In pancreatic β-cells, both lipid and Ca2+ signaling are essential for insulin secretion. The recently characterized lipid transfer protein TMEM24  dynamically localizes to ER–PM contact sites and provides phosphatidylinositol, a precursor of phosphatidylinositol-4-phosphate [PI(4)P] and phosphatidylinositol 4,5-bisphosphate [PIP2], to the PM. β-cells lacking TMEM24 exhibit markedly suppressed glucose-induced Ca2+ oscillations and insulin secretion, but the underlying mechanism is not known. We now show that TMEM24 only weakly interacts with the PM, and dissociates in response to both diacylglycerol and nanomolar elevations of cytosolic Ca2+. Loss of TMEM24 results in hyper-accumulation of Ca2+ in the ER and in excess Ca2+ entry into mitochondria, with resulting impairment in glucose-stimulated ATP production.Endoplasmic reticulum (ER)–plasma membrane (PM) contacts are sites of lipid exchange and Ca Summary: TMEM24 reversibly localizes to ER–PM contact sites and participates in the regulation of both ER and mitochondrial Ca2+ homeostasis and in glucose-dependent ATP production in insulin-secreting cells. These junctions are also important sites for cellular Cantration . The chaustering . TMEM24 ring (2] . It bindlevation .+ (KATP)-channels, causing membrane depolarization, opening of voltage-dependent Ca2+ channels, Ca2+ influx and the fusion of insulin-containing granules with the PM , which triggers Ca2+ release from the ER and may contribute to insulin granule exocytosis, and diacylglycerol (DAG), which together with Ca2+ amplifies secretion by stimulating the PKC activity P2 levels are unaltered in TMEM24 KO cells  microscopy (2+ influx being the trigger for dissociation (2+ from the ER (2+ using the sarcoendoplasmic reticulum (SR) Ca2+ transport ATPase (SERCA) inhibitor cyclopiazonic acid (CPA) also triggered TMEM24 dissociation, although less prominently than in response to carbachol . This resulted in an immediate dissociation of TMEM24 from the PM without apparent change in the cytosolic Ca2+ concentration . To further uncouple DAG formation from Ca2+ changes, we first exposed cells to CPA to deplete ER Ca2+ stores, followed by addition of carbachol to activate PLC and increase PM DAG. CPA caused an increase in cytosolic Ca2+ and a slight dissociation of GFP–TMEM24 from the PM. The subsequent addition of carbachol resulted in a pronounced dissociation of TMEM24 that occurred in the absence of noticeable changes in cytosolic Ca2+ concentration . These results indicate that TMEM24 is spatially controlled by both Ca2+ and DAG. We also noticed that whereas carbachol caused a homogenous dissociation of TMEM24 from the PM . Because of the close interplay between DAG and Ca2+, we determined the effect of known Ca2+ concentrations on TMEM24–mCherry PM binding in α-toxin-permeabilized MIN6 cells in which changes in Ca2+ could easily be uncoupled from those in DAG. We found that dissociation of TMEM24 occurred with an EC50 of 358±70 nM  and was maximal at 30 μM . For comparison, the PM binding of E-Syt1–mCherry, another Ca2+-dependent ER–PM contact protein, was triggered at 1 μM Ca2+ and was maximal at 10 μM Ca2+   (n=31). These results indicate that TMEM24 PM binding might be partially reduced already at resting Ca2+ concentrations. Fluorescence recovery after photobleaching (FRAP) analysis revealed that TMEM24–GFP, although enriched at ER–PM contacts, exhibited pronounced dynamics with a large proportion belonging to a mobile fraction  G. When c P<0.01) H while tfraction I. Taken 2+ influx and insulin release, although the mechanisms are unclear  reduction in TMEM24 expression as assessed by western blotting   due to ATP-driven sequestration of the ion into the ER  and was followed after 3.31±0.11 min  by a sustained rise of Ca2+ with regular oscillations  in the TMEM24 KO cells  A. We nexo the ER , followelevation B–E. Mostesponses B. In TMEllations B,C,E. Thcontrols D. Overexol cells C–E. To aSPR/Cas9 F. Both cKO cells G–I. Simi glucose J,K. Bothllations B,G.Fig.2+ influx observed here, we decided to reinvestigate the previously proposed fundamental role of TMEM24 in insulin secretion  in wild-type cells. This response was increased to 441±8 nM  in TMEM24 KO cells   increase in the cytosolic Ca2+ concentration, reflecting the release of Ca2+ from the ER. This release was increased to 48±2 nM  in TMEM24 KO cells  and when ER Ca2+-store depletion was instead triggered by the addition of thapsigargin in the presence of extracellular Ca2+. Importantly, the response in TMEM24 KO cells returned to normal upon the re-expression of wild-type TMEM24 . Direct measurements of ER Ca2+ using the FRET-based Ca2+ sensor D4ER confirmed that the ER of TMEM24 KO cells contained more Ca2+ than that of wild-type cells under resting conditions, and also that more Ca2+ was released from the ER following SERCA inhibition with CPA  G. SimilaP=0.019) D–F,H. SeKO cells I,J. The ll lines K. Similae TMEM24 L, whereawith CPA M,N. We srvations , preventpe cells N,O. Thispe cells P,Q. Thes2+ influx, extrusion and organellar sequestration all depends on ATP generated primarily via mitochondrial metabolism, we decided to complement the Ca2+ imaging with direct measurement of glucose metabolism using the Seahorse XF technique. As expected from the Ca2+ imaging data, TMEM24 KO had little effect on the resting oxygen consumption rate (OCR)  A. In conte (OCR) B. Howevete (OCR) C–F. Thiste (OCR) G,H. Nextte (OCR) I,J. The see e.g. B. Consisme cells K,L. In cme cells K,L. Simihibition M,N. A pope cells O.Fig. 42+ increases that was reversed when the depolarizing agent was removed . When expressed in MIN6 cells, we observed red fluorescent structures that overlapped with MitoTracker, likely reflecting ER–mitochondria contacts . Co-expression of TMEM24–GFP revealed little overlap under resting conditions, whereas accumulation at sites of ER–mitochondria proximity became apparent after depolarization-induced Ca2+ increases  we found that the sustained glucose-stimulated insulin secretion was reduced following TMEM24 KO. One previous study, in which TMEM24 expression was stably reduced by shRNA, showed impaired glucose-stimulated insulin secretion from both clonal rat INS1 and mouse MIN6 β-cell pseudo-islets. The Ca2+ responses in these cells were normal, and secretion in response to direct depolarization was also unaffected by TMEM24 knockdown  inhibitor thapsigargin causes Ca2+ oscillations in these cells , SERCA, Presenilin-1 and IP3 receptors P2 on the mitochondria surface caused mitochondrial fragmentation, which could be prevented by PKC activation and would also trigger TMEM24 dissociation from the PM to enable interactions with the mitochondria . TMEM24–mCherry was generated by PCR amplification of human TMEM24 with flanking Nhe1 and EcoR1 followed by ligation into the mCherry-N1 vector (gift from Pietro De Camilli) using the following primers: TMEM24-Nhe1-fwd, 5′-CTAGCTAGCATGGATCCGGGCTGGGGGCA-3′; TMEM24-EcoR1-rev, 5′-CCGGAATTCTGAGCTGGGGGCTGGGGTT-3′. RA-Sec61b and GB-Dcp1b Addgene plasmids #153978 and #153979  with 0.2 µg plasmid  and 0.5 µl Lipofectamine 2000 (Life Technologies) and seeded in the center of a 25-mm poly-L-lysine-coated coverslip. The transfection reaction was terminated after 4–6 h by the addition of 2 ml complete culture medium and cells were imaged 18–24 h later. MIN6 pseudo-islets were generated by resuspending 1×106 MIN6 cells in 1 ml complete culture medium and seeding them into a non-stick 12-well cell culture plate  followed by culturing for 48–72 h at 37°C and 5% CO2 in a humidified incubator. This resulted in the spontaneous formation of islet-like cell clusters. Cell cultures were routinely tested for mycoplasma infections (Eurofins genomics).The mouse β-cell line MIN6   was cult2, 1.2 mM CaCl2, 25 mM HEPES, 1 mg/ml BSA with pH set to 7.4) to an intracellular-like medium (see below), the superfusion was interrupted and α-toxin was added directly to the chamber . Permeabilization was considered complete when the Cal520 fluorescence had decreased by >90%, which typically took 2–5 min. Superfusion was then started again and the cells were exposed to intracellular-like buffers containing calibrated Ca2+ concentrations while fluorescence from both remaining Cal520 and mCherry-tagged fusion proteins was recorded. These experiments were performed at ambient temperature (21–23°C).Transfected MIN6 cells, grown on 25-mm poly-L-lysine-coated glass coverslips, were incubated with 0.5 µM of the AM-ester form of Cal520  for 30 min at 37°C. The coverslips were subsequently used as exchangeable bottoms in a modified Sykes–Moore open superfusion chamber that was mounted on the stage of a TIRF microscope (described below) and connected to a peristaltic pump that allowed rapid change of medium. Following a change from normal, extracellular-like, medium  Mg2+, 0–100 μM (free) Ca2+, 1 mM Mg-ATP, 10 mM HEPES, 2 mM  EGTA and 2 mM  nitrilotriacetic acid (NTA) with pH adjusted to 7.00 at 22°C with 2 M KOH. The total concentration of Ca2+ and Mg2+ was calculated using the online version of MaxChelator (http://www.stanford.edu/~cpatton/webmaxcS.htm). Media were made fresh on the day of experiment and kept on ice. To validate the media composition, cells loaded with the Ca2+ indicator Cal520 were mounted on a TIRF microscope, permeabilized and exposed to media with increasing Ca2+ concentrations. From these data a dose–response curve was generated and the EC50 for Ca2+-binding to Cal520 was estimated to be 980 nM, an in situ estimation 3-fold higher than the reported in vitro KD of 320 nM.Intracellular-like medium with buffered pH,  was measured on an epifluorescence microscope setup (described above) using the ratiometric dye Fura-2 or the green-fluorescent dye Cal520. The cells were preincubated for 30 min at 37°C in imaging buffer supplemented with 1 µM of the AM-ester-form of the indicator, followed by repeated washing in imaging buffer and imaging. Mitochondrial Ca2+ was measured using the genetically encoded red fluorescence, low-affinity indicator mito-LAR-GECO .TIRF microscopy and confocal microscopy images were analyzed offline by Fiji . To detet-test were used. All data presented are from at least three biological replicates. For imaging data, n numbers represent individual cells and N numbers represents independent experiments. For all other data presented, n numbers indicate independent observations.One-way ANOVA followed by Tukey's post hoc test , Kruskal–Wallis one-way ANOVA followed by a Mann–Whitney U-test (for non-parametric data) or a two-tailed (paired or unpaired) Student’s


Research shows that trained mentors achieve better results than untrained ones. Their training should particularly address their expectations for their future mentoring. Our study involved 190 preservice teachers, potential mentors of ongoing school mentoring for primary and secondary school students of all grades. They were randomly assigned to one of four conditions in a 2-x-2 between-subjects design of mentoring type  and mentoring context (non-pandemic versus COVID-19 pandemic). Participants assessed mentoring conducted under these four conditions in terms of its appropriateness for achieving four mentoring program targets: learning, key skills, social targets, and problem coping. Participants were also asked to assess the resources available to achieve each program target. Overall, the potential mentors considered the various conditions to be suitable for achieving the four program targets. They were particularly favorable in their assessment of the possibility for the realization of learning targets. Likewise, they assumed that sufficient resources were available to achieve the targets. However, a repeated-measures MANOVA showed that the potential mentors considered more ambitious targets to be possible in traditional mentoring than in e-mentoring and normal  contextual conditions than during the COVID-19 pandemic. In contrast, they estimated the resources available to achieve the targets to be about the same in the four conditions. This indicates a decoupling of mentoring targets from the consideration of the resources needed to achieve them. This assumption was confirmed in correlation analyses and has implications for mentor training. Mentoring is a context-sensitive activity in many respects. This is evident both situationally and transsituationally. Situational context specificity of mentoring means that mentor–mentee interactions are themselves shaped by the particular context in which they take place. Research on this is extensive. Examples include the mentoring format  decision making . For schFrom a transsituational perspective, mentoring is effective to the extent to which is succeeds in preparing mentees to act competently in these target contexts. However, if target settings change, this may substantially impair the effectiveness of mentoring. The COVID-19 pandemic undoubtedly has such potential, particularly with respect to school mentoring. During the first months of 2020, governments around the world implemented various measures, such as mobility restrictions, curfews, and the closing of educational institutions , to slowHowever, interventions to mitigate the COVID-19 pandemic are not only changing transsituational aspects of school mentoring. Situational factors of mentoring interactions are also affected in various ways, such as with respect to the frequency of contact , the incOur research was conducted as part of several ongoing school mentoring programs . These tStudies show that trained mentors are, on average, more successful at providing mentoring , 2014. FThe focus of the NFR-M is the mentoring pathway, which is defined as a sequence of mentoring episodes leading to a mentoring target. However, mentoring can include multiple targets and additional intermediate objectives, which mentors in turn adapt situationally and individually in their activities . Two dimThe regulatory network dimension in the NFR-M refers to the entirety of processes that are regulated and orchestrated to reach a mentoring target. For example, mentors may differ in the relevance they ascribe to mentoring type and mentoring context when it comes to achieving the four mentoring targets . Moreover, mentors may also differ on how they perceive the resources at their disposal to achieve these targets. Successful mentoring requires a variety of resources to regulate mentoring episodes. The NFR-M differentiates between endogenous resources that lie within the mentee  and exogenous resources that lie outside the mentee, that is, in their environment  that can help to achieve mentoring targets. These resources can be combined by mentors based on the situation of the current mentoring episode or based on advanced planning of the mentoring pathway.The COVID-19 pandemic has the potential to influence situational as well as transsituational aspects of mentoring. We hypothesized that this should be reflected in both mentors’ perceived attainability of mentoring targets  and their perceived resources to achieve mentoring targets . Specifically, we were interested in seven research questions.Following the NFR-M , we diffQ1: Is potential mentors’ assessment of the suitability of school mentoring for achieving mentoring targets  influenced by the mentoring type and the mentoring context?Q2: Is potential mentors’ perception of the availability of resources for achieving mentoring targets  influenced by mentoring type and mentoring context?The next two questions address the achievability of the four mentoring targets and the availability of resources to achieve them. Specifically, the questions are as:Q3: In the estimation of potential mentors, which targets  can be achieved by school mentoring?Q4: How do potential mentors perceive the resources for achieving key targets of school mentoring ?Since mentoring type  and mentoring context (non-pandemic versus COVID-19 pandemic) may both influence the assessment of achievable targets and available resources, the next two research questions include these aspects.Q5: In the estimation of potential mentors, which targets  can be achieved by school mentoring depending on the type  and context  of mentoring?Q6: How do potential mentors perceive resources to achieve key targets of school mentoring  delivered either traditionally or as e-mentoring under either non-pandemic or COVID-19-pandemic conditions?The final research question addresses a possible explanation for the decline in mentor engagement during the mentoring process to the point of dropping out . ZieglerQ7: Do potential mentors take the resources into account when assessing the attainability of mentoring targets?The study reports findings from a larger study in which potential future mentors were asked about their expectations regarding school mentoring programs.All undergraduate preservice teachers at the university of the first author were contacted who were potential mentors for one of the school mentoring programs. If they were interested in participating, they were asked to complete an online survey. By participating in the survey, preservice teachers did not commit themselves to mentoring in one of the school mentoring programs. Only 5% of the participants did have previous experience as mentors. The online survey was open 1week before and 1week after the start of the 2020 fall term.Mage=21.25; SD=3.53), of which 69.5% were female and 30.5% male. The sex ratio was identical in the different study conditions . At the end of the survey, a manipulation check was carried out in which participants were asked to indicate for which mentoring type and which mentoring context they had answered. If they answered incorrectly, they were excluded on a case-by-case basis. Informed consent was obtained from all subjects. Anonymity was assured in accordance with EU data-protection regulations.Of the approximately 1,200 preservice teachers for primary and secondary schools contacted, a total of 190 potential mentors participated in the study  and mentoring context  as grouping variables. The groups differed in the introductory text that preceded the completion of the measurement instruments. The introductory text referred to either traditional mentoring or e-mentoring in non-pandemic times  or during the COVID-19 pandemic. The introductory text for the group of respondents being surveyed about e-mentoring during the COVID-19 pandemic was as:At the Department of School Research, School Development, and Evaluation, we are planning e-mentoring in which preservice teachers can participate as mentors. We would therefore like to know in advance what you think about e-mentoring during the COVID-19 pandemic.In the planned e-mentoring, mentor(s) and mentee(s) will meet several times a month online and communicate about current school teaching content. Your participation will help us greatly in designing the e-mentoring during the COVID-19 pandemic.In the introductory text for the group of respondents being surveyed about traditional in-person mentoring, e-mentoring was replaced by mentoring, and instead of online meetings, only meetings were mentioned. In the two survey versions that did not refer to the COVID-19 pandemic, a “general assessment” of the respective mentoring type was requested instead of an assessment “during the COVID-19 pandemic.”The study took place immediately before a second lockdown in Germany. At that time, the 7day incidence of those infected with COVID 19 exceeded the nationally defined critical level of 50 per 100,000 residents. In the city where the study was conducted, the 7day incidence even exceeded a 7day incidence of 100 newly infected persons.The potential mentors were asked about their gender and age. They then completed a standardized questionnaire, which took about 25min to complete.To measure respondents’ “regulatory networks” and “regulatory resources,” we surveyed respondents on 12 items related to four general targets of school mentoring, namely, learning , social relationships , key skills , and coping .not effective at all) to 6 (very effective). Only the endpoints of the scales were labeled.To measure respondents’ regulatory networks, potential mentors were asked to assess how well the 12 targets could be achieved in school mentoring of the respective condition. A sample item to assess respondents’ regulatory networks for e-mentoring in COVID-19 times reads, for example: “How effective is e-mentoring for supporting the following targets during the COVID-19 pandemic?” Each of the 12 targets described above were rated on a six-point Likert-type scale ranged from 1  are available?” Each of the 12 mentoring targets described above were rated on a six-point Likert-type scale with the endpoints labeled “not important at all” and “very important.”α=0.67 to α=0.78 for respondents’ regulatory networks  and from α=0.69 to α=0.79 for regulatory resources .All subscales consisted of three items and were sufficiently internally consistent, with values ranging from F values for each factor. Moreover, the homogeneity of variance–covariance matrix was tested with Box’ M test, and the assumption of sphericity was tested with Mauchly’s test. Correlation analysis was used to explore the bivariate relations between corresponding pairs of regulatory network and regulatory resources variables.In the first place, we conducted repeated-measures multivariate analyses of variance , which hMeans and standard deviations of the dependent variables broken down by mentoring type and mentoring context can be found in F=6.91, p<0.01, partial η2=0.13 for mentoring type; F=26.31, p<0.001, partial η2=0.04 for mentoring context. The interaction of mentoring type and mentoring context did not reach the specified significance level, F=2.07, p=0.15, partial 2η=0.01. Participants in our study thought that mentoring targets were easier to attain in traditional mentoring than in e-mentoring and under non-pandemic contextual conditions than during the COVID-19 pandemic.Considering the repeated-measures multivariate analyses of variance (MANOVA), the Box’s M test values were not statistically significant, showing homogeneity of variance–covariance matrices that were appropriate for the subsequent analyses. The MANOVA showed two statistically significant differences on the combined regulatory network indicators, F=1.72, p=0.19, partial 2η=0.01 for mentoring type; F=0.06, p=0.80, partial 2η=0.00 for mentoring context; and F=0.06, p=0.80, partial 2η=0.00 for the interaction of mentoring type and mentoring context. Thus, participants in our study did not differentiate in terms of perceived resources between traditional mentoring and e-mentoring or between non-pandemic times and COVID-19-pandemic times. These findings may indicate that the potential mentors focused primarily on the targets of their mentoring without thinking more deeply about the resources needed to achieve these targets.The MANOVA showed no statistically significant differences on the combined regulatory resources indicators, with F =51.70, p<0.001, partial η2=0.48, Wilk’s Λ=0.52. Contrasts revealed that respondents viewed learning targets as probably more achievable than the other targets, with F=59.48, p<0.001, partial η2=0.26; respondents deemed coping targets probably less likely to be achieved, with F=49,49, p<0.001, partial η2=0.35.A look at the mean scores of the scales shows that all of them are significantly above the scale mean. Thus, school mentoring is considered likely to achieve the four targets of learning, social relationships, key skills, and coping. However, the MANOVA showed mean differences between the network indicators, with A look at the mean values in X2 =17.04, p=0.004. A repeated-measures MANOVA with a Greenhouse–Geisser correction determined a statistically significant difference between regulatory resources, F=76.81, p<0.001, partial η2=0.31. Contrasts revealed that respondents estimated higher values for learning resources than the other resources, F=75.53, p<0.001, partial η2=0.30. Respondents estimated lower values for social resources than for the other resources, F=109.98, p<0.001, partial η2=0.39.Mauchly’s test indicated that the assumption of sphericity had been violated for regulatory resources indicators, ps>0.05).We calculated difference contrasts for the interaction terms from regulatory network indicators and mentoring type and mentoring context. However, no significant interactions were found , key skills (M=4.52 versus M=4.31), and targets related to coping (M=4.4.7 versus M=4.51) as being more favorable or equally favorable during COVID-19-pandemic conditions in comparison with non-pandemic conditions, they rated the availability of resources for achieving social targets as being less auspicious under non-pandemic conditions than under COVID-19-pandemic conditions (M=3.92 versus M=4.24).We again calculated difference contrasts of the interaction terms. While no significant effects emerged for mentoring type, a counterintuitive finding emerged regarding mentoring context. While respondents assessed the availability of resources for achieving learning targets . For this reason, a replication of this study during this year (or in 2022) would be helpful for drawing more robust conclusions.In a methodological sense, and related to the previous point, a longitudinal model that includes auto-regressive path analyses will provide more objective measures for relations between variables at different time points. Moreover, as it is possible that online tools will be used more often in the future (independent of COVID-19), the reinforcement of e-mentoring could be used to increase sample size and make possible comparisons among latent constructs using structural equation modeling (SEM), reducing the measurement error. Furthermore, a bigger sample size would allow for treating the nested nature of data with multilevel SEM in addition to the analyses we performed in this study.The original contributions presented in the study are included in the article/Ethical review and approval were not required for the study on human participants in accordance with the local legislation and institutional requirements. The patients/participants provided their written informed consent to participate in this study.MM, AZ, and HS contributed to conception and design of the study and wrote sections of the manuscript. MM surveyed participants and organized the data. AV performed the statistical analysis. All authors contributed to manuscript revision, read, and approved the submitted version.This research has been funded by the Hamdan Bin Rashid Al Maktoum Foundation for Distinguished Academic Performance.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. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


NPM1-mutated(mut), chromatin/spliceosome-mut and TP53-mut/aneuploid AML that were confirmed by biofluid analysis. Interestingly, integrated genomic-metabolic profiles defined two subgroups of NPM1-mut AML. One was enriched for mutations in cohesin/DNA damage-related genes (NPM1/cohesin-mut AML) and showed increased serum choline + trimethylamine-N-oxide and leucine, higher mutation load, transcriptomic signatures of reduced inflammatory status and better ex-vivo response to EGFR and MET inhibition. The transcriptional differences of enzyme-encoding genes between NPM1/cohesin-mut and NPM1-mut allowed in silico modeling of intracellular metabolic perturbations. This approach predicted alterations in NAD and purine metabolism in NPM1/cohesin-mut AML that suggest potential vulnerabilities, worthy of being therapeutically explored.Although targeting of cell metabolism is a promising therapeutic strategy in acute myeloid leukemia (AML), metabolic dependencies are largely unexplored. We aimed to classify AML patients based on their metabolic landscape and map connections between metabolic and genomic profiles. Combined serum and urine metabolomics improved AML characterization compared with individual biofluid analysis. At intracellular level, AML displayed dysregulated amino acid, nucleotide, lipid, and bioenergetic metabolism. The integration of intracellular and biofluid metabolomics provided a map of alterations in the metabolism of polyamine, purine, keton bodies and polyunsaturated fatty acids and tricarboxylic acid cycle. The intracellular metabolome distinguished three AML clusters, correlating with distinct genomic profiles:  Current personalized therapeutic approaches in acute myeloid leukemia (AML) are generally restricted to those patients with identifiable and targeteable genomic lesions –3. HowevIDH1/2-mutated (mut) cases [The metabolome is the result of genome- and proteome-wide interactions and is shaped by microenvironmental factors. The biofluid metabolome has been extensively investigated to identify predictive signatures in cardiovascular disorders , divertit) cases , 10. 2-Ht) cases , 12, pret) cases , and is Aberrant enzymatic activity drives cancer metabolic reprogramming and cooperates with mutations of tumor suppressors and oncogenes in pathogenesis. For example, AML cells reduce both host insulin sensitivity and secretion to increase glucose availability for malignant cells . The glyNPM1-mut AML subgroup characterized by mutations of genes involved in DNA damage response and/or chromatid cohesion (NPM1/cohesin-mut) and high levels of serum choline + trimethylamine-N-oxide, and leucine. In silico modeling of the intracellular metabolome based on transcriptomic data highlighted perturbations in the purine and NAD metabolic pathways as NPM1/cohesin-mut-specific alterations.Here we report an integrated genomic-metabolic study in AML that identified, based on intracellular and the biofluid metabolic profile, a specific Participants were included if they were free from infective, autoimmune, celiac, or metabolic diseases such as diabetes and dyslipidaemia. Kidney and liver integrity were also checked. Subjects with acute or chronic renal or hepatic disease, renal or hepatic impairment, cardiovascular disease or a history of neoplasia were excluded from the control cohort. Serum samples from of 119 AML and 145 healthy subjects and urine samples of 103 AML and 139 controls were collected in the fasting state (in the morning). All participants were Caucasian except for 5 (3.4%) healthy controls and six (5%) AML patients. To reduce potential bias and variation unrelated to AML pathogenesis and to ensure that the observed metabolic differences were not due to external confounders, we collected, when possible, two independent serum and urine samples from each patient (more than 50% of cases). Moreover, information on age, gender , race, health status, diet, drug intake, physical exercise was collected along with specimens and used to filter nuclear magnetic resonance (NMR) spectra during the quality control procedures.TP53-mut/aneuploid, NPM1-mut and chromatin/spliceosome-mut samples. Signals in the spectra corresponding to loadings and weights emerging from different tasks were checked for alignment. To minimize the possibility of confounding effects, every step of each classification and clustering task was crossvalidated through suitable k-folds, stratified for gender and age when possible depending on class sizes and sample sizes for the tasks.Serum and urine samples were analyzed by NMR spectroscopy (Supplementary Methods). A stochastic GridSearch was implemented to select the best combination of parameters for dimensionality reduction and classifier performances. Unsupervised and supervised dimensionality reduction were performed using principal component analysis (PCA) and partial least squares discriminant analysis (PLSDA)-sparse(s)PLSDA, respectively. For subset extraction, weights were obtained after signal smoothing via signal-to-noise ratio threshold . The latent components of spectra containing maximum information related to molecular features were identified by a genomic-guided semisupervised approach. This means that the combination of urine and sera latent components used for clustering is extracted with classifiers-derived scores, from classifiers trained with the purpose of discriminating MS-based meabolomics was performed using an ultra-performance liquid chromatography  and a Q-Exactive high resolution/accurate mass spectrometer  interfaced with a heated electrospray ionization (HESI-II) source and Orbitrap mass analyzer operated at 35.000 mass resolution . Raw data were extracted, peak-identified and QC processed. Compounds were identified by comparison with library entries of purified standards or recurrent unknown entities. Peaks were quantified using area-under-the-curve. Metabolite levels were normalized to DNA content.WES was performed on 100 AML cases, 17 belonging to a published dataset  and 83 nhttps://github.com/cladelpino/GenePerturbations.We translated gene expression alterations into constraints reducing the feasible space of a metabolic network model . The imB = 1000,000) simulated Fisher’s exact test. Continuous variables were compared with Mann–Whitney, Kolmogorov–Smirnov, Kruskal–Wallis test, or Welch’s t-test. All tests were performed using either python v3.6.5 [p values were adjusted for multiple comparisons using the Bonferroni or Benjamini–Hochberg method. To investigate the distribution of sera profile according to blast percentage, samples were divided in three classes . In the drug response analysis, the average area-under-the curve values of the two cohorts were compared. NMR peaks, signal integrals (related to metabolite concentration) and intracellular metabolite levels among three groups were compared by Kruskal–Wallis test. For intracellular metabolite levels Welch t-test was also used as post-hoc test. Random Forest analysis was used to estimate the accuracy of individual classification in each group based on metabolomic data. Metabolic pathway analysis was performed using Metaboanalyst  with KEGG annotation. A threshold of five standard deviations from the mean of the control population was used for the identification of outliers.Associations in contingency tables were performed by the Monte Carlo  or Rv3.n v3.6.5 . When apGiven that the metabolite composition of biofluids reflects the real-time activity of all biochemical processes in the body and that leukemic cells alter systemic physiology , we compp < 0.05, Fig. The metabolomic profile provided efficient discrimination between patient and CTRL both at serum and urine level, with an accuracy of 83% Figs.  and S1 aMoreover, we detected increased concentration of 3-aminobutyrate and phenylalanine in the urine of AML patients compared with CTRL Fig.  and S6. p < 0.001), and one of them included 70% of TP53-mut/deleted(del) AML  AML, we found lower levels of threonine and glucose in TP53-mut/del cases  blasts) and compared them with 21 normal cord blood (CB) CD34+ and 21 normal CD33+ peripheral blood (PB) samples from healthy subjects. CD34+ AML and CD33+ AML segregated from their normal counterparts . Moreover, high levels of reduced glutathione , cluster-2 for cases with altered chromatin/spliceosome genes (37.5%), and cluster-3 for TP53-mut/aneuploid AML  and identified 4 NMR clusters , compared with NPM1-mut patients from the other metabolic group .Our data so far described a significant association between genomic and metabolic profiles. However, even within the same genomic category, different subgroups can be identified according to combinatorial mutation patterns and consequently they may show metabolic differences. This hypothesis was confirmed in patients carrying NPM1/cohesion-mut and NPM1-mut AML, we analyzed paired exome and transcriptome  also carried at least one alteration in recurrently mutated genes [NPM1-mut AML, NPM1/cohesin-mut cases were characterized by a lower white blood cell count  and a significantly higher mutation load , with lower frequency of IDH1–2/TET2 mutations , lipid (LPL) and carbohydrate (CHST13) metabolism, energy production (CYP1B1) and transporter/exchanger  and the inhibition of some of them achieved promising results in AML [Second, we integrated biofluid and intracellular metabolomics. We used NMR and MS as complementary techniques for biofluids and primary cell profiling, respectively. The rationale of this approach is twofold: it allowed us to benefit from the reproducibility of NMR, which offers unbiased information and could enable a rapid translation to the clinical practice, and from the high sensitivity of MS in metabolite detection from low cell numbers. Our comprehensive view showed alterations in the TCA cycle and in the metabolism of purine nucleotides, amino acid, fatty acids, keton bodies, polyamine, glutamine and other amino acids. The incomplete overlap between the metabolic alterations observed when comparing CD34s in AML , 45 or is in AML , 47 modeNPM1-mut, chromatin/spliceosome-mut and TP53-mut/aneuploid AML classes. Chromatin/spliceosome-mut and TP53-mut/aneuploid AML shared some metabolic features, according to their clustering. This may be partly explained by the recurrence of aneuploidies in the chromatin/spliceosome-mut class (42.9% of those classified in the TP53-mut/aneuploid AML associated cluster). NPM1-mut AML showed high intracellular levels of N-acetyl-aspartyl-glutamate, that have been previously associated with MYC activation [NPM1 indirectly stabilizes c-MYC protein [MYC mutation was also detected in a NPM1-mut AML case, in line with previous findings [When integrated with genetic features, the metabolic profiles showed association with tivation . Indeed, protein  and an oNPM1-mut AML carrying mutations in cohesin or DNA damage-related genes as a distinct metabolic subgroup. This group does not associate with IDH1–2/TET2 mutations, which are also frequently observed in NPM1-mut cases [NPM1/cohesin-mut-specific metabolic perturbations predicted changes in the balance of leukotrienes. Among the metabolic genes downregulated in NPM1/cohesin-mut compared with NPM1-mut AML, CHST13, ADCY9, PRR16, LPL, and SLC1A3 were confirmed by STAG2-deficient model of NPM1-mut leukemia [NPM1/cohesin-mut-specific ones. Inosine-5′-monosphate, an intermediate in the purine metabolism, showed low levels in NPM1/cohesin-mut compared with NPM1-mut AML cells. Of note, inosine favors spontaneous mutations, since a base transition is introduced when it is incorporated into newly synthesized DNA [NPM1/cohesin-mut leukemic cells. Future studies taking into account leukemic cell metabolism and microenvironmental factors, will further investigate the suggested vulnerabilities. With regard to therapeutics, NPM1/cohesin-mut AML were more sensitive than NPM1-mut AML to EGFR inhibition, which may lead to the release of the differentiation brake [Our data also classified ut cases  but it wut cases . Accordileukemia . Moreoveized DNA . In addiized DNA , 55. Givized DNA  and thatized DNA , the taron brake  and to don brake .Overall, our results provide a map of the crosstalk between metabolic pathways and between genomics and metabolomics in AML, reflecting functional interactions and dependencies that could be therapeutically exploited and provide the rationale for a switch to a genomic- and phenotypic-driven personalized medicine.Supplementary informationTable S1Table S2Table S3Table S4Table S11Table S12Table S13Table S14Table S15Table S16


The α1-acid glycoprotein (AGP) is an abundant blood plasma protein with important immunomodulatory functions coupled to endogenous and exogenous ligand-binding properties. Its affinity for many drug-like structures, however, means AGP can have a significant effect on the pharmokinetics and pharmacodynamics of numerous small molecule therapeutics. Staurosporine, and its hydroxylated forms UCN-01 and UCN-02, are kinase inhibitors that have been investigated at length as antitumour compounds. Despite their potency, these compounds display poor pharmokinetics due to binding to both AGP variants, AGP1 and AGP2. The recent renewed interest in UCN-01 as a cytostatic protective agent prompted us to solve the structure of the AGP2–UCN-01 complex by X-ray crystallography, revealing for the first time the precise binding mode of UCN-01. The solution NMR suggests AGP2 undergoes a significant conformational change upon ligand binding, but also that it uses a common set of sidechains with which it captures key groups of UCN-01 and other small molecule ligands. We anticipate that this structure and the supporting NMR data will facilitate rational redesign of small molecules that could evade AGP and therefore improve tissue distribution. AGP expderstood . To dated review , 10, 11.in vivo across five N-linked sites . Functionally, glycosylation reduces AGP clearance in the kidneys, increases solubility, and may sometimes modulate drug binding (KD ∼ 10−5–10−9) for endogenous and exogenous ligands which may be specific to one AGP variant or bind both with comparable avidity   12). In. In12).  binding , 14, 15. avidity . For exa avidity  and the  avidity .Figure 1To determine their different ligand-binding specificities, high resolution crystal structures of nonglycosylated AGP1  and AGP2In silico docking studies have proposed a binding mode for the tyrosine kinase inhibitor and widely used leukemia treatment imatinib to AGP1 . These properties led to its development as a therapeutic, but multiple clinical trials    were ter0004263) . Nonethe0004263) . These eHere, we present a crystal structure and solution NMR studies of a UCN-01–AGP2 complex. These new data reveal that UCN-01 binds with its aromatic system in a perpendicular orientation to those of AMT and DSP, but with some similarities to CPZ. However, the size and orientation of UCN-01 permits a more extensive array of favorable interactions to be formed within the AGP2-binding pocket and is accompanied by significant stabilization of the structure upon ligand binding. This structure alongside backbone chemical shift assignments of AGP2 will facilitate the studies of further ligands or UCN-01 analogues aimed at abrogating AGP-2 binding.15N-labeled full-length AGP2 (AGP2-FL) from E. coli. This construct incorporated a cleavable N-terminal His6-tag and the C149R mutation shown to improve solution homogeneity of deglycosylated forms used in crystallographic studies  spectrum of 15N-labeled material was collected at 298 K but was of poor quality as judged by the presence of less than 50% of the expected 1H-15N correlations and inhomogeneous peak intensities  internal motions, but there was no evidence of patches of reduced T2 values commensurate with μs-ms exchange motions. The relaxation parameters further suggest a uniformly rigid protein in the UCN-01 bound form.NMR dynamics studies can provide an indicator of the structuring, and internal motions of a macromolecule and main chain amide ect them . Broadenect them . When boprotein. . Both th3APU] and contained a single protein–ligand complex in the asymmetric unit. The electron density for UCN-01 in the AGP2-binding pocket was clearly resolved , 1812, 1B–D). Several aromatic residues line the binding pocket including Tyr37, Phe49, Phe51, His97, Phe112, and Tyr127 and form interactions with UCN-01 which is principally comprised of an extended aromatic ring system. Phe112 forms a CH-π interaction with the central benzene ring of UCN-01 and the Tyr127 hydroxyl group H-bonds to indole nitrogen N2. Neither Phe49 nor Phe51 form aromatic face to face or edge interactions with UCN-01. Together with Leu62 and Leu79, they instead sit, somewhat surprisingly, opposite the polar residues of the five-membered lactam ring. Glu64 and Arg90 sit above the plane of UCN-01 and form a salt-bridge connecting β-strands C and E. This carboxylate – guanidinium group interaction lies perpendicular to the UCN-01 aromatic system allowing the formation of a hydrogen bond from the lactam ring 7R-OH to Glu64 and a cation-π interaction with Arg90. The interaction between Arg90 and the UCN-01 aromatic system is of interest as π-stacking interactions are observed more frequently in published structures than T-shaped cation-π interactions, such as the one observed here  from the bridging oxygen of UCN-01. This oxygen lacks any obvious H-bonding partner. Finally, the pendant methoxy and secondary amine groups both lie on the more solvent exposed face of UCN-01 and lack any discernible packing interactions.Although solved at pH7.4, the UCN-01 bound AGP2 three-dimensional fold is very similar to previous ligated AGP2 structures solved at pH 4.6 . SuperimA) perpendicular to that of DSP and AMT (B and C). CPZ, which otherwise binds in a similar fashion to UCN-01, interacts with Arg90 by a more typical stacking interaction and concomitantly Glu64 also changes in orientation. The π-systems of the two other ligands, for which crystal structures have been solved, are oriented perpendicular to those of UCN-01 and CPZ and so further comparisons of Arg90-π interactions are unavailable. The higher affinity (3.5 nM) of UCN-01 for AGP relative to CPZ may be explained in part by the more extensive aromatic ring system of UCN-01 which occupies parts of the binding pocket closer to Ile44. In the case of DSP, this pocket is occupied by aliphatic chains from the tertiary amine and with AMT, the pocket is occupied by acetate buffer.The conformation of UCN-01 in the binding pocket is most like the drug CPZ. Both CPZ and UCN-01 are bound with aromatic ring systems in a similar plane A perpend and AMT , B and CD). Substituting Phe112 for Leu112 at this position may be accommodated by ligands binding in the mode of UCN-01 and CPZ but would clash with the orthogonal aromatic systems of AMT and DSP. Likewise, UCN-01 is also unlikely to clash with Phe114 if its AGP1-bound form is homologous to the AGP2–UCN-01 complex.Both UCN-01 and CPZ bind to AGP1 and AGP2 with comparable affinity  whereas Fig. 4Kd of 1 - 3.5 nM (Kd = 88 nM ± 30 nM) corresponding to ∼6 to 8 kJ mol−1 difference in binding energy. The structure reveals that the hydroxyl group of UCN-01 is important and is oriented toward Glu64, forming a hydrogen bond and indirectly assisting with orienting Arg90. This key interaction would be lost with staurosporine, assuming a similar binding mode and consistent with loss of the H-bonding interaction and reduced-binding affinity. Reversal of the chirality at C7 to the S-configuration in UCN-02 has a more drastic effect, with >100-fold loss of affinity such that UCN-01 and 02 are essentially the eutomer and distomer pair, respectively. The 100-fold reduction with UCN-02 is of course too large for the loss of a hydrogen bond alone. However, the reversal of the stereochemistry at C7 not only breaks this hydrogen bonding, but is likely to invoke a clash with Phe49 (B) and a further shift from the optimal binding pose. This combination may account for the much greater loss in binding affinity for UCN-02.UCN-01 is the highest affinity ligand for AGP2 determined to date with a - 3.5 nM , 32. Int- 3.5 nM , which p- 3.5 nM . The binth Phe49 B and a fA and B shows the structure of UCN-01 bound to AGP2 compared with the binding pocket of Chk1 aligned to UCN-01’s orientation. UCN-01 binding to Chk1 mimics adenine binding in terms of H-bonding and interaction with nonpolar surfaces. In fact, UCN-01 has greater potency as a Chk1 inhibitor than staurosporine (C). In contrast to the binding mode of the compound in both AGP2 and Chk1, however, there are no hydrogen bond donors or acceptors proximal to the hydroxy-lactam l group of UCN-01. Again, the pyran ring conformation of UCN-01 differs in comparison with the AGP2 complex.UCN-01’s targets include the cell cycle proteins CDK2 and Chk1 for which high-resolution crystal structures have been determined , 34. Togosporine  due to tosporine . In the osporine C. In conTherefore, UCN-01 is surrounded by a β-barrel in AGP2, whereas in Chk1 and CDK2, it is sandwiched between two β-sheets. It may therefore be possible to introduce substitutions on the UCN-01 aromatic system that protrude from these clefts and do not compromise binding Chk1/CDK2 binding affinity but preclude being enveloped in AGP2’s β-barrel. Alternatively, conformational restriction of the pyran ring could potentially prevent the methoxy or amine sidechains adopting the alternate conformation present, and perhaps, required for AGP2 binding.This study has presented the crystal structure of the important antitumour compound UCN-01 bound to AGP2, supplemented with backbone chemical shift assignments and relaxation parameters at 700 MHz. Our NMR studies provide the first indication that AGP2 is significantly more disordered in the free form than would have been assumed based on published crystal structures. The relaxation parameters, on the other hand, show that the main body of the protein displays mostly uniform dynamics when binding UCN-01 in solution. NMR spectral quality is also considerably improved in the presence of UCN-01 relative to the free form, consistent with conformational rigidification upon ligand binding.The crystal structure reveals the binding mode of the highest known affinity ligand for AGP2 and rationalizes how the stereochemistry of the staurosporine hydroxy-lactam ring impacts binding affinity. These findings open new avenues for redesigning UCN-01 to maintain its antikinase activity, whereas avoiding high-affinity plasma binding which may generate useful leads toward new oncology therapeutics.MAHHHHHHSSG LEVLFQGPQIPLCANLVPVPITNATLDRITGKWFYIASAFR NEEYNKSVQEIQATFFYFTPNKTEDTIFLREYQTRQNQCF YNSSYLNVQRENGTVSRYEGGREHVAHLLFLRDTKTLMF GSYLDDEKNWGLSFYADKPETTKEQLGEFYEALDCLRIPR SDVMYTDWKKDKCEPLEKQHEKERKQEEGES. Eighteen N-terminal amino acid residues in italics were removed during purification by 3C proteolytic cleavage (see below). To assist in protein purification and expression, Cys149 of the native sequence was replaced with Arg as in AGP1 and published protocols (15N)- or double (15N and 13C)- labeled protein was produced from the cells grown to an absorbance ∼0.4 at 37 °C in isotope-labeled M9 minimal media containing 2.5 gl−113C6-glucose and 1 gl−115NH4Cl induced with 0.25 mM IPTG. The cells were harvested after 16 h at a reduced temperature of 30 °C. Inclusion bodies were prepared by lysing cells in hypoosmotic buffer  and removing soluble material by centrifugation. The inclusion bodies were washed 2 times with lysis buffer and 2 to 3 times with high salt buffer . The purified inclusion bodies were solubilized in denaturation buffer  with a few drops of NaOH and stored (−20 °C) until further use.AGP2-FL was initially expressed from a pOPINF  construc6-tag was removed by 3C proteolytic cleavage at 2 mg/ml with a 20:1 mass ratio of in-house 3C protease. The protease and His6-tag were removed by reverse IMAC to yield pure and cleaved AGP2.For refolding, the solubilized inclusion bodies were reduced , and excess insoluble material was removed by centrifugation . The reduced denatured AGP2 was added dropwise to refolding solution  and incubated . The folded AGP2 was separated from impurities and denatured material by size-exclusion chromatography on S75 resin eluting at ∼215 ml postinjection . AGP2 was concentrated, and the His15N-labeled AGP2 NMR sample (50 μM and 50 μl) and iodoacetamide (15 mM) in the presence of absence of DTT . The protein was recovered by methanol/chloroform precipitation.Acetylation of AGP2 cysteine residues was performed by incubating a diluted 1-acid glycoprotein model (3APU). The resulting electron density maps were of sufficient quality to autobuild 90% of the model. Iterative rounds of manual rebuilding and refinement in Coot , and the complex was crystallized in 100 mM HEPES and 1.4 M sodium acetate, pH7.4. A dataset of 1800 images was collected at a wavelength of 0.9686 Å, exposure time: 0.01 s, oscillation: 0.10° with cryogenic temperatures maintained throughout data acquisition. The dataset was scaled and merged into P61 using the Diamond Pipeline (Xi2 3dii) with cell dimensions of a = 88.27, b = 88.27, and c = 53.84 Å (α = 90 β = 90 γ = 120°). There was a single molecule in the asymmetric unit with a solvent content of 60%. The structure of AGP2 was determined by molecular replacement with MOLREP  within t in Coot  and Phen in Coot  further  in Coot . The finAGP2 was exchanged into 20 mM NaP and 150 mM NaF, pH 7.2 by Zeba column. The CD experiments were acquired on an Applied Photophysics Chirascan instrument with the manufacturer software v. 4.7.0.194.1H-15N TROSY-HSQC spectra were collected on a 700 MHz Bruker AVANCEHD NMR spectrometer equipped with a 1.7 mm triple-resonance micro-cryoprobe. The samples were composed of 10 mM Na2HPO4/NaH2PO4 and 100 mM NaCl, pH 6.5 with 0.5 or 1 mM AGP2 with at least a 2-fold excess UCN-01 when required. Although the AGP2 samples were not perdeuterated, TROSY-HSQC spectra were collected for assessment of protein folding to allow higher 1H dimension resolution (>4K complex points) without delivering decoupling power to the probe. The chemical shift assignments for 98% of the nonproline backbone residues were obtained by manual analysis of 1H-15N TROSY-HSQC, HNCA, HNCOCA, HNCACB, and HNCOCACB spectra collected at 700 MHz and 1H-15N HSQC-NOESY, 1H-13C HSQC-NOESY, and CBCANH acquired at 950 MHz using standard Bruker pulse sequences.To assess protein quality, 1H dimension and 0.68 ppm in the 15N dimension. Heteronuclear shifts were weighted based on the respective ranges of assigned peak chemical shifts present in the two dimensions (1H:4.504 and 15N:30.616), as described in published methods (1H ppm/15N ppm. All the visible assigned peaks in the UCN-01 spectrum were greater than 5% of the height of the largest peak (His172) and so, signals in the free spectrum were only counted if they were more than 5% of the height of the largest free AGP2 resonance (putative His172).In a reverse minimal shift approach, peaks in the free spectrum were assigned based on their proximity to assigned peaks in the UCN-01-bound spectrum. The free peaks closest to liganded peaks were assigned first, and subsequently, more distant free peaks were assigned to the closest remaining unassigned UCN-01 peak provided that the peaks were closer than 0.1 ppm in the  methods . These rT1) were measured using the standard Bruker sequence, tr1etf3gpsi, with relaxation delays of 10, 50, 100, 200, 400, 600, 800, 1000, 1200, 1500, 1800, 2000, and 2500 ms and a recovery delay of 3 s. Transverse relaxation times (T2) were measured using the standard Bruker sequence, tr2etf3gpsi, with relaxation delays of 10, 48, 64, 80, 96, 128, 160, 192, 240, and 320 ms and a recovery delay of 1.5 s. For 15N steady-state NOE experiments, the data were acquired interleaved using the standard Bruker sequence, trnoef3gpsi, with a saturation period of 6 s. The spectra were processed with NMRPipe (Longitudinal relaxation times ( NMRPipe  and anal NMRPipe .The final model of AGP-2 bound to UCN-01 has been deposited to the PDB with accession code 7OUB. The chemical shift assignments have been deposited in the BMRB with accession number 50946.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Horses are bred for peak athletic performance yet exhibit a high incidence of exertional rhabdomyolysis, with elevated levels of cytosolic Ca2+ proposed as a correlative linkage. We recently reported an improved protocol for isolating SR vesicles from horse muscle; these horse SR vesicles contain an abundant level of SERCA and only trace-levels of sarcolipin (SLN), the inhibitory peptide subunit of SERCA in mammalian fast-twitch skeletal muscle. Here, we report that the in vitro Ca2+ transport rate of horse SR vesicles is 2.3 ± 0.7-fold greater than rabbit SR vesicles, which express close to equimolar levels of SERCA and SLN. This suggests that horse myofibers exhibit an enhanced SR Ca2+ transport rate and increased luminal Ca2+ stores in vivo. Using the densitometry of Coomassie-stained SDS-PAGE gels, we determined that horse SR vesicles express an abundant level of the luminal SR Ca2+ storage protein calsequestrin (CASQ), with a CASQ-to-SERCA ratio about double that in rabbit SR vesicles. Thus, we propose that SR Ca2+ cycling in horse myofibers is enhanced by a reduced SLN inhibition of SERCA and by an abundant expression of CASQ. Together, these results suggest that horse muscle contractility and susceptibility to exertional rhabdomyolysis are promoted by enhanced SR Ca2+ uptake and luminal Ca2+ storage.We have analyzed the enzymatic activity of the sarcoplasmic reticulum (SR) Ca Transport assays were started by the addition of protein samples to reaction tubes. Ca2+ transport was terminated at serial time intervals by vacuum filtering 100 µL of assay solution  through a HA-type glass-fiber filter with a 0.45-micrometer pore size , which was washed twice with 5 mL of ice-cold 150 mM NaCl solution. The loading of 45Ca inside SR vesicles was determined by liquid scintillation counting. Background 45Ca binding (defined as a glass-fiber filter blank loaded with 1 µg of SR protein in the absence of ATP) was subtracted from the experimental values (the glass-fiber filter sample loaded with 1 µg of SR protein in the presence of ATP) to yield the amount of ATP-dependent Ca2+ transport by SR vesicles.Cae tracer ,38. Assa2, and 20 mM MOPS (pH 7.0), with an addition of either 0.1 mM CaCl2  or 2 mM EGTA and 1 μM TG  ..2+ transmax of the horse versus rabbit SERCA orthologs could potentially be attributed to the lack of SLN peptide in horse muscle . Th. Th2+ upby SERCA ,28,69. Factility .2+ storage protein in SR, and CASQ is tightly linked to the regulation of Ca2+ cycling in muscle. Since SR Ca2+ release is sensitized by luminal Ca2+, an increase in luminal Ca2+ stores and an associated RYR1 Ca2+ release could result in a more rapid and more powerful muscle contraction, i.e., producing a positive inotropic effect in vivo. The correlation of a higher rate of SERCA transport in SR vesicles with an increased rate of clearance of cytosolic Ca2+ transient suggests that horse myocytes exhibit a positive lusitropic effect (CASQ is the primary Cac effect .2+ has been detected in (a) primary myocytes isolated from horse muscle during an episode of acute exertional rhabdomyolysis [2+ may provide an advantage that enhances muscle power output, excessively elevated cytosolic Ca2+ may become a disadvantage that decreases contractility, such as through an SR store overload-induced Ca2+ release (SOICR) [Horse breeds have been bred selectively to further enhance their speed and with that has come an incidence of exertional rhabdomyolysis of 3–7% ,75. Elevmyolysis  and (b) myolysis . The lac (SOICR) . We sugg2+ reuptake and the loading level of SR Ca2+ stores, thereby enabling a more rapid and more powerful contraction of horse gluteal muscle in vivo. An increase in the intrinsic rate of Ca2+ transport by horse SERCA , coupled with a robust level of CASQ expression, may also serve as molecular mechanisms that potentiate SR contributions to functional contractility in horse muscle  have 50% faster relaxation times than horses that are not predisposed to recurrent exertional rhabdomyolysis [With the natural selection of horses as fast-moving prey animals, why would they evolve to have a unique SLN amino acid sequence and lack of expression that are not present in closely related ungulates such as rhinoceros? One possible advantage for the lack of a SERCA regulatory peptide inhibitor includes a genetic improvement that generates an increased luminal Camyolysis . Furthermyolysis . Thus, tmdx and mdx/utr-dko [2+ transport regulation and muscular performance in animal models and human disease, which could then provide insights on the utilization of species-dependent mechanisms for contractile and therapeutic control.In human muscular dystrophies, the effect of modulating the SLN expression level and the inhibitory function is unknown. In compelling mouse and canine models, enhancing SLN protein expression has been proposed as an effective therapy, as reported by Babu et al. . Indeed,/utr-dko . However/utr-dko ,78,79. T2+ transport. The results reported here contribute to the understanding of Ca2+ cycling in myocytes, with relevance to muscular performance, adaptability, and disease. The results shown here provide a foundation to further dissect the mechanistic roles of SERCA, CASQ, and SLN in horse muscle contractility, including correlations with gene and protein expression levels. We propose that analyses of horse Ca2+ transport will enhance the basic understanding and therapeutic utilization of Ca2+ control in muscle contractility.Our integrative cellular, transcriptional, and biochemical methodologies ,11,80,81


This review reports about the main steps of development in pediatric acute myeloid leukemia (AML) concerning diagnostics, treatment, risk groups, and outcomes. Finally, a short overview of present and future approaches is given. The treatment of pediatric acute myeloid leukemia (AML) is a success story in improving prognosis. Whereas, in the 1980s, almost all children suffering from AML died, today, up to 75% of the children survive. However, this is only feasible in a well-structured setting of comprehensive diagnostics, intensive therapy, and effective supportive care. This has been achieved by the cooperative study groups in Europe, North America, and Japan. By contrast, even within Europe, the prognosis of children with AML shows an unacceptable level of inequality of survival rates, ranging from less than 50% to 80% . ,4. 3,4].The only new approach within the most recent 20 years was the targeted CD33 antibody gemtuzumab ozogamicin (GO), which showed some advantages in at least one randomized trial, but GO is only approved in North America . The successful therapy of APL with all-trans retinoic acid and arsenic-trioxide is a rare example of curing leukemia targeting specifically leukemia-inducing molecular mechanisms and eradicating the leukemic stem cell ,6. Liposomal drug formulation of daunoribiicn allowed treatment intensification without increasing toxicities, but this has disappeared due to economic reasons, such as a limited pediatric market. The actual approach with a liposomal nanoscale co-formulation of cytarabine and daunorubicin seems to be promising but needs confirmatory trials in pediatric AML and marketing approval thereof. Although stem cell transplantation is still an unspecific treatment option with severe acute and long-term side effects, combined with a better risk group stratification, alloHSCT significantly improved survival in children with high-risk (HR) AML ,8. There are a broad number of new compounds explicitly targeting signaling pathways. However, it is not confirmed in children to what extent these approaches will contribute to curing or be able to reduce toxicities by allowing reduction of treatment intensity of conventional drugs. A fast-growing field are immune and cellular therapies, which show promising results in preclinical and early phase clinical trials (mainly in adults).Although pediatric AML has been described since the 1900s, a formal classification was established, such as in adults, in 1976 by the French-American-British (FAB)-Classification ,11,12,13Until 1968, the remission rate was inferior, and the median survival was about 1.5 months . With thSince 1975 the first clinical trials for pediatric patients with AML were initiated ,17,18. CAlthough most patients were defined just by morphology, due to the high correlation between FAB M2 with Auer rods and t, the presence of atypical eosinophils in FAB M4 Eo with inv(16) or, later, FAB M2 and NPM1-mutated AML allowed event-free survival (EFS) rates of 70% and more ,23. InteWithin the 20th century, the relevance of anthracycline analogs has been discussed. Randomized trials, such as AML-BFM 98 and MRC 12/15, tested idarubicin versus daunorubicin or mitoxantrone. Significant achievements were the identification of idarubicin as the most effective anthracycline if applied in a 1 to 5 conversion rate compared to daunorubicin ,27. In the NOPHO-93, all the patients initially underwent the same treatment (ATEDox), but, dependent on the response after the first induction, an extra AM  induction was recommended . Those pThe tested antileukemic drug in the NOPHO 2004 trial was GO and did not show any significant effect on the recurrence rate of leukemia or overall survival (OS) . An extr2 on days 2, 4, and 6) and etoposide (100 mg/m2 on days 2–6), and high-dose cytarabine (3 g/m2 every 12 hours) [The St. Jude AML02 Study stratified the patients into two subgroups according to morphologic and genetic characteristics. Patients were randomized to receive daunorubicin (50 mg/m2 hours) . The Japanese AML99 study (JPLSG) implemented a risk stratification of three groups. The initial stratification was made for the low-risk patients, and including the HR group criteria of WBC (>100.000 µL) and the age of the patients (<2 years). Additionally, the response after the first induction and the karyotype led to the allocation of the patients in the final stratification group. An alloHSCT was indicated only for the intermediate and HR groups, especially for the last group; a “not familiar” donor was suggested .2) was tested in the low and intermediate-risk patients. At the same time, 50% of the etoposide dose was used in the AML99 protocol. A higher incidence of relapse was noticed, but the OS was not influenced [In the AML05 study (JPLSG), the reduced cumulative anthracycline dose  positive patients . In ItalIn the AML-BFM-87 study, prophylactic irradiation was randomized. However, an increased rate of relapses in children without cranial irradiation led to a premature stop . Since tTo summarize, different intrathecal therapies, such as monotherapy or triple treatment, were administered from the studies mentioned above. Cranial irradiation was excluded from the standard treatment and is now only recommended for children with CNS involvement. In parallel to the intensified therapy, the relevance of genetic risk groups and treatment response became obvious. Improved techniques, such as multicolor immunophenotyping and quantitative PCR, allowed a more precise response detection. Detection of MRD by multicolor flow cytometric immunophenotyping started during the 1990s. The leukemic blasts were selected via different antigens . Langebrake et al. defined different response measurements within AML subtypes, including the prognostics relevance . In contIn the NOPHO 2004 study, a difference in the EFS, but not in OS, comparing the MRD positive with the morphologic positive patients was noticed . Within the Dutch Childhood Oncology Group (DCOG) ANLL, 97, and the MRC 12 trials, MRD levels measured by flow were prognostically favorable for the patients achieving MRD negativity after the first/second induction .Polymerase chain reaction (PCR) is also used to detect MRD for different fusion transcripts. The correlation of expression with the clinical progress of the patients is supported in many studies. The data presented are promising for using this method as a sensitive diagnostic tool. The studies include a small number of patients for the specific subpopulations in the pediatric population, such as t, in(16) .The cooperative trial groups achieved significant improvements in overall survival. The analysis of the AML-BFM trials between 1993 and 2010 revealed a continuous improvement of OS but limited progress of EFS . This suTreatment regimens include initial double induction and 2- or 3-consolidation blocks. Even if the definitions vary, it is evident that about 60% of children with AML can be cured by chemotherapy only. On the other hand, a significant achievement within the ongoing trials was the establishment of a risk group dependent indication for alloHSCT in the 1st CR . Along with the improvements in the transplant procedure and the option to rescue refractory AML, EFS improved . A condiThe AML-BFM Study group and the NOPHO proved that alloHSCT in the 1st CR of high-risk pediatric AML improves EFS, which is not significantly lower than intermediate-risk (IR) or standard risk (SR) ,8. HowevIn case of relapse, all patients indicate alloHSCT in the 2nd CR. Although the prognostic characteristics of relapsed AML are impaired, the survival rate has been maintained or improved ,57. This allows treating the “right” patient group with the “right” intensity. In particular, the precise definition of the high-risk group by genetics and response associated with the indication of alloHSCT in the 1st CR eliminated significant differences in EFS. To achieve this, the results of the AML-BFM 2004 trial have been re-analyzed. Based on the genetic characteristics and augmented by the treatment response to the 1st and 2nd induction, three risk groups could be defined . In the AML-BFM 2012 Registry, this risk group stratification has been implemented. The NOPHO Group and others have published similar reports . In addition to the improved risk group stratification, the selection of conditioning regiments, preparation of the transplanted stem cells, the donor identification and availability, and the graft-versus-host prophylaxis significantly contributed to a better outcome in the 1st/2nd CR but also in children with a refractory AML . Within Most groups have accepted the standard for myeloablative conditioning with busulfan, melphalan, and cyclophosphamide. Earlier studies with less intensive conditioning (busulfan/cyclophosphamide) resulted in unacceptably high relapses . HoweverRegarding stem cell selection, the CD3/CD19-depleted graft transplantation of bone marrow or apheresis cells is the most widely used approach. The donor selection included matched sibling donors (MSD) or matched unrelated donors (MUD), defined as a 9/10 or 10/10 allele match for the HLA loci A, B, C, DR, and DQ, as determined by molecular 4-digit high-resolution typing. For the HR patients without a matched donor, a haploidentical donor is accepted . An unexpected, good outcome has been achieved in children with refractory AML, who got fludarabine/amsacrine (FLAMSA) and reduced conditioning (TBI/DLI). The reported 4-years EFS of 41% seems to be promising because, in the past, almost all patients of this cohort died .The diagnostic of pediatric AML requires morphology, immunophenotyping, and comprehensive cyto- and molecular genetics of the leukemic blasts. All available methods, such as multicolor flow with at least eight colors, panel-next-generation sequencing (NGS), and RNA seq, must be integrated. In general, the risk groups definition can be based mainly on genetics augmented by response measurement by flow and morphology (AIEOP/AML-BFM/FRANCE/UK/COG/Japan), or visa-versa, preferentially MRD-driven augmented by genetics (NOPHO) .−3 should allow direct alloHSCT.Today, measurement of residual disease by immunophenotyping is the most appropriate method to define initial treatment response. The ongoing treatment protocols use residual disease detection either by immunophenotyping only or in combination with morphology for treatment stratification. The different response kinetics of fusion genes  and mutations , measured by quantitative PCR, makes this approach suitable and prognostically relevant only in some subgroups, such as PML/RARA. However, continuous monitoring after remission allows the early detection of molecular relapse. Although it is not entirely proven yet, the treatment of molecular relapse might be feasible with less intensive chemotherapy as bridge to transplant option. The international AmoRe 2017 trial (conducted by GPOH as sponsor) should allow alloHSCT without toxic re-induction in children with a molecular relapse by applying the epigenetically-effective low dose azacytidine. A reduction of MRD -levels to less than 10Until almost the end of the 20th century, patients with AML and Down syndrome (DS) were treated identically with the whole group of pediatric AML . In the All the improvements of the recent decades would be impossible without the progress in supportive care. The introduction of prophylactic antimycotic and, effective antibiotic regimens as well as improved intensive care, including sufficient, sensitive, and specific microbiologic diagnostics, enables intensive treatment with a limited rate of treatment-related death and toxicity ,74,75. IPediatric AML and intensive treatment are associated with relevant long-term sequelae. All organ systems could be involved. Although much attention has been spent on cardiotoxicities, especially anthracycline-induced cardiomyopathy, severe damages of the liver, renal function, and endocrinology must be considered. Recent data showed the increased risk of early-onset cardiovascular diseases, neurology, and mental diseases . In addiThe therapy of pediatric AML is still based on intensive chemotherapy and, if necessary, alloHSCT. Despite significantly improved survival chances, this therapy has severe acute and long-term side effects ,78,79. TEarly deaths from AML in children and adolescents continue to be a significant problem ,81,82. WSeveral achievements will allow more reliable and precise diagnostics, mainly based on NGS, genome mapping, RNA seq, acetylation/methylation assays, and molecular single-cell characterization ,85,86. TRegarding more precise treatment options, the differentiating therapies with all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) in APL are already in use. For the first time, this allows the cure without chemotherapy and with significantly reduced side effects . The use of antigen-mediated therapies was successful, especially with the CD33-specific and ozogamicin-coupled antibody GO (Mylotarg) . HoweverAnother complex area of AML therapy includes various immunotherapies. One approach will be post-HSCT immunomodulation. The effectiveness has already been shown in post-HSCT treatment with donor-lymphocyte infusions (DLI) but definitively provides more options. Cytokine-induced killer (CIK) cells have already been introduced to clinical trials . Other aThe successful cellular therapy approaches in B-cell lymphocytic leukemia/lymphomas raise high hopes for myeloid neoplasms . HoweverOverall, it is unlikely that there will be “the one” effective therapy for a heterogeneous disease like AML. Only the optimized combination of all available options will allow a further, significant improvement of cure rates so that likely different treatments adapted to the AML subtype are needed. This underlines the need for further comprehensive research into the mechanisms of leukemogenesis, specific therapies, and, above all, systematic clinical research to develop scientifically-validated treatments, despite the small number of cases. This will only be possible if the international collaboration between the study groups will be further improved on a global level. It is important to learn more about small subgroups and more precise treatment, as well as reducing inequalities between countries and continents. The recently established Leukemia & Lymphoma Society (LLS), Pediatric Acute Leukemia (PedAL) initiative and the European Pediatric Acute Leukemia (EuPAL) foundation are on the way to launching such a platform in North America, Europe, Australia, and, hopefully, in Japan ,104.In this context, the therapies of AML in children, even if they have low economic impact, should not be considered exclusively as a “waste product” of adult medicine but should have a right to their own, child-specific therapy development. This applies to both, research on pediatric therapies and the timely establishment of therapies that have been successfully used in adult AML.


The advent of novel immunotherapeutic strategies has revealed the importance of immune dysregulation and of a tolerogenic microenvironment for acute myeloid leukemia (AML) fitness. We reviewed the “off-target” effects on the immune system of different drugs used in the treatment of AML to explore the advantages of this unexpected interaction.Acute myeloid leukemia (AML) is considered an immune-suppressive neoplasm capable of evading immune surveillance through cellular and environmental players. Increasing knowledge of the immune system (IS) status at diagnosis seems to suggest ever more attention of the crosstalk between the leukemic clone and its immunologic counterpart. During the last years, the advent of novel immunotherapeutic strategies has revealed the importance of immune dysregulation and suppression for leukemia fitness. Considering all these premises, we reviewed the “off-target” effects on the IS of different drugs used in the treatment of AML, focusing on the main advantages of this interaction. The data reported support the idea that a successful therapeutic strategy should consider tailored approaches for performing leukemia eradication by both direct blasts killing and the engagement of the IS. Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy characterized by an abnormal blast proliferation, variable prognosis and response to treatment, and high mortality . AML is The advent of immune checkpoint inhibitors (ICI) and chimeric antigen receptor-T (CAR-T) cell approaches revealed the importance of immune dysregulation and suppression in the onset of leukemia, encouraging strategies to engage the IS via novel immunotherapeutic approaches . AlthougIn this review, we report the possible effects of the drugs approved for AML treatment on the IS  have been demonstrated to activate, in vitro, an IS-mediated cell death through the translocation of CRT on the neoplastic cell surface  48]. Mo. Mo48]. In line with the high incidence of relapse of AML after conventional chemotherapy-based induction therapy ,53, bothLess evidence is available in regard to cytarabine. The in vitro data demonstrated that cytarabine at low concentrations has, on one hand, as expected, a high antiproliferative effect against AML blasts, and, on the other, it may decrease T-cell function, alter the expression of DAMPs and reduce the release of HSP-90 . InteresThese first observations about cytotoxic agents allowed us to highlight that two drugs, daunorubicin and cytarabine, members of the same category, can exert an opposite immunomodulatory effect. This difference should be considered when they are combined with new drugs, whose mode of action will be discussed below.DAMP: damage-associated molecular pattern molecules, NK: natural killer, HMAs: hypomethylating agents, LAAs: leukemia-associated antigens, HLA: human leukocyte antigens, DC: dendritic cells, T-reg: T-regulatory cells, IS: the immune system.FLT3 mutations occur in approximately 30% of AML patients . In. Infibroated AML . A post-ated AML .FLT3-mutated AML. It has been tested in the post-transplant maintenance setting, yielding encouraging results . Mo. Mo20]. PD-1 (an inhibitory receptor of T cells) is epigenetically regulated . Th. Th96]. The Hedgehog (HH) family of intercellular signaling proteins is of crucial importance in embryonic development, tissue polarity control and stem cell maintenance . AberratThe oral SMO inhibitor glasdegib is the only FDA-approved drug for use in combination with low-dose cytarabine to treat newly diagnosed AML patients over 75 years old or who are not candidates for intensive induction chemotherapy . Its actAberrant HH signaling can induce an immunosuppressive microenvironment through several mechanisms. In basal cell carcinoma, experimental data showed that tumoral keratinocytes secrete transforming growth factor-beta (TGFβ), which can reduce the number of tumor-infiltrating lymphocytes and enhance the recruitment of MDSCs in mice bone marrow ,105.Further data from the breast cancer xenograft model confirmed, on one hand, the MDSC-related observation but, on the other, showed a reduction of MDSCs, T-regs and M2 macrophages upon treatment with vismodegib, the other HH inhibitor .PD-L1; the use of anti-PD-L1 antibodies in this context triggered the apoptosis of tumor cells [Moreover, involvement of the HH effector protein GLI2 in T-cell polarization and CD4+ cell differentiation in immunosuppressive T-regs has been demonstrated ,107,108.or cells . Data fror cells ,111. Lasor cells  and incror cells . These dor cells ; this efor cells . NeverthThese data underline the importance of ICI (PD-1/PD-1L axis) in the context of HH aberrations; therefore, combinations of the ICI and HH inhibitors in the treatment of AML could be considered for further investigation.TP53 has been widely studied in light of its multiple functions and dynamics, being involved in cell fate decisions in response to DNA damage and the consequent modulation of the cell cycle, senescence and apoptosis [TP53 due to deletions of the 17p chromosome occur in 5–10% of de novo AML cases [TP53 aberrations have been shown to modulate both the immune and inflammatory responses in malignancies [TP53 functions could promote innate and adaptive immune responses and increase the immunogenicity of the tumor cells [Often defined as “the guardian of the genome” , TP53 hapoptosis ,116. TheML cases  and in 4ML cases  and are ML cases . TP53 abgnancies , affectignancies ,122, T-rgnancies , PD-1 angnancies ,125 and gnancies . Moreoveor cells .TP53 mutations, the newest data showed an evasive immune phenotype characterized by the overexpression of PD-L1, associated with miR-34a expression [TP53 revealed a high expression of interferon-gamma and inflammation pathway genes [Regarding AML and MDS harboring pression , reducedpression . Accordiay genes .Therapeutic approaches aiming to stabilize both the wild-type and mutant p53 are still investigational, so their contextual immunological effects are as-yet unexplored. APR-246 is a prodrug that covalently binds to the mutant p53 core domain, promoting apoptotic mechanisms and cell cycle arrest pathways ,130. ItsInterestingly, the preclinical data from the melanoma mice model suggest a synergistic action of APR-246 with ICI, resulting in a decreased proliferation and increased cytolytic activity of CD8+ T cells, which was not observed with ICI alone. These findings have been further confirmed in solid cancer patients .p53 transcriptional activity is physiologically regulated by the E3 ubiquitin-protein ligase MDM2; therefore, MDM2 inhibitors are currently being tested in AML , suggestTP53-targeted therapies with ICI.A new oral anti-MDM-2, DS-5272, has been tested in a mouse AML model driven by MLL-AF9 and patient-derived xenograft models of human AML . NotablyConsidering that the results of the clinical trials involving p53-targeted therapies  in AML aThe surveillance by a host’s IS is critical in the control of cancer. The term “immunoediting” was first adopted 20 years ago to explain how a cancer and IS interact in three phases: elimination, in which innate and adaptive immune cells recognize and clear transformed cells; equilibrium, characterized by a high genetic instability in the resistant neoplastic cells with the aim to evade from IS control; escape: the variant clones expand by downregulating (editing) immunogenic molecules and adopting IS-suppressing mechanisms. Therefore, genetic instability and mutations are thought to be the drivers of cancer progression . NotablyMLL-ENL-driven AML have shown that immunoediting has a limited role in leukemic proliferation [AML1/ETO fusion gene, immune escape could even be inhibited by the upregulation of CD48 on the cell surface, thereby allowing NK recognition and killing [FLT3-ITD and NPM1 [IDH1/2 are associated with impairment of the T-cell functions, as discussed above.In line with this concept, the experimental data on feration . Moreove killing . This prand NPM1 . MoreoveOtherwise, the success of allogeneic hematopoietic stem cell transplantation  in AML has demonstrated the efficacy of the immune system in eradicating leukemic cells; the antileukemic response in allo-HSCT is, in fact, mediated by the graft versus leukemia (GvL) effect; donor T CD4+, CD8+ and NK cells recognize and clear residual blasts on the basis of immunological nonidentity, which relies mainly on the cell surface expression of both major and minor histocompatibility complex molecules and leukemia-associated proteins such as Wilms tumor 1, proteinase 3, survivine and immature laminin receptor . FurtherThe ever-expanding armamentarium of therapeutic agents and the refinement of consolidated procedures, such as the aforementioned allo-HSCT, have led to a considerable improvement in the outcome of AML patients. Nevertheless, the RFS and OS rates are still far from satisfactory. The success of allo-HSCT should be seen as two faces of the same coin, illustrating, on one hand, the power and variability of the treatments aiming to eradicate the leukemic clones and, on the other, the efficacy of the GvL immunosurveillance. This concept supports the idea that a successful therapeutic strategy should consider an integrated approach, simultaneously arming both weapons: the antileukemic and the IS-stimulating effects. Consequently, many drugs, both investigational and approved in AML, target or harness the IS: ICI, monoclonal antibodies, bispecific T-cell engagers (BiTEs) and CAR-T cells. It should be noted that, differently from the encouraging results obtained in non-Hodgkin’s lymphoma and acute lymphoblastic leukemia, CAR-T cells struggle to reach success in AML; the data show that unsatisfactory responses may be partly due to the hostile AML microenvironment, where the structural components of the bone marrow niche, soluble factors and suppressive immune cells such as T-regs and myeloid-derived suppressor cells (MDSC) are capable of globally reducing the CAR-T efficacy . Aside fSimilarly, the impairment of apoptosis through BCL-2 inhibition does not only exert its effects on the leukemic clone but also affects the homeostasis of the immune cell. The BCL-2 inhibitor effects on the IS suggest another possible explanation, in line with the data from the drugs inhibiting IDH and HH and with TP53-targeted agents: the eradication of leukemic cells upon the treatment might ultimately lead to the disruption of a tolerogenic microenvironment. Moreover, daunorubicin effects reinforce this concept, since triggering immunogenic cell death is a critical factor for a successful response to chemotherapy.All these considerations highlight the crucial role of the IS in the context of AML therapeutic success. An increasing knowledge of these cellular mechanisms may support the design of tailored therapeutic approaches performing clone eradication by direct blast killing and engagement of the IS. The identification of the biomarkers of IS activation upon drug exposure, such as an increase of CD8+ and NK after treatment with gilteritinib and DNTs after venetoclax/HMA, would provide further information on the IS impact on the prognosis and survival of AML patients.


Most patients with acute myeloid leukemia (AML) have a poor prognosis. Curative therapy of AML requires the complete eradication of the leukemic stem cells (LSCs). One aspect of LSCs that is poorly understood is their low frequency in the total population of leukemic cells in AML patients. After each cell division of LSCs, most of the daughter cells lose their capacity for self-renewal. Investigations into the role of Isocitrate dehydrogenase (IDH) mutations in AML provide some insight on the regulation of the proliferation of LSCs. The primary role of IDH is to convert isocitrate to alpha-keto-glutarate (α-KG). When IDH is mutated, it converts α-KG to 2-hydroxyglutarate (2-HG), an inhibitor of the TET pathway and Jumonji-C histone demethylases (JHDMs). The demethylating action of these enzymes removes the epigenetic gene-silencing markers, DNA methylation, H3K27me3 and H3K9me2 and can lead to the differentiation of LSCs. This enzymatic action is blocked by 2-HG in mutated IDH (mut-IDH) AML patients, who can be induced into remission with antagonists of 2-HG. These observations suggest that there exists in cells a natural enzymatic mechanism that uses demethylation to reverse epigenetic gene-silencing, leading to a loss of the self-renewal capacity of LSCs. This mechanism limits the proliferative potential of LSCs. Epigenetic agents that inhibit DNA and histone methylation exhibit a synergistic antineoplastic action on AML cells. It is possible that the therapeutic potential of this epigenetic therapy may be enhanced by demethylation enzymes, resulting in a very effective treatment for AML. Acute myeloid leukemia (AML) is characterized by the overproduction of abnormal myeloid cells which, when present in the bone marrow, suppress the production of normal blood cells . Chemoth12 cells in about 40 days. Since this explosive growth potential of AML cells is rarely observed in patients, there probably exists an intrinsic molecular mechanism that diminishes the full growth potential of LSCs.LSCs in AML represent a low-frequency subpopulation of leukemia cells that possess stem cell properties, a capacity for self-renewal and propagation of the malignant disease . The LSCInvestigations into the role of isocitrate dehydrogenase mutations (mut-IDH) in AML give an important insight on the mechanisms that regulate self-renewal potential of LSCs . MutatioKey enzymes that have the potential to promote leukemogenesis when they are deregulated are DNA methyltransferase (DNMT) and the histone methyltransferases (HMTs), EZH2 and G9a. DNMT1 silences gene expression by the methylation of cytosine to 5-methylcytosine in the promoter region of genes . EZH2 anEpigenome analyses of AML cells indicate that they contain several thousand additional methylated CpGs compared to normal white blood cells . The mecSupport that EZH2 also plays an important role is leukemogenesis can be seen in the report that its inhibition of EZH2 by 3-deazaneplanocin A (DZNep) reduces the proliferation and induces differentiation of AML cells . The oveThe presence of H3K9me2 gene-silencing marker in hematopoietic cells can also promote leukemogenesis. The methylation of this histone is catalyzed by G9a (EHMT2). The enhanced expression of G9a is involved in the proliferation of cancer cells . In suppOne of the most important enzyme functions that has the potential to suppress leukemogenesis is that of the TET family which can reverse gene-silencing by demethylation of DNA. In support of the anti-oncogenic action of TET2 is the report that AML cells with LOF mutations of this gene exhibit DNA hypermethylation and induction of leukemogenesis . The DNADeregulation of members of the family of JHDMs also favors the development of leukemogenesis. IDH mutations impair histone methylation by the inhibitory action of 2-HG on JHDMs and result in a block of differentiation . One of Another important member of the JHDM family is KDM3B (JHDM2B), the enzyme that demethylates H3K9me2. Demethylation of this histone gene-silencing marker also leads to gene reactivation. The knockdown of KDM3B in myeloid leukemia cells blocks granulocytic differentiation. . The redTSGs that suppress leukemogenesis may be silenced by different epigenetic events. The major epigenetic gene-silencing mechanisms in AML cells are DNA methylation, H3K27me3 and H3K9me2. The interaction between these epigenetic alterations reveals the important role epigenetic gene-silencing markers play in the development of AML . When AMEpigenome analysis revealed that about 25% of DNA methylated genes in cancer cells also contain H3K27me3 modification compared to only about 12% in normal cells . These iA second “double lock” mechanism of gene silencing can also occur by the presence of DNA methylation and H3K9me2 on the promoter of TSGs. Reactivation of these target genes would require the use of 5AZA-CdR in combination with an agent that reduces the level of H3K9me2, such as BIX-01294, which inhibits the methylation of H3K9 by G9a HMT ,33. We oA third “double lock” mechanism can be caused by the presence of H3K27me3 and H3k9me3 in the promoter of TSGs. This type of inhibition can be reversed by a combination of DZNep, an inhibitor of EZH2, and BIX-01294, an inhibitor of G9a HMT. We observed that DZNep in combination with this HMT inhibitor of G9a (EHMT2) exhibited synergistic antileukemic action on AML cells, as determined by colony assay . This reFinally, a “triple lock” mechanism of gene silencing can also be due to the presence of 5-methylcytosine, H3K27me3 and H3K9me2 at the promoter region of TSGs. Gene reactivation, in this case, would require the three inhibitors that reverse three different types of epigenetic gene-silencing. The triple combination of 5AZA-CdR, DZNep and BIX-01294 exhibited greater antineoplastic action against the leukemic cells than any of the double combinations . This “tThese observations indicate that a “cross-talk” exists between the three different types of gene-silencing mechanisms providing a rationale for the use of two to three different epigenetic agents for the therapy of AML to reverse this gene-silencing . From a Studies of AML patients with IDH mutations reveal that DNA methylation and histone methylation play a major role in the development of leukemogenesis. In order for LSCs to retain their capacity for self-renewal they have to silence genes that program differentiation using, primarily, the epigenetic action of DNA methylation, H3K27me3 and/or H3K9me2. This gene suppression can be reversed by the demethylation of DNA by the TET pathway and the demethylation of H3K27me3 and/or H3K9me2 by JHDMs . This inIt was a remarkable discovery that mut-IDH-AML cells produce the oncometabolite 2-HG that inhibits the TET pathway and JHDMs. This action blocks the differentiation of LSCs by the prevention of TSG reactivation via the inhibition of DNA and histone demethylation ,10. The The selection of the most active epigenetic agents is essential to improve the effectiveness of therapy of AML. The epigenetic agent 5AZA-CdR is one of the most potent due to its high selectivity to target cancer cells, its preferential upregulation of genes with promoter DNA methylation, and its preference for targeting genes that are silenced in cancer . In addiThe remarkable antileukemic synergy observed between 5AZA-CdR and DZNep was first observed in human HL-60 myeloid leukemia cells . An impo