Wuchereria bancrofti and Brugia spp. affecting over 120 million people. Heavy infections can lead to elephantiasis, which has serious effects on individuals’ lives. Although current anthelmintics are effective at killing microfilariae in the bloodstream, they have little to no effect against adult parasites found in the lymphatic system. The anthelmintic diethylcarbamazine is one of the central pillars of lymphatic filariasis control. Recent studies have reported that diethylcarbamazine can open transient receptor potential (TRP) channels in the muscles of adult female Brugia malayi, leading to contraction and paralysis. Diethylcarbamazine has synergistic effects in combination with emodepside on Brugia, inhibiting motility: emodepside is an anthelmintic that has effects on filarial nematodes and is under trial for the treatment of river blindness. Here, we have studied the effects of diethylcarbamazine on single Brugia muscle cells by measuring the change in Ca2+ fluorescence in the muscle using Ca2+-imaging techniques. Diethylcarbamazine interacts with the transient receptor potential channel, C classification (TRPC) ortholog receptor TRP-2 to promote Ca2+ entry into the Brugia muscle cells, which can activate Slopoke (SLO-1) Ca2+-activated K+ channels, the putative target of emodepside. A combination of diethylcarbamazine and emodepside leads to a bigger Ca2+ signal than when either compound is applied alone. Our study shows that diethylcarbamazine targets TRP channels to promote Ca2+ entry that is increased by emodepside activation of SLO-1 K+ channels.Filarial nematode infections are a major health concern in several countries. Lymphatic filariasis is caused by  Wuchereria bancrofti, Brugia malayi, and Brugia timori. These parasites are transmitted between hosts through biting insects, and the adults live within the lymphatic vessels. In severe cases, the parasites block lymphatic ducts resulting in swelling of limbs and coarsening of the skin resulting in the condition known as elephantiasis. The swelling of limbs can result in disabilities, societal rejection, and prevent individuals from working. There are no effective vaccines against filarial parasites, nor have the measures to control the spread by their vectors been adequate.Lymphatic filariasis (elephantiasis) is a neglected tropical disease caused by filarial nematode parasites that affects more than 120 million people worldwide, with 40 million people suffering from disfiguration or disability  that bind to β-tubulin, inhibiting microtubule formation as well as metabolism ; macrocyBrugia muscle cells, the effect of diethylcarbamazine opening TRP channels is an inward depolarizing current and contraction with spastic paralysis. The inward current produced by diethylcarbamazine is followed by an outward current produced by the activation of Ca2+-activated SLO-1 K+ channels. The anthelmintic emodepside (Emo), which activates nematode SLO-1 channels  and miconazole (MIC), and have observed that they increase Ca2+ entry into the cytoplasm and TRP channel activation. Finally, we identify a synergistic relationship between diethylcarbamazine and emodepside in potentiating the Ca2+ signal that explains the enhanced paralysis observed in motility assays by disrupting the homeostasis of Ca2+ in the muscle cell. These results support previous observations that diethylcarbamazine interacts with TRP channels and that the combination of diethylcarbamazine and emodepside has potential for the treatment of individuals infected with Brugia malayi.In this study, we have utilized CaBrugia malayi worms were used for the study. Live adult female B. malayi were shipped overnight from the NIH/NIAID Filariasis Research Reagent Resource Center . B. malayi were maintained in non-phenol red Hyclone Roswell Park Memorial Institute (RPMI) 1640 media  containing 10% heat-inactivated fetal bovine serum  and 1% penicillin-streptomycin . Parasites were separated individually into a 24-well microtiter plate containing 2 mL of the RPMI media. Parasites were held in an incubator set at 37°C and 5% CO2. All parasites were used up to 5 days post-delivery.Only female B. malayi was performed as previously described , 11 mM D-glucose, 10 mM sucrose, pH 7.2 using NaOH, ~320 mOsmol). The base of the chamber was a coverslip (24 × 50 mm) coated with a thin layer of Sylgard. The body piece was immobilized by gluing each end to the Sylgard pad using Glushield cyanoacrylate glue  and immobilized by creating a wall down one side under the dissecting microscope. The body piece was cut longitudinally using a tungsten needle, and the “muscle flap” was glued to the coverslip along the cut edge exposing the muscle cells. The intestines and the uterus were removed using fine forceps and the prep was washed with bath solution to remove any eggs or debris.Dissection of escribed –18. Al2+ signals, muscles were injected with 5 µM Fluo-4 penta-potassium salt  as previously described and viewed with DIC optics on a Nikon Eclipse TE300 inverted light microscope (400×) -N,N,N′,N′-tetraacetic acid (EGTA), and 36 mM sucrose, pH 7.2 with KOH, ~315–330 mOsmol). Fluo-4 was added to the pipette solution at the start of each recording day at a concentration of 5 µM and was kept in a dark environment to prevent degradation of the dye. Pipettes with a resistance of 1.5–3 MΩ were used. Giga ohm seals were formed before breaking the membrane by suction. After breaking in, cells were left to allow the Fluo-4 solution to diffuse the entire muscle cell (~5 to 10 minutes) (To record Cae (400×) . Brieflyminutes) .2+ signal recordings were acquired and analyzed using MetaFluor 7.10.2 . Exposure times were 150 ms with 2× binning. Maximal Ca2+ signal amplitudes  for all stimuli applied were calculated using the equation F − F0/F0 where F is the fluorescent value and F0 is the baseline fluorescent value, which was determined as the value immediately before the stimulus was applied to the sample for all recordings analyzed. Representative traces were generated using the same formula, with F0 being the value before the application of the first stimulus. For 1 mM CaCl2 bath solution control experiments , fitted with a Photometrics Retiga R1 Camera . Light control was achieved using a Lambda 10-2 two-filter wheel system with a shutter controller . Filter wheel one was set on a green filter  between the microscope and camera. Filter wheel two was set on the blue filter  between a Lambda LS Xenon bulb light box which delivered light via a fiber optic cable to the microscope . Blue light emission was controlled using a shutter. All Caeriments , F0 was2 before being exposed to either 30 µM DEC for 5 minutes, 10 µM AA for 5 minutes, 10 µM MIC for 5 minutes or 1 µM Emo for 5 minutes. Exposure to diethylcarbamazine and SKF96365 was achieved by exposing muscle cells to 10 µM SKF96365 alone (SKF) for 5 minutes, followed by 10 µM SKF96365 and 30 µM diethylcarbamazine for 5 minutes (SKF + DEC), and finally to 30 µM DEC alone for 5 minutes giving a total exposure time of 15 minutes. For diethylcarbamazine and emodepside combination experiments, muscle cells were exposed to 30 µM DEC for 5 minutes and then 30 µM diethylcarbamazine + 1 µM emodepside (DEC + Emo) for 5 minutes, for a total of 10 minutes. To ensure that each of the preparations was viable after being exposed to any of the stimuli tested, all samples were subjected to 10 mM CaCl2 for 1 minute to act as a positive control. Muscles that elicited Ca2+ signals to 10 mM CaCl2 (>10%) were considered viable, while those that failed to elicit response (<10%) were classified as non-viable.The preparation was constantly perfused with all solutions being delivered to the chamber under gravity feed through solenoid valves controlled with a VC-6 six channel Valve Controller  through an inline heater set at 37°C  at a rate of 1.5 mL/min. New preparations were perfused with bath solution containing 1 mM CaClTrp-2 target T7 promoter labeled primers were amplified using the primers trp-2f2, trp-2f2t7, trp-2r2, and trp-2r2t7 previously described (Table S1)   , 15. Nonable S1) , 20 usintrp-2 and reference gene (Bma-gapdh) primers: trp-2f2, trp-2r2, SSK5F, and SSK5R (Table S1) (cDNA from dsRNA-treated worms were amplified using the following target able S1) , 15. Theable S1) .Emodepside was procured from Advanced ChemBlocks and SKF96365 was procured from Tocris; Sigma Aldrich supplied all other chemicals. All compounds were dissolved in either water or dimethyl sulfoxide (DMSO) and diluted in bath solution to obtain final concentrations. DMSO final concentration: 0.01%2+ amplitudes was done using either unpaired or paired Student’s t-tests with P values <0.05 considered as significant using Prism Graphpad version 9.0 software.Statistical analysis of all data was done using GraphPad Prism 9.0 . We repeated our experiments to ensure reproducibility. The number of experiments are provided in the results and figure legends. The concentrations and the duration of applications of diethylcarbamazine, arachidonic acid, miconazole, SKF96365, and emodepside are provided in the methods and legends of the figures. Analysis of Ca2+ signals can be recorded in the muscles of Brugia malayi by directly injecting a fluorescent dye into the cell via a patch pipette  fluctuations in the fluorescent signal  when 10 mM CaCl2 was applied, and that fell when it was removed . We see that diethylcarbamazine simulates a reversible rise in cytoplasmic Ca2+ in Brugia muscle cells that is consistent with the opening of TRP channels that are permeable to Ca2+.We have reported previously that diethylcarbamazine inhibits motility and produces an initial spastic paralysis in e muscle . This ine muscle . We hypo removed . The aveB. malayi muscles as diethylcarbamazine, but the signal was slower than diethylcarbamazine in onset and reaching a peak suggesting that arachidonic acid metabolites were responsible for the activation of the TRP channels. Here, we investigated the role of arachidonic acid on the Ca2+ signal by exposing the muscles to arachidonic acid. 10 µM arachidonic acid stimulated a Ca2+ signal that had a similar profile to that of diethylcarbamazine  by converting arachidonic acid into biologically active and inactive PUFAs via ω-hydroxylases and epoxygenases enzymes. Verma et al.  showed tBrugia muscle; like arachidonic acid, miconazole produces a slow opening of TRP channels current during its application , stromal interacting molecule (STIM-1), voltage gated Cav channels, and K+ channels  compared to muscles not subjected to the dsRNA, 9%  compared to 5.5%  in LacZ controls , while there was no effect on the control 10 mM CaCl2 Ca2+ signal  which failed to decline after emodepside and diethylcarbamazine were removed  do not kill all adult worms, there are concerns that those that survive administration of therapeutic doses will enhance the rate of development of resistance. Also, if a therapeutic MDA program were able to kill all adults so that microfilariae are no longer produced, elimination would be speeded up. This is because adults can survive for 6–8 years producing millions of microfilariae maintaining the life cycle , 30 if t+ channels  when tested in preclinical investigations  that started in 2020. Emodepside opens nematode SLO-1 Kchannels , 13 and igations –33. Thigations –39. Ad filaria , 35. Als filaria , 12, 15 Ascaris suum (Brugia malayi (Brugia malayi from 395 nM to 114 nM an increase in potency of 3.5-fold (2+ to increase cytosolic Ca2+ and thereby increase emodepside activation of the Ca2+-sensitive SLO-1 K+ channels. Here, we have seen that application of diethylcarbamazine increases cytosolic Ca2+ concentrations by activation of the TRP-2 channel. The involvement of TRP-2 channels mediating the effects of diethylcarbamazine on cytosolic Ca2+ is revealed by inhibition of TRP-2 by the TRP-C antagonist, SKF96365, and by RNAi knockdown of trp-2. Furthermore, we were able to observe that not only does diethylcarbamazine increase cytosolic Ca2+ directly but that activation of SLO-1 K+ channels by emodepside enhances the Ca2+ response to diethylcarbamazine (+ channels has been described previously in mammalian systems (2+ into the muscle cell through the TRP channels opened by diethylcarbamazine. Thus, there is a positive feed-back loop with diethylcarbamazine enhancing the effects of emodepside on SLO-1 K+ channels and emodepside enhancing the entry of Ca2+ produced by diethylcarbamazine (We have observed in ris suum  and in aa malayi , 15 thatbamazine . The pos systems . The opebamazine . This po2+ that is maintained during its application. The increased cytosolic Ca2+ will activate the contractile machinery of the muscle cells and limit the normal vibrating muscle activity that is seen in healthy worms. Emodepside enhances the effects of diethylcarbamazine with a positive feed-back loop: diethylcarbamazine opens TRP-2 channels, increasing cytosolic Ca2+, and activating SLO-1 K+ channels; and emodepside hyperpolarizes the membrane potential of the muscle, increasing the driving force for the entry of Ca2+. The combination of diethylcarbamazine and emodepside may be useful where the potency of either drug is limited by species of parasite or location of the parasite.We have seen that diethylcarbamazine has a rapid effect on increasing cytosolic Ca


Malignant hematopoietic cells gain metabolic plasticity, reorganize anabolic mechanisms to improve anabolic output and prevent oxidative damage, and bypass cell cycle checkpoints, eventually outcompeting normal hematopoietic cells. Current therapeutic strategies of acute myeloid leukemia (AML) are based on prognostic stratification that includes mutation profile as the closest surrogate to disease biology. Clinical efficacy of targeted therapies, e.g., agents targeting mutant FMS-like tyrosine kinase 3 (FLT3) and isocitrate dehydrogenase 1 or 2, are mostly limited to the presence of relevant mutations. Recent studies have not only demonstrated that specific mutations in AML create metabolic vulnerabilities but also highlighted the efficacy of targeting metabolic vulnerabilities in combination with inhibitors of these mutations. Therefore, delineating the functional relationships between genetic stratification, metabolic dependencies, and response to specific inhibitors of these vulnerabilities is crucial for identifying more effective therapeutic regimens, understanding resistance mechanisms, and identifying early response markers, ultimately improving the likelihood of cure. In addition, metabolic changes occurring in the tumor microenvironment have also been reported as therapeutic targets. The metabolic profiles of leukemia stem cells (LSCs) differ, and relapsed/refractory LSCs switch to alternative metabolic pathways, fueling oxidative phosphorylation (OXPHOS), rendering them therapeutically resistant. In this review, we discuss the role of cancer metabolic pathways that contribute to the metabolic plasticity of AML and confer resistance to standard therapy; we also highlight the latest promising developments in the field in translating these important findings to the clinic and discuss the tumor microenvironment that supports metabolic plasticity and interplay with AML cells. Although conventional chemotherapy is known to eliminate bulk tumor cells, its utility is limited in AML, as older patients lack tolerance to chemotherapy and the drug may lack efficacy at eliminating leukemia stem cells (LSCs). Residual clones that survive chemotherapy lead to disease relapse. Moreover, AML exhibits extensive genetic heterogeneity, as it often harbors a complex mixture of heterogenous subclones that possess genetic aberrations in diverse driver genes, with distinct clonal evolutionary patterns existing in single individuals. Cytogenetic abnormalities and acquired somatic mutations are used as surrogate markers for risk assessment and some mutations can be specifically targeted. However, the utility of therapies that target mutations viz. FMS-like tyrosine kinase 3 (FLT3) and isocitrate dehydrogenase enzyme 2 (IDH2) are limited to the context of the particular mutation.Acute myeloid leukemia (AML) is a cancer derived from the myeloid lineage of blood cells. It is characterized by overproduction of leukemic blasts and maturation arrest. With approximately 120,000 cases a year globally, AML is the most common type of acute leukemia in adults. AML cells exhibit unique metabolic dependencies compared with those of their normal counterparts, the noncancerous blasts. Hematopoietic stem cells (HSCs) use mitochondrial oxidative phosphorylation (OXPHOS) during differentiation to meet their higher energy requirements; however, they use anaerobic glycolysis for their routine energy needs and generally avoid aerobic mitochondrial OXPHOS. Of note, mitochondrial OXPHOS generates reactive oxygen species (ROS), which are deleterious for HSCs. In fact, ROS functions as a signaling mediator in the crosstalk between metabolism and stem cell fate decisions by inhibiting the repopulating capacity of HSCs. ROS-high-HSCs are characterized by low self-renewal capacity and high myeloid differentiation capacity compared to ROS-low HSCs.Cancer cells are known to reprogram their metabolism to support their survival and proliferation; this was recently recognized as a hallmark of cancer. Interestingly, mitochondria distribution during stem cell division appears to be crucial to determining the fate of HSCs. Of note, mitochondrial division is asymmetrical during stem cell division and daughter cells that receive fewer old mitochondria maintain stem cell traits. Notably, through a ROS-mediated physiological process, changes in mitochondrial dynamics regulate stem cell fate decisions, triggering a dual program to suppress self-renewal and promote differentiation,7. Evasion from mitochondrial OXPHOS and reliance on glycolysis help prevent HSC pool exhaustion. In contrast, LSCs rely on mitochondrial OXPHOS to generate high-energy compounds while maintaining ROS levels at non-toxic levels by employing a wide repertoire mechanism for ROS mitigation. Therefore, it may be possible to target leukemic cells differently based on their metabolic needs for therapeutic purposes.Upon cell division, HSCs have fate choices when they undergo cell division; they either undergo self-renewal, wherein they produce new HSCs, or differentiate, wherein they produce cells that mature into committed cells. On the basis of the status of the daughter cells, HSC division can occur in one of the following ways: (1) asymmetric division, which maintains the HSC pool, in which one daughter cell remains as stem cell while the other differentiates; (2) symmetric commitment, in which both daughter cells differentiate (stem cell exhaustion); or (3) symmetric division, in which two daughter stem cells are produced, which helps in HSC expansionIn this review, we discuss the role of cancer metabolic pathways in AML development that contribute to the metabolic plasticity of the disease and confer resistance to standard therapy. We also highlight the latest developments in the field including clinical trials for translating metabolic inhibitors to clinic  and disc. In different cancers, including AML, depletion of mtDNA results in OXPHOS deficiency. OXPHOS-deficient cancer cells fail to form tumors unless mtDNA is restored from stromal cells via the horizontal transfer of whole mitochondria-13, suggesting that functional OXPHOS is essential for cancer development, although it is not clear which component of OXPHOS contributes the most to tumor formation. For instance, aspartate supplementation enables cells to proliferate under pharmacological inhibition of electron transport chain (ETC), suggesting that although adenosine triphosphate (ATP) production is the primary function of OXPHOS, proliferating cancer cells obtain oxidizing power and aspartate for pyrimidine biosynthesis through aerobic respiration,15, thus, in addition to ATP or ROS levels, there may be other mechanisms underlying the antiproliferative effects of ETC inhibition. Several compounds block complex I , with high affinity viz. IACS-010759, IM156, BAY87-2243, and ME-344.AML cells are more sensitive to mitochondria-targeted drugs, likely because despite having increased respiratory activity, their mitochondria have lower coupling efficiency and spare reserve capacity due to proton leak. BAY87-2243, an inhibitor of hypoxia-inducible factor-1 (HIF-1) that inhibits mitochondrial complex I activity, causes grade III nausea/vomiting [NCT01297530], while ME-344 showed no clinical efficacy in patients with solid cancers,20.The OXPHOS inhibitor IACS-010759 is currently being evaluated in phase 1 clinical trials in relapsed/refractory AML [16]. BAY. The altered function of this protease contributes to misfolding or degradation of respiratory chain subunits and their accumulation, causing respiratory chain dysfunction. The imipridones ONC201 and ONC212 inhibited the growth and viability of leukemia cells by hyperactivating mitochondrial ClpP and thus selectively proteolyzing certain mitochondrial matrix proteins. A significant reduction in the leukemic burden and improved survival was observed in mice that were administered ONC201 orally. In a clinical trial, a single oral dose of ONC201 resulted in a decrease in circulating blasts and a subsequent increase in platelet counts in patients with relapsed/refractory AML.Oxidative phosphorylation integrity is maintained by a protease, caseinolytic protease proteolytic subunit (ClpP) in AML cells. In vivo, mubritinib delayed AML development without affecting normal hematopoiesis in mice. An isoquinoline alkaloid, berberine, targets mitochondrial ETC complex I, leading to reduced mitochondrial activity and enhanced antileukemic effects in vitro and in vivo when combined with IDH1 mutant inhibitor AG-120.Mubritinib (TAK-165) is a direct and ubiquinone-dependent ETC complex I inhibitor which induces ROS accumulation. Mubritinib also decreases ATP/ADP and NAD/NADH concentration ratios and inhibits the activities of PDH, tricarboxylic acid cycle (TCA), and OXPHOS, which results in the death of AML cells treated with mubritinib. Mechanistically, CPI-613 inhibits the TCA cycle by displacing lipoic acid, a cofactor for the TCA cycle enzymes pyruvate dehydrogenase and α-ketoglutarate dehydrogenase. This displacement, in turn, prevents the enzymes’ activity and halts the TCA cycle.A rationally designed lipoic acid analog, CPI-613, is currently being investigated in clinical trials for patients with pancreatic cancer and acute myeloid leukemia. Metformin was found to synergistically sensitize AML cells to Ara-C by inhibiting the mammalian target of rapamycin (mTOR)C1/P70S6K pathway. By promoting autophagy and apoptosis mediated by mTOR, metformin sensitizes FLT3-ITD-positive acute myeloid leukemia to sorafenib. Additionally, metformin and phenformin are also potentiators of the cytotoxicity of Bcl2 antagonist - ABT-737 against leukemia. By inhibiting protein production, metformin downregulates antiapoptotic Mcl-1 and Bcl-xl expression, resulting in a synergistic anti-tumor effect with ABT-199 in AML. It also exhibited anticancer activity with the MCL1 inhibitor S63845 via redox remodeling. IM156 is another oral biguanide OXPHOS inhibitor of mitochondrial ETC Protein Complex 1, which activates AMPK and is well tolerated at doses active in preclinical models with modest clinical efficacy [NCT03272256] in solid cancer patients. Though the biguanides showed promising efficacy in preclinical studies, they are still under development and warrant further investigation in clinical trials for AML to establish their clinical utility.Metformin is being evaluated in combination with chemotherapy in different cancers; it sensitizes AML cells to Ara-C by reducing the mitochondrial transfer from bone marrow stromal cells (BMSCs) and OXPHOS in the recipient cells. The ability of cytarabine to induce mitochondrial ROS is a major determinant of cytarabine sensitivity in primary AML cells. Resistance to cytarabine is often mediated by SIRT3, a NAD+-dependent protein deacetylase that deacetylates mitochondrial anti‐oxidant enzymes, including IDH2 and superoxide dismutase 2 (SOD2). SIRT3‐mediated deacetylation increases the enzymatic activity of its antioxidant targets, thereby restricting cytarabine-induced mitochondrial ROS production and resulting in reduced apoptosis and increased chemoresistance. In addition, SIRT3 drives OXPHOS and increases the levels of both dihydronicotinamide adenine dinucleotide phosphate (NADPH) and reduced glutathione (GSH) while decreasing the concentrations of their respective oxidized forms. SIRT3 activation conferred resistance to chemotherapy in-vivo and its inhibition showed a synergistic effect with cytarabine. SIRT3 SUMOylation was higher in Ara-C sensitive primary cells, but lower in resistant primary cells. De-SUMOylation of SIRT3 is mediated by SUMO-specific peptidase 1 (SENP1), which enhances its deacetylase activity by inhibiting its protein degradation. When SIRT3 is de-SUMOylated, the expression of HES1, a negative regulator of FAO, is decreased and FAO is upregulated; either FAO inhibitors or overexpression of HES1 can attenuate this effect. The use of Momordin-Ic a, SENP1 inhibitor or HES1 overexpression in vitro and in vivo showed synergy with cytarabine to eradicate AML cells. Although these studies demonstrate a role for SIRT3 in chemoresistance in AML, the number of patients included in these studies is limited, and therefore validation is required in larger cohorts.Cytarabine remains the backbone of AML chemotherapy. Interestingly, pre-existing and persisting cytarabine-resistant cells do not necessarily reside in a quiescent or LSC compartment but rather have higher OXPHOS levels, with polarized mitochondria and an increased mitochondrial massOXPHOS gene signature that is predictive of clinical response to treatment in PDX models and AML patients. Inhibition of mitochondrial protein biosynthesis, electron transfer, or FAO resulted in reduced OXPHOS and strikingly increased efficacy of cytarabine in AML. Another mechanism of cytarabine resistance is mediated by upregulation of CD39, an ectonucleotidase that is localized on the cell surface and hydrolyses extracellular ATP and ADP to produce adenosine. Increased activity of CD39 promotes mitochondrial activity and biogenesis by activating the cAMP-mediated adaptive mitochondrial stress response, which promotes resistance to cytarabine. The activation of cAMP-PKA signaling driven by CD39 through P2RY13 purinergic receptor causes metabolic reprogramming and promotes the survival of chemo-resistant AML cells. Thus, CD39-P2RY13-cAMP-OxPHOS axis plays a key role in cytarabine resistance; inhibiting CD39 ecto-ATPase activity enhanced cytarabine’s cytotoxicity in AML by blocking the mitochondrial reprogramming caused by the drug.Notably, following cytarabine therapy, residual cells showed increased FAO, increased CD36 expression, (a fatty acid translocase) and higher expression of an ,38. IDH1/2 mutations detected in AML are predominantly heterozygous point mutations that not only render the protein incapable of carrying out decarboxylation of isocitrate, but confer it the ability to produce 2-hydroxyglutarate (2-HG), an oncometabolite, by NADPH-dependent reduction of α-KG. 2-HG inhibits several α-KG-dependent dioxygenases, such as demethylases and the TET family of 5-methlycytosine hydroxylases. TET regulates the gene expression of its target genes through DNA demethylation and chromatin modification, such as H2B O-linked N-acetylglycosylation (O-GlcNAcylation) or H3K4 trimethylation. The loss of α-KG in conjunction with the suppression of α-KG-dependent dioxygenases mediated by 2-HG promotes leukemogenesis,43. In addition, loss of NADPH due to IDH mutations perturbs the regulation of redox balance in the cell.The isocitrate dehydrogenases enzymes, IDH1 and IDH2, catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG) in the cell cytoplasm and mitochondria, respectively, and reduce NADP+; this, in turn, contributes to the generation of NADPH. α-KG is essential for the optimal functioning of multiple metabolic and epigenetic processes, while NADPH functions as a reducing agent and plays a crucial role in maintaining the cellular redox state. IDH1/2 mutations have been found in 1 of every 5 AML patients with a higher preponderance among patients with normal karyotypes while many other inhibitors including Olutasidenib (FT-2102), IDH305, Vorasidenib (AG881), BAY1436032, LY3410738 and DS-1001 are currently being evaluated in Phase I/II clinical trials in patients with IDH1/2 mutant AML. The IDH2 inhibitor Enasidenib (AG-221) and Ivosidenib are approved for relapsed/refractory AML in the presence of relevant mutations and ivosidenib was recently approved, in combination with hypomethylating agents, as first-line therapy for IDH1 mutant AML .The emergence of second-site IDH2 mutations in trans, at glutamine 316 (Q316E) and isoleucine 319 (I319M), which are located at the interface of the enasidenib binding site in the IDH2 dimer, were found to confer therapeutic resistance to enasidenib. Although the expression of these mutant proteins alone is not sufficient for the synthesis of 2HG, the presence of R140Q mutation in trans produces 2HG, which is resistant to inhibition by enasidenib, glioma,55, cholangiocarcinoma,56, and AML,57. Conversely, inhibition of mutant IDH1/2 may counteract the cytotoxic effect of chemotherapeutic drugs. In IDH1/2 mutants, D2HG mediates the downregulation of ATM, a DNA damage response gene. Therefore, IDH1/2 mutant AML cells are sensitive to DNA damage-causing agents, such as daunorubicin, and the PARP inhibitors olaparib and talazoparib, while IDH1/2 mutant inhibitors had a protective effect against these treatments. Intriguingly, IDH1/2 mutation sensitized primary AML cells to ABT199 by inhibiting cytochrome c oxidase  by 2HG. The cytochrome c oxidase complex contains two heme moieties (a and a3) and two copper atoms (CuA and CuB). A binding of (R)-2-HG at or near the binuclear center of heme a3 and CuB inhibits cytochrome c oxidase activity, thereby blocking oxygen reduction at that site. Suppression of cytochrome c oxidase activity results in oxygen deprivation, which triggers activation of BAX/BAK and leads to outer membrane permeabilization and apoptosis. When BAX/BAK are bound to BCL-2, permeabilization is prevented, but ABT-199 disrupts this binding so that IDH1/2 mutant cells die, while wild-type IDH1/2 cells are relatively unaffected. IDH1/2 mutant AML exhibited a superior response (36%) to BCL-2 inhibition compared to IDH1/2 wildtype (9%). Patients with IDH-mutant AML show robust responses to venetoclax + azacytidine. This is a great example of a driver mutation that creates a unique metabolic vulnerability.Mutations in IDH1/2 have been reported to confer sensitivity to several chemotherapeutic agents in most cancer types, including colorectal cancer. These mutations often affect transcription factors that are involved in hematopoietic and myeloid differentiation, particularly RUNX1 and CEBPA. An association was found between co-mutation(s) of RUNX1 at baseline and a lower CR rate with IDH inhibitor. RUNX1 mutations were also the most frequently acquired mutations in relapsed disease. In addition, co-occurrence of CEBPA mutation was associated with a lack of response to IDH inhibitor, and the mutation was also reported to be acquired at relapse. Because of their roles as differentiation factors, RUNX1 and CEBPA mutations may interfere with differentiation signals induced by IDH inhibitors, resulting in clinical resistance. Almost 30% of relapse cases were found to have acquired mutations in either NRAS or KRAS during relapse. Interestingly, Tateishi et al. found that IDH1 mutation may cause addiction to NAD+ in solid cancers. The mutant form of IDH1 lowered NAD+ levels through downregulation of the enzyme nicotinate phosphoribosyltransferase (NAPRT1) of the NAD+ salvage pathway and thus sensitized cells to NAD+ depletion when combined with inhibition of nicotinamide phosphoribosyltransferase (NAMPT). Depletion of NAD+ activated AMPK, triggering pro-cell death autophagy, resulting in cell death.Primary resistance to IDH inhibitors is primarily associated with leukemia stemness, while acquired resistance is often conferred by mutations in the genes belonging to the RUNX1/CEBPA or RAS-receptor tyrosine kinase (TK) pathways or genes such as BCOR, and TET2-67, and can drive the TCA cycle by providing glutamate; through glutaminolysis, glutamate is then converted to α-ketoglutarate. In addition, it can serve as a substrate in the synthesis of GSH, a ROS mitigator,68 . Inhibition of glutaminolysis with CB-839 blocks glutamine utilization, resulting in decreased levels of glutamate, aspartate and several TCA cycle intermediates. As described in a previous study. Jones et al. demonstrated that depletion of amino acids, especially cysteine, was strongly associated with response to venetoclax + azacytidine regimen compared to ROS induction. LSCs in patients with de novo AML rely on amino acid metabolism to drive OXPHOS. In LSCs, the depletion of cysteine causes a reduction in GSH levels, which, in turn, leads to decreased glutathionylation of succinate dehydrogenase A (SDHA), a component of ETC complex II. This decreased glutathionylation of SDHA results in OXPHOS inhibition and ATP depletion, ultimately leading to LSC death. BCL-2 inhibition is believed to promote the generation of mitochondrial permeability transition pores, resulting in leakage of mitochondrial membrane and reduced amino acid metabolism. The combination of venetoclax and azacytidine has shown good efficacy in older AML patients, who are generally considered unfit for traditional chemotherapy due to high toxicity and poor outcomes with chemotherapeutic agents. In fact, in a Phase III study, this regimen resulted in higher complete remission compared to the placebo control , the composite complete remission was reported in 66.4% AML patients receiving ven + aza vs. 28.3% in placebo control; P < 0.001. The study reported that almost half of all patients receiving azacitidine plus venetoclax had a first response (complete remission or complete remission with incomplete hematologic recovery) before starting cycle 2, and their remissions lasted an average of 17.5 months. Of note, the remission rate after venetoclax + azacytidine in patients with de novo AML is independent of the genetic background, suggesting that venetoclax targets a unique metabolic program of LSCs.LSCs exhibit increased uptake and catabolism of amino acids. Venetoclax + azacytidine is highly effective in AML patients because of its action at inhibiting amino acid metabolism in LSCs,82. As they lack argininosuccinate synthetase-1 (ASS1), a key enzyme required for the generation of arginine, these cells rely on arginine import. The depletion of extracellular arginine via the use of pegylated arginine deiminase, which converts plasma arginine to citrulline, induced responses in AML through the activation of caspases. Depleting extracellular arginine shows synergy with cytarabine and enhances its cytotoxicity. Arginine and cysteine metabolism are also controlled by the stroma in CLL, which may promote the resistance and growth of leukemic cells; this can be targeted by arginine depletion.Primary AML cells are auxotrophic for glutamine, cysteine, and arginine,86. BCAAs promote cancer growth in de novo AML. LSCs exhibit elevated levels of branched-chain amino acid transaminase 1 (BCAT1), a cytoplasmic aminotransferase that generates glutamate by transferring an amino group from a BCAA to α-KG, which can in turn fuel OXPHOS.The survival of HSCs and LSCs is influenced by branched-chain amino acids (BCAAs), such as leucine, isoleucine, and valine. For instance, valine is essential for survival and maintenance of HSCsde novo nucleotide biosynthesis to meet their nucleotide requirements. As expected, the expression of enzymes involved in nucleotide biosynthesis is elevated in cancer. Uridine monophosphate (UMP) is generated in de novo nucleotide biosynthesis from glutamine. Similarly, dihydroorotate dehydrogenase (DHODH) is a mitochondrial enzyme that converts dihydroorotate (DHO) into orotate through oxidation, in de novo pyrimidine biosynthesis and donates the electrons to ubiquinone to generate ubiquinol, which is then re-oxidized by ETC complex III. In experimental models of AML, inhibition of DHODH enhances myeloid differentiation. In addition, a decrease in leukemia-initiating cells, as well as increased survival in vivo, have also been observed following the inhibition of DHODH. DHODH inhibition promotes myeloid differentiation by depleting uridine, a precursor of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc). The depletion of uridine diphosphate-GlcNAc decreases O-linked N-acetylglycosylation (O-GlcNAc) of several proteins including Akt, TET family of proteins and c-Myc. The loss of O-GlcNAc modification following DHODH inhibition reduced the stability of c-Myc and thereby promoted differentiation in AML. In addition, DHODH inhibition revealed the dysregulation of several key genes that are involved in a maturation block of AML cells, such as HOXA9, FLT3 and c-Myc. Of note, DHODH inhibition using BAY 2402234 induced acute downregulation of HMGA1, a chromatin remodeler that is implicated in stemness, and would otherwise negatively regulate differentiation. In clear cell renal cell carcinoma, the DHODH inhibitor selectively inhibited tumor growth in GPX4-low tumors, while combined treatment with sulfasalazine, which also induces ferroptosis, synergistically inhibited the growth of GPX4-high tumors.Proliferating cells require an abundance of nucleotides. They heavily depend on ,95. ETC complex III helps in maintaining the oxidation of ubiquinol, which is essential to supporting DHODH activity. Interventions targeting respiratory Complex III would increase the generation of ROS and block the activity of DHODH and pyrimidine biosynthesis,98, and could thus be an effective two-pronged strategy involving the exploitation of cell death promoted by ROS and differentiation induced by DHODH inhibition. Alternative oxidase (AOX) can restore DHODH activity and reactivate CoQ redox-cycling following DHODH inhibition.Notably, several clinical trials have reported that brequinar, a DHODH inhibitor, has limited efficacy, likely related to a schedule that did not allow sustained exposure. AML’s dependency on ETC complex I has been well-documented. As a result, there has been an increasing interest in targeting ETC complex I and induction chemotherapy together.Aspartate biosynthesis is a crucial step in pyrimidine biosynthesis, which requires ETC complex I activityde novo AML patients, suggesting that they have alternative resistance mechanisms. Jones et al. (2020) determined whether LSCs from relapsed/refractory AML patients exhibited altered metabolome profiles compared to those from de novo AML; they identified 18 metabolites that were upregulated, including amino acids and nicotinamide. Interestingly, relapsed LSCs exhibited an enhanced rate of NAD+ synthesis driven by the salvage pathway, with no difference in the protein levels of NAMPT, the rate-limiting enzyme responsible for the generation of NAD+ from nicotinamide, suggesting an increase in nicotinamide uptake in relapsed/refractory LSCs. The elevation of nicotinamide metabolism in relapsed LSCs, activates amino acid metabolism and FAO, which in turn drives OXPHOS. An increase in OXPHOS would help LSCs to confer resistance to venetoclax + azacytidine.LSCs from relapsed/refractory AML patients are resistant to venetoclax + azacytidine, in contrast to those from de novo LSCs, relapsed/refractory LSCs exhibit extensive metabolic plasticity, which enables them to adapt alternative mechanisms for activating OXPHOS, a key mechanism for developing resistance to chemotherapy. Given that therapeutic options are limited for relapsed/refractory AML, the inhibition of NAMPT, the rate-limiting enzyme for NAD+ synthesis, may be an effective approach. Notably, relapsed/refractory LSCs were selectively eradicated by the genetic and pharmacological inhibition of NAMPT, while normal hematopoietic stem/progenitor cells were spared. Of note, in secondary transplantation of relapsed/refractory AML LSCs, in-vivo treatment with APO866, a small molecule inhibitor of NAMPT, compromised the engraftment.Unlike vs. exhaustion) by providing the required energy to allow the proper execution of asymmetric division, favoring the maintenance and function of the HSC compartment. Loss of the mitochondrial FAO activator peroxisome proliferator-activated receptor δ (PPAR-δ) perturbs HSC maintenance, whereas treatment with PPAR-δ agonists restores HSC maintenance by promoting asymmetric HSC division. PPAR-δ activates FAO in mitochondria. LSCs from de novo AML patients drive OXPHOS exclusively using amino acid metabolism. In contrast, LSCs from relapsed/refractory AML patients tend to compensate significantly through increased FA metabolism. Despite the importance of FAs for mitochondrial metabolism in LSCs, aberrant lipid metabolism may also promote cancer cell proliferation by increasing its energy availability, structural lipid-based building blocks, and signaling molecules. Vriens et al. demonstrated that cancer cells exhibit extensive plasticity in FA metabolism and can facilitate membrane biosynthesis during proliferation through the FA desaturation pathway. Further investigation is needed to determine whether such metabolic plasticity exists in leukemic cells and if it could be targeted.FA catabolism has been implicated in regulating HSC fate , a key enzyme of the mitochondrial FAO pathway, through its hydrophobic surface. Thus, MCL1 not only suppresses apoptosis in cancer cells, but also regulates β-oxidation in response to stress. Of note, Mcl-1 is essential for the efficient assembly of F1F0-ATP synthase oligomers and thus is important for maintaining mitochondrial cristae structure and efficient ATP synthesis. Another study revealed the bifunctional activities of BAD, demonstrating glucokinase regulation by the proapoptotic BH3 domain of BAD. The findings of these studies have opened a new paradigm, revealing the role of Bcl2 family proteins in not only regulating mitochondrial apoptosis but also energy metabolism in cancer cells. It would be interesting to further explore whether Bcl2 family protein mediated regulation of metabolism plays a role in resistance to standard chemotherapies in AML and to further identify downstream druggable non-canonical targets.Mcl1 is an antiapoptotic protein that is known to interact with proapoptotic proteins in mitochondria. Mcl1 upregulation is common in venetoclax-resistant AML. Ceramidases convert ceramide to sphingosine. Kao et al. found that daunorubicin and cytarabine treatment resulted in the increased expression and activity of enzymes involved in the ceramide pathway, leading to a decrease in ceramide levels and an increase in ceramide 1- and sphingosine 1-phosphate levels. These perturbations induced remodeling of mitochondria in cancer cells and promoted chemoresistance. For instance, FLT3-ITD signaling activation suppresses pro-cell death lipid ceramide generation in AML cells. Intriguingly, LCL-461, a mitochondria-targeted ceramide analog, effectively induced lethal mitophagy in human AML blasts and crenolanib (FLT3-ITD inhibitor)-resistant AML xenografts, suggesting that activation of mitochondrial ceramide synthesis overcomes resistance to FLT3 inhibition.Cellular levels of ceramide are key determinants of response to several chemotherapeutic drugs. Modulation of the ceramide pathway has been implicated in apoptosis induced by daunorubicin in AML cellsde novo cholesterol synthesis through the mevalonate pathway plays a key role in AML. Recently, using genome-scale metabolic modeling, Karakitsou et al. demonstrated that AraC-resistant cells could be targeted by suppressing cholesterol biosynthesis through the inhibition of squalene synthase.A previous study reported that . FASN is significantly elevated in AML blasts compared to healthy granulocytes or CD34+ hematopoietic progenitors at the RNA level. Inhibition of FASN expression on one side accelerated differentiation of APL cells, on the other hand, sensitized ATRA resistant -non-APL AML cells to ATRA, by promoting translocation of transcription factor EB (TFEB) to nucleus and lysosomal biogenesis.Acetyl-CoA is converted to malonyl-CoA by Ac-CoA carboxylases (ACCs). FA synthase (FASN) catalyzes the condensation of malonyl-CoA and Ac-CoA to generate palmitate as the first product of FA synthesis(ACC), namely ACC1 and ACC2. AMPK inhibits ACC under energy stress and thus activates fat synthesis and catabolism. Nutrient abundance, on the other hand, downregulates the activity of AMPK, and ACC1 and ACC2 are no longer repressed. This suggests that ACCs act as a regulatory node in FA metabolism by sensing nutrient availability and balancing the switch between anabolic and catabolic processes. Of note, ACC1 suppressed growth-promoting activity and promoted the generation of ROS in primary BM cultures. In addition, ACC1 has been shown to promote myeloid differentiation and delay the development of AML in mice. Hydroxylation of ACC2 by prolyl-hydroxylase 3 (PHD3) increases the activity of ACC2, which, in turn, prevents FA utilization,118. PHD3 levels are decreased in AML, thus generating a vulnerability due to dependence on FAs, which can make FAO inhibition an attractive strategy to control AML.Cellular stress directly regulates FA metabolism by affecting the enzymatic activity of Acetyl-CoA carboxylases . Venetoclax + azacydine treatment results in a reduction in amino acid uptake by LSCs. Enhanced FA transport facilitates FAO and allows LSCs to compensate for amino acid uptake loss caused by venetoclax + azacytidine treatment, resulting in resistance. However, this resistance can be overcome by the knockdown of very long-chain acyl-CoA dehydrogenase (ACADVL), an enzyme involved in mitochondrial FA beta-oxidation, suggesting that FA beta-oxidation plays an important role in conferring resistance to venetoclax + azacytidine treatment in LSCs.Venetoclax + azacytidine-resistant LSCs exhibit increased levels of several metabolites that are responsible for the transport of FAs into mitochondria at baseline, including acetyl-carnitine, iso-butylryl carnitine, and hexanoyl-carnitine, compared to sensitive LSCs. Pharmacological inhibition of CPT1 by etomoxir depleted refractory LSC function, and eliminated venetoclax+ azacytidine-resistant LSCs. In addition, CPT1A inhibition by etomoxir induced cell death in AML cells and increased cells’ sensitivity to cytarabine. CD36+ LSCs are characterized by an enhanced FAO rate, and disruption of mitochondrial metabolism by targeting CD36-FAO-OXPHOS drives leukemia cells to low OXPHOS and enhances AraC sensitivity. In addition, topoisomerase inhibitor (mitoxantrone)-resistant AML cells showed activation of lipid metabolic pathways. Inhibition of FAO, using etomoxir in these cells, reduced colony formation ability, suggesting that FA metabolism and dependency on OXPHOS are key vulnerabilities, conferring chemoresistance in AML.FAs are conjugated to carnitine by CPT1 to facilitate mitochondrial translocation, and both the PPARs and the coactivator-1 of PPARs modulate CPT1A expression. Of note, BMSCs supply mitochondria to AML cells in vitro, thus providing them with additional energy. AML cells were observed to enhance their mitochondrial mass up to 14% by mitochondrial uptake from BMSCs; this transfer increased in response to chemotherapy and contributed to resistance by reducing mitochondrial depolarization. AML cells exhibited up to a 1.5-fold increase in mitochondrial ATP production following the uptake of mitochondria from BMSCs, were less prone to depolarization of mitochondria following chemotherapy, and showed improved survival. Of note, transfer of mitochondria to AML cells from BMSCs in the presence of chemotherapeutic agents occurs predominantly through endocytosis and requires cell-cell contacts. The NADPH oxidase, NOX2 generates superoxide in AML cells, which promotes mitochondrial transfer to AML blasts from BMSCs through AML-derived tunneling nanotubes. Interestingly, suppression of NOX2 reduced the transfer of mitochondria to AML blasts and resulted in enhanced cell death and improved survival of AML-bearing mice. AML cells show high OXPHOS levels when cocultured with BMSCs. Hou et al. showed that the elevation of OXPHOS levels in AML cells in coculture was dependent on the activation of mitochondrial serine-phosphorylated STAT3 (pS-STAT3). AML cells in coculture induced the secretion of interleukin-6 from BMSCs, which in turn promoted the activation of total and mitochondrial STAT3 in AML cells, resulting in increased proliferation and chemoresistance.BMSCs, which are composed of endothelial cells, osteoclasts, osteoblasts, adipocytes, and fibroblasts, form the BM microenvironment. BMSCs aid the survival of AML cells and mediate resistance to therapy. Cell-cell communications in the BM microenvironment play a key role in aiding AML cell survival, disease progression, and therapeutic response through the transfer of biomolecules or cellular organelles, such as mitochondria. Forte et al. reported that after AML induction, unlike bulk stroma, the nestin-expressing BMSC cell population remains stable in MLL-AF9-driven AML, with no reduction in their number over time. However, these cells undergo changes in their functional behavior to enhance the survival of MLL-AF9 AML cells and promote chemoresistance by promoting TCA cycle and OXPHOS. They also provide a GSH-mediated antioxidant defense, which is critical for ROS mitigation during leukemogenesis and chemotherapy. Forte et al. observed a delay in leukemogenesis, following the depletion of Nestin+ BMSCs in primary AML mice. Most importantly, the depletion of Nestin+ BMSCs in chimeric mice reduces AML, but not normal cells. Thus, BMSCs help AML cells switch their energy sources and provide antioxidant defense mechanisms to survive chemotherapy.AML cells are known to harbor abnormally high levels of ROS. Mesenchymal stem cells, a component of BMSCs, support the survival of AML cells by influencing several key metabolic pathways and eliminating ROS. BMSCs expressing the intermediate filament protein nestin provide HSC niche function,131. This suggests that glutamine metabolism in AML cells could be regulated by the tumor microenvironment,131. Interestingly, van Gastel et al. showed that glutamine metabolism differed between AML and stromal BM cells, especially LepR+ mesenchymal stromal cells. AML cells took up glutamine to maintain GSH but not to promote the TCA cycle. In contrast, in LepR+ mesenchymal stromal cells, glutamine entered the TCA cycle to produce aspartate. Aspartate is transported to and supports pyrimidine biosynthesis in persistent MLL-AF9 AML cells, thus preventing the metabolic collapse triggered by chemotherapy,131. These findings suggest that the tumor microenvironment plays a major role in chemoresistance, independent of cell-intrinsic signaling/apoptosis alterations. However, it is unclear whether aspartate-rich locations in the BM provide a protective niche, and if specific AML subclones compete for these spots. Further niche-specific studies are warranted to delineate whether mutational subclones compete for specific hotspots.Induction chemotherapy causes transient stress in BM, which results in the elevation of glutamine, glutamate, and aspartate with a concomitant increase in the expression of several glutamine transporters, such as Slc1a5, Slc38a1, Slc7a5, and Slc7a6, but no change in the enzymes that metabolize glutamine or glutamate, in residual AML cells,132. When FAO was inhibited pharmacologically with etomoxir or ranolazine, AML cells were sensitized to apoptosis induction by ABT-737, and proliferation was inhibited in the setting of coculture with BMSCs. Interestingly, a recent report described two forms of LSCs, distinguished by differences in their expression of CD36 and in their metabolism and cell cycle status. LSCs expressing CD36 (CD36+ LSCs) are often localized to gonadal adipose tissue (GAT) which is rich in fatty acids, indicating a tropism rich for microenvironments rich in FAs. Specifically, these GAT-resident LSCs are more resistant to conventional chemotherapy because they exist in a quiescent cell cycle state. In the context of these findings, leukemic cells co-opt adipose tissue to create a microenvironment to support leukemia growth and provide a drug-resistant niche. Further, leukemic burden in GAT for the MLL-AF9 model was considerably lower than that in BM, indicating that localization to GAT may be dependent on the specific oncogenes that drive the malignant transformation process. An interesting question for future investigations would be to understand the role of FA metabolism in conferring drug resistance to LSCs in a niche-specific BM microenvironment.Accumulating evidence supports the existence of metabolic symbiosis and bidirectional dialogues between leukemic cells and the BM adipose tissue niche, which provides diverse sources of lipids, such as FAs, cholesterol, and eicosanoids, to resident leukemic cells. Leukemic cells meet their energy needs by using FAs to fuel β-oxidation, which eventually supports anabolismet al., the expression of FGF2 and its receptor, FGFR1, were both increased in several stromal cell lines and primary AML stroma. This heightened FGF2/FGFR1 signaling augmented greater secretion of exosomes. Huang et al. (2021), studied the impact of inhibition of small extracellular vesicle (SEV) secretion from various sources on the progression of MLL-AF9 induced AML, as well as normal hematopoiesis. A significant delay in AML progression occurred when SEV secretion from endothelial cells was inhibited, but not that from perivascular cells, megakaryocytes, or spleen stromal cells. SEVs derived from endothelial cells contained high levels of Angiopoietin-like protein 2 (ANGPTL2) protein, which binds to the leukocyte immunoglobulin-like receptor B2 (LILRB2 receptor) and accelerates leukemia progression. Moreover, Vacuolar protein sorting-associated protein 33B (VPS33B) governed the release of ANGPTL2-SEVs from endothelial cells and this release of ANGPTL2-SEVs was required for primary human AML cell maintenance. These findings demonstrate that SEVs have a role in cancer development and suggest that targeting the release of ANGPTL2-SEVs from endothelial cells would be an effective strategy in the treatment of some types of AML. While IO-108, an antibody targeting LILRB2, is in Phase 1 clinical development for solid tumors; LILRB4 antibody (IO-202) is in phase 1 cohort expansion clinical trial [NCT0437243] in combination with azacitidine and venetoclax, for the treatment of AML and likely to be effective in monocytic AML,137.AML cells alter the BM by releasing exosomes, which promote the proliferation and survival of leukemic cells and suppress normal hematopoiesis. In a study by Javidi-Sharifi . Interestingly, there was an increase in HMGCR levels in SEVs in response to chemotherapy. These HMGCR+ SEVs promoted the growth of AML cells by upregulating intracellular cholesterol levels. This chemotherapy-induced enhancement of SEV secretion in AML cells was prevented by HMGCR inhibition. Thus, HMGCR blockade could potentially provide a therapeutic alternative in AML by inhibiting cholesterol-driven chemoresistance caused by SEV signaling. Another study reported the secretion of exosomes containing vascular endothelial growth factor (VEGF) and VEGF receptor (VEGFR) by AML cells. These exosomes induced glycolysis in human umbilical vein endothelial cells (HUVECs), which resulted in vascular remodeling and chemoresistance.Cytarabine and decitabine treatment resulted in significantly higher intracellular levels of cholesterol and HMGCR (3-hydroxy-3-methyl-glutaryl-coenzyme A reductase), the rate-limiting enzyme in the cholesterol synthesizing mevalonate pathway, in cultured AML and non-malignant cells. Concomitantly, these cells produced higher levels of SEVs. When BM is preconditioned with AML-derived exosomes, AML cell engraftment and growth are significantly accelerated, whereas disruption of exosome secretion in AML cells significantly reduced these cells’ ability to engraft and grow in vivo. In coculture, serum and leukemic cells from patients with NPM1-mutated AML impair CD8+ T cells’ immune function. Through SEVs, leukemic cells secrete miR-19a-3p into the tumor microenvironment. This process was mediated by the NPM1-mutated protein/CCCTC-binding factor (CTCF)/poly (A)-binding protein cytoplasmic 1 (PABPC1). MiR-19a-3p released from SEV was found to be internalized by CD8+ T cells, where it inhibited creatine import by repressing the expression of a transporter solute-carrier family 6 member 8 (SLC6A8). A reduction in creatine levels lowered ATP production and impaired CD8+ T cell immune function, which caused leukemic cells to escape the immune system. Interestingly, the exosomes generated by fully engrafted AML PDX mice were similar to exosomes isolated from the plasma of the patients who had donated the cells for engraftment. These exosomes carried human proteins and leukemia-associated antigens, confirming their origin from AML patients.BM stromal cells exposed to AML-derived exosomes exhibit decreased expression of genes supporting normal hematopoiesis  and osteogenesis  and increased expression of genes supporting AML growth . While LT-HSCs are functional after the recovery, their DNA is damaged for a long time. Hematopoietic stem and progenitor cells (HSPCs) are generally susceptible to SEV entry and use the mTOR pathway, but HSCs selectively enter quiescence because of their unique sensitivity to protein synthesis disruption.AML utilizes SEV miRNAs to target the mTOR pathway which suppresses protein synthesis in the LT‐HSC to elicit their quiescence. The mitostress test using a high-throughput real-time mode approach in Agilent Seahorse XF analyzers is the best assay for measuring basal cell respiratory rate, ATP production, proton leak, maximum respiration, and spare respiratory capacity in an intact cell. This single assay yields a wide range of parameters, providing insight into mitochondrial dysfunction and reveals the functional differences between cell types, cellular metabolic state (quiescent versus active), effect of drug candidates, impact of genetic modifications, or biochemical interactions.A tightly coordinated flux of metabolites occurs in mitochondria, integrating bioenergetics, redox homeostasis, and anabolic metabolism. By assessing mitochondrial function, researchers have gained a better understanding of metabolism in cellular physiology, disease pathology, and etiology. The reduction of oxygen into water is essential to oxidative phosphorylation and measuring oxygen consumption is therefore one way to assess mitochondrial function. As a result of excessive production of ROS and/or decreased antioxidant defense activity, mitochondrial reactive oxygen species (mtROS) accumulate, causing oxidative stress (OS). It leads to oxidative damage that affects several cellular components, including lipids, DNA, and proteins. To detect mitochondrial superoxide selectively, MitoSOX Red and Green are used, which are fluorogenic reagents designed for highly selective detection of superoxide in the mitochondria of viable cells. A notable feature of these probes is that they are readily and specifically oxidized by superoxide alone and not by other ROS or reactive nitrogen species (RNS) generating systems at optimum concentration. However, the user should be aware of the cytotoxic or mitotoxic effect of these probes and their potential impact on mitochondrial morphology at higher concentrations. Furthermore, oxidation of these probes is inhibited by superoxide dismutase. Small, cell-permeant dyes such as TMRM  and TMRE  accumulate in active mitochondria and may be used to quantify mitochondrial membrane potential. They appear bright in healthy cells with functioning mitochondria, but are dimmed or undetectable upon loss of mitochondrial membrane potential.Mitochondria are known to generate approximately 90% of cellular reactive oxygen species (ROS). The metabolomics approach can be divided into two distinct types: untargeted metabolomics and targeted metabolomics. Targeted approaches focus on identifying and quantifying a small number of known metabolites, such as those found in clinical analyses. In untargeted approaches or hypothesis-generating approaches, data are obtained for as many species as possible, metabolites are annotated, and metabolic changes are reviewed regardless of whether they are known or unknown. However, measuring metabolite concentrations by metabolomics only tells half the story. The accumulation of metabolites can be caused not only by increased production, but also by decreased consumption. The metabolic flux, which can be quantified as material flow per unit time, is equally important for understanding pathway activity.Metabolome consists of all the molecules with low molecular weight (metabolites) within a cell, tissue or organism. Analyzing the metabolites within a cell, tissue or organism following a genetic change or physiological stimulus is known as metabolomics. Today, similar studies can be performed using stable non-radioactive isotope tracers, which can be tracked quantitatively and broadly using MS or NMR. Furthermore, an approach called metabolic activity screening integrates the metabolomics data with metabolic pathways and systems biology data, including proteomics and transcriptomics, in order to identify endogenous metabolites that may alter phenotypic characteristics and functionality.Unlike metabolites, fluxes are not physical entities that can be detected by mass spectrometry. But they can be inferred by using isotope tracers. Radioactive tracer studies laid the groundwork for modern metabolism research. Further improvements in our understanding of the disease would further improve outcomes. The genetic makeup of AML, as well as its metabolic requirements for survival and proliferation, are heterogeneous. Diverse types of metabolic vulnerabilities, including dependence on OXPHOS, amino acid metabolism, nucleotide metabolism, and fatty acid metabolism, were reported. In addition, metabolic changes occurring in the tumor microenvironment have also been used as therapeutic targets. However, a thorough understanding of biomarkers of response to these therapies and mechanisms of resistance and combinatorial therapies would pave the way for better clinical management of AML. Understanding how the genetic architecture works with the tumor microenvironment is critical to uncovering new vulnerabilities. In addition, a thorough understanding of the interactions between different cell populations within the BM microenvironment in response to transient stress due to therapy which leads to changes in nutrient availability and metabolic adaptations, will increase the chances of a true cure.Nine therapeutic agents for AML have been developed and approved since 2017. Treatment improvements have increased the overall survival rate of AML patients, from 13% to 55% in those aged younger than 60 years and 8% to 17% in patients older than 60 yearsLSCs have metabolically distinct subpopulations and relapsed/refractory LSCs switch to alternative metabolic pathways to fuel OXPHOS, rendering them therapeutically resistant to conventional treatments. Developing treatment strategies that target these metabolic pathways is key to eliminating relapsed/refractory LSC populations and improving treatment outcomes.


Western blotting was used to detect protein expression levels, and an NO fluorescence probe was used to examine NO production. Intracellular Ca2+ concentrations were measured using a Ca2+-sensitive fluorescent dye under a fluorescence microscope. Compared with those in healthy pregnant women, serum PSG9 levels were significantly decreased in patients with preeclampsia. PSG9 (0.1 μg/mL) treatment of HUVECs significantly enhanced the expression levels of store-operated calcium entry (SOCE) channel proteins Orai1 and Orai2, but not Orai3, and of endothelial nitric oxide synthase (eNOS) and NO production. Pretreatment with an inhibitor of SOCE (BTP2) abolished PSG9-enhanced Orai1, Orai2, and eNOS expression levels and NO production in HUVECs. The mechanisms underlying SOCE that were PSG9 enhanced in HUVECs appear to involve the Ca2+/eNOS/NO signaling pathway. These findings suggest that serum PSG9 levels may be a potential biomarker for monitoring the occurrence or development of preeclampsia in pregnancy and that PSG9 may be a potential therapeutic target for the treatment of preeclampsia.We explored changes in pregnancy-specific glycoprotein 9 (PSG9) levels in the serum of patients with preeclampsia and the effects and underlying mechanisms of PSG9 effects on calcium (Ca Preeclampsia is a hypertensive disorder complicating pregnancy. It presents as hypertension after 20 weeks of pregnancy and is accompanied by abnormal urine protein and damage to the kidney, liver, or other organs ,2. The c2+ in the cytoplasm i) measurement assays, and in vivo blood vessel tension measurement methods to investigate the effect of PSG9 on NO production in human umbilical vein endothelial cells (HUVECs) and on agonist-induced umbilical vein relaxation. Because the placenta is directly linked to the maternal circulation, abnormalities in the expression of placental factors in women with preeclampsia can be detected in maternal serum protein levels. In a previous study by our team, we identified differentially expressed proteins in blood samples derived from patients with preeclampsia by using isobaric tags for relative and absolute quantitation (iTRAQ) combined with high-performance liquid chromatography–tandem mass spectrometry (LC-MS/MS) . CompareAll patients provided written informed consent for this study, which was approved by the Medical Research Ethics Committee of Anhui Medical University . Blood samples were collected from December 2018 to September 2020 at the Maternal and Child Health Hospital affiliated with Anhui Medical University. The criteria for the diagnosis of preeclampsia were consistent with the 2019 American College of Obstetricians and Gynecologists pregnancy hypertension guidelines . All pre2 incubator.HUVECs were purchased from Shanghai Guandao Bioengineering Co., Ltd. . HUVECs were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 10% fetal bovine serum  at 37 °C in a 5% COThe cultured cells were lysed on ice with RIPA buffer  and then centrifuged at 12,000 rpm and 4 °C for 20 min. The resultant proteins underwent electrophoresis using sodium dodecyl sulfate–polyacrylamide gel electrophoresis and were transferred to polyvinylidene fluoride membranes. Then polyvinylidene fluoride membrane was incubated with the respective primary antibodies, including rabbit anti-PSG9 antibody , rabbit anti-Orai1 antibody, rabbit anti-Orai2 antibody, rabbit anti-Orai3 antibody , and rabbit anti-eNOS , overnight at 4 °C. Target proteins were detected using a luminescent agent from an enhanced chemiluminescence system after incubation with horseradish peroxidase-labeled secondary antibody . ImageJ software  was used to analyze the optical density of the resulting bands. The values were normalized to the densitometric values of GAPDH .An NO fluorescent probe  was used to evaluate the generation of intracellular NO. DAF-FM has only weak fluorescence, but it produces a strong fluorescence signal after reacting with NO that can be detected at an excitation wavelength of 495 nm. After the cells were treated with various agents, the cells on a glass slide were processed according to the instructions of the DAF-FM DA kit and eventually photographed under a fluorescence microscope . Cell nuclei were identified by application of 4′,6-diamidino-2-phenylindole. The mean fluorescence intensity of NO was measured using ImageJ software (version 1.53). The mean fluorescence density was calculated by determining the value of the fluorescence intensity of interest divided by the total imaging area.2+ indicator. Before binding to Ca2+, Fluo-8 produces little florescence. However, after Fluo-8 combines with Ca2+, the fluorescence intensity increases at least 200 times. Fluo-8 is excited by light at a wavelength of 488 nm using an argon ion laser, and the emitted light is at a wavelength of 514 nm. We incubated HUVECs for 20 min at 37 °C with 2 μM Fluo-8/AM and 0.02% pluronic F-127 . After the cells were washed with phosphate-buffered saline (PBS) to remove extracellular Fluo-8 dye, the cellular Ca2+ store was depleted by the application of thapsigargin  in a Ca2+-free solution . The Ca2+ influx was initiated by applying 2 mM Ca2+ to the medium. The baseline fluorescence intensity before extracellular Ca2+ application was considered F0. The peak fluorescence intensity after extracellular Ca2+ application was considered F1. The change in Ca2+ concentration in the cells is presented as a fluorescence intensity ratio (F1/F0) before and after the addition of extracellular Ca2+.Fluo-8/AM  is a widely used fluorescent Cap < 0.05 were considered statistically significant differences. The data were analyzed using GraphPad Prism software . Statistical comparisons were performed using the two-tailed Mann–Whitney test or two-way analysis of variance followed by the Games–Howell post hoc tests when more than two treatments were compared. Unless otherwise stated, the data are presented as means with standard errors (mean ± SE). Values of In our previously published study, serum PSG9 levels identified by LC-MS/MS were significantly decreased in the patients with preeclampsia compared with those in the healthy control pregnant women A,B 23]..23]. To Western blotting analyses were used to verify the PSG9 serum level changes in patients with preeclampsia. Our data showed that serum PSG9 protein levels were significantly decreased in the patients with preeclampsia compared with those in the healthy controls C,D. Thes2+ homeostasis i homeostasis includes Ca2+ release from stores in the ER as well as Orai channel-mediated external Ca2+ influx. The former has a short timeframe, and the NO generated is only at trace levels, insufficient to cause biological effects. By contrast, Orai channel–mediated external Ca2+ influx can continuously increase [Ca2+]i. In addition, calmodulin (CaM) combines with Ca2+ to form a Ca2+/CaM complex, which then combines with the CaM binding domain in eNOS to prevent Thr495 of eNOS from being phosphorylated by protein kinase C, thereby more effectively promoting the synthesis of NO in the vascular endothelium i. However, a higher concentration of PSG9 (0.5 µg/mL) did not increase the expression of eNOS, suggesting that there may be dose-dependent effects of PSG9. That is, too high a concentration of PSG9 may produce other effects that ultimately do not promote increased eNOS expression. Takahiro et al. showed that Ca2+-sensing receptors may be involved in the production of NO in HUVECs and may be inhibited by receptor antagonists i changes in HUVECs treated with PSG9. We did not measure resting [Ca2+]i. Thus, the effect of resting [Ca2+]i on cell function remains unknown. Future studies should also assess resting [Ca2+]i. NO is a byproduct of nitric oxide synthase, which generates L-citrulline by using L-arginine and oxygen as substrates and nicotinamide adenine dinucleotide phosphate as a cofactor to provide electrons. NO synthesis is regulated by a variety of factors, including its substrates and products, but is mainly controlled by the enzyme nitric oxide synthase. Endothelial NOS (eNOS) is synthesized primarily by endothelial cells and plays an important role in promoting NO synthesis and regulating vascular tension. In normal pregnancy, blood flow of the maternal placenta must be maintained at a high level to ensure the nutrition and oxygen needed for fetal development and growth. The blood supply to the placenta can be improved by reducing the resistance of peripheral blood vessels. However, in preeclampsia, the ability of the placenta to vasodilate is disrupted. Through clinical experiments, Barr et al. have shown that the severity of preeclampsia is closely related to the diastolic reactivity of the vascular endothelium . In the al cells . An incr nucleus ,39. Normothelium . Our resagonists . Moreovenhibited . The fin2+/eNOS/NO signaling pathway in HUVECs. In addition to clarifying a molecular mechanism whereby PSG9 regulates the function of vascular endothelial cells, these findings suggest that PSG9 may warrant further study for the prevention or treatment of preeclampsia.In summary, we found that serum PSG9 levels were significantly decreased in pregnant women with preeclampsia. Further evidence showed that PSG9 promoted NO release by increasing the expression of Orai proteins and activating the Ca2+/eNOS/NO signaling pathway was mediated by increased Orai1 and Orai2 expression levels to enhance NO release in HUVECs. These findings suggest that PSG9 may have potential value in the prevention or treatment of preeclampsia. However, preeclampsia is a pregnancy-specific disease involving multiple factors and myriad cell types. This study clarified only the role of PSG9 in vascular endothelial cells to suggest its role in the pathogenesis of preeclampsia. The functions of PSG9 in trophoblasts remain uncertain and are also an important direction for future research.In conclusion, this study showed that the levels of PSG9 in the serum of patients with preeclampsia were significantly decreased, and further experimental studies indicated that the Ca


TBCB expression was an independent poor prognostic factor for AML. GO and GSEA enrichment analysis hinted that immune-related signaling pathways were enriched in up-regulated DEGs between two populations separated by the median expression level of TBCB. By constructing a protein-protein interaction network, we obtained six hub genes, all of which are immune-related molecules, and their expression levels were positively linked to that of TBCB. In addition, the high expression of three hub genes was significantly associated with a poor prognosis in AML. Moreover, we found that the tumor microenvironment in AML with high TBCB expression tended to be infiltrated by NK cells, especially CD56bright NK cells. The transcriptional levels of NK cell inhibitory receptors and their ligands were positively related to that of TBCB, and their high expression levels also predicted poor prognosis in AML. Notably, we found that the down-regulation of TBCB suppressed cell proliferation in AML cell lines by enhancing the apoptosis and cell cycle arrest. Finally, drug sensitivity prediction illustrated that cells with high TBCB expression were more responsive to ATRA and midostaurin but resistant to cytarabine, dasatinib, and imatinib. In conclusion, our findings shed light on the feasibility of TBCB as a potential predictor of poor outcome and to be an alternative target of treatment in AML.Acute myeloid leukemia (AML) is a devastating blood cancer with high heterogeneity and ill-fated outcome. Despite numerous advances in AML treatment, the prognosis remains poor for a significant proportion of patients. Consequently, it is necessary to accurately and comprehensively identify biomarkers as soon as possible to enhance the efficacy of diagnosis, prognosis and treatment of AML. In this study, we aimed to identify prognostic markers of AML by analyzing the cohorts from TCGA-LAML database and GEO microarray datasets. Interestingly, the transcriptional level of microtubule-associated protein TBCB in AML patients was noticeably increased when compared with normal individuals, and this was verified in two independent cohorts (GSE9476 and GSE13159) and with our AML patients. Furthermore, univariate and multivariate regression analysis revealed that high  As a quickly and dramatically progressing heterogeneous blood cancer, acute myeloid leukemia (AML) derived from blockage in differentiation and clonal expansion of myeloid blasts + cells, is a potential target antigen for immunotherapy of CML Tumors proliferate rapidly due to their frequent mitoses and shortened cell cycle TBCB in AML patients, which were noticeably increased when compared with those of normal individuals, and found that elevated level of TBCB was an unaided poor prognostic factor. Moreover, we interrogated the relevance between TBCB expression and immune infiltration, expression levels of immune-related molecules, and drug sensitivity in AML.As a microtubule folding cofactor, TBCB is mainly localized in the cytoplasm and centrosomes 2 transformed for intrasample comparison. The data and information of the datasets (TCGA-LAML and GTEx) are available searched on the UCSC XENA In this study, six independent cohorts for mRNA expression were used, namely the TCGA-LAML  Bone marrow mononuclear cells from AML patients and health subjects were extracted by Ficoll-Paque  density gradient centrifugation. The inclusion criteria in this study were: (1) patients with confirmed diagnosis of AML according to WHO-2022 6 cells/mL in RPMI-1640  containing 10% fetal bovine serum , 1% penicillin/streptomycin . In addition to the above components, cell culture medium for SKNO-1 was supplemented with 10 ng/ml GM-CSF . Cells were maintained at 37°C in a humidified incubator .The human AML cell lines  were obtained from State Key Laboratory of Experimental Hematology. THP1, NOMO1 and Kasumi-1 cells were cultured at 1-2 × 106 cells, 5 μL siRNA, 5 μL Lipofectamine 3000, 500 μL cell culture medium and 500 μL Opti-MEM . Transfected cells were harvested for analysis 48 hours after siRNA transfection, including validation of TBCB expression level by RT-qPCR and Western blot, and detection of apoptosis and cell cycle by flow cytometry. The siRNA sequences (5'→3') were as follows: TBCB-siRNA 1: forward GCAUCCACGUCAUUGACCATT, reverse UGGUCAAUGACGUGGAUGCTT; TBCB-siRNA 2: forward GGGAAACGCUACUUCGAAUTT, reverse AUUCGAAGUAGCGUUUCCCTT; and NC-siRNA: forward UUCUCCGAACGUGUCACGUTT, reverse ACGUGACACGUUCGGAGAATT.The siRNA oligonucleotides targeting TBCB (siTBCB) or negative control (NC-siRNA) were synthesized by Suzhou GenePharma Co.,Ltd  and transfected into AML cell lines by Lipofectamine™ 3000 transfection kit . Each well of a 6-well plate contained 1 × 10TM Tubes  were used to extract total RNA TBCB-forward, CTACTGGATTGGTGTCCGCTATG, h-TBCB-reverse, CACGACTGCTGGCTTGACAAAG; 18S-forward, AGTCCCTGCCCTTTGTACACA, 18S-reverse, CGATCCGAGGGCCTCACTA.TRIzol  and PhasemakerWestern blot analysis was performed as previously described 3 cells/well. 10 μL of CCK-8 reagent  was added to the wells after 24 h, 48 h and 72 h, and incubated for 4 hours at 37°C. Absorbance at 450 nm was measured using a microplate spectrophotometer .The CCK-8 assay was performed to assess cell proliferation. Transfected cells were seeded into 96-well plates at 2 × 105/50 μL. 3 μL Annexin V and 3 μL 7-AAD were added to the cell suspension and incubated for 15 minutes at RT in the dark. After that, 200 μL of 1 × Binding Buffer was added to the cell suspension and cells were analyzed by a FACS Canto II Flow Cytometry (BD).The Annexin V Apoptosis Detection Kit  was used to analyze cell apoptosis. Specifically, cells were washed once with PBS and once with 1 × Binding Buffer. Then, cells were resuspended in 1 × Binding Buffer at 2 × 10TM kit  was used to fix and permeabilize the cells. Brieflly, cells were washed with PBS and vigorously resuspended in Reagent A at 3 × 105/100 μL. After incubation for 5 minutes at RT, 1 mL 1 × BD FACS lysing solution was added, vortexed vigorously and incubated 10 minutes at RT. Then, cells were centrifugated at 2000 rpm for 5 min and the pellets were resuspended in 50 μL Reagent B. After that, 3 μL Ki67 was added to the cell suspension and incubated for 30 minutes at RT in the dark. Finally, cells were labelled with 20 ng/μL Hoechst 33342 before analyzed by FACS Canto II Flow Cytometry (BD).Cell cycle progression was measured using APC-Ki67  and Hoechst 33342 . BD IntraSureTBCB expression level. DESeq2 p-adjust < 0.05, |log2FC| ≥ 0.59. The data was originated from TCGA-LAML.Patients were split into two groups gleaned from the median p-adjust < 0.05.ClusterProfiler (v4.4.4) and GOplot (v1.0.2) were used to carry out functional annotation analysis of DEGs p-adjust < 0.05 were defined as statistical significance.ClusterProfiler (v4.4.4) was used to conduct GSEA by the C2 gene sets  from the MSigDB database TBCB and those of hub genes in AML subjects were visualized using ggplot2 (v3.3.6).Raw data of protein-protein interactions (PPIs) network to assess the interrelationships of the up-regulated DEGs were downloaded from STRING TBCB mRNA level and that of immune checkpoint molecules were identified and visualized via ggplot2 (v3.3.6).Based on the ssGSEA algorithm provided in the GSVA (v1.46.0) The pRRophetic p < 0.05). Receiver operating characteristic (ROC) analysis was carried out on the data using the pROC package (v1.18.0) and the results were visualized using ggplot2 (v3.3.6). Hazard ratios (HR) and their 95% confidence intervals (CI) were computed for survival-related genes.The R-studio (v4.2.1) was used for most data processing and statistical analysis. The Kruskal-Wallis test (multiple groups) and Wilcoxon rank sum test (unpaired samples of two groups) were applied to define the differences in clinical characteristics. Unpaired t test was used to compare the means of the two groups for RT-qPCR and drug sensitivity analysis. One-way ANOVA was used to assess differences among multiple groups in biologically experiments. The overall survival (OS) was conducted by Kaplan-Meier, and univariate and multivariate Cox regression were used to determine the significance of prognostic factors  and healthy donors (n = 14). Consistent with the expression profile analysis with database, we noticed a substantially augmentation of transcriptional level of TBCB in AML patients  , only the transcriptional level of ecreased A. Highers Figure A, includcontrol) B.TBCB expression. The calculated area under curve (AUC) value was 0.731  counts , there was a trend that TBCB high expression maybe indictive of poor prognosis of AML  curve. The group with high s of AML K-L. In afindings M-P. The TBCB expression is an independent unfavorable prognostic factor for AML by logistic regression analyses. Univariate Cox regression analysis illustrated that advanced age (p < 0.001), unfavorable cytogenetic risk  and higher TBCB expression (p < 0.001) were associated with adverse outcome  from univariate Cox regression analysis, and we found that older age (p < 0.001) and high TBCB expression (p = 0.002) were independent prognostic factors with clinical meaning for OS  of OS prediction was 0.738  calibration curve presented high concordance between the nomogram predicted survival probability and observed fraction survival probability Figure F. To enhTBCB expression in leukemogenesis, the DEGs analysis was performed in AML populations with high and low TBCB expression. A total of 2316 DEGs (|log2(FC)| > 0.59, p-adjust < 0.05) were identified and displayed in a volcano plot , cytokine signaling in immune system (p < 0.001), adaptive immune system (p < 0.001) and class I MHC-mediated antigen processing and presentation (p < 0.001) in the high TBCB expression group. Accordingly, we extrapolated that high TBCB expression impinged upon AML may be associated with immunoregulation.To further analyze immune response pathways involved in DEGs between AML populations with high and low p < 0.05, Figure TBCB expression  analyses and co-expression modules to explore which set of proteins may interact with TBCB. Fifteen top hub genes were identified by calculating all of the 1068 up-regulated genes using three different algorithms, namely maximal clique centrality  have been reported to be prognostic in various tumors p < 0.05, Figure p < 0.01, Figure  TBCB expression . RT-qPCR Figure A and WesTBCB. Collectively, group with high TBCB expression was shown to sensitive to HSP90 inhibitors , with uncontrolled overproduction of myeloid cells, is one of the most threatening hematological malignancies TBCB is overexpressed, it will lead to microtubule depolymerization and mitosis disorder, thus resulting in disease Microtubules (MTs) are cytoskeletal polymers pivotal for eukaryotic activities, including but not limited to cell division, migration and intracellular trafficking TBCB as a potential prognostic marker in AML. High mRNA level of TBCB was obviously related to unfavorable prognosis, as demonstrated by both univariate and multivariate regression analysis. As predicted, high mRNA level of TBCB was consistent with elevated WBC count, increased proportion of PB and BM blasts, leading us to speculate that the abnormal high level of TBCB acts as a positive regulatory factor of AML for promoting the cell proliferation. The up-regulated DEGs were enriched in signaling pathways relevant for cell proliferation activation and apoptosis inhibition via GO and KEGG analysis. We decreased the expression level of TBCB in AML cell lines by siRNAs and found that the cells obtained slower growth, increased apoptosis and cell cycle arrest. To sum up, it was conceivable that the poor outcome of AML patients bearing high TBCB expression may be related to cell proliferation activation and apoptosis inhibition, but the specific underlying mechanism demands an in-depth study.In this study, we identified TBCB was positively linked to those of immune-related molecules, as revealed by PPI network analysis, GO and GSEA enrichment analysis. The high expression of three hub genes  which are significantly associated with TBCB was also obviously linked to poor prognosis in AML. Moreover, the TBCB expression was associated with immune infiltration cells in AML, particularly by CD56bright NK cells with low cytotoxicity bright CD16- NK cells are the principal components of NK cells in various immune tolerance organs bright NK of AML patients owned high expression of TBCB might cause immune escape of tumor cells, ultimately leading to an unfavorable outcome. Taking a step further, we found that the transcription levels of NK cell inhibitory receptors and their ligands were positively related to that of TBCB, and their high expression also predicted poor prognosis. These results suggest that TBCB may be involved in regulating the function of NK cells and modulating the immune microenvironment of AML. Targeted drugs for NK inhibitory receptors have garnered a great deal of attention TBCB expression by alleviating the immunosuppression of NK cells.Moreover, transcriptional level of TBCB. Our findings exhibited that AML cells highly expressed TBCB were sensitive to ATRA and midostaurin, but resistant to cytarabine, dasatinib and imatinib. Therefore, such knowledge for clinical drug usages should be considered when making a choice in the combined use of AML drugs.We also examined the drug sensitivity of clinical drugs in AML cells with high or low transcriptional levels of TBCB is a potential prognostic marker and therapeutic target for AMLs. Our findings suggested that TBCB might be involved in regulating AML cell proliferation, the immune microenvironment of AML, and the function of NK cells. The underlying mechanism of TBCB in regulating tumor cell proliferation and NK cell-mediated immune escape in AML needs further in-depth studies.In this study, we provided evidence that Supplementary figures.Click here for additional data file.Supplementary tables.Click here for additional data file.


Changes in intracellular Ca2+ concentration ([Ca2+]i) are finely tuned by the concerted interaction of membrane receptors and ion channels that introduce Ca2+ into the cytosol, Ca2+-dependent sensors and effectors that translate the elevation in [Ca2+]i into a biological output, and Ca2+-clearing mechanisms that return the [Ca2+]i to pre-stimulation levels and prevent cytotoxic Ca2+ overload. The assortment of the Ca2+ handling machinery varies among different cell types to generate intracellular Ca2+ signals that are selectively tailored to subserve specific functions. The advent of novel high-speed, 2D and 3D time-lapse imaging techniques, single-wavelength and genetic Ca2+ indicators, as well as the development of novel genetic engineering tools to manipulate single cells and whole animals, has shed novel light on the regulation of cellular activity by the Ca2+ handling machinery. A symposium organized within the framework of the 72nd Annual Meeting of the Italian Society of Physiology, held in Bari on 14–16th September 2022, has recently addressed many of the unexpected mechanisms whereby intracellular Ca2+ signalling regulates cellular fate in healthy and disease states. Herein, we present a report of this symposium, in which the following emerging topics were discussed: 1) Regulation of water reabsorption in the kidney by lysosomal Ca2+ release through Transient Receptor Potential Mucolipin 1 (TRPML1); 2) Endoplasmic reticulum-to-mitochondria Ca2+ transfer in Alzheimer’s disease-related astroglial dysfunction; 3) The non-canonical role of TRP Melastatin 8 (TRPM8) as a Rap1A inhibitor in the definition of some cancer hallmarks; and 4) Non-genetic optical stimulation of Ca2+ signals in the cardiovascular system.Cytosolic Ca InsP3 gent Ca2+ . Repetit(GqPCRs) , or can e (CICR) . Ryanodi not all , cell ty not all . Depletier (NCX)  is prevey (SOCE) . SOCE remembrane . In excimembrane  or spontmembrane  can lead InsP3Rs . In bothresponse . The advachinery . For insachinery  and lysoachinery , to shaplar fate . We now ergetics . Howeverfunction . An unexysosomes . Herein,tivation . Lysosomgression . TRPML1-2+ space , but rec InsP3Rs . An addi InsP3Rs . Indeed, InsP3Rs . For insRs  the role of TRPML1 in Ca2+-mediated water reabsorption in the kidney ; 2) the modulation of the ER-mitochondria distance to fuel cellular metabolism in astrocytes and prevent neurodegeneration in Alzheimer’s disease ; 3) the non-canonical role of TRP Melastatin 8 (TRPM8) in the definition of some cancer hallmarks ; and 4) the use of novel light-sensitive organic actuators to stimulate angiogenesis and control cardiac cells pacing . Herein, we present a full report of the symposium and discuss the implications for the Ca2+ signalling field of the novel findings that were presented during each lecture.A symposium organized within the framework of the 72nd Annual Meeting of the Italian Society of Physiology, held in Bari on 14–16th September 2022, has recently addressed many of the unexpected mechanisms whereby intracellular Ca2+ reservoir that can finely modulate cellular fate through local or global Ca2+ signals  (2+ (0.5 mM) through a mechanism that is still highly debated  P2]  . Lysosom debated . Refillit manner  or by ex or SOCE . Lysosomugh TPCs , of whicugh TPCs . TPCs are (PIP2) . Intriguelective . TPCs caelective , where teceptors . Accordieceptors , the Inslutamate , acetylclutamate , foetal lutamate , and vaslutamate , is initP2]  and reacP2]  or synthP2] . TRPML1 or, TFEB . Furtherogenesis . Recent  InsP3Rs  or RyRs  InsP3Rs . The Ca2duct CD,   channel  with bafilomycin A1 to deplete the lysosomal Ca2+ pool  with cyclosporine A  or eliciting long-lasting transcriptional effects mediated by NFAT   and the associated protein Grp75, and then, into mitochondrial matrix, via a low affinity mitochondrial Ca2+ uniporter. Besides Ca2+ fluxes, MERCS are responsible for a number of key cellular processes, such as lipid and steroid biogenesis, mitochondrial fission and dynamics, autophagosome formation, apoptosis induction, and others  . The mors (MAMs) . Ca2+ trd others . Disruptd others . In AD, d others . Althougd others .2+ homeostasis and signalling, represent early signs of AD pathology   . To inveal cells .2+ transfer in AD pathogenesis.Taken together, these results provide proof of principle that the shortening of ER-mitochondrial distance, observed in AD, may be causative for a number of cellular AD-related alterations. Furthermore, our results suggest that the altered MERCS function in AD astrocytes may result in impairment of CNS homeostasis, BBB and neuronal dysfunction . Further2-terminal domain (N-term) containing ‘TRPM homology regions’ (MHR) involved in channel assembly and trafficking (Ca/PNa = 3.3)  . Intereshe store .TRPM8 gene was first identified and cloned from prostate tissues and described as a new prostate-specific gene due to the peculiar expression pattern shown during prostate cancer (PCa) progression  is highly subject to androgen regulation and thus is significantly downregulated in androgen deprivation and androgen receptor (AR) loss during the late androgen-independent phase of PCa  has been identified and characterized as a mediator of the Ca2+ transfer from the ER to the mitochondria in PCa epithelial cells , in focal adhesion dynamics and consequently in the control of cell adhesion and migration  located within the switch I region of Rap1A, responsible for the transition from the inactive to the active form of the small GTPase  phenotypes. In addition to supporting a potential use of TRPM8 in anti-tumor therapy as a dual target to simultaneously counteract metastatic dissemination and angiogenesis, they also shed new light on the possibility of using TRP channels as targets for the development of peptidomimetics in cancer therapy. In fact, the administration of therapeutic peptide mimicking the channel or part of its structure would further reduce any side effects associated with the wide tissue distribution of TRP channels and the multitude of intracellular signalling pathways regulated by them, directly targeting a specific protein-protein interaction and consequently impairing only its associated cellular pathways . As to T2+ signalling, represents one of the most fascinating insights in modern science  up to the optical stimulation/silencing of neuronal firing . Notablyterfaces , and morterfaces , were al2+]i   , a trulys ECFCs) . TRPV1-mand SOCE . Of noteh factor  and the h factor  induce th factor  , a process where Catraction . Althougtraction .2+ dynamics and ultimately with an increase in the contraction rate , confirmed that Italian Physiology is at the forefront of research in Ca2+ signalling, as also proven by many other oral and poster presentations of the meeting (The Symposium “Ca meeting .