibitors in the treatment of IDH-mutated AML. SIGNIFICANCE: A CRISPR knockout screen identifies a mechanism of resistance to IDH inhibitors in AML involving activated STAT5 signaling, suggesting a potential strategy to improve the clinical efficacy of IDH inhibitors.

Amelioration of methylmercury induced neural damage by essential oil of Selinum vaginatum (Edgew) C. B. Clarke.

Methylmercury (MeHg), an organometallic contaminant is a well spotted cause for a series of disorders, especially in the central nervous system. As there is no proper treatment, Selinum vaginatum (Edgew) C. B. Clarke, a traditional medicinal plant, is taken in the present study for assessing its neuroprotective effect against MeHg induced toxicity using rat brain mitochondrial fractions. The results of 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide dye (MTT) assay indicated that there was a reduction in the mitochondrial viability in MeHg treated sample and IC50 value recorded was 2.5µg/ml. Biochemical analysis showed that there was a significant inhibition of glutathione levels (GSH) and catalase activity and an elevation of thiobarbituric acid reactive substances (TBARS) levels in MeHg treated sample. These changes were prevented by co-incubation with essential oil extracted from Selinum vaginatum. The GSH reduction caused by MeHg is restored by essential oil, endorsing its chelating effect, an important molecular mechanism of defense against oxidative injury. Some of the major compounds are detected in Gas chromatography-mass spectrometry (GC-MS) analysis of essential oil, which could be accountable for its neuroprotection against MeHg.

Smurf2-Mediated Stabilization of DNA Topoisomerase IIα Controls Genomic Integrity.

DNA topoisomerase IIα (Topo IIα) ensures genomic integrity and unaltered chromosome inheritance and serves as a major target of several anticancer drugs. Topo IIα function is well understood, but how its expression is regulated remains unclear. Here, we identify the E3 ubiquitin ligase Smurf2 as a physiologic regulator of Topo IIα levels. Smurf2 physically interacted with Topo IIα and modified its ubiquitination status to protect Topo IIα from the proteasomal degradation in dose- and catalytically dependent manners. Smurf2-depleted cells exhibited a reduced ability to resolve DNA catenanes and pathological chromatin bridges formed during mitosis, a trait of Topo IIα-deficient cells and a hallmark of chromosome instability. Introducing Topo IIα into Smurf2-depleted cells rescued this phenomenon. Smurf2 was a determinant of Topo IIα protein levels in normal and cancer cells and tissues, and its levels affected cell sensitivity to the Topo II-targeting drug etoposide. Our results identified Smurf2 as an essential regulator of Topo IIα, providing novel insights into its control and into the suggested tumor-suppressor functions of Smurf2. Cancer Res; 77(16); 4217-27. ©2017 AACR.

Neuroprotective effect of Tagara, an Ayurvedic drug against methyl mercury induced oxidative stress using rat brain mitochondrial fractions.

BACKGROUND: Methyl mercury (MeHg), an important environmental toxicant is implicated in neurological disorders such as Hunter-Russell syndrome and Autism. Therefore, the present work is in search of new drugs that can alleviate MeHg toxicity. In this connection, Tagara, an ayurvedic drug is used for assessing its neuro protective effect against MeHg toxicity. METHODS: In the present study, we assessed the phytochemical contents of Tagara by colorimetric and HPLC analyses. The neuroprotective effect of Tagara on MeHg induced neurotoxicity was measured in terms of viability by MTT assay and oxidative stress in terms of catalase activity, glutathione and thiobarbituric acid reactive substance levels. Further, the chelating effect of Tagara towards MeHg was performed to identify the molecular mechanism. Statistical analysis was done by statistical package for social sciences (SPSS) version 16.0. RESULTS: The results demonstrated that Tagara contains significant amounts of phenols and flavonoids. Also, HPLC analysis of Tagara revealed the presence of essential oils such as hydroxyvalerenic and valerenic acids. Our results demonstrated that exposure of rat brain mitochondrial fractions to MeHg resulted in a dose dependent death in MTT assay and IC50 value was found to be 10 µM. However, a 250 µg dose of Tagara effectively prevented MeHg induced mitochondrial damage. The oxidative stress caused by MeHg results in elevated levels of reactive oxygen species as evidenced by elevated TBARS (Thiobarbituric acid-reactive substances) levels and diminished catalase enzyme activity and glutathione content. However, Tagara at 250 µg concentration offsets these alterations caused by MeHg. Further, Tagara also diminished GSH oxidation caused by MeHg, confirming its chelating effect, one of the molecular mechanisms that triggers protection against oxidative damage. CONCLUSION: Our results revealed that MeHg induced toxicity is predominantly mediated through oxidative stress mechanism and the propensity of Tagara to abolish such reactions. Hence, we propose that Tagara with a source of potential neuroprotectants may be a useful approach to alleviate MeHg associated neurotoxicity.

Neuroprotective effect of Brahmi, an ayurvedic drug against oxidative stress induced by methyl mercury toxicity in rat brain mitochondrial-enriched fractions.

The present study evaluates the neuroprotective effect of Brahmi against methyl mercury (MeHg) toxicity. The results demonstrated that MeHg-decreased mitochondrial viability in MTT assay and IC50 value was found to be 2.5 µg/mL. However, Brahmi at 250 µg/mL concentration effectively prevented mitochondrial damage caused by MeHg in MTT assay. Our results also demonstrated MeHg significantly inhibited catalase enzyme activity, glutathione content and increased the level of thiobarbituric acid reactive substances in mitochondrial-enriched fractions of rat brain. These alterations were prevented by preincubation with Brahmi. In addition, Brahmi reverted glutathione level to normal that was depleted by MeHg, confirming its chelating effect, one of the molecular mechanisms that underlie protection against oxidative damage. Our study also focused on total phenolic and flavonoid contents of Brahmi, and it was found to contain significant amount of phenols and flavonoids. The presence of saponins was detected by HPLC which might be responsible for neuroprotection against MeHg.