Background and aim: Autophagy, known as cell death type II, is a housekeeping pathway that currently has been worked on in matters of tumorigenesis and leukomogenesis. Therefore, expression levels of ATG7 and LC3 as two key genes in AML patients are targeted in this study.Material and method: This study was performed on 55 de novo AML patients against 17 healthy volunteers, acquired samples from bone marrow (BM) and peripheral blood (PB) sources in different ages and gender. The evaluation was executed by mRNA extraction, cDNA synthesis, real-time PCR and data was analyzed by SPSS. Results: Analyzed data indicate a significant decrease between expression of ATG7 and LC3 in AML patients against control (Pv < 0.05). Decrease in both genes expression was detected in most of the patients, 81.81% and 75.55%, respectively. Also LC3 overexpression was detected in 11.33% of AML patients. Moreover, a positive significant correlation between ATG7 and LC3 genes was detected . Conclusion: This study showed that significant reduction of autophagy genes in de novo AML patients is important to overcome this system and initiate leukomogenesis. It seems a new insight is required for new achievements in diagnosis, prognosis, treatment and monitoring AML patients. AML is a clonal heterogenic disease of hematopoietic progenitor cells and also the most common malignant myeloid disease in adults. The average age of AML occurrence is about 70 years old. In recent years molecular biology studies have been useful in deciphering the pathogenesis of this disease. To determine the treatment response and outcome, genetic abnormalities are considered to be the most significant factors. Notwithstanding, lots of improvements have been made in curing the younger patients; conversely still patients’ condition of older age have not had such success, as overall survival of those is only few months. Such these differences originate from the comorbidity related factors, like age and the disease biology. Moreover, recent efforts in clinical studies have been focusing on assessment of target therapies. Such achievements could probably change treatment success rate. Better understanding of AML pathogenesis would result in target therapy progression intended particularly for elderly adults. Meanwhile as a matter of fact, targeting each of several types of AML genotype variants is a big challenge .For decades, scientific community has been trying to understand not only the molecular mechanisms that are the basis of uncontrolled proliferation of cancer cells, but also to find how these cells are insensitive to the internal and external cell death stimuli.Cancer cell drug resistance is mostly related to abnormally activated or defective programmed cell death (PCD), that mainly occur during apoptosis pathways; as a result, for a long time it was assumed that apoptosis reactivation could be efficient in order to improve the eradication of malignant cells. Classic apoptosis is defined by activation of caspases that account for massive protein degradation. This performance also can be fulfilled by releasing proteolytic enzymes from lysosomes . Programmed cell death (PCD) is referred to apoptosis, autophagy, and programmed necrosis, that all these three pathways can determine the fate of malignant neoplastic cells alongside each other. It gets more complicated when a housekeeping process known as autophagy, that regulates physiologic roles, is also able to expand the cancer cell survival .Autophagy as the second type of programmed cell death (PCD type II)  is evoluIn general, autophagy plays a fatal role in cell homeostasis by inducing pro-survival signals, that are required during starvation periods and stresses such as growth factor deprivation .Autophagy pathway consists of following phases: induction, autophagosome nucleation, elongation and completion, lysosome conjunction, and degradation .During nucleation, proteins and lipids are involved in phagosome membrane formation. Elongation and completion phase ends up with two ubiquitin-like conjugation systems. The first system involves ATG7 and ATG3 accounting for lipid modification in Light Chain 3 (LC3), altering inactive LC3I to active LC3II. LC3 plays its role in mounting the cargo (i.e. proteins and organelles) into the forming phagosome. The second system involves ATG7 and ATG10 accounting for ATG12-ATG5-ATG16L complex formation that is required for LC3 effective lipidation. Then, autophagosomes combine with lysosome to form autophagolysosome, a site for degradation of trapped components and then recycling begins .Despite all, autophagy role in regulation between death and survival of cancer cells  has beenIn recent years, several studies have focused on autophagy in acute leukemia cells, so in this study we assessed the relation between defect in autophagy and AML occurrence by assessment of autophagy key genes expression including ATG7 and LC3 in de novo AML patients.According to these results the other angles of knowledge in AML pathogenesis would be expanded in order to discover novel specific ways in diagnosis, prognosis, targeted therapy, and monitoring.Patients and Healthy VolunteersThe samples were obtained from 55 AML patients in both sources of bone marrow (BM) and peripheral blood and 17 BM and PB samples of healthy volunteers from years 2016 to 2017 with receiving informed consent according to institutional guidelines. The patients were diagnosed at Taleghani Hospital, Tehran, Iran, as diagnosis was determined in accordance with the FAB classification system, which is based on morphology and defining specific immunophenotypes.RNA Isolation and cDNA Synthesis>1.8).Total cellular RNA was extracted from BM using RNeasy Kit . Following the extraction, the integrity of RNA was measured by the NanoDrop . All samples included in the study showed high purity . After synthesizing cDNA, an equal amount of cDNA from each sample of control and patient was used as substrate for qRT- PCR amplification.Subsequently, 1 Real-Time PCR (qRT-PCR)-∆∆ct)  and reverse primer  and reverse primer (5-CTCGTCTTTCTCCTGCTCGT-3), for ABL forward primer (5-AGTCTCAGGATGCAGGTGCT-3) and reverse primer (5-TAGGCTGGGGCTTTTTGTAA). ABL, as a housekeeping gene, was the control gene in this study. Using these primers allowed ATG7, LC3, and ABL cDNA to be specifically amplified. Consequently, ATG7, LC3, and ABL mRNA expression in patient and healthy volunteer samples were analyzed by qRT-PCR . The components in the qRT-PCR reaction for each target consisted of 1 μL of cDNA targeted template, 0.5 μL of each forward and reverse primer, 5 μL of RealQ Plus 2x Master Mix Green- Low ROX , and 3 μL depc-water for a total reaction volume of 10 μL. For each qRT-PCR reaction, a standard curve was provided, using five consecutive 1:20 dilutions of a positive sample  , as previously set up . The the-∆∆ct) .Statistical Analysist-test was used to determine any significant difference in ATG7 and LC3 expression between AML patients and normal control group. Also, Pearson’s chi-squared test was used to measure the linear correlation between ATG7 and LC3 expression.Data analysis was executed using the SPSS Statistics 16.0 and GraphPad Prism 6.07 software. In order to normalize distribution of data in SPSS, log10 was used in analysis. Applying both Shapiro- Wilk and Kolmogorov-Smirnov tests for log10ATG7 and log10LC3 proved normality of distribution in case/control, AML state as M3 and non-M3 AML, age and gender. In addition, α = 0.05 was applied, and the results are expressed as mean ± standard deviation.A significance threshold level of Profile of Patient Sample SpecificationsThe analyzed samples in our study were obtained from 55 de novo AML patients, categorized in two groups of AML M3 and non-M3 AML, and 17non leukemia volunteers varying in gender and age .ATG7 and LC3 gene expression in AML and healthy samplesP < 0.05) between AML and control samples for both ATG7 and LC3 gene expression  of ABL, ATG7, and LC3 were 26.03 ± 2.09, 25.94 ± 1.77 and 27.49 ± 2.17, respectively in the control samples. ABL, ATG7, and LC3 gene expression in AML samples were analyzed using the same technique. The results were as 25 ± 3.82, 27.48 ± 4.20, and 27.76 ± 3.49, respectively for ABL, ATG7, and LC3. Subsequently, the CT values obtained from ATG7 and LC3 were normalized against the internal reference gene, ABL, for both AML positive and normal control group samples  in both AML patients and the control samples, suggesting dependency in their expression . HoweverThis is now obvious that cancer progression is related to autophagy, although its exact roles in different steps of cancer progression are not clearly known and it`s also in controversy for some conditions . Regardi2O3) generates anti-leukemic responses in primary progenitors of AML by utilizing autophagy mechanisms in-vitro . Also, LC3 expression in radioresistant breast cancer cells is found intensively different compared to radiosensitive breast cancer cells, and autophagy is considered to be responsible for radioresistant breast cancer cells’ survival . As in ain-vitro .Our results reveal decrease in expression of LC3 and ATG7 genes for majority of de novo AML patients compared to control group, similar as results in study by As Watson in 2015 that showed decreased autophagy gene expression in human AML (MLL-ENL cell line) . So thesTherefore, autophagy expression changes can be valuable in diagnosis, prognosis, treatment targets, and disease monitoring -21. As wIn many studies autophagy changes were observed in malignancies under chemotherapy and radiotherapy, and they concluded that autophagy genes were overexpressed and resulted in resistance to treatment, so attempted to attenuate autophagy for improvement in treatment outcome , 21, 22.Based on our study, there was no significant correlation between age and these two interested genes expression or between gender and autophagy genes expression. Regarding the decrease in LC3 and ATG7 expression in AML patients compared to control samples and no significant correlation between expression of these genes in AML state as AML-M3 and non-M3 AML, that are naturally different, we suppose that autophagy changes are not directly involved in creating AML by their own, but weakened autophagy mechanisms can be in favor of leukemogenesis.We conjecture that AML patients who indicated lower expression of autophagy genes during AML onset are as the same patients who are therapy sensitive, owing to their de novo weakened autophagy system; on the other hand those patients who indicated expression of autophagy genes the same as the control sample and also those with overexpressed autophagy genes at the AML onset are the same as the patients who indicate therapy resistance, owing to their intact autophagy system.Based on our data, in contrast to the current belief that considers autophagy as a double-edged sword and efforts to find answers to the controversial sides of autophagy behaviors, we suppose that in fact fronting with two different faces of autophagy, one as a weakened housekeeping system on the onset of AML and the other face as the origin of resistance to therapeutic procedures, comes from two sides of autophagy in different patients. Actually, we suppose the former ends up with therapy sensitive patients and the latter initiates with the patients of normal or overexpressed autophagy genes. To prove such a claim another extended study is required to monitor the patients since AML onset till the therapeutic procedure and compare autophagy levels in these two phases of AML together, with the same patients. If such assumption was proved, in future we would be able to characterize our patients into two groups, one as weakened autophagy expression in AML onset accompanied with favor prognosis, and the other as normal or overexpressed autophagy accompanied with poor prognosis. By which we can hope to choose more suitable therapeutic policies in favor of patients.Although abundant efforts have been taken in AML diagnosis, prognosis and treatment, scant progressions has been achieved in recent decades. So, new approach to leukemogenesis and involved related pathways seems to be necessary. Presented results in this study indicate that autophagy genes expression in de novo AML patients are mostly decreased, although in previous studies in relapsed patients, gene overexpression has been detected. Therefore, more studies are required for enhanced utilization of this pathway in diagnosis, prognosis, and treatment.


Recent studies suggest that nuclear-encoded microRNAs (miRNA) are able to translocate in to the mitochondrial compartment, and modulate mitochondrial activities, including [Ca2+]m uptake. Apart from this subset of miRNAs, there are several miRNAs that have been reported to target genes that play a role in maintaining [Ca2+]m levels in the cytoplasm. It is imperative to validate miRNAs that alter [Ca2+]m handling, and thereby alter cellular fate. The focus of this review is to highlight the mitochondrial miRNAs (MitomiRs), and other cytosolic miRNAs that target mRNAs which play an important role in [Ca2+]m handling.Abnormal mitochondrial calcium ([Ca In addition, mitochondria are also a major storage hub of cellular calcium (Ca2+), and Ca2+ homeostasis is fundamental for a wide range of cellular activities such as control of oxidative phosphorylation (OXPHOS), modulation of cytosolic Ca2+ ([Ca2+]cyto) signals, cell death, secretion, and the production of reactive oxygen species (ROS)   uptake. Additional studies have demonstrated a clear role for [Ca2+]m as a pleiotropic signal that regulates many essential functions of cellular physiology. [Ca2+]m concentration is about 10−7 M and it is the result of the dynamic equilibrium between two continuous and opposite processes: mitochondrial Ca2+ influx vs. efflux  cycle that increases the synthesis of reduced substrates (NADH and FADH2), enhances electron transport chain (ETC) activity, and subsequently increases H+ pumping  and isocitrate-dehydrogenases (ICDH) are regulated by direct binding of Ca2+, while pyruvate dehydrogenase (PDH) is regulated by a Ca2+-dependent phosphatase (PDP1) . This happens via the ryanodine receptors (RyR) which increases [Ca2+]m uptake to increase ATP production to meet cellular energetic demands    channels. Activated IP3R can release Ca2+ from the ER, which increases [Ca2+]cyto. Consequently, this cycle further leads to a massive release of cytochrome c, and activation of caspases and apoptosis  as the driving force generated by the electron transport chain   in tissues where mitochondrial NCLX activity is low, such as the liver, kidney, lung, and smooth muscle  that allows the entrance of ions and small proteins (MW < 10 kDa) and an inner mitochondrial membrane (IMM), which is ion-impermeable , recruiting them to the OMM, which leads to the phosphorylation of VDAC , indicating that Ca2+ regulation capacity is not mediated by MCU  is a 10 kDa protein with a mitochondrial target sequence at the 3'-end. Using 2+ sensing EF-hand motifs directed toward the intermembrane space (IMS). It has been demonstrated that MICU1 inhibits [Ca2+]m entry at low resting [Ca2+]cyto concentrations (within the range found in cells at rest), protecting mitochondria from [Ca2+]m overload . Interestingly, results from human skin fibroblasts with MICU1 mutations confirmed the gatekeeper role of MICU1 at low [Ca2+]cyto, but also showed increased [Ca2+]m uptake at higher [Ca2+]cyto . p53 has been shown to influence the oxidative state of SERCA, and thus increase SERCA activity in the ER/mitochondria associated membranes (MAM) fraction. This is related to increased ER calcium release that results in [Ca2+]m overload; a priming step for the release of caspase cofactors—and induction of apoptosis  coronary artery  injury, both myocytes and whole hearts are characterized by increased [Ca2+]m uptake resulting in excessive ROS production and mPTP opening, which ultimately leads to cell death  represent a large set of master regulators of gene expression, which can greatly influence cell death. The binding of miRNAs to their targeted mRNAs can result in translational repression and/or degradation of target genes. These miRNAs are predicted to regulate almost 30–40% of mammalian genes Kim, .2+]m handling, highlighting the role of miR-181c in heart failure  expression. Using rat cardiomyocytes it has been documented that miR-132 could influence [Ca2+]m overload by binding to the 3′-UTR of NCX1 mRNA and modulating [Ca2+]cyto  gets activated. Activated NHE release the H+ from the cell at the cost of Na+ overload. NCX1 plays an important role in releasing the Na+ from the cells to alleviate the intracellular Na+-overload. In cardiac hypertrophy, downregulation of miR-133a has been observed. This leads to higher IP3RII expression and increased [Ca2+]cyto, resulting in left ventricular remodeling and contractile dysfunction  expression (together with other Ca2+ handling proteins), protecting myocytes from apoptosis induced by H2O2  before proposing that a miRNA for regulating the MCU gene could be an effective strategy to achieve a therapeutic goal. However, the gate-keeper of MCU, MICU1, when altered showed very consistent outcomes in multiple groups. It is known that knocking down MICU1 is detrimental when [Ca2+]cyto is low, whereas knocking-down MICU1 is beneficial when [Ca2+]cyto is high. Studies have clearly shown that it is the ratio between MICU1 and MICU2 that plays a pivotal role in [Ca2+]m influx. Therefore, it is fair to predict that miRNA(s) which can effectively bind to MICU1 and/or MICU2 mRNAs can influence the [Ca2+]m. In particular, there is great potential for treatment of conditions such as diabetes and cardiovascular disease with MitomiR(s) manipulation due to their impact on the mitochondria and overall cardiac function.[CaCJDG and BR have performed the literature search. CJDG, BR, and SD wrote the manuscript. All the authors have read and consented to submit this manuscript.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.


Several proteins in the plasma membrane, acidocalcisomes, endoplasmic reticulum, and mitochondria are involved in its homeostasis and in cell signaling roles. The plasma membrane has a Ca2+ channel for its uptake and a plasma membrane-type Ca2+-ATPase (PMCA) for its efflux. A similar PMCA is also located in acidocalcisomes, acidic organelles that are the primary Ca2+ store and that possess an inositol 1,4,5-trisphosphate receptor (IP3R) for Ca2+ efflux. Their mitochondria possess a mitochondrial calcium uniporter complex (MCUC) for Ca2+ uptake and a Ca2+/H+ exchanger for Ca2+ release. The endoplasmic reticulum has a sarcoplasmic-endoplasmic reticulum-type Ca2+-ATPase (SERCA) for Ca2+ uptake but no Ca2+ release mechanism has been identified. Additionally, the trypanosomatid genomes contain other membrane proteins that could potentially bind calcium and await further characterization.Calcium ion (Ca Trypanosoma and Leishmania, with several species that cause severe diseases in humans. Trypanosoma brucei and Trypanosoma cruzi cause African sleeping sickness and Chagas disease, respectively. Leishmania species are the causative agents for cutaneous, mucocutaneous and visceral leishmaniases. T. brucei is an extracellular parasite that replicates in the digestive system of tsetse flies as procyclic form (PCF) and in the tissue fluids and blood of mammals as bloodstream form (BSF). Similar to T. brucei, T. cruzi and Leishmania spp. replicate extracellularly in the midgut of their insect vectors as epimastigotes and promastigotes, respectively. However, once in the mammalian host, they both replicate intracellularly as amastigotes. While Leishmania amastigotes propagate further by infecting other macrophages, T. cruzi amastigotes convert to trypomastigotes before host cell lysis and enters the circulatory system to infect other nucleated cells.Trypanosomatids are a group of unicellular parasitic organisms. This group includes two important genera: 2+ signaling pathways in trypanosomatids are highly divergent from those in the mammalian hosts they infect. As a result, these pathways have been researched thoroughly for identification of potential targets for drugs, vaccines, and diagnostic tools. In the process, we have learned that these parasites possess many unique aspects that are central to their Ca2+ signaling network.CaTrypanosoma and Leishmania species possess a single mitochondrion. Similar to mammalian cells, the mitochondrial Ca2+ uptake is mediated by a mitochondrial Ca2+ uniporter (MCU) complex. The discovery that trypanosomes have a MCU with similar physiological properties as the mammalian uniporter [Both niporter ,2 was esniporter  for the niporter  and the niporter ,6 of theniporter  while thniporter .2+ storage site, trypanosomatids store most of their Ca2+ in an acidic store named the acidocalcisome [2+ export channel, inositol 1,4,5-trisphosphate receptor (IP3R), localizes to the ER in mammals, it localizes to acidocalcisomes in trypanosomatids [2+ homeostasis and signaling pathways in Trypanosoma and Leishmania species as compared to those in their mammalian hosts. We indicate the specific proteins that mediate Ca2+ import and export in various organelles and identify proteins that have the potential to bind Ca2+ and participate in their overall Ca2+ homeostasis.Moreover, while mammalian cells use the endoplasmic reticulum (ER) as a main Caalcisome . Interessomatids ,11. In t2+ signaling. Similar to other eukaryotes, Ca2+ plays a ubiquitous role in trypanosomatids as well.During evolution, two alkali metals, calcium, and magnesium, were present in high concentrations in the primordial soup. Both these elements had similar chemical properties and were chosen to perform many important functions in evolving cells and organisms. However, for inter and intracellular communication, calcium was chosen over magnesium as an important signaling regulator. Both calcium and magnesium can form stable cations by readily losing two electrons. However, calcium, with a larger ionic radius, loses its outermost electrons more easily than magnesium. This enabled calcium to readily participate in multiple chemical reactions that led to the evolution of life. The larger ionic radius also provided calcium with larger polarizability. This meant that calcium ions were highly flexible and therefore able to interact with sites of irregular geometry in various proteins . Additio2+ have been found in trypanosomatid genomes. Putative Ca2+-calmodulin dependent kinases have been identified [T. cruzi a CaM kinase II, which is activated by reactive oxygen species [T. cruzi was also purified and its enzymatic characteristics were analyzed in detail [A variety of proteins that could be modulated by Caentified . In eukaentified . In T. c species , was sho species . A CaM kn detail ,19.2+ activates the T. cruzi phosphoinositide phospholipase C (PI-PLC). This enzyme localizes to the plasma membrane and is upregulated during trypomastigote to amastigote conversion, indicating a potential role for Ca2+ in parasite differentiation [2+ concentration changes occur during differentiation of T. cruzi epimastigotes to metacyclic trypomastigotes [2+ concentration changes also occur during differentiation of T. brucei bloodstream forms to procyclic forms [Cantiation . This isstigotes . Similaric forms .T. cruzi adenylyl cyclase, which interacts with a paraflagellar rod protein is known to be activated by Ca2+ indicating a possible role for Ca2+ signaling in parasite motility [2+ in parasite motility is further corroborated by the parasite centrins. Centrins are well known Ca2+ binding proteins. Out of the five centrins found in T. brucei, three localize to the flagellar basal body [Leishmania donovani localizes to the basal body and is indispensable for basal body duplication and cell division [2+ binding proteins could be playing a pivotal role in trypanosomatid motility and cell division.A motility . The rolsal body . They plsal body  but are sal body  and cellsal body . Similardivision ,28 there2+ in trypanosomatids is the T. cruzi calcineurin [2+ in parasite invasion [2+ is observed during parasites interaction with their host cells [2+ increase in the parasites results in decreased invasion for both T. cruzi [Leishmania amazonensis [2+ also impairs invasion of host cells [2+ has also been proposed to function in parasite osmoregulation [Another protein that is activated by Cacineurin ,29. Inhist cells ,31. PrevT. cruzi  and Leisst cells . Ca2+ hagulation , maintengulation , and reggulation .2+ import through the plasma membrane. These include voltage gated Ca2+ channels (VGCCs), ligand gated Ca2+ channels (LGCCs), transient receptor potential channels (TRPs) and store-operated Ca2+ entry channels (SOCEs).Several different types of transporters can mediate Ca2+ channels that respond to membrane depolarization events. Such channels are classified into high voltage (HVA) L, N, P/Q, R—type or low voltage T-type channels depending on their pharmacological and gating characteristics. Sequences of L, N, P/Q, and T type voltage channels from mammalian cells and sequence of the yeast VGCC named ScCch1p were used to search for VGCCs in parasite genomes. Such a search has identified at least one VGCC ortholog in T. brucei and T. cruzi whereas Leishmania spp. seem to harbor two such orthologs [T. cruzi and Leishmania spp. have not been characterized yet. The T. brucei ortholog for VGCC has been shown to localize to the region where the flagellum attaches to the parasite cell body (known as the flagellar attachment zone) and appears to be necessary for flagellar attachment and proper parasite growth in T. brucei BSF parasites [2+ signaling specifically has not been studied. Evidence has been presented on the presence of an sphingosine-stimulated Ca2+ channel in the plasma membrane of Leishmania mexicana [VGCCs are Carthologs . These oarasites . Howevermexicana  that is mexicana .2+ channels that open in response to a ligand and have not been reported to exist in protozoan parasites [LGCCs are ionotropic Caarasites .2+. Searching the parasite genome with either full-length or N-terminally truncated isoforms of human TRP proteins revealed two orthologs for TRPML/TRPP type in Leishmania spp. and T. brucei. Interestingly, this same search revealed four orthologs of TRPML/TRPP type in T. cruzi CL Brener strain, which has a hybrid genome [T. brucei has been shown to be involved in iron transport [T. cruzi were shown to localize to acidic compartments such as reservosomes and lysosomes [TRP channels open in response to either physical or chemical stimuli. They have been categorized into six families as TRPC , TRPM (melastatin), TRPV (vanniloid), TRPA (ankyrin), TRPML (mucolipin) and TRPP (polycystin) family. Among these, at least TRPC, TRPV, TRPA and TRPP have been shown to localize to plasma membrane and permeate Cad genome . Other sransport  while thysosomes .Trypanosoma and Leishmania spp. seem to lack orthologs for these proteins indicating that SOCE may be absent in trypanosomatids [In mammalian cells, the SOCE is orchestrated by the activity of stromal interaction molecule (STIM) and ORAI proteins . Both Trsomatids .2+ export through the plasma membrane of the mammalian cells is mediated by the co-operative action of a Na+/Ca2+ exchanger (NCX) and a plasma membrane Ca2+ ATPase (PMCA). Orthologs for the Na+/Ca2+ exchanger NCX are missing in the Excavata supergroup [Trypanosoma and Leishmania spp.  and T. cruzi (TcCa1) were able to complement the loss of a vacuolar PMC1 from yeast cells thereby demonstrating the functional capability of these orthologs. An earlier study with vesicles generated from Leishmania spp. plasma membrane has indicated the presence of a vanadate sensitive Ca2+-ATPase pump in the plasma membrane of these parasites [Leishmania spp. genomes [C-terminal calmodulin-binding domain that regulates the activity of PMCA proteins. Although some trypanosomatids apparently lack this C-terminal calmodulin binding domain, it has been shown that calmodulin stimulates the plasma membrane ATPase activity in T. brucei [T. cruzi [L. mexicana [C-terminal region of the PMCA of Trypanosoma equiperdum [T. brucei [In lar Ca2+ . The PMCarasites . In agre genomes . However. brucei , T. cruzT. cruzi  and L. mmexicana . In thisuiperdum ,51, a su. brucei .2+ store in mammalian cells. Uptake of Ca2+ into the ER is mediated by the sarco/endoplasmic reticulum Ca2+ATPase (SERCA). Orthologs for the SERCA protein have been identified and characterized in trypanosomatids  is the major Casomatids . Upon ovsigargin , a specia evansi , a subsp. brucei . Unlike activity . A SERCAparasite . Togethe2+ within the ER is buffered by calcium binding proteins such as calreticulin. T. brucei ortholog for calreticulin is yet to be studied. In T. cruzi the calreticulin localizing to the ER has been identified and its role in quality control of glycoprotein folding has been characterized in detail [T. cruzi calreticulin has also been regarded to possess an inhibitory effect on cancerous cells [Leishmania spp., loss of calreticulin affects the parasite secretory pathway and reduces parasite virulence indicating that ER calcium may have an important role in governing virulence of trypanosomatid parasites [Can detail . Interesn detail . It has n detail . Due to us cells . In Leisarasites ,61.2+ release from ER of mammalian cells is mediated by IP3R or ryanodine receptors (RyR). Trypanosomatids lack orthologs for ryanodine receptors but harbor an ortholog for the IP3R . The MCU complex in mammalian cells comprises of the following components—(1) MCU, which is the pore forming subunit [2+ uptake [Cachondria ,64,65. Amembrane . Further subunit ; (3) Mit subunit  and (4) + uptake . Another+ uptake  has also+ uptake . As a re+ uptake ,72.2+ uptake mechanism is well conserved between mammalian cells and trypanosomatids. An ortholog for the VDAC protein can be found in T. brucei, T. cruzi and Leishmania major (T. brucei VDAC (TbVDAC) has been studied in detail and is essential for parasite growth and mitochondrial ATP production. It has been suggested that TbVDAC acts as a conserved metabolic transporter in the outer mitochondrial membrane of the parasite. However, its direct role in Ca2+ import into the parasite mitochondrion was not tested [T. brucei by bioinformatic analyses [2+ import has not been explored either. Similar to the mammalian mitochondria, an MCU complex situated in the inner mitochondrial membrane is involved in driving Ca2+ into the trypanosomatid mitochondrial matrix. Orthologs of MCU, MICU1, MICU2 and MCUb have been identified and characterized in both T. brucei [T. cruzi [T. brucei did not affect the mitochondrial membrane potential, but reduced the mitochondrial Ca2+ uptake, increased the AMP/ATP ratio and induced autophagy. Based on these results it has been conceived that the mitochondrial Ca2+ uptake is required for activation of dehydrogenases in the mitochondrion of the parasite [T. cruzi. Analyses of these mutants indicate that both MCU and MCUb are necessary for Ca2+ uptake into the mitochondrion of T. cruzi. However, only MCUb was found to be indispensable for parasite growth, metacyclogenesis and infectivity. Additionally, MCUb in T. cruzi does not function as a dominant negative regulator of MCU indicating that this protein differs significantly from its mammalian counterpart [Leishmania spp. indicates that a different and unique role for MICU proteins could exist in trypanosomatid parasites. Orthologs for EMRE and MCUR1 appear to be missing in both Trypanosoma and Leishmania spp [Unlike mammalian cells, trypanosomatids harbor a single mitochondrion that runs the length of the cell body of the parasite and has many peculiar characteristics. Despite, such differences the mitochondrial Caia major . The T. t tested . Later, analyses . However. brucei  and T. cT. cruzi   is also localized to the acidocalcisomes of the parasites [T. brucei, RNA interference (RNAi) mediated downregulation of IP3R reduces parasite growth in both procyclic and bloodstream forms. Additionally, it also reduces the ability of IP3 to release Ca2+ from permeabilized cells [3R in T. cruzi was unsuccessful [3R results in reduced parasite proliferation, differentiation, and infectivity [T. cruzi. This significance is further corroborated by the observation that reduced expression or activity of TcIP3R is associated with differentiation into trypomastigotes [3R or TcIP3R in a chicken B lymphocyte cell line devoid of endogenous IP3Rs indicated that both these proteins could be stimulated by IP3 to release Ca2+ from permeabilized or intact cells [3R in the acidocalcisomes of both T. brucei and T. cruzi.Acidocalcisomes are electron dense acidic organelles that harbor a high concentration of phosphate, pyrophosphate, polyphosphate, magnesium and calcium . Acidocaanalysis . The aciectively . The H+ +-ATPase ,84,85 to tagging  and latearasites . In T. bed cells . Deletioccessful . Howeverectivity  indicatistigotes . Moreovect cells ,86. Toge2+ storage site in mammalian cells [2+ and other biomolecules between these organelles without disturbing the overall cytosolic balance [The ER is the major Caan cells . As the  balance . Similar balance  and plan balance . However2+ transfer to the mitochondria is necessary for cellular bioenergetics and for regulation of cell death processes [2+ uptake has also been demonstrated in T. brucei wherein loss of the MCU affects the AMP/ATP ratio and stimulates autophagy [2+ in trypanosomatids, also house the calcium release channel IP3R. Therefore, the role of mammalian ER-mitochondria MCS might be replaced by acidocalcisome-mitochondrion MCS in trypanosomes. In support of this hypothesis, our group recently demonstrated existence of such contact sites in T. brucei. Using high resolution microscopy and electron microscopy  [Trypanosoma or Leishmania spp. Additionally, trypanosomatids seem to lack orthologs for Golgi specific Ca2+ binding proteins such as Calnuc, Cab45 and Calumenin [2+ store in trypanosomatids. However, new proteins that play a role in Golgi ion homeostasis are still being discovered in mammals that could also be conserved in trypanosomatid parasites.In mammals Cae (SPCA) ,95. An oe (SPCA) . A Golgialumenin . Therefo2+/H+ exchanger. Gdt1p was shown to be indispensable for growth of Saccharomyces cerevisiae under high Ca2+ concentrations. The human ortholog TMEM165 was able to rescue the growth phenotype associated with the loss of Gdt1p indicating functional conservation between these proteins [2+ transporter could play an important role in protein glycosylation. However, more recent studies have indicated that although Gdt1p has higher affinity for Ca2+, it is also able to transport Mn2+ and that its role in Mn2+ homeostasis may be key for efficient protein glycosylation in the Golgi [Leishmania spp. and T. cruzi but cannot be found in the T. brucei genome , termed transmembrane protein 165 (TMEM165) in humans and Gcr1 dependent translation factor 1 (Gdt1p) in yeast, was suggested to be a Caproteins ,99,100. proteins . Furtherhe Golgi . An orthi genome . These oin yeast . It is p2+ release channels have not been identified yet. It is believed that ER localized IP3R [2+ export. Since RyR are absent in trypanosomatids and the IP3R appears to localize to acidocalcisomes instead of ER and Golgi, it is currently unknown how the Ca2+ stored in trypanosomatid Golgi could be exported.Golgi specific Cazed IP3R  or RyR [zed IP3R  also loc2+ in the development of trypanosome flagellum was realized early when it was reported that chelation of Ca2+ resulted in detachment of the flagellum from the cell body of newly formed daughter cells [Crithidia oncopelti, suggested that alterations in the extracellular calcium concentration can alter the flagellum wave pattern of this trypanosomatid [2+ concentrations. In support of its role in Ca2+ signaling, trypanosomatid flagellum harbors several Ca2+ binding proteins. Centrins are well-known calcium binding proteins. Three centrins of T. brucei localize to the flagellar basal body [T. brucei also contain a family of Ca2+ binding proteins that localize to the flagellum and are termed calflagins [2+ has been demonstrated experimentally. RNAi mediated knockdown of entire calflagin family does not reduce parasite motility but results in reduced parasitemia and increased mice survival rate during in vivo infection [2+-binding protein that localizes to the flagellum has been identified in T. cruzi [N-terminal myristoylation and palmitoylation signals to facilitate its transport to the flagellum [2+ binding proteins called calmodulins can also be found in trypanosomatids. At least one such calmodulin has been localized to the T. brucei flagellum [2+ and regulate the development or activity of the flagellum [T. brucei [2+ channel, which localizes to the flagellum of T. brucei, has been demonstrated to be essential for parasite growth and flagellar attachment to the cell body [2+ signaling in the development and maintenance of the trypanosomatid flagellum.The role of Caer cells . Furtherosomatid . Based osal body ,106. Amosal body . T. bruclflagins . Their lnfection . A relatT. cruzi . This prlagellum . Anotherlagellum  and is rlagellum . In addilagellum . The pla. brucei . Additioell body . Overall2+ binding proteins belong to the EF-hand superfamily. More than 800 different proteins have been assigned to this family. Proteins from this family contain at least one EF-hand domain, which is approximately 30 amino acids long, and forms the Ca2+ binding loop. Among the EF-domain containing proteins, CaM is the most conserved Ca2+ binding proteins and is found in all eukaryotes [T. cruzi has been purified and shown to activate the plasma membrane Ca2+-ATPase [T. brucei CaM in activating the plasma membrane Ca2+-ATPase [T. brucei CaM has been localized to the paraflagellar rod. RNAi mediated loss of this protein affected the paraflagellar rod assembly and caused the flagellum to detach from the cell body [A major portion of Cakaryotes ,115,116.+-ATPase  and cycl+-ATPase . A simil+-ATPase . The T. ell body .T. cruzi CaM does not localize to the flagellum but to the spongiome of the contractile vacuole [2+ include the FCaBP in T. cruzi [T. brucei [T. brucei infection in mice [2+ binding domains, which remain to be characterized.Interestingly, the  vacuole ,118. Sev vacuole  but theyT. cruzi  and the . brucei . The cal in mice . In addi2+ homeostasis and signaling  of the mitochondrial Ca2+ uniporter, and the components of the SOCE mechanism.Trypanosomatids have some differences and similarities with mammalian cells regarding Caignaling . Some of2+ signaling [2+ homeostasis and signaling also suggest that they could be exploited as drug, vaccines, or diagnostic targets. In this regard, trypanosomatids, which belong to the Excavata supergroup of eukaryotes, diverged early from the Ophistonkonta, containing animals, and fungi, and the differences pointed out in Ca2+ homeostasis and signaling suggest that the search for new targets is warranted.The presence of TRP in acidic stores  and the ignaling  suggest


However, some Ca2+-dependent effectors that localize away from SOCE microdomains, are activated downstream of SOCE by mechanisms that remain obscure. One mechanism proposed initially in acinar cells and termed Ca2+ tunneling, mediates the uptake of Ca2+ flowing through SOCE into the ER followed by release at distal sites through IP3 receptors. Here we show that Ca2+ tunneling encodes exquisite specificity downstream of SOCE signal by dissecting the sensitivity and dependence of multiple effectors in HeLa cells. While mitochondria readily perceive Ca2+ release when stores are full, SOCE shows little effect in raising mitochondrial Ca2+, and Ca2+-tunneling is completely inefficient. In contrast, gKCa displays a similar sensitivity to Ca2+ release and tunneling, while the activation of NFAT1 is selectively responsive to SOCE and not to Ca2+ release. These results show that in contrast to the previously described long-range Ca2+ tunneling, in non-specialized HeLa cells this mechanism mediates spatially restricted Ca2+ rise within the cortical region of the cell to activate a specific subset of effectors.Agonist-dependent Ca These include contraction, gene transcription, secretion, cellular proliferation, and apoptosis to name a few. Given the disparate often conflicting (cell death vs proliferation for example) cellular functions determined by Ca2+ signals, cells have developed mechanisms to encode specific cellular responses in the spatial, temporal and amplitude dynamics of Ca2+ signals. Spatial encoding can be enforced by co-localization of the Ca2+ source  and the target effector. Ca2+ diffusion around the Ca2+ source is restricted by physical and biochemical barriers, creating a space and time limited microdomain where Ca2+ can effectively and specifically activate a target. The microdomain can range from a few nm (nanodomains) to a couple of µm depending on multiple factors, including but not limited to channel density and conductance and buffering capacity in the microdomain1. Stimulating a Ca2+ sensitive mechanism far from the main Ca2+ source is consequently a signaling challenge since Ca2+ needs to be carried to the effector while avoiding non-specific activation of Ca2+ effectors along the way2.Ca2+-linked agonists mobilize store Ca2+ release typically through activation of IP3-Receptors (IP3Rs), leading to store depletion and to the opening of Store-Operated Ca2+-Entry (SOCE) channels at the plasma membrane (PM). SOCE activation in response to ER store depletion, is mediated by the ER transmembrane Ca2+ sensors of the STIM family (STIM1 and STIM2 and their splice variants), which undergo a conformational change in response to reduced luminal Ca2+ concentrations, leading to their clustering at ER-PM contact sites (MCS), and recruitment and gating of the PM Ca2+ channels of the Orai family 3. Ca2+ flowing through Orai1 channels is subsequently taken up into the ER by the Sarcoplasmic Endoplasmic Reticulum ATPase (SERCA) pump, thus refilling Ca2+ stores and preparing the cell for another round of signaling. However, in addition to store refilling SOCE specifically activates multiple downstream effectors5. A limitation in that regard is the spatial spread of the SOCE Ca2+ microdomain. The assembly of STIM and Orai is not diffuse at the plasma membrane, but highly localized to ER-PM MCS where the two proteins form dense clusters visible at the plasma membrane plane and enhanced in size when SOCE elements are overexpressed6. Accumulating evidence suggests that these clusters are not restricted to a simple STIM/Orai partnership but contain numerous additional proteins, including the SERCA pump. Several studies report that, upon store depletion, SERCA localizes to the SOCE clusters10. This increases the efficiency of Ca2+ store refilling by placing the pump close to SOCE Ca2+ entry source. This particular organization of the SOCE complex defines a spatial Ca2+ microdomain that limits the spread of Ca2+ ions entering through Orai1 to a limited sub-membrane area6. The actual size of this domain is still a matter of debate. The cell type, the extent of store depletion and the physiological state of the cell will generate various cluster sizes. In addition, experimentally STIM-Orai clusters have been measured following overexpression, which is likely to influence their size6. Nonetheless, the most accurate estimates of SOCE clusters come from thin sections studies where the maximal diameter is around 300 nm12. These clusters are also tightly confined in the “z-axis” due to the close apposition of the ER and PM to create a “disc-shaped” microdomain with a 10–20 nm in thickness13. This means that for downstream targets to be specifically responsive to SOCE they have to localize in this spatially restricted and cluttered microdomain. This is the case for adenylate cyclase 8 and for the Ca2+-activated phosphatase calcineurin, which couples to Orai115.Ca2+ in the long-range and expand the SOCE Ca2+ microdomain that has been well described in polarized pancreatic acinar cells and Xenopus oocytes is Ca2+ tunneling16. Ca2+ tunneling involves the transport of Ca2+ ions that flow through SOCE channels into the ER lumen, followed by their release through IP3 receptors at distal sites to activate effectors without inducing a global Ca2+ rise (see recent review2). Here we extend the characterization of Ca2+ tunneling and show that it encodes exquisite specificity in activating effectors downstream of SOCE. Using real time simultaneous imaging of Ca2+ in the ER lumen, cytosol and mitochondria, we show that Ca2+ tunneling differentially signals to downstream effectors. Whereas, Ca2+ influx through Orai1 within the SOCE microdomain effectively activates NFAT translocation, Ca2+ tunneling expands the spatial spread of SOCE by activating other PM effectors such as Ca2+-activated potassium channels. Surprisingly however, tunneling is unable to mediate a Ca2+ rise in the mitochondria arguing that in the non-polarized HeLa cell it results in a spatially restricted cytosolic Ca2+ rise in the cell cortex. This indicates that the long range tunneling mechanism described in polarized cells has been adapted to a preferentially cortical Ca2+ signaling module downstream of SOCE allowing selective activation of Ca2+-dependent effectors.Another mechanism to transport Ca2+ tunneling downstream of SOCE. Conceptually mitochondria represent an ideal target for Ca2+ tunneling, as they form close membrane contact sites with the ER (Mitochondria Associated Membranes or MAMs) where IP3 receptors are enriched17. Furthermore, the mitochondria are a major target of Ca2+ signaling, and play a critical role in buffering cytosolic Ca2+ transients. The relationship between SOCE and the mitochondria is complex and exhibits some cell specific nuances. In immune cells the mitochondria have been shown to act as a Ca2+ sink for Ca2+ flowing through SOCE, which in turn modulates its function20. The mitochondria is further well established as a low pass filter for cytosolic Ca2+ signals and have been shown to functionally interact with SOCE and modulates its signaling21. Furthermore, the subcellular localization of mitochondria affects their response differentially to the source of Ca2+. In both HeLa cells and pancreatic acinar cells the source of Ca2+ was shown to differentially stimulate different populations of mitochondria based on subcellular localization23. In HeLa cells mitochondria localize away from the plasma membrane24, making them an interesting distal target for Ca2+ tunneling.We initially sought to determine the response of mitochondria to Ca2+ tunneling, we imaged Ca2+ dynamics simultaneously in the mitochondria, ER lumen and cytosol. A recently developed family of Ca2+ sensors termed CEPIAs (Ca2+-measuring organelle-Entrapped Protein Indicators), can be targeted to the ER or to the mitochondria, and allow simultaneous imaging of the two compartments25. We thus combined R-CEPIAer (ER lumen Ca2+ sensor) with G-CEPIA2mt  to image Ca2+ changes in the ER and mitochondria respectively, because their spectral properties allow for imaging cytosolic Ca2+ as well using Fura-red  results in a simultaneous rise in Cac2+ and Cam2+ showing that mitochondria readily detect Ca2+ released from stores when the stores are full. Ca2+ release was coupled to a transient depletion of ER stores which refill due to do recycling of released Ca2+ . The relative efficiency of the SERCA pump in capturing released Ca2+ in the absence of Ca2+ influx was variable from cell to cell. As indicated in Fig. 2+ released in the absence of extracellular Ca2+ varied between 7% to 65% with a mean value of 30.1 ± 5.1%   Fig. .2+-free solution followed by Ca2+ addition to maximally activate SOCE and temporally isolate it. TG results in Ca2+ release coupled to emptying of ER Ca2+ stores but little to no increase in mitochondrial Ca2+ (Cam2+) . The rise in Ca2+ recorded in the mitochondria was still significant in both cases  in Ca2+) Fig. . Repleniked Fig. . It shouses Fig. . This co2+ signal was similar in response to Ca2+ release induced by histamine or thapsigargin, or following maximal SOCE induction  to determine the temporal relationship between Cac2+ and Cam2+. The rise in Cam2+ consistently followed the Cac2+ transient with a significant delay  , without any significant increase in Cam2+ . Exposing the cell to histamine after CPA treatment in Ca2+-free solution resulted in a significantly reduced Ca2+ release . CPA induced a store depletion and a Cam2+ Fig. . The peares Fig. , arguingise Fig. . When hi2+ entry through SOCE. However, the experimental conditions above using CPA followed by a wash do not result in the opening of IP3 receptor and as such would not allow for Ca2+ tunneling as would be expected in response to agonist stimulation. To directly test whether the mitochondria act as a downstream effector of Ca2+ tunneling, we devised a protocol to temporally isolate Ca2+ tunneling from the Ca2+ release phase. We depleted Ca2+ stores with CPA (10 min) followed by a wash to release SERCA from inhibition in Ca2+-free conditions . To allow a higher speed of recording (1 Hz), we loaded the cells solely with the Ca2+ indicator Fluo4-AM and performed the same protocol as in Fig. c2+ over time shows a significantly slower rise in the Cac2+ mediated by Ca2+ tunneling as compared to Ca2+ release on full stores, although both signals ultimately reached similar amplitudes  the mitochondria can be positioned close to the plasma membrane as in immune cells where they regulate SOCE20 or deeper in the cell where they preferentially interact with the ER23. We therefore tested whether store depletion affects the relative position of mitochondria in HeLa cells. However, we could not detect changes in mitochondrial localization using either confocal or TIRF imaging  and the target (i.e. the mitochondria) impair the formation of a “Hot Spot” or high Ca2+ domain between the ER and the mitochondria that would allow the MCU to import Ca2+ during tunneling.Taken together our results reveal that given the spatio-temporal dynamics of Ca2+ rise detected following tunneling argues for a spatially localized cytosolic Ca2+ transient. The mitochondria, which tend to localized deeper within HeLa cells, are unable to detect this transient. In Xenopus oocytes Ca2+ tunneling is particularly effective at stimulating Ca2+-activated Cl channels at the PM7. Therefore, to test whether Ca2+-dependent PM localized effectors respond to Ca2+  tunneling, we turned to Ca2+-activated K+ channels (KCa), which are expressed in HeLa cells29. Histamine-dependent Ca2+ release when stores are full induces a transient KCa current   Fig. . InteresOCE Fig. . This hi2+ in the SOCE microdomain and not Ca2+ release from stores is the calcineurin-NFAT signaling pathway32. NFAT1 is a transcription factor, phosphorylated at rest and dephosphorylated following the activation of calcineurin by SOCE, which leads to its translocation to the nucleus 16. Ca2+ tunneling was more recently generalized through studies in frog oocytes, showing dramatic remodeling of the Ca2+ signaling machinery at the PM in response to store depletion to support Ca2+ tunneling, where it targets both Ca2+-activated Cl channels and the IP3R itself to modulate tonic versus oscillatory Ca2+ signaling35. The same mechanism of using ER tunnels to transport Ca2+ to effectors has been alluded to, although not directly investigated, in other studies where it targets the Ca2+-activated K channel, nuclear NFAT activation, and Ca2+ transport from the soma to maintain Ca2+ signaling in dendrites36.Ca2+ tunneling in non-polarized or specialized cells that differ from pancreatic acinar cells, neurons or oocytes for instance, using multiple different effectors of different nature with distinct spatial distribution . We use simultaneous imaging of the three primary Ca2+ signaling compartments in HeLa cells  to assess the functionality of the Ca2+ tunneling mechanism and its ability to specifically and selectively activate downstream effectors. Our results show that Ca2+ tunneling is functional in HeLa cells downstream of store depletion 6. These estimates argue that during Ca2+ tunneling the leak through any open IP3R is orders of magnitude higher than the trickle of Ca2+ that can be fed into the ER by SERCA. This would ensure that the ER does not refill readily allowing Ca2+ tunneling to proceed for tens of seconds. Assuming that IP3Rs are evenly distributed throughout the ER membrane and opening stochastically the first open IP3R encountered would release the Ca2+ fed into the ER by 15,000 SERCA pumps operating at full capacity upstream. This implies that during tunneling Ca2+ entering the cell through SOCE would not be able to travel deep within the cell as the ER acts as a sieve with open IP3Rs resulting in Ca2+ leak from the cortical ER, thus preventing Ca2+ from reaching deep within the ER to activate mitochondria  and streptomycin (100 µg.ml−1). The cells were plated 24 h before transfection on poly-lysine coated glass-bottom dishes . For all live cells experiments, the cells were continuously perfused using a peristaltic pump (Gilson Minipuls) at the speed of 1 ml.min−1. The standard saline contained (in mM) 145 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 Glucose, 10 HEPES, pH 7.2, for Ca2+-free experiments, the Ca2+ was exchanged equimolarly with Mg2+.Hela cells were cultured in DMEM media containing 10% fetal bovine serum supplemented with penicillin (100 units.ml2+ imaging HeLa cells were transfected with the Ca2+ indicators G-CEPIA2mt and R-CEPIAer constructs (0.5 µg per dish) using a standard lipofectamine 2000 (Invitrogen) procedure 24 h to 48 h prior to imaging. Both constructs were obtained from addgene (#58218 and #58216 respectively) and were originally created by Masamitsu Lino’s group25. To image cytoplasmic Ca2+ the cells were loaded for 45 min with Fura Red-AM at 37 °C (1 µM from a 1 mM stock in 20% pluronic acid/DMSO). Imaging was performed on a Leica TCS SP5 confocal system  fitted with a 63x/1.4-06 oil immersion objective using an open pinhole. The G-CEPIA2mt was excited using a 488 nm laser line and the emission collected at 500–590 nm. The same line was used to excite the Ca2+-free form of FuraRed and the emission collected between 600–709 nm. For R-CEPIAer the excitation was performed with a 561 nm laser line and the signal collected between 583–649 nm. The frame rate was set to 0.1 Hz unless stated otherwise. For Fluo4 imaging, the cells were loaded with 1–2 µM Fluo4AM (45 min/37 °C). The excitation was performed using a 488 nm laser line and the signal collected at 500–560 nm with the pinhole at 1 airy unit and the frame rate was set to 1 Hz.To perform organelle Ca−1) (Invitrogen). Confocal images were acquired every 0.5 µm to generate z-stacks. The imaging was performed on a Zeiss LSM880 controlled by Zen Black 2.3  and fitted with a 63x/1.4 objective. The imaging parameters were as follows: for G-CEPIA2mt: λex = 488 nm, λem = 494–568 nm, for R-CEPIAer: λex = 561 nm, λem = 570–622 nm and for WGA-Ax633: λex = 633 nm, λem = 640–747 nm. The distance between the plasma membrane, the ER and the mitochondria was evaluated using the profile of a linear region of interest drawn between both sides of the cell and the distance measured at 50% of the peak amplitudes of the signals.Cells expressing G-CEPIA2mt and R-CEPIAer were fixed  and stained with Alexa633 tagged Wheat Germ Agglutinin (2 µg.ml2+ activated K+ channels were recorded using a standard whole-cell patch-clamp protocol. Patch pipettes (resistance ranging from 4 to 6 MΩ when filled with the pipette solution) were sealed to the plasma membrane and the patch ruptured after the formation of a giga-ohm seal. The cells were voltage-clamped at 0 mV at steady state using an Axopatch 200B amplifier  controlled by pClamp 10. The internal pipette solution contained (in mM) 140 K-Gluconate, 2 NaATP, 2 MgCl2, 10 HEPES, 1 µM EGTA and pH 7.4. The extracellular solutions and perfusion system was the same as the imaging experiments.The Caex = 405 nm, λem = 446–468 nm, for G-CEPIA2mt: λex = 488 nm, λem = 510–555 nm and for Orai1-mCh: λex = 561, λem = 581–679 nm. For TIRF Ca2+ imaging at the plasma membrane, the cells were transfected with the Ca2+ sensor Lck-GCamp5G (Addgene #34924)31. The sensor was excited at λex = 488 nm and the images collected using λem = 510–555 nm, the frame rate was 0.1 Hz. The perfusion system and solutions was identical to the previous experiments.For the localization at the plasma membrane, the cells where transfected with the Orai1-mCh, STIM1-CFP and CEPIA2mt constructs, and the stores depleted with thapsigargin. The membrane plane was identified by the presence of clusters of STIM1 and Orai1, and used to adjust the evanescent wave. The imaging was performed on a Zeiss Cell observer TIRF system using the following parameters for STIM1-CFP: λ41, the imaging was performed using the same settings as the TIRF imaging for GFP-tagged proteins except that the mirror was set vertically to obtain a widefield image. The perfusion saline and system were as previously described.Cells were transfected for 24 h with the NFAT1-GFP construct (Addgene #11107)43 and ZenBlue 2.3 (Zeiss). The patch-clamp data was analyzed with Clampfit 10.0 (Molecular Devices). Statistics and data analysis were performed using Graphpad Prism 7.02 (GraphPad U.S.A). Values are given as means ± S.E.M and statistics were performed using either paired or unpaired Student’s t-test or ANOVA followed by Tukey’s test for multiple comparisons. P-values are ranked as follows *P < 0.05, **P < 0.01, ***P < 0.001.The imaging data was quantified using FIJI/ImageJ 1.51 nSupplementary Information


In this respect, it has been recently proposed that altered astrocytic store-operated Ca2+ entry (SOCE) may underlie aberrant gliotransmitter release and astrocyte-mediated neurotoxicity in ALS. These observations prompted us to a thorough investigation of SOCE in primary astrocytes from the spinal cord of the SOD1(G93A) ALS mouse model in comparison with the SOD1(WT)-expressing controls. To this purpose, we employed, for the first time in the field, genetically-encoded Ca2+ indicators, allowing the direct assessment of Ca2+ fluctuations in different cell domains. We found increased SOCE, associated with decreased expression of the sarco-endoplasmic reticulum Ca2+-ATPase and lower ER resting Ca2+ concentration in SOD1(G93A) astrocytes compared to control cells. Such findings add novel insights into the involvement of astrocytes in ALS MN damage.Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the selective death of motor neurons (MNs), probably by a combination of cell- and non-cell-autonomous processes. The past decades have brought many important insights into the role of astrocytes in nervous system function and disease, including the implication in ALS pathogenesis possibly through the impairment of Ca Such a clustering causes the recruitment and activation of PM pore-forming proteins  PM  and PM (Orai1-3) proteins ,36,37. S the ion , thereby the ion ,40.In light of the results of 2+ homeostasis results from the fine regulation of several mechanisms, we next evaluated whether differences in the resting  Ca2+ levels in different cellular compartments could account for the increased SOCE observed in hSOD1(G93A) astrocytes.Considering that the local cell Ca2+ imaging in hSOD1(WT) and hSOD1(G93A) primary astrocytes by means of different Ca2+ indicators, including fluorescent GECIs (fluorescence resonance energy transfer (FRET)-based cameleons, and GEM-Cepia1ER) and the chemical dye Fura-2 . For GECI-based analyses, cells were transfected with expression plasmids encoding the Ca2+ probes targeted to the cytosol or the ER lumen. Basal [Ca2+] were recorded in 2 mM external [Ca2+] using suitable computer-assisted fluorescence microscopy workstations. Measurements with either the cytosolic-targeted cameleon (D1cpv) or Fura-2 showed that hSOD1(G93A) astrocytes have significantly reduced basal cytosolic [Ca2+] levels compared to non-ALS controls (2+ levels in hSOD1(G93A) astrocytes under resting conditions  astrocytes more sensitive to SOCE activation and cause alterations in other ER-dependent cellular processes (see below).To this purpose, we performed Ca2+ concentrations is guaranteed by an efficient network of Ca2+-transporting pathways and by Ca2+-buffering proteins present in the cytosol and in the lumen of Ca2+-storing compartments . It is worth noting, however, that basal cell Ca2+ levels are tightly controlled by a complex array of Ca2+-transporting/buffering systems. The precise mechanisms leading to such a chronic unbalance of cell Ca2+ homeostasis in hSOD1(G93A) astrocytes, which may weaken ALS astrocytes themselves and contribute to their long-term degeneration , wiyses see  confirmeyses see . Cell mo2+ measurements. AEQ probes (suited for cell population Ca2+ measurements), targeted to cytosolic domains proximal to the plasma membrane (PM) (AEQPM), the bulk cytosol (AEQCyt) and the mitochondrial matrix (AEQmit) were employed to measure Ca2+ fluxes following SOCE stimulation. AEQ-encoding lentiviral vectors were generated, and lentiviral particles purified, as previously described  values, using a computer algorithm based on the Ca2+ response curve of AEQ [Experiments ended by lysing cells with digitonin  in a hypotonic Cae of AEQ .2+ measurements. FRET-based cameleon GECI were used to measure basal Ca2+ levels in the cytosol, the ER lumen and the mitochondrial matrix. Plasmidic vectors coding for the D1cpv and 4mtD3cpv cameleons  were a kind gift by Roger Tsien  following the manufacturer’s instructions. 72 h after cell transfection, coverslips were mounted into an open-topped chamber and maintained under perfusion with a modified KRB  through a temperature-controlled (37 °C) instrument , in order to monitor basal Ca2+ levels in astrocytes.Cameleon-based CaCA, USA; ,80, whilCA, USA; . Astrocy−1). FRET data were then off-line analyzed using the ImageJ software for the calculation of the ratio between the YFP (535 nm) and CFP (480 nm) emission signals, which is taken as a relative measurement of Ca2+ concentration. Before such a calculation, the emission fluorescence intensity of YFP and CFP in proper regions of interest (ROIs) on selected cells was subtracted for the respective background signal. Representative fluorescence micrographs of astrocytes (under resting conditions) expressing the different cameleon probes for both the YFP and the CFP channels are reported in FRET signals were acquired using a computer-assisted DM6000 inverted microscope  with a 40× oil objective , coupled to an IM 1.4C cooled CCD  attached to a 12-bit frame grabber. Excitation light produced by a 410 nm led  was filtered at the appropriate wavelength (425 nm) through a band-pass filter, and the emitted light was collected through a dichroic mirror , and a beam-splitter (OES) with emission filters (Chroma Technology) HQ 480/40M (for CFP) and HQ 535/30M (for YFP). The beam-splitter permits the collection of the two emitted wavelengths at the same time, thus preventing any artefact due to uncontrolled movement of the recording chamber and/or intracellular organelles. Images were acquired using an IM 1.4C cooled CCD  attached to a 12-bit frame grabber. Synchronization of the excitation source and the CCD was performed through a control unit ran by a custom-made software package , which was also used for image acquisition, with an exposure time of 100 ms and a time-shift of 0.5 s. The software allows with-time recording of the FRET-acceptor  and -donor  fluorescence intensity  with Fura-2/AM (1 μM), pluronic F-127 (0.02% (w/v)), and sulfinpyrazone (200 μM) in a modified KRB , and then left 20 min at room temperature in the above modified KRB to allow de-acetylation of the probe. Fura-2-loaded cells were then processed for Ca2+ imaging using an inverted microscope  equipped with a 40× ultraviolet-permeable objective . Alternating excitation wavelengths of 340 and 380 nm were obtained by a monochromator controlled by a custom software . A neutral density UVND 0.6 filter (Chroma Technology) was used in the excitation pathway. The emitted fluorescence was measured at 500–530 nm. Images were acquired every 5 seconds, with a 200 ms-exposure time at each wavelength, by a SensiCam QE camera  controlled by the same software. ROIs corresponding to the entire cell soma were selected for Ca2+ imaging, and the ratio of the emitted fluorescence intensities (F340/F380) was averaged offline after background subtraction.Fura-2-based cytosolic Ca2+ measurements. To monitor basal ER Ca2+ levels by means of the GEM-Cepia1ER GECI [® reagent (Invitrogen). Cells were analyzed 72 h after transfection by means of an inverted microscope  with a 40× oil-immersion objective . Ca2+ recordings were performed in the same modified KRB used for the cameleon probes (see above). Excitation light (395 nm) was provided by a monochromator . The emitted fluorescence was collected at 480 nm and 530 nm for the Ca2+-bound and the Ca2+-free indicator, respectively, and the ratio of the emitted fluorescence at the two emission wavelengths (480/530), after background subtraction, was taken as a measure of resting ER Ca2+ concentration. Images were acquired at 1 Hz, with a 200 ms-exposure time, using a computer-assisted cooled CCD camera , and analyzed as described previously [GEM-Cepia1ER-based Ca1ER GECI , astrocyeviously ,83. Reprm of cultured astrocytes was measured using the membrane-permeant TMRM, a cationic red-orange fluorescent dye  that accumulates electrophoretically into the mitochondrial matrix, thereby allowing to correlate the Δψm amplitude to the fluorescence signal quantification [2 5%) in CaCl2 (1 mM)-containing modified KRB , and then incubated in the above modified KRB devoid of Mg2+. Fluorescence images were collected with an inverted microscope (Olympus IMT-2) equipped with a (75W) Xenon lamp, a cooled 16-bit CCD camera , a 40× oil-immersion objective, and appropriate excitation and emission filters. Several fields were acquired from each coverslip before and after addition of trifluorocarbonylcyanide phenylhydrazone (FCCP)  that, by collapsing Δψm, releases the probe from mitochondria. The TMRM fluorescence intensity in each image was quantified in mitochondria-rich ROIs using the Image J software and reported as the difference between the mean fluorescence intensity before and after FCCP addition. Representative fluorescence micrographs of TMRM-loaded astrocytes, before and after FCCP addition, are reported in The mitochondrial Δψfication . To this® reagent (Invitrogen), according to the manufacturer’s instructions, with a plasmid coding for mtRFP . 72 h after transfection, cells were observed by means of a laser scanning confocal microscope TCS-SP5 (Leica Microsystems) equipped with a 40× oil-immersion objective to evaluate mitochondrial morphology. To this purpose, fluorescence microscopy images of randomly selected fields were acquired (at least 10 fields for each coverslip), and every single detected mitochondrion was analyzed for its morphological characteristics, such as surface area, and major and minor axes by means of the Volocity software . On the basis of such parameters, the mitochondrion circularity shape factor (4π × area/perimeter2) was calculated as an inverse index of mitochondrial elongation [Primary astrocytes, seeded onto 13 mm-diameter glass coverslips at the optimal density of 80,000 cells/well in 24-well plates, were transiently transfected (24 h after plating) using the Lipofectamine 3000ongation ,85, apprw/v), sodium dodecyl sulfate (SDS) 2% (w/v), Tris 62.5 mM (pH 6.8), NaVO4 5 mM, protease and phosphatase inhibitor cocktails (Roche), and cell lysates were boiled (5 min) to accomplish complete protein denaturation. The total protein content was determined by the Lowry method , using BSA as standard. The reducing agent dithiothreitol (50 mM) and bromophenol-blue (0.004% (w/v)) were added to samples just before gel loading. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on precast gels , loaded with 18 μg of proteins per lane and run in a Tris/glycine buffer. Proteins were then electro-blotted onto polyvinylidene difluoride (PVDF) membranes . Membranes were incubated  with a blocking solution of Tris-buffered saline added with Tween-20 (0.1% (w/v)) (TBS-T), containing non-fat dry milk 5% (w/v), or BSA 3% (w/v), and then incubated  in the same blocking solution containing the desired primary antibody (Ab) (see below). Finally, after three 10 min-washes (with TBS-T), membranes were incubated  with a horseradish peroxidase-conjugated anti-rabbit or anti-goat IgG secondary Ab (Santa Cruz Biotechnology), depending on the used primary Ab. After washing in TBS-T, immunoreactive bands were visualized and digitalized by means of a NineAlliance digital camera workstation (UVITEC), using an enhanced chemiluminescence reagent kit . For densitometric analyses, the optical density of the immunoreactive bands of interest was normalized to the optical density of the corresponding lane in the PVDF membrane stained with Coomassie blue [96 h after plating onto 12-well plates , astrocytes were homogenized in a lysis buffer containing glycerol 10%  Abs were used (dilution in parentheses): anti-STIM1 ; anti-STIM2 ; anti-Orai1 ; anti-Orai3 ; anti-SERCA2 ; anti-MCU ; anti-CRT , anti-CLNX , anti-Grp78 . All pAbs were raised in rabbit, with the exception of the anti-SERCA2 pAb (raised in goat).n) is indicated in the figures and/or figure legends). Statistical significance was evaluated by unpaired two-tailed Student’s t-test, a p-value < 0.05 being considered statistically significant .Data were analyzed using Origin 8.0 SR6  and Microsoft Excel 2010  software. Values are reported as mean ± SEM (the number of experimental replicates (


It remains unknown how the sensitivity of TRPV4 to this agonist is regulated. The spatial and temporal dynamics of receptors are the major determinants of cellular responses to stimuli. Membrane translocation has been shown to control the response of several members of the transient receptor potential (TRP) family of ion channels to different stimuli. Here, we show that TRPV4 stimulation with GSK1016790A caused an increase in [Ca2+]i that is stable for a few minutes. Single molecule analysis of TRPV4 channels showed that the density of TRPV4 at the plasma membrane is controlled through two modes of membrane trafficking, complete, and partial vesicular fusion. Further, we show that the density of TRPV4 at the plasma membrane decreased within 20 min, as they translocate to the recycling endosomes and that the surface density is dependent on the release of calcium from the intracellular stores and is controlled via a PI3K, PKC, and RhoA signaling pathway.TRPV4 is a non-selective cation channel that tunes the function of different tissues including the vascular endothelium, lung, chondrocytes, and neurons. GSK1016790A is the selective and potent agonist of TRPV4 and a pharmacological tool that is used to study the TRPV4 physiological function  Further, we elucidate the importance of this trafficking on global changes in [Ca2+]i following GSK1016790A stimulation.Here, using the combination of Total Internal Reflectance Fluorescence (TIRF) microscopy, Ca2+ imaging experiments, cells were immersed in standard Hanks' balanced salt solution (HBSS) containing: 140 mmol/L NaCl, 5 mmol/L KCl, 10 mmol/L HEPES, 11 mmol/L D-glucose, 1 mmol/L MgCl2, 2 mmol/L CaCl2, and 2 mmol/L probenecid, adjusted to pH = 7.4 .For the CaGSK1016790A (TRPV4-selective agonist), brefeldin A (an inhibitor of intracellular protein transport), cytochalasin D (an inhibitor of actin polymerization), and GSK690693 (Akt inhibitor) were purchased from Sigma-Aldrich . HC067047 (TRPV4-selective antagonist) and NSC23766 (Rac1 inhibitor) were purchased from Santa Cruz Biotechnology . Pipstop2 (Clathrin inhibitor) was purchased from Abcam. Cpd 22 (ILK inhibitor) was obtained from Calbiochem.2O or 0.1% v/v DMSO) and diluted in HBSS prior to experimentation, and corresponding vehicles (0.1% v/v dH2O or 0.1% v/v DMSO) were used as controls.All drugs were dissolved according to the supplier's instruction . RhoA, RhoA (T19N), CDC42, CDC42 (T17N), Rac1, and Rac1 (T17N) cloned in vector pcDNA3.1 were obtained from cDNA Resource Center, University of Missouri-Rolla, MO. HEK293 T-Rex cell lines  stably expressing TRPV4 (TRPV4-Venus) were generated as described in Poole et al.  were grown in EGMTM-2 media supplemented with SingleQuotse et al.  and Barae et al.  and grow2+ bound) 380/520 nm (Ca2+ unbound) data represented at a ratio of 340/380 nm (Ca2+ bound/Ca2+ unbound). Microscopy experiments utilized FLUO-4AM (1 μM), ex/em: 450/520 nm, data represented as F1/F0, the ratio of fluorescence compared to fluorescence at the zero time point. Cells were imaged for baseline [Ca2+]i then agonists were administered at the points indicated on the graphs.For calcium imaging experiments, media was removed, and cells were washed with HBSS, then loaded with calcium-sensitive dyes in HBSS. Dyes were then washed off, and cells were treated with inhibitor buffer or corresponding vehicle controls, cells were either imaged on a plate reading fluorimeter  or using confocal microscopy  using effectene (QIAGEN). Rab11-Rluc8 and Kras-Rluc8 were from Nevin A Lambert . At 48 h post-transfection, TRPV4 expression was induced using 0.1 μg/ml of tetracycline for 16 h. Before the BRET assay, the medium was replaced with HBSS 30 min before the assay. Cells were loaded with coelenterazine H (Nanolight Tech.) (5 μM), RLuc8 luminescence (480 nm) and Venus fluorescence (530 nm) were measured for a 2–3 min to establish a baseline, and at different times after addition of 100 nM GSK1016790A, using a CLARIOstar microplate reader (BMG LABTECH).HUVECs were grown in a 24-well plate to confluence, incubated with inhibitors or the appropriate controls for at least 30 min. After TRPV4 stimulation with GSK101, EZ-Link Sulfo-NHS-SS linked biotin (0.5 mg/ml) was added in PBS, and incubated on ice for 30 min at 4°C. Following stimulation, the reaction was quenched with 50 mmol/L glycine and excess biotin was removed with PBS. Lysates were prepared in standard lysis buffer , and 1% Triton-X-100). Samples were normalized for the protein concentrations and incubated with streptavidin beads overnight at 4°C, and the SDS page and Western Blotting was performed as reported previously . First, to test the specificity of GSK1016790A, we used non-transfected HEK293 cells or treated cells with a selective antagonist of TRPV4, HC067047 produced a near maximal effect, so we used this concentration for the rest of the study , while the BRET ratio between TRPV4-Rluc8 and Rab11-Venus increased by . Together, these data showed that within 30 min post- TRPV4 stimulation with GSK1016790A, TRPV4 was internalized and transferred into recycling endosomes   in endocytosis of TRPV4 post GSK1016790A treatment in HUVECs using both TIRF microscopy and cell surface biotinilation assay and using both approach found that depletion of Ca2+ from intracellular stores or removal of Ca2+ from the buffer reduced the endocytosis of TRPV4 post-GSK1016790A treatment  Figure . We inve Figures .2+ release from the intracellular stores or Ca2+ influx from the extracellular environment, we depleted intracellular Ca2+ stores by using thapsigargin or by removing Ca2+ from the buffer (extracellular space). Under both conditions, TRPV4 stimulation with GSK1016790A failed to induce cytoplasmic aggregation of TRPV4 channels, suggesting that this phenomenon is dependent on both Ca2+ release from intracellular stores as well as Ca2+ influx from the extracellular environment , and depletion of calcium from the stores reduced the aggregation by , while the inhibition of TRPV4 did not have any effect  within the first minute   Figure , and theWe next investigated which signaling molecule is required for the TRPV4 channel partial fusion and endocytosis following TRPV4 stimulation with GSK1016790A. Rho GTPases play an important role in the regulation of different endocytic pathways. For example Rac1, CDC42, and RhoA have been postulated to regulate clathrin-mediated endocytosis  and non-transfected control group (p < 0.05), 20 min post TRPV4 stimulation with GSK1016790A  that was significantly higher than the cells transfected with the wild-type RhoA , and in return, PKC phosphorylation leads to the activation of different TRP channels including TRPV4  fold. In contrast, pre-treatment with PKA and PKG inhibitors did not have any effect of on the ratio of BRET signal between TRPV4 and Kras. This result is consistent with our previous observation that PKA and PKG inhibitors do not affect the elevation of [Ca2+]i following TRPV4 stimulation with GSK1016790A   Figure . It shou2+ ion channels in maintaining Ca2+ homeostasis has been defined before  controls trafficking Cav1.2 in a mouse atrium cell line (HL-1) (Peng et al., In distal nephrons (Mamenko et al., PI3K plays a regulatory role in clathrin-dependent endocytosis of different types of membrane proteins, including renal outer medullary potassium channels (Cheng and Huang, 2+ influx may play a regulatory function in the mechanotransduction of blood flow and the development of atherosclerosis in regions where blood flow is disturbed. Our finding suggests that the vasoprotective effect of GSK1016790A on endothelium could be attributed at least in part to its regulatory role in controlling the basolateral expression of cadherins and β-catenin.Overnight endothelial TRPV4 stimulation with GSK1016790A has been shown to protect the endothelium from atherosclerotic plaque formation at the regions of arteries that blood flow is disturbed (Sullivan et al., SB and PM: designed the experiments; SB, PK, WD, AL, PV, and KE: performed the experiments; SB, PK, WD, KK, and PT: analyzed the data; SB and PM: wrote the manuscript.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.


Acute myeloid leukemia (AML) is the most common type of adult leukemia. Several studies have demonstrated that oncogenesis in AML is enhanced by kinase signaling pathways such as Src family kinases (SFK) including Src and Lyn, spleen tyrosine kinase (SYK), and bruton’s tyrosine kinase (BTK). Recently, the multi-kinase inhibitor ArQule 531 (ARQ 531) has demonstrated potent inhibition of SFK and BTK that translated to improved pre-clinical in vivo activity as compared with the irreversible BTK inhibitor ibrutinib in chronic lymphocytic leukemia (CLL) models. Given the superior activity of ARQ 531 in CLL, and recognition that this molecule has a broad kinase inhibition profile, we pursued its application in pre-clinical models of AML.The potency of ARQ 531 was examined in vitro using FLT3 wild type and mutated (ITD) AML cell lines and primary samples. The modulation of pro-survival kinases following ARQ 531 treatment was determined using AML cell lines. The effect of SYK expression on ARQ 531 potency was evaluated using a SYK overexpressing cell line (Ba/F3 murine cells) constitutively expressing FLT3-ITD. Finally, the in vivo activity of ARQ 531 was evaluated using MOLM-13 disseminated xenograft model.FLT3 ITD mutation. We demonstrate decreased phosphorylation of oncogenic kinases targeted by ARQ 531, including SFK (Tyr416), BTK, and fms-related tyrosine kinase 3 (FLT3), ultimately leading to changes in down-stream targets including SYK, STAT5a, and ERK1/2. Based upon in vitro drug synergy data, we examined ARQ 531 in the MOLM-13 AML xenograft model alone and in combination with venetoclax. Despite ARQ 531 having a less favorable pharmacokinetics profile in rodents, we demonstrate modest single agent in vivo activity and synergy with venetoclax.Our data demonstrate that ARQ 531 treatment has anti-proliferative activity in vitro and impairs colony formation in AML cell lines and primary AML cells independent of the presence of a Our data support consideration of the application of ARQ 531 in combination trials for AML targeting higher drug concentrations in vivo. Acute myeloid leukemia (AML), the most common form of acute leukemia in adults, is a rapidly progressing aggressive hematopoietic malignancy arising from the bone marrow myeloid progenitor cells  characteFLT3 mutations, either as an internal tandem duplication (FLT3-ITD) or tyrosine kinase domain point mutation (FLT3-TKD), occur in 25% and 7% of AML, respectively, and constitutively activate the FLT3 proliferation and cell survival pathway [FLT3 mutations in AML patients has prompted the development of FLT3 inhibitors such as quizartinib, midostaurin, and gilteritinib. Despite the impressive clinical response with FLT3-ITD selective inhibitor, quizartinib, 50% of patients relapsed within 3 months [ pathway . FLT3-IT pathway , increas pathway . The pro3 months  due to a3 months –20. Howe3 months , and gilOther kinases have been shown to be relevant to AML and to potentially cooperate with FLT3, such as the SFK –26. SpecTmax) after a single 10 mg/kg oral dose with 72% oral bioavailability [ArQule 531 (ARQ 531) is an orally bioavailable kinase inhibitor with favorable pharmacokinetic (PK) properties. In cynomolgus monkeys, ARQ 531 achieved 9 μM maximum concentration within 6 h . Quizartinib, midostaurin, and dasatinib were purchased from LC Laboratories . Gilteritinib and venetoclax were purchased from Chemietek . Entospletinib (GS-9973) was purchased from Selleckchem . All chemical compounds were dissolved in Dimethyl sulfoxide (DMSO) to obtain a 10 mM stock solution.MOLM-13, MV4-11, and OCI-AML3 AML cell lines were purchased from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (Germany). THP-1 and U937 AML cell lines were purchased from the American Type Culture Collection . HS-5 GFP were obtained from Dr. William Dalton (H. Lee Moffitt Cancer Center & Research Institute).All cell lines were cultured in Roswell Park Memorial Institute 1640 medium  supplemented with 10% fetal bovine serum , 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin . The luciferase expressing MOLM-13 cell line was a generous gift from Dr. Ramiro Garzon . Drug-reThe pMSCV-IRES-Tomato (pMIT-Empty) and pMIT-SYK-TEL vectors were a generous gift from Dr. Kimberly Stegmaier (Dana-Farber Cancer Institute) and previously described in Puissant et al. . HEK293TAML patient samples were obtained from The Ohio State University Comprehensive Cancer Center Leukemia Tissue Bank under an Institutional Review Board-approved protocol.For AML cell lines and primary patient samples, genomic DNA was extracted using the DNeasy Blood and Tissue Kit . The mutational status of 91 protein-coding genes was determined using Illumina TruSeq Custom Amplicon library preparation. Libraries were pooled and sequenced using the MiSeq platform . DNA library preparations were performed according to the manufacturer’s instructions. Illumina amplicon sequenced reads were aligned to the hg19 genome build using the Illumina Isis Banded Smith-Waterman aligner. Single nucleotide variant and indel calling were performed using MuTect and VarScan, respectively. The MuCor algorithm was used as the baseline for integrative mutation assessment. We only considered non-synonymous variants not listed in either the 1000 Genome database or dbSNP142-common variants. After variant calling, variants were annotated using SnpEff and vcfanno along with the dbsnp, COSMIC, 1000 genomes, and 6500 exomes variant databases. The Mucor3 algorithm was used as the baseline for integrative mutation assessment. All called variants underwent visual inspection of the aligned reads using the Integrative Genomics Viewer.31. Variants in regions of high discrepancy, low quality, tandem repeats, or mononucleotide runs were excluded.Antibodies purchased from Cell Signaling Technology  including anti-FLT3 (Cat.#3462), anti-phospho-FLT3 Tyr589/591 (Cat.#3464), anti-STAT5 (Cat.#9363), anti-phospho-STAT5 (Cat.#9351), anti-ERK (Cat.#4695), anti-phospho-ERK (Cat.#4377), anti-Src family kinase (Cat.#2109), anti-phospho-Src family kinases Tyr416 (Cat.#2101), anti-SYK (Cat.#13198), anti-phospho-SYK Tyr525/526 (Cat.#2711), anti-BTK (Cat.#8547), anti-phospho-BTK Tyr223 (Cat.# 87457), anti-MCL1 (Cat.# 94296), anti-BCLXL (Cat.# 2764), and anti-HSP90 (Cat.#4874). Anti-β-Actin (Cat.# SC-1616) was purchased from Santa Cruz Biotechnology  and anti-GAPDH (Cat.# MAB374) from EMD Millipore . HSP90, β-Actin, and GAPDH were used as loading controls.Cell lines were serum starved overnight followed by a 10-min treatment. Cells were washed in ice-cold PBS then lysed in 1X Cell Lysis Buffer (CST) supplemented with proteinase and phosphatase inhibitors cocktail (Roche). Pierce BCA Protein Assay was used to quantify the protein lysates . For Immunoblotting, 15–25 μg of protein lysate was prepared in 2X or 4X Laemmeli’s sample buffer  and heated for 5 min at 100 °C. Samples were separated on 8% sodium dodecyl sulfate polyacrylamide gel and transferred onto nitrocellulose membrane . The membranes were blocked for 1 h in Blocker BLOTTO Blocking Buffer  or 5% non-fat milk followed by overnight incubation at 4 °C with primary antibody followed by HRP-conjugated secondary antibodies and visualized with chemiluminescent Pierce ECL Substrate  on X-ray films.50 was determined.Biochemical inhibition of FLT3-WT and FLT3-ITD fusion was measured using recombinant protein constructs of kinase domains (Reaction Biology). ARQ 531 was tested in a 10-point concentration mode with 3-fold serial dilutions starting at 1 μmol/L in the presence of 10 μmol/L ATP, and the IC50) was calculated using GraphPad Prism 7  and SAS/STAT software . For the MOLM-13 resistant and the Ba/F3-FLT3-ITD/TKD cell lines, cells were treated for 72 h and cell viability was evaluated using MTT assay (Roche Diagnostics. Mannheim. Germany) according to the manufacturer’s instructions.A total of 5000–30,000 cells from the lines above were treated in a 96-well plate for 72 h. CellTiter 96 Aqueous MTS reagent  was added to cells followed by a 3-h incubation at 37 °C, 5% CO2. The absorbance was measured at 490 nm using DTX 880 plate reader and the half maximal inhibitory concentration  containing Annexin V-FITC and Propidium Iodine . Live and apoptotic cells were measured using FC 500 flow cytometer and analyzed on Kaluza software .Primary AML patient samples were treated and plated at 5000–10,000 cells in 1.1 ml methylcellulose supplemented with recombinant human stem cell factor, interleukin 3, granulocyte colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor  per dish in duplicates. Colonies were counted after 14 days when using primary patient samples.All experiments were conducted after approval of The Ohio State University Institutional Animal Care and Use Committee. Mice were randomized and enrolled for daily oral gavage (Q.D.P.O) treatment of vehicle or 50 mg/kg ARQ 531. Mice were treated until reaching early removal criteria (ERC) defined as 20% weight loss, lethargy, hind limb paralysis, moribund, anorexia, dehydration, hunched posture, severe diarrhea, breathing difficulties, or any abnormal neurological symptoms.A total of 10,000 luciferase expressing MOLM-13 cells  were eng50 with 95% confidence intervals (CI) were calculated using 4-parameter logistic models. Mixed effects models were used in experiments described in Additional file p values have been adjusted for multiple comparisons using Holm’s procedure. p values from experiments with multiple endpoints were adjusted for multiple comparisons using either Holm’s procedure or Dunnett’s test . All analyses were performed using SAS/STAT software .For in vitro cytotoxicity experiments Fig. b–d, repe+, DNMT3A-mutated OCI-AML3 cell line at low μM concentrations, while U937 and THP-1 cell lines were resistant to concentrations up to 10 μM  in addition to the NPM1c50  cell line previously described in Zimmerman et al. . In thisTo verify these results in primary cells, AML patient blasts   was trann = 7) or 50 mg/kg ARQ 531 (n = 8) daily, a dose shown previously effective in TCL1 adoptive transfer mouse model of CLL [n = 3) injected with luciferin and imaged weekly for tumor burden . Additionally, it strongly inhibited SFK phosphorylation suggesting the importance of modulating SFK or a downstream target of SFK in AML and potentially explaining the activity of ARQ 531 in FLT3-WT cells. To confirm the role of SFK inhibition in response to ARQ 531, we examined SYK phosphorylation, a critical SFK downstream target. Indeed, phosphorylated SYK levels were reduced by ARQ 531, and SYK constitutive overexpression caused a shift in ARQ 531 ICCss) in the μM range unlike in CLL where BTK phosphorylation inhibition is achieved in nM range. Additionally, the PK profile for ARQ 531 is very different between primates and rodents. In Macaca fascicularis cynomolgus monkeys, a single oral dose of 10 mg/kg demonstrated favorable PK and oral bioavailability achieving maximum concentration (Cmax) of 9 μM and 72% bioavailability. Due to the absence of elimination phase, the half-life (t1/2) was not calculated where 75% of peak concentration was present at 24 h, suggesting that ARQ 531 has a long plasma half-life [Css plasma concentration in patients and the potential use of ARQ 531 in AML. At the previous cleared 65 mg daily dose of ARQ 531, the mean Cmax of ~ 3 μM was obtained [ARQ 531 also potently inhibits BTK and previously demonstrated a remarkable survival advantage in two immunocompetent adoptive transfer mouse models of CLL, Eμ-TCL1, and Eμ-MYC/TCL1 when compared with ibrutinib  resultinalf-life . The comalf-life  will be obtained  suggestiTo date, ARQ 531 demonstrated a manageable safety profile with no reported treatment emergent adverse events in the dose escalation study . This isIn summary, we present the first preclinical assessment of the multi-kinase inhibitor ARQ 531 in AML. We report in vitro anti-leukemic activity independent of FLT3 mutation status and modest in vivo activity that might be more evident in larger animal models based upon observed interspecies pharmacokinetic differences. Further optimization studies are needed to identify an in vivo dose and schedule capable of achieving a sustained, favorable PK profile in mice for its evaluation in AML. Collectively, our findings suggest that ARQ 531 could have a role in AML therapy especially with the high plasma concentration reached in patients, but likely will be most impactful if administered in rational combinations with drugs that effectively synergize with ARQ 531.Additional file 1: Table S4. ARQ 531 and Gilteritinib efficacy in patient samples.Additional file 2: Table S1. Estimated absolute IC50 based on a 4-parameter logistic model in AML cell lines.Additional file 3: Table S2. Estimated absolute IC50 based on a 4-parameter logistic model in MOLM-13 resistant and Ba/F3 FLT3-ITD cell lines.Additional file 4: Table S3. Patient samples mutation information.Additional file 5: Table S5. Comparison of colony formation in patient samples.Additional file 6: Figure S2. Immunoblot analysis for ARQ 531 comparison with ibrutinib irreversible BTK inhibitor and entospletinib SYK inhibitor.Additional file 7: Table S6. Estimated IC50 calculations in SYK overexpressing cell lines.Additional file 8: Figure S1. Bioluminescence imaging for ARQ 531 in vivo monotherapy.Additional file 9: Table S7. ARQ 531 in vivo activity in MOLM-13 disseminated AML xenograft model.Additional file 10: Figure S3. Bioluminescence imaging for ARQ 531 in vivo combination therapy.Additional file 11. Supplementary Information 1.