Acute myeloid leukemia (AML) accounts for around 20% of diagnosed childhood leukemia. Cytarabine (CYT) is involved in the AML treatment regimen. AML and CYT showed impairment in spermatogenesis in human and rodents in adulthood. We successfully developed an AML disease model in sexually immature mice. Monocytes and granulocytes were examined in all groups: untreated control, AML alone, CYT alone and AML+CYT (in combination). There was a significant increase in the counts of monocytes and granulocytes in the AML-treated immature mice (AML) compared to the control, and AML cells were demonstrated in the blood vessels of the testes. AML alone and CYT alone impaired the development of spermatogenesis at the adult age of the AML-treated immature mice. The damage was clear in the structure/histology of their seminiferous tubules, and an increase in the apoptotic cells of the seminiferous tubules was demonstrated. Our results demonstrated a significant decrease in the meiotic/post-meiotic cells compared to the control. However, CYT alone (but not AML) significantly increased the count of spermatogonial cells (premeiotic cells) that positively stained with SALL4 and PLZF per tubule compared to the control. Furthermore, AML significantly increased the count of proliferating spermatogonial cells that positively stained with PCNA in the seminiferous tubules compared to the control, whereas CYT significantly decreased the count compared to the control. Our result showed that AML and CYT affected the microenvironment/niche of the germ cells. AML significantly decreased the levels growth factors, such as SCF, GDNF and MCSF) compared to control, whereas CYT significantly increased the levels of MCSF and GDNF compared to control. In addition, AML significantly increased the RNA expression levels of testicular IL-6 (a proinflammatory cytokine), whereas CYT significantly decreased testicular IL-6 levels compared to the control group. Furthermore, AML alone and CYT alone significantly decreased RNA expression levels of testicular IL-10 (anti-inflammatory cytokine) compared to the control group. Our results demonstrate that pediatric AML disease with or without CYT treatment impairs spermatogenesis at adult age (the impairment was more pronounced in AML+CYT) compared to control. Thus, we suggest that special care should be considered for children with AML who are treated with a CYT regimen regarding their future fertility at adult age. Acute myeloid leukemia (AML) is a heterogeneous blood disorder characterized by immature myeloid cell proliferation and bone marrow failure . AlthougA significant reduction in sperm parameters, such as sperm counts, concentration and motility, has been shown in adult leukemia patients compared to healthy donors and other examined adult cancer patients ,5,6. RecThe treatment of AML patients under the age of 60 often includes two chemotherapeutic medications: cytarabine (CYT) and anthracycline drugs, such as daunorubicin, idarubicin and/or mitoxantrone ,17. In gSpermatogenesis is a complicated biological process of spermatogonial cell division and maturation into highly specialized haploid spermatozoa . SpermatThe microenvironment that surrounds the germ cells plays a key role in the regulation of spermatogenesis. Somatic cells  secret proinflammatory and immunoregulatory cytokines (IL-10 and IL-6) under normal conditions and in response to inflammatory stimuli , in addiIn the present study, we developed an AML-treated immature mouse model and evaluated the direct effect of AML and cytarabine (CYT), alone and in combination, on the development of different stages of spermatogenesis post treatment.Two-week-old mice were injected i.p. with AML cells. Two weeks later, the mice were sacrificed, and blood was immediately collected from the heart (by cardiac puncture) for counting of monocytes and granulocytes. There was a 10-fold increase in monocytes and a 30-fold increase in granulocytes in the AML cell-injected mice compared to uninjected control mice A,B. AddiThe survival of the AML- and CYT-treated immature mice was examined for 6 weeks post treatment. Our results show that AML-injected immature mice survived around three weeks post injection, whereas the AML+CYT-injected immature group survived for 36 days post injection G. The CTTo evaluate the effect of AML and CYT on the testes, we collected testes from all study groups at different time points  post treatment with AML, CYT, AML+CYT or control. We weighed the body and testes of all mice involved in the study. We found that none of the treatments affected the body weight of the treated mice compared to the control group (data not presented). Additionally, injection of AML did not significantly affect testicular weight compared to control A. HoweveSections from testes collected from all groups at several time points, depending on the survival of each treated group , were stained with hematoxylin and eosin for histological analyses. They were analyzed according to the classification scale of normal (without damage of the tubules), moderate damage of tubules  and severe damage of the tubules , as shown in B. The quOur results showed a significant decrease in the percentages of normal histology of the seminiferous tubules (STs) at all examined time points post treatment in the AML- (1–2.5 weeks), CYT- (1–4.5 weeks) and AML+CYT (1–4 weeks)-treated groups compared to control C. AdditiHistological analysis showed a clear impairment effect of AML and CYT on the cellular composition/structure of the seminiferous tubules. Therefore, we examined the effect of all treatments on the apoptosis process and the proliferation of cells in the seminiferous tubules of the treated groups. Testicular sections from all treated groups were examined by TUNEL assay to identify apoptotic cells in the seminiferous tubules B. For quThese results may suggest that AML and CYT treatments increase the percentage of tubules with positive apoptotic stained cells. It is known that CYT has the ability, like other cytotoxic medications, to induce damage in the proliferating cells, which leads to an increase in the percentage of apoptotic cells.Following the examination of the effect of AML and CYT on the apoptosis process in testes, we investigated the effect of those treatments on cells of the seminiferous tubules at different stages of spermatogenesis, such as SALL4 and PLZF (premeiotic stage), CREM (meiotic) and acrosin (meiotic/post-meiotic). Following immunohistochemical staining (IHC) for SALL4 see  and PLZFNext, we investigated the effect of AML and CYT alone or in combination on the meiotic (CREM) and post-meiotic (ACROSIN) stages. The quantification analysis of immunofluorescent staining and expression levels of CREM and ACROSIN was performed A–D. For In parallel, the analysis of the meiotic/post-meiotic stage was performed by evaluating the immunofluorescent staining of ACROSIN in the same AML and CYT immature treated mice see . The tubRNA expression results showed that AML significantly decreased the expression levels of ACROSIN in testicular homogenates of immature treated mice in the first week post treatment but increased its expression in the second week and was similar 2.5 weeks post treatments compared to control D. HoweveOur results show that AML, CYT and AML+CYT significantly decreased the meiotic and post-meiotic cells at several points post treatment compared to control. Furthermore, the AML+CYT-treated group showed a similar effect to that of the CYT only group on meiotic and post-meiotic cells at almost all examined time points A–D.Testicular sections from all treatment groups were examined for the presence of PCNA-positive cells (PCNA is a proliferation cell marker) by immunofluorescent staining to identify the localization of proliferating cells in the seminiferous tubules of testes from mice 2 weeks post treatment (the last period that mice survived post AML treatment) . PositivThus, our results show that CYT treatment significantly decreased proliferative cells 2 weeks post treatment, whereas AML significantly increased proliferative compared to control.In order to identify the proliferating cells counted in previous immunostaining assays, we conducted double immunostaining using specific antibodies against (PCNA) and spermatogonial stem cell marker (PLZF) D. Our re6/mL. To examine the effect of CYT and CYT+AML on the generation of sperm, we sacrificed the mice from all treated groups at the last possible time point of their survival (4 weeks post treatment) and isolated sperm from their epididymis. Our results show the complete absence of sperm from CYT- and CYT+AML-treated groups compared to the control group (CT), which showed normal generation of sperm at a concentration of (1.07 ± 0.13) × 10Following the examination of the effect of AML and CYT on spermatogenesis, we investigated the effect of those treatments on the testicular growth factors and cytokines mainly secreted from testicular somatic cells (microenvironment of the SSCs) involved in the regulation of spermatogonial cell maintenance, proliferation and differentiation. Our results show that AML significantly decreased the protein levels of GDNF, MCSF and SCF but not LIF compared to control A,B.In addition, our results show that AML treatment significantly increased the RNA expression level of the proinflammatory cytokine (IL-6), whereas CYT treatment decreased it significantly A. FurtheIn this study, we demonstrated that the AML disease model in sexually immature mice is successfully developed. There was an increase in monocytes and granulocytes in the AML-cell-injected mice, which is consistent with the analysis used to diagnose pediatric AML in humans . In addiAML increased the RNA expression levels of testicular IL-6 (a proinflammatory cytokine), which is secreted by testicular somatic and spermatogenic cells and involved in the regulation of spermatogenesis under normal conditions and in response to inflammatory stimuli ,35,36,37In parallel, we demonstrated that CYT decreases the testicular weight in adult-age immature treated mice. Our results are in harmony with previous studies that showed that cytotoxic agents such CYT and busulfan decreased the weight of developing organs, such as testes and placenta, in rodents ,43,44. HCYT alone and AML alone or in combination (AML+CYT) significantly increased the apoptosis of germ cells in the seminiferous tubules, as examined by TUNEL assay. However, the RNA expression levels of the examined intrinsic and extrinsic apoptotic factors  in the testicular tissues were not affected by any of the treatments. The discrepancy between the results of TUNEL assay and those of RNA expression could be related to the counting of cells in the seminiferous tubules that were stained positively in the TUNEL assay, whereas the RNA analyses were performed on testicular tissue. An additional option is the possibility of differences in the regulatory processes (transcription/translation and RNA stability) between RNA expression and the final process of apoptosis (dead cells). Furthermore, CYT increased the count of SSCs (SALL4 and PLZF cells), and testicular growth factors, including GDNF. We suggest that CYT increased GDNF within the niche of the spermatogonial cells, which had a critical role in SSC self-renewal and maintenance by binding to its receptors, GFRa1, that were mostly expressed in undifferentiated spermatogonia and RET. This regulation could involve RET phosphorylation that induces SRC family and PI3K/AKT activation, allowing for the proliferation of SSCs ,46,47. TOur results show that the effect of AML and CYT together (AML+CYT) showed a similar effect to that of CYT in most of the examined parameters and factors, except SCF and percentage of apoptotic cells. This may reflect the severe effect of CYT on spermatogenesis and that the mechanism of CYT is major factor. However, this mechanism is different in some functions of the spermatogenesis (cellular apoptosis and expression of SCF). In conclusion, our results show that AML alone, in the prepubertal age, affects male fertility in the adult age. In addition, treatment of boys with AML with CYT could adversely affect their fertility at adult age.Two-week-old C57Bl/6 mice were used in the present study. The mice were purchased from Harlen Laboratories Ltd., Jerusalem, Israel. They were appropriately handled at the animal house of the Faculty of Health Science at Ben-Gurion University in Negev, Israel, according to the established handling protocols. The mice were sacrificed at several time points post injection, blood was collected from the heart, and the body and testes were weighed. This study was performed in accordance with the Guiding Principles for the Care and Use of Research Animals promulgated by the Society for the Study of Reproduction and was confirmed by the Ben-Gurion University Ethics Committee for Animal Use in Research (IL-70-11-2016). 4 cells/100µL PBS according to the protocol of the murine C1498 Cell Line used in adult mice  was used. We intraperitoneally (i.p) injected 3 × 10ult mice ,50,51. Bult mice . As a co2 sacrifice. Monocyte and granulocyte levels were measured by an Exigo H400 veterinary hematology analyzer according to the manufacturer instructions. Giemsa staining was used to morphologically identify AML cells in blood samples.Blood samples were collected from the heart of the mice immediately after COImmunofluorescent staining was performed as described previously . TesticuImmunohistochemistry staining was performed as described in the protocol . Briefly, the slides were prepared exactly as mentioned above. Then, the slides were deparaffinated and dehydrated by xylene and ethanol, respectively . After washing with a phosphate-buffered solution (PBS) , antigen retrieval of the sections was performed in heated sodium citrate solution  in a warm microwave for 7 min (twice). After washing, the nonspecific adhesion sites in the tissues and cells were blocked for 90 min at room temperature using a horse serum. Following the removal of the blocking buffer, the first antibodies were added as follows: PLZF , SALL4 . After overnight incubation at 4 °C, the slides were washed, and the specific secondary antibodies were added compatibly to the first antibodies for 60 min at room temperature. After washing, the slides were dried. The specificity of the staining was also examined in the testicular tissue using the relevant IgG isotype as the negative control. The slides were examined for staining using a fluorescence microscope .This staining was performed as described previously . BrieflyRNA was extracted according to the company protocol ) and according to our previous publication . cDNA syt) values were defined, representing the cycle number at which the sample fluorescence rises statistically above the background and crossing points (CP) for each transcript. The relative quantity of gene expression was analyzed using the 2−ΔΔCt method. The results are expressed as the fold of increase related to the GAPDH of the same examined sample.The qPCR reaction was performed following the method described our previous study . The “thImmunohistochemical apoptotic detection was carried out using a commercial kit . The TUNEL assay was performed according to the manufacturer’s instructions. The system directly detects apoptotic cells in situ at the single-cell level in paraffin-imbedded tissue. The positive staining was examined using a light microscope. Mice were sacrificed four weeks post treatment, and sperm was extracted according to the method described in our previous study .The protein levels of growth factors  in testicular homogenates were evaluated by specific Duoset ELISA for each examined growth factor. The following kits were used. according to the manufacturers’ instructions: GDNF , MCSF , LIF  and SCF . Briefly, 96-well plates were separately coated with 100 µL capture antibodies against each growth factor overnight at room temperature, washing 3 times with wash buffer , followed by blocking with 300 µL of BSA 1% at room temperature for at least 1 h. The washing step was repeated, as mentioned previously. Samples and the standards were then added and incubated at room temperature for 2 h. After the washing step, 100 µL of detection antibodies, anti-GDNF, anti-MCSF or anti-LIF and anti-SCF, were added and incubated at room temperature for 2 h. The plates were then washed as mentioned before. A volume of 100 µL of the working dilution of streptavidin-HRP  was added and incubated for 20 min at room temperature, and another washing procedure was performed. A volume of 100 µL of substrate solution was added to each well, and the plate was incubated for 20 more minutes. At the end of the incubation, 50 µL of stop solution was added, and the optical density of each well was measured using a microplate reader set to 450 nm.Protein assay dye reagent concentrate  was diluted in PBS (1:5) and filtered. Each well was covered with 200 µL of the diluted dye reagent. The protein standard, albumin bovine, was purchased from MP Biomedicals, Irvine, CA, USA. The standard protein was diluted in duplicates . Each well was covered with 10 µL of standard protein/sample. The optical density of each well was then measured by a microplate reader set to 595 nm.t-test and was shown as p-value: *,@,$ p < 0.05, **,@@,$$ p-value < 0.01, ***,@@@,$$$ p-value < 0.001.The statistics values were calculated according to Av. ± SEM. The statistical significance was examined by two-tailed


TP53 aberrations are found in approximately 10% of patients with acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) and are considered early driver events affecting leukemia stem cells. In this study, we compared features of a total of 84 patients with these disorders seen at a tertiary cancer center. Clinical and cytogenetic characteristics as well as immunophenotypes of immature blast cells were similar between AML and MDS patients. Median overall survival (OS) was 226 days  for the entire cohort with an estimated 3-year OS rate of 11% . OS showed a significant difference between MDS  and AML patients  which is likely due to a different co-mutational pattern as revealed by next-generation sequencing. Transformation of TP53 aberrant MDS occurred in 60.5% of cases and substantially reduced their survival probability. Cox regression analysis revealed treatment class and TP53 variant allele frequency as prognostically relevant parameters but not the TP53-specific prognostic scores EAp53 and RFS. These data emphasize similarities between TP53 aberrant AML and MDS and support previous notions that they should be classified and treated as a distinct disorder.The online version contains supplementary material available at 10.1007/s00277-022-04766-2. TP53 gene, located at chromosome 17p13.1, encodes p53—a fundamental tumor suppressor highly conserved during evolution. Among a multitude of different physiological functions, p53 is activated by several extrinsic and intrinsic stress signals including DNA damage, oncogene activation, hypoxia, and nutrient deprivation. Dependent on the activation signal, p53 induces a multitude of downstream signals aimed at sustaining cellular homeostasis. Importantly, by pursuing these functions, p53 acts in a cell context–specific manner. There is tight regulation of p53 at the transcriptional, post-transcriptional, and post-translational levels, respectively, conferring fine-tuning of this essential cellular protein [The  protein .G12D, p53 loss led to indefinite self-renewal of these cells with a propensity for transformation into leukemia-initiating cells [p53 displays pivotal functions in normal hematopoietic stem and progenitor cells being involved in their proliferation, differentiation, and apoptosis . Murine TP53 aberrations are constantly observed at a frequency of approximately 10% with steep increases in older patients and those with therapy-related myeloid neoplasms. Predominantly, they constitute missense mutations located in the DNA binding domain of the gene. However, other types of mutations—nonsense variants, small insertions and deletions—as well as chromosomal losses encompassing the TP53 locus and combined aberrations are also observed [TP53 mutations are somatically acquired in the majority of patients affecting hematopoietic stem and progenitor cells; however, germline mutations characterizing the Li-Fraumeni (LF) and LF-like syndromes are also observed in these disorders [TP53 mutations exhibit a high risk of progression to AML [TP53 aberrations usually have a dismal prognosis even when treated with intensive regimens including allogeneic hematopoietic stem cell transplantation (HSCT) [Acute myeloid leukemia (AML) is an aggressive hematopoietic malignancy derived from transformed HSPCs ultimately leading to bone marrow failure. Myelodysplastic syndromes (MDS) are clonal bone marrow disorders characterized by ineffective hematopoiesis and peripheral blood cytopenias progressing to AML in a substantial number of cases. Both disease entities are highly heterogeneous with respect to biological and clinical features , 7. In Aobserved , 9. Thesisorders . Recentln to AML . Patientn (HSCT) , 18.TP53 aberrations should be regarded as a distinct disease entity [Based on biological and clinical features, we recently proposed that AML and MDS with e entity . In thisTP53 aberrations, seen at our institution—a tertiary hematology center for a population of approximately 1.5 million people—between November 2014 and June 2021. Only those patients whose TP53 aberrations were detected at diagnosis were included. Diagnosis and risk stratification of myeloid neoplasms were performed according to standard criteria including conventional karyotyping as well as fluorescent in situ hybridization (FISH). For molecular studies, genomic DNA extracted from bone marrow biopsies was analyzed for mutations in up to 44 myeloid-associated genes using an Ion Torrent next-generation sequencing (NGS) platform (Thermo Fisher Scientific) as described previously [TP53, all coding exons and flanking exon–intron boundaries were sequenced with a lower limit of detection set at 5% mutant allele reads. Variants were classified according to the VarSome (https://varsome.com) and TP53 databases (https://p53.iacr.fr), and only pathogenic and likely pathogenic ones were included. Treatment categories comprised intensive chemotherapy and allogeneic hematopoietic stem cell transplantation (HSCT), non-intensive therapies , and best supportive care including hydroxyurea. All patient data were retrieved from openMEDOCS, a regional hospital–based documentation system.The study cohort consisted of adult patients suffering from AML and MDS with U and Fisher’s exact tests were used for continuous and categorical parameters, respectively, and median (range) or numbers  are used to descriptively summarize the data. Kaplan–Meier analysis and the log-rank test were used to describe and compare overall survival (OS), calculated as time from diagnosis to either death or last-follow up, and the median OS with 95% confidence intervals (CI) is presented. To identify relevant prognostic parameters, Cox regression analyses were performed taking the particular disease , age, sex, the TP53 variant allele frequency (VAF), TP53-specific scores, and the treatment class into account. The TP53 scores were explored as previously described by our group [TP53 variant. Then, we investigated the evolutionary action score (EAp53) focusing on the evolutionary sensitivity to sequence variation and amino acid conservation of missense mutations. Finally, the relative fitness score (RFS) based on in vitro growth properties of particular TP53 variants was evaluated. For the multivariable analysis, all parameters with p < 0.05 in the univariable analysis as well as age and sex were considered. The analyses were performed using R version 4.1.0 (https://www.R-project.org/).To compare clinical and biological parameters, the Mann–Whitney ur group . BrieflyTP53 aberrations. Multicolor flow cytometry was performed using a Navios cytometer  with harmonized baseline settings as described previously [dim/SSClow gating with additional backgating using CD34− and CD117− expression to improve the identification of the blast population when appropriate. For all markers, isotype controls were used to define the percentage of marker positive cells. In addition, the mean fluorescence intensity (MFI) ratio was calculated by dividing the geometric MFI of the antibody-stained sample by the geometric MFI of the respective isotype control. MFI ratio values > 1.5 were considered as significant [TP53 aberrant AML and MDS samples using the Mann–Whitney U test. Multiple testing correction was performed using the Benjamini–Hochberg procedure to control the false discovery rate [p value below 0.05 was considered statistically significant. Principal component analysis (PCA) was performed on scaled MFI ratio and PPC data and visualized as a biplot of principal components 1 and 2, respectively, using the R package ggbiplot (v. 0.55) (https://github.com/vqv/ggbiplot). Samples with missing values were removed prior to PCA. Statistical analysis was performed with GraphPad Prism software version 9.1 (GraphPad Software) and R (v. 4.1.2).The immunophenotype of blast cells was compared between AML and MDS patients showing eviously . After enificant . MFI ratery rate . A p valTP53 aberrations were included, 46 of them with AML and 38 with MDS. The median observation period for the total cohort was 149 days . Demographic data of the patients are depicted in Table p = 0.004 and p = 0.005, respectively, Mann–Whitney U test). Intensive chemotherapy treatments (“3 + 7” regimen) were more frequently given to AML patients, and allogeneic HSCT was performed in 5 AML (10.9%) and 3 MDS (8.1%) patients, respectively. In MDS patients, non-intensive therapies, preferably the hypomethylating drug azacitidine, were most commonly applied.A total of 84 patients with a median age of 71 years (range 34–86) suffering from myeloid malignancies with TP53-specific risk scores are shown in Table TP53 gene, were similar in both disease entities. Also, the number of patients with multiple TP53 variants and the median TP53 VAF was comparable between AML and MDS. The number of cooperating gene mutations as revealed by NGS of a panel of myeloid-associated genes was low in both groups. However, the genes affected differed between both groups with mutations in KRAS, SRSF2, DNMT3A, and CEBPA being more common in MDS. Due to the small number of cases, we refrained from statistical evaluation here.Cytogenetic and molecular genetic data including TP53 aberrant AML and MDS samples could not be separated by immunophenotyping ; non-intensive treatment, 463 versus 98 days (p < 0.001); and best supportive care, 187 versus 32.5 days (p < 0.001).The median OS of the entire cohort was 226 days  Fig.  and was TP53 VAF as well as the TP53-specific scores. In both uni- and multivariable analyses, the presence of AML and best supportive care as treatment as well as higher TP53 VAFs were associated with a significantly shorter OS. However, neither of the TP53-specific risk scores reached statistical significance and were therefore not considered in the multivariable analysis.Table TP53 aberrant AML and MDS evaluated and treated at a tertiary cancer center. In agreement with our recently published postulation that they constitute a distinct disease entity [TP53 aberrant AML and MDS are undistinguishable by this approach. With a median OS of 226 days and an estimated 3-year survival rate of 11%, our study confirms previous results reporting an exceedingly adverse outcome of such cohorts [TP53 mutations are early driver lesions affecting preleukemic/leukemic stem cells. As a sole event, TP53 mutations are unable to induce neoplastic transformation as has been exemplified by their occurrence in healthy individuals with clonal hematopoiesis of indeterminate potential [TP53 mutated AML and MDS, preferably constitute chromosomal aberrations and copy number alterations leading to pronounced expression changes of co-operating gene [TP53 aberrant myeloid disorders although their frequency is substantially lower than in AML and MDS with a TP53 wild-type status. In our NGS analysis, co-mutational patterns were different between TP53 aberrant AML and MDS although the number of cases studied was too small to allow for an adequate statistical comparison. Several previous reports have unambiguously determined typical mutational patterns associated with MDS and secondary AML affecting genes like SRSF2 and SF3B1 [. We, therefore, speculate that the basis for manifestation as either TP53 aberrant AML or MDS as well as their different survival probabilities is mainly due to different co-mutational events occurring secondary to the TP53 aberration. In this regard, it would also be interesting to investigate molecular events responsible for transformation of TP53 aberrant MDS to AML occurring within short periods of time.Here, we present data comparing patients with e entity , a multi cohorts , 25, 26.otential . Secondaing gene . Co-opernd SF3B1 –32. We, TP53 VAFs were also comparable between AML and MDS. With a median value of 53% in MDS patients, it indicates that the mutant clone size is considerably exceeding the percentage of bone marrow blast cells in line with previous data on this issue [TP53 VAF has been shown to be a statistically significant prognostic parameter in this study with higher values having a more pronounced adverse impact on survival. In a study of 1537 AML patients treated intensively within protocols of the AMLSG study group, we were able to demonstrate that even TP53 VAFs of less than 20% represent significant adverse risk factors for both OS and event-free survival [TP53 mutation and its functional consequence has an impact on treatment response [TP53 load was associated with a significantly increased risk for treatment failure [TP53 VAF as an independent prognostic parameter for survival with a 40% VAF as cutoff for high and low clonal burdens [TP53 allelic state” as an important biological and clinical parameter. Ideally, the TP53 allelic states should be assessed by combining conventional karyotyping + / − FISH and NGS analysis, respectively. Patients with TP53 mono-allelic mutations were comparable to TP53 wild-type patients, whereas multi-hit aberrations consistent with TP53 bi-allelic events showed complex karyotypes and poor outcomes [TP53 allelic state in AML are not available yet.The is issue . Interessurvival . Furtherresponse . In cont failure . A syste burdens . Recentloutcomes . HoweverTP53 aberrant AML and MDS remains unsatisfactorily; however, novel promising drugs and strategies are being tested in several clinical trials. So far, the only curative approach is allogeneic HSCT indicating that restoration of intact immune surveillance mechanisms mediating graft-versus-leukemia takes a central role in combating the disease [TP53 mutations were 55% treated with that drug combination versus 0% in the azacitidine group. However, long-term survival did not improve and TP53 mutations could be shown to be a major determinant of resistant disease [TP53 aberrant subgroups [TP53 mutated AML and MDS with encouraging CR and OS rates [TP53 mutant MDS failed to meet the primary endpoint of a significantly increased CR rate (Press Releases | Aprea Therapeutics). A comprehensive discussion of ongoing clinical trials in this particular patient cohort has recently been published [Treatment of patients with  disease , 37. Rec disease –40. Evalubgroups , 42. APROS rates , 44. A cublished .TP53 aberrant AML and MDS, obtained at a tertiary cancer center, revealed a high concordance of biological and clinical features between these two disease entities. A significantly increased OS rate in MDS patients is likely due to a different co-mutational pattern. As TP53 aberrations are early leukemogenic events in AML and MDS, these disease entities may, indeed, be classified and treated as a distinct disorder.In conclusion, our data on Supplementary file1 (PDF 487 KB)Below is the link to the electronic supplementary material.


HCMD is efficiently cleared by local mitochondria that avidly take up Ca2+ through their uniporter (MICU), then released back to the cytosol through mitochondrial Na+/Ca2+ exchanger (MNCX). We found that newly synthesized derivative ITH15004 facilitated the release of catecholamines triggered from high K+-depolarized bovine CCs. Such effect seemed to be due to regulation of mitochondrial Ca2+ circulation because: (i) FCCP-potentiated secretory responses decay was prevented by ITH15004; (ii) combination of FCCP and ITH15004 exerted additive secretion potentiation; (iii) such additive potentiation was dissipated by the MICU blocker ruthenium red (RR) or the MNCX blocker CGP37157 (CGP); (iv) combination of FCCP and ITH15004 produced both additive augmentation of cytosolic Ca2+ concentrations ([Ca2+]c) K+-challenged BCCs, and (v) non-inactivated [Ca2+]c transient when exposed to RR or CGP. On pharmacological grounds, data suggest that ITH15004 facilitates exocytosis by acting on mitochondria-controlled Ca2+ handling during K+ depolarization. These observations clearly show that ITH15004 is a novel pharmacological tool to study the role of mitochondria in the regulation of the bioenergetics and exocytosis in excitable cells.Upon depolarization of chromaffin cells (CCs), a prompt release of catecholamines occurs. This event is triggered by a subplasmalemmal high-Ca Milder 0.5–1 µM [Ca2+]c elevations, as a result of Ca2+ redistribution, occur at inner cytosolic areas to regulate pre-exocytotic steps, such as Ca2+-dependent vesicle transport to reload the secretory machinery c transients elicited by BZATP stimulation of human P2X7R-expressing HEK293 cells c available for exocytosis during CC depolarization.Here, we report the pharmacological effects of compound ITH15004 on catecholamine release and Ca+ solution . + pulses (P1 to P22) applied to each cell batch. In this experiment, the initial peak secretion amounted to 200 nA equivalents of catecholamines, that gradually decreased to reach 45.7% of P1 at P22. Pooled data from 14 cell batches from six different cell cultures show that the decay of exocytosis along the experiment reached 45.7 ± 2.2% of P1 at P22  of the black circle curve in + pulse in the presence of the compound), augmented secretion was estimated to be concentration-dependent at 0.3–1 µM; at higher concentrations (3–10 µM), facilitation of secretion was still present, but to a lesser extent. This suggests that two distinct mechanisms of action could be present at lower and higher ITH15004 concentrations, as argued in the Discussion.In the experiment shown in 2+ store of CCs c depend not only on Ca2+ entry, but also on its redistribution into the ER c fluorescence, cells were challenged with 35K+ pulses (one pulse per well in 96-well plates). Changes of [Ca2+]c were expressed as arbitrary fluorescence units (AFU) [2+]c transient curves in the absence (control) and presence of increasing ITH15004 concentrations. At 0.3, 1, and 3 µM the compound augmented the [Ca2+]c peaks elicited by 35K+. At 10 µM the [Ca2+]c transient was also augmented but to a lesser extent. This is more clearly shown in 2+]c by 71.6 ± 11.1%, 101.2 ± 9.8% and 87.6 ± 10.1%, respectively. At 10 µM, the [Ca2+]c increase was 40.8 ± 22.8 but it did not reach the level of statistical significance when compared to control.After recording for a minute to estimate basal [Cats (AFU) . Figure 2+]c was elevated by ITH15004, as shown in the original full traces shown in + addition. Baseline elevations (recording of traces just before applying the 35K+ pulse) are averaged in 2+]c is compatible with an effect of ITH15004 on Ca2+ mobilisation from an intracellular organelle. This was explored next.Of interest was the observation that baseline [Ca2+ movements was explored in cells with their ER Ca2+ store irreversibly depleted by CRT. The effects of the compound on [Ca2+]c were explored with experimental protocols like those presented in The potential effect of ITH15004 on ER Ca2+]c signals control response. Added on top of CRT, ITH15004 further augments the response  and H (FCCP+JNJ+ITH15004) show quite similar effects on [Ca2+]c elevations, suggesting that P2X7Rs are not involved in the effects of compound ITH15004 on the [Ca2+]c transients elicited by 35K+ in BCCs. Additionally, JNJ did not affect the facilitation of secretion elicited by 35K+ (data not shown).Finally, we investigated whether compound JNJ-47965567 (JNJ), a potent blocker of P2X7Rs, was mimicking somehow the effects of ITH15004 on mitochondrial Ca+-elicited exocytotic release of catecholamines from BCCs by the novel purine derivative compound ITH15004  in mitochondria [2+-dependent ATP production [2+ handling by mitochondria. This was proven through the monitoring of [Ca2+]c elevations of basal [Ca2+]c and [Ca2+]c elevations triggered by 35K+. While ITH15004 did not exert major changes of [Ca2+]c under conditions of ER Ca2+ depletion, its effects on mitochondrial Ca2+ were notorious. Thus, under cell exposure to FCCP when mitochondria are unable to trap cytosolic Ca2+ because of the dissipation of the proton gradient and mitochondrial depolarization [+-induced [Ca2+]c elevations, even above the levels previously augmented by FCCP alone. Furthermore, additional observations showed that the interruption of mitochondrial Ca2+ circulation, either by RR (blockade of Ca2+ influx) or CGP (blockade of Ca2+ efflux), cancelled the augmentation of [Ca2+]c elicited by ITH15004 in the presence of FCCP. This suggests two interesting mechanisms involved in ITH15004 effects. First, the compound requires active mitochondrial Ca2+ circulation even in the presence of FCCP and is reinforced by ITH15004, that might contribute to maintain ATP synthesis. In so doing, it is plausible that ITH15004 is facilitating on the one hand, vesicle transport and priming of the secretory machinery by maintaining for longer time the elevated basal [Ca2+]c during the resting periods in between the repeated 35K+ pulses. On the other hand, through its effects in facilitating mitochondrial Ca2+ circulation, ITH15004 may contribute to maintain ATP synthesis and hence, the ATP required for maintaining healthy secretory responses upon repeated stimuli of BCCs. How these effects are exactly exerted, and which are the mitochondrial targets on which ITH15004 is acting, is a matter of further investigation.In sharp contrast, ITH15004 deeply affected the secretory responses under conditions of pharmacological manipulation of Cachondria . The decoduction ,18 that oduction . Compounrization ,18, ITH150 of 14 µM c, cells were plated at a density of 2 × 105 cells per well into 96-well black plates, and the experiments were performed 48 h later. Cells were loaded with a Krebs-HEPES solution  with 10 µM fluo-4-AM and 0.2% pluronic acid. Cells were incubated for 45 min at 37 °C in the dark. After this incubation period, cells were washed twice with the Krebs-HEPES solution at room temperature in the dark. Changes in fluorescence  were measured using a fluorescent plate reader . Basal fluorescence levels were monitored before adding the stimulation solution (35K+) with an automatic dispenser. After stimulation of the cells, changes in fluorescence were measured for 60 s. To normalize fluo-4 signals, responses from each well were calibrated by measuring maximum and minimum fluorescence values. At the end of each experiment, 5% Triton X-100 (Fmax) was added, followed by 2 mM MnCl2 (Fmin). Data were calculated as a percentage of Fmax–Fmin.To monitor the changes of [Ca−2 M) were prepared in dimethyl sulfoxide (DMSO) and protected from light. Final drug concentrations were obtained by diluting the stock solutions directly into the extracellular solution. At these dilutions, solvents had no effect on the parameters studied.The following chemicals were used: collagenase type I, FCCP (carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone), ruthenium red (RR), caffeine, ryanodine (Ry), thapsigargin, and CGP37157, were from Sigma/MercK ; Dulbecco’s modified Eagle’s medium (DMEM), bovine serum albumin fraction V, foetal calf serum, and antibiotics were from Gibco . Fluo-4-AM from Life Technologies . JNJ-47965567 was provided by Janssen Pharmaceutica NV . All the other chemicals used were reagent grade from Merck and Panreac Química . FCCP, RR, Ry and Thapsigargin mother solutions  and different cultures used (N). Mathematical analyses were done using the GraphPad Prism software, version 5.01 . Comparison between means of group data were performed by one-way analysis of variance (ANOVA) following by the Tukey’s post hoc test when appropriate.


Drosophila. In this study, we report CG9911, the Drosophila homolog of ERp44 (endoplasmic reticulum protein 44) plays a role in regulating adipose tissue fat storage. Using the CRISPR/Cas9 system, we generated a CG9911 mutant line deleting 5 bp of the coding sequence. The mutant flies exhibit phenotypes of lower bodyweight, fewer lipid droplets, reduced TAG level and increased expression of lipolysis related genes. The increased lipolysis phenotype is enhanced in the presence of ER stresses and suppressed by a reduction of the ER Ca2+. Moreover, loss of CG9911 per se results in a decrease of ER Ca2+ in the fat body. Together, our results reveal a novel function of CG9911 in promoting fat storage via regulating ER Ca2+ signal in Drosophila.Fat storage is one of the important strategies employed in regulating energy homeostasis. Impaired lipid storage causes metabolic disorders in both mammals and  Fat is stored in the form of lipid droplets (LDs) formation of which is regulated by a variety of lipases on the LD surface . The dynCG9911 is a predicted Drosophila homolog of ERp44 which was first identified as a member of the protein disulfide isomerase (PDI) family residing in the ER of mammalian cells [2+ homeostasis [3R1) dependent Ca2+ release from ER to cytoplasm by specifically binding to the third luminal loop (L3V) of IP3R1 [an cells . The cryan cells . Recent eostasis . As a pHeostasis , serotoneostasis  and FGE/eostasis . ER-resieostasis  and Ero1eostasis  oxidaseseostasis  and hypoeostasis . Furtherof IP3R1 .2+ is a key signal which plays roles in a variety of cellular processes such as fertility, cell proliferation and apoptosis [2+ storage place where the cellular Ca2+ homeostasis is maintained via transmembrane Ca2+ channels such as the inositol 1,4,5-trisphosphate receptors (IP3Rs), the ryanodine receptor (RyR), the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) and the STIM1  [2+ signaling is also an important regulator involved in multiple adipocytic activities including adipogenesis and lipid storage. For example, Drosophila IP3R mutants are obese and hyperphagic [Capoptosis . Endoplalecule1) . Ca2+ sierphagic ; STIM1 nerphagic ; and adierphagic .Drosophila adipocytes. CG9911 knockout causes elevated lipolysis which is suppressed by RNAi of ER Ca2+ channels. Ca2+ imaging shows that ER Ca2+ store is decreased in CG9911 mutant flies. ER stress is induced by the mutation of CG9911 and CG9911 mutant flies exhibit aggravated lipolysis in the fat body. We propose that decreased ER Ca2+ store induces ER stress which is responsible for the lipolysis alteration in the fat tissues.Here, we identify CG99AA/ERp44 as an important regulator of lipid storage in CG9911 with the N-terminal signal peptide, KDEL motif and an ER resident signal, on the C-terminal are depicted in CG9911 to inject Drosophila embryos , we analyzed the expression pattern of CG9911 in lization . It is elization , the winlization , the oenlization  and the lization  at the llization . Semi-qulization , which slization . Taken tf20CG9911 is significantly reduced compared to that of the wild type flies  in the fat body (Drosophila fat tissue) at different developmental stages. At the larval stage, no significant differences of LD size or TAG levels were detected between the CG9911 mutant and 1118w wild type animals  under fed condition. CG9911 mutant did not alter the lipid mobilization compared with wild type under starvation  and lipolysis related genes  of whole body were examined by real time PCR  are transcription factors which regulate the synthesis of enzymes involved in sterol biosynthesis [CG9911 mutation. However, as the key enzymes catalyzing the hydrolysis [Lip3) and adipose triglyceride lipase (bmm) show increased expression by different folds. The expression of bmm in f20CG9911 is ~1.6 folds of that in wild type animals, and Lip3 is ~9 folds. These results indicate that CG9911 loss of function results in an increase of lipolysis in the fat body of adult flies. Lipolysis is a process in which triacylglycerol is hydrolyzed into glycerol and free fatty acids (FFAs) in adipocytes and affects energy homeostasis [The bodyweight of both male and female adult flies ofpe flies , but thenditions . Given t animals . Consistarvation . Though nt stage , TAG detnt stage , 3C. Intpe flies , 2D. Mor control . To veritime PCR . Fatty a mammals . Drosophynthesis . As showdrolysis , lipase3eostasis . Lipolyseostasis , TNF-α aeostasis . Lipolyseostasis . Thus, wCG9911 is associated with ER stress, tunicamycin (TM) was used to induce ER stress in the adult flies. The survival curves show that f20CG9911 is more sensitive to TM-induced ER stress as compared to the wild type , and Atf4 expression, three ER stress markers  [3RIP depletion leads to a phenotype of obesity and increased fat storage [3RIP in fat body promotes both the size of LDs  . IP3R de storage . Our rese of LDs , 4B and e of LDs . IP3R knets size , 4B and ets size . STIM kn mutants , 4C. Intand Atf4 . These rCG9911 mutant is suppressed by the down-regulation of Ca2+ channel, we reasoned that CG9911 may play a role in ER Ca2+ homeostasis. To test this hypothesis, Ca2+ imaging experiments were performed with the fat body of CG9911 mutant and the wild type files. Cells with Fluo-4 AM, Ca2+ indicator, were treated with 10 μM ionomycin (an ionophore that binds Ca2+) to stimulate Ca2+ release from ER to cytosol , the half time of rise (t1/2on) and half time of decay (t1/2 off) are shortened by 32.6%, 29.1% and 37.1%, respectively  is reduced by 58.4%  as indicated by the activation of three effectors  [Drosophila adipocytes  , which lipocytes , 6B. OurCG9911 mutants whose phenotypes include: 1) CG9911 mutant flies exhibit lower bodyweight and more sensitivity to starvation; 2) both the size and number of lipid droplets are decreased and the mutant animals exhibit lower TAG level than the wild type; 3) The mRNA level of lipolysis related genes, bmm and Lip3, is elevated in adult flies. Some of the phenotypes except for the body weight are observed only in the fat body of adult flies. Apart from the fat body, oenocytes are the tissue where lipolysis occurs as well. However, our data indicated that CG9911 played roles only in the adult fat body.By using the CRISPR/Cas9 system, we generated 2+ signaling by interaction with IP3R1 in mammalian cells [2+ concentration, and redox state, further to inhibit the Ca2+ overload [2+ signal in Drosophila. Consistently, in our present study, we find that phenotypes of CG9911 mutants are fully rescued by knockdown of 3RIP in fat body in Drosophila. This finding suggests that CG9911 has a genetic interaction with 3RIP and thus potentially possible to regulate Ca2+ homeostasis by physical interaction with 3RIP.Previously we reported that ERp44 (CG9911 homolog) modulates intracellular Caan cells . Specialoverload , 43. As 2+ imaging experiments show that CG9911 loss of function leads to down regulation of ER Ca2+ without affecting cytosol resting Ca2+ level in adult fat bodies. Without CG9911, decreased ER Ca2+ acts as a trigger to ER stress. ER stress has been connected to transcriptional activation in the regulation of lipid metabolism [2+ homeostasis has been discussed for years due to their collective regulation of transcription and protein maturation [2+ homeostasis by CG9911 is important, other ways through which CG9911 affects ER stress cannot be ruled out. A few questions remain to be explored. For example, lipolysis is largely regulated by the intracellular concentration of cyclic adenosine monophosphate (cAMP) and by the activation of cAMP-dependent protein kinase A (PKA) in mammalian cells [Catabolism . ER stretabolism  and fat turation . Althougan cells . In our 2+ signaling and lipid metabolism. It may also be a hint to study the function of ERp44 and the mechanism of relevant metabolic diseases. Our study also sheds new light on explaining the interaction between Ca2+ signaling and lipid metabolism.Our findings may contribute to understand the interactions of Cappl-GAL4 (#105013) and Oe-GAL4 (#113874) stocks were obtained from Drosophila Genomics Resource Center (DGRC). Other stocks used were from the Bloomington Stock Center.All flies were kept on standard medium containing cornmeal, yeast agar, soybean, syrup and molasses . The culin vitro as previously described [1118w embyos. Inheritable F0 which yielded enough F1 were collected for screen by using single fly PCR with the pair of primers: 5’-TGCAGCGTCTAATGAATTGG-3’ and 5’-GTCCCTACGATCGAAGTAGC-3’. Positive PCR fragment from homozygous flies were used for sequencing.By CRISPR/Cas9 system, we selected two target DNA binding site on the third exon according to the rules . We follescribed , the mRNdrosophila embryos and then subcloned into pGEX-6p-1  by EcoR I and Sal I. An N-terminal, GST-fused CG9911 protein was expressed in E.coli BL21 (DE3) strain  induced by 40 μM IPTG at 16° C for 18 h. The protein was purified using Glutathione Sepharose 4B  according to a standard protocol. GST-tag was removed by PreScission Protease  in cleavage buffer  at 4° C overnight. The purified CG9911 protein was used as antigen at the concentration of 400 μg/ml in PBS and mixed with 20 μg/ml CpG ODN (5’-tccatgacgttcctgacgtt-3’thiophosphorylated) as adjuvant. Each of 8 week-old female BALB/C mouse was immunized with 500μl antigen by intraperitoneal (i.p.) injection. The second booster injection was 30 days after the primary immunization dose. The third, fourth and fifth booster injection went 15, 7 and 3 days after the last injection, respectively. After five booster injections, the spleen B lymphocytes isolated from immunized mice and SP2/0-Ag14 myeloma cells were fused in the presence of PEG4000 (Merck) at 37° C overnight. The hybridoma cells were cultured in complete IMDM medium  supplemented with 20% FBS , 1% penicillin/streptomycin  and HT . After 15 days feeding in 96-well plates, the hybridomas were screened by using ELISA. To gain monoclones, the cells from positive wells were recloned by limiting dilution and identified under an inverted microscope as previously described . The positive monoclonal hybridoma cells tested by ELISA again were expended to 24-well plates and incubated for 1 week at 37° C, 5% CO2. Monoclonal antibodies were purified from collected cell supernatant by using protein G column .The CDS fragment of CG9911 isoform C was cloned from cDNA of 2SO4and liquid color was changing from blue to yellow. The absorbance was measured at 450 nm using a Spectrophotometer  and Multiskan GO v1.00.40.Polystyrene 96-well plates were coated with250μg purified CG9911 protein in 50μl PBS at 4° C overnight and blocked with block buffer  for 1 h at room temperature. After 3 times washing with PBST (0.05% Tween-20 in PBS), the plates were loaded with 100μl supernatant of hybridoma cells for 1 h. Subsequently,100μlgoat anti-mouse Ig-HRP (1:10000 diluted in block buffer) was added to each well and incubated for 1h.After 3 washes, the plate was developed by using 100μl TMB substrate  per well for 5 min. The reaction was stopped by adding50 μl 2N HFlies of different developmental period were homogenized in cold RIPA lysis buffer  in the presence of 1 mM PMSF and protease inhibitor cocktail  and centrifuged at 12000 rpm, 4° C for 20 min to remove insoluble precipitates. The concentration of total protein was determined by Bradford  and was boiled in 2×SDS loading buffer for 15 min. The detected samples have the same total protein concentration and were separated by SDS-PAGE, transferred onto PVDF membranes (Millipore), and incubated with primary antibodies against CG9911 , β-actin . The membranes were incubated with the appropriate HRP-conjugated secondary antibodies , and the signals were analyzed using the SuperLumina ECL HRP Substrate Kit . The position of the target protein is referenced to the color prestained marker . A minimum of three independent analysis were performed, and typical example is presented.1118w were collected and dissected in cold PBS. Wing discs, fat body, oenocytes, muscles, ovary, testis and other tissues were separately fixed in 4% paraformaldehyde in PBS for 40 minutes at room temperature followed with 3 washes of PBST (0.3% Triton X-100 in PBS). After blocked in 10% goat serum in PBST for 1 h, tissues were incubated with CG9911 antibody (1:100) at 4° C overnight. Subsequently, fluorescent secondary antibody was used for signal detection. For Lipid droplet staining in adult abdominal fat bodies, 5 days adult flies of the correct genotypes were collected and dissected as previously described. After 3 washed in PBS, fat bodies were incubated in PBS with 1 mg/ml BODIPY 493/503  and 2 ng/ml DAPI  for 40 min at room temperature. The embryos of 1118w were stained with CG9911 antibody according to standard protocol. All fluorescent images were captured by a Leica confocal microscope SP5 .Wandering third instar larvae and adult flies of Whole adult flies glyceride quantification was determined as described. 10 adult flies were collected (three groups per genotype) and homogenized in PBST (0.5% Tween in PBS). The supernatants were measured with TAG reagent .1118wand CG9911 mutant flies (1 day after eclosion) were dissected in hemolymph-like (HL) buffer  as previously described. Individual cells were loaded with 5 mMFluo-4 AM  on poly-L-Lysine  coated wells at 37° C for 30 min in the dark. After perfused with calcium-free HL buffer , cells were stimulated by 10 μM ionomycin  or 10 μM thapsigargin  to cause Ca2+ release from ER. The fluorescence signal was recorded by using a SP5 confocal microscope  connected to an inverted microscope . The Ca2+dependent fluorescence intensity ratio (ΔF/F0) was used to present the Ca2+release signal in 30 fat cells. The resting Ca2+ measurement were performed as previously described. Fat cells were incubated with 10μM Fura-2 AM  in HL buffer at 37° C for 30 min. Photometric measurements were performed by using cellˆR system  and operated at an excitation wavelength of 340 and 380 nm. The relative resting Ca2+ signal was presented by a ratio of 340/380 nm by using Olympus cellˆR Software.Fat body cells fromβ-actin and rp49 were used as endogenous control. See Total RNA was extracted from adult flies by using TRIzol reagent . 2μg of RNA was reverse transcribed to cDNA by using PrimeScript™ RT reagent Kit with gDNA Eraser . Quantitative real-time PCR was performed using EvaGreen 2× qPCR Mix  on a Roter-Gene machine  following the manufacturer’s instructions. 1118wand CG9911 mutant (3 days after elcosion) were kept in vials only with PBS at normal condition. The number of dead flies was recorded every 3 hours with 3 independent groups employed. Data was analyzed by Log-rank (Mantel-Cox) test.50 flies of To induce ER stress, 100 files with different genotypes were transferred to vials containing 1% agar, 5% sucrose and 12 μM tunicamycin  as previously reported. The flies were kept in normal conditions and counted every 3 hours.Statistical analysis was performed using GraphPad. Data were tested for significance using the Student’s test and shown as mean ± SEM. Data from three groups were compared by one-way ANOVA.Supplementary FiguresSupplementary Table 1


Obtaining clinically relevant genetic data at diagnosis is the basis for the success of patient-tailored therapy. The Italian specialists also confirm the role of multidisciplinary diagnostics for AML, now mandatory and expected to become more important in the future context of “precision” medicine.Acute myeloid leukemia (AML) is a heterogeneous disease with a wide variety of clinical presentations, morphological features, and immunophenotypes. The diagnostic approaches to AML that are adopted in Italy have been explored using an online Delphi-based process to expand the global discussion on mandatory tests for the correct diagnosis and, consequently, for optimal management of AML in clinical practice. The final results of the panel of Italian hematologists involved in this work highlight the importance of genetic evaluation for classification and risk stratification and firmly establish that karyotyping, fluorescence  FLT3) and differentiation/apoptosis pathways , play a pathogenetic role in AML, provide prognostic criteria, and can guide therapy (Acute myeloid leukemia (AML) is an uncontrolled clonal proliferation derived from progenitor/precursor hematopoietic cells. Abnormal differentiation of myeloid cells results in high levels of immature malignant cells (blasts) and fewer normally differentiated red blood cells, white blood cells and platelets , 2. From therapy . In rece therapy .The most recent World Health Organization (WHO) classification, published in 2016, compared with that published in 2008 , includeSeveral organizations, including the National Comprehensive Cancer Network (NCCN), the European Leukemia Net (ELN), and the European Society for Medical Oncology (ESMO), recently developed guidelines and risk score systems to help physicians to design ab initio the best “patient-tailored” therapeutic strategy –11. InteThe diagnostic process of a heterogeneous disease such as AML is complex and requires high levels of expertise. A panel of Italian hematologists explored the diagnostic approaches that are adopted across the national territory through a survey that involved a representative sample of the Italian Centers of Hematology.Information on how Italian hematologists manage the diagnostic workup of AML may contribute to expand the global discussion on mandatory tests for the correct diagnostic approach and, consequently, for optimal management of AML in clinical practice. The diagnosis of the different genetic subtypes of AML is a subject of intense debate within the international scientific community because it has relevant prognostic and therapeutic implications.To gather expert opinion on the implementation of accurate diagnosis of AML subtypes, we used an online Delphi-based process . This is a group-facilitative method designed to verify the convergence of opinion of a panel of experts in a given area of uncertainty within health-related research , 13.The process was developed over nearly 7 months using the following steps: (1) establishment of a scientific steering committee of five experts who were in charge of reviewing the literature and developing the survey items; (2) selection of a panel of AML specialists; (3) administration of online survey to the panel of AML specialists; (4) collection and analysis of the results; (5) final meeting.Five experts from Italian hematology institutions were identified as representatives of the specialists involved in the medical care of patients with AML. The scientific steering committee defined 26 questions covering four main topics:Topic 1: application of the WHO 2016 classification (8 items)Topic 2: confirmatory diagnostic tests (4 items)Topic 3: implementation of the guidelines (7 items)Topic 4: therapeutic implications (7 items)The possible answers to each question were chosen from multiple options, all possible and applicable in clinical practice.Forty-eight specialists  selected from 42 centers among the Italian hematology institutions, were invited to participate in the project during a virtual meeting where the project objectives and methodology were shared.The survey elaborated by the steering committee was delivered to the panel of AML specialists: 34 participants from 31 sites (29 hematologists and 5 cytogeneticists/molecular biologists) expressed an opinion on each of the 26 questions, independently and blindly. All 34 specialists were affiliated to centers with availability of a diagnostic lab, and with an allogeneic or autologous stem cell transplantation unit in 27 and 7 cases, respectively. The survey was performed online on a secure website using an online platform.The scientific steering committee collected and analyzed the results, then discussed the real-life AML diagnostic approach adopted in Italy in the light of the international recommendations.The panel of specialists answered each of the 26 questions based on their own clinical practice, experience, and facilities available Table 1.In recent decades, the diagnostic workup for AML, initially based solely on morphological examination, has integrated more and more disciplines and methodologies. The WHO 2016 classification adopted such fundamental tools as cytomorphology, cytochemistry, immunophenotyping, cytogenetics, and molecular genetics. The integration of all these techniques allows for a comprehensive and complementary characterization of each patient with AML, which is a prerequisite for optimal diagnosis and management.The aim of the questions in the first topic of the Delphi questionnaire was to investigate the specialists’ attitude in applying the WHO diagnostic criteria in clinical practice, focusing on which tests they would consider crucial for the correct identification of AML subtypes Table 1.NPM1, CEBPA, and RUNX1 genes only in young patients. However, the specialists who provided comments in the open field stated that the use of tests is driven more by fitness to undergo intensive chemotherapy than by age. On the other hand, 35% of the specialists perform all of the above diagnostic tests, regardless of age. Finally, 39% of specialists stated that assessment for CEBPA and RUNX1 is performed in selected cases only.Almost all experts participating in the survey stated that they always refer to the WHO 2016 classification Table 2 When collecting anamnestic information, most of the specialists (81%) declared that they carefully seek information about previous exposure to chemo-/radiotherapy to exclude therapy-related myeloid neoplasms. An accurate anamnesis, also asking about history of a previous transfusion support or a diagnosis of antecedent myelodysplastic syndrome (MDS), was considered a fundamental step for the identification of AML-MRC.RUNX1-RUNX1T1, CBFB-MYH11, PML-RARalpha, BCR-ABL1). The remaining two thirds were equally divided between those who request all possible PCR assays (32%) and those who urgently require only qualitative PCR for PML-RARalpha (32%) to exclude a diagnosis of acute promyelocytic leukemia.The turnaround time to obtain information on cytogenetics can be as long as 7–15 days, whereas it can be no longer than 72 h for molecular testing. The specialists were asked to state which molecular changes they considered critical for the diagnosis of AML with recurrent genetic abnormalities. There was no convergence on which molecular assay to request. About one third (36%) of the panelists claimed to request only qualitative polymerase chain reaction (PCR) for the main fusion genes (CEBPA mutation (CEBPA mutations (Sanger sequencing (29%) and next-generation sequencing (NGS) (29%) are the most frequently used techniques for detecting AML with biallelic mutation Figure 1utations .NPM1 and biallelic CEBPA mutational analysis, in line with WHO recommendations. Sixty percent of the specialists mentioned fluorescence in situ hybridization (FISH) as a valid substitute for karyotyping to clarify difficult diagnosis such as AML-MRC as soon as possible. Such an approach is deemed reliable in situations of punctio sicca, insufficient number of metaphases, or when the turnaround time to obtain information about the karyotyping is too long.Over 70% of panelists believed that morphological assessment alone is not sufficient to support the diagnosis of AML-MRC. Morphology assessment should always be associated with clinical history, cytogenetics https://www.gimema.it/ricerca/labnet/) for centralized activities.The outcomes from the first set of answers demonstrated the specialists’ attitude to applying the 2016 WHO AML classification and the recommended diagnostic workup. Nevertheless, such an intention is affected by the technological heterogeneity among the different centers and the use or not of reference laboratories  and cytotoxic treatments, there is increasing evidence of a frequent association between bone marrow dysplastic features and autoimmune disorders . TherefoWith regard to age, the participants stated that it is not an absolute discriminating factor. The impact of age should be evaluated in the light of co-existing co-morbidities, with the aim of selecting patients eligible for intensive chemotherapy and, if needed, allogeneic transplantation. Nevertheless, most new targeted agents are also indicated for the treatment of older patients with AML –18, therRUNX1-RUNX1T1, CBFB-MYH11, PML-RARalpha, BCR-ABL1 are also clinically relevant because they serve to track measurable residual disease (MRD) during and after treatment  is highly variable (The turnaround time for the cytogenetic reports was variable. Results are shown in The scientific steering committee emphasized the importance of bone marrow (BM) blast count. In patients with specific genetic alterations, cytomorphological examination of BM, and accurate evaluation of blast infiltration, according to the current guidelines, is of paramount importance to guide diagnosis and assess response to treatment . The comNPM1 and FLT3 mutation should be 48–72 hours, 96 hours for further gene rearrangements, 24 hours for molecular reporting of PML-RARalpha, and 5–7 days for cytogenetics  are fundamental for prognostic stratification. According to the 2017 ELN guidelines , and 36% and 3% stated that they refer to the ESMO  and NCCNFLT3 mutation, 80% of the clinicians opted for targeted induction treatment (“7+3” regimen plus midostaurin) , APL and AML-MRC, has a significant impact on clinical decisions. For patients considered eligible for intensive chemotherapy, with a diagnosis of AML-MRC and concomitant staurin)  rather tstaurin) .FLT3 re-testing is critical upon relapse to offer FLT3 inhibitor treatment if the mutation occurs   , even ifFLT3-mutated cases, both midostaurin and CPX-351, despite their diverse mechanisms of action, are effective  is the product of an integrated approach, primarily based on international guidelines, including patient history, and morphological, immunophenotyping, and cytogenetics/molecular findings. The opinions of the panelists and experts suggest that only a holistic approach to patients with AML might lead to long-lasting therapeutic success.de novo AML are characterized by a chromatin-spliceosome mutational signature and, from a clinical and prognostic perspective, resemble a secondary more than a de novo entity, despite the absence of specific morphological and cytogenetic abnormalities , to harmonize reports, increase the quality of technologies, and share knowledge. We emphasize the role of multidisciplinary diagnostics in AML, which is mandatory today and will gain even more importance in the future, especially in the context of precision medicine. This approach, together with the identification of centralized laboratories that could help centers not technically fully equipped, will overcome the possible problem of turnaround times, which sometimes vary from some hours to a week in different laboratories. Recent data confirm that it may be a feasible strategy to wait for genetic and other laboratory test results in clinically stable patients to assign them to the best available treatment option . TherefoDemostrating that the diagnostic approaches in AML are not uniform across the Italian territory, this survey highlights the fundamental role of the GIMEMA LabNet and, in general, of a diffuse lab network to support the clinical Centers which are not technically fully equipped, and to finally ensure an adequate management of AML.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.MV designed the project, selected the literature, wrote and reviewed the manuscript. FF designed the project, selected the literature, wrote and reviewed the manuscript. SG designed the project, selected the literature, wrote and reviewed the manuscript. AR designed the project, selected the literature, wrote and reviewed the manuscript. AV designed the project, selected the literature, wrote and reviewed the manuscript. All authors contributed to the article and approved the submitted version.AR has received travel expenses and fees for consultancies and participation in meetings, boards and symposia sponsored by Jazz, ABBVIE, Amgen, Pfizer, Celgene, Novartis, Incyte, Omeros, Astellas, and Roche. AV has served on an advisory board, been an invited speaker, and taken part in a speakers’ bureau for Novartis, Jazz, Pfizer, Astellas, Gilead, Helsinn, and Amgen. SG has received speaker fees for events supported by Amgen, Jazz, Astellas, and Novartis. MV has served on an advisory board, been an invited speaker, and taken part in a speakers’ bureau for Novartis, Jazz, Astellas, BMS/Celgene.The remaining author declares 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.


Perspective, we briefly review the recent advancements with immune cell-based strategies against adverse genetic risk AML and discuss how such approaches could favorably impact on patients’ outcomes.Adverse genetic risk acute myeloid leukemia (AML) includes a wide range of clinical-pathological entities with extremely poor outcomes; thus, novel therapeutic approaches are needed. Promising results achieved by engineered chimeric antigen receptor (CAR) T cells in other blood neoplasms have paved the way for the development of immune cell-based therapies for adverse genetic risk AML. Among these, adoptive cell immunotherapies with single/multiple CAR-T cells, CAR-natural killer (NK) cells, cytokine-induced killer cells (CIK), and NK cells are subjects of ongoing clinical trials. On the other hand, allogeneic hematopoietic stem cell transplantation  still represents the only curative option for adverse genetic risk AML patients. Unfortunately, high relapse rates (above 50%) and associated dismal outcomes  even question the role of current allo-HSCT protocols and emphasize the urgency of adopting novel effective transplant strategies. We have recently demonstrated that haploidentical allo-HSCT combined with regulatory and conventional T cells adoptive immunotherapy (Treg-Tcon haplo-HSCT) is able to overcome disease-intrinsic chemoresistance, prevent leukemia-relapse, and improve survival of adverse genetic risk AML patients. In this  TP53, RUNX1, ASXL1, and FLT3 genes , epigenetic modifiers , and cell-cycle regulators (TP53)  and new-generation cytotoxic treatments, like CPX-351  (High risk (or adverse risk) acute myeloid leukemias (HR-AML) include a number of clinical and biological AML subsets which are usually characterized by poor response to conventional treatments and dismal long-term survival, even when conventional allogeneic hematopoietic stem cell transplantation  is performed . Such AMT3 genes ] that coT3 genes  convergeedistat)  are provImmune-based adoptive cell therapies (ACTs) rely on the infusion of immune cells that aim to kill the tumor. These therapeutic platforms are revolutionizing treatment of blood neoplasms . In receversus-host disease (GvHD) damaging  may serve as “discovery platforms” that can help to reveal the complex interplay between AML and the immune microenvironment and to set the base for pioneering studies of AML immune-targeting –16. In odamaging . In partve study .Perspective, we will review molecular mechanisms underlying HR-AML biology that drive disease relapse, as well as the potential impact of the newly developed approaches with target therapies on patients’ outcomes. We will describe immune-based strategies against HR-AML in ongoing trials and discuss how refined transplantation approaches with adoptive immunotherapy might represent the “ultimate” therapeutic option for a definitive eradication of HR-AML.In the present AML with high-risk cytogenetics, a subset of AML with different pathologic and clinical features that include the following:HR-AML includes many distinct biological entities, often characterized by an aggressive phenotype and intrinsic resistance to conventional treatments . HR-AML - AML with complex karyotype (CK): its definition is not clear yet; however, it might be identified by the presence of ≥3 chromosomal abnormalities not included in defined WHO categories, and not associated with favorable prognosis , 19, 20- AML with monosomal karyotype (MK): it is defined by the presence of at least two autosomal monosomies or one single autosomal monosomy in combination with at least one structural abnormality GATA2,MECOM(EVI1); -5 or del(5q); -7; -17/abn(17p) q21.3q26.2) or t; 1.3q26.2 typical and atypical cases by the presence  of 5q, 7q, and/or 17p losses  more frequently and, thus, it is associated with poorer outcomes . On the other hand, atypical CK AMLs are characterized by different mutational onco-prints and more frequently display mutations of RAS pathway–associated genes, NPM1 and/or FLT3 genes (TP53 pathogenic mutations (NOTCH1 (rarely reported in AML), BCOR/BCORL1, or RUNX1 genes ] could be related to a distinct  clinical-biological entity  are common features of trAML (EVI1-rearranged (EVI-r) AML (MECOM and IKZF1). EVI-r AML is associated with very poor overall survival (OS)  AML , 29. Impval (OS) , 29.NPM1 and FLT3-ITDhigh, mutated RUNX1, and mutated ASXL1. The molecular pathogenesis of each distinct genetic entity is very poorly understood, and future studies are needed to investigate such biological complexity.Furthermore, the 2017 European LeukemiaNet (ELN) adverse-risk category  molecules with killer immunoglobulin receptors (KIRs) on NK cells plays a major role in regulating NK cell function and activity. NK-cells kill leukemia cells when a mismatch between KIRs and their ligand on target cells is present . Such acin vitro activated by anti-CD3, OKT3, INF-gamma and subsequently expanded with IL-2. Other than T cell markers, they express surface protein similar to NK-cells, such as CD56, the inhibitory NK receptors, and the natural killer group 2 member D (NKG2D) receptor, one of the most important receptors involved in NK-mediated cancer cell killing (CIK cells derived from cytotoxic T lymphocytes (CTL) that are  killing . In clin killing , last st killing , 52, 53.HL) on cell surface with high specificity for malignant cell antigens (in vitro studies and xenografts demonstrated the effectiveness of anti-CD33 and anti-CD123 CAR-T cells (In vitro ed in vivo (xenograft) preliminary data showed an increased cytokine release and decreased tumor burden using anti-CD123 CAR-T cells. FLT3 receptor is typically expressed on myeloid blasts, independent of FLT3 mutational status. Anti-FLT3 CAR-T cells showed in vitro ed in vivo promising antileukemic effect  lack of NK-cell activating molecules (CD16 and NKp44); iii) potential in vivo tumorigenicity  for different relapsed hematologic malignancies after allo-HSCT  showed that 80% of patients (4/5) with AML achieved a stable complete remission . No significant survival benefit was observed between fully myeloablative conditioning and RIC regimen for patients with CK AML (34 and 28%). RFS rate was 39.9, 33, and 18.3% for patients ages <40, 40 to 60, and >60 years, respectively  compared a total of 584 patients carrying HR-AML in first complete remission (CR1) from 151 transplantation centers. It showed a median 3-year OS of 45% (range 38–52%), 37% (range 31–44%), and 31% (range 22–41%) in patients undergoing matched sibling donor (MSD), HLA-well-matched and partially-matched unrelated donor (MUD) transplantation, respectively. Myeloablative or reduced-intensity conditioning (RIC) regimens were used. Cumulative incidence (CI) of relapse at 3 years was 37% for MSD, 40% for well-MUD, and 24% for partially-MUD, while 3-year relapse-free survival (RFS) was 42, 34, and 29%, respectively. No significant differences in relapse were observed among the various cytogenetic subsets . Anotherectively . Such stThe recent advancements in T-cell manipulation and in GvHD prophylaxis make haplo-HSCT a valuable transplantation strategy to overcome intrinsic chemotherapy resistance of high-risk leukemias. Haplo-HSCT procedures can be mainly divided in two major categories: T-cell depleted (TCD) peripheral-blood stem cells (PBSCs) haplo-HSCT and unmanipulated haplo-HSCT.Chiusolo P et al.  have been tested to improve outcomes of acute leukemia patients who underwent haplo-HSCT. While traditional TCD procedure based on positive selection of CD34+ cells was associated with delayed immune reconstitution and increased risk of NRM  from PBSC preserves hematopoietic engraftment and allows for the transfer of donor-derived memory T cells, that can confer immunity against pathogens with low risk of GvHD . Moderate/severe cGvHD occurred in only one patient. Only two patients relapsed (4%). Consequently, at a median follow-up of 29 months, the probability of moderate/severe cGvHD/relapse-free survival was 75%  is able to overcome disease-intrinsic chemoresistance , 76. We  was 75% , 76. TML was 75% . HoweverHR-AMLs are usually characterized by a very poor response to conventional treatments and to conventional allo-HSCT. Indeed, relapse rates are high (often above 50%) and result in very low survival (often below 10–20%). Thus, novel effective strategies are needed. Recent studies on new adoptive cell strategies  bring new hopes for the treatment of such unfavorable diseases. Indeed, immune-cell-based therapies may represent a powerful tool to successfully treat chemoresistant HR-AML. NK cell adoptive immunotherapies are a promising therapeutic, but their efficacy is still limited and fine-tuning of the approach is still required for larger clinical use –35. The The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.All procedures were in accordance with the ethical standards of the institutional research committee. The patients/participants provided their written informed consent to participate in this study.RL and AMar contributed equally to the figures and writing. AMan, SB, and EH reviewed the manuscript. AV provided guidance, and AP wrote the manuscript and provided critical review. SH and LR provided critical review. All authors contributed to the article and approved the submitted version.These studies were supported by the grant from the “Associazione Italiana per la Ricerca sul Cancro (AIRC)”, START-UP Grant n. 20456 to AP.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.


In this review, we discuss how these Ca2+ signals control procoagulant platelet formation and whether this process can be targeted pharmacologically to prevent arterial thrombosis.Platelets are the major cellular contributor to arterial thrombosis. However, activated platelets form two distinct subpopulations during thrombosis. Pro-aggregatory platelets aggregate to form the main body of the thrombus. In contrast, procoagulant platelets expose phosphatidylserine on their outer surface and promote thrombin generation. This apparently all-or-nothing segregation into subpopulations indicates that, during activation, platelets commit to becoming procoagulant or pro-aggregatory. Although the signaling pathways that control this commitment are not understood, distinct cytosolic and mitochondrial Ca Platelets play a critical role in arterial thrombosis, a major cause of myocardial infarction, stroke, and ultimately death. However, current anti-platelet therapies carry increased bleeding risk, have substantial inter-individual variation, and may have lower and reduced effectiveness in some high-risk groups such as patients with diabetes ,2. ThesePro-aggregatory platelets have active integrin αIIbβ3, which allows them to bind fibrinogen and aggregate with other pro-aggregatory platelets to form the body of the thrombus. Current anti-platelet therapies reduce platelet aggregation, either by blocking the signals that lead to αIIbβ3 activation, or by directly blocking αIIbβ3cyt) is a regulator of many platelet actions during thrombosis including PS exposure and release of PS-exposing EVs. Ca2+ activates the phospholipid scramblase, TMEM16F , and inactivates an aminophospholipid flippase cyt signals between individual platelets may commit them to form different subpopulations.This apparently all-or-nothing segregation into subpopulations indicates that during activation, platelets flippase ,23. Thisprotease ,25, compprotease ,26. Cyto2+ signaling also has a central role in procoagulant platelet formation. Opening of the mitochondrial permeability transition pore (PTP) is an important event in procoagulant platelets and is regulated by mitochondrial Ca2+ concentration ([Ca2+]mito). Although less is known about mitochondrial Ca2+ signaling, [Ca2+]mito signals are also likely to be heterogenous in activated platelets.Mitochondrial Ca2+ signals, highlighting how this might lead to the all-or-nothing commitment to becoming procoagulant, and consider the implications for therapeutic targeting of procoagulant platelets.In this review, we discuss the interplay between platelet cytosolic and mitochondrial Ca2+]cyt from resting levels <100 nM to up to 1–2 μM. Collagen fibers cluster GPVI, triggering a cascade of protein tyrosine phosphorylation to activate phospholipase Cγ2 cyt is amplified and sustained by Ca2+ entry across the plasma membrane. Ca2+ store depletion is detected by STIM1, which activates the channel ORAI1 in the plasma membrane. This process is called ‘store-operated Ca2+ entry’ (SOCE) and is a major mechanism for Ca2+ entry in platelets cyt, depends on the cytosolic Na+ concentrationcytcyt dynamicscyt in whole platelet populations, such as in cuvettes or microplate wells. This approach, although convenient, blurs the dynamics of such signals in individual platelets and the differences between them. Insights from studies of Ca2+ signaling in individual platelets  s2+ concentration ([Ca2+]mito) is a key regulator of mitochondrial oxygen consumption and ATP synthesis in many cells, and also regulates procoagulant platelet formationcyt due to inhibition by MICU2. When [Ca2+]cyt increases, MICU2 is inhibited and MICU1 is activated, leading to Ca2+ entry and an increase in [Ca2+]mitomito in response to increased [Ca2+]cytmito. Ca2+ le[Ca2+]cyt.2+ uptake is a key step in procoagulant platelet formation. Platelets from −/-Mcu mice, or human platelets treated with the MCU inhibitor, Ru360, generate fewer procoagulant platelets in response to physiological stimuli without effect on integrin activation[2+]cyt with a Ca2+ ionophore bypasses the requirement for MCU[Mitochondrial Cactivation. Notablyt for MCU.2+]mito triggers the permeability transition pore (PTP), a large conductance pore of the IMM resulting in depolarization of ∆Ψm and movement of ions and metabolites up to 1.5 kDa[2+]mito[2+]mito for PTP opening is much higher. CypD-deficient mouse platelets (−/-Ppif) generate significantly fewer procoagulant platelets than wild-type in response to physiological agonistscyt. However, in response to physiological stimulation, it is clear the PS exposure and procoagulant platelet formation requires mitochondrial Ca2+ entry and PTP opening.A large rise in [Cao 1.5 kDa. Cyclopha[2+]mito. In the  agonists. Cyclosp agonists. As with2+ entry is likely to occur in all activated platelets, PTP opening only occurs in platelets that become procoagulant. Panteleev and colleagues proposed an elegant model in which PTP opening is the step that commits platelets to becoming procoagulant [2+]cyt through Ca2+ release and Ca2+ entry. Some of this Ca2+ is taken into mitochondria via MCU leading to an increase in [Ca2+]mito. However, since cytosolic Ca2+ signaling varies between individual platelets, the increase in [Ca2+]mito will also vary between individual platelets. In some platelets, [Ca2+]mito will be above the threshold for PTP opening. In these platelets, PTP opening commits the platelet to become procoagulant. They lose ∆Ψm (because of PTP opening), expose PS, and inactivate integrin αIIbβ3. In contrast, activated platelets without PTP opening maintain αIIbβ3 activation and are pro-aggregatory. In this model, the Ca2+ threshold for PTP opening is therefore the commitment point that converts heterogenous Ca2+ signaling into an all-or-nothing response.Although mitochondrial Caoagulant ,51. In t2+ signaling in such a way that it can now activate TMEM16F, or PTP opening releases an additional signal that regulates TMEM16F in addition to cytosolic Ca2+ .How does PTP opening commit a platelet to becoming procoagulant? Specifically, how is this mitochondrial event communicated to TMEM16F in the plasma membrane? We envisage two possibilities: either PTP opening changes cytosolic Ca2+ signaling[2+]cyt that varied in frequency and amplitude between individual platelets. However, a transition to high, sustained [Ca2+]cyt during procoagulant platelet formation coincided with loss of ∆Ψm and PS exposure. The extent of this high, sustained [Ca2+]cyt is difficult to estimate, however, as [Ca2+]cyt was mostly measured using Fura Red in these experiments. Fura Red’s fluorescence decreases with Ca2+ binding. The high sustained [Ca2+]cyt levels that coincide with PTP opening resulted in very low fluorescence. PTP inhibition with CsA reduced the percentage of platelets that became procoagulant but did not affect the thrombin-induced spikes in [Ca2+]cyt. Similarly, Arachiche et al. also found that Fura Red fluorescence was lower in PS-exposing platelets, and this was also reversed by CsA[2+]cyt, or transition from spiking to sustained cytosolic Ca2+ signaling.Obydennyy and colleagues demonstrated that procoagulant platelet formation was accompanied by a change in cytosolic Casignaling. Thrombied by CsA. These o2+]cyt and [Ca2+]mito were higher in procoagulant platelets, they found no difference in cytosolic or mitochondrial Ca2+ signaling in CypD-deficient platelets[2+ uptake reduced [Ca2+]mito, ∆Ψm loss and PS exposure, but had no effect on [Ca2+]cyt. In this study, [Ca2+]cyt was measured with Fluo-4, a commonly-used probe with high affinity for Ca2+. From these results it appears that PTP opening is downstream of cytosolic and mitochondrial Ca2+ signaling, but that PTP opening does not further affect [Ca2+]cyt, in clear contrast to the conclusions of the studies described above. However, whilst we can readily repeat these resultscyt in procoagulant platelets is much higher than previously appreciated, and much higher than in pro-aggregatory (non-coagulant) platelets[2+]cyt signal a 2+ signalsupramaximal Ca, that is, much higher than normally considered maximal. Our observations are based on the use of fluorescent Ca2+ probes with different affinities for Ca2+. The Kd (dissociation constant) of Fluo-4 in vitro is approximately 390 nM. Although the Kd will be affected by the intracellular environment, the high affinity of Fluo-4 means that it is likely to be saturated by Ca2+ in the low micromolar range. In contrast, Fluo-5N has a very low affinity for Ca2+ (Kd = 90 μM in vitro). This low affinity means that changes [Ca2+]cyt in the nanomolar and low micromolar range will have little effect on Ca2+ binding to Fluo-5N, whereas [Ca2+]cyt of tens of micromolar will increase Ca2+ binding and give a fluorescent signal. The fluorescence of platelets loaded with either Fluo-4 or Fluo-5N showed very different patterns in procoagulant and non-coagulant platelets. The Fluo-4 fluorescence in stimulated platelets was much higher than in unstimulated platelets and was similar in procoagulant and non-coagulant platelets. In contrast, in Fluo-5N fluorescence of non-coagulant platelets was similar to unstimulated platelets whereas pro-coagulant platelets had brighter fluorescence. Our interpretation is that [Ca2+]cyt in procoagulant platelets is very high compared to non-coagulant platelets. In both subpopulations, [Ca2+]cyt is high enough to saturate Fluo-4, which is why there is no apparent difference in Fluo-4 fluorescence between the two subpopulations. This would normally lead to the conclusion that [Ca2+]cyt is similar in the two subpopulations. However, although [Ca2+]cyt is not high enough in non-coagulant platelets to give a signal with Fluo-5N, [Ca2+]cyt is much higher in procoagulant platelets because it is high enough to give a signal with Fluo-5N. The low affinity of Fluo-5N means that [Ca2+]cyt must be very high indeed. We estimated [Ca2+]cyt as approximately 160 μM.34 Although this fairly crude estimate needs further refining, it shows that [Ca2+]cyt in procoagulant platelets may be much higher than previously thought.Recently, we proposed that [Caplatelets. We term2+]cyt signals have been reported in other cells. For example, [Ca2+]cyt was estimated as greater than 100 μM using low-affinity dyes in ATP-depleted renal proximal tubular cells [2+]cyt was estimated as >5 µM by a low-affinity dye but only 0.3–0.4 µM with high-affinity fura-2[2+]cyt signal in these procoagulant platelets is not simply due to loss of plasma membrane integritycyt for activation mito reaches threshold for PTP opening. Differences in mitochondrial number between healthy platelets could result from small differences in packaging mitochondria into megakaryocyte proplatelets, or release of mitochondria into the circulationmito [2+. Necrosis often involves lysis of intracellular lysosomes, for example[2+ entry, including MLKL in necroptosis[2+ could identify new anti-thrombotic targets.How the supramaximal Cae cytosol. PTP opechondria). Alternar example. In addicroptosis, gasdermcroptosis,70 and gcroptosis. Clarify2+ release and inhibition of this step is unlikely to lead to selectivity. There may be some selectivity achieved by inhibiting Ca2+ entry channels, such as ORAI1 or TRPC6. Ca2+ entry is required to amplify cytosolic Ca2+ signaling to a sufficient level to lead to PTP opening, whereas aggregation can take place in the absence of extracellular Ca2+. Although procoagulant platelet formation is reduced in −/-Orai1 mouse platelets and with ORAI1 inhibitors [in vitro was reduced, even under non-coagulating conditions, suggesting that platelet aggregation is inhibited. Moreover, loss of ORAI1 in T cells results in severe combined immunodeficiency (SCID)[Selective inhibition of procoagulant platelet formation, without affecting platelet aggregation, might be a therapeutic approach to prevent arterial thrombosis. In our model, the first step uses the same molecular machinery in procoagulant and pro-aggregatory subpopulations, such as the receptors  and Cahibitors ,32,34,35hibitors ,72. Howecy (SCID), limitin2+ uptake into mitochondria via MCU is another potential target. Mcu−/- platelets have reduced procoagulant platelet formation with no apparent effect on aggregation[−/-Mcu mice are viable and appear grossly normal[2+ and subsequent mPTP opening also underlies cardiac ischemia-reperfusion injury, making MCU a potential target to limit myocardial injury following arterial thrombosis.Cagregation. Mcu−/- ly normal, suggest2+ sensitivity of PTP opening. However, CsA also has other targets, including cyclophilin A, leading to immunosuppression through inhibition of calcineurin, a Ca2+-dependent phosphatase. More selective PTP inhibitors might prevent procoagulant platelet formation without immunosuppression.Similarly, PTP inhibition is a potential target. CsA reduces procoagulant platelet formation ,34. CsA 2+ signal that we have described. As noted above, better understanding of the mechanism of the supramaximal signal may identify new anti-thrombotic targets, but we are not yet at that stage. At present, the more promising avenues for inhibiting Ca2+ signaling in procoagulant platelet formation appear to be to inhibit mitochondrial Ca2+ uptake or PTP opening.PTP opening leads to the supramaximal Ca−/-Tmem16f mouse strains have been reported with divergent effects on bleeding time, from no difference to severe bleeding[2+ signaling and on αIIbβ3 activation[Alternatively, platelet procoagulant activity could be directly blocked by inhibiting TMEM16F. Loss of TMEM16F causes Scott Syndrome, a moderate, very rare bleeding disorder. Several bleeding. Inhibit bleeding, and thoctivation. Similarctivation,79, (thoctivation), these ctivation. Underst2+ channel modulator, and a related molecule, 3-(diphenylphosphino)-1-propylamine (DP3A). This inhibition was not due to inhibition of PS exposure or calpain activity, indicating that there are as yet unidentified components to the EV release machinery that can be targeted[Finally, procoagulant platelets release PS-exposing EVs, which further promote coagulation. Indeed, EVs contribute approximately 5% of the volume of an arterial thrombi. Their m targeted.2+ signals is required, leading to permeability transition pore opening and an overload of cytosolic Ca2+. In this, procoagulant platelet formation resembles a rapid form of regulated necrosis [2+ signaling code that commits a platelet to becoming procoagulant.The mechanisms that commit a platelet to becoming procoagulant are still incompletely understood. A complex interplay between cytosolic and mitochondrial Canecrosis ,67,90. Lnecrosis . Inhibit