Several ion channels and pumps cooperate in a complex machinery that controls the influx, release, and efflux of Ca2+. Probably one of the most interesting and most complex players of this crosstalk is the Na+/Ca2+ exchanger, which represents the main Ca2+ efflux mechanism; however, under some circumstances, it can also bring Ca2+ into the cell. Therefore, the inhibition of the Na+/Ca2+ exchanger has emerged as one of the most promising possible pharmacological targets to increase Ca2+ levels, to decrease arrhythmogenic depolarizations, and to reduce excessive Ca2+ influx. In line with this, as a response to increasing demand, several more or less selective Na+/Ca2+ exchanger inhibitor compounds have been developed. In the past 20 years, several results have been published regarding the effect of Na+/Ca2+ exchanger inhibition under various circumstances, e.g., species, inhibitor compounds, and experimental conditions; however, the results are often controversial. Does selective Na+/Ca2+ exchanger inhibition have any future in clinical pharmacological practice? In this review, the experimental results of Na+/Ca2+ exchanger inhibition are summarized focusing on the data obtained by novel highly selective inhibitors.Life-long stable heart function requires a critical balance of intracellular Ca They contribute to the long-lasting plateau phase of the ventricular action potential, trigger the opening of the ryanodine receptors (RyRs), contribute to the feed-back regulation of Ca2+-influx, induce the activation of the contractile machinery of myocytes, and, finally, are involved in the regulation of many cardiac signal transduction pathways. Under physiological conditions, cardiac cells are in equilibrium, i.e., the entry and efflux of Ca2+ are equal on a long time scale. This is also the case in the diseased heart; however, equilibrium can be reached only at a higher cytosolic Ca2+ level. This leads to cardiac Ca2+ overload, which may be considered as adaptation to the compromised contractility, but also results in the impaired relaxation and development of life-threatening cardiac arrhythmias i, causing considerable changes in both the reverse and forward modes, and the former may contribute to frequency-dependent shortening of the action potential [2+ level in the subsarcolemmal space [Egan and Twist (1989) described NCX current as an inward current during the action potential, calculated from experimental results . In linerization . Using arization . Faber aotential . The Beral space . This seal space .CaL. The authors reported a small and brief outward current corresponding to phase 1 repolarization and a small inward current in the entire action potential. Both the reverse and forward mode operation of NCX increased when intracellular Na+ concentration was increased by activation of late sodium current [The first experimental evidence applying a relatively selective NCX inhibitor SEA-0400 (3 µM) was published by Bányász et al. in 2012 in guinea pig ventricular myocytes . They fo current . Horvath current .2+ extrusion from the intracellular space with concomitant suppression of the NCX-induced inward current during the action potential, which might tend to shorten the action potential  exerted non-selective behaviour, the interpretation of the results was difficult . Especia50 of the NCX block was 3.35 ± 0.82 µM, for forward mode, and 4.74 ± 0.69 µM for reverse mode operation [+ or Na+ currents; however, it decreased ICaL with EC50 of 3.6 µM. This means that the typically used 1 µM concentration causes approximately a 20% reduction in ICaL amplitude. Still, the submicromolar concentrations of SEA-0400 could be considered relatively selective for NCX [SEA-0400 phenoxy)-5-ethoxyaniline) was the first NCX inhibitor which was shown to exert improved selectivity. The ECperation . SEA-040 for NCX .CaL; however, a 20% blocking effect on IKr was identified. ORM-10103 equally inhibited both the reverse (EC50: 960 nM) and the forward (EC50: 780 nM) modes of NCX. Importantly, IK1, IKs, Ito, and Na/K pump were not influenced by 3 µM ORM-100103.ORM-10103, a novel compound with improved selectivity, was investigated in detail in 2013 . The maj50 values  [The next compound, ORM-10962, was further improved, displaying complete selectivity to NCX. ORM-10962 equally inhibited both the reverse and forward mode NCX currents with considerably lower EC: 55 nM) .2+ balance is crucial for the Ca2+ handling integrity; therefore, the Ca2+ influx and Ca2+ efflux must be equal, causing no Ca2+ gain or loss within beats [During ECC, a stable Cain beats .2+ largely increases and considerably facilitates the activity of the forward NCX [2+ removal by the NCX generates inward Na+ current that could lead to membrane depolarization causing triggered activity either during phase 2 and phase 3 of repolarization  or at resting membrane potential . These triggered activities can lead to the development of extrasystole and—in the presence of arrhythmogenic substrate—may cause life threatening arrhythmias. Additionally, the actual intracellular Na+ level also significantly alters the function of NCX. Intracellular Na+ concentration can be increased due to suppressed Na+/K+ ATPase activity, increased Na+/H+ function, or via enhanced late Na+ current during the action potential plateau. The increased Na+ level activates the reverse mode of NCX, causing net Ca2+ entry, which, in turn, facilitates the activity of the forward mode leading to development of triggered activity [2+ entry that causes secondary reduction in the forward mode) could be antiarrhythmic [In several diseases, the intracellular Caward NCX ,41. The activity ,41. Therrhythmic .2+-extrusion could increase the intracellular Ca2+ level, which is able to accelerate the Ca2+-dependent inactivation of the L-type Ca2+ channels to match the Ca2+-influx to the Ca2+ efflux. Therefore, presumably, NCX inhibition is able to set a new Ca2+ balance where NCX activity is reduced and intracellular Ca2+ is elevated, leading to a consequent increase in contractility.Another aspect of the selective NCX inhibition is the proposed positive inotropic effect. Considering that the contribution of the reverse mode during the action potential is restricted, NCX inhibition mainly prevailed on the forward mode. The decreased Ca2+ overload and DAD, alternans, depolarized membrane potential, shift in the refractory period, etc. Furthermore, arrhythmia experiments could also differ in experimental conditions. Accordingly, Ca2+ overload-related arrhythmias could be caused by Na+-induced Ca2+ overload following Na/K ATPase blockade, or may be due to increased sympathetic tone, hypokalaemia, etc. In this part of the review, the effects of selective NCX inhibition on elementary arrhythmogenic events are summarized. Since, among NCX inhibitors, only the SEA-0400 and ORM-compounds can be considered more or less selective, the next sections are restricted to summarizing the results obtained with these inhibitors.In most cases, cardiac arrhythmias are complex phenomena involving several cellular mechanisms at the same time, such as EAD, increased repolarization dispersion, Ca2+ or increased external Ca2+. The phenomenon was termed as “potentiels tardifs,” or late potentials. The first direct measurements of DADs were carried out in 1977 by Crenfield et al. [2+ content can be increased via increased sympathetic tone, or can develop as a consequence of several diseases, such as catecholaminergic polymorph tachycardia, hyperthyreosis, etc., when the cells are overloaded with Ca2+. Ca2+ overload increases Ca2+ reuptake to the SR leading to spontaneous diastolic Ca2+ releases. These events drive the forward mode of the Na+/Ca2+ exchanger, causing spontaneous depolarizations [The first observation of DAD-related events was reported by Segers in 1940 and 1947. In frog, turtle, and rabbit hearts, spontaneous activity was observed which could be facilitated by external Bad et al. . It was izations .+- and the consequent Ca2+ overload [+ gain was induced by oubain/strophantine administration in guinea pigs and dogs.Since NCX function is considered to be a crucial player of the DAD development, selective NCX inhibition was a promising candidate to suppress arrhythmias related to DADs . SEA-040overload ,46,47. I2+ releases, DADs, and extra beats following rapid pacing [2+ waves.Fujiwara et al. showed a close link between spontaneous Cad pacing . In thisAmong new NCX inhibitors, the effect of ORM-10103 was tested against DAD development . DAD was2+ releases were investigated in isolated dog ventricular myocytes during rest after a rapid pacing period. In the pacing-free period, several spontaneous Ca2+ releases were observed which were significantly suppressed by 10 µM ORM-10103. In this paper, the effect of strophantin on steady-state Ca2+ transients was also investigated: 1 µM strophantin markedly increased the amplitude of Ca2+ transients; however, when the cells were pre-treated with 10 µM ORM-10103, strophantin failed to increase their amplitude [In a further study, strophantin-induced spontaneous Camplitude .2+ transient [+ shifted the NCX reversal potential toward more negative values and increased the time for reverse mode operation.Similar results were obtained by using ORM-10962 . Dog rigransient ,49. The 2+ concentration and reduced forward mode NCX activity. Since, in these experiments, the DADs were evoked by Na/KATPase inhibition, it is possible that the increased intracellular Na+ facilitated the reverse mode activity of NCX, resulting in increasedCa2+ entry. This scenario is further supported by the fact that NCX inhibition prevented Ca2+ accumulation caused by strophantin, while the magnitude of the Ca2+ transient was unchanged [2+ releases via reverse mode NCX inhibition during Na+-induced Ca2+ overload.These results suggest that selective NCX inhibition effectively suppresses the occurrence of the DADs. Regarding the mechanism, it is feasible that DAD can be suppressed either directly, i.e., by inhibition of the forward mode, or indirectly via reduction of reverse mode leading to decreased intracellular Canchanged . Therefo2+ waves induced by rapid pacing in dog atrium were found to activate the forward mode of NCX and markedly depolarized the membrane. The application of SEA-0400 or ORM-10103 effectively abolished the Ca2+ waves [Ca2+ waves .+-induced Ca2+ overload, it seems feasible that NCX inhibition reduces DAD development, which is presumably the consequence of reverse mode block, and the concomitant decreased Ca2+ indirectly reduces the forward mode NCX. This effect resulted in decreased intracellular Ca2+ and reduced depolarizing events.It appears that selective NCX inhibition displays a beneficial effect against DAD formation. However, it is uncertain whether the reverse or the forward mode inhibition dominates the effect. Data show that, when DADs are the consequence of Na2+ current during a long-lasting action potential [+ current [+ channel inhibition and/or during slow pacing rate. Also, several diseases, such as chronic heart failure or long QT-syndrome, are also reported to be associated with the development of EADs [The term EAD indicates a spontaneous depolarizing event that occurs before the completion of repolarization. The classical proposed mechanism for EAD development is the reactivation of the L-type Caotential ; however current ,53, the  current , CaMKII, current  were als current . EADs ty of EADs . Torsade of EADs .+ current using aconitine, where SEA-0400 was unable to suppress ventricular tachycardia and triggered activity [In a guinea pig model, LQT3 was pharmacologically mimicked by the enhancement of late Naactivity . In anotactivity . A loweractivity .In Langendorff-perfused rabbit hearts with atrioventricular block, NCX inhibition by SEA-0400 did not suppress the incidence of dofetilide-mediated TdP .In contrast, in Langendorff-perfused rabbit hearts, LQT2 and LQT3 were pharmacologically evoked by sotalol or veratridine, respectively . The hea2. It was found that 3 and 10 µM ORM-10103 decreased the amplitude of EADs in a dose-dependent manner, indicating the role of forward mode NCX in the development of EAD. A further  possibility for the mechanism of action is that ORM increased the intracellular Ca2+ level, inducing faster inactivation of Ca2+ channels. In this case, if EADs were mediated by Ca2+ channels, reopening of the channel performing window Ca2+ current—as it was claimed previously in [2O2-induced EADs [The effect of NCX inhibition on EAD incidence was investigated also in canine right ventricular papillary muscle . EADs weously in —NCX inhiously in . In rabbced EADs .Kr inhibition, leading to enhanced EAD development and Tdp inducibility in both control and failing hearts. When ORM-10103 was applied, the number of premature beats was reduced; however, Tdp inducibility remained unchanged. It was found that ORM-10103 increased action potential duration and inhomogeneity in failing hearts. These findings are consistent with previous results by Farkas et al., where SEA-0400 failed to reduce Tdp incidence in rabbit hearts [Kr inhibitory action [Chang et al. investigated the antiarrhythmic effect of ORM-10103 in normal and failing rabbit hearts . QT-intet hearts . It is iy action , which cCaL, INaL, and INCX are known to contribute to EAD formation. It is conceivable that the participation of these currents in the EAD formation highly influences the effect of NCX inhibition. Conditions enabling a larger contribution of NCX to EAD development, such as higher cytosolic Ca2+ concentration, predict a more effective beneficial influence of selective NCX inhibition on EAD incidence.It seems possible that selective NCX inhibition has a limited effect against EAD development. However, this is probably not surprising since changes in several ion currents, such as IThe aerobic metabolism of the heart is seriously compromised during ischemia, which occurs typically after regional coronary artery occlusion. The concomitant decline in intracellular ATP level, intracellular acidosis, deliberation of reactive species, etc., may influence the vast majority of ion channels and transporters , leading+ concentration due to suppression of the Na+/K+ pump is a consequence of ATP depletion. This may facilitate the reverse mode operation of NCX, leading to net Ca2+ gain [It Is well-known that NCX has a key role in arrhythmogenesis related to ischemia/reperfusion. The rise of the intracellular Naa2+ gain ,69,70. Ta2+ gain ,72.In vivo studies on ischemia-/reperfusion-related arrhythmias obtained with NCX blockers yielded controversial results, since the majority of experiments failed to show any antiarrhythmic effect of SEA-0400 ,74,75. IIn Langendorff-perfused rat hearts, SEA-0400 improved the cardiac function and energy metabolism  but fail+ and lactate concentrations, but no glucose, combined with acidic pH and hypoxia. Intracellular Ca2+ level, action potentials, and cell viability were recorded in these experiments.The effect of ORM compounds in ischemia/reperfusion injury was investigated on both isolated cells and Langendorff-perfused hearts. Simulated ischemia was induced by a modified external solution containing unusually high K2+ level, decreased the amplitude of the Ca2+ transient, caused depolarization, shortening, and triangulation of action potentials, and ultimately cell death. An amount of 10 µM ORM-10103 prevented the rise of diastolic Ca2+ during both ischaemia and reperfusion  without any statistical difference between the results .+-induced Ca2+ load, it has a limited protective effect against other symptoms of ischemia/reperfusion injury. It is likely, therefore, that the antiarrhythmic effect observed against the Na+-induced Ca2+-overload may be attributable mainly to the reverse mode blockade of NCX. However, in a whole heart aspect, several other factors are present that could be unaffected by the NCX inhibitors, such as changes in repolarization, reduction in impulse propagation, depolarization of the resting membrane potential, etc.Although selective NCX inhibition clearly exerts beneficial effects against some pathophysiological components of the ischemia/reperfusion injury, such as the NaThe equilibrium potential of the NCX predicts that both outward and inward current could be developed during an action potential. However, under normal conditions, the reverse mode is suggested to be restricted in the first section of the action potential ; therefoMilberg et al. recorded monophasic action potentials from Langendorff-perfused rabbit hearts. When SEA-0400 was applied alone, the drug shortened action potentials within the range of 0.8 and 1.6 µM. Similarly, the QT interval was also shortened in this concentration range . SEA-040Tadros and Cheung reported no change in the duration of rat action potentials after the down-regulation of NCX . NamekatIn the study where ORM-10962 was used for NCX inhibition, the action potential duration was unaltered after administration of 1 µM of the compound . Slight An important additional effect of selective NCX inhibition is that it increased the effective refractory period . This efIn the study of Nagy et al., the Purkinje fibre and ventricular muscle action potentials were simultaneously recorded from a Purkinje-ventricular preparation using two microelectrodes, where the two preparations were connected . The appMilberg et al. demonstrated that the sotalol- or varatridine-induced dispersion between epicardial and endocardial monophasic action potentials was significantly reduced by the application of 1 µM SEA-0400 . This ef90 was unchanged when SAR296968, a novel, selective NCX inhibitor compound, was used. Similarly, action potential amplitude and resting potential remained unaltered after selective NCX inhibition  Tpromised ,95,96. S2+ and Vm mapping in control rabbits and with one-month myocardial infarct revealed that SEA-0400 suppressed the pacing-induced ventricular premature beats. SEA-0400 steepened the slope of the restitution curve and enhanced spatially discordant alternans. It was concluded that, in one-month infarct hearts, SEA-0400 suppresses pacing-induced ventricular extrasytoles but promotes the development of discordant alternans . It was found that 1 µM ORM-10962 significantly decreased the amplitude of both types of alternans, while the slope of the restitution curve remained unaltered. Furthermore, the ORM-10962 increased the refractory period of the action potential. The underlying mechanism of the “anti-alternans” effect was supposed to be a consequence of establishing a new balance in the Ca2+ homeostasis when SR Ca2+ loading is increased and the SR release refractoriness is reduced. These data also suggest that the main driver of the alternans is the intracellular Ca2+ rather than the slope of the restitution curve.In 2021, the effects of selective NCX inhibition on action potential duration and Ca2+ extrusion from the cells in each cycle; therefore, its selective inhibition was a promising novel therapeutic tool to modify intracellular Ca2+ handling and improve cardiac performance, especially in heart failure. The seminal work of Hobai et al. [2+ transient magnitude near to the normal level in canine failing hearts. However, the application of XIP has several limitations. Subsequently, several experimental approaches were performed by using novel inhibitors to address this issue.NCX has a crucial role in Cai et al.  demonstrRegarding the inotropic action of NCX inhibition, a species-dependent effect seems to be delineated: the results obtained from rat or mice are different from those recorded from larger mammals developing longer, plateau-forming action potentials. In order to better illustrate this discrepancy, the results have to be discussed according to the experimental animal applied.2+ transients [2+ current.In 2005, Tanaka et al. investigated the effects of 1 and 10 µM SEA-0400 in rats. An amount of 1 µM SEA-0400 caused about 25% inotropy in tissue preparations, and a similar increase was found in cell shortening and Caansients . Single 2+ transients and cell shortening were recorded in the absence and presence of SEA-0400 [2+ transient amplitude (by ~30%) were increased after selective NCX inhibition. Parallel with increasing the amplitude of the Ca2+ transient, the magnitude of L-type Ca2+ current decreased. This change could be the consequence of the Ca2+ current-inhibitory side effect of SEA-0400; however, additionally, some indirect effect of the increased intracellular Ca2+ also could reduce Ca2+ current by a Ca2+-dependent inactivation process [In 2007, Acsai et al. investigated the effect of selective NCX inhibition in isolated rat myocytes by using 0.3 µM SEA-0400. CaSEA-0400 . It was  process .2+ transients [2+ transient. Ozdemir et al. (2008) reported that 1 µM SEA-0400 facilitated cell shortening and decreased the rate of relaxation in murine myocytes [Similar results were obtained in 2008 by Farkas et al. . In thisansients . The autmyocytes . A contrmyocytes , was obtIn 2019, Primessnig et al. investigated the effect of ORM-11035 on heart failure with preserved ejection fraction in rats. It was found that chronic application of the compound markedly attenuated the cardiac remodeling and diastolic dysfunction .2+ content and, in 300 nM and in 3 µM, increased the developed tension in human atrial trabeculae.In 2022, Hegner et al. used a novel, selective NCX inhibitor SAR296968 on human atrial myocytes . It was Despite the mostly positive results regarding the inotropic effect of NCX inhibition in rats, studies using guinea pigs, rabbits, or dogs mainly reported that NCX inhibitors failed to influence contractility.2+ transients, cell shortening, and SR Ca2+ content in canine myocytes [In 2008, Farkas et al. reported no effect of 1 µM SEA-0400 on the maximal systolic pressure of the rabbit heart measured in a Langendorff-perfusion system . This stmyocytes   could exert a relatively high (~80%) effect; however, this could be markedly decreased under normal Ca2+ handling. Importantly, the relaxation of caffeine-induced Ca2+ transient was delayed by 1 µM SEA-0400, suggesting that NCX inhibition could be more pronounced when NCX operates with nearly its full capacity. In line with these results, Szentandrassy et al. (2008) also reported no alteration in the systolic and diastolic pressure and intracellular Ca2+ concentration in Langendorff-perfused guinea pig hearts after the application of 0.3 and 1 µM SEA-0400 [In spite of the results of current measurements, indicating that SEA-0400 effectively inhibited the NCX current, no change was found in the transient and cell shortening irrespective of the applied concentration. The results were explained by a CaSEA-0400 .2+-transient or the rate of cell shortening. When cells were pre-treated by 2 nM ATX-II (activator of late Na+ current), the magnitude of the Ca2+-transient was reduced by ORM-10103. Similar results were obtained when the Na/K pump was inhibited by strophantin prior to the application of ORM-10103 [A novel NCX inhibitor, ORM-10103, was also tested in canine cardiomyocytes. Under normal conditions, ORM-10103 failed to change the amplitude of the CaRM-10103 .2+ transient and cell shortening under normal conditions. In contrast, a positive inotropic effect was observed when intracellular Ca2+ was increased by forskolin prior to ORM administration [2+ handling, the effect of NCX-inhibition may be markedly less than 80%. In order to overcome this, NCX must be considerably burdened by Ca2+. In contrast, when reverse NCX was facilitated by a low (70 mM) external NaCl concentration, ORM-10962 reduced the amplitude of Ca2+ transients, indicating a reverse NCX-mediated Ca2+ loss.Kohajda et al. studied an improved NCX inhibitor, ORM-10962 (1 µM), in canine ventricular myocytes. In spite of the approximately 80% NCX inhibition in both the forward and reverse modes, ORM-10962 failed to influence the Castration . This re2+ transient and cell shortening in canine ventricular myocytes under normal conditions. Augmentation of the reverse mode NCX activity by low external Na+ decreased, while facilitation of the forward mode activity increased the effect of NCX inhibition on the amplitude of Ca2+ transients [Similar results were obtained in a study by Oravecz et al., where 1 µM ORM-10962 failed to influence the Caansients .2+ transient and also the Ca2+ content of the SR in left atrial myocytes but failed to alter the parameters of the Ca2+ transient and SR Ca2+ content in right atrial cells. This discrepancy could be attributable to the shift of NCX toward reverse mode in the case of right atrial cells. The NCX blockade ratio was found to be approximately 60% in both the left and right atriums [Jin et al. used 3 µM ORM-10103 in porcine atrial myocytes, where 11-deoxycorticosterone acetate was used to induce hypertension. In these cells, it was found that NCX inhibition increased the amplitude of Ca atriums .50 values  for NCX [Otsomaa et al. used a brand new ORM compound, ORM-11372, which exerted extremely low EC for NCX . It was Pelat et al., in 2021, by using a novel inhibitor, SAR340835, found that selective NCX inhibition improved the systolic function and sympathovagal balance in a dog heart failure model .Taking together inotropic data obtained from SEA-0400, ORM-10103/10962/11372, and SAR296968/340835 experiments, it seems likely that selective NCX inhibition shows a species-dependent effect. In experiments performed in rats ), primarily performed using SEA-0400, mainly positive inotropic effects were found. In contrast, when SEA-0400 or ORM compounds were applied in animal models having long lasting action potentials with a pronounced plateau, the vast majority of the results reported no effect of NCX inhibition.2+ is near to its peak or it is still high; thus, the Ca2+ extrusion function is boosted. In this case, NCX inhibition decreases the portion of active exchanger molecules, leading to exhaustion of the Ca2+ extrusion capacity of the exchanger that could be manifested in intracellular Ca2+ gain. In contrast, in dogs or rabbits, the plateau phase maintains the membrane potential at a depolarized level for a relatively long period of time where the speed of Ca2+ extrusion by the NCX is restricted. When the membrane potential drops to more negative values, and forward NCX is boosted, the intracellular Ca2+ is in strong decline due to SERCA function. Therefore, the lower intracellular Ca2+ may exhaust the NCX capacity less; thus, the NCX-inhibition may be “better tolerated” and unable to shift the Ca2+ balance toward higher cytosolic Ca2+ levels. However, this assumption could stand for only the compounds that have a limited NCX-inhibitory effect under normal Ca2+ cycling . It is +-induced Ca2+-overload. In this case, it is feasible that the antiarrhythmic effect is driven by the reverse mode inhibition, mediated by a reduction in cytosolic Ca2+ concentration which suppresses triggered potentials induced by the forward mode activity of NCX. The inhibition of NCX could also be promising to attenuate action potential duration and Ca2+-transient alternans. However, it is important to note that these ‘beneficial’ effects of NCX inhibition could have important side effects. For instance, reverse mode inhibition could lead to decreased Ca2+I; however, it may have a detrimental effect to the contraction. Action potential shortening and shifts in the Ca2+ handling attenuate alternans; however, they could facilitate the development of atrial fibrillation and increase the propensity of the heart to the development of triggered arrhythmias. These potential unwanted effects could limit the possible future clinical application of NCX inhibitors and require further studies on large animals.The data suggest that selective NCX inhibition could be a promising novel therapeutic tool against triggered arrhythmias, especially during Na2+ extrusion.In contrast, selective NCX inhibition seems to be ineffective to induce significant, clinically useful inotropy in ventricular myocardium of larger mammals, including humans. The lack of effect could be based on the peculiar time course of NCX current during the action potential and the relatively low degree of NCX blockade that leaves enough capacity for NCX to maintain normal Ca


The diagnosis of mixed phenotype acute leukemia (MPAL) with T/megakaryocyte or T/myeloid lineages accompanied by t is always a challenge. Therefore, multiple experimental methods are usually required to avoid misdiagnosis. In this report, we presented a rare case of MPAL with T/myeloid lineages accompanied by t and discussed the experience of differential diagnosis and our appreciation of the MPAL with T/megakaryocyte and T/myeloid lineages accompanied by t.A 31-year-old woman was admitted to our hospital due to recurrent fever for 20 days. Two distinct blast populations were detected by flow cytometry analysis: one population fulfills the immunophenotypic criteria for T-lymphoblastic leukemia, while the other population is highly suggestive of megakaryoblasts. These immunophenotypic features support the diagnosis of MPAL (T/megakaryocyte), which is rarely reported. Interestingly, a complex karyotype was detected afterward by cytogenetics with t, indicating a diagnosis of AML with t, a subset of which is also characterized by megakaryocytic markers such as CD41 and CD61. It seems that the second blast population detected by flow cytometry could not be classified into either diagnosis based on the morphology, immunophenotyping, and even cytogenetic findings, posing a real diagnostic problem because of the lack of clear-cut cytogenetic morphological defined criteria to distinguish between acute megakaryocytic leukemia and AML with t. Combining all of the examination data, this case was ultimately diagnosed as MPAL (T + My)-NOS with t through differential diagnosis. Before the cytogenetic results were available, the patient received an acute lymphoblastic leukemia (ALL) regimen for MPAL treatment, but the effect was unsatisfactory. After the diagnosis was clear, she received an AML-like regimen with azacitidine for 7 days and venetoclax for 14 days, and achieved complete morphological remission.MPAL with either T/megakaryocyte or T/myeloid lineages accompanied by t is rare, and it is difficult to make a clear diagnosis. Thus, comprehensive examinations, including bone marrow cell morphology, flow cytometry analysis, cytogenetics, and molecular analysis are recommended to avoid misdiagnosis. AML-like regimen including azacitidine and venetoclax may be effective for treating MPAL (T + My)-NOS with t. Ethical lot number: KT2020004-EC-2.Mixed phenotype acute leukemia (MPAL) is rare, accounting for 2–5% of all acute leukemia cases . It has bri+ CD38bri+ CD2+ CD34+ CD117+ HLA-DR+ CD33+ CD11b+ CD123+ CD56+ cCD3par+ CD13 par+ CD19 par+ and CD5− CD36− TdT− MPO− cCD79a− CD19− CD10− CD203c− CD235a− CD41− CD61− CD42b−, which fulfills the immunophenotypic criteria for T-lymphoblastic leukemia. These immunophenotypic features support the diagnosis of MPAL (T/megakaryocyte), which is rarely reported. Interestingly, a complex karyotype  examination identified a strong positivity (93%) of EVI-1 rearrangement  and AML with t.MPAL can contain distinct blast populations of different lineages or one population with multiple antigens of different lineages. This case fits into the former category as two atypical blast populations were detected by flow cytometry. As shown in Fig. AMKL or AML-M7 as defined by FAB classification is a rare subtype of AML . A limitMPAL-NOS T/megakaryocytic type is exceptionally rare. To date, there have been very few reports of B or T/megakaryocyte. Gupta et al. reported a rare case diagnosed as MPAL-NOS (T/megakaryocyte) with a complex karyotype . In theiOf note, megakaryocytes are not specific for AML-M7. Some other cases are also accompanied by increased or abnormal megakaryocytes, including patients with AML with t3;3) or invIt seems that the second blast population detected by flow cytometry could not be classified into either diagnosis based on the morphology and immunophenotyping data. So, could the cytogenetic finding of t3;3) do the job? The answer may be no. Although AMKL is not characterized by any specific chromosome abnormality, it may represent at least three separate disease entities. The first is pediatric patients with Down syndrome AML. The second is patients with t or other cytogenetic abnormalities involving 22q13. The third is the remaining patients with AMKL who can carry cytogenetic abnormalities in chromosome 3 [Monosomy 3, Inv;3 do the. However14q32/BCL11B rearrangement is identified in ~ 20–30% of T/myeloid MPAL, and is increasingly recognized as the cytogenetic hallmark of this entity. The new WHO classification has classified such cases as acute leukaemia of ambiguous lineage with BCL11B rearrangement for its importance . Given tMPAL with either T/megakaryocyte or T/myeloid lineages accompanied by t is rare, and it is difficult to make a clear diagnosis. Thus, comprehensive examinations, including bone marrow cell morphology, flow cytometry analysis, cytogenetics, and molecular analysis, are recommended to avoid misdiagnosis. AML-like regimen including azacitidine and venetoclax may be effective for treating MPAL (T + My)-NOS with t.


Cardiac calcium regulation plays a critical role in the cardiomyocyte and is often dysregulated in cardiovascular disease. Due to the limited availability of human cardiac tissue, calcium handling and its regulation have most commonly been studied in the context of animal models. hiPSC-CMs can provide unique insights into human physiology and pathophysiology, although a remaining limitation is the relative immaturity of these cells compared to adult cardiomyocytes Therefore, this field is rapidly developing techniques to improve the maturity of hiPSC-CMs, further establishing their place in cardiovascular research. This review briefly covers the basics of cardiomyocyte calcium cycling and hiPSC technology, and will provide a detailed description of our current understanding of calcium in hiPSC-CMs.Human induced pluripotent stem cell cardiomyocytes (hiPSC-CMs) are based on ground-breaking technology that has significantly impacted cardiovascular research. They provide a renewable source of human cardiomyocytes for a variety of applications including  This review focuses on the current understanding of Ca2+-handling proteins expression and function in hiPSC-CMs, and how this relates to existing knowledge from other model systems. Because this is a nascent and rapidly changing field with little standardization, much of this information is limited and scattered, so direct apples-to-apples comparisons are often difficult.This review briefly details the major mechanisms of cardiac Ca2+ cycling directly mediates cardiac muscle contraction and relaxation. Briefly, sarcolemmal depolarization triggers a Ca2+-induced Ca2+ release through opening and release of Ca2+ through the Cav1.2 subunit of the L-type Ca2+ channel (LTCC), which activates the release of Ca2+ stores from the sarcoplasmic reticulum (SR) through opening of the ryanodine receptor 2 (RyR2)  and the plasma membrane Ca2+ ATPase (PMCA) (2+ uniporter (MCU)  . Elevateo the SR , which iugh RyR2 . Ca2+ cae (PMCA) , and inter (MCU)   . In humant cells , highlig2+ cycling is physiologically regulated by the adrenergic system for the fight-or-flight response, through increases in Ca2+ release and uptake capabilities. SERCA is inhibited by the 52-aa transmembrane protein phospholamban (PLB), which lowers the binding affinity of Ca2+  is also expressed  binds PLB, enhancing its inhibition of SERCA2a, and it  is respoxpressed . SLN is turnover .2+ transport apparatus. In chronic heart failure, a state characterized by pathological chronic sympathetic activation, there is reduction in SERCA2a expression and activity, as well as an increase in the inhibitory function of PLB. These alterations in the heart failure state lead to increased intracellular cardiac Ca2+ and reduced SR Ca2+ stores, and consequently reduced systolic calcium concentrations . Although RaM allows for more rapid calcium influx, it is inactivated at calcium concentrations well below those seen in active cardiomyocytes and can take well over a minute to recover from inactivation. As a result, the likelihood of its involvement in beat-by-beat cardiac calcium regulation is slim (+/Ca2+-exchanger (mNCX), and although Na+-independent means of mitochondrial calcium efflux are more prominent in other tissues , their activity has been found to be relatively low in the heart  have been identified as the most essential auxiliary components for optimal differentiation of hiPSC-CMs , Wnt, Nodal/Activin A and bone morphogenic proteins (BMPs), served as the foundation for directed differentiation . It was iPSC-CMs .The increased utilization of hiPSC-CMs has also renewed the interest in understanding cardiac development. During heart development, cells undergo specification, morphogenesis, and lastly maturation to form an adult heart. Maturation is defined by a developmental program that leads to the conversion of cardiomyocyte gene expression, function, cell structure, and metabolism from an immature fetal to mature adult cardiomyocyte  Figure . While h2+ handling in hiPSC-CMs. To this end, Ca2+ transient parameters, assessed by real-time detection of fluorescent Ca2+ dyes and indicators, are often used as positive markers for this improved cellular physiology.Thus, the cardiovascular stem cell field is highly invested in harnessing our understanding of cardiovascular development to drive maturity enhanced maturity, and importantly more robust CaThe initial approach to maturation of hiPSC-CMs was to increase the duration of time from differentiation to mimic temporal maturation of the human cardiomyocyte . Charact2+ handling including metabolic, hormonal, electrical, and engineered heart tissues   , and supand NCX) . Specifiand NCX) . While Tin vivo , while control cells continued to display a negative FFR  have been used as markers to assess improved differentiation maturity  is known to arise from mutations to RyR2. CPVT1 is characterized by abnormal intracellular Ca2+ handling and in conjunction with increased catecholamine levels result in DADs and ventricular arrhythmias. It is unknown whether the arrhythmic activity in CPVT1 is due to SR Ca2+ leak following RyR2 phosphorylation or increased Ca2+ sensitivity leading to diastolic Ca2+ release. Numerous groups have utilized hiPSC-CM to study specific RyR2 mutations causing CPVT1, from cells directly collected from patients, or through gene editing to introduce known CPVT1 mutations.RyR2 is a well established locus for arrhythmogenic mutations, through perturbation of CaF2483I variant in RyR2 exhibited diminished Ca2+ release upon exposure to caffeine, indicating smaller stores of Ca2+, as well as a similarly diminished INCX , C-terminal (Q4201R), and central-domain (F42831). hiPSC-CMs from all three RYR2 variant lines had wandering sparks with abberrant focal Ca2+ release. The magnitude of Ca2+ leak was larger and SR content was larger in the F2483I and Q4201R variants than the R420Q mutant hiPSC-CMs. The increase in spark frequency and large leaks in the F2783I could be due to its position near the FK506-binding protein, that results in dissociation of the FK506-binding protein from RyR2 to result in larger leaks. The location of the mutation impacts the calcium induced calcium release gain, SR Ca2+ content, leakiness, and resposne to arrhythmic medications  o hiPSC-CMs led to the elimination of the whole cell Ca2+ transient, which suggests that Ca2+ entry via the LTCC is important for generation of the whole cell transient in hiPSC-CMs , or Timothy Syndrome, is caused by a missense mutation in iPSC-CMs .2+ current inactivation and alterations to intracellular Ca2+ handling as evidenced by Ca2+ transients with larger and more inconsistent amplitudes as well as longer durations. LQTS hiPSC-CM, also showed a prolonged action potential and DADs. The LQTS8 electrical and Ca2+ handling abnormalities could be ameliorated by roscovitine, a small molecule, that increases voltage-dependent inactivation of CaV1.2.  and cytoplasmic Ca2+ buffer, namely cardiac troponin C, will be discussed including the impact of dysregulated Ca2+ buffering and insights from hiPSC-CM disease modeling.As outlined in prior sections, Caomyocyte , and a mytoplasm . In this2+ binding protein, that is the major cardiac SR Ca2+ buffer  , a high-capacity, low-affinity Ca+ buffer . The abi (CPVT2) . In bothherapies .2+ transient kinetics , myosin heavy chain (MYH7), and cardiac troponin T mutations (CNNT). . .2+ signaling. Cardiac troponin T, encoded by TNNT2, is a common DCM mutation that has altered Ca2+ handling and response to adrenergic signaling  exhibited similar arrhythmic Ca2+ activity and responses to isoproterenol and propranolol as seen in the DMD-hiPSC-CMs derived from reprogrammed patient cells  biosensors for SERCA2a which have typically been utilized in non-cardiac cells, can inform the field about the structure and function of these calcium handling components at baseline and in disease states. Additionally, hiPSC-CMs that express FRET biosensors for SERCA2a can be used for high-throughput drug screening to identify novel therapeutics that could be used to increase SERCA2a activity, which is reduced in heart failure states . These hSimilarly, other biosensors can be utilized in hiPSC-CMs to understand the contribution of mitochondrial calcium in the context of cytosolic calcium at baseline and in disease states, which is not currently well established .Gene editing hiPSC-CMs with CRISPR/Cas9 and newer precise editing methods can be utilized for both basic investigation and translational development, by assessing the impact of mutations in various calcium handling genes and their regulators, systematically assessing novel therapies, and for precision medicine to determine if specific mutations result in a phenotype. Genetic engineering strategies have great potential to help elucidate mechanisms of early cardiac calcium regulation and is an exciting area for the field.


Introduction: Ca2+ levels in adult skeletal muscle fibers are mainly controlled by excitation-contraction (EC) coupling, a mechanism that translates action potentials in release of Ca2+ from the sarcoplasmic reticulum (SR) release channels, i.e. the ryanodine receptors type-1 (RyR1). Calsequestrin (Casq) is a protein that binds large amounts of Ca2+ in the lumen of the SR terminal cisternae, near sites of Ca2+ release. There is general agreement that Casq is not only important for the SR ability to store Ca2+, but also for modulating the opening probability of the RyR Ca2+ release channels.The initial studies: About 20 years ago we generated a mouse model lacking Casq1 (Casq1-null mice), the isoform predominantly expressed in adult fast twitch skeletal muscle. While the knockout was not lethal as expected, lack of Casq1 caused a striking remodeling of membranes of SR and of transverse tubules (TTs), and mitochondrial damage. Functionally, CASQ1-knockout resulted in reduced SR Ca2+ content, smaller Ca2+ transients, and severe SR depletion during repetitive stimulation.The myopathic phenotype of Casq1-null mice: After the initial studies, we discovered that Casq1-null mice were prone to sudden death when exposed to halogenated anaesthetics, heat and even strenuous exercise. These syndromes are similar to human malignant hyperthermia susceptibility (MHS) and environmental-exertional heat stroke (HS). We learned that mechanisms underlying these syndromes involved excessive SR Ca2+ leak and excessive production of oxidative species: indeed, mortality and mitochondrial damage were significantly prevented by administration of antioxidants and reduction of oxidative stress. Though, how Casq1-null mice could survive without the most important SR Ca2+ binding protein was a puzzling issue that was not solved.Unravelling the mystery: The mystery was finally solved in 2020, when we discovered that in Casq1-null mice the SR undergoes adaptations that result in constitutively active store-operated Ca2+ entry (SOCE). SOCE is a mechanism that allows skeletal fibers to use external Ca2+ when SR stores are depleted. The post-natal compensatory mechanism that allows Casq1-null mice to survive involves the assembly of new SR-TT junctions (named Ca2+ entry units) containing Stim1 and Orai1, the two proteins that mediate SOCE. Calsequestrin, a member of the macromolecular complex that regulates EC coupling. Calcium ions (Ca2+) are powerful second messengers in all cell types. The electrochemical gradient existing between the cytoplasm and the extracellular space or intracellular stores  allows release channels to generate elevations in cytoplasmic Ca2+ concentrations (transient or prolonged) that may control gene transcription, cellular differentiation, communication between different intracellular organelles, cellular metabolism, etc. (2+ ([Ca2+]i) regulate contractile function and are generally caused by a large and rapid Ca2+ release from internal stores, i.e., sarcoplasmic reticulum (SR), that follows the depolarization of exterior membranes and that may be supplemented, especially in cardiac and smooth muscle, by Ca2+ entry from the extracellular space. The mechanism that links depolarization of the sarcolemma to Ca2+ release, known as excitation-contraction (EC) coupling (2+ release units (CRUs). CRUs are formed by the association of two separate and well-organized membrane systems that come in close contact with one another  also known as CaV1.1, and the SR Ca2+ release channel . DHPRs and RyRs are the two key elements in the EC coupling machinery , are clustered in ordered arrays forming usually double rows in the junctional face of SR  pumps. This is especially important in fast-twitch fibers, where the amount of Ca2+ cycled in and out of the SR is greater than in slow fibers  Elegans wall muscles (which express a single Casq isoform) and in mammalian striated muscles suggested that Casq is not absolutely essential for muscle function  proved to be (C. Elegans (J. Physiol. (London). The absence of Casq1 was confirmed by both WB analysis and immunocytochemistry in extensor digitorum longus (EDL) and Soleus muscle, respectively, a predominantly-fast and a predominantly-slow twitch muscle  in these multi-layered CRUs were not organized in two rows as in normal triads but formed by multiple rows similar the arrays of RyR in dyads of cardiomyocytes  the SR terminal cisternae is filled with an electron-dense matrix (anchored to the SR membrane by thin strands) and proposed that this matrix was polymerized Casq ; b) the 2+ , which in turn would trigger compensatory mechanisms aiming to increase junctional SR volume and/or the number of release sites (i.e. more RyR1 channels available for Ca2+ release). Multilayered CRUs do not form in Soleus fibers even when both Casq1 and 2 were missing   promote mitochondriogenesis to provide additional energy and/or also cytosolic Ca2+ buffering capacity: the volume of mitochondria in Casq1-null fibers was indeed increased (2+ entry that could damage mitochondria: studies conducted in single fibers from double Casq-null mice - which lack both Casq1 and 2 (dCasq-null) - revealed that the mitochondrial [Ca2+]free at rest was higher compared to WT  , which increased , possibled to WT .2+ and also of a redox imbalance. Indeed, the decrease in GSH/GSSG ratio and increase in mSOF activity in muscle from Casq1-null mice were both indicative of increased oxidative stress , leading to a dangerous feed-forward mechanism that may underline mitochondrial and cellular damage. Membrane structures of the cells can be affected by increased oxidative stress which is known to cause lipid peroxidation , Casq1-null fibers were still functionally impaired. Studies in whole muscles showed that Casq1 ablation caused compromised Ca2+ release and reuptake by the SR, i.e. prolonged time-to-peak and half-relaxation time. Both parameters were more pronounced in EDL than in Soleus (2+ efflux from the SR in isolated single flexor digitorum brevis (FDB) fibers  greatly reduced amount of Ca2+ stored in the SR, as shown by application of 20 mM caffeine in permeabilized fibers.n Soleus , a fact ) fibers  revealed2+ handling in fibers lacking Casq1. Using Ca2+dyes of new generation that allowed independent measurements of [Ca2+]free in myoplasm and SR lumen:2+ during repetitive EC coupling activation. The authors also underlined the fact that in Casq1-null fibers the intermediate step of reuptake, directly dependent on the SR [Ca2+]free, is the mostly affected, both in terms of amplitude and rate constant (a) Canato and coll. (2010) demonstrated that CASQ1-knockout results in deep SR depletion under high-frequency stimulation. These findings were explained by the absence of Casq1 in proximity of release sites which would lower the amount of releasable Caconstant .2+ efflux from the SR in presence or absence of Casq , they stated that: a) E is directly proportional to SR Ca2+ permeability and inversely to its Ca2+ buffering capacity; and b) E starts low and increases progressively as the SR is depleted in WT fibers, while E starts high and decreases upon SR Ca2+ depletion in dCasq-null fibers.b) Royer and coll. (2010), using fibers from dCasq-null mice (lacking both Casq1 and 2), while confirming SR depletion in dCasq-null fibers, added elegant information regarding the rate-velocity of Ca of Casq . In theiA few years later two other papers were published and added additional information toward the full understanding of the mechanisms underlying CaStudies of contraction kinetics in dCasq-null mice were also used to investigate possible differences between fast- and slow-twitch muscles . Analysi2+ transient. The corrected  functional parameters were ascribed only to Casq1 re-introduction, as all other SR proteins that could participate in Ca2+ homeostasis did not vary their expression level after re-expression of exogenous Casq1 , also the functional parameter of Caus Casq1 .Increased spontaneous mortality rate in Casq1-null mice. During the years that brought to the first publication in J. Physiol. , which is caused by excessive SR Ca2+ leak , the gold standard protocol used to diagnose MH in humans (1984). Muscles of Casq1-null mice exhibited sensitivity to both temperature and caffeine and developed contractures more easily than muscles from WT mice  by mitochondria, which in turn would exacerbate SR Ca2+ leak via RyR1 nitrosylation (in-vivo in Casq1-null mice. N-acetylcysteine (NAC) and Trolox were respectively administrated in drinking water or by intraperitoneal injection. Both treatments were quite successful in reducing the mortality rate of Casq1-null mice in response to exposure to heat  and halothane : from about ∼80% to only ∼20%. Animals treated with antioxidant also showed a decreased rise in core temperature during the heat challenge and a delayed onset of lethal crises (+ and Ca2+ concentrations); b) rescued structural alterations observed in Casq1-null mice at rest, reducing mitochondrial damage , and finally lowered reduced/oxidized glutathione ratio (GSH/GSSG) in comparison to muscle of untreated Casq1-null mice. As SOD-1 is one of the main antioxidant enzymes  activity after the treatment with NAC in dissociated FDB muscle fibers : mSOF fr enzymes , 3-NT ar enzymes , and GSH enzymes .2+ levels, the effect of NAC treatment on Ca2+ transients was also tested directly in isolated FDB fibers loaded with a ratiometric Ca2+ dye in experiments at rest and during prolonged high-frequency stimulation   .(a) In the first study to investigate the gender difference in the phenotype of Casq1-null mice , we changed the hormonal profile of mice . Many st(b) In the second paper published in 2018, as aerobic training is known to increase endogenous antioxidant protection , we subjIn two later studies published between 2017 and 2018, additional evidence pointing to a central role of oxidative stress in HS crises was collected .(a) In tIn short, both estrogen administration  and aeroExertional stress is also a factor of risk for hyperthermic crises in Casq1-null mice. Humans experience hyperthermic crises with very similar symptoms, either as a result of the administration of volatile anesthetics , or when exposed to environmental heat or to intense physical/emotional stress   . Some exonal HS) ; b) MH ponal HS) ; c) lifeonal HS) .Y522S/WT and Casq1-null mice to an exertional-stress (ES) protocol, i.e. incremental running on a treadmill placed in an environmental chamber in which temperature and humidity were kept at 34°C and 40% respectively .(a) Lethal crises occurred in about 80% of both RYR1(b) Pretreatment of susceptible mice 60min prior the ES protocol with a single injection of Azumolene  was effective in preventing exertional HS: the mortality rate was indeed almost completely abolished in both mouse models, and also core temperature was reduced in pre-treated animals.2+ levels during exposure to increasing [caffeine].c) Azumolene reduced also muscle damage during ES protocol, and normalized IVCT and intracellular Ca2+ release , and even more following ES; though, pretreatment with Azumolene was effective in controlling the increase in 3-NT levels during exposure to the ES protocol.(d) Finally, as in MH crisis triggered by either exposure to halothane and heat ROS and RNS had a pivotal role in promoting a feed-forward mechanism that led to excessive SR Ca release , we meas release . MusclesWhile susceptibility to anesthetics and environmental heat was tested between 2006 and 2009 in knock-in mice carrying RYR1 mutations linked to MH in humans  and in Cectively . We alsoThe data collected in this study  suggeste2+ entry in skeletal muscle fibersThe role of Store Operated Ca. Ca2+ homeostasis in skeletal fibers is the result of a complex interplay between several mechanisms: release of Ca2+ by EC coupling, Ca2+ re-uptake by the SR, Ca2+ binding to sarcomeric and cytoplasmic proteins , mitochondrial Ca2+ uptake, etc. (2+ in the extracellular space via plasma membrane Ca2+ ATP-ases (PMCA). In turn, this may result in a reduction of the total amount of Ca2+ stored in the SR, which is one the main mechanism underlying muscle fatigue  in excitable and non-excitable cells is known to be the trigger for the activation of store-operated Ca2+ entry (SOCE). SOCE is a ubiquitous Ca2+ influx mechanism in which Stim1 , a Ca2+ sensor in the lumen of the ER/SR (2+ release-activated Ca2+ (CRAC) channel of external membranes  (SOCE activity in muscle is important to limit fatigue during sustained and repetitive activity . As SOCEs (CEUs) . These js (CEUs) . We demo2+, which is lost when extracellular Ca2+ is removed or Ca2+ entry blocked by supplementation in the extracellular solution with SOCE blockers (BTP-2 and 2-APB)  accelerated rate of Mn2+ quench, the gold standard technique used to assess SOCE function (Assembly of CEUs during exercise have been correlated with: a) increased fatigue resistance in presence of external Cad 2-APB) ; b) redufunction . We alsofunction .2+ Entry UnitsCasq1-null fibers contain constitutively assembled Ca. As Casq was considered one of the three key proteins on EC coupling (together with RyR1 and DHPR), when we planned to generate Casq1-null mice we expected a lethal phenotype. We were not expecting that muscle fibers could function in absence of Casq1, yet surprisingly mice were alive and developed quite normally to adulthood . As detailed above, EM allowed us to discover that lack of Casq1 induced morphological adaptations to sites of EC coupling: CRUs were formed by multiple SR-TT-SR junctions and fibers contained an almost doubled amount of RyR1  contained preassembled junctions between SR-stacks and TT extensions within the I band virtually identical to the CEUs formed in WT mice during acute exercise  increased SR refilling during repetitive stimulation, which helped to normalize Ca2+ transients .Compelling evidence published in exercise . Quantit2+ influx  and verified the protective effect of antioxidants and estrogens  - in an apparently unrelated project - allowed us to revisit some structural modifications induced by ablation of Casq1 , requires the presence of extracellular Caeactions . Hence, l stress . One puz


Determining the mechanisms, traits, and pathways that regulate microbial transformation of natural organic matter (NOM) is critical to informing our understanding of the microbial impacts on the global carbon cycle. The capillary fringe of subsurface soils is a highly dynamic environment that remains poorly understood. Characterization of organo-mineral chemistry combined with a nuanced understanding of microbial community composition and function is necessary to understand microbial impacts on NOM speciation in the capillary fringe. We present a critical review of the popular analytical and omics techniques used for characterizing complex carbon transformation by microbial communities and focus on how complementary information obtained from the different techniques enable us to connect chemical signatures with microbial genes and pathways. This holistic approach offers a way forward for the comprehensive characterization of the formation, transformation, and mineralization of terrestrial NOM as influenced by microbial communities. Yet, thA holistic, systems biology approach, defined here as coupling NOM chemistry with microbial community omics, provides an important avenue to connect microbial pathways to carbon transformations and environmental conditions. Integration and synthesis of all the necessary analytical approaches requires expertise in many different fields, including physical chemistry, organic chemistry, microbial physiology, and bioinformatics, among others. Fortunately, the increasing application of state-of-the-art instruments to probe NOM chemistry, related edaphic factors, and microbial genomic and metabolic data provide opportunities to examine the role of microbial community transformations of NOM on terrestrial subsurface carbon dynamics. However, there are still missed opportunities in the employment of these analytical techniques to collect data in a continuous or non-destructive manner .Here, we review several state-of-the-art analytical tools used to explore the relationship between microbial community composition and function and NOM transformations by highlighting key technologies and frameworks related to NOM in both dissolved and mineral-bound forms. We then discuss the necessary development of novel tools for performing non-destructive experiments that capture microbial impacts on carbon cycling in the capillary fringe of the terrestrial subsurface and other terrestrial/aqueous interfaces.Establishing mechanistic links between microbial community function and NOM transformation requires a sound understanding of NOM structure and composition. NOM exists as a heterogenous mix of compounds with a wide variety of sizes, chemical compositions, structures, functional groups, and polyelectrolytic characteristics that govern unique reactivities . These rFor soil samples, NOM must be separated from the mineral phase using solvents and extraction methods that vary in their effective isolation of defined mineral-NOM fractions . NOM chaGeneral patterns on the bioavailability and age of the compounds from these extractions can be made, though estimates may vary depending on the environment from which the samples were collected from. In soil, SOM from organo-metal complexes has been measured to be younger than organic matter extracted from more crystalline mineral phases . Furthervia electrospray ionization or in combination with other complementary ionization techniques (Fourier transform ion cyclotron mass spectrometry (FTICR-MS) is a type of ultra-high-resolution mass spectrometry that can detect and assess the elemental formulas of hundreds to thousands of compounds in NOM . To emplchniques . Detectichniques , and thechniques . The verFTICR-MS has been successfully applied to NOM characterization in several terrestrial environments. FTICR-MS metabolite profiling has been used to identify origins of soil NOM, particularly as a result of bacterial transformations , and was13C/12C. There are several methods to direct samples into the IRMS such as gas chromatographs or elemental analyzers. Elemental analyzers in combination with IRMS (EA-IRMS) produces representative data of an average isotopic signal of the sample (13C) of NOM, which is measured by elemental analysis – isotope ratio mass spectrometry (EA-IRMS), provides insight into the source, composition, and turnover of organic carbon (OC) in soils and sediment. The δ13C of OC in soils has been linked to associated plant δ13C signatures. For example, soil 0-30 m predominantly reflects the original isotopic signal of the former vegetation (13C(OC) of subsurface rhizosphere soil is usually higher than that of surface soil, as the δ13C of root material is generally higher than that of the above ground biomass, such as leaves (13C(OC) variations in soils. Microbes, invertebrates, and other soil fauna are typically enriched in δ13C compared to source-substrates, and their activities and biomass may also affect the turnover and δ13C of soil OC  can be indicative of microbial preferences for 12C vs. 13C in decomposing substrates, which in turn increases the 13C/12C of residual OC fractions (13C(OC) in soil and sediment tends to increase with depth until it reaches a maximum value at which point it approaches an asymptote.Isotope ratio mass spectrometers (IRMS) enable the measurement of variations between isotopic ratios, such as e sample . Stable getation , and thes leaves . However soil OC . Thus, tractions . TherefoIt is important to note that, although MS techniques provide high resolution data on the diversity of compounds found in NOM, there are some limitations. MS techniques provide mostly qualitative and semi-quantitative information on the composition of NOM. For example, in EA-IRMS, the data represent an average of the isotopic ratio and is not able to differentiate the contribution of each isotope to the total, making it largely a qualitative method . The rel1H, 13C, 15N, and 31P with minimal disruption of the sample. Several studies have utilized NMR to determine NOM composition of solid, gel, or solution-state samples (Nuclear magnetic resonance (NMR) characterizes NOM in soils and sediments quantitatively. This technique can focus on significant atoms within NOM, such as  samples . Simpson samples , with si samples . Similar1H atoms before using 13C or 15N to concentrate on specific chemical groups within the same sample. This application is critical for multidimensional experiments, such as Heteronuclear Single Quantum Coherence or Heteronuclear Multiple Quantum Coherence (HSQC/HMQC) that use cross-peaks of two atoms (such as 1H connected to either 13C or 15N) to provide more information of the specific chemical groups in NOM. Larger macro molecules or aggregated NOM can be characterized via Diffusion-edited 1H NMR (DE) that takes advantage of molecular self-diffusion in a sample solution  has proven useful in characterizing the chemical quality of NOM . FluoresSeveral studies have utilized EEMs in groundwater and terrestrial sediment NOM characterization in combination with other physicochemical analyses to draw connections among NOM qualities and environmental conditions . Huang eOne of the advantages of fluorescence technology is its high sensitivity and selectivity . FluoresFourier transform infrared spectroscopy (FTIR) is a technique that analyzes the vibrations of molecular bonds at infrared wavelengths to understand the molecular composition of compounds. FTIR can be used to identify organic matter functional groups because each spectrum is the result of unique absorption bands of different molecular bonds . Further−1 indicated the presence of phenolic, carboxylic groups, and amides, suggesting a high degree of stabilization of microbial-derived components that could be instrumental in forming stable mineral-organo complexes  is an element-specific technique that yields electronic and structural information about the element of interest . Air-dried samples can be analyzed by XANES, and this minimally disruptive sample preparation makes this analytical technique increasingly popular. An average XANES spectrum is produced by calculating the weighted sum of many reproducible spectra containing intense resonance features representing chemical attributes  of all compounds in a sample at a specific energy edge . There a+ or O−) to excite the sample surface in order to produce secondary ions (+)]. NanoSIMS imaging, can detect secondary ions to a lateral resolution of up to 50 nm  can be an effective way to address critical characteristics of soil microscale processes, such as the source of mineral associated NOM, relative importance of biotic and abiotic processes in NOM stabilization in soils, and spatial variance in these processes . NanoSIMary ions . These sto 50 nm . Althougto 50 nm .in situ information on the molecular class and elemental quantity of mineral-associated NOM. When combined with isotope labeling, STXM/NEXAFS and NanoSIMS imaging can be used to infer the molecular and spatial fate of the added labeled material in a mineral matrix, and can contribute to a mechanistic understanding of sorption, occlusion, and decomposition processes that operate at fine spatial scales in natural environments. Applied in sequence to the same regions of interest, NanoSIMS is used to determine the spatial fate of the isotopic label while STXM/NEXAFS is used to determine the chemical transformation of the label. Isotopic data are expressed in atom percent excess (APE), with higher APE representing higher isotopic enrichment. Similar to bulk XANES, linear combination fitting of the average C and N NEXAFS spectra using reference helps to identify the molecular composition of NOM while simultaneously providing its spatial distribution with other NOM moieties. This technique has been successfully used to look at microbial transformations of organic residues from root exudates in natural samples (Far more uncommon is the application of NanoSIMS in concert with Scanning Transmission X-ray Microscopy (STXM) coupled with Near Edge X-ray Absorption Fine Structure spectroscopy NEXAFS; . In STXM samples . OverallThe final tool to investigate surface chemistry of soil and sediment particles we will highlight here is X-ray photoelectronic spectroscopy. Within a vacuum, samples are mounted on holders and particles from soils or sediments can be irradiated with X-rays, which causes photoelectron emissions of the atoms on the top 2–10 nm of the surfaces . This alSubsurface environments contain over half of all global microorganisms, yet less than 8% of available 16S rRNA gene sequences have been derived from subsurface organisms and even fewer are represented by complete genomes, severely limiting our capacity to understand the interconnected microbial metabolic processes that drive carbon transformations . TherefoThe connections between microbial community function and the dynamic fluctuations in capillary fringe water content, oxygen availability, and substrates are difficult to identify without merging analytical chemistry and an omics-level perspective . These iMetagenomics provides an opportunity to understand the functional potential of difficult-to-cultivate microorganisms and can illuminate potential interactions among microbial community members that drive biogeochemical cycles of carbon in subsurface environments . Genome-In a multi-omics study that also employed FTICR-MS to determine chemical diversity of sediment NOM, Idiomarina and Alteromonas spp., relative to an unamended control. These results indicate taxa-specific resource partitioning of carbon sources that have downstream consequences for succession, metabolic pathways, and the carbon cycle. One application of this type of analysis in the subsurface is to examine relationships between specific organic matter compounds in various redox conditions to identify consequences to microbial community transcription. Such a study would provide a glimpse of how microbial community activity changes in response to redox-related impacts on carbon resources. In fungal microbiology, Transcriptomics can be used to examine the pattern of gene expression during monitored physiological activities and in response to environmental conditions . There aGeobacter cells proliferated while middle stages, were characterized by an increased carbon flux through respiratory pathways and high abundance of related proteins for energy generation proteomics can be used to characterize all proteins expressed at a given time point to understand how microbial isolates and communities respond physiologically to the environment . Proteinneration . Here, Wneration . Althoug13C or 15N) to track incorporation into different biomolecules  in a series of microcosm studies. Kieft et al. found that although taxonomy remained the same throughout the microcosms, there were strong differences in protein labeling that were indicative of differentiated carbon use capabilities. Bacteroidetes preferentially increased ribosomal protein expression in response to DOM from diatom lysates, and many newly synthesized proteins in response to diatom lysates were associated with degradation of carbon-rich high molecular weight DOM  and various mass spectrometry techniques (including FTICR-MS) to investigate the biological transformation of freshwater NOM by a Janthinobacterium isolate. Similarly, EEMS was applied to analyze the composition of dissolved organic carbon (DOC) and the impact of various recharge waters on microbial respiration in a shallow aquifer. Metabolomics, the final member of the -omics analysis suite, involves the comprehensive profiling of the compendium of small molecules in a biological sample . Metabolin situ data.The development of omics tools enabled unprecedented abilities to unravel microbial community responses and feedbacks to environmental conditions. Metagenomics is ideal for capturing the genomic capabilities of microbial communities , particuin-situ experimental approaches is paramount for linking microbial activity to terrestrial biogeochemistry.While the current approaches that integrate analytical chemistry with omics tools have advanced our understanding of microbial NOM transformation in many environments, one primary obstacle with most of these analytical approaches as applied to the terrestrial subsurface is that they are destructive, e.g., samples must be extracted or experimental replicates sacrificed to obtain data for a specific timepoint during the course of an experiment. The further development of non-destructive and continuous, 13C/15N-labeled organic compounds) coupled to trace-gas analysis have a long history of use in soil biogeochemistry and microbiology  will prompt clear changes in the activity of different pathways, thus strengthening the signal:noise ratio. In addition to redox dynamics, mVCs can also vary based on environmental parameters including temperature, humidity, and pH , a Science Focus Area Program at Lawrence Berkeley National Laboratory is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Biological & Environmental Research under contract number DE-AC02-05CH11231 and the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research, Early Career Research Program to NB (#FP00005182).This work was supported by ENIGMA – Ecosystems and Networks Integrated with Genes and Molecular Assemblies (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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Both NCX1 and TRPC1 play critical roles in maintaining cytosolic free Ca2+ ([Ca2+]cyt) homeostasis in mammalian cells. Although either TRPC1 channel or Ca2+ entry mode of NCX1 is implicated in some tumorigenesis, it has not been explored if a coordination of NCX1 and TRPC1 involves in the pathogenesis of H. pylori-associated human gastric cancer (GC). Here we found the protein expression of NCX1 was significantly enhanced in human GC specimens, which correlated with tumor progression and poor survival in GC patients. TRPC1 and NCX1 were parallelly enhanced, co-localized and bound in human GC cells. By a functional coupling, TRPC1 drives NCX1 to the Ca2+ entry mode, raising [Ca2+]cyt in GC cells. Moreover, CaCl2, H. pylori and their virulence factors all enhanced expressions and activities of NCX1 and TRPC1, and evoked aberrant Ca2+ entry to promote proliferation, migration, and invasion of GC cells through AKT/β-catenin pathway. Tumor growth and metastasis also depended on the enhanced expression of NCX1 in subcutaneously xenografted GC mouse model. Overall, our findings indicate that TRPC1/NCX1 coupling may promote H. pylori-associated GC through the Ca2+/AKT/β-catenin pathway. Since the Ca2+ exit mode and the Ca2+ entry mode of NCX1 play different roles under mostly physiological and pathological conditions respectively, targeting TRPC1/NCX1 coupling could be a novel strategy for selectively blocking Ca2+ entry mode to potentially treat digestive cancer with less side effect.Plasma membrane Na Hp) infection in the stomach is a well-known risk factor for GC and ammonia/ammonium is the major Hp virulence factor [Hp-associated GC. Cytosolic free Ca2+ ([Ca2+]cyt) is a pivotal second messenger in eukaryotic cells to maintain critical cellular processes, including the energetic metabolism, cell signaling, and cell motility, etc [2+]cyt signaling is involved in GC, though the occurrence and progression of cancer are complex [2+-permeable channels and transporters play important roles in the regulation of [Ca2+]cyt, their aberrant expression and function are associated with GC development cyt homeostasis in other cancer cells cyt homeostasis cyt under the restricted membrane space and induce membrane depolarization, switching NCX1 to Ca2+ entry mode cyt imaging experiments were performed as previously described [2+]cyt imaging solution were described in the [Caescribed , 50. The3, CaCl2 (4 mM), KB-R7943 (30 μM), or CaCl2 plus KB-R7943 were injected into the tumors in one side of the armpits once a day, and 0.1% DMSO into the other side as controls. Similarly, the shNCX1 SGC7901 cells or negative control NC was separately injected into each side, and CaCl2 was injected into the tumors of each side once a day. Four weeks later, xenografted tumors were quantified. For the peritoneal dissemination assay, 1 × 106 shNCX1 SGC7901 cells and the NC cells were injected into the abdominal cavity of nude mice. CaCl2 (4 mM) was injected into abdominal cavity once a day. Five weeks later, xenografted tumors were quantified.Tumor xenograft assay was performed as previously described . The mal*P < 0.05) are expressed in the figures and figure legends.SPSS Statistics 26.0  and GraphPad Prism 7.0  software were used to analyze the data. All data shown are means ± SD. All experiments were repeated for at least three times. Student’s unpaired, two-tailed t test or one-way ANOVA were used to analyze statistical significance differences of experimental groups. The patient survival was examined by the log-rank test using the Kaplan-Meier method. Significant differences (Supplementary materialsSupplementary figure 1-5Supplementary table 1


In MLL1-rearranged AML (MLL1-r), the MLL1 fusion protein (MLL-FP) causes dysregulated expression of HOXA9, MEIS1, PBX3, MEF2C and CDK6 . Additio−/−, as compared to MOLM13 cells  dose-dependently inhibited cell growth of MOLM13 (expressing MLL1-AF9) and OCI-AML3 cells   from 6 samples of bone marrow aspirate (BMA) or peripheral blood (PB) AML cells expressing MLL1-FP or mtNPM1   and FLT3-TKD (sample #1). As shown in Fig. 9 and FLT01) Fig. . Notably01) Fig. G, H. Ind01) Fig. . Neither+/+ cells, ziftomenib drives more lethality in MOLM13-TP53-R175H and MOLM13-TP53−/− cells. Finally, in a PDX model of AML cells harboring MLL-AF9 and FLT3 mutation, co-treatment with ziftomenib and venetoclax or OTX015 yielded significantly greater reduction in AML burden and improvement in survival compared to each agent alone without significant host toxicity. Overall, these findings support further in vivo testing and development of these MI-based combinations to not only improve the efficacy of ziftomenib, but also to undermine resistance to ziftomenib monotherapy in AML with MLL1-r or mtNPM1 that is encountered in the clinic.In summary findings presented here highlight preclinically for the first time that ziftomenib treatment triggers Menin protein degradation through the ubiquitin-proteasome pathway and depletes Menin levels, as well as reduces protein expressions of MEF2C, MEIS1, FLT3, CDK6 and BCL2. This was associated with reversal of the differentiation block, induction of differentiation and loss of viability of AML cell lines and patient-derived AML cells harboring MLL1-r or mtNPM1 –10. Our Supplemental Data FiguresSupplemental Figure LegendsSupplemental Materials and Methods


Therapeutic developments targeting acute myeloid leukemia (AML) have been in the pipeline for five decades and have recently resulted in the approval of multiple targeted therapies. However, there remains an unmet need for molecular treatments that can deliver long-term remissions and cure for this heterogeneous disease. Previously, a wide range of small molecule drugs were developed to target sub-types of AML, mainly in the relapsed and refractory setting; however, drug resistance has derailed the long-term efficacy of these as monotherapies. Recently, the small molecule venetoclax was introduced in combination with azacitidine, which has improved the response rates and the overall survival in older adults with AML compared to those of chemotherapy. However, this regimen is still limited by cytotoxicity and is not curative. Therefore, there is high demand for therapies that target specific abnormalities in AML while sparing normal cells and eliminating leukemia-initiating cells. Despite this, the urgent need to develop these therapies has been hampered by the complexities of this heterogeneous disease, spurring the development of innovative therapies that target different mechanisms of leukemogenesis. This review comprehensively addresses the development of novel targeted therapies and the translational perspective for acute myeloid leukemia, including the development of selective and non-selective drugs. Acute myeloid leukemia (AML) is a hematologic malignancy that is characterized by excessive growth of immature white blood cells (myeloblasts) in the bone marrow and in circulation, intrinsically affecting the process of hematopoiesis . AML is FLT3, which is attributed to the signaling gene category, along with mutations in KIT, PTPN11, RAS, JAK2, and PPM1D . Ov. Ov45]. IDH1-mutated AML [IDH2 R140 and IDH2 R172 variants [IDH1 mutation, as detected by an FDA-approved test [IDH inhibitors are being used for specific patients, based on their biological phenotype regarding white blood cells and the patients’ response to the previously approved drugs, with a strong commitment to seek the best outcomes for patients of AML. IDH inhibitors can block mutant IDH proteins to help leukemia cells differentiate normally, and for this reason, they can also be called differentiation agents. In a study of ivosidenib with subcutaneous or intravenous chemo (azacitidine), common side effects include febrile neutropenia (28% ivosidenib vs. 34% placebo) and neutropenia (27% ivosidenib vs. 16% placebo) [Isocitrate dehydrogenase (IDH) inhibitors have emerged as a promising therapeutic strategy for the treatment of acute myeloid leukemia. The enzymes IDH1 and IDH2 are responsible for the conversion of isocitrate to alpha-ketoglutarate within the TCA cycle . In 2018ated AML . This drvariants , was appvariants , with thved test . This drplacebo) . IDH inhplacebo) .FLT3), which is found in one-third of patients with de novo AML [FLT3 mutations. Midostaurin and sorafenib belong to the class of first-generation FLT3 inhibitors, whereas quizartinib and gilteritinib belong to the class of second-generation inhibitors. The second-generation inhibitors were developed with a potential need to avoid off-target effects, such as nausea, febrile neutropenia, mucositis, vomiting, headache, petechiae, and fever. Thus far, the second-generation inhibitors currently in trials have shown promising effects in the treatment of FLT3-mutated AML [FLT3 mutation, as detected by an FDA-approved test. Overall, these FLT3 inhibitors represent a new standard of care for patients with FLT3 mutations in both the first-line and salvage settings.The most frequent mutations in AML are those in the gene for FMS-like tyrosine kinase 3 , that comprise a novel class of BCL-2 inhibitors have shown promising results in several hematological malignancies, both alone and in combination with other cancer therapies. However, these inhibitors exhibited some inadequacies, such as insufficient bioavailability and on-target toxicity of BCLxL inhibition. Hence, the other BCL-2 inhibitors came into existence to fill this gap ,55. BCL-Hypomethylating agents (HMAs) are an important treatment option in patients with acute myeloid leukemia (AML) who are not suitable for intensive chemotherapy. To date, hypomethylating agents have been extensively tested in hematologic malignancies, due to their critical role in reversing harmful DNA aberrations in crucial regions. Recently, azacitidine was tested in two separate phase 3 studies in myelodysplastic syndrome (MDS) patients. Azacytidine is used either alone or in combination with other drugs for AML patients. This drug reduces the formation of leukemia cells and subsequently induces the bone marrow to produce more healthy cells. Mechanistically, azacytidine inhibits DNA methyltransferase by the formation of covalent bonds between DNA-cytosine methyltransferase and DNA containing 5-azacytosine, thus resulting in DNA hypomethylation . Azacytip = 0.78). Both groups also had similar median OS rates  with a critical role in the primary stages of development for myeloid and lymphoid lineages. Additionally, the FLT3 ligand activates for FLT3 ,74. The for FLT3 . The secfor FLT3 .KMT2A-rearranged AML.The advent of new technological approaches in recent years has helped unravel the complexity of AML. Genomic approaches, including single-cell RNA sequencing, CRISPR gene editing, multi-targeted tissue imaging studies, and several other technological advances, have provided a better understanding of the complex genomic landscape of this disease. These recent developments have produced key discoveries in developing small molecular drugs that can potentially target specific genetic abnormalities or mutant proteins in AML . These tKMT2A (MLL1) occurs in up to 10% of acute leukemias and is especially common in infant leukemia, with an occurrence rate of at up to 80% [KMT2A. The transcriptional regulator KMT2A contains binding domains for menin, where it forms the menin-KMT2A-LEDGF complex by menin binding to menin binding domains (MBDs). This complex links KMT2A to chromatin, and the menin-KMT2A complex plays a crucial role in the regulation of HOX genes of hematopoiesis, specifically the leukemogenic HOXA9 and MEIS1 co-factor in myeloid progenitor stem cells [KMT2A rearrangement (KMT2Ar), this AML-sustaining fusion protein occurs and causes the overexpression of HOXA9/MEIS1 [KMT2Ar AML carries a poor prognosis and is associated with a higher relapse rate [KMT2A to bind to HOX gene promoters, and this fact has pushed the development of KMT2A-menin interaction-targeting small molecule inhibitors. Notably, menin inhibitors may be beneficial for the treatment of overexpression of other HOX genes in other subtypes of leukemia [The rearrangement of p to 80% . Menin iem cells . In KMT2A9/MEIS1 . KMT2Ar pse rate . As a coleukemia ,80.KMT2Ar or NPM1-mutated AML [KMT2Ar leukemia, the overall response rate was 23 of 38 patients (61%), with complete remission in 9 of 38 patients (24%). For patients with the NPM1 mutation, the overall response rate was 5 of 13 (38%), and complete remission was achieved in 3 of 13 patients (23%). KO-539 is another small molecule inhibitor of the binding site of menin-KMT2A. MI-3454 is a structural analog of the KO-539 clinical compound. MI-3454 was found to have a selectivity greater than 100-fold for KMT2Ar cell lines compared to non-KMT2Ar. Unlike SNDX-5613, there was no evidence of QT prolongation or other electrocardiographic abnormalities with KO-539 [Menin inhibitors currently undergoing clinical trials include SNDX-5613 and KO-539, among others. The SNDX-5613 phase 1 study enrolled both adult and pediatric patients with relapsed or refractory acute leukemia, with a focus on patients with ated AML . SNDX-56ated AML . For path KO-539 . The comh KO-539 . These tTP53 tumor suppressor gene is frequently inactivated in cancers by loss-of-function or missense DNA binding domain mutations, seen in almost 50% of tumors [TP53 allows for the enhanced proliferation of cancerous cells [The f tumors ,83, in uf tumors . These mf tumors . The p53us cells . Targetius cells . APR-246us cells .TP53 mutations, as well as in vivo models [APR-246 has been tested in two clinical trials, in combination with azacitidine, showing synergistic cytotoxicity in the AML cell lines with o models . Of the o models . The phaAnother class of targets are MDM2 (murine double minute 2 or human homolog HDM2) inhibitors that act as a physiologic antagonist of p53, which activates p53 and induces apoptosis or cell cycle arrest in wild-type p53-expressing AML. However, this could not be employed for the mutant p53. Despite this, these inhibitors have potential as p53 enhancers to initiate a tumor suppression effect in AML patients ,88.MCL-1, as a member of the BCL-2 family of proteins, is a pro-survival regulator of apoptosis. Proteins in this family bind to pro-apoptotic BH-3-only activators, bestowing MCL-1 with a role in cell death avoidance. Hematological malignancies were shown to be dependent, not only on MCL-1, but on BCL-2 as well . DistingMCL-1, while mTOR inhibitors block MCL-1 translation [Development of MCL-1 inhibitors can be approached indirectly; for example, CDK inhibitors lead to decreased transcription of nslation . Multiplnslation . S63845 nslation .XPO1’s cargo-binding groove, blocking the interaction of XPO1 with any cargo, therefore inhibiting nuclear export [SF3B1 responded better to XPO1 inhibition [XPO1 inhibition via selinexor has shown promising results in hematological malignancies in in vitro and in vivo studies [Eltanexor (KPT-8602) is a second-generation inhibitor of XPO1-mediated nuclear export. This compound covalently binds to cysteine 528 in r export . Comparer export . A recenr export . Unlike hibition . Moreove studies . Hence, In recent years, immune checkpoint inhibitors have shown promising results in various cancers, and they are becoming a strong option in the targeting of cancer cells. For example, programmed cell death protein 1 (PD-1) and its ligands PD-L1 and PD-L2 are revolutionizing cancer treatment in lung cancer, melanoma, Hodgkin’s lymphoma, and several other forms of cancer . AnotherFLT3 inhibitors in combination with selinexor or eltanexor could be tried to address the complexity of this disease as tested in multiple myeloma [To improve upon single agent therapies, a combinatorial approach seeks an advantage to strategically treat AML patients by targeting parallel pathways or providing synergistic tumor death . The opt myeloma . The optp < 0.001). Additionally, the incidence of remission rose among patients who received this combination therapy when compared to azacitidine alone. Hence, based on a thorough understanding of genomic heterogeneity, the use of different permutations in combination with specific inhibitors could be promising in order to address the complexity of this disease. Another advantage of developing combinatorial therapy is to act against the different mutations or against leukemic clone variations seen within the same patient.Recently, other combination therapies have been tried, including pro-apoptotic inhibitors and hypomethylating agents. In a recent study that included randomly assigned and previously untreated patients (n = 431) with confirmed AML, a combinatorial therapy approach of venetoclax and azacitidine was employed . The VIAThe drug-like compounds, which have shown to be effective in other cancers, introduce a translational approach to the treatment of AML. Drugs that are known to be selective and non-selective inhibitors  have beeSince these inhibitors may have non-specific effects, they can be more difficult to use in combination therapies and can pose a great disadvantage in treating older patients. These drawbacks of non-selective drugs have led to the exploration of new therapeutic drugs that could be more reliable and exhibit fewer side effects, making them applicable to the specific morbidities of AML patients.AML is a disease of a highly complex nature with a varied genomic landscape with a number of different mutations, and this complexity has presented a challenge to drug development for nearly five decades. However, the recent development of targeted therapies seeks to resolve this complexity. The main role of targeted therapy is to target the specific abnormality with maximum efficacy. The improved overall survival (OS) rate seen in patients with some of these agents is evidence of positive results that can grant hope to patients, scientists, and physicians. Despite the fact that not all targeted agents were discussed within the contents of this review, we have covered numerous promising agents of interest.Overall, these targeted therapies show promising potential for AML patients. The broader and deeper molecular understanding of this disease has paved the way to address the core problems of treatment. However, there are challenges in regards to designing a proper scientific and clinical trial approach to attain the most accurate efficacy of these targeted drugs to deliver fuller benefits to patients. The phenotype of AML disease has been developed as a consequence of complex genetic and biological pathway changes, hence, addressing its complex nature would not be possible with one specific target, whereas a combinatorial approach could potentially include the various facets of this disease. The continuous and determined focus on understanding the underpinnings of molecular genetics and epigenetics, as well as the persistent surveillance of clonal evolution before and after the treatment of these targeted therapies, could potentially introduce novel changes to the treatment strategies, offering the maximum beneficial outcomes to patients of all ages.


Autophagy is an evolutionarily conserved and tightly regulated lysosome-mediated intracellular bulk degradation pathway by which intracellular macromolecules are sequestered in autophagosomes and delivered to lysosomes for degradation and recycling. Identification of autophagy-related (ATG) genes in yeast has promoted the understanding of the molecular mechanism of autophagosome formation.2+) is well known as an essential second messenger in eukaryotic cells.2+ levels are distinct in different subcellular compartments and are built up by Ca2+ channels and pumps located in the plasma membrane and organelles. Due to the resulting highly localized gradients, cytoplasmic Ca2+ signals display spatiotemporal heterogeneity in the form of sparks, transients, oscillations, and waves. It is well known that rapid dynamic changes of the local Ca2+ concentration ([Ca2+]) in the cytoplasm and organelles are capable of initiating a variety of physiological signals in the cell. Mounting evidence has shown that cytosolic and endoplasmic reticulum (ER) Ca2+ transients are the core signals in the regulation of autophagy activity. One of the important differences in autophagy between metazoans and yeast is the location of autophagosome formation. In yeast, autophagosomes fuse with the tonoplast and are formed at a single site,2+]c and [Ca2+]ER could regulate autophagy by affecting the activity of proteins in the autophagic machinery.2+ signals and the molecular mechanisms involved in the regulation of the autophagosome during the process of autophagy are completely unknown. Therefore, identification of how Ca2+ signals regulate autophagosome formation on the ER is an unresolved scientific question in the field of autophagy.Calcium  and Atlastin 2/3 (ATLs). These proteins are localized at the ER and become autophagosome initiation sites  revealed that Ca2+ transients or oscillations, which could be blocked by BAPTA-AM, occurred on the ER surface under starvation or Torin1 treatment. Further experiments using pharmacological inhibition of Ca2+ release channels, or knockdown strategies, showed that FIP200 condensates on the ER were significantly reduced.2+ transients on the ER surface are essential for autophagosome initiation. In contrast, Ca2+ release channel activator treatment increased the frequency, amplitude, and duration of local/global Ca2+ transients and oscillations on the ER. However, autophagic flux activity was inhibited in this scenario, suggesting that persistent activation of calcium release channels suppress the formation of functional autophagic structures. This conclusion was also supported by transmission electron microscopy studies demonstrating that ER-associated small unacidified autophagosomes exist in Ca2+ release channel activator-treated cells.Zheng et al.EPG-4/EI24.2+ signals on the ER surface in EI24 knockout cells was similar to what was observed in the cells treated with a calcium channel activator. Moreover, EI24 knockout caused autophagy defects, which could be attenuated by the application of Ca2+ release channel inhibitors or knockdown. Most importantly, Zheng et al.2+ release channels and pumps on the ER membrane, causing clustering of a subtype of inositol 1,4,5-trisphosphate receptor (IP3R)-IP3R3 to modulate ER Ca2+ transients. Additionally, they found that Ca2+ release from lysosomes was also involved in the formation of FIP200 puncta on the ER. Ca2+ transients on the ER surface were further shown to promote the assembly of the ULK1 complex by triggering the formation of ATG13-dependent FIP200 liquid–liquid phase separation, in which VAP and ATL2/3 proteins play an important role. Finally, ATG9 vesicles were found to regulate this phase separation and the spatial organization of FIP200 puncta on the ER. This indicates an important functional role of ATG9–ULK1 interaction in autophagosome formation.Zhang and colleagues previously identified a series of autophagy genes unique to multicellular organisms such as 2+ release has now been shown to significantly determine the ER Ca2+ transients that trigger the formation of FIP200 puncta upon autophagy induction, but it will be important to extend the work of Zheng et al. by exploring the possible role of other Ca2+ channels, such as Ca2+ entry channels, which may modulate the ER Ca2+ transients. It would also be interesting to investigate the potential mechanism of the generation of ER Ca2+ transients stimulated by starvation and explore how these signals trigger formation of FIP200 puncta.Ca