NKA consists of one α subunit and one β subunit, with α1 and α2 being the main α isoforms in cardiomyocytes. Substantial evidence suggests that NKAα2 is the primary regulator of cardiac contractility despite being outnumbered by NKAα1 in cardiomyocytes. This review will mainly focus on differential regulation and subcellular localization of the NKAα1 and NKAα2 isoforms, and their relation to the proposed concept of subcellular gradients of Na+ in cardiomyocytes. We will also discuss the potential roles of NKAα2 in mediating cardiac hypertrophy and ventricular arrhythmias.Cytosolic Na + concentrations regulate cardiac excitation-contraction coupling and contractility. Inhibition of the Na The membrane depolarization activates the L-type Ca2+ channels, and the resulting Ca2+ influx leads to the opening of the ryanodine receptors (RyRs), causing a substantial Ca2+ release from the sarcoplasmic reticulum (SR). The ensuing rise in cytosolic [Ca2+] eventually leads to cardiac contraction when free cytosolic Ca2+ binds troponin-C in the myofilaments, where the contraction’s strength depends on the levels of cytosolic Ca2+ (2+ entering the cytosol during the excitation-contraction coupling must be extruded from the cytosolic space. The Ca2+ extrusion is mainly executed by either the SR Ca2+ ATPase (SERCA2), which pumps Ca2+ into the SR, or the Na+/Ca2+- exchanger (NCX), which extrudes Ca2+ over the cell membrane in exchange for 3 Na+ ions.Cardiac contraction is initiated by the opening of voltage-gated Nalic Ca2+ . For the2+ for Na+, NCX indirectly couples intracellular [Na+] to regulate cardiac contractility. NCX can operate in two modes: A forward mode with Ca2+ efflux and Na+ influx and a reverse mode with Ca2+ influx and Na+ efflux. Whether NCX operates in forward or reverse mode depends on the transmembrane gradients for Na+ and Ca2+ and the membrane potential. Most of the time, NCX extrudes Ca2+ (forward mode), and Ca2+ entry through NCX (reverse mode) only occurs briefly during the early stages of a regular action potential, where increased [Na+]i , low [Ca2+]i, and positive membrane potential all favors NCX-mediated Ca2+ influx , which uses the energy from ATP to extrude 3 Na+ for 2 K+. NKA is ubiquitously expressed in all cell types and contributes to a wide range of cellular tasks in addition to the regulation of cardiac contractility, including secondary active transport, volume regulation, and pH regulation  increased Ca2+ transients and cardiac contractility in mice, while NKAα1+/- mice were hypocontractile  in the extracellular region  of NKAα1+ and K+ . In cont+ and K+ . A doubl+ and K+ , allowinal [Na+] .2+], but overexpression of NKAα1 reduces the expression of NKAα2 and vice versa  compared to NKAα2 (k0.5=2.9 mM) (+] (∼4.5 mM). This could have some critical physiological and pathophysiological implications. Diffusion of K+ could be restricted in the T-tubules  , meaning-tubules , which c-tubules , as dischythmias . As discgh NKAα2 .+] and extracellular [K+]  is a member of the FXYD family of small, membrane-spanning proteins  that asslar [K+] , which rlar [K+] . A thirdlar [K+] , but pholar [K+]  of beta-lar [K+] , while plar [K+] . Followi[K+] (+] . Althoug or both .+] was affected similarly in both isoforms (Two early reports found no effects of β-adrenergic stimulation on NKAα2 activity . In thesisoforms .+ ions out and 2 K+ ions in), NKA activity becomes dependent on the membrane potential. The current-voltage relationship for NKAα2 is different compared to NKAα1. NKAα1 is activated over a broad range of physiological potentials, whereas NKAα2 is nearly inactive at resting membrane potentials, indicating that NKAα2 only is active during phases one to four of the action potential when the membrane potential is more positive  (As NKA moves one positive charge out of the cell per pumping cycle (3 Napositive . Some sue period . Anotherto 0 mV) , indicat2+domains in the T-tubules where sarcolemmal L-type Ca2+ channels are located near ryanodine receptors (RyRs), allowing effective Ca2+-induced Ca2+ release into the cytosol , the total amount of NKAα1 in the T-tubules is equal to or even higher compared to NKAα2 despite a low T-tubule/surface sarcolemma ratio for NKAα1  was higher in heterozygous NKAα2+/- mice. Similarly, by using NKAα2 selective doses of oubain, Swift et al. later showed that NKAα2 regulated NCX-sensed (Na+) and NCX activity  effect on the [Na+] in more distant compartments. A prerequisite for this hypothesis is the presence of intracellular Na+ gradients between different compartments in cardiomyocytes.Any effect of NKAα2 on intracellular , came from a landmark paper by Leblanc and Hume (+ current activation was sufficient to induce Ca2+-induced Ca2+ release (CICR) through activation of reverse NCX, a mechanism that localized elevations of [Na+] in an undefined compartment coined “fuzzy space”  Genetic models are impure systems because genetic modification of one protein leads to several secondary changes with unpredictable effects. Mice with overexpression of either NKAα2 or NKAα1 have reduced levels of the other isoforms, and direct functional interpretation is thus difficult. In addition, the expression of PLM and the Ser-63 and Ser-68 phosphorylation were reduced in the NKAα2 overexpression mice . 2) It ioverload . While ie course . In conted to WT , which c+], reduces NCX-mediated Ca2+ extrusion, increases intracellular [Ca2+], and increases the risk of triggered ventricular arrhythmias in hypokalemia (2+] following reduced NKA activity increases the frequency of arrhythmogenic Ca2+ waves (+ and Ca2+ currents (2+ transient amplitude and increased the frequency of Ca2+ waves, which was abolished following NKAα2 inhibition (2+] and cellular arrhythmias following disruption of NKA from Ankyrin B, a proposed mechanism for ventricular arrhythmias in the Ankyrin B syndrome (+, reduced Ca2+ extrusion through NCX, and increased the frequency of Ca2+ sparks and Ca2+ waves (+/- mice (2+] and CaMKII activity downstream. Specific activation of NKAα2 might thus represent a future anti-arrhythmic target that warrants further investigation.Reduced NKA activity increases intracellular [Naokalemia , digitalokalemia , and theokalemia . The inc2+ waves  but also2+ waves , which fcurrents . The spehibition . We alsosyndrome . NKA/Ank2+ waves , thus mi+/- mice , and all+/- mice . These d


The curative potential of allogeneic hematopoietic cell transplantation  for acute myeloid leukemia (AML) relies on the graft-versus-leukemia (GVL)-effect. Relapse after allo-HCT occurs in a considerable proportion of patients, and has a dismal prognosis with very limited curative potential, especially for patients with FLT-ITD-mutated AML. Since the first description of sorafenib for treatment of FLT3-ITD-mutated AML, several clinical trials have tried to determine the efficacy of FLT3 inhibitors for preventing and treating AML relapse after allo-HSCT, but many questions regarding differences among compounds and mechanisms of action remain unanswered. This review provides an overview on the established and evolving use of FLT3 inhibitors to prevent or treat relapse of AML in the context of allo-HCT, focusing on the recently discovered immunogenic potential of some FLT3 inhibitors and addressing the possible mechanisms of leukemia drug-escape. Acute myeloid leukemia (AML) harboring an activating mutation in FLT3 represents around one third of AML cases and is characterized by high relapse rate and dismal prognosis despite undergoing allogeneic hematopoietic cell transplantation  , 2.FLT3 is a transmembrane receptor tyrosine kinase physiologically expressed by hematopoietic stem and progenitor cells. The most common mutation is an internal tandem duplication (ITD) within the juxtamembrane domain, which constitutively activates the receptor, promoting proliferation and cell survival mainly through the activation of the PI3K, MAPK (ERK) and STAT5 signaling pathways Fig. . The secMutations within FLT3 occur late in leukemogenesis, are strong driver mutations and have been identified as a druggable target in FLT3-mutated AML , 9. EffoFLT3 inhibitors are classically divided in first-generation multi-target inhibitors  and next-generation selective inhibitors . Furthermore, inhibitors can be classified based on the ability to bind to the active conformation of the receptor , which are functional against FLT3-ITD and TKD-mutated forms or to bind only the inactive or ITD-mutated conformation of the target , which are therefore intrinsically inert toward TKD-mutated receptors . Since oThis review provides an overview on the established and evolving potential use of FLT3 inhibitors to prevent or treat AML relapse in post-transplantation context and addresses pending questions regarding its immunogenicity and the mechanisms of leukemia relapse during or after treatment.Relapse incidence is highest within the first 2 years after allo-HCT and survival is better if incipient relapse is detected earlier. Therefore, regular monitoring for MRD markers including chimerism is recommended , 23. In Sorafenib, a first-generation multi-target kinase inhibitor approved for treatment of advanced renal and hepatic cell carcinoma, has been studied since more than a decade in FLT3-ITD-mutated AML patients. First results of small retrospective studies and case series of sorafenib monotherapy in relapse/refractory AML were controversial –26. Metzp = 0.007)  [As donor lymphocyte infusions (DLI) and hypomethylating agents are widely used for the treatment of post-transplant AML relapse, some patients received sorafenib in combination with DLI or azacytidine . Treatme= 0.003) . Interes= 0.003) . DespiteAs first on the market next-generation potent FLT3-inhibitor , quizartp < 0.001) and event-free survival . Compared to other inhibitors, gilteritinib had a better safety profile as severe adverse events occurred less frequently in the gilteritinib group compared to the salvage therapy group. Seventy-four patients included in the study underwent gilteritinib regimen for relapse after allo-HCT and, interestingly, relapse in the first 6 months after allo-HCT was associated with a high benefit from gilteritinib therapy compared to salvage chemotherapy . Based upon these results, gilteritinib was approved by the FDA for relapsed/refractory AML in 2018. These findings were confirmed in the Japanese subpopulation in an open-label phase I trial [The highly selective type I next-generation FLT3-inhibitor gilteritinib was analyzed in a phase I–II trial in seven dose escalation cohorts of relapse/refractory AML and showed favorable safety profile and consistent FLT3 inhibition in most patients receiving a daily dose of 80 mg or higher . Based o I trial , and in  I trial . Of noteBased on the high response rates, gilteritinib may be the most promising FLT3-TKI and ongoing trials are testing gilteritinib in combination with venetoclax and hypomethylating agents in relapsed/refractory AML . The role of TKI in post-allo-HCT setting is not tested here as the number of patients with post-transplantation relapse is usually very low in these studies: Maiti and colleagues included only 4 transplanted patients in their phase II trial on triple combination therapy with gilteritinib–venetoclax–decitabine, but obtained a promising CRc of 62% in relapse/refractory patients [Despite the defined role of midostaurin in first-line therapy of FLT3-ITD-mutated AML , this fiBased on the promising results in relapse/refractory AML, treatment with FLT3-TKI was tested as maintenance therapy in clinical trials to elucidate its potential in preventing the occurrence of relapse after allo-HCT. Phase II–III trial on maintenance treatment after allo-HCT are listed in Table Induction therapy and maintenance with Midostaurin in FLT3-ITD-mutated AML had shown a very low relapse rate , 43, thop = 0.002) and overall survival  in the sorafenib group compared to the placebo group [Several early phase studies and case reports have reported evidence for the efficacy of sorafenib maintenance therapy after allo-HCT in patients with FLT3-ITD AML –47. The bo group . These rbo group , and by bo group –52. A rebo group .The efficacy of sorafenib is connected to side effects. A prospective single-arm pilot study  and the Subgroups analyses to identify variables that correlate with better outcome were conducted in both randomized trials. In the SORMAIN trial, patients who reached a MRD negativity before transplantation showed a higher benefit from sorafenib maintenance, compared to MRD-positive patients . InteresBased on these studies, the Acute Leukemia Working Party of the European Society for Blood and Marrow Transplantation recommends post-transplant maintenance therapy with a FLT3 inhibitor, preferably sorafenib, for patients who undergo allo-HCT for FLT3-ITD-mutated AML .Phase I data on quizartinib show acceptable tolerability and feasibility of FLT3-TKI maintenance after allo-HCT and, even though very preliminary, these data show early evidence of low relapse rate with only one relapse in 13 treated patients (8%) .Currently, a prospective phase III randomized, double-blind, multicenter trial (NCT02997202) using gilteritinib versus placebo for patients with FLT3-ITD AML as maintenance after allo-HCT is ongoing . StratifA comparison between sorafenib and midostaurin was conducted in a multicenter retrospective cohort study by Shimony and colleagues . Here, eThe optimal duration of maintenance therapy is also subject of debate. In the SORMAIN trial on sorafenib and in the ongoing clinical trials on gilteritinib and crenolanib, the maintenance therapy was conducted for up to 2 years. Midostaurin was administered in the trial for 12 months. Xuan and colleagues reduced the period of sorafenib treatment to 6 months claiming concerning about risk of developing drug resistance. Currently, the Leukemia Working Party recommends a duration of 2 years for maintenance therapy, to be adapted to tolerability .One last frequently debated issue is the need of treatment of MRD positivity. FLT3 inhibitors offer a valid and relatively safe therapy in the context of MRD positivity after transplantation. Both the phase II  and the As previously discussed in this review, sorafenib showed clinical evidence of synergism with allo-immunity against leukemia cells and enhanced graft-versus-leukemia effect. These synergism-motivated studies in mouse models which showed that FLT3 inhibition combined with T cell infusion can lead to complete elimination of leukemia cells. Mechanistically, FLT3 inhibition reduced the expression of the transcription factor ATF4. ATF4 normally blocks interferon regulatory factor 7 (IRF7) activation and this effect was antagonized by the FLT3 inhibition. Taking away the ATF4-mediated blockade, sorafenib allowed IRF7 activation and caused IL-15 transcription in the leukemia cells which caused an increase of CD8+ CD107a+ IFNγ+ T cells, which was connected to the increased elimination of leukemia cells by activated donor T cells  Increased IL-15 was detected in the blood of responder patients and caused increased mitochondrial spare respiratory capacity in T cells, which is consistent with previous reports indicating that IL-15 causes mitochondrial reprogramming in T cells , 62. SorWithin few months from initial response, the development of resistance to FLT3 inhibitors can occur and this typically limits the use of FLT3-TKI as monotherapy. The first predicted mechanism of resistance against type II inhibitors is the on-target mutation of TKD at activation loop D835 or at gatekeeper F691 . AccordiBased on the results obtained in clinical trials, FLT3-inhibitor maintenance is the recommended therapy for FLT3-ITD AML after allo-HCT. Gilteritinib has shown remarkable efficacy and acceptable toxicity in FLT3-ITD AML patients outside of the allo-HCT setting. However, since the ADMIRAL trial was conducted before the addition of midostaurin to induction therapy became standard of care, gilteritinib’s role in relapsed/refractory disease has to be re-evaluated carefully. For post-allo-HCT maintenance therapy, the Leukemia Working Party recommends sorafenib based to the large amount of available data and the stimulation of GvL effect. The ongoing trials on gilteritinib as maintenance and as combination therapy will clarify the importance of FLT3 inhibitors in this clinical context. In future, more data are needed to further unveil the potential advantages of these multi-kinase inhibitors, to understand and overcome possible relapse mechanisms and to fine-tune the immunogenic capacity through synergism with allo-HCT and combination with DLI.


However, we lack detailed knowledge of the molecular identity, cellular distribution, and functional role of Ca2+ channels expressed during folliculogenesis. In the neonatal period, ovarian maturation is controlled by protein growth factors released from the oocyte and granulosa cells. Conversely, during the early infantile period, maturation becomes gonadotropin-dependent and is controlled by granulosa and theca cells. The significance of intracellular Ca2+ signaling in folliculogenesis is supported by the observation that mice lacking the expression of Ca2+/calmodulin-dependent kinase IV in granulosa cells suffer abnormal follicular development and impaired fertility.The postnatal mammalian ovary undergoes a series of changes to ensure the maturation of sufficient follicles to support ovulation and fecundation over the reproductive life. It is well known that intracellular [Ca2+ channel alpha subunits and InsP3 and ryanodine receptors in the postnatal period from 3 to 16 days. During the neonatal stage, oocytes from primordial and primary follicles show high expression of various Ca2+-selective channels, with granulosa and stroma cells expressing significantly less. These channels are likely involved in supporting Ca2+-dependent secretion of peptide growth factors. In contrast, during the early and late infantile periods, Ca2+ channel expression in the oocyte diminishes, increasing significantly in the granulosa and particularly in immature theca cells surrounding secondary follicles.Using immunofluorescence in frozen ovarian sections and confocal microscopy, we assessed the expression of high-voltage activated Ca2+ channel expression from the oocytes to the perifollicular cells likely reflects the vanishing role of the oocytes once granulosa and theca cells take control of folliculogenesis in response to gonadotropins acting on their receptors.The developmental switch of CaThe online version contains supplementary material available at 10.1186/s13048-022-01015-y. Ion channels perform a diversity of essential functions in mammalian cells. However, we still lack a detailed description of most ovarian ion channels' molecular identity, cellular distribution, and functional role . This in2+ concentration ([Ca2+]i) (∼100 nM). [Ca2+]I transient elevations, termed Ca2+ signals, carry out many specific functions i. Most TRP channels are Ca2+-permeable with varying Ca2+ selectivity i, a signal that controls migration, proliferation, growth, maturation, and hormone secretion. [Ca2+]i is also intimately linked to apoptosis and cytotoxic cell death. In the ovary, [Ca2+]i. works as a universal activator of fertilization, egg activation, and egg-to-embryo transition i. in granulosa cells i. This FSH-induced [Ca2+]i rise is inhibited by removing external Ca2+ and after incubation with the Ca2+-channel blocker diltiazem, suggesting that FSH can increase [Ca2+]i directly through the opening of L-type Ca2+ channels i occur in several cell types before and during mitosis [2+ fluxes underlying these [Ca2+]i signals are mediated by CaV1.2 and CaV1.3 Ca2+ channels i signals are essential during fertilization and have critical roles during early events of egg activation and egg-to-embryo transition [One could speculate that Can slices . We planrtal PND 3-37, and2+ selective channels capable of supporting Ca2+- dependent exocytosis of autocrine and paracrine growth factors. In contrast, the early infantile period coincides with a diminished expression of Ca2+- permeable channels in the oocyte and increased expression in the granulosa and immature theca cells, which control folliculogenesis in response to gonadotropins.Oocytes and granulosa cells in the neonatal ovary express an assortment of CaCD1 mice from postnatal days (PND) 3, 8, and 16 were employed. Three mice of each age were used. They were maintained under controlled conditions of light and temperature  and free access to food and water. Mice were maintained in the animal facility and fed ad libitum. All animals were sacrificed at noon. For the morphologic descriptions of follicles and the immature ovary, we used the definitions from Picut .o C and then placed in PBS with 10%, 20%, and 30% sucrose (overnight incubation with each) to prevent freeze damage through  for sectioning . Frozen sections (10 μm in thickness) were mounted on Superfrost glass slides (Fisher Scientific), washed with PBS and incubated with Saline-Sodium Citrate 2x solution at 65°C for 45 min, and placed in PBS blocking solution  with 0.1% triton 100 for 60 min at room temperature for permeabilization. Three sections from each ovary were mounted and immunostained from at least two different animals. Then, sections were incubated for 24 hrs. in a humid chamber at 4°C with one of the primary rabbit antibodies from Alomone Labs : anti-CaV 2.1 , anti-CaV 2.2 , anti-CaV 1.2  or anti-CaV 1.3 , rabbit anti-InsP3R  H-300 Santa Cruz Biotechnologies  and mouse anti-RyR C3-33,  from ABCAM. Frozen sections were washed and incubated at room temperature for 2 hrs with F (ab')2 Alexa 488 fraction of donkey anti-rabbit IgG  or with the F (ab')2 Alexa 647 fraction of donkey anti-mouse IgG (H + L) in PBS  Finally; the sections were washed and mounted with Dako Glycergel . Several controls were required for reliable immunolabeling: Anti-CaV primary antibodies were pre-adsorbed with the corresponding synthetic antigenic peptide (2 μg peptide per 1 μg antibody). This procedure completely blocked specific immunofluorescence. Also, incubation with the secondary antibody alone gave a weak, nonspecific fluorescence , perfused transcardially with phosphate-buffered saline PBS, 0.1M), and then ice-cold 4% paraformaldehyde in PBS. Ovaries were removed, cleaned, and post-fixed in 4% paraformaldehyde in PBS for 3 hrs at 4M, and thFiji is Just software (Image J 1.53c). For this purpose, areas of interest were drawn by hand to analyze the mean fluorescence of all primordial, primary, and secondary oocytes, granulosa cells, theca cells, and interfollicular stroma separately. Primordial follicles have a single layer of flattened pre-granulosa cells which are difficult to measure; therefore, granulosa cells belong to primary, secondary, and preantral follicles. In Fig. Ovarian sections were viewed by confocal microscopy. Two representative sections from each age and different mice were chosen for display and quantitative analysis. Images were acquired with an LSM 800 inverted microscope  with a 40X Plan APO objective . Samples were excited with either a 488 nm or 647 nm laser and acquired with GaAsP detectors. For the reconstruction of each ovarian section, between 70 and 120 images (512 × 512 pixels) were acquired using the "tile scan mode" and a programmable motorized stage. The confocal microscope settings  were the same for all images. Raw image intensity was normalized so that no more than 0.01% of pixels were saturated. Some figures were digitally processed  to emphasize certain aspects or weakly stained structures. As a result, small areas in some images may appear saturated. The fluorescence intensity of the different ovarian structures was measured in two slices from each age and antibody , using Additional file 1: Supplementary Figure 1. Comparison of nonspecific and specific fluorescence. A: CaV1.2 (α1C) immunostaining of a frozen ovarian section at PND 16. After 24 hrs incubation with the anti-CaV1.2 primary rabbit antibody, the section was incubated for 2 hrs with the Alexa 488 donkey anti-rabbit IgG. B: Another ovarian section from the same stage was incubated for 2 hrs with the donkey anti-rabbit IgG alone to characterize nonspecific staining. C: RyR immunostaining of a frozen ovarian section at PND 3. After 24 hr incubation with the anti-RyR primary mouse antibody, the section was incubated with the Alexa 647 donkey anti-mouse IgG. D: Another ovarian section from the same stage was incubated for 2 hrs with the secondary donkey anti-mouse IgG alone to demonstrate nonspecific staining. Confocal microscope settings  were the same for all images, and pixel values of raw images were multiplied by the same factor (1.5). Calibration bars: 100 μm.


In this review, we focused on mitochondrial Ca2+ alterations in AD, their causes and bioenergetic consequences in neuronal and glial cells, summarizing the possible mechanisms linking detrimental mitochondrial Ca2+ signals to neuronal death in different experimental AD models.Alzheimer’s disease (AD) is a hereditary and sporadic neurodegenerative illness defined by the gradual and cumulative loss of neurons in specific brain areas. The processes that cause AD are still under investigation and there are no available therapies to halt it. Current progress puts at the forefront the “calcium (Ca In these processes, their tight interaction with other organelles, in particular the endoplasmic reticulum (ER), is fundamental ) is maintained at ~100 nM, much lower compared to that of the extracellular space, which is 1.2–2 mM. The [Ca2+] gradient is ensured by a complex cell-specific molecular Ca2+ toolkit constituted by channels, pumps and exchangers present both in the plasma membrane (PM) and in membranes of intracellular Ca2+ stores (mainly the ER), as well as by buffering systems  is tightly controlled by several processes: (1) the activity of Sarco-Endoplasmic Reticulum ATPase (SERCA), which pumps back Ca2+, when released, from the cytosol to the ER lumen at the expenses of ATP hydrolysis  within the cell needs to quickly return to resting baseline levels to prevent cytosolic Ca2+ overload and cell death. SERCA and PM Ca2+ ATPase  at the PM also aids in restoring baseline cytosolic Ca2+ levels. PMCA displays a higher Ca2+ affinity but lower capacity for Ca2+ transport, while the NCX shows the opposite features  upon stimulation is generally between 1–3 μM, which cannot efficiently trigger the opening of the MCU. However, near the mouth of Ca2+ permeable channels and receptors, such as PM channels, IP3Rs and RyRs, microdomains of high [Ca2+] occur, enabling the rapid opening of the MCUC  typically generated at sites where, upon stimulation, Ca2+ either enters the cell at the PM or is released from intracellular stores. Their formation is crucial for cell physiology and for mitochondrial Ca2+ homeostasis. Indeed, efficient mitochondrial Ca2+ uptake requires Ca2+ hotspot formation, and the buffer of Ca2+ by mitochondria at these sites is required for the spatiotemporal modulation of intracellular Ca2+ dynamics  are essential for mitochondrial Ca2+ uptake and ATP synthesis in all brain cells. On the other hand, mitochondria are also reported to play a role in determining the formation of other Ca2+ microdomains spatially restricted within the cell and occurring spontaneously in glial cells, especially in astrocytes. These localized Ca2+ events were first described as Ca2+ microdomains in the Bergmann glia processes  in this sub-compartment. Additionally, several mitochondrial carriers located in the IMM are regulated by Ca2+. This is the case of flavin adenin nucleotide-glycerol phosphate dehydrogenase (FAD-GPDH), Aralar and the ATP-Mg/Pi carrier. FAD-GPDH transfers reducing equivalents from cytosolic NADH to the ETC, sustaining mitochondrial respiration; Aralar is a glutamate/aspartate antiporter, involved in glutamate-dependent mitochondrial respiration, and ATP-Mg/Pi carrier exchanges ATP-Mg (or ADP-Mg) and phosphate (Pi), controlling the content of mitochondrial adenine nucleotides. Furthermore, Ca2+ is emerging as a key regulator of nucleotide, metabolite and cofactor shuttling, mediated by L-CAMCs  and S-CAMCs , both able to sense changes in IMS [Ca2+]. Despite debate, some works have reported a direct effect of Ca2+ on ETC complexes and ATP-synthase. In line with all these findings, the constitutive, low level ER-mitochondria Ca2+ transfer has been reported to be pivotal in maintaining cell ATP production .2+ entrance into neuronal mitochondria regulates energy production in an activity-dependent manner  by inhibiting PMCA and NCX .PSEN mutations to Ca2+ alterations and cell death. Presenilin 1 and 2 (PS1 and PS2) form the catalytic core of γ-secretase complex, an enzyme mainly located in the ER membranes and participating in APP cleavage  causes both altered APP processing, with an accumulation of Aβ toxic peptides, and deregulated kinase activity, leading to tau hyper-phosphorylation and eventually to a Ca2+-dependent neuronal death , compared to controls [2+]. This cytosolic Ca2+ overload has been attributed to purinergic P2X7 receptor activation, whose expression increases in AD microglia  caused by the mutated protein.Overall, an increase in cytosolic Ca2+ influx across the PM or its release from the ER, described above in different AD samples, can impact mitochondrial Ca2+ handling. However, mitochondrial Ca2+ can also be altered per se and this alteration could affect the global Ca2+ signaling. Generally, the accumulation of Ca2+ into the mitochondrial matrix can trigger cell death by the release of cytochrome c and the activation of caspase 9 and 3  a blunted mitochondrial Ca2+ uptake, mainly in FAD-PS2-based mouse models  and IP3R1 and IP3R3 [Analyses of postmortem cerebellar tissues from patients with nd IP3R3 . Similarnd IP3R3 .PSEN1 mutations [Different AD models display an increased physical interaction between ER and mitochondrion ,155,156,utations . Generalutations .2+ signaling deregulation in AD. Jadiya et al. showed that AD progression, in the 3xTg AD mouse model , is associated with the progressive loss of the mitochondrial NCLX. It is noteworthy that this loss started before Aβ plaque deposition, and neuronal overexpression of NCLX completely restored brain Aβ plaque load, tau hyperphosphorylation and cognitive decline [2+ in a similar model, only after Aβ plaque deposition, i.e., at 8–9 months of age. This phenotype has been attributed to an increase in MCU expression levels, as its lowering rescued most of the AD hallmarks [Only recently have in vivo studies been performed to address mitochondrial Ca decline . Furtherallmarks .2+ dynamics in vivo in PS2-based mice, as well as in astrocytes or microglia in any AD model. Instead, in vitro primary cultured astrocytes lacking APP show fragmented mitochondria with altered Ca2+ uptake after ATP stimulation [2+ and mitochondria in glial cell physiology, this aspect deserves future proper studies.To the best of our knowledge, no studies have been performed to assess mitochondrial Camulation . Conside2+ deregulations in AD reported so far . Hed so far . Indeed,2+ handling and, thus, mitochondrial bioenergetics. The two mitochondrial Ca2+ phenotypes reported in AD models  can both culminate in cell death, thus contributing to neurodegeneration via two different mechanisms. In this context, the Ca2+ shuttling between the ER and mitochondria is of particular interest. Indeed, it has been shown that even a reduced basal shuttling of this cation between the two organelles can increase the phosphorylation of PDH, inhibiting the enzyme and slowing down the TCA cycle, reducing the availability of NADH and FADH2, thus impairing ETC activity. This process reduces ATP production, culminating in autophagy activation .2+ alteration and bioenergetic deficits is lacking in most of the studied AD models. Some of this information can be inferred by combining data obtained in different studies and performed in AD mouse models based on the same mutations. For example, mitochondrial functional analyses in the 3xTg-AD mouse model, characterized by an age-dependent reduction in NCLX expression [However, a direct correlation between mitochondrial Capression , displaypression . Interespression .2+ level has been found to be increased after Aβ plaque depositions [Severe mitochondrial impairments were observed in neurons in proximity of Aβ plaques of an APP/PS1 AD mouse model . In a siositions .2+ defects induced by PSEN2 mutations to bioenergetics. In particular, Rossi et al. showed that in different cell types expressing mutated PSEN2, blunted mitochondrial Ca2+ uptake, together with decreased mitochondrial pyruvate import, contributed to reduced ATP production .2+ efflux, the NCLX inhibitor CGP-37157 is widely used despite its lack of specificity [As for mitochondrial Cacificity .2+ handling dysfunction, by overcoming the mitochondria hypometabolism caused by impaired pyruvate import [Furthermore, different therapeutic possibilities are expanding over time, showing promising tools for modulating mitochondrial function (reviewed here ). Gene te import .2+ homeostasis in the brain, with the organelle Ca2+ signal not only crucial for cell metabolism and bioenergetics but also fundamental for shaping overall cytosolic Ca2+ handling. Very little is known about the role of mitochondrial Ca2+ buffering in the regulation of global Ca2+ signal in glial cells. Even in neurons, this aspect is overlooked. Recent works highlight a close correlation between mitochondrial Ca2+ buffering and neuronal activity, pointing toward neuronal subtype-specific regulation of mitochondrial Ca2+ signaling. In this context, mitochondrial functional analysis in AD is even more puzzling because of the difficulties of fully phenocopying AD traits and progression, the lack of systematic transversal and longitudinal studies of mitochondrial Ca2+ handling and related pathological consequences in physiologically relevant contexts, and the absence of correlative studies between cytosolic and mitochondrial Ca2+ dynamics. Despite the multiple hypothesis, nowadays, AD is considered a multifactorial pathology and mitochondria could have a pivotal role. The continuous development of indicators that enable the measurement of the dynamic changes of intracellular ligands in real time and in complex systems, together with the possibility of pharmacologically and genetically modulate mitochondrial Ca2+ handling and bioenergetics, constitute and will constitute a great number of tools to investigate the pure role of mitochondrial (dys) function in the pathogenesis and progression of AD.A very complex scenario is emerging on mitochondrial Ca


The angiomyolipoma (AML) is a hamartoma tumor, most of the times of kidney origin, having the liver as the second most common site; often times associated with tuberous sclerosis. Angiomyolipomas originating from other sites are much rarer, and there are very few descriptions of cases in the mediastinum, heart, spermatic cord, vaginal wall, Fallopian tube, oral cavity, pharynx, nasal cavities and the skinWe found only seven reports of this tumor arising from the nasal cavities in the literature; ours would be the eighth case.One male, 54 year-old patient came to our ENT service in 2006, complaining of 20 years of spontaneous and recurrent epistaxis, always on the left side. These were quick and self-contained episodes, which happened from one to two times per month and had worsened in the last year. He did not have nasal obstruction, hyposmia, rhinorrhea or local injury.Upon nasal endoscopy we noticed a round, red and smooth surface lesion in the anterior region of the left inferior meatus. The right nasal cavity and the remaining of the physical exam were normal. We ordered a CT scan of the paranasal sinuses which showed a soft tissue lesion in the left inferior meatus, shifting medially to the inferior turbinate, towards the nasal septum, without signs of bone erosion . The patHe was submitted to nasal endoscopic surgery in October of the same year, in which we found a macroscopically round, smooth lesion in the left inferior meatus, near the outlet of the nasolacrimal duct, and the lesion was resected without complications.Pathology revealed a tumor made up of well-differentiated typical leiomyocites, pervaded by proliferated vessels with thick and twisted walls, besides well differentiated fat tissue, suggesting angiomyolipoma . HMB-45 The patient remains in periodic follow up in our clinic, He has been asymptomatic and he does not have changes upon nasal endoscopy.The kidney angiomyolipoma (AML) is a hamartoma tumor made up of smooth muscle, vessels and fat tissue in different ratios. It is closely associated with the occurrence of tuberous sclerosis (TS), and about 80% of patients with TS have AML and half of the kidney AML patients have TSOf all the AML described from sites other than the kidney and liver, the most commonly found are those on the skin. Skin AML has been described on the penis, head and neck and limbs. Histopathologically speaking, the skin and nasal AML are identical, as are the oral cavity and pharyngeal types. Nonetheless, these have important pathology differences from kidney and liver AML; and Watanabe & SuzukiMucocutaneous AML are different in many aspects from those arising from the kidneys or liver. First, they are not associated with TS, they usually affect older men and are usually small, contrary to what happens to kidney and liver tumors, which are, often times, large. However, the most striking difference is that the nasal cavity and skin AML are made up only of mature smooth muscle cells which are negative to the HMB-45 melanoma-specific antigen, contrary to those which are not mucocutaneous.Of the 7 patients with nasal cavity AML found in the literature, 5 were men and 2 were women. Mean age was 45 to 88 years and three in seven (42%) had DM associated. Our patient follows such incidence, an adult young man without diabetes.AML is a very rare tumor, especially among those which affect the nasal cavity. Nonetheless, it is necessary to be attentive to the possible differential diagnosis in nasal cavity tumors, especially when facing a unilateral nasal tumors.


TP53 mutations, complex karyotypes) of well-recognized prognostic significance. In this review, we aim to discuss the latest updates on intermediate-risk definition and highlight the therapeutic advances and challenges that warrant refining the prognostic classification of this category.Risk stratification models, including the European LeukemiaNet 2017 and 2022 guidelines, categorize newly diagnosed acute myeloid leukemia (AML) patients into several subgroups of distinct genetic characteristics and disease outcomes. The intermediate-risk group remains the most heterogenous group, as most AML patients fall into it  by virtue of not fulfilling criteria that identify specific entities  represents a heterogeneous group of hematopoietic neoplasms deriving from the abnormal proliferation of myeloid progenitors in the bone marrow. Patients with AML may have highly variable outcomes, which are generally dictated by individual clinical and genomic characteristics. As such, the European LeukemiaNet 2017 and 2022 guidelines categorize newly diagnosed AML into favorable-, intermediate-, and adverse-risk groups, based on their molecular and cytogenetic profiles. Nevertheless, the intermediate-risk category remains poorly defined, as many patients fall into this group as a result of their exclusion from the other two. Moreover, further genomic data with potential prognostic and therapeutic influences continue to emerge, though they are yet to be integrated into the diagnostic and prognostic models of AML. This review highlights the latest therapeutic advances and challenges that warrant refining the prognostic classification of intermediate-risk AML. FLT3 mutants, most patients usually fall into this group (an umbrella category) by virtue of not fulfilling criteria that identify specific entities  with well-recognized prognostic significance.Acute myeloid leukemia (AML) is a diverse group of hematopoietic clonal disorders characterized by the accumulation of immature myeloid progenitors . DespiteAML classification and prognostic criteria are based on cytogenetic and molecular features at the time of diagnosis. At least 50% of cases exhibit karyotypic abnormalities by conventional cytogenetic analysis; in addition, mutational screening reveals at least one somatic alteration in virtually all patients ,8. TradiFLT3, NPM1, CEBPA, IDH1, IDH2, DNMT3A, TET2, and KIT genes provided important information that improved AML diagnosis, treatment stratification, and monitoring ..19].Interestingly, machine learning (ML) techniques have been applied in leukemia research and represent a promising auxiliary tool for developing new approaches that aim to improve AML risk stratification. Several studies have implemented unsupervised and unbiased ML methods that demonstrated high accuracy in terms of genomic classification . These aFLT3 allelic ratio, as demonstrated in Most recently, the 2022 ELN guidelines for diagnosis and management of AML were published . In thisThe current AML treatment paradigm recommended by the major societies, including NCCN and ESMO, is heavily based on four factors: age, performance status, organ function , and risk classification per ELN 2017 criteria . Of note2 and 90 mg/m2) show a survival benefit with higher doses of anthracyclines in both younger and older patients (at least up to the age of 65 years) [Treatment of intermediate-risk ELN patients eligible for intensive therapy has not substantially changed over the past few decades, and “7 + 3” induction therapy still represents the standard of care. Data from three randomized trials comparing anthracycline doses (daunorubicin 45 mg/m5 years) ,22,23. T5 years) ,25,26.2 (high-dose) by 3 h infusion, every 12 h on days 1, 3, and 5, was associated with a lower relapse rate and improved OS compared to intermediate- and low-dose regimens. However, there was no subgroup analysis according to cytogenetics in the original trial [p < 0.05) [al trial . A follo < 0.05) .Since most studies have focused on stratifying patients to various first-line therapies, little is known about the outcomes of different consolidation regimens in the intermediate-risk AML group. As mentioned, patients who remain in remission may be considered for HSCT as a potentially curative option, when suitable for the procedure. However, randomized trials investigating the benefit of transplant in intermediate-risk AML are lacking. Studies investigating the benefit of allogeneic and autologous HSCT have used donor availability as a criterion for transplant ,32; hencNPM1 mutations [p = 0.713) or disease-free survival (DFS)  rates between the MRD-negative and -positive groups. Remarkably, MRD-positive intermediate-risk AML patients treated with allogeneic HSCT had OS and DFS rates similar to favorable-risk patients. The same group confirmed the role of MRD when risk categorization according to the ELN 2017 recommendations was applied to the AML1310 trial [p = 0.234), whereas allogeneic HSCT did significantly increase survival compared to autologous HSCT in MRD-positive patients .The role of MRD in treatment stratification is also evolving. MRD refers to the detection of malignant cells by molecular biology or flow cytometry techniques in patients in complete remission, defined as hematologic recovery and an absence of AML blasts by morphological bone marrow examination. Studies have demonstrated the usefulness of monitoring MRD in patients with RUNX1-RUNX1T1 rearrangements and utations ,35. Patiutations . Howeverutations ,38,39. Outations . Neverthutations . No sign10 trial . Indeed,2 subcutaneously days 1–5 every four weeks vs. observation. While patients treated with azacitidine had improved 12-month DFS , OS was similar . Of note, the study design did not require patients to receive consolidation with cytarabine, and no subgroup analysis based on risk stratification was provided in this trial [p < 0.05) [Maintenance therapy is used to decrease the risk of disease relapse. In an open-label phase III study, patients ≥ 60 years with AML or refractory anemia with excess blasts who achieved remission post induction were randomized to receive azacitidine 50 mg/mis trial . In anot < 0.05) . A subgrIn patients who do not achieve remission with induction or relapse after treatment, the current NCCN and ESMO guidelines recommend targeted therapy or clinical trial enrollment whenever possible. As discussed, meticulous clinical research from bench to bedside has led to a better understanding of the pathophysiology of AML and, subsequently, to the discovery of novel therapeutic agents. Nevertheless, the molecular heterogeneity of AML continues to remain a challenge. Indeed, the significant advances in elucidating the genomics of AML have not yet led to ameliorating outcomes, which remain dismal in relapsed and refractory patients (R/R AML). Moreover, the situation is even hazier when considering the treatment of older and frail (unfit) AML patients.In the United States, more than 300 clinical trials are currently assessing the safety and efficacy of various therapeutic agents in AML patients. A detailed review of ongoing studies is beyond the scope of this review; however, in IDH1/2 or FLT3 inhibitors, it is uncertain if this classification would still apply [The entire schema of ELN risk classification currently used in the clinic is derived from the experience of patients treated with intensive chemotherapy. Over the past few years, with the advent of many new drugs for AML, including venetoclax combinations and a molecularly defined class of targeted therapies, ll apply .FLT3 gene are among the most common mutations in intermediate-risk AML, most commonly associated with a normal karyotype [FLT3 receptor tyrosine kinase and multiple downstream signaling cascades involved with AML cell survival, proliferation, and differentiation. In the ELN 2017, the allelic burden and the pattern of co-mutations (in particular in NPM1 and DNMT3A) modulated the prognostic significance of the more common and prognostic FLT3 mutation, FLT3-ITD [FLT3-ITD with a high allelic ratio (>0.5) conferred adverse prognosis; however, the simultaneous presence of an NPM1 mutation made it instead recognized as intermediate-risk [FLT3 mutants to the intermediate-risk category [FLT3-mutated AML patients has been transformed by the advent of small molecule tyrosine kinase inhibitors  for both newly diagnosed and R/R AML [FLT3-mutated AML patients to the azacitidine/venetoclax combination are comparable to those of patients with wild-type FLT3. In light of response improvements from doublets, the need to study rational combinations of triplets or sequential therapies to continue improving the outcomes of patients with AML with FLT3-ITD is a current topic of research.Mutations in the aryotype . These mFLT3-ITD . For insate-risk . The newcategory . The tre R/R AML ,62. Furt R/R AML ,63. RespIDH1 and IDH2 mutations in AML led to the development of targeted oral therapies. This represents a remarkable bench-to-bedside journey. IDH1/2 mutations are present in ~20% of AML cases (IDH1: ~5%–8% and IDH2: 9%–12% [IDH1 (ivosidenib) [IDH2 (enasidenib) [IDH2-mutant R/R AML patients (after the failure of two or three lines of therapy) reported no survival benefit with enasidenib compared with standard of care [IDH1-mutant newly diagnosed AML unfit for intensive treatment showed a 12-month event-free survival rate significantly higher in the experimental group, along with a benefit in OS , opening new therapeutic opportunities in such a setting. [IDH1-mutated cells, and less transfusion support was needed, with safety signals similar in the two groups if excepting the higher rate of differentiation syndrome in the experimental arm.Characterization of the unique mechanism of leukemogenesis resulting from : 9%–12% ). These sidenib)  and IDH2sidenib)  are FDA-sidenib) . Similar of care . These tsetting.  ParticulFLT3 inhibitor, with potential for anti-tumor activity in AML, that is currently in the early phases of therapeutic development (NCT04278768). In adult patients with FLT3-mutated R/R AML, combinations with experimental oral agents such as TL-895 (tyrosine kinase inhibitor) and KRT-232 (MDM2 inhibitor) are being evaluated for safety and efficacy in an early phase clinical trial (NCT04669067). Minnelide is a potent heat shock protein 70 inhibitor that showed potent preclinical anti-tumor activity in AML [CA-4948 is a potent interleukin-1 receptor-associated kinase 4 (IRAK-4)/NCT042787. In adulFLT3-positive cases, now representing a good fraction of the intermediate-risk group according to the latest classification, especially in light of the availability of specific targeted treatment options. A second challenge is represented by AML trial designs, which beg the question of what is the best setting to test these novel compounds. Indeed, since the intermediate-risk group is the most heterogeneous category, issues of clonal evolution and genomic and/or epigenomic complexity, after receiving standard of care therapies, and the emergence of subclones with distinct resistance mechanisms are only some of the challenges to be faced. A third challenge is the incorporation of novel agents and existing targeted therapies in the frontline regimens, which are likely to increase the proportion of patients achieving and maintaining CR for longer periods [Many challenges remain unsolved in intermediate-risk AML. A first challenge is to define the precise scenarios for using the plethora of novel targeted drugs . The ELN periods . As a co periods ,72) may Over the last decade, immunotherapies have shown encouraging anti-tumor activity across most cancer types and may offer a therapeutic opportunity also in intermediate-risk AML, in a way that is mutation-agnostic. Immune-based therapeutics, including monoclonal antibodies, immune checkpoint inhibitors (ICIs), bispecific T cell engagers (BiTE) antibodies, and chimeric antigen receptor T-cell therapy (CAR-T) are being extensively investigated to improve the outcome of AML patients.FLT3, RUNX1, and TET2 mutations [Immune checkpoints, namely cytotoxic T lymphocyte antigen 4 (CTLA-4), programmed death-1 (PD-1), and programmed death-ligand 1 (PD-L1), play a crucial role in regulating T cell immune response to the tumor cells . ICIs, sutations ,76,77. Hutations . Of noteutations . Out of utations . Out of Ravandi et al. evaluated nivolumab in combination with Idarubicin and cytarabine in newly diagnosed AML patients. In this single-arm, phase 2 clinical trial, 44 patients were enrolled, of whom 17 (39%) were intermediate-risk AML based on ELN 2017 classification . The medCD33 is highly expressed on AML cells, and unconjugated and conjugated CD33 monoclonal antibodies are in the early phase of drug development . CurrentAnother active area of experimental drug development is CAR-T cell therapy . In AML,AML is a disorder characterized by a puzzled molecular landscape. As a result, patients are characterized by different outcomes according to their individual genomic makeup, which, together with host factors such as age and performance status, contribute to the diverse therapeutic and survival scenarios. Intermediate-risk cases, as per ELN 2017, and more recently ELN 2022, represent the most heterogeneous group because of the broad features included in the definition of this category . While new genomic information is generating a more detailed dissection of the disease, as in the recent two classifications of myeloid disorders ,72, an uPerhaps, as our understanding of AML pathogenesis and risk-stratification modalities continues to improve, we anticipate that the intermediate-risk group definition will continue to change. In fact, the exponential growth in the targeted therapeutic options utilized in AML continues to alter the prognostic significance of many molecular derangements. Nonetheless, in harmony with the 2022 ELN guidelines, we recommend judicious incorporation of the patient’s fitness for the intensive regimens and disease molecular characteristics used as criteria for treatment selection.


NPM1 mutation achieve remission with intensive chemotherapy. However, many patients subsequently relapse, which occurs frequently within the first 2–3 years after therapy, while late relapse after more than 10 years is rare and can also represent secondary/therapy-associated AML without the NPM1 mutation. Here, we present a case of NPM1-mutated AML that developed medullary and extramedullary relapse 17 years after allogeneic stem cell transplantation, maintaining the NPM1 mutation and all other genetic alterations detected at first diagnosis. This exceptionally long latency between diagnosis and relapse of a genetically highly related leukemic clone implies the existence of therapy-resistant, persisting dormant leukemic stem cells in NPM1 mutant AML.The majority of patients with acute myeloid leukemia (AML) with the  NPM1 mutations are among the most frequent genetic alterations in acute myeloid leukemia (AML) and are associated with distinct morphological, genetic, and clinical features. Intensive chemotherapy with or without allogeneic stem cell transplantation  leads to remission in 70–90% of patients with NPM1mut AML, but about 30–50% of patients relapse due to persistent leukemic stem cells [em cells , 3, withem cells , 4–7. Reem cells , 9. Extrem cells . NeverthNPM1-mutated AML who developed a bone marrow (BM) and EM relapse 17 years after the first diagnosis  in 2003. A BM biopsy showed 80% blasts with monocytic features without evidence of extramedullary disease. Conventional cytogenetics revealed a 46, XY, del(9q) karyotype. Flow cytometry revealed the expression of CD33 and HLA-DR, without CD34 or CD117 expression. Molecular genetics and MRD assessment were initially not performed as they were not available at that time. No relevant comorbidities, no family history of malignancies, and no known environmental exposures to substances that damage DNA were reported. The patient received intensive induction therapy with cytarabine, daunorubicin, and thioguanine and achieved a complete remission. He then underwent allogeneic HSCT from an HLA-identical sibling, which was well tolerated without the development of GvHD. Bone marrow assessment revealed a CR with complete donor chimerism one month after transplantation until the end of follow-up seven years after transplantation.A 23-year-old male patient was diagnosed with AML  scan of the chest revealed lesions in both lungs suspicious of metastases. However, histologic analysis of the tumor that infiltrated the colon mucous membrane showed myeloid leukemic cells that were strongly positive for CD33 and CD117 and negative for CD34.NPM1-Type A mutation and a DNMT3A R882H mutation, while no mutations were found in FLT3, IDH1/IDH2, RUNX1, TP53, or ASXL1. Cytogenetics revealed the same del(9) (q22q34) as 17 years before, in addition to further chromosomal aberrations in the same metaphases 47, XY, t , del(9) (q22q34)x2, add(14) (q22), add(15) (q22), add(16) (p13.3),and +21.Next, a BM biopsy was taken, which revealed a 30% infiltration of the BM by myeloid leukemia blasts with monocytic differentiation positive for CD13, CD33, CD34, and CD117. By molecular diagnostics, we detected an DNMT3A R882H and NPM1 Type A mutations were already present at initial diagnosis, while no other mutations were found in FLT3, IDH1/IDH2, RUNX1, TP53, or ASXL1. A drop in donor chimerism revealed that leukemia originated from the host and not the donor. The persistence of two identical gene mutations and one chromosomal aberration highly implies that relapse evolved directly from persisting cells of the initial leukemic clone rather than an independent clone with newly acquired aberrations.Retrospective analyses of the diagnostic AML sample obtained 17 years before revealed that the After induction therapy with cytarabine and daunorubicin, followed by consolidation therapy with intermediate-dose cytarabine and radiation of the pelvic chloroma, a second complete hematologic remission was achieved with complete regression of the pelvic tumor. Radiotherapy of the lung and pelvis was performed. A second HSCT from an HLA haploid donor was planned, but unfortunately, the patient died due to neutropenic sepsis during consolidation therapy.NPM1 mutation at both diagnosis and relapse. The presence of the same gene mutations, NPM1mutA and DNMT3AR882H, as well as a del(9q), strongly argues that the second AML directly evolved from the initial AML clone [TP53 [NPM1mut AML often represents a genetically distinct, therapy-associated AML without NPM1 mutation [This case represents one of the latest relapses described in AML with a confirmed ne [TP53  were abs as a delq, strongNPM1 mutation than in AML without the NPM1 mutation [NPM1 mutation. Bolli et al. described a case of a myeloid sarcoma with an NPM1 mutation occurring 20 years after the first diagnosis of AML but no genetic information was available at the initial diagnosis [NPM1 mutations [Bertoli et al. showed that “true,” genetically stable, very late relapses after 5 years are more common in patients with the mutation . Watts emutation  that weriagnosis , 12. Chautations , 10, 12,The underlying pathophysiologic mechanisms of such long quiescence of a leukemic stem cell are unclear. The microenvironment has a strong influence on normal and leukemic stem cells, and the transition of such a cell outside the bone marrow may have induced an awakening from dormancy. Decreased immunosurveillance may be a possible factor. Graft versus leukemia might not be that effective outside the BM, explaining the higher rates of EM relapse after allo-HSCT –15.NPM1mut AML has to be considered and warrants awareness of the possibility of EM relapse. AML cases with VLR further demonstrate that leukemia stem cells (LSCs) may persist for many years, most likely in a dormant state that is invisible to the immune system, even after allo-HSCT. It is conceivable that many patients in long-term remission also have persisting, dormant LSCs, as also suggested by the detection of low MRD levels in patients in long-term remission [Very late relapse AML with clear proof of a genetic relationship, as in our case, has several implications. From a clinical perspective, the occurrence of very late hematologic and extramedullary relapse in emission . The rea


ABCA13, ANXA3, ARG1, BTNL8, C11orf42, CEACAM1, CEACAM3, CHI3L1, CRISP2, CYP4F3, GPR84, HP, LTF, MMP8, OLR1, PADI2, RGL4, and RILPL1) were found to be related to AML patient survival time. We then compared the hub gene expression levels between AML peripheral blood (PB) samples (n = 162) and control healthy whole blood samples (n = 337). Seventeen of the hub genes showed lower expression levels in AML PB samples. The gene expression analysis was also done among AML BM (bone marrow) samples of different stages: diagnosis (n = 142), posttreatment (n = 42), and recurrent (n = 12) stages. The results showed a significant increase of ANXA3, CEACM1, RGL4, RILPL1, and HP in posttreatment samples compared to diagnosis and/or recurrent samples. Transcription factor (TF) prediction of the hub genes suggested LTF as the top hit, overlapping 10 hub genes, while LTF itself is just one of the hub genes. Also, 3671 correlation links were shown between 128 mRNAs and 209 lncRNAs found in survival time-related modules. Generally, we identified candidate mRNA biomarkers based on CN-AML data which can be extensively used in AML prognosis. In addition, we mapped their potential regulatory mechanisms with correlated lncRNAs, providing new insights into potential targets for therapies in AML.The prognosis of acute myeloid leukemia (AML) remains a challenge. In this study, we applied the weighted gene coexpression network analysis (WGCNA) to find survival-specific genes in AML based on 42 adult CN-AML samples from The Cancer Genome Atlas (TCGA) database. Eighteen hub genes ( The malignant hematologic disease, acute myeloid leukemia (AML), is a heterogeneous clonal disorder of myeloid progenitors that accumulates due to a blockage in their differentiation and infiltration into other organs of the body , leading to death –3. The pNPM1 and CEBPA, in which mutations have been proposed as good prognostic factors, as well as PLT3, RUNX1, ASXL1, and TP53, in which mutations have been considered to be correlated with poor prognosis [dimCD117+ phenotypical abnormal cell ratio > 2.055% within 2 weeks after the first complete remission (CR) is considered to be an independent risk factor for recurrence, which also is an adverse factor for relapse-free survival (RFS) and overall survival (OS) in adult AML patients [Cytogenetically normal acute myeloid leukemia (CN-AML) presents without microscopically detectable chromosomal abnormalities and contributes to approximately 50% of the observed AML cases . Heterogrognosis , 5. Trearognosis . Also, iTo better understand the complex prognostic gene expression signatures of CN-AML and investigate potential targeted therapies, we performed the weighted gene coexpression network analysis (WGCNA) on the RNA-seq data of adult patients with CN-AML, available from The Cancer Genome Atlas (TCGA). Our study identified survival-specific genes and provided system-level evidence of genetic networks that contribute to the prognosis of adult CN-AML patients. What is more, the survival-specific genes we found based on CN-AML samples also showed prognostic values in AML samples regardless of any clinical characteristics .https://portal.gdc.cancer.gov/) (project TCGA-LAML [CGA-LAML ) for thehttp://www.ncbi.nlm.nih.gov/geo/; accession number GSE12417 [To perform the survival analysis among the hub genes obtained after the WGCNA, we chose 148 adult (≥18 years) CN-AML patients with an OS > 30 days from the Gene Expression Omnibus (GEO) database  [To compare the expression levels of hub genes and correlatedly expressed lncRNAs in AML BM samples of different stages, we chose 196 samples of 163 patients from an independent cohort in the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) database (/target) . The samhttps://portal.gdc.cancer.gov/) and 29 samples from the TARGET (https://ocg.cancer.gov/programs/target) database (samples from TCGA database do not include the 42 CN-AML used for WGCNA). The AML PB sample screening criteria were primary AML PB samples of the diagnosis stage with RNA expression profiles; AML PB samples were selected regardless of their clinical characteristics  to serve as normal controls. The healthy whole blood sample screening criteria were healthy whole blood samples with RNA expression profiles.To compare the expression levels of hub genes and correlatedly expressed lncRNAs between primary AML PB samples at the diagnosis stage and normal whole blood samples, we chose 133 samples from TCGA database (We collected the fragments per kilobase of exon model per million (FPKM) mapped reads  and stanβ values for mRNAs, miRNAs, and lncRNAs was 5, 4, and 4, respectively ) was applied to statistically identify enriched pathways and gene ontologies (GO) [Q value < 0.05 [The ToppGene database (ies (GO) . The cute < 0.05 . The rese < 0.05 , 18.https://string-db.org/) was used to construct the PPIs [The online database Search Tool for the Retrieval of Interacting Genes (STRING) (Version 11.0)  [The survival analysis based on the hub gene mRNA expression levels and patient OS was analyzed by an online tool, GenomicScape (pe.com/) . The prohttps://xenabrowser.net/) [The expression matrices with RSEM (RNA-Seq by Expectation Maximization)  normalizer.net/) .https://amp.pharm.mssm.edu/chea3/) website [TF prediction for the hub genes was done via the ChEA3 ( website .http://www.rstudio.com), Microsoft Excel 2007, the Cytoscape software (Version 3.7.0), and GraphPad Prism 7 were used for all statistical analysis or graphic drawings in this research. P values < 0.05 were considered statistically significant [The RStudio software  1 module of mRNAs, as well as ME2, ME3, and ME4 modules of lncRNAs, showed positive associations with the survival time of adult patients with CN-AML Figures , suggestP = 1.2e − 12) and the ME3 module of lncRNAs  and strongly correlated in ME2  and ME4  modules of lncRNAs, indicating that genes significantly associated with survival time were also key elements of modules associated with survival time. Thus, we considered genes from the ME1 module of mRNAs, together with those in the ME2, ME3, and ME4 modules of lncRNAs, as survival-specific in adult patients with CN-AML.To further explore the association of these four modules with patient survival time, we used GS and MM measures to identify the genes with both high GS for “survival time,” as well as high MM in the selected modules. As shown in Figure To explore the survival-specific protein-coding genes, the GO analysis of BP, MF, and CC, as well as pathway analyses, was performed on the 131 mRNAs of the ME1 module. The top 20 GO terms of each category are shown in Next, we established a PPI network of the ME1 mRNAs recognized in STRING, as shown in Figure ABCA13, ANXA3, ARG1, BTNL8, C11orf42, CEACAM1, CEACAM3, CHI3L1, CRISP2, CYP4F3, GPR84, HP, LTF, MMP8, OLR1, PADI2, RGL4, and RILPL1.We obtained 18 hub genes from 131 mRNAs of the ME1 module by the method we described above, using the cytoHubba plugin of the Cytoscape software. These were C11orf42) had a decreased expression level in AML PB samples compared to healthy samples (P < 0.05).The expression levels of the hub genes in PB samples of primary AML patients from the diagnosis stage and healthy whole blood samples were analyzed. We separately compared TCGA AML PB samples vs. GTEx healthy samples Figure S A and TANXA3 and CEACM1 are significantly upregulated in posttreatment samples compared to diagnosis samples (P < 0.05). RGL4 and RILPL1 are significantly upregulated in posttreatment samples compared to both diagnosis and recurrent samples (P < 0.05). ABCA13, ARG1, CRISP2, and CYP4F3 showed higher expression levels in recurrent samples than in diagnosis samples (P < 0.05). Also, the expression level of HP in posttreatment samples is significantly higher than that in recurrent samples (P < 0.05).Next, we compared the hub gene expression levels in primary AML BM samples of three different stages: diagnosis stage, posttreatment stage, and recurrent stage . From ARG1, CEACAM1, CHI3L1, CRISP2, and CYP4F3, were significantly correlated with a longer OS (P < 0.05)  .Lactotransferrin (LTF) ranked the first place with the lowest mean rank [CEACAM3, CEACAM1, ANXA3, ARG1, CYP4F3, CHI3L1, PADI2, RGL4, MMP8, and ABCA13). Also, LTF is just one of our 18 hub genes.We then predicted TFs for the 18 hub genes by ChEA3 website. The top 10 TFs were listed in ean rank  and the R| > 0.5, P < 0.05). In particular, 127 mRNAs and 28 lncRNAs formed 224 very strong [R| > 0.8 (P < 0.05)  . The top cancers . We anal samples . No sign samples . Since wPatients with AML without chromosomal changes are diagnosed as CN-AML . Having WGCNA, an algorithm for a scale-free network introduced in 2005, has been used to propose candidate therapeutic targets or predict diagnosis, classification, progression, or prognosis in various types of cancers –37. As aAfter constructing a PPI network of 131 mRNAs and mRNA-lncRNA network carried out by Pearson's correlation analysis, we used the cytoHubba plugin of Cytoscape software to find hub genes. CytoHubba provides 12 topological analysis methods, which are MCC, DMNC, MNC, Degree, EPC, BottleNeck, EcCentricity, Closeness, Radiality, Betweenness, Stress, and ClusteringCoefficient, to rank nodes in a network by the network features . These nARG1, CEACAM1, CHI3L1, CRISP2, and CYP4F3 with a longer OS.In expression analyses of the hub genes in different cohorts of AML samples and healthy whole blood samples, 17 of the 18 hub genes showed higher expression levels in AML PB samples than in healthy whole blood samples. Nine genes showed higher expression levels in AML BM samples in the posttreatment stage, compared to the diagnosis and recurrent stages. These results were consistent with our expectation of prognostic values of these genes. And it also proved that these potential biomarkers extracted based on CN-AML sample data may be extensively applicable to all kinds of AML samples, regardless of clinical traits. Also, survival analysis of the 18 hub genes in 148 GEO CN-AML patients showed the correlation of higher expression levels of CEACAM1, CRISP2, and CYP4F3 showed their strong competitiveness in both expression analyses  and the survival analysis. They can be key study genes in our further research. Their relationship with tumor progression has been reported in previous studies. Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) mediates the direct interaction between tumor and immune cells as a cell-cell communication molecule [Cysteine-rich secretory protein-2 (CRISP2) has been reported to be less expressed in high-grade squamous intraepithelial lesions than in other histological grades, making it a novel biomarker for the detection of cervical cancer [Cytochrome P450 family 4 subfamily F member 3 (CYP4F3) has been reported to have good diagnostic values for osteosarcoma [CYP4F3 (rs4646904) may contribute to the etiology of lung cancer [CYP4F3A was upregulated in all-trans-retinoic acid- (ATRA-) treated AML cell line HL-60 [In the 18 hub genes, molecule . It has molecule –57, whill cancer . Cytochrosarcoma , and a pg cancer . Mizukamne HL-60 .LTF  was predicted to be a transcription factor to 10 of the 18 hub genes. It is a member of the transferrin family of genes, and its protein product is found in the secondary granules of neutrophils. Its relationship with various types of tumors including AML has been widely reported. Back in 1988, Davey et al. reported a quantitative decrease in LTF staining in AML and myelodysplasia, which supports the concept that abnormal neutrophils and bands are derived from a malignant clone of myeloid precursor cells [LTF to be a candidate biomarker for AML prognosis.or cells  and alsoDNA methyltransferase 3 alpha (DNMT3A) mutation positive and negative AML samples  [The pathway enrichment analysis suggested innate immune system, neutrophil degranulation, and ensemble of genes encoding ECM-associated proteins  as the top three hits with the hit gene number > 10% of the ME1 mRNAs . The innate immune system has been widely reported to be closely related to various kinds of cancers including AML , 64. Neuomarker) . ECM-assomarker) –68. EMC-omarker) . Berdel omarker) .LINC00671 is one of the lncRNAs revealing a high expression correlation with mRNAs in our PPI network. It was previously found to be a tumor suppressor in multiple cancers including renal cell cancer, pancreatic cancer, and papillary thyroid tumor by inhibiting the growth and metastasis of cancer cells [er cells –29. AlthThere are previous studies investigating AML based on the WGCNA method. Wiggers et al.  identifiAdmittedly, this work was limited by the sample size and statuses of our WGCNA—42 samples (deceased patients only) were included. More comprehensive studies of larger sample sizes should be performed in the future. Additionally, our study was a bioinformatics analysis. The mRNAs and their potential regulatory lncRNAs identified in this study for their prognostic values should be further investigated by in-depth mechanical approaches such as RT-PCR validation and gene function experiments. To use these results in clinical prognosis prediction, prediction models would be constructed, and PCR-based quantifications might be used in risk grading of adult AML patients.ABCA13, ANXA3, ARG1, BTNL8, C11orf42, CEACAM1, CEACAM3, CHI3L1, CRISP2, CYP4F3, GPR84, HP, LTF, MMP8, OLR1, PADI2, RGL4, and RILPL1) were downregulated in AML PB samples compared to healthy whole blood samples; ANXA3, CEACM1, RGL4, RILPL1, and HP showed increased expression levels in AML BM samples of the posttreatment stage compared to the diagnosis and/or recurrent stage. Also, the expression levels of ARG1, CEACAM1, CHI3L1, CRISP2, and CYP4F3 were demonstrated to be positively correlated with OS in an independent cohort. One of the hub genes, LTF, appeared on top of the TF prediction list, overlapping 10 hub genes. lncRNA-mRNA networks were constructed to exhibit the possible genetic regulatory mechanisms of adult CN-AML. LINC00671, which was linked to 17 mRNAs, has been widely reported as a tumor suppressor in various solid tumors. Clearly, this study identified the prognosis-specific biomarkers and the potential lncRNA-related regulatory mechanisms in AML. Our findings suggest CN-AML samples as good sources to investigate the relationship of RNA profiles and AML prognosis, and also provide a necessary groundwork for further exploration of the function and potential applications of these biomarkers as therapeutic targets for AML.In this study, we identified AML survival-specific mRNAs and lncRNAs using the WGCNA methodology based on CN-AML data. Eighteen mRNAs were screened as hub genes of the survival-specific mRNAs. Expression analyses in different cohorts of AML samples revealed 17 of the hub genes (


Spontaneous remissions (SRs) in acute myeloid leukemia (AML) are infrequent, poorly documented and transient. Similarly, morphological and cytogenetic complete remissions (CR) under azacitidine treatment are scarce. We report a 71-year-old man with a secondary AML arising from essential thrombocythemia (ET), who developed an SR after discontinuation of azacitidine following a respiratory infection (four courses were administered). The distinctive feature of our case is the depth of the achieved CR, documented by next-generation sequencing (NGS) techniques. We also detected persistence of molecular lesions that might already have been present in the previous ET clone. Our patient relapsed 5 months after achieving CR. We conclude that our patient showed a spontaneous remission of his AML rather than an exquisite response to azacitidine. We hypothesize that the concurrent respiratory infection, or any other unknown trigger, might have activated his immune system forcing the leukemic stem cell to enter a quiescent state through a yet unexplained mechanism. Acute myeloid leukemias (AML) arising from previous myeloproliferative neoplasms (MPN) are marked by a dismal prognosis . SpontanAzacitidine therapy has shown an increase in overall survival (OS) compared to previous conventional care regimens . HoweverHerein, we report a patient diagnosed with AML arising from essential thrombocythemia (ET), who developed disease remission once azacitidine treatment was discontinued while suffering a respiratory infection.V617FJAK2 mutation, diagnosed 10 years prior and treated with hydroxycarbamide, who presented to our department complaining of constitutional syndrome in November 2019. There were no relevant findings in the physical examination. Complete blood count (CBC) showed leukocytosis  with 27% of blasts, anemia (Hb 7.4 g/dL) and thrombocytopenia . Bone marrow aspirate was a dry tap so a biopsy was performed, showing massive blast infiltration, consistent with diagnosis of AML secondary to MPN.We report a 71-year-old man with a medical history of ET with FLT3 (ITD or TKD) nor NPM1 mutations were detected, yet an NGS panel revealed mutations in V617FJAK2 : 60.9%), R248QTP53 (VAF: 60.9%) and Y158_159dupU2AF1 (VAF: 42.5%) .Flow cytometry showed a 25% of cells CD45 + dim, CD34+, CD117+, HLA-DR+, CD13+, CD33+, CD71 + (10%), CD61 + (8%) and MPO + (16%). Cytogenetics found a complex karyotype: 46XY, del(5) (q13q33), der(7) t , del(9) (q22q32), del(13) (q14) [12]/46XY [2). Between the 3rd and 4th cycles he was admitted to hospital with an upper respiratory infection, requiring intravenous antibiotics for 5 days. Due to high transfusional requirements (18 red cell concentrates and 2 platelet pools), limited tolerability and absence of response, treatment was discontinued in April 2020, following the 4th cycle of azacitidine.The patient underwent geriatric assessment and started hypomethylating therapy (azacitidine 75 mg/m3 platelets and 9000/mm3 neutrophiles without immature cells). Bone marrow examination showed morphological CR (<5% of blasts in BM) with CCR  and negative minimal residual disease (MRD) by flow cytometry . NGS showed JAK2 VAF: 44.3%, U2AF1 VAF: 40.8%, and TP53 VAF: 8.7% (NGS sensitivity: 3%). Monthly follow-ups as an outpatient were established, until in September 2020, cytopenias  and blasts in PB smear reappeared (11%). At this time, the patient received supportive care at home until his death in December 2020.Since drug discontinuation, the patient’s CBC progressively improved, reaching peripheral blood (PB) counts similar to those observed previous to AML development (i.e., Hb 13 g/dL, 550,000/mmThe AML’s timeline from diagnosis to death is depicted in This report was a retrospective chart review. The patient had passed away at the time of publication. Fundación Jiménez Díaz Institutional Review Board approved submission of the article for publication in the journal.TP53, TET2, ASXL1, IDH1/2, U2AF1, SRSF2 and RAS family genes suggest leukemic evolution in MPN [R248QTP53 and Y158_159dupU2AF1 mutations at time of leukemic transformation. NGS showed a marked reduction in TP53 VAF, while only a minimal reduction was seen in the JAK2 and U2AF1 clones. This finding was consistent with documented AML remission with persistence of mutations that might be already present in the ET clone. Regrettably we had no sample available from the ET phase of the patient.Our patient had an AML arising from an MPN. It is well known that these kind of patients have an extremely dismal prognosis, even worse than in other secondary AMLs . Diseasen in MPN ,7,8. In TP53 mutant AML achieving SR [To our knowledge, there are only 3 SR reported cases with a complex karyotype and we report the first case of eving SR ,9. Additeving SR . TherefoAzacitidine implementation in AML treatment has shown that achievement of CR is not mandatory to improve survival of AML patients . In factNPM1 mutation has been suggested to facilitate SR in AML due to a greater sensibility to oxidative stress [SRs in AML are an uncommon phenomenon, rarely well documented. In total, 90% of reported SRs achieve CRs . Response stress .At the present time, the underlying mechanism of SRs is unclear. However, we could hypothesize that somehow the infection, or any other previous unknown trigger, induced immune activation against AML . A possiSR is an uncommon and transient phenomenon thought to be the consequence of different triggers that might force the LSC to enter in a quiescent state. A prospective study of these cases, measuring the cell cycle of the LSC and cytokines of the patient, could help us to decipher the mechanism of regulation of the quiescence of LSC which could have interesting therapeutic implications.


Altered mitochondrial morphology and function is a common feature of cancer cells and the regulation of mitochondrial homeostasis has been identified as a key to the response to chemotherapeutic agents in human leukemias. Here, we explore the mechanistic aspects of cytotoxicity produced by thioridazine (TR), an antipsychotic drug that has been investigated for its anticancer potential in human leukemia cellular models. TR exerts selective cytotoxicity against human leukemia cells in vitro. A PCR array provided a general view of the expression of genes involved in cell death pathways. TR immediately produced a pulse of cytosolic Ca2+, followed by mitochondrial uptake, resulting in mitochondrial permeabilization, caspase 9/3 activation, endoplasmic reticulum stress, and apoptosis. Ca2+ chelators, thiol reducer dithiothreitol, or CHOP knockdown prevented TR-induced cell death. TR also exhibited potent cytotoxicity against BCL-2/BCL-xL-overexpressing leukemia cells. Additionally, previous studies have shown that TR exhibits potent antitumor activity in vivo in different solid tumor models. These findings show that TR induces a Ca2+-mediated apoptosis with involvement of mitochondrial permeabilization and ER stress in leukemia and it emphasizes the pharmacological potential of TR as an adjuvant in antitumor chemotherapy.Mitochondria have pivotal roles in cellular physiology including energy metabolism, reactive oxygen species production, Ca Simultaneously, a progressive increase in the mitochondrial calcium levels ([Ca2+]m) was observed, which suggests mitochondrial uptake of the cytosolic Ca2+. To test whether this TR-induced disruption of Ca2+ homeostasis was required for cell death, we preincubated K562 cells with EGTA or BAPTA-AM to chelate extracellular and intracellular Ca2+, respectively, and following TR addition, measured cell viability with the MTT assay. Both chelators prevented cell death  fluorescence emission. TR promoted a sustained increase in the production of ROS  in the presence of 5 mM potassium succinate as respiratory substrate and the ΔΨ was kinetically recorded. TR abrogated ΔΨ formation and this effect was prevented by the Ca2+ chelating activity of EGTA, showing the role of Ca2+ in TR-induced mitochondrial permeabilization in K562 leukemia cells  mouse embryonic fibroblasts (MEF) with TR and then measured both cell viability and changes in ΔΨ. The absence of BAX/BAK expression completely abolished the TR-induced dissipation of ΔΨ  and BAX/BAK double-knockout . One downstream target of PERK, namely, the transcriptional factor CHOP, the chief mediator of ER stress-induced cell death, was elevated after 24 h of incubation with TR. To test the requirement for this protein, we knocked down CHOP expression in K562 cells  on TR-induced cell death, the results suggest that Ca2+ increase is an upstream event in TR-induced cell death that can trigger the downstream effects. A high-throughput drug screening identified TR as a highly selective drug for the MLL-AF6 rearrangement in AML, which is associated with a very poor prognosis. It was found that TR promoted the remodeling of the cytoskeleton in blasts, resulting in apoptosis-related Ca2+ influx [2+ to the cytosol.We showed that TR produces an immediate increase of cytosolic Ca+ influx . NeverthAside from the involvement of mitochondria in TR-induced cell death, additionally, we showed that TR increased the expression levels of the primary ER stress sensors, PERK and IRE1α, and CHOP, the downstream target of PERK. CHOP induces the expression of GADD34 and increases levels of ERO1α, which results in the production of cytotoxic ROS . As downBCL2 gene [2+ concentrations [Bax−/−Bak−/− DKO cells, thereby indicating that other cellular processes/pathways contribute to cell death. The expression of Bcl-2 antiapoptotic proteins is upregulated in hematological malignancies, including leukemia [As an intricate network, CHOP can modulate Bcl-2 proteins , inhibitCL2 gene , and enhCL2 gene . Additiotrations  and can trations . Althougleukemia , and sevleukemia , with soleukemia ,65. In tleukemia ,67,68.2+-mediated cellular responses that result in mitochondrial permeabilization, ER stress, and apoptosis in K562 cells (Taken together, our data demonstrate that TR induces complex, Ca62 cells . Among o


According to the in vitro research, FLVCR2 silencing suppressed AML cell growth and promoted their apoptosis. This study provides insights into FLVCR2’s effect on tumor immunity, indicating that it might serve as an independent prognostic biomarker and was related to immune infiltration within AML.Acute myeloid leukemia (AML) is one of the most common hematopoietic malignancies in adults. The tumor microenvironment (TME) has a critical effect on AML occurrence, recurrence, and progression. The gene feline leukemia virus subgroup C cellular receptor family member 2 (FLVCR2) belongs to the major facilitator superfamily of transporter protein members, which is primarily involved in transporting small molecules. The potential role of FLVCR2 in the TME in AML has not been investigated. To clarify the expression and role of FLVCR2 in AML, we analyzed the Gene Expression Omnibus and The Cancer Genome Atlas databases and found that FLVCR2 mRNA expression significantly increased among patients with AML. Furthermore, based on an analysis of the Gene Expression Profiling Interactive Analysis database, FLVCR2 upregulation predicted dismal overall survival of patients with AML. Our validation analysis revealed the significant upregulation of FLVCR2 within the bone marrow of AML relative to healthy controls by western blotting and qPCR assays. Gene set enrichment analysis was conducted to explore FLVCR2’s related mechanism in AML. We found that high FLVCR2 expression was related to infiltration degrees of immune cells and immune scores among AML cases, indicating that FLVCR2 possibly had a crucial effect on AML progression through the immune response. Specifically, FLVCR2 upregulation was negatively related to the immune infiltration degrees of activated natural killer cells, activated memory CD4 Acute myeloid leukemia (AML) is a frequently observed and highly heterogeneous adult hematologic malignancy, and its features include clonal expansion, impaired differentiation, and apoptosis evasion in normal hematopoietic stem cells . TherapeThe causes of AML are typically related to genetic mutations and immune system deficiency. The tumor microenvironment (TME) affects AML pathogenesis and development . TME comFeline leukemia virus subgroup C cellular receptor family member 2 (FLVCR2) belongs to the major facilitator superfamily (MFS) of transporter protein members that transport small molecules . MFS is This work comprehensively analyzed the FLVCR2 level within AML by referring to several public databases on public bioinformatics platforms. The results showed that FLVCR2 had high expression within AML, which predicted low survival rates. Pathway analysis showed that FLVCR2 was related to TME and immune-related pathways. Moreover, high FLVCR2 expression was related to infiltration degrees of immune cells and immune scores for AML cases, indicating that FLVCR2 possibly had an important effect on AML progression through immune reaction. FLVCR2 silencing suppressed AML cells proliferation and promoted their apoptosis. Our results provide insights into the AML tumorigenesis mechanism and identify FLVCR2 as the significant independent predictor of AML.https://portal.gdc.cancer.gov) and then eliminated cases that had incomplete clinical data. This study gained approval from the Ethics Committee of Qilu Hospital . TCGA database is publicly available under specific guidelines, and the analysis in this study was conducted following the Declaration of Helsinki. This study also obtained the GSE12417 dataset (http://www.ncbi.nlm.nih.gov/geo/) to assess the relation between FLVCR2 expression and prognosis.This study obtained transcriptome RNA sequencing (RNA-seq) and relevant clinical data for the tumor and normal samples from The Cancer Genome Atlas (TCGA) database ( dataset  from thehttp://gepia.cancer-pku.cn/index.html) to evaluate the association of FLVCR2 level with clinicopathological features of AML  online database  to identify the potential biological processes and pathways related to FLVCR2 expression in AML progression . This stwww.kmplot.com), including clinical and gene expression profiles (http://timer.comp-genomics.org/) (http://dna00.bio.kyutech.ac.jp/PrognoScan/)  ; and PronoScan/)  to evaluhttps://david.ncifcrf.gov/summary.jsp) , cellular component (CC), and molecular function (MF) terms. Pathways related to FLVCR2 activity along and its co-expression genes were identified by KEGG analysis. The results of enrichment analysis are shown using corrected p values and normalized enrichment scores.Differentially expressed genes (DEGs) were conducted using Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis with the DAVID database  : p < 0.0https://string-preview.org/), used for retrieving interacting genes and PPI networks, to map the FLVCR2 gene and protein interactions. Significant enrichment was assessed by the thresholds of FDR < 0.05 and p < 0.01.This study applied the database Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) (http://cibersort.stanford.edu/) was used  sequences were designed based on the appropriate design principles. GenePharma  was responsible for preparing the negative control (NC) and FLVCR2-targeting small interference RNA (siRNA). Lipofectamine RNAiMAX (Invitrogen) was used for transfection within the 6-well plates.This study used TRIzol (Invitrogen) to extract total RNA according to specific protocols and measured it using UV spectrophotometry (DeNovix). Subsequently, an RNA sample  was collected to prepare cDNA through reverse transcription in 10 μl reaction volume using Evo M-MLV RT Premix for qPCR (AG11706). The SYBR Green Premix Pro Taq HS qPCR Kit (AG11701) was applied in RT-qPCR on the Light Cycler 480 Ⅱ (Roche). The results were calculated by the 2^-ΔΔCT method. GAPDH was used as endogenous control.M-PERTM Mammalian Protein Extraction Reagent (Thermo Fisher) was used for cell lysis. After separation through SDS-PAGE, proteins were transferred onto nitrocellulose membranes (Millipore). This study used GAPDH as the reference. Anti-FLVCR2 (proteintech) and anti-GAPDH (ZSGB-BIO China) primary antibodies were used. Next, this assay was incubated with HRP Substrate Luminol Reagent (Millipore), and exposure was performed with ChampChemi (SageCreation).in vitro was analyzed by EdU cell proliferation assay. For EdU assays, treated cells were incubated by the iClick™ EdU Andy Fluor™ 647 Flow Cytometry Assay Kit  and tested by flow cytometry (FCM).The proliferation of transfected cells After transfection, supernatants of the cell culture were eliminated before cell collection. After washing with phosphate-buffered saline twice, the cells were added with 1× binding buffer (300 μl). Next, the cells were subject to Annexin V/PI staining  and FCM (Beckman Coulter) analysis of cell apoptosis within 1 h.p < 0.05 (two-sided) denoted statistical significance.This study used R software (version 3.6.3) for statistical analysis. The relation between FLVCR2 level and clinical features was analyzed by logistic regression analysis. OS was defined as the duration between the diagnosis date and the all-cause mortality date. This study conducted Cox proportional hazards regression to identify OS-related clinical characteristics in patients with AML. p < 0.05, |log2FoldChange| > 1). We focused on the function of FLVCR2 in the tumorigenesis of AML, and FLVCR2 showed upregulation within AML (p < 0.05). Additionally, the proportion of AML cases with low FLVCR2 expression was higher than that with high FLVCR2 expression without treatment or neoadjuvant. However, no other differences were observed between low- and high-FLVCR2 expression groups.For the exploration of key genes within AML, DEGs were analyzed in the TCGA dataset, and the significance of these genes was tested using immune scores and AML prognosis. FLVCR2 was one of the six overlapping genes among the MFS members . The oththin AML . Furtherthin AML . The FLVn = 76) and low FLVCR2 expression group (n = 75). We found that the low FLVCR2 expression was correlated with superior OS in the median cutoff groups in AML , biological process (BP), and molecular function (MF) categories based on the following criteria: DR <0.05 . The GO-DR <0.05  indicateTo understand FLVCR2’s functional implications within AML between two groups, we conducted GSEA for FLVCR2, based on LinkedOmics database, to investigate the possible BPs and pathways. Based on the normalized enrichment scores, the most significantly enriched signaling pathway in terms of FLVCR2 gene expression was selected. As shown in FLVCR2 level has been suggested to be associated with immune pathways in AML. Because immune and stromal cells represent the main TME components, this study analyzed the effect of FLVCR2 on TME. First, the stromal and immune scores of AML cases were determined. Next, the effects on the prognostic outcome were assessed. The results showed that low immune scores predicted improved prognosis, but stromal scores were not significantly related to OS . Further+ T cells, resting NK cells, CD4+ memory T cells, monocytes, mast cells, and M2 macrophages represented the main immune cells affected by FLVCR2 level. For AML cases, FLVCR2 upregulation was significantly related to the high proportion of monocytes. However, FLVCR2 upregulation was negatively associated with some immunocyte types, such as plasma cells, activated CD4 T cells memory, activated NK cells, CD8+ T cells, M1 macrophages, and activated dendritic cells , compared to that in matched healthy samples , and FLVn = 30) and healthy subjects (n = 24). p < 0.01, n = 15, p < 0.01) than in those achieving complete response  and NC siRNAs (si-NC). The FLVCR2 level markedly elevated within THP-1and MOLM-13 cells in comparison with that of heathy controls (AML is a highly heterogeneous, malignant tumor with varied prognoses. The pathogenic mechanisms underlying AML have not been fully elucidated. Therefore, novel biomarkers are necessary to understand mechanisms related to AML. FLVCR2 was reported to be associated with Fowler syndrome . SubsequThe study examined FLVCR2’s level, prognostic significance, and potential mechanism in AML by using TCGA datasets. The results showed that FLVCR2 mRNA levels increased more in AML samples than in normal samples according to the results obtained through GEPIA and GEO database. FLVCR2 level elevated in relapsed/refractory AML cases relative to complete remission AML cases, suggesting that FLVCR2 level might be related to treatment efficacy and prognostic value. We then performed prognostic analysis and found that FLVCR2 upregulation was associated with poor prognosis, suggesting that FLVCR2 functions as an oncogene. DEGs associated with increased FLVCR2 levels were enriched in immunity-associated pathways. Our findings indicate that FLVCR2 may be an independent prognostic factor that influences tumorigenesis and tumor immunology in AML progression. However, its biological involvement in AML remains to be explored.Furthermore, we investigated the biological functions and mechanisms of action of FLVCR2 in AML. The literature  showed tMoreover, PPI network analysis revealed that CORO1A and HMGCR had a low but significant correlation with FLVCR2. CORO1A, responsible for encoding the Trp-Asp repeat protein, is related to different cell events. CORO1A depletion possibly results in compromised T-cell activity and homeostasis due to the defect in the T-cell receptor pathway . Additio+ T cells, and CD8+ T cells, whereas a positive relation was observed with Tregs and M2 macrophages in AML. Tumor eradication by CD8+ T cells involves tumor antigens, antigen presentation, T-cell activation, and tumor cell killing (+ T cell proportions within tumors predict a dismal AML prognosis (in vitro results demonstrated that FLVCR2 interference inhibited cell proliferation activities and increased the incidence of apoptosis, verifying that FLVCR2 was important for regulating cell apoptosis and maintaining cell proliferation within AML.Immune-based therapies have become important in cancer treatment . However killing . The lowrognosis . By contrognosis . Therefoin vivo.Previous studies have confirmed the importance of identifying a biomarker for tumorigenesis and development of particular cancer by combining bioinformatics analysis and experimental demonstration. In conclusion, this study first identifies the prognostic value of FLVCR2 in patients with AML. FLVCR2 expression is associated with immunocyte infiltration, such as NK cells, T cells, or macrophages in AML. Our results suggest that FLVCR2 may predict treatment outcomes and prognosis in patients with AML and play a critical role in regulating cell apoptosis and maintaining cell proliferation within AML. With an improved understanding of its function, FLVCR2 may be developed as a component of targeted therapies for AML treatment.