The proton pump dissociates periplasmic AGP-glycomodules that bind Ca2+. Thus, as the immediate source of cytosolic Ca2+, an AGP-Ca2+ capacitor directs the vectorial exocytosis of cell wall precursors and auxin efflux (PIN) proteins. In toto, these components comprise the Hechtian oscillator and also the gravisensor. Thus, interdependent auxin and Ca2+ morphogen gradients account for the predominance of AGPs. The size and location of a cell surface AGP-Ca2+ capacitor is essential to differentiation and explains AGP correlation with all stages of morphogenetic patterning from embryogenesis to root and shoot. Finally, the evolutionary origins of the Hechtian oscillator in the unicellular Chlorophycean algae reflect the ubiquitous role of chemiosmotic proton pumps that preceded DNA at the dawn of life.Morphogenesis remains a riddle, wrapped in a mystery, inside an enigma. It remains a formidable problem viewed from many different perspectives of morphology, genetics, and computational modelling. We propose a biochemical reductionist approach that shows how both internal and external physical forces contribute to plant morphogenesis via mechanical stress–strain transduction from the primary cell wall tethered to the plasma membrane by a specific arabinogalactan protein (AGP). The resulting stress vector, with direction defined by Hechtian adhesion sites, has a magnitude of a few piconewtons amplified by a hypothetical Hechtian growth oscillator. This paradigm shift involves stress-activated plasma membrane Ca Cell shape is one aspect of the more general problem of differentiation. Consider the primary cell wall, how it grows, and there is evidence enough for the primary wall playing a decisive role in influencing the shape of a cell. Therefore, the primary cell wall must be studied, but how?” Lamport 1963) [963 [1].+, and Ca2+ gradients and how their interaction creates morphogenetic patterns. We therefore approach the formidable problem of plant morphogenesis from an empirical biochemical perspective based on the dynamic role of AGPs  in a novel Hechtian growth oscillator. A cell surface AGP-Ca2+ capacitor is a crucial component that depends on the complex carbohydrate chemistry of classical AGPs [2+ by paired glucuronic carboxyl groups of numerous small glycomodules. Not surprisingly, after their initial discovery as an arabinogalactan polysaccharide [2+ capacitor [2+ and tip-localised AGPs with Hechtian adhesion and rapid tip growth of pollen tubes [In 1952, Turing proposed that “morphogens acting together and diffusing through a tissue is adequate to account for morphogenesis.” His classic paper  “On Growcal AGPs ,8 that cccharide , it has ccharide  to unravapacitor . The rolen tubes .2+. We propose that mechanotransduction generates both gradients, although each has a different biochemical origin; thus, auxin gradients originate from biosynthesis and transport by tissue-specific auxin efflux PIN proteins. On the other hand, cytosolic Ca2+ gradients originate from Ca2+ stored in a cell surface AGP-Ca2+ capacitor [2+ at the periplasmic cell surface seems inescapable to us, the role of AGPs in Ca2+ gradient formation in tissues is less obvious because it depends on a membrane-bound H+-ATPase that dissociates AGP-Ca2+. That shows the close link between auxin and Ca2+ signalling with the primal proton pump. Paired glucuronic carboxyls of AGP glycomodules bind Ca2+ tightly [2+ chelation by dicarboxylic acids). This effectively scavenges less tightly bound Ca2+ of pectin and also free Ca2+ in the transpiration stream. Thus, abundant cell surface AGP-Ca2+ acting as a Ca2+ sink would enable both cells and tissues to compete for Ca2+, so that Ca2+ supply meets demand where it is greatest, such as in meristem primordia. The “choice” of Ca2+ as a universal signalling ion can be ascribed to the size (0.9 Å) of its ionic radius; thus, Ca2+ sheds water more rapidly and is therefore more reactive than other divalent ions—notably Mg2+ [2+.Here, we extrapolate these recent results to morphogenesis and propose two new avenues: Firstly, we propose the avenue of cell walls and mechanoperception involving Hechtian transduction as described in detail here but not considered by Turing; secondly, we propose postulated chemical morphogen gradients  identifiapacitor . AGPs arapacitor ; that exapacitor  and why apacitor . Many AGapacitor ,16,17,18 tightly   cell wall mechanotransduction and the developmental sequence, (2) the embryogenesis of the fertilised egg cell, (3) roots, (4) shoots, and finally (5) the evolution of morphogenesis.2+ capacitor.Most models of morphogenesis, particularly computer-generated models, generally favour a genetic perspective with an emphasis on signalling cascades. However, they ignore two “unknown unknown” missing pieces of the morphogenetic puzzle, identified here as Hechtian mechanotransduction and the AGP-Ca−12 N) forces  forces . 2+ channels and ATPases?How does it distinguish between the huge forces  exerted on the membrane by osmotic pressure and the minute piconewton forces that activate CaHow does it provide a directional signal in response to anisotropic stress? Why does the wall need strong adhesion to the plasma membrane although turgor pressure ensures it? Is the elusive mechanosensor a Boojum or a Snark?However, the Hechtian transduction hypothesis creates three dilemmas: Hechtian adhesion must satisfy several criteria: minimally, it must involve strong, stable and specific interactions with both the cell wall and plasma membrane. Several candidates frequently suggested such as wall-associated kinases and integrins lack hard data and can be eliminated. For example, integrins recognise the RGD motif of the mammalian extracellular matrix but are absent from plants. Other possible candidates include formins and many AGPs anchored securely to the plasma membrane ,28,29,30At-AGP57C (At3g45230) is covalently linked to pectic RG-I homogalacturonan  and is a2+ channels and H+-ATPase; both membrane components are stretch-activated by just a few piconewtons. Wall stress and strain is thus focussed at the molecular level on CaPiezo proteins responsive to mechanical force are well-documented in animal systems . FinallyAlthough osmotic pressure is equally distributed at the plasma membrane, wall pressure exerts an equal and opposite force. However, a direct covalent connection between pectin and the plasma membrane would enable instant transmission of the wall stress–strain status by fibrous macromolecules.Two extreme examples, stomata and pollen tubes, demonstrate the efficacy of Hechtian adhesion: the stomatal guard cell wall elasticity transmits stress while the rapid tip growth of a pollen tube involves wall plasticity; when stretched, it transmits strain arguably via AGP57C  or its h2+ crosslinking forms much stronger gels. A frequently cited pectic paradigm [2+ for crosslinking but ignores the regulation of Ca2+ in muro by AGPs which, with their demonstrated higher affinity for Ca2+, compete for pectic Ca2+. Such competition effectively strips Ca2+ from pectin. The concomitant electrostatic repulsion of negatively charged pectic carboxylates explains the increase in wall plasticity by pectin methylesterase [2+. Significantly, small AGP peptides are upregulated by auxin [The pollen tube tip has the simplest primary cell wall—almost exclusively pectin and with fast tip growth—so is an ideal system to test hypotheses involving the Hechtian transduction of wall stress–strain. Pectin rheology is deceptively simple and depends largely on the degree of methyl esterification of pectic polygalacturonic acid residues, in contrast to AGP glucuronic acid, which is never esterified. Highly methyl-esterified pectins form gels, while de-esterification followed by Caparadigm  assumes esterase . This efesterase  that posby auxin , suggest2+ oscillations and exocytosis (2+ and borate crosslinking of the RG-II pectic component. Crosslinking of the cellulosic component (notably absent from pollen tube tips) may also be regulated by expansin [Driven by turgor pressure, cells expand in a direction orthogonal  to the direction of stress that strains the pectic matrix; the concomitant Hechtian transduction of pectic strain initiates Caocytosis . The strexpansin ,61.2+ in the exocytosis of wall plasticisers as detailed in [2+ by increasing the size of their cell surface AGP-Ca2+ capacitor; this can increase the amplitude of Ca2+ oscillations, thereby enhancing exocytosis.Such complexity explains why the molecular basis of cell wall plasticity remains recalcitrant since first described . “The diailed in  rather tailed in . Cells a2+ channels [+-ATPase [2+ influx as follows: the plasma membrane ATPase [Consider the primary receptors of force transduction. What are they, and how do they propagate tension to the cell interior? Stretch-activated Cachannels  and auxi+-ATPase  control e ATPase ,65 is sie ATPase .2+ of the cell surface capacitor that supplies the Ca2+ channels. The evidence [2+ during the rapid tip growth of pollen tubes.These powerful proton jets dissociate the AGP-Caevidence  for this2+ fluxes translate into morphogenesis depends on the local wall plasticity that determines the direction and magnitude of the wall stress vector; its amplification depends on the size of the AGP-Ca2+ capacitor (How Caapacitor  and its Plant cells exploit a “thermodynamic loophole” based on their ability to trap ionised auxin in the cytosol (pH 7.4) where it cannot freely permeate the plasma membrane. However, in the wall at ~pH 5, auxin is protonated and thus freely permeates the plasma membrane of adjacent cells. Thus, auxin wins the Sysiphean uphill struggle.2+ channel activity and the plasma membrane H+-ATPase  to the outer protoderm (tension). The concomitant rearrangement of auxin efflux proteins in the protoderm under tension results in lateral auxin export from its apex outwards, with consequential slow growth and anisotropy at the torpedo stage that splits the tissue into the two cotyledons [The above description of stress-dependent auxin and Catyledons   with apically polarised PIN4 and PIN7 auxin efflux proteins [The Hechtian growth oscillator generates morphogenetic patterns; arguably, these depend on localised adhesion sites that result in vectorial exocytosis of wall precursors. This includes auxin efflux PIN proteins directed to the sites of the highest strain in expanding cells,  thus ensproteins  highligh2+, thus increasing cytosolic Ca2+ that triggers vectored exocytosis. We infer that Hechtian adhesion and AGPs amplify both the direction and magnitude of stress vectors resulting in growth orientation. Not surprisingly, mutants of Gal31 are lethal [2+ glycomodule [Stress also predicts the plane of cell division marked by the preprophase band of microtubules  as putate lethal , possiblcomodule .2+-signalling was unexpected yet surprisingly close to reality, with AGP-Ca2+ belatedly seen here as the major dynamic source of Ca2+ regulated by auxin. Significantly, transformed plants that overproduce auxin show excessive xylem and phloem development [The morphogenesis of root xylem tissues begins with the specific expression of the JIM13 AGP epitope in a single metaxylem initial just above four central cells of the quiescent centre . The howelopment  and bushelopment .Mechanical stress patterns involved in root morphogenesis have yet to be modelled. Nevertheless, a recent comprehensive experimental approach  correlat2+ [2+ influx” [. Nevertheless, the Cholodny–Went explanation of auxin redistribution remains unquestioned dogma [The presence of the cell wall is essential for gravity perception . These o2+  and the 2+ : “Thus, 2+ ,92,93,94 influx” ,51. Neveed dogma : “The abed dogma . This deThe root–shoot paradox: root tip gravitropism increases auxin at the lower side that decreases growth while less auxin at the upper side increases growth. However, in shoots, a similar auxin distribution in response to gravity reverses the direction of growth. We resolve this paradox by asking: How does auxin inhibit growth at the lower side of the root? Extensin peroxidase  providesWhile the above approach to a complex problem is consistent with Occam’s razor, it contrasts with the complexity of recent papers that deal with the dynamic rearrangement of PIN proteins transporting auxin from both root tip and the shoot via xylem and root epidermis .2+ signalling although [2+ waves,” the major source being the Hechtian oscillator via auxin-activated H+-ATPase dissociation of AGP-Ca2+ that releases Ca2+ to the cytosol via Ca2+ channels. How that translates into increased cell extension in the root tip elongation zone remains a conundrum, as discussed above. Although the prevalent statolith hypothesis is now textbook dogma, the precise molecular details of statolith mechanotransduction remain quite unknown. Mutants that lack amyloplast statoliths do not resolve the problem: while they decrease the gravitational response, they do not eliminate it [+-ATPases [Arabidopsis) and PIN proteins recycle in response to mechanotransduction, thus adding an additional level of complexity.However, no convincing molecular mechanism has been offered for auxin-increased cell extension. Auxin has also not been successfully connected with Caalthough  “auxin sinate it . Finally-ATPases   to redistribute auxin by recycling the auxin efflux PIN proteins. As the root tip derives its auxin from transport through central root tissues and also local biosynthesis  proteins maintained by the cell wall ,108. How2+ by auxin. Thus, auxin efflux PIN proteins transport auxin to target sites, where the activation of H+-ATPase releases Ca2+ from AGP-Ca2+, triggering exocytosis. This paradigm shift can be traced back to the primordial soup and unifies the chemiosmotic proton motive force with auxin and Ca2+ signalling. By analogy with inscriptions on the Rosetta stone, once decoded, these three fundamental transport phenomena translate the same growth message into morphogenesis.Phylogeny and morphogenesis are inextricably linked , and so 4, CO2, and water to yield HCHO [Morphogenesis begins with prebiotic synthesis from CHeld HCHO , a precueld HCHO , a univeeld HCHO  remain tChlamydomonas provides the most striking example of evolutionary conservation and diversification, with a large complement of 182 different proteins related to hydroxyproline-rich extensins [Chlamydomonas wall is devoid of cellulose and consists almost entirely of glycoproteins that self-assemble as a crystal lattice [Emiliana huxleyi of the cretaceous that built the chalk hills and cliffs of the present era.From the cell wall viewpoint, the unicellular flagellate xtensins . The Chl lattice , a remar lattice . The lea3 coccoliths of Coccolithophores represented by E. huxleyi are the probable evolutionary precursor to dynamic cell surface AGP-Ca2+, hence Ca2+ signalling. Algae increased the tensile strength of a primary cell wall prototype by co-opting cellulose, thus enabling turgor pressure and the major evolutionary pathway to metaphytes. Indeed, the composition of algal pectin-rich cellulosic walls is remarkably similar to those defined as primary walls in the cambial tissues of higher plants [2+ signalling. This implies incredible evolutionary conservation and an early origin of the Hechtian pscillator in unicellular Chlorophycean algae such as Closterium [Static cell surface CaCOr plants . That rar plants , we extrosterium  and Peniosterium , as bothSpirogyra [Spirogyra rhizoids, as well as stretch-activated Ca2+ channels, nicely represent the earliest evolutionary progression from marine via freshwater to land plants  that retains the fertilised diploid egg cell until “germination”. Coleochaete cell walls are also significantly enriched in hydroxyproline-rich glycoproteins. Alkaline hydrolysis yields a hydroxyproline glycoside profile  culminated in its complete dominance in higher plants with their increasingly complex division into root, shoots and leaves. Root-like structures appeared as rhizoids in the Bryophytes, but true roots defined by their gravitropic response, endogenous branching, root hairs, and a protective root cap originate from the suspensor hypophysis during angiosperm embryogenesis. While sharing much in common with true shoots, roots clearly lack many shoot characteristics and have therefore been described as “partial shoots”  that evo2+ release and exocytosis. The ultimate reduction of the gametophyte generation to just a few cells, namely pollen and the ovule as an integumented megasporangium, completes this brief summary based on a novel growth oscillator unifying other growth regulators that control CaIon transport dominates this paper, which is greatly influenced (1955–1961) by personal acquaintance (DTAL) with the Cambridge pioneers who included Peter Mitchell (proton pump), David Keilin (cytochrome discovery) and Robert “Robin” Hill Reaction (photolysis of water). The cell wall protein was undoubtedly inspired by Fred Sanger in the adjacent lab , and Joseph E. Varner  on his s


However, the interconnection between OST1 and Ca2+ signaling in ABA‐induced stomatal closure has not been fully resolved.During drought, abscisic acid (ABA) induces closure of stomata via a signaling pathway that involves the calcium (Ca2+ signals were monitored in intact Arabidopsis leaves, which express the ratiometric Ca2+ reporter R‐GECO1‐mTurquoise and the Ca2+‐dependent activation of S‐type anion channels was recorded with intracellular double‐barreled microelectrodes.ABA‐induced Ca2+ signals that occurred during the initiation period, as well as in the acceleration phase of stomatal closure. However, a subset of stomata closed in the absence of Ca2+ signals. On average, stomata closed faster if Ca2+ signals were elicited during the ABA response. Loss of OST1 prevented ABA‐induced stomatal closure and repressed Ca2+ signals, whereas elevation of the cytosolic Ca2+ concentration caused a rapid activation of SLAC1 and SLAH3 anion channels.ABA triggered Ca2+ signals are evoked during the acceleration phase of stomatal closure, which is initiated by OST1. These Ca2+ signals are likely to activate Ca2+‐dependent protein kinases, which enhance the activity of S‐type anion channels and boost stomatal closure.Our data show that the majority of Ca Several lines of evidence show that the stress hormone abscisic acid (ABA) plays a central role in drought‐induced stomatal closure  channels are activated, anions and K+ are released by guard cells; this reduces their osmotic content and causes stomatal closure in less than 20 min  in guard cells (Roelfsema 2+)‐independent OST1 pathway, guard cells are also likely to exhibit a Ca2+‐dependent signaling chain that activates SLAC1, as well as the homologous channel SLAH3  guard cells  chain  and grown for another 2–3 wk in the same conditions.All t et al. , using t2) and 10 mM potassium citrate, pH 5 and illuminated with white light (100 μmol m−2 s−1) for at least 2 h before the start of the experiment.Measurements were carried out on stomata, either in isolated epidermal peels or in intact leaves that were excised with a sharp razor blade from 4‐ to 5‐wk‐old plants and gently fixed in a petri dish (diameter 35 mm) with the adaxial side attached to double‐sided adhesive tape. The leaves were immersed in the following solution: 10 mM potassium chloride (KCl), 1 mM calcium chloride  with medical adhesive  and placed in the following bath solution: 10 mM KCl, 1 mM CaClhttp://www.hilgenberg-gmbh.com) on a horizontal laser puller . The electrode tips were filled with ABA at the standard concentration of 50 μM, or concentrations ranging from 0.5 to 100 μM to obtain a dose–response curve, and the electrodes then further filled with 300 mM KCl. Control experiments were conducted with 50 μM benzoic acid. The electrodes were connected via silver/silver chloride (Ag/AgCl) half‐cells to a headstage of a custom‐made amplifier . A glass capillary that was filled with 300 mM KCl and sealed with 300 mM KCl in 2% agarose served as a reference electrode. The microelectrodes were mounted to a piezo‐driven micro‐manipulator  and slowly moved towards the guard cell wall. The tip potential was monitored during manipulation of the microelectrode, and when the electrode came into contact with the guard cell wall it suddenly changed to values more negative than −15 mV. After establishment of a connection between the guard cell wall and microelectrode, ABA, or benzoic acid as control, was ejected from the electrode with a current of −0.8 nA for a period of 20–30 s. Directly after termination of current ejection, the microelectrode was removed from the guard cell wall.Stomata in the abaxial epidermis of intact leaves, or epidermal strips, were visualized with a water immersion objective  mounted to an upright microscope . ABA was applied with single‐barreled microelectrodes that were pulled from borosilicate glass capillaries  and recorded at 1 kHz, using USB‐6002 interfaces . A dual low‐pass Bessel filter  was used to low‐pass filter the electrical signals at 0.5 kHz.The plasma membrane conductance of guard cells was studied in voltage clamp experiments with double‐barreled microelectrodes. These microelectrodes were fabricated from two borosilicate capillaries , which were aligned, heated, twisted 360°, and pre‐pulled on a vertical puller . Subsequently, the joint capillaries were pulled on a horizontal laser puller . The double‐barreled electrodes were backfilled with 300 mM KCl and had a tip resistance that ranged from 180 to 280 MΩ. Both barrels of the microelectrode were connected by Ag/AgCl half cells to the Ulliclamp01 amplifier, which enables voltage clamp experiments with an internal differential amplifier. Voltage pulses were applied with 2+‐imaging experiments were obtained from regions of interest in the central part of guard cells that included the nucleus. The measurements were carried out with a charge‐multiplying charge‐coupled device camera  that was mounted to a CARV, Crestoptics, Rome, Italy confocal spinning disc unit. Within the CARV unit, three filter wheels were used, while the spinning disc was moved out of the light path. The R‐GECO1 and mTurquoise subunits in RG‐mT were excited with light of an LED illumination system  at 435 nm and 580 nm, respectively. The emission signals were passed through dichroic mirrors with cut‐off wavelengths of 450 nm  and 590 nm  and band filters at 475/28 nm  and 628/40 nm .Fluorescence signals of Ca2+‐imaging experiments, with light provided by a halogen bulb in the microscope lamp, filtered through a far‐red light bandpass filter (713/30 nm). All images were analyzed offline with the image‐j/fiji software package  and origin pro 8 .Changes in stomatal aperture were monitored during the Caet al., −, can be ejected from the glass microcapillary into the guard cell wall by application of current pulses.In a previous study, rapid stomatal closure was induced by nanoinfusion of ABA‐containing solution, which was pressure‐injected through open stomata into intact leaves (Guzel Deger n = 9), the stomata closed with a maximal velocity of 0.28 μm min−1 . By contrast, application of benzoic acid as control  did not affect the aperture of stomata  using the same conditions as already described for ABA Fig. . Indeed,2+ reporters have become available in recent years, of which RG‐mT was chosen, since it exhibits a strong Ca2+‐dependent change in fluorescence emission ratio and it does not dramatically affect plant growth ; however, the impact of ABA on the cytosolic Ca2+ level split into three guard cell populations . On average, stomata with a transient Ca2+ rise displayed a half‐maximal closure within 289 s , whereas this value was reached later  in the absence of Ca2+ signals.Several genetically encoded fluorescent Casee Fig. , and farsee Fig. –S6. Currted Fig. . In a se2+ concentration observed, and these Ca2+ signals had a smaller amplitude than those elicited by ABA  was 55 times more likely than the model with one Gaussian function , whereas a model based on the sums of three Gaussian functions was very unlikely . This analysis thus strongly supports that ABA‐induced Ca2+ signals in guard cells occur in two time windows; some are elicited early (before the stomata start to close), whereas others are evoked later .Our data thus indicate that ABA‐induced Ca2+ levels were shown to activate plasma membrane anion channels in guard cells of several species  after the cytosolic Ca2+ peak  should be directed to the Fig. S1 Quantification of ABA concentrations evoked by current‐ejection.Fig. S2 Calibration of RG‐mT with FURA2 in Arabidopsis guard cells.Fig. S3 Current‐ejection of benzoic acid only induces Ca2+ signals in 2 out of 24 guard cells.Fig. S4 Inward currents triggered by hyperpolarization of wild type, slah3‐1 and slac1‐3 guard cells.Fig. S5 Current‐ejection of benzoic acid only induces Ca2+ signals in 1 out of 21 ost1‐3 guard cells.Click here for additional data file.Methods S1 Procedures to estimate the ABA concentration in the guard cell wall that was imposed by current‐ejection and calibration of R‐GECO1‐mTurquoise with FURA2.Click here for additional data file.Videos S1 Stomatal closure induced by current‐ejection of ABA.Videos S2 Current‐ejection of Lucifer Yellow CH (LY) into the wall of an Arabidopsis guard cell.Videos S3 Calibration of R‐GECO1‐mTuquiose (RG‐mT) with FURA2.Videos S4 ABA‐induced rise in the cytosolic Ca2+ concentration of a guard cell, during stomatal closure.Videos S5 ABA‐induced rise in the cytosolic Ca2+ concentration of a guard cell, before stomatal closure.Videos S6 ABA‐induced stomatal closure in the absence of a cytosolic Ca2+ signal in the guard cells.Videos S7 Voltage‐induced Ca2+ signals in an Arabidopsis guard cell.Videos S8 ost1‐3 stoma exposed to ABA, which did neither evoke stomatal closure, nor Ca2+‐signals.Videos S9 ABA‐induced Ca2+‐signals in an ost1‐3 stoma that were not linked to stomatal closure.Videos S10 Voltage‐induced Ca2+ signals in an ost1‐3 guard cell.Click here for additional data file.


AML holds a unique place in the history of immunotherapy by virtue of being among the first malignancies in which durable remissions were achieved with “adoptive immunotherapy,” now known as allogeneic stem cell transplantation. The successful deployment of unselected adoptive cell therapy established AML as a disease responsive to immunomodulation. Classification systems for AML have been refined and expanded over the years in an effort to capture the variability of this heterogeneous disease and risk-stratify patients. Current systems increasingly incorporate information about cytogenetic alterations and genetic mutations. The advent of next generation sequencing technology has enabled the comprehensive identification of recurrent genetic mutations, many with predictive power. Recurrent genetic mutations found in AML have been intensely studied from a cell intrinsic perspective leading to the genesis of multiple, recently approved targeted therapies including IDH1/2-mutant inhibitors and FLT3-ITD/-TKD inhibitors. However, there is a paucity of data on the effects of these targeted agents on the leukemia microenvironment, including the immune system. Recently, the phenomenal success of checkpoint inhibitors and CAR-T cells has re-ignited interest in understanding the mechanisms leading to immune dysregulation and suppression in leukemia, with the objective of harnessing the power of the immune system via novel immunotherapeutics. A paradigm has emerged that places crosstalk with the immune system at the crux of any effective therapy. Ongoing research will reveal how AML genetics inform the composition of the immune microenvironment paving the way for personalized immunotherapy. Acute myeloid leukemia (AML) is a disease of the myeloid lineage in which rapidly proliferating progenitors are transformed, leading to a block in terminal differentiation and the ability to self-renew, the latter normally being the privilege of only hematopoietic stem cells. Two decades of research have demonstrated that these expanded clones are the result of a differentiation hierarchy which mirrors normal hematopoiesis, with “stem-like” progenitors giving rise to partially committed myeloid progenitors, themselves giving rise to committed but undifferentiated myeloid precursors . This dyAML is an aggressive disease; in the U.S. from 2009 to 2015, the 5-years survival rate was 28.3% . For patEfforts over the last years to understand the impact of recurrent genetic alterations in AML on prognosis have resulted in genetic classification systems that summarize the risk of relapse following intensive induction chemotherapy , 6. A siIt has been appreciated for some time that outcomes with intensive induction chemotherapy are quite variable, and that a minority of patients attain durable remissions. Older age (>60 years old) and certain genetic alterations predict for markedly inferior outcomes. Multiple studies have demonstrated the prognostic utility of classifying patients by genetic mutations and cytogenetic abnormalities –14. The de novo AML samples by The Cancer Genome Atlas Network revealed that AML is characterized by few mutations in coding genes, on average 13 per patient, 5 of these recurrent mutations  or t ; AML with biallelic CEBPA mutations; AML with t ; AML with MLL fusion genes, AML with inv (3); t, GATA2, MECOM; AML with IDH2-R172 and no other class-defining lesions; AML with t . The chromatin-spliceosome and TP53-aneuploidy groups in particular represent new genomic subgroups with their respective class defining lesions imparting a deleterious effect on survival. Interestingly, the initial, recently reported findings of the Beat AML programme found that TP53 and ASXL1 (one of the recurrent mutated chromatin genes) were associated with a general pattern of drug resistance in an ex vivo 122 small molecule inhibitor screen  enabling a more detailed view of the mutational landscape of AML, prompting a new proposed genomic classification scheme for diagnosis beyond the current WHO subgroups, and allowing the authors to tackle the problem of the prognostic implications of co-occurring mutations. Analysis of allele frequencies allowed for establishment of clonal relationships identifying mutations in the epigenetic modifiers escribed –20 with escribed . The proith t 8, ; AML witNPM1-mutated AML is noted to be contingent on a low FLT3-ITD mutational burden. At the present time, the ELN 2017 system does not include other interactions between/among genes in its risk stratification algorithm and this remains a frontier in AML that is actively being explored  together with activating co-stimulatory signals and immunologic memory . In AML,+ (MHC I bound antigen) or CD4+ (MHC II bound antigen) TCR. MHC class I is expressed by most cells and presents self-antigens, whereas MHC class II expression is normally restricted to professional antigen presenting cells, such as dendritic cells, B cells and macrophages and binds extracellular antigens. The critical role of antigen presentation in AML is highlighted by a recent publication reporting that relapse following allogeneic stem cell presentation is associated with down-regulation of MHC class II genes  class I or II, in conjunction with co-stimulatory signals to a cognate CD8II genes . NeoantiII genes , 29. WitII genes , AML is blockade . In addiIn addition to the challenges imposed by low mutational/neoantigen burden, multiples studies have reported on defective antigen presentation by AML cells and APC in the leukemia microenvironment . AML celAML has been demonstrated to induce suppressive populations derived from the myeloid lineage, such as myeloid derived suppressor cells (MDSC) and M2 macrophages. MDSC are associated with a poor prognosis in solid and hematologic malignancies and have been associated with a blunted response to immunotherapy , 42. TheThe nomenclature M1/M2 macrophages, refers to the distinction between “classically activated” macrophages, M1, that engage in effective anti-microbial or tumor responses and “alternatively activated” macrophages, M2, that have anti-inflammatory activity and tumor supporting properties. AML promotes the polarization of leukemia-supporting macrophages . Yang anRegulatory T cells (Treg) constitute another part of the immune suppressive leukemia microenvironment. Treg restrain immune competent helper T and cytotoxic T cells through inhibition of T cell proliferation and cytokine production and play an important role in preventing autoreactivity , 50. TreAnother means by which T cell effector function is restrained is T cell exhaustion, which was initially characterized in the context of chronic viral infection, but is by now a well-established mechanism cancer immune evasions . Cell suAs noted above, soluble factors, such as arginase 2 have been implicated in the induction of a tolerogenic environment by AML blasts and M2 macrophages . ArginasAlongside the recognition that immune evasion is a hallmark of cancer  has emerA small subset of chemotherapeutic agents and fractionated radiation therapy can induce “immunogenic cell death” , 80. Imm+ CD33+ cells from healthy volunteers displayed exposed calreticulin, HSP70 and HSP90 independent of exposure to induction anthracycline-based chemotherapy suggesting a baseline level of ER-stress response in a subset of AML; in line with this, surface exposed calreticulin correlated with the expression of genes involved in ER stress response. Noting that surface calreticulin is best known for promoting phagocytosis by antigen-presenting cells and thus initiating anti-tumor immunity, the authors showed that upon recovery from chemotherapy, the normal PBMCs from patients expressing high surface calreticulin, as compared to low surface calreticulin, showed a gene expression pattern reflecting enrichment of TH1 polarization, T cell activation, CD8+ T-cell cytotoxicity and NK-related genes. Consistent with the gene expression data, surface calreticulinhi patients had increased circulating NK cells at the time of recovery from chemotherapy. Additionally, they had an increased fraction of CD8+ and CD4+ T cells responding to leukemia associated antigens both prior to and following chemotherapy as well as an increased percentage of T central memory cells, again both prior to and at the time of recovery from chemotherapy  i.e., azacitadine and decitabine. Both drugs are cytidine analogs and were originally developed as cytotoxic agents before being found, at low doses, to cause DNA demethylation by inhibition of DNA methyltransferase-1 (DNMT1) . DNA demClinically, the optimal response to HMA has been noted to require multiple cycles of treatment in line with a cell-intrinsic mechanism involving slow kinetics, potentially related to the differentiation of leukemic cells  and akinPML-RARA APL (in vitro and in vivo by inducing the expression of pro-oxidative genes in Treg with concomitant induction of increased levels of nitric oxide and reactive oxygen species (ROS). ATO-induced Treg depletion appeared to be dependent on “oxidative and nitrosative bursts.” Consistent with these observations, the authors found that in a model of colon cancer, a reduction in tumor volume was observed in immunocompetent but not athymic NUDE mice  and arsenic trioxide (ATO) for the treatment of APL is widely considered to constitute the first targeted therapy for cancer as well as the first example of differentiation therapy. ATRA targets the RARA moiety and ATO the PML moiety of the PML-RARA fusion protein for degradation in RARA APL . The pheRARA APL  was infoRARA APL . The roaRARA APL –119 inclRARA APL , 121. ATRARA APL , 121. InRARA APL . As regaUDE mice . Xu et aUDE mice . ATO hasUDE mice  modulateUDE mice . Taken tated AML , 128. AsIt has become apparent that all categories of anti-neoplastic drugs can modulate the immune response. Approved targeted therapies possess highly specific immune modulatory activities pertinent to their potential roles in the induction, consolidation and maintenance phases of therapy. Ongoing delineation of the immunologic mechanisms of action of targeted therapies is expected to inform the rationale combination of targeted therapies, as well as, novel combinations with chemotherapy, HMA, cellular therapies and biologics.NPM1, FLT3-ITD, IDH1/2, and TP53 can modulate the immune microenvironment in AML, are steadily being elucidated   . NPM1c A (NPM1c) , 133. FL (NPM1c) , 134–137 (NPM1c)  underscoCRi [91% ]. This r relapse .NPM1-mutant AML have been investigated as potential leukemia specific antigens that may elicit endogenous T cell responses and could also serve as targets for immunotherapy. Noting that aberrant localization of mutant NPM1 in the cytoplasm could lead to processing by the class I degradation pathway and subsequent presentation by HLA, Liso et al. tested and confirmed that mutated-NPM1 peptides can be presented by HLA molecules  or as an internal tandem duplication of the juxtamembrane domain (ITD), with the latter more frequently occurring than the former  is a receptor tyrosine kinase, normally involved in hematopoietic stem and progenitor cell proliferation and survival . It is me former . It is kia SOCS1 . It is aia SOCS1 . In addiia SOCS1 .Constitutive activation of FLT3 via internal tandem duplication (ITD) mutation or point mutations in the tyrosine kinase domain (TKD) has been shown to mediate cell-extrinsic changes in the immune landscape of AML. Mice with a total body ITD mutation demonstrate expanded populations of dendritic cells (DCs), dendritic cell precursors (pre-DCs), and regulatory T cells (Treg) as a result of the ITD mutation . The exp+, CD25+ cells, and in the mRNA level of FOXP3, consistent with a decrease in Treg, was observed following treatment in AML patients treated with Midostaurin, a possible consequence of ITD inhibition  in first remission  account for between 9 and 20% of mutations in AML  and decreasing the fraction of double positive thymocytes, unlike that of other mutations in IDH1 and IDH2, as a result of the exceptionally high levels of intracellular R-2-HG this mutation produces (The production of the oncometabolite R-2-Hydroxyglutarate (R-2-HG) mediates the proto-oncogenic effects of mutant IDH1 and IDH2. Human T cells express multiple transporters capable of importing R-2HG, including SLC13A3 and SLC22A6 . The impeg cells . Subsequphocytes , 171. BoH status . Interesproduces .IDH1-mutated gliomas  as the two types of genetic alterations are closely correlated  has been reported as 69–78% , GATA2 and MECOM(EVI1) mutations in terms of the increased risk of death associated with the mutation , regulation of senescence and apoptosis, regulation of fatty acid oxidation –205. MorJAK2V617F mutation, most commonly associated with the Myeloproliferative Neoplasm (MPN) Polycythemia Vera (PV) which can progress to AML, has been shown to regulate expression of PD-L1 cell-intrinsically  and inflammatory signaling in bone marrow HSPCs  that can converge on vulnerabilities, including metabolic, of a given subtype, resulting in elimination of leukemia.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.PP serves in the Scientific Advisory Board of Agios Pharmaceuticals. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To the Editor: Urticaria can be associated with significant functional impairment.Complete details of GBD methodology are available elsewhere.The global age-standardized DALY rate for urticaria is 55.49/100,000 population. Among skin disease, the global burden from urticaria in 2016 ranks only behind acne vulgaris , dermatitis , viral skin disease  and psoriasis .Limitations of the GBD Studies have been previously described.


In mammals, it is accepted that Ca2+ influx through the L-type Ca2+ channels occurs during AP phase 2. However, in murine models, experimental evidence shows Ca2+ influx takes place during phase 1. Interestingly, Ca2+ influx that activates contraction is highly regulated by the autonomic nervous system. Indeed, autonomic regulation exerts multiple effects on Ca2+ handling and cardiac electrophysiology. In this paper, we explore autonomic regulation in endocardial and epicardial layers of intact beating mice hearts to evaluate their role on cardiac excitability and contractility. We hypothesize that in mouse cardiac ventricles the influx of Ca2+ that triggers excitation–contraction coupling (ECC) does not occur during phase 2. Using pulsed local field fluorescence microscopy and loose patch photolysis, we show sympathetic stimulation by isoproterenol increased the amplitude of Ca2+ transients in both layers. This increase in contractility was driven by an increase in amplitude and duration of the L-type Ca2+ current during phase 1. Interestingly, the β-adrenergic increase of Ca2+ influx slowed the repolarization of phase 1, suggesting a competition between Ca2+ and K+ currents during this phase. In addition, cAMP activated L-type Ca2+ currents before SR Ca2+ release activated the Na+-Ca2+ exchanger currents, indicating Cav1.2 channels are the initial target of PKA phosphorylation. In contrast, parasympathetic stimulation by carbachol did not have a substantial effect on amplitude and kinetics of endocardial and epicardial Ca2+ transients. However, carbachol transiently decreased the duration of the AP late phase 2 repolarization. The carbachol-induced shortening of phase 2 did not have a considerable effect on ventricular pressure and systolic Ca2+ dynamics. Interestingly, blockade of muscarinic receptors by atropine prolonged the duration of phase 2 indicating that, in isolated hearts, there is an intrinsic release of acetylcholine. In addition, the acceleration of repolarization induced by carbachol was blocked by the acetylcholine-mediated K+ current inhibition. Our results reveal the transmural ramifications of autonomic regulation in intact mice hearts and support our hypothesis that Ca2+ influx that triggers ECC occurs in AP phase 1 and not in phase 2.The relationship between cardiac excitability and contractility depends on when Ca The ANSdynamics  and elecdynamics , throughP levels . The cAMP levels , inducin channel , ryanodi channel  and phos channel , a proteGi protein   from postganglionic cholinergic intracardiac neurons. The locally released ACh binds to muscarinic (M2) receptors which in turn inhibit AC through a  protein . The AC  (IKACh) . Althoug obscure .2+ in ventricular myocytes is driven by the activation of L-type Ca2+ channels during an AP depolarization. In large mammals including humans, the prolongation of the AP plateau phase (phase 2) increases the time L-type Ca2+ channels are open leading to a sustained influx of Ca2+ into the myocyte. Small mammals like mice have a high metabolic demand. Thus, the heart needs to beat at higher rates to handle their metabolic needs. To cope with a higher heart rate, the mouse heart has developed a short duration ventricular AP. These characteristic kinetics promoted the idea that mouse ventricular AP lacks a plateau phase (phase 2) (in situ mouse ventricular APs display a well-defined phase 2 (+ through the Na+-Ca2+ exchanger (NCX) (2+ dynamics in mouse hearts.The influx of Caphase 2) . Recentl phase 2 . Interes phase 2  and it wer (NCX) . However2+ that triggers excitation–contraction coupling (ECC) does not occur during phase 2. Our results reveal for the first time the transmural effects of autonomic regulation in intact mice hearts and confirm our previous observation that Ca2+ influx that triggers ECC occurs in the AP phase 1 and not in phase 2.As sympathetic and parasympathetic drives impact the kinetics of both phase 1 and phase 2  mimickin2, 1 MgCl2, 0.33 NaPO4H2, 10 HEPES, and 10 glucose, pH 7.4 and equilibrated with 100% O2. Experiments were conducted at physiological temperatures of 35–37°C using a Peltier unit. The myosin ATPase inhibitor, blebbistatin (10 μM) was added to the tyrode solution to inhibit the heart’s mechanical activity. Blebbistatin was continuously perfused throughout the whole duration of the experiment to avoid introducing movement artifacts, breaking the glass electrode and photolability. All salts and drugs were obtained from Sigma . Tertiapin was purchased from Tocris .Experiments were conducted on 8-week-old, male Balb/c mice . Mice were maintained in accordance to the National Institutes of Health Guide for the Care and Use of Laboratory Animals  and the Institutional Animal Care and Use Committee guidelines of the University of California, Merced (Protocol # 2008–201). Mice were injected intraperitoneally with sodium heparin 15 min before euthanasia. Hearts were extracted by thoracotomy and perfused in a Langendorff apparatus with tyrode solution containing (in mM): 140 NaCl, 5.4 KCl, 2 CaClA new experimental approach, the micro-hemodynamic analyzer, was developed to measure left ventricular pressure in the whole intact heart . A commo2+ transients and membrane potential, respectively. Rhod-2 AM and Di-8-ANEPPS were prepared with 20% Pluronic  in tyrode solution as previously described  technique has been previously used by our group . Brieflyescribed . All dyeEpicardial electrical recordings of the action potentials (APs) were obtained by means of sharp glass microelectrodes filled with 3M KCl that were connected to a high input impedance differential amplifier . Glass microelectrodes were fabricated with a micropipette puller  and had a resistance of 10–20 MΩ. All the AP recordings were performed in hearts that were externally paced at 6–7 Hz.2+-driven. The LPP was used to measure any changes in the Ca2+-driven currents in the epicardium under either continuous β-adrenergic or cholinergic stimulation. In addition, for transient β-adrenergic stimulation, we used the UV-sensitive caged cAMP .Our developed LPP technique was used to measure membrane currents during a triggered AP, at the intact heart level . LPP comg 15 min 4°C. The clear phase was transferred to a new tube and 500 μL of isopropanol per mL of Trizol was added and centrifuged at 12,000 × g for 10 min at 4°C. The supernatant was removed and 1 mL of 75% ethanol per mL of Trizol was added and centrifuged at 7,500 × g for 5 min at 4°C. The dried pellet was dissolved in 30 μL of RNAse free water. For real time PCR, RNA was quantified (NanoDrop 1000 Spectrophotometer) and 2 μg was used for the reverse transcription reaction. The 2 μg of RNA was mixed with 1 μL of random primers (200 ng/μL) and water up to 12.5 μL. 7.5 μL of a MIX solution containing 4 μL of reaction buffer, 0.5 μL of RNAse free water, 2 μL dNTPs mix and 1 μL of RevertAid was added before the thermocycle cycles began. cDNA samples were diluted with water, 6.25 μL of Mix (FastStart Roche), 0.37 μL of forward primers, and 0.37 μL of reverse primers (see Hearts were perfused with RNAlater (Qiagen) to inhibit the RNAses. Then, left ventricular square pieces of 4 × 4 mm were included in agarose to facilitate slicing. Specimens were sliced in 200 μm sections using a vibratome (Leica VT1000S). For RNA extraction, individual slices were placed in a 1.5 mL Eppendorf containing 0.5 mL of Trizol reagent. Tissue was homogenized with an ultrasonic cell disruptor. The homogenate was incubated for 5 min at room temperature and 0.2 mL of chloroform per mL of Trizol was added. After 2–3 min of incubation, the sample was centrifuged at 12,000 × mers see . The rea2+ transients and APs in the same region (the mid region of the left ventricle), it is impossible to perform the recordings in the same precise location between different hearts. Thus, the data is presented as the measured times with their standard error (SEM) in Tables and then as percentage change in Graphs, where data was normalized to the control values. To assess electrical changes, AP traces were evaluated at certain repolarization times. Specifically, the time it takes for the AP to reach 30%, half of phase 2, or 90% repolarization was assessed and referred to as APD30, APD half phase 2, or APD90, respectively. To evaluate the kinetics of the recorded Ca2+ transients, these were normalized between zero (minimum fluorescence) and one (maximum fluorescence). The kinetics parameters of the Ca2+ transients evaluated were the rise time (time for the transient to rise between 10% and 90%), time-to-peak (time for the transient to reach a maximum amplitude), half duration , and fall time . Each recorded parameter for AP and Ca2+ transient kinetics, control and non-control experiments, were evaluated and normalized to the control values for each heart used. After this normalization, data was compiled and statistical analysis was performed. Data is presented as multiple measurements  recorded on different hearts (N) with the mean ± SEM (solid lines). The statistical significance was tested using a two-sample Kolmogorov–Smirnov test (OriginPro 2019). The difference was considered to be significant if the p-value was <0.01.It is important to mention that in whole heart experiments there are two main sources of variance. First, the hearts are not identical between each other and second, although we are measuring CaIn the heart, both excitability and contractility are tightly controlled by the ANS. Although there is substantial information about sympathetic and parasympathetic regulation of the electrical and mechanical function, it is not clear how autonomically driven changes in the AP kinetics impact contractility in the mouse heart, which is often used as a model system.N = 4 hearts p < 0.01), showing that the perfused hearts used here respond to a β-adrenergic drive.In a beating heart, the classical way to assess the degree of autonomic regulation is to evaluate the developed pressure during the cardiac cycle. Using intact perfused mouse hearts, we measured the effect of catecholamines in the developed pressure during the cardiac cycle. 2+ transients in those particular regions using the PLFFM technique. 2+ transients recorded simultaneously at the endocardial and epicardial layer. Perfusion of the hearts with 500 nM isoproterenol increased the amplitude of Ca2+ transients in both layers . As expected, the time to peak of the Ca2+ transients follow the same trend (11.1 ± 1.3% for endocardium and 5.9 ± 1.70% for epicardium). Isoproterenol induced a reduction in Ca2+ transients’ half duration. The half duration is statistically significant shorter in the presence of isoproterenol (5.9 ± 0.42% endocardium and 8.8 ± 0.45% epicardium). However, the changes in the fall time of the relaxation (20.3 ± 0.96% endocardium and 26.3 ± 0.92% epicardium) were larger than those observed in the half durations. The large effect of isoproterenol on the fall time compared to the half duration could be explained by the fact that isoproterenol not only has a positive lusitropic effect but also changes the morphology of the Ca2+ transients as shown in p < 0.01) than those in the endocardial layer  occurs during phase 1  technique. We simultaneously acquired Ca2+-driven currents with the corresponding APs before and after isoproterenol. In 2+ currents, activated by the photolysis of nifedipine on the membrane patch surface , were recorded before and after the heart was perfused with isoproterenol. We observed a clear increase in the amplitude of the Ca2+-driven currents, displayed as an early fast component (earlyi) and a late slower component (latei). Unfortunately, the simultaneous measurement of Ca2+ driven currents in the endocardium and epicardium was not possible due to the size of the patch pipettes. To reach the endocardial layer with the patch pipette would require a large size hole that would result in electrical conduction alterations. earlyi that increased from 13.9 ± 0.2 nA/nF to 27.6 ± 0.2 nA/nF. In a recent work (2+ released from the sarcoplasmic reticulum (SR), activated the NCX in the forward mode, resulting in latei . To test the hypothesis that β-adrenergic stimulation directly increases the amplitude of the Ca2+ current occurring during phase 1, we performed an identical experiment to the one presented in earlyi. The simultaneous recording of the corresponding APs shows that isoproterenol only altered the kinetics of phase 1  indicating that most of the nifedipine was photolyzed.The decrease of the repolarization rate of phase 1 with isoproterenol can be attributed to an increase in the amplitude of L-type Ca phase 1  and that phase 1 . To direent work , we showin ilate . This ilbolished  when we earlyi. It is well known that catecholamines activate PKA, which in turn leads to the phosphorylation of multiple targets involved in Ca2+ signaling . In order to determine which target is phosphorylated first, the effect of catecholamines on the time course of Ca2+-driven currents was evaluated. We performed experiments in which we mimicked a transient β-adrenergic stimulation by photolyzing caged 3′,5′-cyclic adenosine monophosphate (cAMP). A typical response is illustrated in 2+-driven current, earlyi and latei, began to increase over time. To better illustrate the changes of earlyi and latei, in earlyi and latei as a function of time during consecutive cardiac cycles, highlighted that both currents changed over time, but with a greater effect on earlyi . These results suggest that in the absence of a β-adrenergic stimulus, carbachol by itself does not induce a decrease in the developed ventricular pressure during the cardiac cycle.Sympathetic stimulation is highly antagonized by the other branch of the ANS, the parasympathetic nervous system. Parasympathetic stimulation is thought to generally predominate over an existing sympathetic activation. Next, we evaluated the response of endocardium and epicardium to muscarinic cholinergic agonists. 2+ transients across the ventricular wall in the presence of a cholinergic agonist and antagonist  in 2+ transients were significantly increased in endocardium (5.5 ± 1.17%) but not in epicardium (2.5 ± 1.83%). Carbachol induced a significant increase in the half duration  and fall time of the Ca2+ transients . Although the changes in the fall times are significantly different, they were smaller than the kinetic effects observed with isoproterenol  it was possible to observe significant values above the control, for rise time (epicardium 5.3 ± 3.14%), time to peak , half duration  and fall time .To assess the cholinergic regulation of contractility, we examined the properties of Catagonist . Figure ocardial  and epicocardial  layer wiicardium . Howeveroterenol . Thus, w2+ transients indicates that the Ca2+ influx that triggers Ca2+ induced Ca2+ release may not occur during phase 2. To further explore this possibility, we then evaluated the properties of Ca2+ currents driven by its own AP, when the heart was exposed to a parasympathetic drive. Indeed, the decrease of APD half phase 2 with carbachol could be due to a decrease in the amplitude of L-type Ca2+ currents. For example, a reduction in the L-type Ca2+ current will lead to a decrease in the Ca2+ released from the SR. In turn, this will result in less activation of the NCX in its forward mode. To test this scenario, we evaluated the effect of carbachol on the time course of the L-type Ca2+ current during an evoked AP. In 2+-driven currents that were activated by the photolysis of nifedipine before and after carbachol perfusion. 2+-driven current traces before and after the carbachol perfusion. The Ca2+-driven currents displayed an earlyi and latei component, but they were not affected by the presence of carbachol. earlyi.Since the effects induced by carbachol in the contractility were minor, we decided to explore how the activation of a parasympathetic pathway affects the electrophysiological behavior of the endocardial and the epicardial layers. In 2+ currents, we examined if a repolarizing outward current (associated with the cholinergic stimulation) was involved in the shortening of the APD half phase 2. Although it is well established in the atrium that the current of an acetylcholine (ACh) activated potassium channel, IKACh, can severely affect the AP repolarization, the role of this current in the ventricle is still a matter of debate. The role of the IKACh was evaluated by perfusing the hearts with tertiapin, a bee venom that can block with high affinity GIRK1/GIRK2 hetero-tetramers (+ channels responsible for IKACh. The perfusion of tertiapin prevented the carbachol shortening of the AP in the epicardium N = 5 hearts) shows tertiapin induced statistically significant differences in all of the AP phases . Although these differences are statistically significant, the magnitude of the change is so small , and the higher systemic demands will be met by increasing the systolic pressure (inotropism) as well as the rate of relaxation (lusitropism) of the developed pressure. In our mouse model, exposure to isoproterenol generated an increase in the left ventricular developed pressure . This inl layers . Althougl layers . For exais layer . In addiis layer .+ channel explains the faster rate of repolarization of AP phase 1 in epicardium. In addition, β-adrenergic stimulation affected the AP’s morphology by increasing the duration of the AP phase 1 and the amplitude of phase 2 across the ventricular wall  components increased the amplitude of this fast repolarizing current. This possibility is unlikely to occur in our experimental conditions. Increase of Ito, would accelerate instead of slowing the repolarization rate of phase 1. In contrast, a reduction of the amplitude of Ito by norepinephrine and alpha adrenoceptor stimulation has been reported in healthy rat ventricular myocytes  during phase 1 will maintain a depolarization of this phase for a longer time, increasing APD30. Additionally, in the presence of nifedipine, the effect of isoproterenol on APD30 was smaller (2+ currents under isoproterenol stimulation. These findings agree with previous experiments from our group in which we showed that the transmembrane Ca2+ influx that triggers Ca2+-induced Ca2+-released (CICR) enters during phase 1  through the L-type Ca2+ channels and a significant increase in the amplitude of the late NCX current as reflected by latei. These results suggest that changes in the electrical excitability induced by the β-adrenergic stimulation across the ventricular wall are highly mediated through a Ca2+-driven mechanism.A final proof of concept was to demonstrate that isoproterenol mediates an increase of the amplitude of the L-type Ca phase 1 . This couggested . The val2+ currents and consequently, Ca2+ release from the SR. However, PKA activation can affect other targets. For example, PLN  activity  an agonistic effect on a cationic outward current. In principle, the muscarinic effect on the L-type Ca2+ is very unlikely because, as we showed, there were no effects on the developed pressure and very minor effects  on the amplitude of the Ca2+ transients in the presence of carbachol. Even more, this first hypothesis was discarded when epicardial Ca2+ currents were not affected by carbachol (+ current was tested in IKACh blocker (tertiapin) a small shortening of the total duration of the AP was observed. Moreover, this apparent blocking effect was corroborated when the same hearts were perfused with carbachol after tertiapin was washed out  inhibitory effect on the Caarbachol . The secshed out . Togethe2+ currents triggered during the AP phase 1; (2) PKA phosphorylates L-type Ca2+ channels before the SR Ca2+ release activates NCX current; (3) cholinergic stimulation by carbachol decreased the duration of the late AP repolarization. However, this cholinergic stimulation did not substantially modify per se the intracellular Ca2+ signals when compared with β-adrenergic stimulation; (4) cholinergic stimulation decreased the total duration of the ventricular AP through activation of IKACh.In conclusion, our results indicate that: (1) in both endocardium and epicardium, the increase in contractility by isoproterenol was driven by an increase in the amplitude of intracellular Ca2+ currents that occur in phase 1 but not phase 2 of the APs. In contrast, a cholinergic input does not directly modulate Ca2+ dynamics, but rather alters inward rectifier potassium channels in the isolated heart.In summary, the results presented here demonstrate that, in a mouse heart, β-adrenergic input acts across the ventricular wall by modulating the L-type CaMice were maintained in accordance to the National Institutes of Health Guide for the Care and Use of Laboratory Animals  and the Institutional Animal Care and Use Committee guidelines of the University of California Merced (Protocol # 2008–201).AE and JR-F designed the research. YA-S, AR, MA, AE, and JR-F performed the research. YA-S, AR, AE, and JR-F analyzed the data. YA-S, AE, and JR-F wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Recent studies have revealed that long noncoding RNAs (lncRNAs) may hold crucial triggers of the pathogenesis of hematological malignancies, while the studies evaluating the expression pattern of lncRNA in acute myeloid leukemia (AML) are few. Thus, this study aimed to investigate the implication of lncRNA expression pattern in AML development and progression.Bone marrow samples from four AML patients and four controls were subjected to lncRNA sequencing. Then, bone marrow samples from 110 AML patients and 40 controls were proposed to real‐time quantitative polymerase chain reaction (RT‐qPCR) validation for 10 candidate lncRNAs. Clinical data and survival profiles were recorded in AML patients. Furthermore, lncRNA RP4‐576H24.2 expression in AML cell lines and its effect on AML cell proliferation and apoptosis were detected.LncRNA expression pattern by sequencing clearly distinguished AML patients from controls, and 630 upregulated and 621 downregulated lncRNAs were identified in AML patients compared to controls, which were mainly enriched in AML oncogene‐related biological process and pathways . RT‐qPCR validation observed that six lncRNAs correlated with AML risk, one lncRNA associated with risk stratification, and three lncRNAs correlated with survivals, among which lncRNA RP4‐576H24.2 was the only one correlated with AML susceptibility, risk stratification, and survivals. Further in vitro experiments showed that lncRNA RP4‐576H24.2 was upregulated in AML cell lines compared to normal bone marrow mononuclear cells (BMMCs), and promoted proliferation while inhibited apoptosis in HL‐60 and KG‐1 cells.LncRNA expression pattern is closely involved in the development and progression of AML, and several specific lncRNAs exhibit potential to be biomarkers for AML risk and prognosis. Besides, lncRNA RP4‐576H24.2 might be a potential oncogene in AML pathogenesis. This study aimed to investigate the implication of long noncoding RNA (lncRNA) expression pattern in acute myeloid leukemia (AML) pathogenesis. It showed that lncRNA expression pattern is closely involved in the development and progression of AML, and several specific lncRNAs exhibit potential to be biomarkers for AML risk and prognosis. Besides, lncRNA RP4‐576H24.2 is discovered to be a key oncogene in AML pathogenesis. The inclusion criteria were: (a) Diagnosed as de novo AML patients according to the World Health Organization (WHO) Classification of Tumors of Hematopoietic and Lymphoid Tissues (2008); (b) Age above 18 years. The exclusion criteria were: (a) Secondary or mixed AML; (b) History of other solid tumor or hematological malignancies; (c) Previous treatment with chemotherapy or radiotherapy. Meanwhile, 40 participants who were adult bone marrow donors or adult patients with nonmalignancy diseases (such as thrombocytopenic purpura and myelofibrosis) who underwent bone marrow biopsy were enrolled as controls. This present study was approved by the Ethics Committee of our hospital, all participants (AML patients and controls) provided written informed consents.2.29/L, and platelet count above 100 × 109/L. Patients were then followed up, the last follow‐up date was June 31, 2018 and the median follow‐up duration was 17.0 (range 1.0‐36.0) months, and event‐free survival (EFS) as well as overall survival (OS) were calculated. The EFS was defined as the time interval from initiation of treatment to disease recurrence, progression, or death, and the OS was defined as the time interval from initiation of treatment to death. Besides, bone marrow samples were also acquired from controls by biopsy as well.Age, gender, French‐American‐British (FAB) classification, white blood cell (WBC), cytogenetics, molecular genetics, and risk stratification  of AML patients were documented. And, bone marrow samples were obtained from AML patients during biopsy. Patients were treated in accordance with NCCN Clinical Practice Guidelines in Oncology of AML . Treatment remission status of induction chemotherapy was assessed, and complete remission (CR) was defined as bone marrow with at least 20% cellularity and BM blasts below 5% at steady state after treatment, without cytological evidence of leukemia, no transfusion requirement, leukocyte count above 1 × 102.3Four de novo AML patients and four age‐ and gender‐matched controls were randomly selected from total participants, and their bone marrow samples were subjected to RNA sequencing by Genergy Bio Company. In brief, (a) Total RNA was extracted from bone marrow using PureZOL RNA isolation reagent (Bio‐Rad), and then concentration, purity, and integrity were assessed and adjusted; (b) Ribosomal RNA (rRNA) was removed by Epicentre Ribo‐zero™ rRNA Removal Kit (Epicentre) and the remaining RNA was proposed to generate sequencing library according to the methods in a previous study2.4P value <.05 and the biological significance defined as a difference of fold change (FC) above 2.0 times; (d) Heatmap analysis of dysregulated genes  was performed by Pheatmap package; (e) Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of dysregulated lncRNAs were performed using DAVID web server according to their regulated mRNAs, afterward, GO and KEGG enrichment analysis of dysregulated mRNAs were also performed; (f) Regulatory network of top 40 dysregulated lncRNAs (20 upregulated and 20 downregulated) was drawn by Igraph package based on their regulated mRNAs; (g) Circos graph for transcription and regulation information was drawn by RCircos package. R Software (Version 3.5.3) (Lucent Technologies) was used for bioinformatic analysis. The genes (lncRNAs and mRNAs) which were identified in 50% or above samples were included in the bioinformatics analysis.The following bioinformatic analyses were carried out based on the sequencing data of lncRNA and mRNA expression patterns: (a) Principal component analysis (PCA) of gene expression pattern  was performed by Stats package; (b) Heatmap analysis of gene expression pattern  was performed by Pheatmap package; (c) Dysregulated genes  were analyzed by DeSeq2 package and showed as volcano spot with the statistical significance defined as 2.52FC, which included top 5 upregulated and top 5 downregulated lncRNAs in AML patients compared to controls in RNA sequencing as follows: lncRNA CES1P1, lncRNA RP4‐576H24.2, lncRNA LINC01262, lncRNA SIGLEC16, lncRNA OR7E140P, lncRNA DIO3OS, lncRNA MEG3, lncRNA ST3GAL6‐AS1, lncRNA OR51A10P, and lncRNA RP5‐983L19.2. Then, their expressions in bone marrow samples from 110 de novo AML patients and 40 controls were validated by RT‐qPCR.So as to validate the correlation of some potential lncRNA expressions with disease susceptibility, risk stratification, and prognosis, 10 candidate lncRNAs were chosen according to their absolute value of Log2.6Human AML cell lines HL‐60, HT‐93, KG‐1, and AML‐193 were purchased from Leibniz Institute DSMZ‐German Collection of Microorganisms and Cell Cultures. HL‐60, HT‐93, and KG‐1 cells were cultured in 90% Roswell Park Memorial Institute (RPMI) 1640 Medium (Gibco) and 10% fetal bovine serum (Gibco), AML‐193 cells were cultured in 90% Iscove's Modified Dulbecco's Medium (Gibco) and 10% fetal bovine serum (Gibco). Besides, human normal bone marrow mononuclear cells (BMMCs) were isolated from a healthy donor as controls. Then, the relative expression of lncRNA RP4‐576H24.2 was detected in HL‐60, HT‐93, KG‐1, AML‐193 cell lines and controls. Afterward, control overexpression, lncRNA RP4‐576H24.2 overexpression, control shRNA, and lncRNA RP4‐576H24.2 shRNA plasmids were constructed by Shanghai QeeJen Bio‐Tech Company and transfected into HL‐60 cells as well as KG‐1 cells, which were divided into NC(+), Lnc(+), NC(−), and Lnc(−) groups. Subsequently, lncRNA RP4‐576H24.2 relative expression in each group was detected by RT‐qPCR at 24 hours posttransfection; cell proliferation in each group was detected by Cell Counting Kit ‐ 8 (Sigma) at 0, 24, 48, and 72 hours posttransfection; finally, the cell apoptosis rate in each group was detected by Annexin V Apoptosis Detection Kit FITC (Thermo) at 24 hours posttransfection. All the cell experiments were performed in triplicate.2.7−△△Ct formula using U6 or GADPH as an internal reference. And, the primers used in RT‐qPCR are listed in Table First of all, the total RNA was isolated from bone marrow samples or cells using PureZOL RNA isolation reagent (Bio‐Rad) and was evaluated by a spectrophotometer. Then, the total RNA was reversely transcribed into complementary DNA using the QuantiNova Reverse Transcription Kit (Qiagen). Subsequently, PCR was performed by QuantiNova SYBR Green PCR Kit (Qiagen) and the results were calculated with 22.8t test or Wilcoxon rank sum test. Predictive value of candidate parameters for disease risk was detected by receiver operating characteristic (ROC) curve and assessed by area under the curve (AUC). Comparison of EFS and OS was detected by Kaplan‐Meier curve and log‐rank test. P value <.05 was considered as significant.Statistical analysis was performed by SPSS Software 21.0 (IBM), while the statistical image was drawn by GraphPad Prism Software 7.01 . Comparison among multiple groups was detected by one‐way analysis of variance (ANOVA) followed by multiple comparison test or Kruskal‐Wallis H test. Comparison between two groups was detected by 33.19/L. Other detailed information of the four AML patients’ characteristics is shown in Table A total of four AML patients and four controls were randomly selected and included in RNA sequencing. For AML patients included in RNA sequencing, the mean age was 42.5 ± 10.3 years, and the numbers of females as well as males were both 2 (50.0%) , cellular component , and molecular functions  Figure A, and th3.42FC, which are presented in Table Top 20 upregulated lncRNAs and top 20 downregulated lncRNAs were selected based on the rank of the absolute value of Log3.59/L.Ten candidate lncRNA expressions in bone marrow including top 5 upregulated lncRNAs and top 5 downregulated lncRNAs by RNA sequencing were selected for RT‐qPCR validation in 110 AML patients and 40 controls. As for the AML patients included in RT‐qPCR validation, the mean age was 43.1 ± 13.4 years, with 63 (57.3%) males and 47 42.7%) females , lncRNA RP4‐576H24.2 (P < .001), and lncRNA SIGLEC16 (P = .003) were elevated in AML patients compared with those in controls; however, no difference of lncRNA LINC01262 (P = .122) or lncRNA OR7E140P (P = .662) expressions was found between AML patients and controls , LncRNA MEG3 (P = .002), and lncRNA RP5‐983L19.2 (P < .001) were downregulated in AML patients compared with controls, while the expressions of lncRNA ST3GAL6‐AS1 (P = .080) and lncRNA OR51A10P (P = .227) were similar between AML patients and controls  (Table P = .074), lncRNA SIGLEC16 (P = .924), lncRNA DIO3OS (P = .445), lncRNA MEG3 (P = .158), lncRNA ST3GAL6‐AS1 (P = .171), lncRNA OR51A10P (P = .283), or lncRNA RP5‐983L19.2 (P = .109) were of no difference among patients at better‐risk, intermediate‐risk, and poor‐risk.Only lncRNA RP4‐576H24.2 expression in bone marrow was correlated with AML risk stratification, to be exact, the level of lncRNA RP4‐576H24.2 was the highest in patients with poor‐risk followed by patients with intermediate‐risk, and was the lowest in patients with better‐risk (3.8P = .003)       , LncRNA RP4‐576H24.2 (P = .640), LncRNA LINC01262 (P = .802), LncRNA SIGLEC16 (P = .656), LncRNA OR7E140P (P = .178), LncRNA DIO3OS (P = .602), LncRNA MEG3 (P = .792), LncRNA ST3GAL6‐AS1 (P = .269), LncRNA OR51A10P (P = .334), or LncRNA RP5‐983L19.2 (P = .235) expressions among patients with different FAB classifications was found (Table No difference of LncRNA CES1P1 (3.10P < .001), KG‐1 cells (P < .05), and AML‐193 cells (P < .001), while was of no difference in HT‐93 cells (P > .05) compared with control cells , and was downregulated in Lnc(−) group compared with NC(−) group   and 72 hours  while was repressed in Lnc(−) group compared with NC(−) group at 48 hours  and 72 hours  , while was upregulated in Lnc(−) group than that in NC(−) group  at 24 hours after transfections in HL‐60 cells and KG‐1 cells  Figure B,F in bos Figure C,D,G,H. 4Profiting from the development of RNA sequencing and bioinformatics analysis, investigation of the heterogenetic pathology of AML has become more profound, among which the role of ncRNAs in development and progression of AML has been increasingly revealed in recent decades, including forming regulatory networks that could mediate multiple pathways related to AML.In terms of the studies including RNA sequencing in AML, in a study using miRNA sequencing and transcription factor (TF) activity array, 308 dysregulated miRNAs and 84 dysregulated TFs are detected, then 1462 miRNA‐target gene pairs, 982 TF‐target gene pairs, and 196 TF‐miRNA pairs are identified subsequently; after emerging as a regulatory network of these dysregulated miRNAs and TF, the KEGG pathway analysis finds that the network is markedly enriched in 33 pathways with the AML‐related pathways the most significant.The poor prognosis of AML has highlighted the need for searching more novel and reliable biomarkers which can assist in the management of AML patients, and several specific lncRNAs have been proposed to exhibit prognostic value. For instance, the lncRNA LINC00265 is reported to be upregulated in bone marrow in AML patients than that in controls and is associated with worse OS in AML patients.In addition, there were several issues that need to be illustrated in this study. First, there were patients with FAB classification of M1 and M6 in the 110 AML patients in RT‐qPCR validation; however, no patients with FAB classification of M1 or M6 were included in the four AML patients in RNA sequencing. This heterogeneity might result from that the sample size was much larger in RT‐qPCR validation and all the patients were enrolled consecutively, thus, there were patients with more diverse FAB classifications in RT‐qPCR validation. However, we further analyzed the difference of candidate lncRNA expressions among patients with different FAB classifications, which disclosed that candidate lncRNA expressions did not vary among patients with different FAB classifications. These results indicated that the FAB classification was not a factor affecting the candidate lncRNA expressions in AML patients. Second, lncRNA LINC01262 and lncRNA OR7E140P expressions were markedly dysregulated in AML patients compared with controls in RNA sequencing, while did not show any difference between AML patients and controls in RT‐qPCR validation, which might derive from that: (a) the results in RNA sequencing might be affected by a singular value due to that the sample size for RNA sequencing was small; (b) the RNA sequencing technique was less accurate than RT‐qPCR, which might also contribute to this conflict.Furthermore, the underlying mechanisms of several specific lncRNAs in AML pathogenesis are also reported previously. For instance, knockdown of lncRNA ZFAS1 represses the AML progression through mediating the miR‐150/Sp1 and miR‐150/Myb pathways.In conclusion, the lncRNA expression pattern is closely involved in the development and progression of AML, and several specific lncRNAs exhibit potential to be biomarkers for AML risk as well as prognosis. Besides, lncRNA RP4‐576H24.2 might be a potential oncogene in AML pathogenesis. These data would shed light on the potential role of lncRNA in AML pathology and the application of lncRNA as a novel biomarker for AML management.The authors declare that they have no competing interests.


FLT3 juxtamembrane domain (FLT3-ITD)-positive acute myeloid leukemia (AML) leads to poor clinical outcomes after chemotherapy. We aimed to establish a cytarabine-resistant line from FLT3-ITD-positive MV4-11 (MV4-11-P) cells and examine the development of resistance. The FLT3-ITD mutation was retained in MV4-11-R; however, the protein was underglycosylated and less phosphorylated in these cells. Moreover, the phosphorylation of ERK1/2, Akt, MEK1/2 and p53 increased in MV4-11-R. The levels of Mcl-1 and p53 proteins were also elevated in MV4-11-R. A p53 D281G mutant emerged in MV4-11-R, in addition to the pre-existing R248W mutation. MV4-11-P and MV4-11-R showed similar sensitivity to cabozantinib, sorafenib, and MK2206, whereas MV4-11-R showed resistance to CI-1040 and idarubicin. MV4-11-R resistance may be associated with inhibition of Akt phosphorylation, but not ERK phosphorylation, after exposure to these drugs. The multi-kinase inhibitor cabozantinib inhibited FLT3-ITD signaling in MV4-11-R cells and MV4-11-R-derived tumors in mice. Cabozantinib effectively inhibited tumor growth and prolonged survival time in mice bearing MV4-11-R-derived tumors. Together, our findings suggest that Mcl-1 and Akt phosphorylation are potential therapeutic targets for p53 mutants and that cabozantinib is an effective treatment in cytarabine-resistant FLT3-ITD-positive AML.Internal tandem duplication of  Acute myeloid leukemia (AML) is a disorder of clonal hyperproliferation of abnormal myeloid progenitor cells emerging from a heterogeneous genetic background. Standard induction therapy for patients with AML consists of cytarabine and anthracycline (idarubicin or daunorubicin) treatment. After achieving complete remission, patients are given a consolidation therapy involving a high dose of cytarabine. Among the 50–75% of those who achieve complete remission after induction therapy, 80% suffer disease relapse attributed to drug resistance ,2. HenceMultiple mechanisms have been shown to relate to chemoresistance in AML. Cytarabine resistance in AML cells was shown to be linked to aberrant expression of equilibrative nucleoside transporters (ENT1) and metabolic enzymes deoxycytidine kinase (DCK) and cytosolic 5′-nucleotidase-II (NT5C2) ,4,5. EnhFLT3-ITD) is one of the most frequently found mutations in AML and is detected in 20–30% of adults with AML and in 10–15% of pediatric AML cases . No. No50 vaMV4-11-R A–D.FLT3-ITD-dependent signaling in MV4-11-R cells, including phosphorylation of FLT3, STAT5, and Akt; however, ERK phosphorylation was only transiently and weakly inhibited  both in vitro and in vivo. Patients with AML often relapse after chemotherapy, and 50 of cytarabine was 3.37 μM, which is approximately 13-fold higher than that for MV4-11-P (0.26 μM). Previous studies examining the plasma level during cytarabine treatment showed that plasma concentrations of 5–40 μM were reached during high-dose (3 g/m2) cytarabine infusion [2) treatment yielded 10–15 μM steady-state plasma concentrations [2) treatments generated steady-state plasma concentrations between 0.1 μM and 1.0 μM [50 of MV4-11-R corresponds to the plasma concentration in patients receiving between conventional and intermediate-dose infusion. Since 3.37 μM is the IC50 value obtained for a 72-hour culture, a considerably higher concentration of cytarabine is required to completely eliminate MV4-11-R. Therefore, MV4-11-R can be evaluated as a cytarabine-resistant cell line suitable for assessment of changes in the development of drug resistance. MV4-11 cell line expresses an MLL-AF4 rearranged gene resulted from the t translocation. MLL gene-rearranged leukemia was reported to be more sensitive to cytarabine treatment [MLL-AF4 expression. Therefore, MLL-rearrangement would hardly be considered a driving force for cytarabine resistance.For MV4-11-R, the IC4-11-P 0. μM. Prevtrations ,28; in c4-11-P 0. μM. Prevd 1.0 μM ,28. The reatment ,30. In aFLT3-ITD-dependent growth and are therefore more sensitive to cytarabine, whereas a fraction of cells with ER-bound and less phosphorylated FLT3 exhibits FLT3-ITD-independent growth and develops cytarabine resistance. Therefore, targeted inhibition of ERK and Akt can be a promising strategy to overcome cytarabine resistance in AML.We demonstrated that the FLT3 protein exists in an immature, underglycosylated form in MV4-11-R. Glycosylation of the FLT3 receptor starts in the endoplasmic reticulum (ER) and is completed in the Golgi apparatus, from where it is delivered to the cell membrane. It was reported that the constitutive kinase activity of the FLT3 protein is linked to its inefficient folding and ER retention , leadingFLT3-ITD-mediated drug resistance of cytotoxic agents including cytarabine [FLT3-ITD-positive AML [Our data show that the anti-apoptotic Mcl-1 is increased at the protein level but not at the transcription level in MV4-11-R. The Mcl-1 protein has a short half-life and its stability can be controlled by post-translational regulation such as ubiquitination ,33,34. Itarabine . A furthtive AML ,38.TP53 mutations were reported to associate with poor prognosis in de novo AML [MYC, PCNA, EGFR, and EGR1; the D281G mutant shows an even greater transactivating activity than R248W in these studies [FLT3-ITD-positive AML.We observed the emergence of a D281G p53 mutant in MV4-11-R, in addition to the original R248W mutation in MV4-11-P. novo AML . D281G anovo AML . Previou studies ,42,43,44Our results showed that p53 phosphorylation at Ser15, Ser46, and Ser392 was increased in MV4-11-R. We reason that the increase in p53 phosphorylation is due to a higher amount of total p53 protein expressed in MV4-11-R. The Ser15 and Ser46 phosphorylation sites are both located in the N-terminal transactivation domains of p53. Although phosphorylation at these two sites enhances apoptosis triggered by wild-type p53 , Ser15 pProlonged drug exposure can result in selective survival in favor of certain pre-existing subclones within the heterogeneous cancer cell pool. It can also induce certain events including mutations, epigenetic changes, or non-genetic transcriptional variability in a limited number of cells . Our pyrTP53 was the most frequently mutated gene found in t-AML. An existing TP53 E286K mutant clone (2.5%) was identified at first relapse and expanded to more than 30% at second relapse, compatible with t-AML [TP53 mutations were significantly enriched in t-AML compared with de novo AML. These TP53 mutations were presented at low frequency before any chemotherapy or years before the diagnosis of t-AML [TP53 mutation is an important therapeutic target for chemoresistance and t-AML.Exposure to chemotherapy agents may lead to the development of therapy-related AML (t-AML) in cancer patients. th t-AML . Anotherof t-AML . These cNT5C2 mRNA. In vitro data showed that SAMHD1 decreased cytarabine sensitivity through its hydrolyzing activity to ara-CTP [SAMHD1 expression correlated with better response to cytarabine therapy [SAMHD1 expression between MV4-11-P and MV4-11-R cells, demonstrating SAMHD1 did not play important role in this resistance. In order to mimic cytarabine resistance evolved in AML patients, a number of studies were done using resistant AML cell lines established by prolonged cytarabine exposure. The resistant cell lines exhibit increased ERK and Akt activation compared to the parental cell lines ,52, as s ara-CTP . Clinica therapy ,56. Howe therapy . In the MDR1 gene and loss of histone methyltransferase EZH2 [MDR1 and MRP1 expression, and the two drugs are metabolized differently within the cells [Altered phenotypes were described in cytarabine-resistant cells derived from parental AML cells. It was shown that the resistant cells possess enhanced susceptibility to NK-mediated lysis  and incrase EZH2 ,60,61. Whe cells . Mechani50 data, we showed that cabozantinib, sorafenib, and MK2206 are as potent for MV4-11-R as for MV4-11-P. Using a mouse xenograft model, we demonstrated that cabozantinib effectively inhibits the FLT3-ITD pathway along with MV4-11-R-derived tumor growth. These observations strongly support the therapeutic potential of cabozantinib in combination with cytarabine to treat FLT3-ITD-positive AML. Our data revealed that Akt and ERK phosphorylation are significantly elevated in MV4-11-R. We speculate that the effectiveness of the anti-cancer drug correlates with its ability to inhibit Akt phosphorylation in MV4-11-R. The decreased sensitivity of MV4-11-R to CI-1040, idarubicin, and cytarabine may result from the failure of these drugs to inhibit Akt phosphorylation. On the other hand, our data suggest that the ERK phosphorylation level does not correlate with the decreased sensitivity of MV4-11-R to these anti-cancer drugs. We hypothesize that strong Akt phosphorylation, but not ERK phosphorylation, results in cytarabine resistance in MV4-11-R. According to the IC2. Cytarabine was purchased from the the Pharmacia & Upjohn Company LCC . To establish a cytarabine-resistant line, MV4-11 cells were grown in gradually increasing concentrations of cytarabine. Before each adjustment of cytarabine concentration, the growth status of cells was confirmed to reach the growth rate in the parental line using the trypan blue exclusion method. The established cell line that was named MV4-11-R and the parental line was termed MV4-11-P. Both MV4-11-P and MV4-11-R cells were authenticated with 16-marker short tandem repeat (STR) by the Food Industry Research and Development Institute, Hsinchu, Taiwan. The genetic profiles of these two cells were identical to the reported genetic profiles. MV4-11 cells were kindly provided by Yen-Chun Chen of the Industrial Technology Research Institute, Hsinchu, Taiwan, and were cultured in RPMI 1640 media supplemented with 10% fetal bovine serum at 37 °C in 5% CO® Aqueous One solution (20 μL in each well) at 37 °C for 2 h. Absorbance at 490 nm for each well was obtained. For each experimental set, a standard curve representing the relationship between the absorbance and the actual viable cell number was done in parallel. Cell viability was calculated as a percentage of the control .MV4-11-P or MV4-11-R cells were seeded in 96-well plates at 2500 cells/well. After drug treatment for 72 h, cells were incubated with CellTiter 96Chromosome analysis was performed as described previously . Briefly® Reagent  followed by DNase I treatment. Reverse transcription was performed using High-Capacity cDNA Reverse Transcription kits  according to the manufacturer’s instructions. qPCR was performed using synthesized cDNA and SYBR Green PCR Master Mix (Applied Biosystems) with initial denaturation at 95 °C for 10 min followed by 40 thermal cycles comprising 95 °C for 15 s and 60 °C for 1 min in an ABI 7500 Fast Real-Time PCR system (Applied Biosystems). Dissociation curves were obtained after the qPCR cycles to detect melting temperatures of the products by descending from 95 °C to 60 °C. HPRT expression was analyzed as the internal control. Relative target gene expression was calculated as 2–ΔCT where ΔCT = CT(target gene) − CHPRT)T(. The primers used for qPCR reactions are 5′-CCACCCCGCCCAAGAG-3′ (forward) and 5′-CCTTCCCTGCAGCGATGTTCCC-3′ (reverse) for DCK; 5′-CCCGTGAAGAGTTCCCTGTC-3′ (forward) and 5′-GTAGGGGGCTAGAAGGGTGA-3′ (reverse) for SAMHD1; 5′-TGCGCTGGAGCCGAATAC-3′ (forward) and 5′-CCCTACTTCCTTCATCCGGC-3′ (reverse) for NT5C2; 5′-TGCATCCTGAAAGCTGCGTA-3′ (forward) and 5′-GGCATTCTCTGGCTGTCACT-3′ (reverse) for CDA; 5′-GCAGCTGGAAGACAAATACACAAA-3′ (forward) and 5′-CCCCAACATCGTGCACATC-3′ (reverse) for MDR1, 5′-TGTTTCCAGCCGTGACT-3′ (forward) and 5′-CAGGCCACATGAATACAG-3′ (reverse) for ENT1; 5′-AGGTCAAGCTTTTCCGTGTACTG-3′ (forward) and 5′-GGACTTTCGTGTGCTCCTGA-3′ (reverse) for MRP; 5′-GCAATTTAGGTATGAAAGCCAGC-3′ (forward) and 5′-CTTTCAGCATTTTGACGGCAACC-3′ (reverse) for FLT3-ITD; 5′-AAGAGGCTGGGATGGGTTTGTG-3′ (forward) and 5′-TTGGTGGTGGTGGTGGTTGG-3′ (reverse) for Mcl-1; 5′-TGACACTGGCAAAACAATGCA-3′ (forward) and 5′-GGTCCTTTTCACCAGCAAGCT-3′ (reverse) for HPRT.Cellular RNAs were extracted using TRIzolFLT3-ITD mutation, PCR fragments were amplified with fluorescence-labelled primers, diluted with distilled water, and mixed with deionized formamide and GeneScan Size Standard (Applied Biosystems). Mixtures were electrophoresed on an ABI 3100 Genetic Analyzer (Applied Biosystems), and fluorescence signals were analyzed by GeneScan, Volume 3.1 software (Applied Biosystems). For DNA sequencing of TP53, we purified PCR products amplified from primers 5′-GCCAAGTCTGTGACTTGCACG-3′ (forward) and 5′-TCAGTCTGAGTCAGGCCCTTCT-3′ (reverse), and then sequenced with the primers 5′-GCGTGTGGAGTATTTGGATG-3′ using the BigDye terminator v3.1 Cycle Sequencing kit (Applied Biosystems) and a 3730xl DNA Analyzer (Applied Biosystems).The QIAamp DNA Mini kit  was used for cellular DNA extraction according to the manufacturer’s instructions. For GeneScan analysis of the PCRs for pyrosequencing were performed using cDNA and the primers 5′-TTATCCGAGTGGAAGGAAATTTG-3′ (forward) and 5′-TCTTCTTTGGCTGGGGAGAGG-3′ (reverse). PCR products were immobilized on streptavidin-coated beads, denatured, and mixed with sequencing primers 5′-TGGTGAGGATGGGCC-3′ and 5′-TCTTCCTCTGTGCGC-3′ for R248W and D281G, respectively. Sequencing reactions were performed using the PyroMark Q24 instrument (QIAGEN) and analyzed by the PyroMark Q24 software (QIAGEN).TM phosphatase inhibitor mixture (Roche Applied Science). Protein concentrations were determined using the Quick StartTM Bradford Protein Assay . Lysates containing equal amounts of protein were subjected to SDS-PAGE and transferred to PVDF membranes . Target proteins on the membranes were detected with specific primary antibodies and matching horseradish peroxidase-conjugated secondary antibodies and visualized using Western Lightning® Plus-ECL  and a LAS 4000 image reader . The primary antibodies used were as follows: anti-FLT3, anti-MEK1/2, anti-p-MEK1/2, and anti-p53 antibodies ; anti-p-FLT3, anti-ERK1/2, anti-p-ERK1/2, anti-Akt, anti-p-Akt, anti-p-STAT5, and anti-β-actin antibodies ; anti-CREB and anti-p-CREB antibodies ; anti-Mcl-1 and anti-GAPDH antibodies .Cultured cells were lysed in lysis buffer containing 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, protease inhibitor cocktail , and PhosStopCell lysates were analyzed using the Human Phospho-Kinase Array kit  and the Human Apoptosis Array kit (R&D Systems) according to the manufacturer’s instructions. Chemiluminescent signals were captured and analyzed by a LAS 4000 image reader (Fujifilm).6 MV4-11-R cells in 100 μL of 50% BD MatrigelTM Matrix . When the tumor size reached 200 mm3, mice were randomly grouped and orally administered cabozantinib-malate or vehicle by gavages. Body weights and tumor sizes were measured every 1–2 days after inoculation. When tumor volume reached 2000 mm3, animals were euthanized. Tumors were collected for Western blot analyses and immunohistochemical staining as described previously [Female BALB/c athymic (nu+/nu+) mice (6–8 weeks old) were obtained from the National Laboratory Animal Center, Taipei, Taiwan. The animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of National Taiwan University in accordance with the approved guidelines. The animal experimental protocol was modified from our previous report . Each moeviously . For immt-tests. Survival of mice was analyzed by Kaplan–Meier survival curves and log-rank tests. p values less than 0.05 were considered statistically significant.Quantitative data of mRNA expression, tumor sizes, and Ki-67-positive cells between experimental groups were analyzed by two-tailed Student’s FLT3-ITD-positive cell line, MV4-11-R. A number of major alterations have been identified in MV4-11-R including decreased glycosylation and phosphorylation of the FLT3-ITD protein, increased ERK, Akt, and p53 phosphorylation, accumulation of the Mcl-1 protein, and emergence of the D281G gain-of-function p53 mutation. The multi-kinase inhibitor cabozantinib was shown to effectively inhibit the growth of cytarabine-resistant FLT3-ITD-positive AML cells in vivo. We propose here that down-regulation of mutant p53 activity, down-regulation of the Mcl-1 protein, and inhibition of aberrant Akt phosphorylation can provide promising therapeutic strategies for combination treatment with chemotherapy in a subset of patients with FLT3-ITD-positive AML. In this study, we have successfully established a cytarabine-resistant