Wild-type IDH2 is a therapeutic target for triple-negative breast cancer.

Mutations in isocitrate dehydrogenases (IDH) are oncogenic events due to the generation of oncogenic metabolite 2-hydroxyglutarate. However, the role of wild-type IDH in cancer development remains elusive. Here we show that wild-type IDH2 is highly expressed in triple negative breast cancer (TNBC) cells and promotes their proliferation in vitro and tumor growth in vivo. Genetic silencing or pharmacological inhibition of wt-IDH2 causes a significant increase in α-ketoglutarate (α-KG), indicating a suppression of reductive tricarboxylic acid (TCA) cycle. The aberrant accumulation of α-KG due to IDH2 abrogation inhibits mitochondrial ATP synthesis and promotes HIF-1α degradation, leading to suppression of glycolysis. Such metabolic double-hit results in ATP depletion and suppression of tumor growth, and renders TNBC cells more sensitive to doxorubicin treatment. Our study reveals a metabolic property of TNBC cells with active utilization of glutamine via reductive TCA metabolism, and suggests that wild-type IDH2 plays an important role in this metabolic process and could be a potential therapeutic target for TNBC.

Overexpression of TNFSF11 reduces GPX4 levels and increases sensitivity to ferroptosis inducers in lung adenocarcinoma.

BACKGROUND: Lung adenocarcinoma (LUAD), the most common and lethal subtype of lung cancer, continues to be a major health concern worldwide. Despite advances in targeted and immune therapies, only a minority of patients derive substantial benefits. As a result, the urgent need for novel therapeutic strategies to improve lung cancer treatment outcomes remains undiminished. METHODS: In our study, we employed the TIMER database to scrutinize TNFSF11 expression across various cancer types. We further examined the differential expression of TNFSF11 in normal and tumor tissues utilizing the TCGA-LUAD dataset and tissue microarray, and probed the associations between TNFSF11 expression and clinicopathological parameters within the TCGA-LUAD dataset. We used the GSE31210 dataset for external validation. To identify genes strongly linked to TNFSF11, we engaged LinkedOmics and conducted a KEGG pathway enrichment analysis using the WEB-based Gene SeT AnaLysis Toolkit. Moreover, we investigated the function of TNFSF11 through gene knockdown or overexpression approaches and explore its function in tumor cells. The therapeutic impact of ferroptosis inducers in tumors overexpressing TNFSF11 were also investigated through in vivo and in vitro experiments. Through these extensive analyses, we shed light on the potential role of TNFSF11 in lung adenocarcinoma, underscoring potential therapeutic targets for this malignancy. RESULTS: This research uncovers the overexpression of TNFSF11 in LUAD patients and its inverse correlation with peroxisome-related enzymes. By utilizing gene knockdown or overexpression assays, we found that TNFSF11 was negatively associated with GPX4. Furthermore, cells with TNFSF11 overexpression were relatively more sensitive to the ferroptosis inducers. CONCLUSIONS: Our research has provided valuable insights into the role of TNFSF11, revealing its negative regulation of GPX4, which could be influential in crafting therapeutic strategies. These findings set the stage for further exploration into the mechanisms underpinning the relationship between TNFSF11 and GPX4, potentially opening up new avenues for precision medicine in the treatment of LUAD.

Modulation of energy metabolism to overcome drug resistance in chronic myeloid leukemia cells through induction of autophagy.

Tyrosine kinase inhibitors (TKIs) such as imatinib (IM) are key drugs for treatment of chronic myeloid leukemia (CML). Development of drug resistance to TKIs due to BCR-ABL mutation, especially T315I mutation, poses a major challenge in the clinical treatment of CML. The purpose of this study was to test metabolic modulation as a potential strategy to overcome imatinib resistance based on the possible crosstalk between BCR-ABL signaling and metabolic changes in CML. 2-deoxy-d-glucose (2-DG) was used to modulate the glucose metabolism in CML cells sensitive to IM (KBM5 cell line) and resistant to imatinib with BCR-ABL T315I mutation (KBM5-T315I cell line). Seahorse XFe24 extracellular flux analyzer to quantify oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) was used to measure cellular energy metabolism. Cell proliferation was analyzed by CCK-8 assay and MTS assay. Annexin V/PI staining was used to evaluate cell apoptosis. Autophagy-related proteins and enzyme/proteins were detected by Western blotting. Cellular ATP concentration was detected using an ATP-based Cell Titer Kit. The combined action of 2-DG and IM was evaluated by calculating the drug combination index. Our results found that inhibition of glucose metabolism by 2-DG significantly impaired the viability of CML cells and co-treatment with 2-DG and imatinib induced a synergistic inhibition of KBM5 and KBM5-T315I cells. 2-DG induced cell death by autophagy, not by apoptosis, as evidenced by increased expression of Beclin1 and LC3AII and lack of annexin V/PI-positive cells. At the biochemical level, 2-DG inhibited glycolysis and mitochondrial oxygen consumption manifested by a significant decrease in ECAR and OCR, and a depletion of ATP. The severe metabolic stress induced by 2-DG in CML cells led to autophagic cell death. Our results suggested a metabolic vulnerability of CML cells that could be targeted by a combination of 2-DG and imatinib as an alternative treatment for imatinib-resistant CML.

Reductive TCA cycle catalyzed by wild-type IDH2 promotes acute myeloid leukemia and is a metabolic vulnerability for potential targeted therapy.

BACKGROUND: Isocitrate dehydrogenase-2 (IDH2) is a mitochondrial enzyme that catalyzes the metabolic conversion between isocitrate and alpha-ketoglutarate (α-KG) in the TCA cycle. IDH2 mutation is an oncogenic event in acute myeloid leukemia (AML) due to the generation of 2-hydroxyglutarate. However, the role of wild-type IDH2 in AML remains unknown, despite patients with it suffer worse clinical outcome than those harboring mutant type. METHODS: IDH2 expression in AML cell lines and patient samples was evaluated by RT-qPCR, western blotting and database analyses. The role of wild-type IDH2 in AML cell survival and proliferation was tested using genetic knockdown and pharmacological inhibition in AML cells and animal models. LC-MS, GC-MS, isotope metabolic tracing, and molecular analyses were performed to reveal the underlying mechanisms. RESULTS: We found that wild-type IDH2 was overexpressed in AML and played a major role in promoting leukemia cell survival and proliferation in vitro and in vivo. Metabolomic analyses revealed an active IDH2-mediated reductive TCA cycle that promoted the conversion of α-KG to isocitrate/citrate to facilitate glutamine utilization for lipid synthesis in AML cells. Suppression of wild-type IDH2 by shRNA resulted in elevated α-KG and decreased isocitrate/citrate, leading to reduced lipid synthesis, a significant decrease in c-Myc downregulated by α-KG, and an inhibition of AML viability and proliferation. Importantly, pharmacological inhibition of IDH2 showed significant therapeutic effect in mice inoculated with AML cells with wt-IDH2 and induced a downregulation of C-MYC in vivo. CONCLUSIONS: Wt-IDH2 is an essential molecule for AML cell survival and proliferation by promoting conversion of α-KG to isocitrate for lipid synthesis and by upregulating c-Myc expression and could be a potential therapeutic target in AML.

LY294002 Is a Promising Inhibitor to Overcome Sorafenib Resistance in FLT3-ITD Mutant AML Cells by Interfering With PI3K/Akt Signaling Pathway.

Internal tandem duplications (ITD) mutation within FMS-like tyrosine kinase 3 (FLT3), the most frequent mutation happens in almost 20% acute myeloid leukemia (AML) patients, always predicts a poor prognosis. As a small molecule tyrosine kinase inhibitor, sorafenib is clinically used for the treatment of advanced renal cell carcinoma (RCC), hepatocellular carcinoma (HCC), and differentiated thyroid cancer (DTC), with its preclinical and clinical activity demonstrated in the treatment of Fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) mutant AML. Even though it shows a rosy future in the AML treatment, the short response duration remains a vital problem that leads to treatment failure. Rapid onset of drug resistance is still a thorny problem that we cannot overlook. Although the mechanisms of drug resistance have been studied extensively in the past years, there is still no consensus on the exact reason for resistance and without effective therapeutic regimens established clinically. My previous work reported that sorafenib-resistant FLT3-ITD mutant AML cells displayed mitochondria dysfunction, which rendered cells depending on glycolysis for energy supply. In my present one, we further illustrated that losing the target protein FLT3 and the continuously activated PI3K/Akt signaling pathway may be the reason for drug resistance, with sustained activation of PI3K/AKT signaling responsible for the highly glycolytic activity and adenosine triphosphate (ATP) generation. PI3K inhibitor, LY294002, can block PI3K/AKT signaling, further inhibit glycolysis to disturb ATP production, and finally induce cell apoptosis. This finding would pave the way to remedy the FLT3-ITD mutant AML patients who failed with FLT3 targeted therapy.

Wild-type IDH2 protects nuclear DNA from oxidative damage and is a potential therapeutic target in colorectal cancer.

Although the role of isocitrate dehydrogenase (IDH) mutation in promoting cancer development has been well-characterized, the impact of wild-type IDH on cancer cells remains unclear. Here we show that the wild-type isocitrate dehydrogenase 2 (IDH2) is highly expressed in colorectal cancer (CRC) cells, and plays an unexpected role in protecting the cancer cells from oxidative damage. Genetic abrogation of IDH2 in CRC cells leads to reactive oxygen species (ROS)-mediated DNA damage and an accumulation of 8-oxoguanine with DNA strand breaks, which activates DNA damage response (DDR) with elevated γH2AX and phosphorylation of ataxia telangiectasia-mutated (ATM) protein, leading to a partial cell cycle arrest and eventually cell senescence. Mechanistically, the suppression of IDH2 results in a reduction of the tricarboxylic acid (TCA) cycle activity due to a decrease in the conversion of isocitrate to α-ketoglutarate (α-KG) with a concurrent decrease in NADPH production, leading to ROS accumulation and oxidative DNA damage. Importantly, abrogation of IDH2 inhibits CRC cell growth in vitro and in vivo, and renders CRC cells more vulnerable to DNA-damaging drugs. Screening of an FDA-approved drug library has identified oxaliplatin as a compound highly effective against CRC cells when IDH2 was suppressed. Our study has uncovered an important role of the wild-type IDH2 in protecting DNA from oxidative damage, and provides a novel biochemical basis for developing metabolic intervention strategy for cancer treatment.

Oncogenic K-ras Induces Mitochondrial OPA3 Expression to Promote Energy Metabolism in Pancreatic Cancer Cells.

K-ras (Kirsten ras GTPase) mutations are oncogenic events frequently observed in many cancer types especially in pancreatic cancer. Although mitochondrial dysfunction has been associated with K-ras mutation, the molecular mechanisms by which K-ras impacts mitochondria and maintains metabolic homeostasis are not fully understood. In this study, we used two K-ras inducible cell systems, human pancreatic epithelial/ K-rasG12D (HPNE/K-rasG12D) and human embryonic kidney cells with tetracycline repressorT-Rex/K-rasG12V, to evaluate the role of oncogenic K-ras in regulating mitochondrial function. Among a panel of genes known to affect mitochondria, only the expression of OPA3 (optic atrophy protein 3) was consistently up-regulated by K-ras activation in both cell lines. Importantly, high expression of OPA3 was also observed in clinical pancreatic cancer tissues. Genetic knockdown of OPA3 caused a significant decrease of energy metabolism, manifested by a suppression of oxygen consumption rate (OCR) and a decrease in cellular ATP content, leading to inhibition of cell proliferation capacity and reduced expression of epithelial-mesenchymal transition (EMT) markers. Our study suggests that OPA3 may promote cellular energy metabolism and its up-regulation in K-ras-driven cancer is likely a mechanism to offset the negative impact of K-ras on mitochondria to maintain energy homeostasis. As such, OPA3 could be a potential target to kill cancer cells with K-ras mutations.

Imidazolopiperazines: hit to lead optimization of new antimalarial agents.

Starting from a hit series from a GNF compound library collection and based on a cell-based proliferation assay of Plasmodium falciparum, a novel imidazolopiperazine scaffold was optimized. SAR for this series of compounds is discussed, focusing on optimization of cellular potency against wild-type and drug resistant parasites and improvement of physiochemical and pharmacokinetic properties. The lead compounds in this series showed good potencies in vitro and decent oral exposure levels in vivo. In a Plasmodium berghei mouse infection model, one lead compound lowered the parasitemia level by 99.4% after administration of 100 mg/kg single oral dose and prolonged mice survival by an average of 17.0 days. The lead compounds were also well-tolerated in the preliminary in vitro toxicity studies and represents an interesting lead for drug development.