This review aims to provide an overview of the current knowledge of the genetic lesions driving pediatric acute myeloid leukemia (AML), emerging biological concepts, and strategies for therapeutic intervention. Hereby, we focus on lesions that preferentially or exclusively occur in pediatric patients and molecular markers of aggressive disease with often poor outcome including fusion oncogenes that involve epigenetic regulators like KMT2A, NUP98, or CBFA2T3, respectively. Functional studies were able to demonstrate cooperation with signaling mutations leading to constitutive activation of FLT3 or the RAS signal transduction pathways. We discuss the issues faced to faithfully model pediatric acute leukemia in mice. Emerging experimental evidence suggests that the disease phenotype is dependent on the appropriate expression and activity of the driver fusion oncogenes during a particular window of opportunity during fetal development. We also highlight biochemical studies that deciphered some molecular mechanisms of malignant transformation by KMT2A, NUP98, and CBFA2T3 fusions, which, in some instances, allowed the development of small molecules with potent anti-leukemic activities in preclinical models . Finally, we discuss other potential therapeutic strategies that not only target driver fusion-controlled signals but also interfere with the transformed cell state either by exploiting the primed apoptosis or vulnerable metabolic states or by increasing tumor cell recognition and elimination by the immune system. Here, wThe TARGET-AML study suggested that the association of pediatric AML with different fusion oncogenes strongly correlates with age of the patient  Figure . WhereasKMT2A  gene on the long arm of chromosome 11 (11q23) encodes a SET-domain histone methyltransferase that is important for the maintenance of the hematopoietic stem cells  fusions are often associated with therapy-related AML, in pediatric patients, these fusions are found in congenital and perinatal leukemia. Interestingly, some infants with KAT2A-CREBBP+ AML were reported to go into spontaneous remission; however, the underlying biology remains poorly understood  or 6B [KAT6B a.k.a. Moz-related factor (MORF) or MYST4] to the histone acetyl transferases EP300 or CREBBP in some rare cases of pediatric myelodysplastic syndromes (MDS) and AML –16. In cderstood , 18.CBFA2T3 (a.k.a. ETO2 or MTG16) gene on the long arm of chromosome 16, encoding a transcriptional co-repressor, is targeted by two recurrent AML-associated chromosomal rearrangements, the t and the cytogenetically silent inv(16) (p13q24) leading to expression of RUNX1–CBFA2T3 and CBFA2T3–GLIS2 fusions, respectively. Whereas, the first is more prevalent in therapy-related adult AML and rarely found in pediatric patients, the second appears to be an exclusive pediatric lesion (de novo acute megakaryoblastic leukemia (non-DS AMKL) (ERG (carried by chromosome 21) and GATA1 CBFA2T3 (DS AMKL) . CBFA2T3DS AMKL) . Geneticnd GATA1  26, 27), 27CBFA2nd GATA1 , 29. CBF the t16;nd GATA1 .NUP98 gene on the short arm of chromosome 11 (11p15) encodes a structural component of the nuclear pore but the protein can also function as a transcriptional regulator  gene on the long arm of chromosome 7 (7q36) encodes a homeobox transcription factor that is essential for pancreatic organogenesis, and moto-neuron differentiation, and is involved in a t translocation that is a hallmark of infant AML with poor outcome  and RAS-related molecules  are found in adult and pediatric AML . These mRAS proteins are a family of highly homologous low-molecular-weight proteins that bind GTP, located at the inner face of the plasma membrane. In the normal situation, the activity of RAS is controlled by hydrolysis of bound GTP by GTPase activating proteins (GAPs) and the replacement of bound GDP with fresh GTP, which is catalyzed by the family of guanine nucleotide exchange factors (GEFs) (The s (GEFs) . About 2s (GEFs) . The TARs (GEFs) . Most cas (GEFs) .FLT3 protein is a class III receptor tyrosine kinase (RTK) family that contains an extra-cellular domain made up of five immunoglobulin-like regions, a single transmembrane region, an intracellular juxtamembrane domain (JMD), and two kinase domains at the carboxyl terminus. Inactivation studies in mice have shown that FLT3 signaling is central to the development of HSPC, B-cells, dendritic cell progenitors, and natural killer cells , IDH1 or IDH2, Enhancer of Zeste Homolog 2 (EZH2), DNMT3A, and Additional Sex Combs like-1 (ASXL1) are much less prevalent, affecting only about 1–2% of pediatric patients . NeverthIn vitro experiments measure the impact of an AML-associated mutation on proliferation and survival but also self-renewal and differentiation of bone marrow (BM)-derived HSPC. The latter two are often determined by measuring the serial replating capacity in growth factor-containing semisolid medium such as methylcellulose (MC). Expression of many KMT2A, NUP98, CBF, or RARA fusions support serial HSPC replating in MC associated with variable blockage of normal maturation. This assay also allows structure-function studies to dissect critical domains of a given gene product. To determine the transforming potential in vivo, researchers aim to express the respective cDNA/mini gene in HSPC of their animal model. In one widely used approach, mouse BM-derived cells are virally transduced to overexpress the respective genetic lesion and transplanted into irradiated syngenic recipients [often referred to as BM transplant (BMT) or reconstitution model]  mostly by recombinant retroviruses. Based on the limited access to primary material of a genetically rather heterogeneous disease as pediatric AML, the majority of functional studies are performed in animals, mostly mice. n model] . Despiten model] , 59. Nown model] .As the hematopoietic system of the mouse is not identical to the situation in humans, researchers developed transgenic mouse lines with a humanized hematopoiesis expressing several human hematopoietic regulatory genes in combination with defects of a normal immune response such as the MISTRG strain . These h+ Sca1+(LSK) cells, common myeloid progenitor (CMP), or granulocyte-macrophage progenitor (GMP) cells with virus expressing KMT2A–MLLT3 or KMT2A–MLLT1 followed by transplantation resulted in a very similar disease  carrying an activating mutation (W515L) induced leukemia with morphologic characteristics of AMKL; however, such a combination is rarely seen in patients  a driver and cooperating lesion might not occur and/or be active at the same developmental stage; (3) a given genetic lesion  has its optimal expression level; and (4) particular developmental cell stages or identity are likely more permissive to transformation than others  driver  mutations most likely need to be active n others .Functional cooperation studies in cellular and animal models suggest that we can group AML-associated mutations into drivers that are essential for induction and maintenance of the disease, and cooperating mutations that support expansion of the malignant clone or may facilitate transformation by mostly metabolic modifications . As a co+ APL patients, who are often considered separately for therapeutic reasons, they make up to of 5–10% of pediatric AML patients in the United States (2O3) induce different molecular mechanisms that ultimately lead to proteasome-dependent degradation of the PML–RARA fusion protein  that is, in the vast majority of the cases, driven by the PML–RARA fusion resulting from t. Although, the TARGET pediatric AML study did not report PML–RARAd States . Notablyd States . Severald States . Pioneer protein . A phasepatients . Pediatr of >95% . CollectMultiple mouse models demonstrated that several KMT2A fusions are strong leukemogenic oncogenes that drive the disease in the presence of few, if any, cooperating mutations , 110. Wein vitro and in vivo in preclinical models (KMT2A fusion complexes recruit the DOT1L histone H3 lysine 79 (H3K79) methyltransferase that positively regulates expression of the target genes most likely by preventing association of the sirtuin-1 (SIRT1) deacetylase complex resulting in loss of H3K9 and H4K16 acetylation, and reduced SUV39-mediated methylation on H3K9m3 . The mail models . Howeverl models .In addition to MENIN/LEDGF or DOT1L, targeted interference with other components of the KMT2A fusion complexes  have been explored in various preclinical models . Interesin vivo. In addition, KMT2A-dependent gene expression signatures from murine NUP98–HOXA9 transformed cells resembled human NUP98–NSD1-derived profiles, suggesting at least overlapping pathways. These observations suggest that therapeutic targeting of KMT2A would also be effective against NUP98 fusion-driven AML. However, more translational studies found significant differences in gene expression signatures of pediatric AML cases harboring either KMT2A fusions or NUP98–NSD1. Indeed, whereas the first group is often characterized by increased expression of EVI1, the latter is associated with increased expression of another PRDM family member PRDM16 (a.k.a. MEL1)  and Trithorax (KMT2A) protein complexes . More rea. MEL1) , 130. Neactivity , 131. Innd HoxB5 .+ AML. They developed a small molecule (AI-10-49) that selectively binds the CBFB–MYH11 fusion protein, resulting in RUNX1-mediated repression of the potent oncogenic driver MYC (+ human AMKL cell line in immunodeficient mice (Although the use of high-dose cytarabine has improved the outcome of core binding factor (CBF) AML, we still lack efficient strategies to selectively inactivate the RUNX1–RUNX1 partner transcriptional co-repressor 1  or CBFB–MYH11 driver fusions. Earlier work found that oligomerization of RUNX1–RUNX1T1 through the nervy homology 2 (NHR2) domain of RUNX1T1 was essential for its activity as a transcriptional corepressor and mediator of self-renewal to BM cells . Interfeiver MYC , 139. CBent mice . Collectde novo FLT3-mutated AML (+ AML particularly affecting the gatekeeper (F69L) or activation loop (D835/I836) residues , or Lestaurtinib (a.k.a. CEP-701), to very selective compounds such as Quizartinib (a.k.a. AC220), Tandutinib (a.k.a. MLN518), Crenolanib, or Gliternitinib (a.k.a. ASP2215) . Based oated AML . Very reated AML . Severalated AML . Promisiated AML . Howeverresidues . Future in vitro and in vivo cooperation in AML, targeted inhibition of constitutive active mutant RAS should be of therapeutic benefit. Although long viewed to be undruggable, recent observations suggest that pharmacological inhibition of RAS could be achieved  in refractory or relapsed adult AML patients show overall response rates >40% with about 20% complete remission over several months underlining the potential for these compounds for personalized therapeutic strategies . However, small molecules were generated that potently and selectively inhibit mutant IDH1 or IDH2 through binding in an allosteric manner at the interface of the dimerized enzymes , 150. ID benefit . Phase Irategies , 153. Horategies . Despite50 as low as 10 nmol/L (It is well-known that malignant transformation leads to various cellular dependencies that may offer targets for therapeutic intervention . A very 0 nmol/L . A phase0 nmol/L . Small-m0 nmol/L , 162. In0 nmol/L –165. Mor0 nmol/L . Very re0 nmol/L . These o0 nmol/L .Intensive research is currently ongoing that aims to therapeutically target the capacity of pediatric leukemia cells to escape destruction by the immune system . Some stBoth authors have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Interestingly, the presence of the mitochondrial NCX inhibitor CGP-37157 prevented these alterations. The specific involvement of the mitochondrial NCX was confirmed by the inability of the plasma membrane inhibitor SN-6 to counteract such phenomenon. Of note, using a siRNA approach, we found that NCX1 was the isoform specifically involved. These findings suggested that NCX1, operating on the mitochondrial membrane, may have a critical role in the maintenance of ionic Ca2+ homeostasis in PD and that its inhibition most likely exerts a protective effect in the toxicity induced by α-syn and Rot.Progressive accumulation of α-synuclein (α-syn) and exposure to environmental toxins are risk factors that may both concur to Parkinson’s disease (PD) pathogenesis. Electrophysiological recordings of field postsynaptic potentials (fEPSPs) and Ca The histopathology of PD is also characterized by the presence of Lewy bodies, which are mainly composed of aggregates of the α-synuclein (α-syn) protein2. Accordingly, many studies showed that in PD patients the presence of α-syn is increased in the brain4. Mutations and multiplication of the α-syn gene (SNCA) are associated with familial PD5. Several studies have tried to understand the role of fibrillary and oligomeric forms of α-syn on neuronal damage6. Conversely, only a few studies are available on the mechanisms underlying α-syn-induced synaptic dysfunction. The striatum, a subcortical nucleus receiving major excitatory inputs from the cortex and the thalamus, is a brain region particularly involved in PD. We previously demonstrated that exogenous α-syn application, applied at nanomolar concentrations, directly affects striatal neurotransmission by targeting N-methyl-D-aspartate (NMDA) receptors and reducing synaptic currents7. This α-syn-triggered NMDA receptor-dependent alteration might in turn participate in intracellular spreading of α-syn, also affecting NCX1 function expressed in the striatum at the mitochondrial level. It has also been reported that α-syn alters the hippocampal synaptic transmission in normal rodents8.Parkinson’s disease (PD) is a multifactorial neurodegenerative disorder mainly characterized by the damage of neurons of basal ganglia and four cardinal motor symptoms such as bradykinesia, rigidity, resting tremor, and postural instability. These pathological features are induced by the slow and progressive death of dopaminergic neurons of the substantia nigra2+ levels10. Emerging findings suggest that exposure to environmental toxins, such as pesticides, can increase the levels of α-syn11. Among these drugs, rotenone (Rot), an inhibitor of mitochondrial respiratory chain complex I, induces a parkinsonian-like phenotype in rats through α-syn accumulation, caspase activation, dose-dependent ATP depletion, and oxidative damage14. In vitro studies have shown that acute application of Rot on rat striatal slices induces a widespread degeneration of striatal neurons13. Moreover, experiments on human neuroblastoma SH-SY5Y cells reported that combined exposition to α-syn and Rot deregulates Ca2+ homeostasis by opening voltage-gated Ca2+ channels16.Accumulation of α-syn in human dopaminergic neurons leads to different neurotoxic mechanisms such as the reduction of mitochondrial complex I activity, the increase of production of reactive oxygen species, and the deregulation of mitochondrial Ca2+ homeostasis, both at cytosolic and mitochondrial levels, might contribute to the pathophysiology of neurodegenerative diseases including PD20. Both in in vitro and in vivo models of PD, it has been demonstrated that NCLX is an essential regulator of mitochondrial Ca2+ homeostasis18. However, several studies seem to indicate that other transporters may also play a role22. Among them, Na+-Ca2+ exchanger (NCX) has recently gained much attention. NCX is a key regulator of ionic homeostasis at both the plasma membrane and mitochondrial levels, as it can facilitate Na+ and Ca2+ flow in bidirectional way24. NCX family encompasses three members NCX1, NCX2, and NCX3 differently expressed in the brain26. NCX1 is particularly expressed in the basal ganglia where it represents a key player in controlling ionic homeostasis at both plasma membrane and mitochondrial levels in the striatum26. A role of plasma membrane NCX in the pathogenesis of PD has also been suggested27. In particular, the administration of an inhibitor of plasma membrane NCX attenuated the degeneration of dopaminergic neurons in a PD model27.Indeed, the deregulation of CaThe aim of the present study was to analyze the involvement of both plasma membrane and mitochondrial NCX1 in the toxicity induced by combined application of α-syn and Rot in different neuronal models. Specifically, as in vitro model we used SH-SY5Y human neuroblastoma cells differentiated with retinoic acid to a neuron-like state; as ex vivo model we used striatal slices from adult rats.For electrophysiological experiments we used 2–3-month-old male Wistar rats . All experimental procedures were conducted in conformity with the European Directive 2010/63/EU, in accordance with protocols approved by the Animal Care and Use Committee at the University of Perugia. All efforts were made to minimize the number of animals used as well as their suffering.2, 1.2 NaH2PO4, 2.4 CaCl2, 10 glucose, and 25 NaHCO3) using a vibratome. The slices were maintained in aCSF, bubbled with an O2 95% and CO2 5% gas mixture (pH = 7.4) at room temperature (RT). Single coronal slices including the cortex and the striatum were transferred to a recording chamber and submerged in a continuously flowing aCSF  bubbled with a 95% O2–5% CO2 gas mixture28. Glutamatergic field excitatory postsynaptic potentials (fEPSPs) were evoked every 10 s by means of a bipolar electrode connected to a stimulation unit (Grass Telefactor) and located in the white matter between the cortex and the striatum to activate glutamatergic fibers. The recording borosilicate glass electrode filled with 2 mM NaCl (resistance 10–15 MΩ) was placed in the dorsolateral striatum. All drugs were dissolved in aCSF and bubbled with O2 95% and CO2 5% gas mixture during all the experiments. Then, 10 µM SN-630 or 3 µM CGP-3715731 was sent in perfusion alone for 20 min and in co-application with 0.3 µM Rot. The slices treated with 3 nM α-syn were incubated for 1 h before the application of Rot.The brain was rapidly removed and coronal corticostriatal slices (270 μm) were cut in artificial cerebrospinal fluid (aCSF) solution . SH-SY5Y cells were cultured in 100 ml Petri dishes using Eagle’s minimum essential medium (MEM)/Nutrient Mixture Ham’s F-12 (1:1) media supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin. The cell culture medium was replaced every 2 days. The cells were maintained in a humidified incubator at 37 °C and 5% COtrans retinoic acid (RA)33 that was added to the cell culture medium every 3 days for 1 week prior to performing the experiments.Differentiation into neuron-like cells was achieved by treatment with 10 µM all-35 with minor modifications. Specifically, silencing of NCX1 isoform was performed according to Qiagen manufacturer’s instruction using HiPerfect Transfection Kit (Qiagen) and FlexiTube small interference RNA (siRNA) for NCX1 , and FlexiTube siRNA for NCX3 (Qiagen Hs_SLC8A3_7). The validated irrelevant Allstars siRNA (Qiagen) was used as a negative control. Target sequences of the FlexiTube NCX1 siRNA was Hs_SLC8A1_9 (5′-CAGGCCATCTTCTAAGACTGA-3′), and of the NCX3 siRNA sequences was Hs_ SLC8A3_7 (5′-ACCATTGGTCTCAAAGATTCA-3′). The transfection protocol was as follows: SH-SY5Y cells  were differentiated with 10 µM RA in 6-well plates for 7 days. After differentiation protocol, SH-SY5Y cells were incubated 48 h with 2.3 ml of MEM7F-12 media containing 100 µl of MEM/F-12 (without FBS and antibiotics), 12 µl of HiPerfect Transfection Reagents, and 80 nM of siRNA oligonucleotide (each well). At 48 h after transfection, cells were subjected to specific treatments. The yield of RNA silencing was assessed by western blot analysis using specific antibody.RNA interference (RNAi) was performed as described earlier2+ levels were monitored by single-cell computer-assisted video imaging using a LSM 510 confocal system 36. The slices were loaded with 5 µM Rhod 2-AM  in aCSF solution bubbled with O2 95% and CO2 5% gas mixture for 1 h in the dark at RT37. The slices were then washed once in aCSF solution and placed into a perfusion chamber submerged in a continuously flowing aCSF solution  bubbled with O2 95% and CO2 5% gas mixture, mounted onto the stage of an inverted Zeiss Axiovert 200 microscope. Mitochondrial Ca2+ levels were evaluated as fluorescence increase. Bath solution was changed with a peristaltic pump and images were acquired every 5 s. Excitation light was provided by argon laser at 488 nm and the emission was time-lapsed recorded at 505–530 nm. Analysis of fluorescence intensity was performed off-line after image acquisition, by averaging the fluorescence intensity values within selected areas overlying the cell somata as previously described39. There are 5 experimental groups: control, 3 nM α-syn, 0.3 µM Rot, 3 µM CGP-37157, α-syn plus Rot, CGP-37157, and α-syn plus Rot. Before the application of drugs to acquire a stable baseline, the slices were perfused with aCSF for 5 min, then were perfused for 25 min with aCSF to control, and for 25 min for other drugs dissolved in aCSF.Mitochondrial Ca2+ levels were monitored by single-cell computer-assisted video imaging using a LSM 510 confocal system 36. After being differentiated into neuron-like cells on 25 mm coverslip, SH-SY5Y were loaded with 5 µM Rhod 2-AM  in MEM/F-12 media for 1 h in the dark at 37 °C. Coverslips were then washed once in phosphate buffer solution (PBS), placed into a perfusion chamber mounted onto the stage of an inverted Zeiss Axiovert 200 microscope, and maintained in buffer solution  and maintained at 37 °C using a heated microscope stage and climate box from PeCon GmbH. [Ca2+]m was evaluated as fluorescence increase. Bath solution was changed with a peristaltic pump and images were acquired every 5 s. Excitation light was provided by argon laser at 488 nm and the emission was time-lapsed recorded at 505–530 nm. Analysis was obtained as previously described40. Pharmacological modulation of plasma membrane or mitochondrial NCX was performed by exposing to 1 µM SN-6 or 3 µM CGP-37157, respectively35. Before drug application, to acquire a stable baseline, RA-differentiated SH-SY5Y cells were perfused with buffer solution for 5 min and for 25 min with buffer solution to control, and for 25 min for other drugs dissolved in buffer solution.Mitochondrial Ca39 . To detect NCX3 protein we used a rabbit monoclonal IgG anitibody 35.NCX1 protein was detected using a commercially available mouse monoclonal IgG antibody31 for 30 min at 37 °C and then fixed with PBS and 3.7% formaldehyde for 30 min at RT and then permeabilized with PBS-Triton X-100 for 5 min at RT. After permeabilization, cells were incubated with NCX1 or NCX3 primary antibody for 20 min. Immunoreactions were revealed by incubation with secondary antibody conjugated .Differentiated SH-SY5Y cells were loaded with MitoTracker 300 nM (MitoTracker Red CMXRos M7512 Invitrogen)2-[[4-[(4Nitrophenyl) methoxy] phenyl] methyl]-4-thiazolidinecarboxylic acid ethyl ester (SN-6) and the mitochondrial NCX inhibitor 7-chloro-3,5-dihydro-5-phenyl-1H-4,1-benzothiazepine-2-on (CGP-37157) were obtained from Tocris. Human recombinant α-syn and Rot were purchased from Sigma.n represents the number of slices used for each electrophysiological recording. Changes in the evoked fEPSP amplitudes induced by drugs were expressed as a percentage of the baseline, the latter representing the normalized fEPSP mean amplitude acquired during a stable period (10 min) before drug administration. Two-way analysis of variance (ANOVA), followed by Bonferroni’s post hoc test, were used for statistical analysis. The significance level was established at P < 0.05.Data analysis was performed off-line using Clampfit 10 (Molecular Devices) and GraphPad Prism 6 . Values given in the text and figures are mean ± S.E.M., where 2+ levels data were expressed as mean ± S.E.M. The n represents the number of independent experiments and 100–200 cells were recorded in each different session. Values less than 0.05 were considered to be significant. Differences among means were assessed by one-way ANOVA followed by Dunnett’s post hoc test.Ca2+ levels13. In order to test whether α-syn affected per se the basal synaptic transmission, we recorded the fEPSP for 40 min in the presence of 3 nM α-syn in control striatal slices. In this condition we found no effect of α-syn on the fEPSP .It has been previously shown that α-syn and Rot impact neuronal cell viability, in particular they reduce mitochondrial complex I activity causing changes of CaPSP Fig. . AfterwaPSP Fig.   and the mitochondrial NCX (NCXm) inhibitor CGP-37157 (3 µM). SN-6 affected neither the striatal response to Rot alone nor response to the combined application of Rot and α-syn , suggesting that the plasma membrane NCX1 was not involved in the toxic effects of α-syn and Rot. To investigate the role of NCXm isoform, striatal slices were exposed to CGP-37157 for 20 min. While this drug did not affect the toxic effect induced by Rot alone, it was able to reduce the detrimental effect produced by the combined application of α-syn and Rot on the fEPSP amplitude . This increase was statistically significant with respect to control and to all other groups. Interestingly, incubation with CGP-37157 was able to reduce the increase of mitochondrial Ca2+ levels induced by combined application of α-syn and Rot .It has been reported that α-syn and Rot increase mitochondrial Caons Fig. . We foun31% Fig.   in the presence of α-syn plus Rot and this effect was statistically significant with respect to control and all other groups . In line with the results obtained on fEPSP in striatal slices, SN-6 did not counteract the mitochondrial Ca2+ level deregulation induced by α-syn plus Rot, suggesting that plasma membrane NCX1 was not involved in such response .We found that mitochondrial Caups Fig.  , we showed that mitochondrial NCX1 is involved in the cell toxicity induced by the concomitant exposure to α-syn and Rot. In particular, this finding is supported by three main results. Firstly, the alteration of striatal electrical activity induced by α-syn and Rot is prevented by pharmacological inhibition of mitochondrial NCX with CGP-37157, but not by using the plasma membrane NCX inhibitor SN-6. Secondly, α-syn and Rot increased mitochondrial Ca2+ levels in striatal slices through a mechanism that involved NCXm, since CGP-37157 partially prevented this response. Finally, the silencing of NCX1 in RA-differentiated SH-SY5Y cells prevented the increase of mitochondrial Ca2+ levels and mimicked the effect of CGP-37157.The regulation of Ca44, little is known on the mechanisms underlying the convergent detrimental effects induced by the concomitant application of α-syn and Rot. Previous reports have demonstrated that in PC12 cells and in neurons of the substantia nigra, Rot raised intracellular Ca2+, leading to increased α-syn aggregation45.While several studies have investigated the toxic effects induced by α-syn and Rot on neurons, including synaptic dysfunction, oxidative stress, and mitochondrial complex I deficiencym could represent an important target for the control of acute synaptic dysfunction induced by the concomitant action of α-syn and Rot. Growing evidence suggest that, in the presence of toxic stimulus, the mitochondrial Ca2+ uptake may induce apoptosis in a variety of pathological conditions38. In particular, Ca2+ dysregulation has been previously reported in α-synopathy models of PD. Accordingly, evidence from many studies suggest that α-syn and Ca2+ may be related in several ways. For instance, it has been reported that α-syn induces a reduction of ΔΨm and a disruption of electronic transport complex (ETC), thus accelerating the mitochondrial permeability transition pore (mPTP) opening10. In addition, an increase in cytosolic Ca2+ levels may also occur since α-syn may form plasma membrane pores, allowing extracellular Ca2+ to pass into the cytosol46. This in turn may worsen mitochondrial function through the increased Ca2+ uptake46. Moreover, Angelova et al.47 showed that the overexpression of intracellular α-syn in neuroblastoma cells altered basal and depolarizing-stimulus-evoked Ca2+ signals and confirmed that α-syn per se can induce a Ca2+ flux across neuronal membranes through pore formation47. Several Ca2+ transporters are implicated in the ionic regulation, and between them NCXs  represent critical proteins contributing to Ca2+ homeostasis in the brain26. Accordingly, the expression of all three NCX isoforms has been already reported in isolated mitochondria20. Recently, the main role of plasma membrane and mitochondrial NCX1 in controlling energy metabolism in several cell types, including neurons and astrocytes, has been demonstrated48. Interestingly, a metabolic stimulus, such as glutamate, could induce the reverse mode of NCXm, as a consequence of glutamate-induced Na+ influx through excitatory amino acid transporters31. α-Syn and Rot interact with Na+/K+-ATPase with a reduction of its ability to pump out Na+, thus leading to Na+ accumulation. Consequently, Ca2+ overload may occur50. In line with these observations, it is possible to speculate that under our conditions, the Na+ influx and the disruption of ETC may contribute to ΔΨm perturbation, ultimately forcing the mitochondrial NCX1 to work on the reverse mode and leading to Ca2+ increase. Our data showed that in SH-SY5Y cells, Ca2+ increase can be prevented by silencing of NCX1, mimicking the effect of CGP-37157 in striatal slices. An interesting study has already proposed that plasmalemmal NCX isoforms, in particular NCX2 and NCX3, contribute to mitochondrial Na+/Ca2+ exchanger in human DAergic neurons acting downstream PINK1, a pathway implicated in a recessive form of PD, preventing neuronal death induced by mitochondrial Ca2+ accumulation20. Notably, the role of another mitochondrial Ca2+ antiporter, NCLX, has been reported51. Palty et al.17 and Luongo et al.18 demonstrated that in SH-SY5Y undifferentiated cells and transgenic mice, NCLX is an essential regulator for mitochondrial Ca2+ homeostasis. Nevertheless, the same authors showed that the pharmacological modulation of NCLX (using CGP-37157) did not fully inhibit mitochondrial Ca2+ efflux. Thus, NCLX may play an important role but its contribution is not sufficient to regulate mitochondrial Ca2+ levels. Therefore, as suggested by several evidences, it is reasonable to hypothesize that other transporters, including NCX1, play a critical role in controlling mitochondrial Ca2+ homeostasis21.Here, we presented electrophysiological evidence that in striatal rat slices, α-syn plus Rot led to enhanced neurotoxic effects causing progressive irreversible loss of excitatory striatal transmission and electrical signals. This detrimental effect could be prevented by blocking mitochondrial NCX1, suggesting that NCX52. Furthermore, our data confirmed that α-syn has an important role in controlling neuronal mitochondrial dynamics, and in particular in regulating Ca2+ signals in animal models of PD as well as in PD patients52.In line with our findings and existing literature, we can assert the relevance of mitochondria in the pathology of PD. The involvement of mitochondrial defects in PD pathology is also supported by pharmacological evidence showing deleterious effects of inhibitors of the mitochondrial electron transport chain, such as Rot, mPTP, or paraquatIn conclusion, our study shows for the first time a critical interaction among α-syn Rot-induced toxicity and mitochondrial NCX1, suggesting that NCX1 might represent a possible target for disease modifying therapy in PD.Supplementary informationFig. S1


Invasion of an action potential (AP) to presynaptic terminals triggers calcium dependent vesicle fusion in a relatively short time window, about a millisecond, after the onset of the AP. This allows fast and precise information transfer from neuron to neuron by means of synaptic transmission and phasic mediator release. However, at some synapses a single AP or a short burst of APs can generate delayed or asynchronous synaptic release lasting for tens or hundreds of milliseconds. Understanding the mechanisms underlying asynchronous release (AR) is important, since AR can better recruit extrasynaptic metabotropic receptors and maintain a high level of neurotransmitter in the extracellular space for a substantially longer period of time after presynaptic activity. Over the last decade substantial work has been done to identify the presynaptic calcium sensor that may be involved in AR. Several models have been suggested which may explain the long lasting presynaptic calcium elevation a prerequisite for prolonged delayed release. However, the presynaptic mechanisms underlying asynchronous vesicle release are still not well understood. In this review article, we provide an overview of the current state of knowledge on the molecular components involved in delayed vesicle fusion and in the maintenance of sufficient calcium concentration to trigger AR. In addition, we discuss possible alternative models that may explain intraterminal calcium dynamics underlying AR. In most cases, Ca2+ concentration collapses below the threshold level for triggering vesicle fusion within a few milliseconds of the AP reaching the presynaptic terminal. Generally, even high frequency bursts of APs result in highly synchronized postsynaptic activity. However, at some synapses high frequency stimulation can trigger not only synchronized phasic transmitter release but can also generate vesicle fusion that lasts for tens or hundreds of milliseconds after the end of the AP burst . Usually synaptic delay, the time between the peak of the AP and the onset of the postsynaptic response, does not exceed a few milliseconds. This holds true even for synapses where APs can trigger multi vesicular release . When these channels open during AP, short lasting and spatially restricted elevation of intraterminal Ca2+ ([Ca2+]i) occurs in close vicinity to the channels or the cluster of the VGCCs known as nano- or microdomains  affinity of the calcium sensor mediating AR should be high enough so that fusion events may be triggered by the remaining bulk [Ca2+]i; (2) invasion of APs to the presynaptic terminals, besides opening VGCC, triggers additional long lasting Ca2+ entry; and (3) a combination of both high-affinity calcium sensors and an additional calcium source is necessary for AR generation.According to the most widely accepted model, synaptic release is triggered in the active zones by Caxtrusion . The rap2+ sensors (2+ microdomains and high affinity of the sensor mediating this mode of release. It is commonly accepted that the role of vesicular Ca2+ sensor is played by proteins belonging to the synaptotagmin (Syt) family consisting of 17 members. They differ in their expression pattern and Ca2+ binding properties ; in knockout animals, using flash photolysis of caged Ca2+, release rate progressively increases reaching a peak about 100–200 ms after the flash that triggered Ca2+ uncaging . In addition, both drugs failed to block AR evoked by stimulation of parallel fibers. Thus, additional experiments are necessary to determine the impact of Ca2+ release from presynaptic intracellular stores on AR generation.In frog neuromuscular junctions, long-lasting amplification of Ca2+-dependent activation of these channels resulting in long-lasting Ca2+ influx. Indeed, this current might be sufficient to trigger vesicle fusion. However, since the peak amplitude of the Ca2+-induced current is about 10% of the peak amplitude of the depolarization-induced Ca2+ current, the amplitude of asynchronous events should be substantially smaller than that of synchronous fast responses. This is probably the case at synapses formed by cerebellar parallel fibers, but seems to be unlikely in the case of AR from hippocampal CCK-positive basket cells. In the latter connection, the integral of asynchronously released IPSC detected after the bust of Aps is in the same range as the cumulative phasic response evoked during the AP train  and high Ca2+ affinity of Syt7, the Ca2+-induced tail current through N- and P- type Ca2+ channels is unlikely to be sufficient to trigger AR at CCK-positive synapses. In addition to that, activation of CB1 receptors expressed on these terminals leads to suppression of VGCC reducing overall Ca2+ entry; this mostly affects synchronous release and has a weaker effect on the asynchronous component  and the sodium/calcium exchanger  and in the Ca2+ elevations generated by cell stimulation (in the μM range). For instance, PMCA2, in particular, PMCA2a, exhibits exceptionally rapid activation in response to a rise in [Ca2+]i  substantially slows down NCX-mediated Ca2+ extrusion resulting in elevation of residual [Ca2+]i. Presynaptic Na+ dynamics are not well studied, however, several lines of evidence suggest that the decay time constant of Na+ extrusion is in the range of hundreds of milliseconds (Regehr, + concentration can rapidly build up in the terminal and then slowly decay to the basal level; in this period NCX will extrude Ca2+ at a substantially slower rate (2+]i due to Na+-dependent decelerating of NCX-mediated Ca2+ extrusion is even more pronounced at synapses with reduced PMCA function (Roome et al., +]i may reverse NCX and result in Ca2+ influx into the cell via this exchanger (Roome et al., 2+-clearing protein or Ca2+ source, depending on the intensity of presynaptic activity (2+]i sufficient for AR generation. Selective suppression of AR at these GABAergic synapses and at some excitatory terminals by moderate concentration of EGTA strongly suggests that residual Ca2+ and Ca2+ extrusion machinery, determining the kinetics of [Ca2+]i are involved in delayed release generation (Hefft and Jonas, NCX is a plasma membrane transport protein that exchanges 3 Na Regehr, . Thus, dwer rate . Elevatiactivity . In the 2+ with Sr2+ or application of lanthanides reduces phasic release and greatly promotes AR (Dodge et al., 2+ and La3+ on the timing of synaptic release can be explained by a reduction in the functioning of extrusion pumps.Substitution of extracellular Cavia interaction with Syt1, although in a way that does not involve activation of SNARE (Shin et al., 2+, clearance of “residual Sr2+” from presynaptic terminals is significantly slower (Xu-Friedman and Regehr, 2+-driven release. The rapid effect of La3+ does not require La3+ entry into the terminal, or binding to Syt1 and is independent of extracellular Ca2+ concentration. Nevertheless, the delayed component of an La3+-evoked increase of spontaneous release frequency can be blocked by intracellular loading of Ca2+ buffers (Chung et al., 3+ to block PMCA (Shimizu et al., 2+]i and promotes vesicle fusion. Thus, one can assume that the lack or reduced function of one of the extrusion proteins may result in slowed presynaptic [Ca2+]i dynamics leading to prolongation of vesicle release.Strontium can enter the terminals and trigger synaptic vesicle fusion 2+ sensors involved in fast and AR are different and that they have different Ca2+-binding kinetics. Syt7 has been proposed to perform the function of high affinity Ca2+ sensors for AR generation that is spatially and temporally located outside Ca2+ domains. However, Syt7 was found on the presynaptic plasma membrane and other internal membranes, but not on synaptic vesicles suggesting a non-canonical mechanism of Syt7-mediated of vesicle exocytosis. Thus, recruitment of Syt7 into evoked delayed release needs to be more thoroughly studied. In addition to that, involvement of other synaptotagmins to AR generation, which have high Ca2+ affinity and neuronal expression, has to be investigated. Importantly, experiments with EGTA loading clearly show that AR requires the presence of long-lasting elevation of free intraterminal Ca2+. In this respect, it might be promising to study the possible role of modulation of Ca2+ extrusion proteins. The role of NCX should be investigated, since the NCX-mediated extrusion rate depends on [Na2+]i, which is determined by the rate of presynaptic AP activity. Thus, Na+ dependent modulation of NCX functioning might provide an alternative mechanism not only for AR generation but also for short-term plasticity.Currently, there is an agreement that the CaAll authors listed have made substantial, direct, and intellectual contribution to the work and approved it for publication.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.


Thus, the CaM–peptide interface is an important determinant of the kinetic responses of GECIs; however, the topology of the structural link to the fluorescent protein demonstrably affects the internal dynamics of the CaM–peptide complex. In the dendrites of hippocampal CA3 neurons, f-RGECO1 indicates calcium elevation in response to a 100 action potential train in a linear fashion, making the probe particularly useful for monitoring large-amplitude, fast signals, e.g. those in dendrites, muscle cells, and immune cells.Genetically encoded calcium indicators (GECIs) are useful reporters of cell-signaling, neuronal, and network activities. We have generated novel fast variants and investigated the kinetic mechanisms of two recently developed red-fluorescent GECIs (RGECIs), mApple-based jRGECO1a and mRuby-based jRCaMP1a. In the formation of fluorescent jRGECO1a and jRCaMP1a complexes, calcium binding is followed by rate-limiting isomerization. However, fluorescence decay of calcium-bound jRGECO1a follows a different pathway from its formation: dissociation of calcium occurs first, followed by the peptide, similarly to GCaMP-s. In contrast, fluorescence decay of calcium-bound jRCaMP1a occurs by the reversal of the on-pathway: peptide dissociation is followed by calcium. The mechanistic differences explain the generally slower off-kinetics of jRCaMP1a-type indicators compared with GCaMP-s and jRGECO1a-type GECI: the fluorescence decay rate of f-RCaMP1 was 21 s Chameleon) (––Monitoring Caameleon)  were folameleon) , 6. Furtameleon) , 8. Thosameleon) –11. Sincmeleon) –, intensimeleon) ––16.Red GECIs (RGECIs) follow the design of GCaMPs because they are composed of a cp red fluorescent protein (cpRFP) that is fused to the smooth muscle myosin light-chain kinase peptide RS20 at its N terminus and to calmodulin (CaM) at its C terminus. The RGECO variants are based on the cp mApple, whereas the RCaMPs contain cp mRuby, with the exception of R-CaMPs, which are also based on cp mApple , 17. Pro2+, the intrinsic chromophore exists mostly in its protonated neutral state that shows no fluorescence. Upon Ca2+ binding to CaM, a hydrophobic interface is exposed that binds to the RS20 peptide forcing changes in the conformation of the β-barrel structure of the cpRFP, leading to deprotonation of the chromophore and formation of the anionic state that has high fluorescence emission. Previous studies on GCaMP3 and GCaMP6f showed slow fluorescence rise and decay in response to Ca2+ concentration changes, to a large extent caused by the steric constraints imposed by cpFP interjecting between CaM and the RS20 peptide as well as their strong binding with a dK in the nanomolar range  and the W4Y mutation in the RS20 target sequence (termed RS-1). The novel variant RGECI probes were analyzed in detail and showed significantly faster transients than their parental variants in ATP-stimulated HEK293T cells. To obtain information about the mechanism of the formation and decay of the highly fluorescent state, the Ca2+ response kinetics were analyzed in detail. The results offer a deeper insight into the mechanisms and the effects of the mutations on them for better understanding of GECI and the rational design of new and faster probes.Following the same design principle that led to GCaMP3GCaMP6fu , 24, we GCaMP6fu  to weake2+ dynamics. The kinetics of the fast jRGECO1a and jRCaMP1a variants were determined in detail and where possible an analytical solution was found. The remaining 14 variants are shown in the supporting information .Following our hypothesis that weakening the interaction of CaM and RS20 will generate probes with faster kinetics, we disabled single EF hands of CaM by mutating the first conserved aspartate into alanine –27 and a–2+-dependent fluorescence was ∼570 nm for all RGECIs. For Ca2+-bound jRGECO1a and its variants, the peak wavelength was 562 nm with a shoulder at 530 nm. The absorption maximum for Ca2+-bound jRCaMP1a and variants lay at 570 nm with a shoulder at 538 nm . For both jRGECO1a and jRCaMP1a, the peak at ∼570 nm corresponds to the anionic state of the chromophore, more prominent in the presence of Ca2+. The absorption peak at 450 nm in the absence of Ca2+ is assigned to the neutral state of the chromophore for all RGECIs (2+ binding (F+Ca2+/F−Ca2+) were determined from three different kinds of measurement: (i) the emission spectra, (ii) Ca2+ equilibrium titrations, and (iii) the ratio of brightness values from quantum yield and extinction coefficient measurements, and were in good agreement . For jRGECO1a and f-RGECO1, the paK values at saturating Ca2+ were 6.4 ± 0.1 and 6.6 ± 0.1. The RS-1 EF-4 mutation shifted the paK to 7.4 ± 0.1. In the absence of Ca2+, the paK values of jRGECO1a and its variants were ∼8.5 ± 0.2. A more detailed look at the pH dependences of jRGECO1a variants in the absence of Ca2+ revealed increasing fluorescence intensity with the pH almost reaching the value measured in 1 mm CaCl2 at pH 10 . For the jRCaMP1a variants all Ca2+-saturated paK values were 6.5 ± 0.2, and in the absence of Ca2+ paK = 7.5 ± 0.2. In contrast to jRGECO1a, normalized fluorescence for jRCaMP1a and its variants in the absence of Ca2+ remained low across the pH range . The differences in the pH dependences in the absence of Ca2+ are thus based on the intrinsic properties of the cp red fluorescent proteins derived from mApple and mRuby .p2+ binding in one of the binding motifs . jRGECO1a had a high affinity for Ca2+ with a dK of 161 ± 2 nm and a Hill coefficient n of 1.8 ± 0.1 measured  with an essentially linear response to Ca2+ .The EF-hand mutations disabled Cag motifs –27, wher2+] (20 μm) at 37 °C show monophasic fluorescence rises with limiting rates (kon(lim)) of 544 ± 5 s−1 for jRGECO1a and 100 ± 1 s−1 for f-RGECO1 (C). f-RGECO2 had biphasic on-kinetics with kon(lim) of 457 ± 7 and 86 ± 2 s−1, with relative amplitudes of 65 and 35%, respectively. Thus, the mutations applied did not improve the rise kinetics for f-RGECO1, whereas f-RGECO2 fast phase had similar kinetics to its parental GECI. The dissociation rate for jRGECO1a at 37 °C was 14.1 ± 0.1 s−1. f-RGECO1 and f-RGECO2 had 8-fold faster off rates of 109 ± 1 and 108 ± 1 s−1, respectively (E).Representative records obtained at saturating [Caf-RGECO1 C. f-RGECectively E.D). Rates obtained at saturating [Ca2+], kon(lim) for jRCaMP1a were 153 ± 7 and 4.0 ± 0.1 s−1 with relative amplitudes of 19 and 81%, respectively. f-RCaMP1 showed similar rates of 152 ± 2 s−1 and 12 ± 1 s−1, but with the faster rate representing the majority of the relative amplitude of 68%. f-RCaMP2 association kinetics were a bit slower with limiting rates of 64 ± 2 and 5.2 ± 0.1 s−1 and amplitudes of 35 and 65%, respectively.The association kinetics of jRCaMP1a variants were all biphasic at 37 °C, revealing the formation of two fluorescent states D. Rates 2+ off-kinetics of the two new fast variants of jRCaMP1a were biexponential, both showing faster decay rates (koff) compared with their parental probe. For jRCaMP1a the dissociation rates at 37 °C were 5.8 ± 0.1 and 1.6 ± 0.1 s−1 with relative amplitudes of 35 and 65%, respectively (F). f-RCaMP1 had a three times faster rate of 15.8 ± 0.1 s−1 and a similar slow rate of 1.4 ± 0.1. However, the fast rate represented the majority of the relative amplitude with 78%. For f-RCaMP2 koff was 4-fold faster with a value of 21 ± 1 s−1 (86%) and a slower rate of 0.9 ± 0.1 s−1 (14%).The Caectively F. f-RCaM2+ dynamics and fluorescence dynamic ranges (ΔF/F0) of RGECIs and their fast-decay variants were monitored in ATP-stimulated HEK293T cells. After stimulation the fluorescence rapidly rises and then decays over several seconds. The data were fitted with monoexponential decays resulting in time constants τ of 42 ± 2 s for jRGECO1a, 31 ± 2 s for f-RGECO1, and 5.0 ± 0.5 s f-RGECO2, with ΔF/F0 of 0.81 ± 0.37, 0.59 ± 0.25, and 0.29 ± 0.20, respectively (G). For the jRCaMP1a variants the obtained time constants τ were 17.5 ± 0.3 s for jRCaMP1a, 10.4 ± 0.7 s for f-RCaMP1, and 6.0 ± 0.6 s for f-RCaMP2, with ΔF/F0 of 1.00 ± 0.50, 0.42 ± 0.26, and 0.14 ± 0.06, respectively (H). Thus, faster response kinetics enabled the RGECI variants to more faithfully report Ca2+ dynamics in the cells.Intracellular Caectively G. For thectively H. Thus, 2+ transients simultaneously optically recorded from a single spine with two-photon imaging (A and B). The signals in response to 10 bAPs at 100 Hz from six to nine spines were averaged and corrected for bleaching. By fitting these signals with monoexponential decays the following time constants (τoff) were obtained: jRGECO1a 314 ± 49 ms, f-RGECO1 83 ± 17 ms, f-RGECO2 77 ± 13 ms, jRCaMP1a 327 ± 49 ms, f-RCaMP1 211 ± 46 ms, and f-RCaMP2 140 ± 34 ms (C and D). Thus, the novel RGECI variants showed up to 4-fold faster off rates compared with their parental sensors with comparable dynamic ranges. It must be noted that in dendritic spines, because of their lower affinities (dK = ∼5 times higher), the signal amplitudes and signal to noise ratios are lower for the fast sensors than for the parent probes . The advantage of the fast probes is, however, evident when monitoring Ca2+ transients in the dendrites, where the f-RGECO1 fluorescence on-response to 100 bAPs displayed linearity, indicating that because of the variant's reduced Ca2+ affinity, the saturation commonly observed with higher affinity sensors did not occur with f-RGECO1 (E). It should be noted that RGECIs based on mApple, like jREGECO1a and its variants, showed strong photoswitching as already reported in earlier studies  were induced by somatic current injections and the Ca imaging , A and B ± 34 ms , C and Df-RGECO1 E. It sho2+]. The observed association rate, kobs, was, however, dependent on [Ca2+]. When kobs was plotted against [Ca2+], a variety of patterns were observed. Kinetic models were developed where possible and are introduced through the highlighted fast variants with further examples shown in the supporting information . Three types of behavior are described using the fast-decay variants as class leading examples.The above described association kinetics at 37 °C were obtained at saturating  (B). At low [Ca2+] kobs, tended to 0 and then, showing a cooperative pattern, saturated as a function of [Ca2+]. The simplest model to fit the data depicts a two-step process in which rapid, cooperative Ca2+ binding is followed by isomerization involving the binding of the RS20 peptide to Ca2+n.CaM leading to the development of the highly fluorescent state . The reversal of the process, peptide dissociation from the Ca2+-bound complex would be extremely slow. However, an alternative dissociation pathway allows rapid decay via Ca2+ dissociation followed by peptide dissociation. The on-reaction therefore can be treated kinetically as an essentially irreversible process. The plot of kobs against [Ca2+] is fitted to dK  .(Eq. 1)dK1 of 0.35 ± 0.07 μm, k+2 of 150 ± 2 s−1 and n of 2.6 ± 0.1 were derived (B). The fitted dK1 value was similar to the measured dK (162 ± 2 nm); therefore it is assumed that Ca2+ and jRGECO1a are in a rapid equilibrium (k−1 ≫ k+2), with k+1 probably close to diffusion limited (∼108m−1 s−1). On Ca2+ sequestration, the fluorescent state decays via a different pathway from that of its formation: Ca2+ dissociation from the fluorescent complex is followed by the weak peptide complex rapidly falling apart. The measured dissociation rate for jRGECO1a was 3.4 ± 0.1 s−1 (C). Similar patterns were observed for jRGECO1a EF-3, jRGECO1a EF-4, and jRGECO1a RS-1 EF-3 variants . jRGECO1a RS-1 EF-1  had a limiting association rate (kon(lim)) of 23 ± 1 s−1 and a dissociation rate (koff) of 64 ± 1 s−1 (Table S1). Their Ca2+-dependent observed association rate showed an all-or-none pattern. Their mechanisms may be consistent with Scheme S1, with the amplitudes at low [Ca2+] too small to detect.From the fit  derived B. The fi 0.1 s−1 C. SimilaE), and the observed association rates, kobs, were plotted as a function of [Ca2+] (F). Below 0.51 μm free [Ca2+], no fluorescence increase was observed. In contrast to jRGECO1a, both association rates decrease with increasing [Ca2+], suggesting a mechanism in which a slow pre-equilibrium precedes Ca2+ binding. Two forms of the apo state, with only one binding Ca2+, could be in equilibrium. The biphasic behavior may indicate two independent populations of the variant. However, dissociation was monoexponential with a rate of 34 ± 1 s−1 (G).Association kinetic records were biexponential E, and thf [Ca2+] F. Below  ± 1 s−1 G.H and I). Initially, at [Ca2+] lower than 1 μm, a biphasic process was observed, indicating the existence of two fluorescent intermediates. The amplitude of the faster rate decreased with increasing [Ca2+], and at [Ca2+] > 2 μm, a single phase was observed apparently at the saturating rate of the slow phase (kon(lim)) of 79 ± 1 s−1. We attribute the fast phase to CaM N-lobe Ca2+ binding leading to a fluorescent state. The plot of the second, slow phase was fitted to Scheme S1, giving dK1 of 1.2 ± 0.1 μm, k+2 of 79 ± 2 s−1 and n of 2.7 ± 0.3. The fitted dK1 of the slow phase is in good agreement with the measured dK of 1.26 ± 0.02 μm.A more complex underlying mechanism was observed for fast-decay variant f-RGECO2 , H and IFig. S6) showed a similar behavior with a bell-shaped appearance of kobs for a fast phase as a function of [Ca2+] and a sigmoidal saturating curve for the slow phase . In contrast, the fast and slow phases for jRGECO1a RS-1 EF-2 had different saturation values at high [Ca2+], suggesting two final fluorescent states . For jRGECO1a RS-1 and jRGECO1a RS-1 EF-2, the fitted dK1 values of the fast phase were in the same range as their measured dK values, 428 ± 4 and 429 ± 5 nm, respectively. However, for jRGECO1a EF-2, they are in less good agreement (dK1 of 1.1 ± 0.2 μmdversus K of 436 ± 5 nm), indicating that the fast phase has a bigger impact and cannot be neglected for the estimate.jRGECO1a RS-1, jRGECO1a EF-2, and jRGECO1a RS-1 EF-2 . The two phases presented with equal amplitudes. This behavior can be explained with a mechanism in which binding is followed by two reversible isomerizations with two fluorescent states . The plot of the fast rate, kobs1, against [Ca2+] is fitted to Scheme S1 with a reversible second step, whereas the Ca2+-independent slow rate, kobs2, represents the sum of the rate constants for the second isomerization dK1 of 0.35 ± 0.14 μm, a k+2 of 53 ± 2 s−1, a k−2 of 5.3 ± 0.8 s−1, and n of 2.0 ± 0.2. For k+3 + k−3, 2.3 ± 0.1 s−1 was obtained. If we assume that K2K3 is negligible a dK of 105 ± 34 nm is calculated, which is in good agreement with the measured one (dK = 141 ± 3 nm). The Ca2+ off-kinetics of jRCaMP1a were biexponential, with rates of 2.2 ± 0.1 and 0.32 ± 0.01 s−1 and relative amplitudes of 17 and 83%, respectively, consistent with two successive reversible steps. Similar behavior was observed for jRCaMP1a EF-1, jRCaMP1a EF-2, and jRCaMP1a EF-4 .The fit to the fast phase gave a 2+ dependence of the observed association rate of f-RCaMP1 showed a sigmoidal pattern; however, in contrast to jRGECO1a, the rates at low [Ca2+] did not tend to 0 but to a finite minimum rate (E and F). The observed association rates saturated at 22 ± 1 s−1, and the intercept tends to 5.1 ± 0.5 s−1. This behavior is consistent with a two-step mechanism in which cooperative Ca2+ binding is followed by a reversible isomerization  and the intercept corresponds to k-2. Fluorescence develops in the second step. kobs is expressed in dK  .(Eq. 6)dK1 of 0.41 ± 0.15 μm−1 and K2 of 3.3 ± 0.6 gave an dK of 0.27 ± 0.08 μm, which is close to the measured value of 0.52 ± 0.02 μm . Variants jRCaMP1a RS-1 EF-1 and jRCaMP1a RS-1 EF-2 also fit well to Scheme S1, with the fitted dK (250 and 291 nm) a bit smaller than the measured ones (702 and 607 nm) .Using  0.02 μm . In term 3.9 s−1 G. Varian2+-dependent association rates, with biphasic dissociation kinetics . The mechanism for these variants is too complex for an analytical solution.Variants jRCaMP1a RS-1 EF-3 and jRCaMP1a RS-1 EF-4 show an all-or-none pattern of Ca−1 (I and J). For both, kobs decreases with increasing [Ca2+], similar to f-RGECO1. Thus, there is a slow pre-equilibrium preceding Ca2+ binding with two forms of the apo state, with only one binding Ca2+, that could be in equilibrium. The biphasic behavior may indicate two independent populations of the variant. However, dissociation was monoexponential with a rate of 6.6 ± 0.1 s−1 (K).Fast variant f-RCaMP2 is characterized by two fluorescent states of equal amplitude with saturating rates of 15 ± 1 and 2.1 ± 0.1 s−1 , I and J 0.1 s−1 K.e.g. f-RGECO2 in faithful monitoring of even slow intracellular Ca2+ dynamics.We generated fast-decay variants of jRGECO1a and jRCaMP1a using the rational design strategy previously applied to GCaMPs , 24. In 2+ transients are used to visualize action potential firing. 10 bAPs were detected by our fast variants, with an up to 4-fold faster decay rate (τoff of 77 ms for f-RGECO2) compared with jRGECO1a. Fast jRCaMP1a variants f-RCaMP1 and f-RCaMP2 showed similar improvements in the decay kinetics with time constants of 211 and 140 ms, respectively, with the added advantage of not showing the photoswitching characteristic of jRGECO1a. Moreover, it should be noted that the in situ decay rates were ∼10-fold slower than those measured in solution, indicating that the probe kinetics are not limiting the detection of the Ca2+ transient.The advantage of the fast response kinetics of the probes is clearly seen in hippocampal CA3 pyramidal cells, in which rapid Ca2+ consistent with the equilibrium of the chromophore shifting to the protonated form. A small shift of 4 nm seen in the Ca2+-free state for jRGECO1a and jRCaMP1a to 566 and 574 nm, respectively, is similar to that observed for GCaMP variants and is caused by a change in the local electric field at the chromophore caused by Ca2+-dependent rearrangements of the surrounding residues  or had slightly lower affinities, with dK values of ∼700 nm. However, comparing the specific site mutation combinations best improving the kinetics and the fluorescence brightness and dynamic range reveals that interactions with the fluorescent proteins affect the properties of the Ca2+/CaM–RS20 peptide complex. For jRGECO1a mutation of the EF1 hand leads to faster Ca2+ dissociation rates but slows down the limiting association rates significantly (f-RGECO1 and jRGECO1a RS-1 EF-1). Although the mutation of the EF3/EF4 hands and RS-1 alone had little effect on the association and dissociation kinetics, the combination of RS-1 and EF-4 (f-RGECO2) generated a probe with ∼8-fold accelerated Ca2+ dissociation kinetics. For jRCaMP1a the EF-3, EF-4 and RS-1 mutation and the combination of them generated the fastest probes with up to 13-fold faster decay kinetics. This behavior resembles GCaMP3 and GCaMP6f with the combination of RS-1 and EF-3 mutations generating the fastest variants  showed a fast phase with a bell-shaped dependence on Ca2+ concentration and a slow phase the rate of which tended to 0, indicating that the dissociation process occurs by a different pathway from the association, by Ca2 dissociation followed by the peptide . It was thus hypothesized that two fluorescent states were formed in parallel processes. The fast phase could correspond to the Ca2+-bound CaM N-lobe forming a fluorescent complex with the peptide. This complex then would be able to bind two more Ca2+ at the CaM C-lobe. In parallel, the fully Ca2+-bound CaM forms a peptide complex at the slower rate determined by the slower C-lobe Ca2+ binding . This finding is in agreement with previously proposed models, where a partially bound fluorescent complex (Ca2+2N.CaM.RS20*) was predicted, with the possibility of binding two further Ca2+ to the C-lobe to generate the fully bound and highly fluorescent complex (Ca2+4·CaM.RS20*)  , 24. The .In contrast to the jRGECO1a variants with bell-shaped Ca2+] dependence of the observed association rates. The parental variant and all others that do not contain the RS-1 mutation had two phases, with the fast one following a sigmoidal saturating Ca2+ dependence and the slow one Ca2+-independent, suggesting the formation of two fluorescent states, which show biexponential dissociation kinetics. In contrast, all jRCaMP1a variants carrying the RS-1 mutation seem to miss the Ca2+-independent slow rate. However, measurements at 37 °C for f-RCaMP1 revealed a slow phase with small amplitude. Furthermore jRCaMP1a RS-1 EF-3 and jRCaMP1a RS-1 EF-4 show a biexponential dissociation like their parental GECI. These results indicate that the weakening of the interaction of the CaM C-lobe with the N terminus of the RS20 peptide intensively slows down the second isomerization until it becomes undetectable at 20 °C. Although this model explains the observed kinetics of almost all jRCaMP1a variants and the influence of the RS-1 mutation, f-RCaMP2 and f-RGECO1 seem to follow their own specific pattern. Both observed rates decrease slightly with increasing [Ca2+] and are almost [Ca2+]-independent. Such behavior may be explained with a model involving two Ca2+-free states that are in equilibrium with each other, with only one able to bind Ca2+. Thus, the association rates would not increase with increasing [Ca2+] but are rather entirely dependent on this pre-equilibrium. This model describes similar kinetics observed for enzymes binding to ligands, that have an open (ligand can bind) and closed (ligand cannot bind) conformation  is much2Y (3WLD ) with reode 3U0K ), the he2+ indicators, similarly to GCaMPs. However, RGECI variants differ from GCaMPs in their mechanism with the N-lobe binding as a required step for fluorescence increase. Furthermore, the red GECIs show a far more complex variety of kinetic responses, which were explained by and fitted to simple models. jRGECO1a and jRCaMP1a are able to detect a single action potential in vivo  . jRCaMP1Escherichia coli. Mutations in the EF hands and the RS20 peptide were performed using the QuikChange XL mutagenesis protocol using the following 5′ to 3′ primers: for jRCaMP1a and jRGECO1a: RS-1 W44Y, CGACTCATCACGTCGTAAGTACAATAAGGCAGGTCACGCAG; EF-1 D323A, GCTTTCTCCCTATTTGCCAAGGACGGGGATGGG; EF-2 D359A, CATGATCAATGAAGTAGCTGCCGACGGTGACGGC; and only for jRGECO1a: EF-3 D396A, GCGTTCCGCGTGTTTGCTAAGGACGGCAATGGC; EF-4 D432A, GATCAGGGTAGCAGCCATCGATGGGGATGG; and only for jRCaMP1a: EF-3 D396A, GCGTTCGGCGTGTTTGCTAAGGATGGCAATGGC; EF-4 D432A, GATCAGGGAAGCAGCCAGCGATGGGGATGG. Mutations were confirmed by DNA sequencing (Genewiz®).The pET30b-jRCaMP1a and pET30b-jRGECO1a plasmids were used for heterologous expression in E. coli BL21 (DE3) gold cells. Cells were grown at 37 °C, and protein expression was induced at A600 of 0.8 with 0.4 mm isopropyl β-d-1-thiogalactopyranoside overnight at 20 °C. The cells were harvested, resuspended in 50 mm Na+-HEPES, 200 mm NaCl, pH 7.5, containing protease inhibitors . The cells were lysed by sonication on ice . Protein was purified using a HisTrap column with an Äkta Explorer system . Protein was eluted in 50 mm Na+-HEPES, 200 mm NaCl, pH 7.5, containing 500 mm imidazole. Fractions containing the protein were dialyzed overnight against storage buffer  at 4 °C. Purity was checked by SDS-PAGE (∼90% pure), and aliquots were stored at −80 °C. Protein concentrations were determined spectroscopically using extinction coefficients calculated from their amino acid sequence (0(280 nm) = 40,680 m−1 cm−1, all jRGECO1a variants containing RS-1 mutation ϵ0(280 nm) = 36,270 m−1 cm−1, jRCaMP1a and all EF mutants ϵ0(280 nm) = 32,430 m−1 cm−1, all jRCaMP1a variants containing RS-1 mutation ϵ0(280 nm) = 28,020 m−1 cm−1).Proteins were expressed using sequence   at different pH levels were recorded at a Fluorolog3 (Horiba©). Buffer solutions with 50 mm buffer , 100 mm KCl, and 2 mm MgCl2 containing either 1 mm CaCl2 or 2 mm BAPTA were prepared and protein to a final concentration of 1 μm was added. BAPTA was chosen as Ca2+ chelator because of its stable Ca2+ affinity over a wide pH range.To determine the pm K+-HEPES, 100 mm KCl, 2 mm MgCl2 pH 7.5) containing either 1 mm CaCl2 or 2 mm EGTA were measured with a Lambda Bio40 (PerkinElmer Life Sciences) and the fluorescence emission spectra  were recorded at a Fluorolog3 (Horiba). The maximum absorption was plotted against the integrated fluorescence, and the points were fitted with a linear regression line. The quantum yield of the variants was calculated by using the slope and the reported quantum yield of the parental GECIs: jRGECO1a Φ(+Ca2+) = 0.220, Φ(−Ca2+) = 0.120; RCaMP1a Φ(+Ca2+) = 0.515, Φ(−Ca2+) = 0.308 . Extinction coefficients ϵ0 at 570 nm were determined using the protein concentration calculated from the absorption at 280 nm and the ϵ0(280 nm) values (see “Protein purification and expression” above). The brightness was calculated by multiplying the extinction coefficient by the quantum yield.The fluorescence quantum yield (Φ) was determined relative to the parental red GECIs jRCaMP1a and jRGECO1a . The absdK) values were determined by following the change in fluorescence emission  on a Fluorolog3 (Horiba) with increasing Ca2+ concentrations. Ca2+ solution (325 mm) was titrated using an ALLADIN syringe pump (10 μl/min) into the protein solution in assay buffer containing 5 mm BAPTA, pH 7.5, while constantly stirring. The fluorescence was corrected for the dilution, and the free [Ca2+] was calculated using MaxChelator® software. The data were fitted with the Hill equation for specific binding using GraphPad Prism 6 (y = Bmax*nx/(dnK + nx)).The dissociation constant  from the maxima of the spectra obtained in the pH titrations in assay buffer with 1 mm CaCl2 or 2 mm BAPTA, (ii) calculated from the ratio of brightness obtained from the quantum yield measurements, and (iii) the ratio of the end point (1 mm [Ca2+]) and the starting fluorescence values (5 mm EGTA) in the titration experiments.The fluorescence dynamic range TM double-mixing stopped-flow apparatus was used in single mixing mode. Fluorescence was excited at 577 nm, and emission was detected using a long pass filter (>590 nm). For Ca2+ association kinetics, RGECI (concentration in the 0.1–1 μm range) in assay buffer  containing 10 mm EGTA was rapidly mixed with assay buffer containing increasing concentration of [Ca2+]. For dissociation kinetics, RGECI in assay buffer containing 0.5 mm CaCl2 was rapidly mixed with assay buffer containing 12.5 mm EGTA (concentrations in mixing chamber). [Ca2+] concentrations were calculated using MaxChelator® software. The data were fitted either with a monoexponential or a biexponential function.A Hi-Tech Scientific KinetAsyst2 in a black 24-well glass-bottomed plate in Dulbecco's modified Eagle's medium containing nonessential amino acids (Life Technologies), 10% heat-inactivated fetal bovine serum (Life Technologies) and penicillin/streptomycin . Transfection of cells was carried out with 0.5 μg of DNA and 2.5 μl of Lipofectamine 2000 in 100 μl of OptiMem® for 16–48 h at 37 °C. The cells were imaged 16–48 h post-transfection in 2 or 1 ml of OptiMem®. The cells were imaged at 37 °C (OKO lab incubation chamber) with a 3i Marianas spinning-disk confocal microscope equipped with a Zeiss AxioObserver Z1, a 40×/NA1.3 oil immersion objective, and a 3i Laserstack as excitation light source (568 nm). Emitted light was recorded through a band bass filter (Yokogawa CSU-X filter wheel) by a CMOS at 1152 × 1656 pixel size. Time-lapse images were recorded at 2-s intervals for 3 min . Elliptical regions of interest were stacked, and fluorescence intensities were analyzed using ImageJ program. The data obtained from 13–30 cells was plotted and analyzed in OriginPro 9. Fluorescence decay times were determined from single exponential fits to the data.The cells were cultured at 37 °C and 5% COCCATGGTGCAGAACGAGCTTGCTCTTAAG; and EcoRI-pCI-RGECO-fw, CATGCAGAATTCATGCTGCAGAACGAGCTTGCTCTTAAG.The RGECI variants were each subcloned into an expression vector (pCI) under the control of the human synapsin1 promoter by PCR using the following primers  and restriction ligation using NcoI/NotI for jRCaMP1a variants and EcoRI/NotI for jRGECO1a variants and T4 DNA ligase. Successful cloning was confirmed by DNA sequencing (Genewiz®): NcoI-pCI-RCaMP-fw, TCCAm potassium gluconate, 4 mm MgCl2, 4 mm Na2-ATP, 0.4 mm Na-GTP, 10 mm Na2-phosphocreatine, 3 mm ascorbate, and 10 mm HEPES, pH 7.2. CA3 pyramidal neurons were co-transfected by single cell electroporation at days 10–15 in culture with a mixture of the RGECI and the green morphological marker. For the electroporation procedure, slice cultures were kept in 145 mm NaCl, 25 mmd-glucose, 2.5 mm KCl, 1 mm MgCl2, 2 mm CaCl2, and 10 mm HEPES, pH 7.4 (sterile filtered). Thin-wall glass pipettes with a resistance of 12–14 MΩ were filled with a potassium gluconate–based solution containing the mixture of the two plasmids. An Axoporator 800A (Molecular Devices) was used to deliver 50 voltage pulses  at 50 Hz , NBQX (10 μm), and picrotoxin (100 μm) were added to the recording solution. Patch pipettes with a tip resistance of 3.5–4.5 mΩ were filled with 135 mm potassium gluconate, 4 mm MgCl2, 4 mm Na2-ATP, 0.4 mm Na-GTP, 10 mm Na2-phospho-creatine, 3 mm ascorbate, and 10 mm HEPES, pH 7.2. Whole-cell recordings from transfected CA3 pyramidal neurons were made with a Multiclamp 700B amplifier (Molecular Devices) under the control of Ephus software written in Matlab (The experiments were performed 8–10 days after electroporation when 50 ng/μl of red GECI was electroporated or 3–5 days after electroporation when 100 ng/μl of red GECI was electroporated. Recordings were performed in a recording solution containing 135 mn Matlab . The anaThe custom-built two-photon imaging setup was based on an Olympus BX51WI microscope controlled by a customized version of the open-source software package ScanImage  written S. K. and C. C. data curation; S. K., C. C., and C. D. D. formal analysis; S. K. and C. D. D. investigation; S. K. and K. T. methodology; S. K. and K. T. writing-original draft; S. K. and K. T. writing-review and editing; T. G. O. and K. T. conceptualization; T. G. O. and K. T. supervision; T. G. O. validation; K. T. resources; K. T. funding acquisition.


The identification of heterozygous neomorphic isocitrate dehydrogenase (IDH) mutations across multiple cancer types including both solid and hematologic malignancies has revolutionized our understanding of oncogenesis in these malignancies and the potential for targeted therapeutics using small molecule inhibitors. The neomorphic mutation in IDH generates an oncometabolite product, 2-hydroxyglutarate (2HG), which has been linked to the disruption of metabolic and epigenetic mechanisms responsible for cellular differentiation and is likely an early and critical contributor to oncogenesis. In the past 2 years, two mutant IDH (mutIDH) inhibitors, Enasidenib (AG-221), and Ivosidenib (AG-120), have been FDA-approved for IDH-mutant relapsed or refractory acute myeloid leukemia (AML) based on phase 1 safety and efficacy data and continue to be studied in trials in hematologic malignancies, as well as in glioma, cholangiocarcinoma, and chondrosarcoma. In this review, we will summarize the molecular pathways and oncogenic consequences associated with mutIDH with a particular emphasis on glioma and AML, and systematically review the development and preclinical testing of mutIDH inhibitors. Existing clinical data in both hematologic and solid tumors will likewise be reviewed followed by a discussion on the potential limitations of mutIDH inhibitor monotherapy and potential routes for treatment optimization using combination therapy. The discovery of mutations in isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2) in over 80% of low-grade gliomas (LGGs) and secondary glioblastomas has revolutionized pharmaceutical approaches to targeted therapies and the overall glioma classification schema –6. LimitIDH1 and IDH2 are homodimeric isoenzymes involved in a major pathway for cellular NADPH generation through the oxidative decarboxylation of isocitrate to α-ketoglutarate. IDH1 is found in the cytosol and in peroxisomes, while IDH2 is a mitochondrial enzyme. Mutations in IDH3 isoforms, which form heterotetrameric complexes in mitochondria, are rarely seen in cancer, but there is some evidence that upregulation of wild-type IDH3 may contribute to various tumorigenic metabolic pathways , 15. TheAccumulation of 2HG, increasingly well-characterized as an oncometabolite, disrupts multiple regulatory cellular pathways involving α-ketoglutarate-dependent dioxygenases including those involved in epigenetic remodeling and DNA repair  21–23).–23.21–23Here, we provide an overview of the current literature on IDH mutations in cancer with a particular emphasis on glioma and AML and the potential for mutIDH as a therapeutic target in these contexts. We describe the current evidence for the various generations of mutIDH inhibitors through the drug-discovery, preclinical, and clinical stages and systematically review related past and ongoing clinical trials. We furthermore describe the possible adverse effects of IDH inhibitors, such as “differentiation syndrome,” and conclude with a discussion on the potential for enhancing the efficacy of IDH inhibitors in combination with epigenetic modification-based therapies.Ten years ago, our understanding of the molecular landscape in glioma was transformed by the first genome-wide analysis of somatic mutations in glioblastoma (GBM) and the identification of recurrent mutations in IDH1 nearly exclusively in secondary GBM . MutatioIDH-mutant gliomas are generally further categorized into two major subtypes: those with chromosome 1p/19q co-deletion, historically termed oligodendrogliomas; and those without 1p/19q co-deletion, also known as astrocytomas . These tConsistent with other IDH-mutant cancers, IDH-mutant glioma is characterized by high levels of 2HG and the resulting “CIMP” hypermethylator phenotype described previously. In glioma specifically, these genome-wide DNA methylation changes have been shown to establish “insulator dysfunction” or disruption of topologically-associated domains (TADs) and thereby directly influence key transcriptional regulatory pathways related to gliomagenesis , 39. As R172 residue was mutated  is the most common, with the IDH1R132 and IDH2R172 mutations also appearing frequently in the literature  is an orally available pan-inhibitor of both mutIDH1 and mutIDH2 and was the first pan-inhibitor developed under the Celgene and Agios Pharmaceuticals collaboration  60–62).–62.60–62 periods . IC50 fontiation .50 ranging from 0.6 to 17.1 μM—for further evaluation. Optimization of a lead compound based on differential inhibition of mutIDH1 and wild-type IDH1 enzymes resulted in BAY-1436032, an allosteric inhibitor that binds at the IDH dimer interface  and genetically engineered cell lines representative of solid tumors (IC50 13–135 nM) . In. In50 ra–135 nM) , 63. Adda models , 63. In nographs , 64. TwoR132H U87 xenografts (IC50 0.07 μM in vitro)  , 67. Rohn vitro) . Early an vitro) . Work byantiomer . The preantiomer .R132H colorectal cancer cell line (IC50 24 nM), low liver microsomal clearance values, and substantial brain penetrance in murine models . ID. ID71). e models .R140Q mutant enzyme specifically and exhibits non-competitive inhibition with respect to substrate, and uncompetitive inhibition with respect to the NADPH cofactor, operating at an allosteric site at the enzyme's dimer interface (50 11nM in IDH2R140Q-overexpressing U87 cells) , 73R140Q ex vivo . Lack ofin silico computational screening and validated in vitro in murine bone marrow cells transduced with IDH1R132C to deplete 2HG production with an IC50 of 1μM and in vivo by prolonging survival in a leukemia mouse model  or AG-221, developed by Celgene Corporation under a license from Agios Pharmaceuticals. Enasidenib is a first-in-class, oral, selective inhibitor of the mutIDH2 enzyme. A precursor form of Enasidenib was originally selected from a high-throughput screen for inhibitors of the most prevalent form of mutIDH2 in AML, IDH2R140Q .50 of 0.03 μM for the most prevalent oncogenic enzyme, the IDH2R140Q/WT heterodimer, and also for the IDH2R172K/WT enzyme (IC50 = 0.01 μM)  , 86. Robutations . Furtherutations . In vivoe models . These re models .®), the mutIDH1-specific counterpart to Enasidenib, was developed  out of efforts to optimize AGI-5198 for human therapeutic applications (Ivosidenib (Tibsovoications . Poor phications –68. Broaications  (68).R132 mutants in a series of cell lines in a selective manner, showing no inhibition of wild-type or mutIDH2 isoforms (R132H glioma xenografts (IC50 range 5–13 nM for various IDH1 mutants in vitro)  , 88, 89.n vitro) . These en vitro) .A summary of the completed, ongoing, recruiting, and planned clinical trials of mutIDH inhibitors in AML and other hematological malignancies can be found in R140Q patients and up to 94% in IDH2R172K patients. In patients with relapsed or refractory AML, 38.5% of patients receiving the expansion dose demonstrated a clinical response with a median 5.6 months response duration   paved the way for FDA approval of the first small molecule mutIDH inhibitor in cancer and propelled global clinical research efforts for IDH-based targeted cancer treatments  . In thisesponse) . Unlike esponse) . A similSince the success of the Enasidenib phase 1/2 trial in myeloid malignancies and FDA approval for the indication of relapsed/refractory IDH2-mutant AML was obtained in 2017, clinical trials investigating the efficacy of mutIDH2 inhibitors, mostly Enasidenib, in various AML subpopulations have become abundant. One, which is actively recruiting, is analyzing the safety of Enasidenib when given in combination with standard AML induction and consolidation chemotherapy in newly diagnosed AML . Another recruiting phase 1 trial is investigating Enasidenib as a maintenance therapy for IDH2-mutant AML or CML following allogeneic hematopoietic stem cell transplantation . Two trials, a phase 1b/2 and a phase 2, are evaluating Enasidenib in combination with a DNA methyltransferase (DNMT) inhibitor, azacitidine, in newly-diagnosed and relapsed/refractory AML, respectively . Another phase 1b/2 trial is studying biomarker-based treatment of AML more broadly by using genomic screening to identify specific subtypes of AML (including IDH2-mutant) and assign each subtype to a distinct treatment regimen—IDH2-mutant AML will be assigned to Enasidenib treatment . There is one phase 3 randomized, open-label study comparing Enasidenib to conventional care specifically in patients age 60 or older with relapsed/refractory IDH2-mutant advanced AML, also known as IDHENTIFY . The IDHENTIFY protocol was presented at the 2017 American Society of Clinical Oncology (ASCO) Annual Meeting . A few cShortly after the approval of Enasidenib, its mutIDH1 counterpart, Ivosidenib, similarly demonstrated safety and clinical efficacy in a phase 1/2 dose-escalation/dose-expansion trial of relapsed/refractory IDH1-mutant AML  . In thisThere are several trials studying Ivosidenib in AML in various treatment contexts. The previously mentioned currently recruiting phase 1b/2 study investigating biomarker-based treatment of AML also includes an Ivosidenib arm for those patients with IDH1 mutations . Another trial in the recruitment phase is examining simultaneous treatment with Ivosidenib and the chronic lymphocytic leukemia drug venetoclax in patients with relapsed or refractory AML (NCT03471260). In another currently-recruiting phase 3 study azacitidine is combined with either Ivosidenib or placebo as therapy for previously untreated AML with IDH1 mutations (NCT03173248). This study is planning on enrolling 392 patients, but data as of September 2017 indicated that out of 11 patients who received both Ivosidenib and azacitidine, 8 of them achieved a response, including 4 who had complete remission . PrelimiR132-mutant AML started in 2017 with no results published to date . A phase 1/2 study is evaluating the use of FT-2102 alone or with azacitidine in patients with AML or MDS carrying IDH1 mutations . Results presented at the 2018 ASCO Annual Meeting from data on 57 patients  demonstrated a complete response rate of 38% with monotherapy and 27% with combination therapy (n = 24), complete remission was achieved in 2 patients (9.5%), complete remission with incomplete recovery in 1 patient (4.8%), and partial remission in 4 patients (19%)  . Notablyts (19%) .Several clinical trials studying mutIDH inhibitors in glioma and other solid tumors are ongoing or in planning stages and involve both pan and specific inhibitors . Early rIn 2015, the first phase 1 data on the safety of Ivosidenib in patients with advanced glioma and other IDH-mutant solid tumors was presented at the annual EORTC-NCI-AAR Molecular Targets and Cancer Therapeutics Symposium and indicated that Ivosidenib treatment was well-tolerated with a positive pharmacokinetic profile  . This muAG-881 (Vorasidenib), a pan-inhibitor, is the only other mutIDH inhibitor currently with supporting clinical data in glioma. In a similarly designed, phase 1, multicenter, open-label, dose-escalation and expansion trial, the safety and pharmacokinetic profiles of AG-881 is being investigated in both mutIDH1 and mutIDH2 gliomas and other solid tumors  , 98. AccOther ongoing clinical trials of mutIDH inhibitor compounds in glioma (currently without early results) are also in phase 1 and are mostly in the early recruitment phase. These studies are evaluating safety and pharmacology of mutIDH-specific inhibitors such as DS-1001b, IDH305, and FT-2012, Enasidenib, and BAY-1436032 . There is a single phase 3, multicenter, randomized, double-blind study in solid tumors comparing Ivosidenib (500 mg daily) to placebo in advanced or metastatic mutIDH1 cholangiocarcinoma, also known as the “ClarIDHy” trial  , nausea (26.9–35.5%), and dose-dependent transaminase elevation (without elevated bilirubin) (34.4–44.2%) , 98. AllPatients with hematologic malignancy, on the other hand, experience a greater incidence of more critical treatment-related adverse events that are more critical in nature. As previously discussed, 2017 and 2018 saw the respective publications of the results of two landmark clinical trials in AML: first, a phase 1/2 study of the selective IDH2 mutant inhibitor Enasidenib, and second, a phase 1 dose-escalation and dose-expansion study of the selective IDH1 mutant inhibitor Ivosidenib , 13. In trans retinoic acid (ATRA) (Differentiation syndrome (DS), previously called “retinoic acid syndrome,” was originally described over a quarter-century ago as an adverse event in patients with acute promyelocytic leukemia (APL) undergoing treatment with all-d (ATRA) . This lid (ATRA) . A simild (ATRA) .IDH-DS can first develop up to several months following the initiation of IDH mutant inhibitor treatment for hematologic malignancy and shares distinctive symptomology with the retinoic acid syndrome: culture-negative fever, rapid weight gain or edema, respiratory symptoms with or without infiltrates, pleural or pericardial effusions, hypotension, and acute renal failure , 106. DiWhile exact mechanisms and pathophysiology remain poorly understood, differentiation syndromes are generally thought to be inflammatory phenomena resulting from cytokine release during widespread drug-induced differentiation of immature blasts into mature cell types . While nMutIDH inhibitors have demonstrated some efficacy in IDH-mutant AML and early results have validated their safety in glioma patients. However, given the extent of hypermethylation induced early in oncogenesis by the IDH mutation, mutIDH inhibitor monotherapy may be insufficient to thoroughly undo these preexisting and persistent global epigenetic alterations keeping the tumor cells in a dedifferentiated state. Preclinical evidence, particularly in glioma, supports the hypothesis that mutIDH assumes a “passenger,” rather than a “driver,” role soon after the development of the IDH mutation likely due to initiating methylation changes being preserved by epigenetic memory and consequent acquisition of additional driver mutations , 81.in vitro human IDH1R132H TF-1 erythroleukemia cells demonstrated increased cell differentiation by a factor of 25 after Ivosidenib treatment alone, which improved to a 46-fold increase with concurrent azacitidine treatment  inhibitors, to a mutIDH inhibitor regimen may generate a synergistic response by dually targeting the CIMP phenotype. In AML, it is known that patients with IDH mutations are up to 14 times more likely to response to DNMT inhibitors . In precreatment . Severalreatment . In the reatment . A phasereatment .In IDH-mutant glioma, multiple DNMT inhibitors have demonstrated efficacy in preclinical studies to induce cellular differentiation, reduce global methylation and inhibit growth , 122. A Epigenetic memory in IDH-mutant cancers is likely not limited to DNA methylation alone. RNA methylation patterns have also been shown to be critical for glioblastoma stem cell self-renewal and tumorigenesis specifically, and as previously discussed, the early molecular insults of IDH mutations and 2HG include competitive inhibition of histone demethylases , 125. WhR132C-mutant AML that, after achieving remission on Ivosidenib, both recurred with new IDH2R140Q mutations , a mutIDH2-specific inhibitor, and Ivosidenib AG-120), a mutIDH1-specific inhibitor, have demonstrated safety and efficacy in phase 1 clinical trials for relapsed or refractory AML and recently earned FDA approval for this indication 0, a mutI. They arIn the context of this narrative review article, no consent, or IRB approval was required by the authors' institution given that no human subjects were involved in this research.DG, NI, SD, TW, KT, and AM manuscript drafting. DG, NI, SD, TW, and DB figure and table design. DG, NI, SD, TW, DB, KT, AM, and DP manuscript editing and revision. DG, NI, SD, and TW literature review. DG, NI, and SD clinical trials review. DG, DB, KT, AM, and DP research team management and oversight.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.