Early diagnosis of acute myeloid leukemia (AML) in the pre-leukemic stage remains a clinical challenge, as pre-leukemic patients show no symptoms, lacking any known morphological or numerical abnormalities in blood cells. Here, we demonstrate that platelets with structurally abnormal mitochondria emerge at the pre-leukemic phase of AML, preceding detectable changes in blood cell counts or detection of leukemic blasts in blood. We visualized frozen-hydrated platelets from mice at different time points during AML development in situ using electron cryo-tomography (cryo-ET) and identified intracellular organelles through an unbiased semi-automatic process followed by quantitative measurement. A large proportion of platelets exhibited changes in the overall shape and depletion of organelles in AML. Notably, 23% of platelets in pre-leukemic cells exhibit abnormal, round mitochondria with unfolded cristae, accompanied by a significant drop in ATP levels and altered expression of metabolism-related gene signatures. Our study demonstrates that detectable structural changes in pre-leukemic platelets may serve as a biomarker for the early diagnosis of AML. Wang et al characterise platelets in a mouse model of acute myeloid leukemia (AML) using Cryo-electron tomography (Cryo-ET) and find that 23% of platelets in pre-leukemic cells exhibit abnormal, round mitochondria with unfolded cristae. They also detect reduced ATP levels and altered expression of metabolism-related genes, altogether suggesting that platelet alterations may serve as a biomarker for AML. AML is the most common type of leukemia diagnosed in adults and accounts for the highest percentage of leukemic deaths2. AML develops quickly with only a 2-month median overall survival (OS) for untreated patients3, emphasizing the need for early diagnosis. For younger patients, the OS and complete remission (CR) rate decrease dramatically when the time from diagnosis to treatment (TDT) is more than 5 days4. To improve the prognosis of AML patients, early diagnosis and immediate initiation of therapy are critical to reduce morbidity and mortality. Generally, the diagnosis of AML and specific subtypes are based on blood tests, bone marrow tests, and chromosome testing5. Diagnosis of the early stage of AML (defined as pre-AML) can be challenging because early in development AML is often asymptomatic, with no changes in blood tests, and no immature platelets detected in the bloodstream. By studying the early stages of AML, it may be possible to develop new diagnostics capable of early detection.Acute myeloid leukemia (AML) is a heterogeneous malignancy characterized by defective hematopoietic differentiation, leading to the accumulation of immature leukemic blasts and suppression of normal hematopoiesis7. Previous studies have shown that hemostasis disorders and bleeding tendency in AML patients could be due to not only reduced platelet counts, but also functional platelet defects11. Some AML patients exhibited decreased platelet size and expression of the activated membrane receptor (GPIIb/IIIa and GPIb), which led to reduced in vivo platelet activation13. Conventional transmission electron microscopy (TEM) was applied in structural studies of platelets from AML patients, which discovered decreased numbers of α-granules and an absence of dense granules13.In AML, genetic lesions in hematopoietic stem/progenitor cells (HSPCs) produce developmental defects in cell lineages arising from them, including megakaryocytes that produce platelets16. Unlike conventional EM, vitrification can arrest cellular dynamics in intact platelets within milliseconds, thereby providing a physiological “snapshot” of whole platelets without artifacts produced by chemical fixation19. Facilitated by the development of electron cryo-tomography (cryo-ET), ultrastructural alteration of platelets has been found to be associated with diseases such as ovarian cancer17. In this study, we performed an ultrastructural analysis of platelets in an AML mouse model21 at different time points after inducing AML. We characterized morphological changes and quantitively measured subcellular organelles within platelets providing observations of early-stage AML.However, the current understanding of platelet ultrastructure is derived mainly from TEM studies with conventional chemical fixation known to cause structural artifacts in platelet organelles and macromolecules23. Five populations of platelets were visualized in this study, drawn from un-irradiated wild-type (WT) mice, irradiated mice transplanted with MLL-AF9-transformed HSPCs at two time points (1-week: pre-AML and 3-week: AML), and irradiated mice with normal bone BMT at the same two time points(1-week control and 3-week control)  and AF9 genes (producing MLL-AF9) is often found in AML, we used a murine AML model driven by MLL-AF9 oncogene in our study, which is widely used to recapitulate AML in humann = 3) mice (designated as un-irradiated WT platelets hereafter), and a total of 94 platelet tilt series were collected and automatically reconstructed using EMAN224. Reconstructed tomograms showed all un-irradiated WT platelets were in a quiescent state appearing as a discoid with a small number of pseudopods  was found randomly dispersed on the otherwise featureless platelet exterior. Platelets and organelle membranes were clearly resolvable, indicating that we achieved spatial discrimination at nm resolution. Microfilaments in the cytoplasm could be identified in thinner regions of the tomograms, such as those near the plasma membrane. A coiled circumferential marginal band of microtubules is clearly observed. The cytoplasm of platelets was rich in glycogen and granules were randomly distributed in the cytoplasm. α granules with a single membrane had high morphological variability and the peripheral zone had less contrast than the central zone. The most distinguishing feature of dense granules was the electron-opaque spherical body within the organelle, separated from the enclosing membrane by an empty space producing a “bull’s-eye” appearance27, giant platelets28, and Medich’s giant cell disease29 using TEM. This structure was previously named as membranous inclusions27 or scroll-like structure30 because the membranes are flat sheets wrapped around each other once or several times forming a scroll. We named this feature TS because we found the membranes were repeatedly folding to form a tubule-like structure. This subcellular feature is clearly resolved in the tomograms, with a wide range of diameter and direction in cells. In contrast to previous studies, we found TS contained various types of organelles including α granules, dense granules, gamma granules, mitochondria, or glycogen. The function of TS is not yet clear: it may be the membrane storage to meet the maximum abduction of platelet activation, accumulation of glycogen particles to store energy or a channel for quick movement of organelles in platelets.In addition, 75.5% 71/94) of platelets had a tubule-like structure (TS) , decreased red blood cells (RBC) and platelets 3 weeks after transplantation. In contrast, mice with normal bone marrow cells exhibited normal numbers of blood cells  and empty vacuole vesicles or absent vesicles, called ‘ghost platelets’ , and no other structural abnormalities were detected  of AML platelets contain only glycogen and OCS with roughly 21% of the normal cell size (15.6 ± 1.00 n = 3) 1 week after transplanting MLL-AF9-induced AML cells and analyzed them with cryo-ET. Recipient mice at this time point exhibited reduced blood cell counts due to the effects of sub-lethal irradiation, but showed improvement at 3 weeks33. In contrast to mice 3 weeks after transplantation, which exhibit leukocytosis and severe anemia, mice at 1-week post-transplantation did not show leukocytosis or anemia according to the blood count  and decreased number of α granules  compared to un-irradiated WT mice , so was the average area   ) of pre-AML platelets had clearly abnormal mitochondria with a round shape and less reduced cristae and matrix Fig. .Fig. 3Abvs. 0.6 ± 0.01 105 nm2, P = 0.023) and BMT control mice   Fig. . The numlet Fig. . To bettKey to its possible use as a diagnostic, not even a single instance of an abnormal mitochondria was observed in either un-irradiated mice or mice transplanted with normal bone marrow cells at the same time point as pre-AML mice Fig. . The lac2 surrounding each mitochondrion  containing all subcellular features including α granules, dense granules, gamma granules, mitochondrion, the plasma membrane, microtubules, glycogen particles, OCS, and TS; (2) pre-AML platelets with a similar size (~73.0 105 nm2) as un-irradiated WT and BMT control platelets but with a fraction of mitochondria (~25%) that have a round shape and diluted matrix; and (3) AML platelets, in which the majority appear as “ghost platelets” with signs of apoptosis, are five times smaller than BMT control platelets (~15.6 105nm2), and only contain glycogen storage and an open canicular system with empty vacuole vesicles.Based on cryo-ET imaging, platelets can be classified into three distinct stages based on the phase of AML development: (1) Healthy platelets from un-irradiated WT mice that have a normal size  in bone marrow is a major cause of abnormal platelets such as thrombocytopenia43. There have been intensive studies on platelet mitochondria, demonstrating that the mitochondria impairment is associated with platelet dysfunction and diseases, such as sepsis, Alzheimer’s disease (AD) and Parkinson’s disease (PD)46. In our study, by taking advantage of cryo-ET, we are able to detect the ultrastructural alteration of mitochondria in platelets in pre-AML phase. More importantly, the function of mitochondria was also impaired, with cellular ATP level decreased by more than 50%. GSEA analysis reveals downregulation in genes related to mitochondria function such as oxidative phosphorylation. Since the rate of ATP turnover and oxidative phosphorylation is tightly regulated by mitochondria during platelet’s activation to meet the energy demand, the incapability of platelet in AML would be likely attributed to the dysfunction of mitochondria47. In pre-AML, mitochondria with unfolded inner membranes did not appear different in density than the surrounding cytosol, suggesting that swelling and dilation of the inner membrane compartment caused matrix dilution or efflux of matrix components. Taken together, we demonstrate that both the structure and function of mitochondria exhibit early signs of impairment in the pre-AML phase. Mitochondrial apoptosis and/or mitophagy may lead to cell shrinkage and shedding of platelet microparticles49. We speculate that the abnormal mitochondria that lacks spherical cristae could be the product of apoptosis or mitophagy and the small empty platelets we found in fully developed-AML phase are the outcome after platelet apoptosis.Mitochondria, referred as the “powerhouse of the cell”, play critical roles in various essential biological processes including proliferation, differentiation, and metabolismThe progressive depletion of normal platelets and accumulation of abnormal platelets parallels the development of AML from the pre-AML stage without detectable changes in blood cell counts to frank AML. Our data suggest that the emergence of abnormal mitochondria and surrounding empty vesicles could be an early sign of AML initiation. These features can be detected in raw cryo-ET images, but our semi-automatic reconstruction pipeline allows clear distinction and quantification of different compartments and changes in the mitochondrion  or C57BL/6 mice was used as transplant recipient mice. Female mice (8–12 weeks old) were used and housed in AAALAC-accredited, specific-pathogen-free animal care facilities at Baylor College of Medicine (BCM). All procedures were approved by the BCM Institutional Animal Care and Use Committee. The BCM Institutional Biosafety Committee approved all other experimental procedures.The mouse alleles used in this study were Ubc-GFP (C57BL/6-Tg(UBC-GFP)30Scha/J, JAX Stock #004353)31. Hematopoietic stem and progenitor cells (Lin−c-kit+Sca-1+) were sorted and incubated in X-Vivo 15  supplemented with 50 ng/ml SCF, 50 ng/ml TPO, 10 ng/ml IL-3, and 10 ng/ml IL-6  for 24 h prior to retrovirus spin infection. For primary recipients, 50 000 cells were transplanted into lethally irradiated C57BL/6 mice . Secondary transplantation was performed by transplanting 100 000 GFP + cells from primary recipient mice into sub-lethally irradiated mice (650 cGy). For the control, mice underwent transplantation of normal bone marrow cells.MLL-AF9 retrovirus was prepared by transfecting the pMIG-MLL-AF9-IRES-GFP construct with pCL-ECO into HEK293T cells as previously describedPlatelet-rich plasma (PRP) was separated from drawn blood samples and then vitrified for cryo-ET. The mice were in supine position after inhalation anesthesia by diethyl ether. The abdomen and thoracic cave were open to expose heart. Then, 100 μL whole blood was obtained by puncturing right atrium and put in 500 μL EP tube with 3.8% sodium citrate (SC) as the anticoagulant (blood vs SC 9:1). The whole blood is centrifuged at 500 g to collect PRP. PRP (2–4 μL) mixed with 10 nm fiducial gold marker was applied to the glow-discharged Quantifoil grid and then blotted with calcium-free blotting paper using a Vitrobot , and immediately plunged into liquid ethane at liquid nitrogen temperature to vitrify the platelets . PRP purification and cryo-preservation were maintained at room temperature. All platelet samples were vitrified for cryo-ET study within 2 h after blood draw.53, tilt series were recorded at /300 keV on a Gatan 4096 × 4096 pixels K2 Summit direct election camera in counting mode with 0.2 s per frames exposure in . Single-tilt image series  were collected at a defocus range of 15–20 μm and a magnification range of 3000–4 000×, with a pixel size of 12.06 Å. The total electron dose per tomogram was 90–100 electrons/Å2, as typical for cellular cryo-ET. Each tilt series has 51 tilt images with a fixed 2-degree increment.The frozen grids with platelets were then transferred into one of three electron microscopes  equipped with K2 Summit camera for imaging. Low-dose conditions were used to preserve structural integrity. Using SerialEM54, yielding 3D reconstructed tomograms . Gold fiducials were present but not used explicitly, as the software uses any high contrast features for alignment. To enhance the contrast, tomograms were averaged by two (bin2) and filtered uniformly using a low-pass filter (set up at 33 Å) to reduce the noise and visualized in UCSF Chimera55. Structural features such as α granules, dense granules, gamma granules, mitochondria, microtubules, membrane systems, glycogen particles, and tubule-like system (TS) that was formed by membrane repeated folding were identified as previous study26, segmented, and manually annotated using EMAN2, UCSF Chimera55, and UCSF ChimeraX  were manually quantified for each platelet tomogram .P = 0.05, we can state that false positive rate can be no more than 2.8%.In our study, un-irradiated WT and control cells never exhibited abnormal mitochondria. We performed a post-hoc power analysis to put an upper bound on the probability of observing such false positives. With an N of 106 non-malignant cells and 57, which was followed by differential expression analysis using DESeq2 (version 1.12.4)58. The PCA plot was generated with deep Tools.Whole blood from control and pre-AML mice were collected and PRP was separated via Lymphoprep (Stemcell Technologies). Platelets were further purified from the plasma by staining with CD45 and Ter119 antibodies then depleting the CD45 or Ter119 positive cells through autoMACS (Miltenyi Biotec). Purified platelets were resuspended in Trizol and RNA purified according to the manufacturer’s instructions. DNase I-treated RNA samples were purified using the QIAGEN MinElute kit before cDNA was made and amplified with a SMART-Seq2 protocol. The cDNA was then fragmented and barcoded for sequencing using a Nextera XT kit . RNA-seq libraries were sequenced on an Illumina Sequencer . Reads were mapped to mm10 using STAR (version 2.5.2b)Platelets were collected from control or AML mice and resuspend in PBS. Platelet ATP level was measured using CellTiter-Glo® 2.0 Cell Viability Assay (Promega). Luminescence was determined by Infinite M200 PRO from Tecan.t test or Mann–Whitney U-test and statistical significance was taken at P < 0.05.Experimental data were analyzed by SSPS 16.0 for Windows . Data are presented as the means ± standard error of the mean (SEM). Differences between groups were assessed using the Student’s t test or Mann–Whitney U-test was used to assess differences. P values equal to or less than 0.05 were considered statistically.The platelet preparation, Cryo-ET imaging, ATP measurement and RNA-seq were all done from three independent mice each time under same treatment. For subcellular feature measurement and analysis, data were presented as means ± SEM. The Student’s Further information on research design is available in the Nature Research Reporting Summary linked to this article.Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Data 1Supplementary Data 2Supplementary Data 3Reporting Summary


This remodelling promotes cytosolic/mitochondrial Ca2+ oscillations and elevates mitochondrial Ca2+ load, mitochondrial oxygen consumption rate and oxidative phosphorylation. Re-expression of TRPC3 in senescent cells diminishes mitochondrial Ca2+ load and promotes escape from OIS-induced senescence. Cellular senescence evoked by TRPC3 downregulation in stromal cells displays a proinflammatory and tumour-promoting secretome that encourages cancer epithelial cell proliferation and tumour growth in vivo. Altogether, our results unravel the mechanism contributing to pro-tumour behaviour of senescent cells.Cellular senescence is implicated in a great number of diseases including cancer. Although alterations in mitochondrial metabolism were reported as senescence drivers, the underlying mechanisms remain elusive. We report the mechanism altering mitochondrial function and OXPHOS in stress-induced senescent fibroblasts. We demonstrate that TRPC3 protein, acting as a controller of mitochondrial Ca Mitochondrial Ca2+ homeostasis is reported to influence cellular senescence. Here the authors show that TRPC3 limits senescence by inhibiting IP3R-mediated Ca2+ release and ER mitochondria Ca2+ transfer and that the downregulation of TRPC3 in stromal cells affects SASP production and tumour progression. Senescent cells are depicted by specific characteristics such as morphological modifications, cell cycle arrest, apoptosis resistance and altered gene expression. Despite the irreversible loss of their proliferative ability, senescent cells remain metabolically active and secrete several soluble factors, referred to as senescence-associated secretory phenotype (SASP)6, which includes secreted proteins like metalloproteases, inflammatory cytokines and growth factors.It is now well established that various cell-stressing factors, such as DNA-damaging radiations, oxidative stress or oncogene activation, are able to initiate an alternative cellular response to DNA damage that engages the p53 and pRB tumour suppressor pathways culminating in the induction of premature senescence3. The induction of CIS in the tumour microenvironment alters the cross-talk between tumour and stromal cells via development of the SASP, thus supporting tumour relapse and the resistance to further antineoplastic therapies. Indeed, stromal cell CIS is able to boost neoplastic epithelial cell proliferation by paracrine mechanisms. This involves molecules such as FGF, HGF, amphiregulin and WNT16B recently identified as components of the SASP in the tumour microenvironment and capable of promoting tumour cell survival/disease progression and attenuating the effects of cytotoxic chemotherapy10. It is therefore vital to identify molecular actors regulating senescence in stromal cells and the underlying molecular mechanisms.Chemotherapeutic agents have also been shown to induce so-called chemotherapy-induced senescence (CIS)11) and more recently, NAD+ metabolism has been related to the acquisition of a proinflammatory senescence-associated secretome12. In addition, mitochondrial reactive oxygen species (ROS) production is known to induce/stabilise the DNA damage response (DDR), leading to a permanent growth arrest13. It is now clear that mitochondrial metabolism is tightly coupled to cellular Ca2+ homoeostasis. Indeed, mitochondria both encode and decode Ca2+ signals which in turn have a direct impact on oxidative phosphorylation (OXPHOS) and ATP production. Indeed, mitochondrial Ca2+ transients have been reported to increase mitochondrial dehydrogenase (DHase) activity that in turn enhances ATP synthesis. Taking into account that three key DHases of the tricarboxylic acid (TCA) cycle are Ca2+ dependent, it was suggested that mitochondrial Ca2+ regulates the NADH/NAD+ ratio and, hence, controls ATP synthesis16.In recent years, emerging evidence indicate that the mitochondrial activity is coupled to cellular senescence. Mitochondrial dysfunction has been described to induce a specific subset of senescence called “mitochondrial dysfunction-associated senescence –mitochondrial Ca2+ transfer due to constitutive activity of inositol 1,4,5-trisphosphate receptors (IP3R)17. We and others have recently shown that IP3R-mediated Ca2+ release from the ER and subsequent Ca2+ accumulation in mitochondria lead to cellular senescence in normal human cells21. It is important to note that a great number of partner proteins not only regulate  the IP3R activity, but also determine the molecular targets of IP3R-mediated Ca2+ release22. Among these, a number of oncogenes and tumour suppressors proteins have been identified25 shedding light on the Ca2+ dependence of the cancer hallmarks. Whether alteration of the IP3R activity in senescence is mediated by partner protein(s), however, remains unknown.Nevertheless, the mechanisms underlying mitochondrial Ca2+ signatures are altered in senescent cells and whether these alterations affect the SASP composition and tumour progression. Our results provide original insights into the determinants of Ca2+-dependence in pro-tumour senescence.Here we show how cytosolic and mitochondrial Ca2+-permeable channels known to affect IP3R activity and to be recruited during capacitative Ca2+ entry are transient receptor potential (TRP) channels26.One of the most widespread families of Ca2+-permeable channels in senescence, we used several experimental models: (1) human prostate fibroblasts (HPrFs) and prostate cancer-associated fibroblasts (CAFs) treated with docetaxel (Doce)—a microtubule poison commonly used in prostate cancer therapy28 and known to induce senescence in several cell types9; (2) HPrFs treated with hydrogen peroxide, H2O230 and (3) MRC-5 fibroblasts in which senescence was induced by oncogenic RAS (hRASG12V31). The induction of senescence was confirmed by SA-β-galactosidase staining  was not affected.Knowing that, on the one hand, Ca34, was downregulated in tumours from patients undergoing anticancer treatments  conducted on the data from the gene expression omnibus (GEO) dataset GSE35988 from prostate cancer tissues showed a strong enrichment of the genes upregulated in senescence , utilising ratiometric fluorescence wide-field Ca2+ imaging with Fura-2/AM  in senescent cells. Application of histamine  to cells bathed in a Ca2+-free solution revealed that Ca2+ release from the ER is dramatically increased in senescent cells. This was associated with a significant increase in capacitative Ca2+ entry (SOCE) following Ca2+ readmission only in cells with CIS  resulted in senescence induction, reported by proliferation arrest , at least in this model.To check whether TRPC3 downregulation affects ER Catry Fig. . The lat2+ oscillations  region on the C-terminal fragment of TRPC3 and the N-terminal fragment of the IP3R39. Using absolute qPCR quantification, we found that prostate fibroblasts express all three IP3R isoforms, of which type 3 accounts for almost 80%  in fibroblasts  were briefly exposed to 40 µM digitonin (to permeabilize the plasma membrane) during x–y time series imaging , a well-known inhibitor of IP3Rs42. As shown in Fig. 3Rs reduces the permeabilization-unmasked Ca2+ release from the ER by about 80%. This indicates that even without exogenous IP3R stimulation, the observed release is mediated mainly via IP3Rs and thus depends on TRPC3 expression levels.For further verification, we conducted confocal Ca it Fig. . To prob3 concentration ([IP3]) following cell membrane permeabilization with digitonin, the cells were superfused with a solution supplemented with IP3 at different concentrations ranging from: 0 to 10,000 nM  obtained from TRPC3-overexpressing membranes  which disrupts the interaction between the two proteins44. The expression of this mutant, in contrast to the expression of the wild-type TRPC3, had virtually no effect on the IP3-induced Ca2+-release  or E630K (“pore-dead”)45 were overexpressed instead of wild-type TRPC3 . Comparison of the mean values of GEM-GECO1mito fluorescence ratio at the two wavelength bands in a number of x–y confocal images revealed that TRPC3 knockdown resulted in significant elevation of [Ca2+]mito  by mitochondria visualised with MitoTracker Deep Red, showed that TRPC3 kD leads to augmented genesis of ROS, which was notably prevented by the concurrent suppression of IP3R3  Fig. . Moreove3R3 Fig. .2+ transfer to mitochondria by Ru360, (2) IP3R3 knockdown or (3) mitochondrial ROS scavenging by MitoTempo , providing an additional evidence for the role of TRPC3 in modulation of the ER–mitochondrial Ca2+ transfer and mitochondrial metabolism observed in senescence.Our flow cytometry analyses revealed that the percentage of senescent cells observed after TRPC3 knockdown was reduced following: (1) inhibition of Ca4. In this context, mitochondrial metabolism seems to play a central role12. However, mitochondrial dysfunction has also been associated with another secretory phenotype, lacking the IL-1-dependent inflammatory arm and altering the differentiation of preadipocytes and keratinocytes11. To discriminate between these two distinct phenotypes, we analysed the SASP secreted upon TRPC3 downregulation. Antibody arrays covering a large spectrum of cytokines, chemokines and growth factors, showed that TRPC3 downregulation is associated with a pro-inflammatory and pro-tumour SASP, notably including IL-8, ENA-78, GRO-alpha and other soluble factors associated with tumour progression and aggressiveness  only and cultured for an extra 48 h. Cell viability was then measured for all six conditions. The chemotherapy-resistance was expressed as a ratio of the cell viability observed after treatment with docetaxel to that observed with vehicle only Fig. . This ap3R–mediated Ca2+ release thus limiting the ER–mitochondria Ca2+ transfer, while the development of senescent phenotype is associated with TRPC3 downregulation. This, in turn, results in augmented mitochondrial Ca2+ uptake/load, mitochondrial depolarisation, elevated ROS production and mitochondrial metabolism alteration.Our study uncovers a molecular mechanism underlying mitochondrial dysfunction that contributes to pro-tumour effects of senescence. In particular, we demonstrated that, in proliferating cells, TRPC3 inhibits the IP3Rs by TRPC3 appeared to be unrelated to its potential activity as Ca2+-permeable channel. Indeed, on the one hand, downregulation of TRPC3 was associated with significant increase in the ER Ca2+ release, while its overexpression had the opposite effect  or SERCA activity, since the measurements were performed on permeabilized cells superfused with Mg2+/ATP-free solution with [Ca2+] clamped at 50 nM. On the other hand, the observed increase of the mitochondrial Ca2+ uptake/load upon TRPC3 downregulation  in the ER membrane by TRPC3. IP3R is commonly recognised as the major conduit contributing to the ER–mitochondria Ca2+ transfer51. Inhibition of the ER Ca2+ release by the IP3R inhibitor XeC42 indicates that IP3Rs are responsible for about 80% of the Ca2+ release from the ER of prostate stromal cells  following xestospongin application compromised cell bioenergetics and evoked either pro-survival macroautophagy in normal cells, or cell death in transformed or tumour cells. However, the extent of mitochondrial Ca2+ uptake alteration in these cells was not reported53. On the other hand, a sustained increase of mitochondrial Ca2+ concentration induces apoptosis, since mitochondrial Ca2+ overload leads to mitochondrial swelling and, hence, to perturbation/rupture of the outer mitochondrial membrane, release of mitochondrial apoptotic factors into the cytosol and activation of the caspase cascade54.The ER–mitochondria Ca55. Our results confirm further the role of mitochondrial homoeostasis and bioenergetics in senescence, since TRPC3 downregulation leads to increased OXPHOS (reported by augmented NAD+/NADH and ADP/ATP levels) and augmented mitochondrial ROS production.Another possible consequence of mitochondrial dysfunction is the induction of senescence. Indeed, several papers associate ROS production with the induction and stabilisation of senescence, as well as with OXPHOS2+ signalling is an important determinant of cell fate56. One of the major findings of our paper is that the relief of the TRPC3-mediated IP3R inhibition caused by TRPC3 downregulation facilitates [Ca2+] oscillations in the cytosol and mitochondria rather than sustained elevation of [Ca2+]mito. This specific oscillatory Ca2+ signalling in prostate stromal cells seems then to be a crucial point for the choice of the induction of senescence as an alternative to autophagy or apoptosis. Although senescent cells are more prone to Ca2+ oscillations than their proliferative counterpart, we observed that SOCE amplitude in senescence may vary. This is not surprising, since it is generally recognised that different Ca2+ signatures can lead to a same cell fate33. However, understanding and deciphering these differences is a challenge that deserves to be explored to better characterise the role of Ca2+ signalling in senescence.The spatio-temporal pattern of Ca60. However, the discrepancies observed between the rate of cell proliferation in vitro and tumour growth in vivo strongly suggest the contribution of some other factors to the latter.Senescence in the microenvironment exerts a well-documented pro-tumour effect. Indeed, SASP content is enriched in soluble factors which facilitate (1) inflammation-related cancer progression and immune escape and (2) cancer cell proliferation and resistance to anticancer therapies. Our results demonstrate that senescence is associated with mitochondrial dysfunction depending on TRPC3 bioavailability and is characterised by the release of pro-inflammatory chemokines, cytokines and other tumour-promoting molecules Fig. . Besides2+ events engaged in senescence affecting SASP production and thereby tumour progression.Altogether, our findings pinpoint TRPC3 as a key regulator of intracellular Ca-1. Human primary prostate fibroblasts  were cultured in MCDB131 medium  supplemented with 10% foetal bovine serum, 10 mM Hepes, 1 ng ml−1 bFGF and 0.1 ng ml−1 EGF. CAFs from human prostate cancer biopsies were obtained by explant as described in our previous paper61.Human primary lung embryonic fibroblasts MRC5  and Virus-packaging GP293 cells  were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal bovine serum and penicillin/streptomycin 100 U mll-glutamine. HEK293T (Dharmacon) and GP293 packaging cells were cultured in DMEM containing 10% FBS. The cells were maintained at 37 °C under a 10% CO2 atmosphere.PC3, PC3-Luc and DU145 epithelial prostate cancer cells (ATCC) were cultured in RPMI 1640 supplemented with 10% FBS and 1% Sf-9 insect cells (Novagen) were cultured in Grace’s Insect Medium, supplemented with 5% FBS at 28 °C in a ventilated incubator. All the media and supplements, if not stated otherwise, were from Gibco .G12V:ER, a gift from Masashi Narita (Addgene plasmid # 67844) was used to transfer the mutated RAS oncogene in MRC5 cells. The endoplasmic reticulum Ca2+ fluorescent genetic reporter “CEPIA1-er”, a gift from Masamitsu Iino (Addgene plasmid # 58217), was PCR modified to insert the StuI restriction site and then cloned into the pLNCX2 retroviral vector (Clontech) by HindIII/StuI. The mitochondria Ca2+ fluorescent genetic reporter “CMV-mito-GEM-GECO1”, a gift from Robert Campbell (Addgene plasmid # 32461), was cloned into the pLNCX2 vector by BamHI-BglII/HindIII.The plasmid pNLCX2-hRASEGFP-TRPC3 was produced by E-Zyvec (France) and cloned into the pLNCX2 vector by SalI/ClaI.44 and the pore mutants “TRPC3 E630K-E630Q” were cloned into the baculoviral vector pFastBac LIC, a gift from Scott Gradia (Addgene plasmid # 30111), previously modified by LIC cloning to insert the CMV promoter (pFastBac-CMV).Wild-type “TRPC3-Myc”, a gift from Craig Montell (Addgene plasmid # 25902), CIRB mutants “TRPC3-MRK/AAA” To produce retroviral particles, GP-293 packaging cells were transfected with the corresponding pLNCX2 plasmid and the pVSV-g vector (Clontech) using PEIpro Transfection reagent . After 48 h, viral supernatant was then filtered and used to transduce target cells. Bacmids, obtained after transformation of DH10Bac bacteria (Life Technologies), were transfected into Sf9 cells (Novagen) by the Cellfectin-II reagent (ThermoFisher) to produce P1 baculoviral stock. P2 stock, obtained by infecting fresh Sf9 cells, was then titrated by qPCR and target cells were transduced at MOI = 500.Lentiviruses encoding shRNA, siRNAs and controls were purchased from Dharmacon . Target cells were transduced at MOI = 1.Cells stably expressing the construct were selected by G418 (400 µg/ml) or puromycin (1 µg/ml) for 7 days.3) and incubated for a further 72 h with the conditioned medium. Cell viability/proliferation was calculated by MTS assay.Conditioned medium was obtained by incubating HPrFs, previously treated with doxycycline for 1 week to induce TRPC3-mediated senescence,  for 24 h in RPMI 1640 medium 0% FBS. PC3-Luc or DU145 cells were plated into 96-well plates .Xe − log  Xb) + S, where Xb = cell number at the beginning of the incubation time, Xe = the cell number at the end of the incubation time, and S is the starting PDL of the inoculum.To calculate cumulative population doublings, the following formula was applied: PDL = 3.32 .SA-β-Gal activity was determined according to Debacq-Chainiaux F et al.® RNA Plus (Macherey Nagel). Two μg of RNA were reverse-transcribed using random hexamers, MuLV and dNTPs (Life Technologies) in a final volume of 20 μl according to manufacturer’s instructions. Quantitative Real-time PCR reactions were performed using the CFX96 Real-time PCR system (Biorad). Primers were designed with the qPrimerDepot software (http://primerdepot.nci.nih.gov/) and are listed in Supplementary Table ® Supermix, Biorad). Experiments were performed in triplicates for each data point. Results were analysed with the CFX Manager version 3.1 software (BioRad). Gene expression was normalised to GAPDH, ACTIN or 18S gene, and fold expression relative to the control is shown.Total RNA was prepared using the NucleoSpin3R1, IP3R2 and IP3R3 and for GP64 viral glycoprotein were serially diluted to obtain a calibration curve and used in a standard reaction.For absolute qPCR, plasmids containing the sequences for human IP3R3 (5 µg/mg lysate) for 3 h at +4 °C. Proteins were then incubated overnight at +4 °C with the Ab-coupled beads and washed five times in IP buffer followed by western blot.For western blots, cells lysis was performed with different solutions depending on the analysed proteins and protocols. The total protein content was measured using the BCA assay. Immunoprecipitation was performed on cell lysates prepared as above and diluted in 1 ml of lysis buffer. Agarose-ProteinA/G beads (SantaCruz) were incubated either with rabbit anti-TRPC3 (5 µg/mg lysate) or rabbit anti-IPProteins were resolved by SDS–PAGE, transferred to PVDF or nitrocellulose membranes  and incubated with the following primary antibodies: rabbit polyclonal anti-TRPC3 (1/200) Alomone Labs Cat# ACC-016; mouse monoclonal anti-GAPDH clone 6C5 (1/2000) Abcam Cat# ab8245; mouse monoclonal anti-Beta-Actin clone AC-74 (1/2000) Sigma Aldrich Cat# A2228; mouse monoclonal anti-IP3R3 clone 2/IP3R-3 (1/1000) BDBiosciences Cat# 610313; rabbit monoclonal anti-N-Cadherin clone H-63 (1/200) SantaCruz Biotech. Cat# sc-7939; mouse monoclonal anti-Calnexin clone C8.B6 (1/2000) Millipore Cat# MAB3126; mouse monoclonal anti-VDAC1 clone B-6 (1/200) SantaCruz Biotech. Cat# sc-390996; mouse monoclonal anti-Cytocrome C clone A-8 (1/200) SantaCruz Biotech. Cat# sc-13156; rabbit monoclonal anti-MCU clone D2Z3B (1/1000) Cell Signalling Cat# 149975; mouse monoclonal anti-P53 clone DO-1 (1/200) SantaCruz Biotech. Cat# sc-126; mouse monoclonal anti-PCNA1 clone PC10 (1/200) SantaCruz Biotech. Cat# sc-56; mouse monoclonal anti-Cyclin D1 clone G124-326 (1/1000) BD Pharmingen. Cat# 554180; goat polyclonal anti-p16INK4a/CDKN2A (1 µg/mL) R&D systems Cat# AF5779; rat monoclonal anti-hRAS clone 342404 (5 µg/mL) R&D systems Cat# MAB3429.® Human Cytokine Array C2000 (RayBioTech). Briefly, conditioned medium was generated from cells  following a PBS wash and incubation in serum-free RPMI for 48 h. Conditioned medium was then filtered (0.2 μm) and incubated on the array overnight at 4 °C. Washes and detection procedures were performed as indicated by the manufacturer, and chemiluminescence signals were analysed with the ImageJ/Fiji version 1.53 software.Secondary antibodies used were peroxidase-conjugated . Peroxidase activity was revealed using an enhanced chemiluminescence (ECL) or ECL advanced kit . Band densities were quantified using the ImageJ/Fiji version 1.53 software. Density of each band was divided by the density of the reference protein band or related control band, and the obtained value was normalised with respect to that obtained under control conditions. Full uncropped and unprocessed blots are available in the Source Data file. Secreted proteins were detected using a RayBioWhere applicable, cells were permeabilized “online” during imaging protocol by 20 s exposure to 40 µM Digitonin using home-made local perfusion system.Confocal imaging was performed with LSM 510 META confocal workstation using a Plan-Neofluar ×40 1.3 NA objective .Fluo-4, Mitotracker Green and eGFP were excited by the 488 nm line of 500 mW Argon ion laser  and fluorescence was captured at wavelengths 505–545 nm. TMRE and ER-Tracker Red were excited by the 543 nm line of 5 mW He/Ne laser  and fluorescence was captured at wavelengths >560 nm. CellMask Deep Red was excited by the 633 nm line of 15 mW He/Ne laser  and the fluorescence was captured at wavelengths >650 nm.2+]ER or [Ca2+]mito imaging, fluorescence emission spectra of GEM-CEPIA1er and GEM-GECO1mito expressed in fibroblasts were measured either after cell permeabilization (with 40 µM Digitonin) and incubation in the solution with [Ca2+] buffered (EGTA) to 50 nM or 1 mM (for GEM-CEPIA1er), or in control and after application of 2.5 µM ionomycin (for GEM-GECO1 mito). Fluorescence was excited by 405 nm line of blue diode laser and captured by META unit of LSM 510 META confocal workstation. Upon time-series [Ca2+] imaging, fluorescence then was captured at the two channels: green—470–500 nm and red—>520 nm in line-by-line mode of the multitrack configuration of the confocal scanner.To verify optical channel setting for ratiometric confocal [CaIllumination intensity was attenuated to 0.6–4% (depending on fluorophore used) with an acousto-optical tunable filter.Image processing was carried out using LSM 5 software  and with custom routines written in IDL .Fura-2 was alternatively excited at 340 and 380 nm with a monochromator  and was captured after filtration through a long-pass filter (510 nm) by a 5 MHz charge-coupled device camera . Acquisition and analysis were performed with Metafluor 4.5 software .43.The collection of the ER-containing fractions from prostate fibroblasts transduced either with empty vector or TRPC3, the preparation of GUVs and the patch-clamping were performed following the protocol described in our previous paper6 cells were incubated with 5 µl of Pacific Blue Annexin-V and 5 μl of 7-AAD for 15 min at +4 °C. Collected data were exported to and analysed with the FlowJo 7.0 software .Flow cytometry was performed using a CYAN AD  and the Summit Software version 4.3. Briefly, 1 × 10Mitochondrial membrane potential flickers were analysed by utilising the TMRE probe (0.1 μM). Cells were incubated with TMRE for 1 min and analysed by confocal microscopy to determine the number of flickers over a time period of 5 min.+/NADH ratio was determined using the NAD/NADH-Glo Assay  according to the manufacturer’s instructions. The ADP/ATP ratio was determined using an ADP/ATP Ratio Assay kit  according to the manufacturer’s instructions. Luminescence signals were captured using a TriStar² S LB 942 microplate reader (Berthold).MRC5 cells were transfected with siTRPC3 or siCTL and cultured for 7 days. Cells were then plated in 96-well plates for measurements. The NAD2-free chamber at 37 °C for 30 min. Baseline OCR was measured: (i) at the basal state, (ii) after oligomycin (Oli) injection (1 μM), (iii) after FCCP injection (0.27 μM and then 0.34 μM), and (iv) after rotenone (Rot)/antimycin A (AA) mix injection (1 μM).Oxygen consumption rates (OCR) were measured using the XFe24 Extracellular Flux Analyser . Briefly, culture medium was removed and cells were washed once with DMEM (Sigma-Aldrich), pH buffered at 7.35, supplemented with 10 mM glucose and 2 mM glutamine. Then, cells were incubated with DMEM in COMitochondrial ROS levels were measured by incubating cells with MitosoxGreen and Mitotracker Deep Red probes (Life Technologies) for 30 min in culture medium. Cells were washed twice in phenol-red free culture medium and analysed by confocal microscopy. ROS levels were estimated by calculating the mean fluorescence ratio Mitosox/Mitotracker.NOD-SCID mice (NOD.CB17-Prkdscid/NCrHsd) 5 weeks old, male, mean weight) were purchased from Envigo , maintained in laminar-flow boxes under standard conditions (standard diet and water ad libitum) at 23 °C with 12-h light and 12-h dark cycles at Animal Facility Core  under Specific Pathogen Free (SPF) conditions. After one week of quarantine and acclimatisation, the mice were earmarked and randomly separated into 2 groups of 10 subjects, 5 animals/cage, by an independent person. Animal study was approved by the Animal Experimentation Ethics Committee CEEA Nord—Pas de Calais no. 75 (Permit No. 2017062015327270), and all procedures were conducted in accordance with the French National Chart for Animal Experimentation and the ARRIVE guidelines.6 PC3-Luc cells and 0.5 × 106 HPrFs stably expressing doxycycline-inducible shRNA against TRPC3 or scramble sequence (ratio 2:1) and previously treated for 7 days with doxycycline (1 μg ml−1) to induce shRNAs expression, in vivo animal imaging was performed one day after injection and weekly by I.P. injection of 150 mg/kg D-XenoLight d-Luciferin–K+ salt bioluminescent Substrate  using the IVIS Spectrum optical imaging system (PerkinElmer) for a total of 6 weeks. Bioluminescence recording was performed with the Living Image software version 4.5 from PerkinElmer.To evaluate the effect of TRPC3 down regulation in HPrFs on the proliferative potential of prostate epithelial cancer cells, NOD-SCID mice were injected s.c. on the right flank with a mix of 1 × 10Microarray datasets from Oncomine (“Grasso Prostate” and “Tamura Prostate”) were used to analyse the expression of TRPC3 in prostate cancer patients undergoing or not chemotherapy or radiotherapy. Log2 median-centred ratios for each patient were used to create the scatter plot.Raw data (accession number GSE130727 and GSE35988) were downloaded from Gene Expression Omnibus. For GSE130727 dataset, normalisation and differential expression was done with ‘DESeq2’ R-package version 1.24.0 and R version 3.6.0. Lastly, we extracted normalised counts for TRPC3 on log scale for each condition and treatments .For GSE35988 dataset, patients were classified using the median expression of the TRPC3 gene. Differentially expressed genes (DEGs), were identified using the Linear Models for Microarray and RNA-Seq Data limma package v3.46.0 and R 3.6.0. Fold changes were calculated after loess within-normalisation and quantile between-normalisation. Replicate probes were replaced with their average using the limma ‘avereps’ function. A total of 1585 up-regulated and 1801 down-regulated genes were identified with a cut-off value of absolute log fold change (logFC) > 0.5 and an adjusted 35. All probe sets were pre-ranked using a metric defined as the sign(logFC)* − log1062; thereafter, the ordered probe set list was used as the pre-ranked GSEA input. Detailed GSEA parameters are as follows: the number of permutations is 1000, min size is 15 and max size is 500.Gene Set Enrichment Analysis (GSEA) was performed using the GSEA software version 4.1.0 from UC San Diego and Broad Institute, using the Human Cellular Senescence Gene Database (HCSGD)n, is included in the associated figure legend. Statistical significance was determined by the Student’s t test and the one-way ANOVA using GraphPad Prism 8.0. p values < 0.05 were considered significant. p values for each experiment are also included in the associated figure legends.Error bars represent either the SD or SEM, as described in figure legends. Sample size for each experiment, Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Chromosome instability (CIN) is an increased rate where chromosome acquire alterations due to errors in cell division. CIN creates genetic and cytogenetic diversity and is a common feature in hematological malignancies such as acute myeloid leukemia (AML). Low to moderate levels of CIN seems to be well tolerated and can promote cancer proliferation, genetic diversity, and tumor evolution. However, high levels of CIN seems to be lethal, where enhancing CIN could improve AML treatment. However, little is known about CIN in AML. Our review focus on CIN studies in AML, their prognostic results, as well as the use of CIN as a therapeutic target in AML.Chromosomal instability (CIN), the increasing rate in which cells acquire new chromosomal alterations, is one of the hallmarks of cancer. Many studies highlighted CIN as an important mechanism in the origin, progression, and relapse of acute myeloid leukemia (AML). The ambivalent feature of CIN as a cancer-promoting or cancer-suppressing mechanism might explain the prognostic variability. The latter, however, is described in very few studies. This review highlights the important CIN mechanisms in AML, showing that CIN signatures can occur largely in all the three major AML types . CIN features in AML could also be age-related and reflect the heterogeneity of the disease. Although most of these abnormalities show an adverse prognostic value, they also offer a strong new perspective on personalized therapy approaches, which goes beyond assessing CIN in vitro in patient tumor samples to predict prognosis. Current and emerging AML therapies are exploring CIN to improve AML treatment, which includes blocking CIN or increasing CIN beyond the limit threshold to induce cell death. We argue that the characterization of CIN features, not included yet in the routine diagnostic of AML patients, might provide a better stratification of patients and be extended to a more personalized therapeutic approach.  Since Boveri’s theory that chromosome abnormalities promote cancer, studies have attempted to elucidate the mechanisms behind the origins of chromosomal aberrations . ChromosCIN not only occurs as a tumor-promotor mechanism but also as a tumor-suppressor mechanism. This observation comes from the evidence showing that different levels of CIN lead to distinct outcomes. Moderate or low levels of CIN are associated with increased rates of genetic cancer-promoting features. On the other hand, extreme levels of CIN could lead to decreased cell fitness or apoptosis . The levThe current models for CIN involve telomere dysfunction, defective spindle assembly, sister chromatid cohesion, DNA double-strand breaks (DSB) repair, genes involved in the cell cycle, and epigenetic regulators. These CIN mechanisms and their signatures can be largely found in acute myeloid leukemia (AML), a heterogeneous disease characterized by abnormal proliferation and accumulation of myeloid precursor cells in the bone marrow . AML canOne type of nCIN is aneuploidy, characterized by an abnormal number of chromosomes in the cell, which is a result of chromosome mis-segregation errors . These eTP53 [TP53 arise before or after the first aneuploidy event [TP53 may result in the continued proliferation of aneuploidy cells or can trigger off apoptosis [TP53 has a role in suppressing nCIN and sCIN by inducing apoptosis in cells that have a long pause in the mitotic checkpoint, which indicates DNA damage [TP53 is one of them [TP53 mutations in AML varies between 10% and 15% [TP53 mutations are highly present in AML patients with CK (60%) [A key gene to maintain diploid karyotype in normal cells is TP53 . This prTP53 ,40. Mutady event ,42 Thesedy event ,44. Altepoptosis . TP53 haA damage . Mutatio of them . The inc and 15% ,47,48. ICK (60%) ,50,51.TP53 mutations in AML is low in comparison with other cancer types. This observation highlights that additional mechanisms are affecting p53 function for AML patients [TP53-knockout have CIN phenotype and karyotype heterogeneity [TP53 mutations, can promote genome and chromosomal instability to block normal hematopoietic differentiation and is significantly associated with CK and poor prognostic [TP53, this alteration is frequently found in AML patients with CK (60–69%) [TP53 and the ones located at 5q), suggesting an important role of dysfunctional cell cycle checkpoint in AML.Interesting, the incidence of patients . Many papatients . Li et apatients . Additiopatients ,56,57. Tpatients . Cazzolaogeneity . The delognostic ,60,61. I(60–69%) ,63,64. TJin and Burkard 2018) have associated CIN in AML patients with defects in the spindle assembly checkpoint (SAC). SAC is a mitotic checkpoint mechanism used to prevent transition to anaphase when there is an error on the kinetochore-microtubules attachments ,66. When8 have asBUB1B, where the SAC protein BubR1 is expressed. BubR1 stabilizes the kinetochore-microtubule and corrects the proper chromosome positioning. It prevents cell division until forming an appropriate bi-oriented mitotic spindle [BUB1B leads to CIN with consecutive constitutional mosaicism for chromosomal gains and losses and subsequent predisposition to several types of cancer [BUBR1 is an anaphase-promoting complex (APC/C) inhibitor gene, AML cells with defects in this gene usually allow chromosomal alterations to bypass mitosis [Mosaic variegated aneuploidy (MVA) is a rare human chromosomal instability disorder. Patients with MVA present germline mutations in the mitotic checkpoint components and 25% of the cells show aneuploidy . The mos spindle . In MVA,f cancer . In AML,f cancer . Since B mitosis . NUP98) is another gene associated with SAC defects in AML. NUP98 regulates the timely destruction of securin by APC/C [NUP98 translocation contain aberrant securin (a regulatory protein of the metaphase-anaphase transition), leading to aneuploidy [Cdh1, an antagonist regulator of SAC (which activates and mediates securin degradation). Therefore, the high number of SAC alterations in AML cells can be associated with other dysfunctional chromosome segregation features. The most common chromosomal abnormality found in AML is t . Boyapatby APC/C . Cells weuploidy . Anotherde novo AML patients [NPM1 (Nucleophosmin 1) mutations [NPM1 mutation results in the inactivation of the nuclear factor-κB (NF-κB) in the cytoplasm [NPM1 mutations leads to favorable prognostic and chemosensitivity. Sister chromatids are kept together until the proper formation of the bipolar spindle. If cohesion between sister chromatids is lost, chromosomes mis-segregate . Cohesinpatients . Howeverutations . Interesutations ,88. NPM1ytoplasm . Since aAnother important mechanism related to CIN is centrosome dysfunction ,92,93,94+ blast cells compared to CD34+ from normal individuals with no evidence of hematologic diseases [The aurora kinases, a family of serine-threonine protein kinases, have a key role in centrosome dynamics, mitotic spindle, and mitotic centrosomes . Two comdiseases ,101,102.diseases . Yang etdiseases . MYC overexpression [MYC is a proto-oncogene located at the long arm of chromosome 8 (8q24). MYC deregulation leads to DNA damage [MYC-mediated suppression of the NHEJ (Non-homologous end-joining), an important pathway that repairs DSBs in normal cells [DSBs can lead to translocations and DNA deletions ,105,106.pression . MYC is A damage , the indA damage . The indal cells ,116,117.Trisomy 8 can be found in the blood of normal individuals ,119. GroTelomeres are TTAGGG repetitive sequences directly associated with capping proteins, shelterin proteins, that protect the ends of chromosomes . The linTERT, or hTERT in humans) is a catalytic subunit of the enzyme telomerase, which, together with the telomerase RNA component (TERC), comprises an important unit of the telomerase complex [Telomeres become shorter at each cell division and, without telomerase, an enzyme that adds TTAGGG repetitive sequences to elongate the telomeres, cells undergo senescence . The sen complex . A decre complex ,135.hTERT expression correlated with chromosomal abnormalities in AML patients. They found that telomere length in mononuclear cells of AML patients was significantly reduced compared to controls . In addition, patients with abnormal karyotype presented shorter telomeres than those with normal karyotype. In contrast, extremely short telomeres  were found in AML patients showing multiple chromosomal abnormalities. Furthermore, hTERT continued to be associated with an increase in the karyotype complexity [Swiggers et al. (2006) demonstrated that critically short telomeres in blasts AML patients lead to nCIN. The authors reported an increased rate of loss or gain of chromosome parts after telomere shortening . Hartmanmplexity .hTERT expression were reported in 87% of AML patients in another study [Capraro et al. (2012) have also shown that an abnormal karyotype was associated with shorter telomeres and extremely low telomerase activity in AML . Intereser study . Swiggerer study ,144. Telomeres are also responsible for preventing end-to-end fusions of chromosomes, one of the major mechanisms that can trigger both nCIN and sCIN . CriticaDicentric chromosomes (DC) are one of the major signatures of telomere dysfunction. The incidence of dicentric chromosomes varies among AML types, but in general is present in 8–15% of all AML cases ,150. DCsTP53 mutated patients, ∼47% of cases presented CCRs. The occurrence of CCRs was associated with a poor prognosis [TP53 [TP53 on CIN in AML. Hence, Fontana et al. (2018) have found an incidence of 6.6% CCRs in a large cohort of AML patients (N=395). It was also reported that AML cells with CCRs also presented signatures of CIN, such as TP53 alteration, a higher mean of copy number alteration (CNA), CK, 5q deletion, alterations in DNA repair, and cell cycle. They also observed that AML cells with CCRs had marker chromosomes with the MYC gene [MYC (8q24.2) or lysine methyltransferase 2A (KMT2A) (11q23.3) [MYC amplicons in AML. Their results provide evidence that CCRs are not related to a single catastrophic event as the chromothripsis model describes it but rather to an accumulative evolution [Complex chromosome rearrangements (CCR) have been extensively reported in AML ,155,156.rognosis . Rücker is [TP53 . Once mo11q23.3) . L′Abbatvolution . Marker volution . In AML,volution . Marker volution . (TET2) and Enhancer of zeste homolog 2 (EZH2) could induce CIN through the deregulation of histone modifications, which alters the chromatin structure and affect gene expression [TET2 are among the most common mutations in AML [EZH2 is located in 7q36.1, a chromosomal region affected by the loss of chromosome 7 (-7) or deletion of 7q, which reduces its gene expression [TET2 is hypermethylated (downregulated transcription) in 30% of AML patients. Alterations in the expression of TET2 or EZH2 also protects against apoptosis by an unknown mechanism [EZH2 loss of function induced resistance to multiple drugs in AML [TET2 and EZH2 were also positively correlated to the CIN MAD2 and CDC20 genes expression levels [CDC20 is more present in aneuploid than euploid AML [Abnormalities in the epigenetic regulator Tet methylcytosine dioxygenase 2 pression ,165,166.s in AML ,168,169.pression ,171. Thepression ,172. Wanechanism . Göllners in AML . Interesn levels . The pron levels ,176. Schn levels . Overexploid AML . MYC, and TP53 abnormalities are all age-related in AML [de novo AML. However, the de novo AML patients older than 60 years of age and adverse cytogenetics present a similar prognosis of sAML and tAML [A summary of CIN features and the studies associating them to AML pathogenesis, evolution to worst outcomes, and relapse are shown in d in AML ,183,184.and tAML . The thrTP53 mutations achieved complete remission with the use of alisertib [Beyond assessing CIN in vitro in patient tumor samples to predict prognosis, current and emerging AML therapies are exploring CIN to improve AML treatment. Small-molecule inhibitors of aurora kinases are in phase I/II clinical trials for AML ,218,219.lisertib . wild-type p53 cancer cells leads to increased p53 protein level and expression of p53 target genes to inhibit tumor growth [TP53 are present in only 10–15% of AML patients. Interestingly, in acute megakaryocytic leukemia (AMKL), characterized by the presence of abnormal megakaryoblasts, aurora inhibitors can induce blasts differentiation due to their properties of inducing polyploidy . Aurora inhibitors in AMKL induce the upregulation of CD41 and CD42 expression, two characteristic markers of differentiated megakaryocytes [Moreover, Ikezoe et al. (2010) have shown that the induction of apoptosis by barasertib (AZD1152), an aurora B inhibitor, is dependent on functional p53. One of the consequences of using barasertib is the generation of polyploid cells. Aurora B inhibition leads to cells directly re-entering the S-phase without the cytokinesis. Barasertib aurora B inhibition in r growth ,239. Takryocytes ,241.BCL-2 inhibitor used to treat chronic lymphocytic leukemia, showed a synergistic effect on apoptosis both in cell lines and patient samples in vitro, with a prolonged survival in xenograft models [MYC overexpression can also be a target, as was demonstrated in preclinical studies using molibresib (GSK525762), an orally bioavailable drug, which reduced c-MYC expression and its downstream transcriptional effects [MYC inhibition could overcome resistance to cytotoxic drugs in AML cells by promoting differentiation [MYC is JQ1 [MYC and hTERT, could be useful especially for patients with telomere dysfunction [With respect to telomere dysfunction, AML cells could be targeted by telomerase inhibitors ,207. Brut models . DiNardot models . MYC ove effects . This dr effects . MYC inhntiation . AnotherC is JQ1 . JQ1 comC is JQ1 . The comfunction . TET2 function can be restored by using ascorbate, a potentially non-toxic therapy that promotes DNA demethylation, differentiation, and cell death in leukemic cells [TET2 mutations [Moreover, ic cells ,247,248.ic cells . The useutations ,249.TP53, a phase II study performed by Cluzeau et al. (2019) demonstrated that the combination of APR-246 (a drug that reactivates the mutated p53) with azacitidine (a DNA methyltransferase inhibitor whose cytotoxicity interferes with DNA synthesis) showed a response rate of 75% including 56% of complete remission (CR) [TP53 cells with complex karyotype in AML [Regarding patients with mutations in ion (CR) ,196. Polion (CR) ,251,252.e in AML . PLK1 ine in AML .Targeting CIN as an approach to AML therapy is another attractive strategy, but some drugs could increase CIN beyond the “accepted” threshold and actually induce cell death. Jin & Burkard (2018)  measuredBcl-2 inhibition (an anti-apoptotic protein) and p53 activation overcome apoptosis resistance in AML [BRCA1 are more dependent on functional poly (ADP-ribose) polymerase (PARP) proteins than normal cells. They depend on PARP’s DNA repair role by homologous recombination (HR) [BRCA1- or BRCA2-mutated cells in AML BRCA1 mutated patients (BRCA1 loss is reported in 12% of AML patients) [AML1-ETO fusion, a product of the chromosomal translocation t , results in the repression of genes that are essential to the DNA damage response [AML1-ETO or PML-RARa cells leads to sensitivity to PARP inhibitors [High degrees of CIN can also induce cell death through a mechanism known as synthetic lethality. Synthetic lethality is characterized by simultaneous mutations in two different genes, resulting in a significant decrease in cell fitness compared with the same mutations occurring independently ,261. Syne in AML . Leukemiion (HR) . PARP inatients) . Furtheratients) . In AML,response ,267. Esphibitors . Faraonihibitors . They alhibitors .de novo AML, sAML, and tAML. The higher incidence of chromosomal abnormalities found in tAML can be correlated to the prevalence of CIN features in this AML entity and results in the poor prognostic among the AML types. The CIN features in AML are strongly related to ageing, prior disease characterized by CIN, and previous exposure to cytotoxic therapy. Although most of these features show poor prognostic value, they also offer strong new perspectives on personalized therapeutic approaches to decrease the toxic effects of chemotherapy and relapse rates in AML patients. For each CIN feature occurring in AML, there are studies in different research phases showing promising results on targeting those specific mechanisms. Therefore, these studies open the perspective of combining multiple therapies based on the occurrence of the different CIN features individually or on their co-occurrence. The characterization of CIN features that are not in the current routine analyses of AML patients might provide a better stratification of patients. Studies based on the CIN approach with a more extensive characterization of CIN signatures may provide better therapy strategies, especially for those patients with a high risk of relapse or who do not respond properly to current chemotherapy. Future studies evaluating the association among the mechanisms cited here comprise venues for further exploration.The clinical outcome of AML is very heterogeneous and can range from few days of median survival to complete cure, depending on AML subgroups. In AML, the karyotype is the most powerful predictor of treatment outcome. Approximately 30% of cases of AML have an unfavorable karyotype and, if treated with conventional chemotherapy, a 5-year overall survival of 10% to 20% is expected. The best chance of cure for those patients seems to be an allogeneic transplant, but elderly patients are not eligible. Several studies had highlighted CIN as an important mechanism related to the origin, evolution, and relapse of AML. AML patients with CK can present various CIN features, such as defects in the spindle assembly checkpoint, centrosome dysfunction and assembly of multipolar mitotic spindles, defective DNA damage response, telomere dysfunction, and chromosomal abnormalities known to trigger CIN . CIN sig


Despite increasing use of machine-learning algorithms in deciphering the content of biological and medical data, Ca2+ spark images and data are yet to be deeply learnt and analyzed. In the present study, we developed a deep residual convolutional neural network method to detect Ca2+ sparks. Compared to traditional detection methods with arbitrarily defined thresholds to distinguish signals from noises, our new method detected more Ca2+ sparks with lower amplitudes but similar spatiotemporal distributions, thereby indicating that our new algorithm detected many very weak events that are usually omitted when using traditional detection methods. Furthermore, we proposed an event-based logistic regression and binary classification model to classify single cardiomyocytes using Ca2+ spark characteristics, which to date have generally been used only for simple statistical analyses and comparison between normal and diseased groups. Using this new detection algorithm and classification model, we succeeded in distinguishing wild type (WT) vs RyR2-R2474S± cardiomyocytes with 100% accuracy, and vehicle vs isoprenaline-insulted WT cardiomyocytes with 95.6% accuracy. The model can be extended to judge whether a small number of cardiomyocytes (and so the whole heart) are under a specific cardiac disease. Thus, this study provides a novel and powerful approach for the research and application of calcium signaling in cardiac diseases.Ca To datem noises . A connem noises , which m reduced .2+via the L-type Ca2+ channels, which triggers SR Ca2+ release by activating type 2 ryanodine receptor Ca2+ release channel (RyR2) , and catecholaminergic polymorphic ventricular tachycardia (CPVT)—as the cause or consequence of cardiac dysfunction  converts electrical stimuli to mechanical forces in cardiomyocytes. Impaired ECC are closed associated with cardiac pathology . ECC stal (RyR2) . A numbefunction . As elemmyocytes . While m2+ spark images is expected.With recent advances in machine learning and deep learning, a collection of algorithms with impressive abilities to decipher the content of images is now available to researchers in medical and biological fields . Machine2+ spark detection, analysis, and event-based classification of single cardiomyocytes, including the following. (1) To better characterize Ca2+ sparks in cardiomyocytes under diseased conditions, we developed a deep-learning method for automated spark detection that is free of arbitrarily defined thresholds and can detect a greater number of weak signals. (2) Based on the characteristics of Ca2+ sparks detected above, we proposed an event-based binary classification model to identify single cardiomyocytes into normal or diseased groups with promising accuracy.In this study, we proposed a deep-learning framework for automated Ca± (RS)   or wild-2+ imaging as described previously  for 15 min and subjected to line-scan imaging at a speed of 400 lines/s on a Leica TCS SP8 confocal microscope with 40× magnification and a 1.3-NA oil immersion objective. The scan zoom was adjusted to fit the cells, and the scan line was along their long axis. The excitation for Fluo-4 was 488 nm, and emission was collected at 505–530 nm. Ca2+ spark detection and analysis were performed using either the traditional method  or one-way ANOVA with Tukey’s multiple comparison test (for three or more groups) was carried out using GraphPad Prism . Differences were considered statistically significant at P < 0.05.Data are reported as mean ± standard error. Student’s 2+ sparks compared to the WT group  that were omitted by the traditional method were detected by the proposed deep-learning method, especially in RS cardiomyocytes  stimulation or vehicle (VEH) treatment. The amplitude and rise time of the detected Ca2+ sparks differed significantly between the ISO and VEH groups, while the other parameters were similar  system is a common and important mechanism of various cardiac diseases.  similar . Our met similar .2+ release events in cardiomyocytes, Ca2+ sparks were widely used as readouts to distinguish normal and diseased cardiomyocytes , whereas attempts at deep digging and how to use these learnt data have rarely been reported. In the present study, we proposed an event-based logistic regression and binary classification model to classify single cardiomyocytes using Ca2+ spark characteristics. With deep learning, our classification model succeeded in distinguishing WT and CPVT-mutated cardiomyocytes with 100% accuracy, and it distinguished isoprenaline-insulted WT cardiomyocytes from vehicle ones with 95.6% accuracy. Chronic overactivation of the β-AR system is a common and important mechanism of various cardiac diseases . Therefore, the proposed classification model should work well to identify whether a heart is under a specific disease or quantify the disease development using Ca2+ spark images of a small number of cardiomyocytes, through presetting the classification model with Ca2+ spark properties of cardiomyocytes under normal and the specific diseased conditions.As elementary SR Camyocytes . Despite2+ sparks are events with small spatial size and fast kinetics, traditional methods have usually introduced an arbitrarily defined threshold (μ+3.8σ) to exclude noises from the true tiny signals in the confocal line-scan Ca2+ images. However, because of the differences in opening times and channels of local Ca2+ release units, the SR Ca2+ content, and the distance between focal plane and release sites, the amplitudes of Ca2+ sparks vary widely. Thus, cutoff with a threshold has resulted in omission of weak but true Ca2+ sparks, while lowering the threshold can lead to false positive detection. The missed and false-positive events can distort the distribution of measured Ca2+ spark properties. Despite continuous progression in confocal microscopy, fluorescent indicators, and denoising algorithms in recent decades that have greatly improved the SNR of line-scan Ca2+ images, the detection problem awaits further resolution, especially under diseased conditions with many Ca2+ sparks in cardiomyocytes. The deep-learning method proposed in this study is free of any hard threshold for better detecting low-amplitude spark events and is shown to be more accurate for characterizing the difference of Ca2+ sparks between normal and diseased cardiomyocytes.Because Ca2+ sparks remains to be solved for this fields, the labeling process of Ca2+ sparks in the training set is done manually with the assistance of a spark detection and characterization tool set to exclude false positive events, which could not guarantee that all low-amplitude events in the training images have been included and might lead to false negative detection. However, with these labeled low-amplitude events in the training set, our network can detect many more low-amplitude events in the test set, which is expected to improve further toward the final resolution of detecting low-amplitude Ca2+ sparks by self-adaption training procedure. Our training set included experimental line-scan Ca2+ spark images with a signal-to-noise ratio (SNR) of between ∼1.75 and ∼3.30, thereby allowing our model to detect spark events from images with SNR values in the same range. As our proposed approach is currently much slower than the traditional threshold-based algorithms, a further optimization in our algorithms will be required.As how to detect the low-amplitude CaTaken together, the present study provides a novel and powerful approach for the research and application of calcium signaling in diseased hearts.https://github.com/BJUT-XJTU-DigitalCell/Software.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: The animal study was reviewed and approved by Institutional Animal Care and Use Committee of Xi’an Jiaotong University.SY and WX designed the research. SY, RL, JC, ZL, and WX performed the research. SY, RL, JC, and WX analyzed the data. SY, ZH, and WX wrote the manuscript. All authors contributed to the article and approved the submitted version.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 patients diagnosed with intermediate or high-risk acute myeloid leukemia (AML) allogeneic hematopoietic cell transplantation  remains in most cases the only curative option. However, despite major improvements in allo-HCT, disease relapse remains the main cause of treatment failure and mortality. AML characteristics  and disease status, including measurable residual disease level at the time of allo-HCT are well-known risk factors for early AML relapse after transplantation. Nevertheless, it is complex to decipher the respective impact of these parameters and individual prediction of relapse risk remains suboptimal.Zhang et al. develop a nomogram based on these parameters to predict the cumulative incidence of relapse risk after allo-HCT. Such an approach is crucial to identify patients at increased risk of relapse after allo-HCT and may implement some therapeutic strategies to prevent relapse. These include the use of adoptive immunotherapies, i.e. donor lymphocyte infusions (DLI) either alone or in association with chemotherapy or targeted agents. The latter strategies are well summarized in the Review by Ye et al. focusing on the different approaches in the use of DLI  and their association with other agents such as hypomethylating agents or FLT3-inhibitors. In patients with AML relapse, therapeutic strategy includes the combination of treatment with direct anti-leukemic effect including intensive reinduction therapy, hypomethylating agents and BCL2 inhibitors and immunotherapy with DLI, second allo-HCT but also checkpoint inhibitors. In addition, in patients with particular molecular abnormalities, IDH1/2 inhibitors and FLT3 inhibitors are important options. These different strategies are extensively reviewed in this Research Topic by Webster et al., Abou Dalle et al., and Ciotti et al. Finally, donor choice can also be an important parameter to decrease AML relapse risk after alloHCT. Whether choosing a Haploidentical (Haplo) donor could implement the graft-versus-leukemia effect has not been formally demonstrated yet. However, in this Research Topic Chang et al. comprehensively review the use of Haploidentical donors for AML, allowing the accessibility of allo-HCT to almost all patients. They also report data from recent studies, showing that for some subgroups of AML patients, such as those with measurable residual disease prior to allo-HCT, Haplo-HCT may improve both leukemia-free and overall survival compared to other donor types.In this Research Topic, Overall, allo-HCT for AML is a comprehensive platform that must take into account disease characteristics and status prior to transplant to build the most effective strategy to achieve an AML cure starting with the choice of the donor. In the post-transplant setting, close monitoring of measurable residual disease is crucial to driving prophylactic therapeutic strategies and initiating early preemptive treatment to avoid hematological relapse. Finally, even in patients with overt AML relapse, effective therapeutic strategies based on targeted agents and immunotherapy can be offered to the patient to achieve complete remission and AML cure.GB, FS, and FM equally contributed to the Editorial. All authors contributed to the article and approved the submitted version.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.


Mounting evidence suggests that the electrochemical gradient that powers mitochondrial function, the mitochondrial membrane potential (Δψm), is a core regulator of the UPRmt. Here we tested this notion directly by pharmacologically dissipating Δψm and monitoring UPRmt activation. We found that chemical dissipation of Δψm using FCCP indeed activated UPRmt dose-dependently in C. elegans assayed by the HSP-60::GFP reporter strain.Mitochondria are ATP-producing organelles that also signal throughout the cell. Mitochondrial protein homeostasis is regulated through membrane potential-dependent protein import and quality control signaling. The mitochondrial unfolded protein response (UPR In addition to energy production, mitochondria are recognized as signaling organelles (Chandel 2015), and Δψm powers diverse signaling outputs that mitochondria coordinate. One such signaling response is the mitochondrial unfolded protein response (UPRmt). Some ETC complex components are encoded by mitochondrial or nuclear genes and require coordination between the genomes for proper stoichiometry (Shpilka and Haynes 2018). The UPRmt is a protein homeostasis response that is activated when nuclear and mitochondrial gene expression are not correctly coordinated (Melber and Haynes 2018). When the expression of mitochondrial and nuclear encoded proteins is mismatched, the UPRmt is activated. In C. elegans, the UPRmt is controlled by the transcription factor ATFS-1, which is normally trafficked to mitochondria and degraded. When nuclear:mitochondrial gene expression is perturbed, ATFS-1 is blocked from entering mitochondria and is instead trafficked to the nucleus where a host of chaperones and stress-response genes are activated and transcribed.Mitochondria are organelles that make ATP by generating a proton gradient across the inner membrane through the action of the electron transport chain (ETC). The gradient is composed of a membrane potential . In vivo evidence supported this assertion, and implied that Δψm is a main regulator of mitochondrial protein homeostasis through its role in driving ATFS-1 import to mitochondria . Based on this premise, we sought to directly test if targeting Δψm could alter UPRmt signaling. We predicted that decreased Δψm would result in activation of UPRmt due to decreased driving force for ATFS-1 to enter mitochondria and be degraded. This would lead to ATFS-1 localization to the nucleus and activation of the UPRmt target gene hsp-60 . We used the protonophore FCCP at various doses to decrease Δψm in live C. elegans. Δψm was monitored in live, adult worms using the potentiometric fluorescent indicator, TMRE.At all doses tested Δψm was decreased compared to untreated control . Using a similar dose range, we found that one intermediate dose was sufficient to activate UPRmt signaling, assayed through the characterized transcriptional hsp-60 reporter strain  (mt positive control (Kim and Sieburth 2018), was used to confirm our results with FCCP. This study was repeated on at least 3 independent days.Recently, it was suggested that ATFS-1 entry to mitochondria for degradation depended on sufficient Δψl. 2019) . Paraquamt is linked to many stress-resistance signaling responses, including hypoxia resistance . We previously showed that FCCP protected against hypoxia , and this work suggests that UPRmt may have been involved. Our other work, however, implicated energy-sensing signaling downstream of decreased Δψm and hypoxia resistance . The requirement of UPRmt for hypoxia resistance downstream of decreased Δψm remains untested. Further, the UPRmt has been implicated in many disease models that are not fully characterized . It is likely that many changes occur with decreased Δψm, and it will be necessary to characterize changes in Δψm and other mitochondrial functions in a wide range of contexts to uncover molecular mechanisms of mitochondrial signaling. For example, it is unclear why only a certain dose (10 nM) of FCCP activated the UPRmt here when all doses resulted in mitochondrial depolarization. This result suggests that other cellular mechanisms likely control UPRmt activation in addition to Δψm. In addition, the level of mitochondrial depolarization may differentially result in UPRmt or mitochondrial autophagy, a process that is also responsive to decreased Δψm. The mechanism that controls which cellular response is elicited likely depends on the degree of mitochondrial stress. This work is complementary to the established role of mitochondrial ETC stress and UPRmt. Inhibition of the ETC results in activation of UPRmt, but only if initiated before the L3/L4 stage in C. elegans . Our results suggest that the mechanism of ETC mediated UPRmt activation may be somewhat distinct from the Δψm control over UPRmt, because decreased Δψm was able to induce UPRmt after the L3/L4 stage.The UPRFluorescence imaging. C. elegans were fed OP50 bacteria on nematode growth media (NGM) plates. L4 animals were synchronized by egg lay and were stained by exposure to 100 nM TMRE for 24 hours that was applied to plates . Fluorescence was photographed in day 1 adult animals on 2% agarose pads with tetramisole (0.1% w/v) for anesthetic. Red TMRE fluorescence was captured using TexasRed filter sets, and green GFP was captured using GFP filter sets on an epifluorescence microscope (Olympus MVX) equipped with a Lumenera camera and acquisition software . Fluorescence of individual animals was quantified using ImageJ. ROIs were whole animals, and background was manually subtracted from final measurements in ImageJ. Mean intensity was measured, and after background subtraction, was normalized to control intensity. Paraquat was dissolved in water and applied to plates at a final concentration of 4 mM. FCCP was dissolved in ethanol  and applied to plates at various concentrations (see figure). Animals were exposed to paraquat or FCCP for 24 hours.