In this work, the dynamics of the intracellular Ca2+ signal were optically mapped in a Langendorff guinea pig heart. We found that DOXO treatment: (1) Delayed the activation of the Ca2+ signal. With the reference time set at the peak of the action potential (AP), the time lag between the peak of the Ca2+ signal and AP (Ca-AP-Lag) was significantly prolonged. (2) Slowed down the intracellular Ca2+ releasing and sequestering process. Both the maximum rising (MRV) and falling (MFV) velocity of the Ca2+ signal were decreased. (3) Shortened the duration of the Ca2+ signal in one cycle of Ca2+ oscillation. The duration of the Ca2+ signal at 50% amplitude (CaD50) was significantly shortened. These results suggested a reduced level of intracellular Ca2+ after DOXO treatment. Furthermore, we found that the effect of tachypacing was similar to that of DOXO, and, interestingly, DOXO exerted contradictory effects on the tachypaced hearts: it shortened the Ca-AP-Lag, accelerated the MRV and MFV, and prolonged the CaD50. We, therefore, concluded that DOXO had a different effect on intracellular Ca2+. It caused Ca2+ underload in hearts with sinus rhythm; this might relate to the contractile dysfunction in DOXO cardiomyopathy. It led to Ca2+ overload in the tachypaced hearts, which might contribute to the Ca2+-overload-related toxicity.The severe doxorubicin (DOXO) side effect of cardiomyopathy limits it clinical application as an effective anticancer drug. Although Ca Its toxicity on the heart has been drawing research attention for decades ,2,3. Dil53-mediated apoptosis [2+ homeostasis [2+ concentration is synergically regulated by several consecutive events: the entry of the extracellular Ca2+ via the voltage-gated Ca2+ channel (VGCC), which is activated following the generation and propagation of the action potential (AP); interaction of the Ca2+ ion with several intracellular molecules, eventually causing intracellular Ca2+ release via the ryanodine receptor Ca2+-releasing channel (RYR), which is located on the sarcoplasmic reticulum (SR); and the SR Ca2+-ATPase (SERCA) and Na+-Ca2+ exchanger (NCX) which are the major proteins that handle Ca2+ sequestering [2+ regulatory proteins have been confirmed in rodents with DOXO injection [2+ level in cardiomyopathy caused by DOXO are discordant. Most of the current evidence tends to relate Ca2+ overload with the cardiomyopathy caused by DOXO [2+ level after DOXO incubation in isolated rat ventricular cardiomyocytes [2+ level with slowed rising velocity of the Ca2+ signal after 15 weeks of DOXO injection. This is concomitant with their observation of the reduced ejection fraction [DOXO is believed to cause cardiomyopathy via several ways, including the downregulation of genes for the contractile proteins, oxidative stress, and Ppoptosis . Concerneostasis . It is westering . Changesnjection ,9. The d by DOXO ,11,12,13myocytes , and LIa2+ handling is one of the proposed mechanisms underlying the pacing-induced cardiac dysfunction. A downregulation of sarcoplasmic reticulum Ca2+ transport ATPase and myofibrillar Ca2+ ATPase has been documented in tachycardia-induced cardiomyopathy [2+ handling is a potentially unifying mechanism, controversy exists with regard to systolic dysfunction. Some groups suggest a decreased Ca2+ sensitivity [Tachycardias can lead to ventricular contractile dysfunction, and rapid pacing is widely used to produce animal models of cardiomyopathy  and atrimyopathy . Althougsitivity , while ositivity . 2+ on an ex vivo Langendorff guinea pig heart, and the effects of DOXO and tachypacing were evaluated. We found that DOXO decoupled the temporal connection between the signals of AP and Ca2+. DOXO also decreased the rising and falling velocity of the Ca2+ signal, suggesting detrimental effects on machineries corresponding to both Ca2+ releasing and sequestering. Similar to the effect of DOXO, tachypacing also decoupled the AP-Ca2+ connection and slowed down the rising and falling velocity of the Ca2+ signal. In the rapid-paced group, however, the effects of DOXO were in contrast to that of the sinus group: 1 µM and 3 µM DOXO promoted the coupling between AP and Ca2+, and accelerated both the rising and falling velocity of the Ca2+ signal. The decreasing effect of DOXO on the intracellular Ca2+ signal might reveal, at least partially, the contractile dysfunction which occurs in DOXO-related cardiomyopathy, and the different effects of DOXO on the sinus and tachypaced heart suggested that DOXO might exert different effects on the heart with physiological and pathological status.In this study, we simultaneously optically mapped the dynamic signals of membrane potential and intracellular CaThe guinea pig hearts were harvested from male adult animals (300~400 g) which were housed in the animal center of Tongji Medical College, Huazhong University of Science and Technique, and fed ad libitum . All experimental procedures with guinea pigs were approved by the Animal Welfare Committee of Huazhong University of Science and Technology, and experiments in this study were carried out in accordance with the rules of this committee.2 1.8, MgCl2 1, NaH2PO4 1.2, NaHCO3 25, Glucose 10. pH = 7.4) for 15 min. The flow rate was set at 30 mL/min. Blebbistatin (6 mg/L), an excitation–contraction uncoupling agent, was loaded to arrest the heart, which ensures mechanical stabilization for consequential optical sampling. Heparin (3125 IU/Kg) was administrated  15 min before inhaling of Isoflurane. After being overdosed, the animal was scarified and their heart exposed. The heart was Langendorff-perfused with 37 °C KH solution  was used as a dye solubilizer to facilitate their loading. These agents were circulating the heart for 15 min before sampling. The dyes Rhod-2AM (1 mg/L) and RH273 (1 mg/L) were used for labelling Ca2+ and voltage signals were filtered at 590 nm and 700 nm before being sampled with two separate CMOS cameras . The sampling frequency was 0.8 kHz. The software package for recording was OMapScope4.0 . The Ca2+ and voltage signals were recorded simultaneously, so that their temporal properties could be evaluated. A 530 ± 25 nm laser light source, LEDC-2001 , was applied for signal excitation. The emission light was detached via a dichroic mirror at 630 nm, and the CaThe sampling framing was 150 × 150 pixels. Each sampling episode lasted for one or two seconds, followed by a 5 min interval. The heart was subjected to two different excitation patterns: uninterfered sinus excitation and paced excitation. To pace the heart, a pair of stimulatory electrodes was placed on the apex, and a 5 Hz 2 mA current was applied. As illustrated in 2+ intensity signal was consecutively mapped onto 1600 frames; for clarity, only three of them  are displayed. The fluorescence intensity of each pixel can be extracted and plotted along the time axis (the frames). Thus, for each of the 150 × 150 segmented regions, the periodic oscillation of both Ca2+ and membrane potential can be monitored. As an example, 2+ signal of the mapping region. The peaks were detected; each of them marked one rhythmic activity. From each of these activities, the following information was retrieved: the maximum rising velocity (MRV), maximum falling velocity (MFV), and the 50% signal duration (CaD50) (the duration with intensity ⩾50% amplitude). Because voltage and Ca2+ signals were recorded simultaneously, the time difference of peak occurrence of Ca2+ and membrane potential (Ca-AP-Lag) could be calculated, which denotes the property of action potential–Ca2+ coupling. Data were retrieved with custom-written Python code (Python 3.9) and analyzed with Matlab 2017a. As exampled in Blebbistatin and Rhod-2 AM were from Abcam  and Rh-237 was from Univ . All other agents were from Sigma . p < 0.05 was regarded as significant. Figures were plotted with Prism or Matlab. The data were analyzed with the built-in statistical tool in GraphPad Prism 9.3 , and presented as mean ± SEM. An unpaired t-test, if not stated, was applied to compare the differences between groups, and the difference with 2+ level is a subsequent event following the generation of AP. Several events are involved in the AP-Ca2+ coupling: the entry of the extracellular Ca2+ via the voltage-gated Ca2+ channel and binding of the Ca2+ with Ryanodine receptors which induced Ca2+ release from the SR. The time lag between the Ca2+ and AP  reflects the dynamics of the underlying AP-Ca2+ coupling events. It is well known that the rise in the intracellular Cap < 0.01, control vs. DOXO treatment). We found that in hearts with sinus rhythm, DOXO prolonged the Ca-AP-Lag in a concentration-dependent manner. As shown in p < 0.01, sinus control vs. paced control). In other words, fast pacing hampered the proper coupling between AP and Ca2+ signaling.Compared to sinus rate excitation, pacing significantly increased the Ca-AP-Lag A. Althoup value did not reach the significant level, the low concentration of DOXO (1 uM and 3 µM) showed a trend of concentration-dependent decreasing effect on the Ca-AP-Lag , opposite to its increasing effect on CA-AP-LAP in the sinus group. The higher concentration of DOXO (10 µM) in the tachypaced group, however, consistent with its effect in the sinus group, increased the Ca-AP-Lag (p < 0.01). In other words, a low concentration of DOXO tends to alleviate the pacing damaged Ca-AP coupling, while a high concentration of DOXO exacerbates the damage.In the paced group, however, DOXO showed different effects compared with those in the sinus group. Although the 2+ (MRV_Ca) signal reflects the process of Ca2+ release, and the maximum falling velocity of the Ca2+ signal (MFV-Ca) denotes the Ca2+-sequestering process. In the sinus group, DOXO treatment damaged both processes. For instance, 10 µM DOXO significantly reduced the MRV-Ca from 3520 ± 24 to 3072 ± 177 ΔF/ms (p < 0.01), and the MFV_Ca from 3460 ± 25 to 3094 ± 255 ΔF/ms (p < 0.05).The maximum rising velocity of the Ca2+. It reduced MRV-Ca from 3520 ± 24 (sinus rhythm) to 2225 ± 330 ΔF/ms (p < 0.01), and MFV_Ca from 3460 ± 25 (sinus rhythm) to 1541 ± 382 ΔF/ms (p < 0.01), respectively (Tachypacing (300 bpm) is detrimental to both the releasing and the sequestering process of Caectively B,C. 2+ overload. The higher concentration (10 µM), however, fostered the slowing effect of pacing and might reduce the risk of Ca2+ overload. DOXO treatment had contrary effects on the pacing-interfered MRV_Ca and MFV_Ca. In the pacing group, 1 µM and 3 µM DOXO increased the MRV_Ca and MFV_Ca in a concentration-dependent manner, an action opposite to that observed in the sinus group. The higher concentration of DOXO (10 µM), however, pulled both the MRV_Ca and MFV_Ca down B,C. Thus2+ signal  was significantly reduced by DOXO (3 and 10 µM) treatment. For example, the CaD50 before and after 10 µM DOXO was 126.5 ± 1.4 and 112.8 ± 0.9 ms (p < 0.01). In comparison with the sinus group, pacing (300 bpm) significantly shortened the CaD50 from 126.5 ± 1.4 to 97.3 ± 1.5 ms (p < 0.01). Within the paced group, however, DOXO 3 µM increased the CaD50 from 97.3 ± 1.5 to 118.5 ± 0.4 (p < 0.01), and DOXO 10 µM pulled the CaD50 down to 99.9 ± 0.5 ms. In the sinus group, DOXO treatment increased the duration of the Ca+ signal D. The du2+ signals were retrieved for each of the mapped pixels (150 × 150); as a result, the heterogeneity of these parameters and the effects of tachypacing and DOXO treatment can be evaluated along the dimension of the spatial frame. The standard deviation (STD) was employed as a describer of the heterogeneity. As an example, As explained in the method , the parp < 0.01, in both cases). In the tachypaced group, DOXO 10 µM also increased the heterogeneity of CaD50. As shown in DOXO and pacing did not affect the heterogeneity property of MRV and MRF (data not shown).2+ properties. All the correlations in the results displayed in p < 0.05), and each were successfully fitted with a simple linear regression method: Y = A × X ± B, where X and Y represent the x and y axis data, and A and B are the slope factor and the Y-intercept, respectively. In order to unveil the possible causes and/or effects of the change in Ca-AP-lag, a series of correlation analyses (Pearson) were performed between different CaThe MRV_Ca was positively correlated with MFV_Ca2+ signal were positively correlated with each other in both the sinus  suggested that, under the paced circumstance, a unit decrease in MRV corresponded to a more prominent decrease in MFV. This effect might contribute to, at least partially, the Ca2+ overload observed in the fast-paced heart model. The correlation analysis revealed that the MRV and MFV of the Cahe sinus A and thehe sinus B groups.2.2+ signalThe Ca-AP-Lag was negatively correlated with the peak amplitude of Ca2+ in both groups of sinus rhythm  to −259 (the tachypaced group). Therefore, similar change in the Ca-AP-LAG corresponds to a more prominent change in the peak Ca3.2+ signalThe Ca-AP-Lag was positively correlated with duration of the Ca2+ signal also displayed an increased Ca2+ activation window. This might contribute to the reported DOXO-related Ca2+ overloading which underlines the toxic effect of DOXO treatment. The Ca-AP-Lag was positively correlated with CaD50 in both the sinus E and pac4.2+ MRV and MFVThe Ca-AP-Lag was negatively correlated with the Ca2+ MRV and MFV in both sinus and fast-paced groups  and falling  of the intracellular Ca+ signal A. Togethmyopathy . This wamyopathy  and LIacmyopathy  whose wo of DOXO ,21 mightmyopathy ,12,22,232+ signal was directly measured with optical procedure at the whole-heart level. Many previous works were performed on the isolated cardiomyocytes [2+ transient and increased the sarcomere contraction [2+ signal was determined at the tissue level, and the static total Ca2+ amount was measured due to a technique limitation at the time of the experiment [2+ level might not precisely reflect the Ca2+ level in the cytosol rather than the total amount in all compartments of the cell, and the essential dynamic characteristics of the cytosol Ca2+ were overlooked. Additionally, for some research, the conclusion of an increased intracellular Ca2+ might be based on assumptions from some ‘indirect’ observations; for instance, in isolated rat cardiomyocytes, whole-cell patch clamp recording revealed that DOXO increased the amplitude of the L-type Ca2+ current [Several possible factors might contribute to this discrepancy between the current research and the available data. The first consideration is the recording method. In the current experiment, the Camyocytes ,23; theitraction . More imperiment ,12. As a current ,9. 2+-releasing channel incorporated in an artificial lipid bilayer. The initial exposure of DOXO increased the opening probability of the channel; after a mean of 8 min DOXO, however, it irreversibly inhibited the channel. Because the DOXO-related cardiomyopathy occurred after chronic treatment, the general effects of DOXO would more likely inhibit the SR Ca2+ channel. This was consistent with our observations. Interestingly, the pretreatment of dithiothreitol (DTT) prevented the inhibiting effect of DOXO on the SR Ca2+ channel [2+-releasing channel, and, thus, reduce the intracellular Ca2+; calcium overloading might be an indirect effect of DOXO. For instance, DOXO might promote the release of ROS [Importantly, the work of Ondrias, K. et al.  confirme channel .Because e of ROS  which ale of ROS .2+-overload-related DOXO cardiotoxicity, the increased heterogeneity of the Ca2+ signal might play a role. The heterogeneities of the heart electrical properties were greatly concerned, because the abnormal increase in many of them was proarrhythmic [2+ handling underlined the alternans in AP morphology [Although the current work did not support the mechanism of Carhythmic ,27,28,29rphology . Thereforphology ,32. Simirphology , with a 2+ overload in DOXO-related cardiomyopathy cannot be ruled out based on the current research. Firstly, the slowing effect of DOXO in the falling velocity of the Ca2+ (MFV_Ca)  C might cl of PLB ,9,34. Sel status ,35. For 2+ level, further investigations, however, need to be carried out to clarify the underlying mechanisms. As a series of cascade molecules are involved in the fine-tuning of the intracellular Ca2+, it was valuable to test the effects of these molecules on the DOXO-meddled hearts. For instances, DOXO is known to affect Ca2+ homeostasis by modulation of SERCA and NCX, and the NCX inhibitor ameliorates DOXO toxicity in cardiomyocytes [2+ handling proteins, such as RYRs and SERCA [2+ level. Limitations and future works: Although the current work stated a general reducing effect of DOXO on the intracellular Camyocytes . Thus, ind SERCA ,9, it wa2+ level, providing experimental evidence for elucidating the long-existing controversy in terms of the contractile dysfunction in DOXO-caused cardiomyopathy. Furthermore, DOXO in the tachypaced hearts had the opposite effect to that in hearts with sinus rhythm. This suggested the importance of Ca2+-overload-related cardiotoxicity in a heart undergoing pathological states. Although more affirmative research still needs to be performed, the current research might shed some light on the clinical strategies for treating and/or preventing DOXO-induced cardiomyopathy.The results of this study demonstrate the effect of DOXO in reducing the intracellular Ca2+ underload; this might contribute to the contractile dysfunction in DOXO-related cardiomyopathy. In the tachypaced hearts, however, DOXO might increase the intracellular Ca2+ level and cause Ca2+-overload-related toxicity.In heart with sinus rhythm, DOXO led to Ca


In acute myeloid leukemia (AML), a small cell population that contains stem cell features such as lack of differentiation, self-renewal potential, and drug resistance, can be identified. These so-called leukemic stem cells (LSCs) are thought to be responsible for relapse initiation after initial treatment leading to successful eradication of the bulk AML cell population. Since many studies have aimed to characterize and eliminate LSCs to prevent relapse and increase survival rates of patients, LSCs are one of the best characterized cancer stem cells. The specific elimination of LSCs, while sparing the healthy normal hematopoietic stem cells (HSCs), is one of the major challenges in the treatment of leukemia. This review focuses on several surface markers and intracellular transcription factors that can distinguish AML LSCs from HSCs and, therefore, specifically eliminate these stem cell-like leukemic cells. Moreover, previous and ongoing clinical trials of acute leukemia patients treated with therapies targeting these markers are discussed. In contrast to knowledge on LSCs in AML, insight into LSCs in acute lymphoid leukemia (ALL) is limited. This review therefore also addresses the latest insight into LSCs in ALL. Acute leukemia can be subdivided into two main classes: acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL). AML is a heterogeneous malignancy characterized by the proliferation and accumulation of myeloid progenitor cells in the bone marrow (BM) and peripheral blood,2, while in ALL lymphoid progenitor cells accumulate in the BM and peripheral blood. Based on the immunophenotype, ALL can be subdivided into different types; B-cell acute lymphoid leukemia (B-ALL) and T-cell acute lymphoid leukemia (T-ALL), with B-ALL being the most common type. The median age of onset differs between AML and ALL. While ALL is most commonly diagnosed in children, AML occurs most frequently in patients older than 65 years of age,5. Despite standard treatment with intensive cytotoxic induction chemotherapy and various clinical trials, five-year overall survival (OS) rates remain poor, especially in adults-8. For treated AML patients between 60 and 70 years old, the OS rate is about 25%, and for ALL patients aged 70 years and older, it is only 15%. This is much lower compared to OS rates in children: around 80% in ALL and 70% in AML, depending on the risk group classification,10. A major cause of dismal outcomes for both AML and ALL patients is the high relapse rate,8. The prevention of relapse remains one of the most complicated challenges in the treatment of acute leukemia. However, the discovery of a rare so-called leukemia stem cell (LSC) population in AML has led to more insight into the mechanisms of relapse development and thereby novel therapeutic opportunities.Acute leukemia is a rapidly progressing hematological malignancy that causes more than ten thousand deaths per year in the United States alone,12. This subpopulation of cells shares several properties with normal hematopoietic cells (HSCs). For example, AML LSCs are, similar to HSCs, self-renewing cells that remain undifferentiated themselves but are capable of giving rise to both a stem cell copy and more differentiated progeny cells through mitotic cell division. In addition, direct evidence shows that AML consists of three distinct LSC classes with heterogeneity in their self-renewal potential: short-term, long-term, and quiescent long-term LSCs. Since HSC compartments have a similar hierarchical structure of heterogeneous cell classes, it is indicated that LSCs in AML originate from normal HSCs. In concordance with these functional characteristics, it is suggested that AML LSCs share specific stem cell transcriptional programs with HSCs. Altogether, this provides strong evidence for a hierarchical organization in AML with LSCs at the apex. Only these LSCs have the ability to initiate and fuel the disease, distinguishing them from more differentiated non-tumorigenic leukemic cells and healthy cells,16. The stem cell features that distinguish LSC from healthy cells or more differentiated leukemic cells not only provide the capacity to initiate and maintain leukemia but are also thought to contribute to relapse. For example, drug-resistant properties due to changes in the expression of drug resistance genes are attributed to the stem cell phenotype,17-19. The clinical relevance of LSCs is underlined by studies that showed an increased chance of relapse and worse overall survival in AML patients with a high CD34+/CD38- LSC frequency at diagnosis and after induction therapy compared to patients with a low LSC frequency. These data suggest that eliminating LSCs during or after induction therapy will be crucial in improving the clinical outcome of AML patients.In the 1990s, a detailed investigation of AML subpopulations provided the first proof of a rare LSC population, with a CD34+/CD38- phenotype, within AML capable of establishing leukemia in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice: leukemic stem cells (LSCs)vs. HSCs. Examples of such signaling pathways include JAK/STAT, Wnt/β-catenin, Hedgehog, and Notch-24. The dysregulation of these key signaling pathways in LSCs not only contributes to their oncogenic potential and cancer progression, but some of them also contribute to drug resistance, illustrating the importance of therapeutically targeting these pathways.Besides functional similarities, LSCs and HSCs also differ in many characteristics, such as cell surface protein expression or activation of intracellular signaling pathways, which may be exploited to specifically eliminate LSCs while sparing HSCs. Some of the key signaling pathways that play a role in the regulation of self-renewal, survival, proliferation, and differentiation are dysregulated in LSCs ,26. In addition, the overexpression of drug efflux transporters such as ATP-binding cassette transporters is also suggested to be an important intrinsic resistance mechanism (comprehensively reviewed in), but treatment with specific inhibitors remains controversial for acute leukemia. Recently, there has been a revival of the research into the metabolic rewiring of resistant cancer cells. For LSCs specifically, the pathway of mitochondrial oxidative phosphorylation seems to be very distinct, as comprehensively reviewed by de Beauchamp et al.. Extrinsic factors, including proteins involved in the cell-to-cell interactions between LSCs and the tumor microenvironment, could sensitize LSCs for eradication,30. In addition to these intrinsic and extrinsic mechanisms, identification of aberrantly expressed surface markers on LSCs in AML is also a technique of interest in order to specifically eradicate these cells. Due to surface markers uniquely expressed on AML LSCs and not on HSCs, specific drug delivery to the rare population of LSCs is feasible without harming normal stem cells,32.Other factors suggested to be involved specifically in drug resistance of LSC and therefore promising targets are intracellular enzymes such as aldehyde dehydrogenase (ALDH) and histone deacetylase (HDAC),34. This model states that all tumor cells are biologically equal and have the same tumor initiating potential. Their intrinsic characteristics cannot predict their behavior, so enrichment of these cells by sorting them based on these characteristics is impossible according to this model. Therefore, besides summarizing the most important AML LSC specific markers and recent clinical trials targeting these markers, evidence and important markers of a stem cell population in ALL are also discussed.While knowledge on LSCs in AML has rapidly increased over the past few years, less is known about the nature of LSCs in ALL. There are even contradictory results on the existence of an LSC population in ALL. Several studies support a similar hierarchical organization in ALL as AML, while other studies provide evidence suggesting that the stochastic model may fit better,35. These markers can be used to isolate LSCs for further characterization, as well as monitored during therapy. In addition, these markers are investigated for specific targeting by therapeutics, such as antibody-drug conjugates (ADCs) and chimeric antigen receptor (CAR)-T cells directed to these LSC-specific markers. The most important LSC specific cell surface markers are detailed below.Similar to what is seen for AML LSCs, HSCs also have a CD34+/CD38- phenotype. The challenge in eliminating LSCs while sparing HSCs is therefore to find unique markers on LSCs that distinguish them from HSCs. Several studies have shown markers that are aberrantly expressed on LSC but not or very lowly expressed on normal HSC. Despite the lack of CD123 expression on HSCs in healthy subjects, several other studies did find CD123 expression on HSCs and more differentiated hematopoietic cells such as monocytes,38. For instance, NOD/SCID mice injected with patient cells from cord blood and BM revealed that not only human AML LSCs expressed CD123, but also a small proportion of normal human HSCs were positive for CD123. However, since the percentage of CD123+ HSCs in the bone marrow was relatively low and only a relatively small part of HSCs expressed CD123, most HSCs should not be harmed by CD123-targeted therapy.One of the surface markers that has been shown to be overexpressed in primary AML blasts and LSCs is CD123 [interleukin-3 receptor alpha (IL-3Rα)]. CD123 expression was not detectable on normal CD34+/CD38- hematopoietic cells, HSCs, discriminating LSCs from HSCs. Moreover, high CD123 expression was associated with more cell cycle activity in the leukemic blasts, apoptotic resistance, and elevated signal transducer and activator of transcription 5 (STAT5) activation by IL-3. The biological basis explaining this poor survival, when blasts have high CD123 expression, is enhanced signaling via the IL-3R in the CD123 overexpressing AML LSCs, resulting in increased proliferation and cell viability and decreased CXCR4 expression. CXCR4 is a receptor expressed by HSCs/LSCs that interacts with stromal-derived factor 1 (SDF1), a chemokine constitutively expressed by BM stromal cells. This interaction plays a major role in the homing and preserving of the stem cells in the BM niche. Downregulation of CXCR4 in vitro impaired the migration of LSCs to SDF1, suggesting that high CD123 expression and downregulated CXCR4 in LSCs releases them from the chemoprotective BM niche into the circulation. A promising strategy to eliminate CD123-expressing AML LSCs could therefore be to combine CXCR4 antagonists with CD123 antibodies, since the antagonists would release more cells from the BM, leading to more effective targeting of LSCs by CD123 antibodies.Elevated levels of CD123 in AML are correlated to an increased number of leukemic blasts at diagnosis, a decreased chance to achieve complete remission and poor survival rates. Since CD33 shows similar homogeneous expression in relapsed AML samples as CD123, it was suggested that both these surface markers are promising drug targets. Gemtuzumab ozogamicin (GO), a humanized anti-CD33 monoclonal antibody attached to a cytotoxic agent, has been used in the clinic and has shown clinical efficacy in the treatment of AML. However, since CD33, similar to CD123, shows expression on HSCs, healthy BM myeloid progenitor cells, and more differentiated myeloid cells, the risk of unwanted on-target off-tumor toxicity increases after targeting these molecules. This is especially seen in ADCs targeting CD33, since the expression of CD123 is more restricted in healthy BM cells compared to CD33 expression. In addition, the clinically effective GO revealed severe toxicities such as liver and hematological toxicities,42. Therefore, targeting of more specific surface markers is required to reduce such toxicities.Another surface marker identified on LSCs is CD33, also known as sialic acid-binding Ig-like lectin 3. TIM-3 is expressed on multiple immune cells, such as regulatory T cells, natural killer cells, dendritic cells, monocytes, and macrophages-46. Transcriptional profiling of LSCs and HSCs derived from human AML samples showed that TIM-3 is highly expressed on most LSCs, with the exception of acute promyelocytic leukemia LSCs, but not expressed on normal HSCs. In addition, only TIM-3-positive AML cells, and not TIM-3 negative ones, were able to regenerate AML in immune-deficient mice. A similar differential expression of TIM-3 between LSCs and HSCs was observed in a study performing flow cytometry on primary human AML samples. This differential expression allows for prospective separation of LSCs from normal HSCs, and it is also promising for the successful elimination of LSCs in AML. Moreover, the number of TIM-3 expressing LSCs after allogeneic stem cell transplantation seems to be predictive for relapse.T cell immunoglobulin mucin-3 (TIM-3), a transmembrane protein initially found on differentiated CD4+ Th1 and CD8+ Tc1 cells, is a membrane marker expressed on AML cellsin vivo experiments in human AML xenograft mice using the anti-human TIM-3 mouse antibody named ATIK2a. This antibody induces antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity, which resulted in the effective eradication of LSCs without harming HSCs. The outcome of this was a strong decrease in leukemic burden in treated mice. Altogether, these studies provide evidence that TIM-3 is a promising target in the elimination of LSCs and suggest that targeting of TIM-3 results in fewer side effects compared to the targeting of, e.g., CD33.That LSCs can effectively be eliminated by targeting TIM-3 is supported by . CLL-1 is expressed in 92% of primary AML samples and expressed on LSCs in the majority of AML patients, but it is absent on HSCs from healthy and regenerating BM from patients who received chemotherapeutic treatment,52. It was recently published that CLL-1 expression can also be bimodal in AML samples, which warrants further investigation into effective elimination of LSCs including those negative for CLL-1. Despite this, CLL-1 is still a promising target in the treatment of AML. Zheng et al. studied the efficacy and safety of an anti-CLL-1-ADC in which a CLL-1 antibody is conjugated via a self-immolative disulfide linker to a pyrrolobenzodiazepine (PBD) dimer. Xenograft mice and cynomolgus monkeys were treated with this anti-CLL-1 ADC and showed an effective decrease in AML cells. Despite the fact that CLL-1 is expressed on healthy myeloid progenitors, its expression pattern is more restricted on healthy cells than that of CD33, resulting in less toxicity and faster recovery from side effects such as cytopenia. Anti-leukemic effects were also observed in xenografted mice engrafted with human AML and treated with CAR-T cells targeting CLL-1. The results show both in vitro and in vivo compelling anti-leukemic effects.C-type lectin-like molecule-1 (CLL-1), a transmembrane glycoprotein, was first identified on AML cells in 2004. The interaction between CD47 and SIRPα, a protein expressed on phagocytic cells such as dendritic cells and macrophages, leads to inhibition of phagocytosis. The CD47 surface marker is, compared to the other mentioned surface markers, not as stem cell-specific and therefore not used to identify and monitor stem cells. However, since it was found that CD47 has elevated expression on AML LSCs compared to normal HSCs, blocking monoclonal antibodies have been used in multiple studies as a strategy to eliminate LSCs,57. The results show that these blocking anti-CD47 antibodies enable phagocytosis, resulting in the eradication of LSCs without affecting normal cells-57.CD47 is a surface marker widely expressed on both hematopoietic cells and other cell typeset al. showed that this is valid for the CD33/TIM-3 and CLL-1/TIM-3 combinations.Targeting LSCs via one protein is a big challenge, since even a low expression of a cell surface marker on HSCs leads to unwanted toxicities; thus, more specific targeting of LSCs is required to prevent this. One option to increase efficacy and decrease toxicity is by targeting surface marker combinations that are highly co-expressed on AML cells but not co-expressed on healthy cells. Haubner Different therapies have been developed to target these surface markers specifically expressed on LSCs . Besidesin vivo,88. Since CD9 is hardly expressed on normal HSCs, it was suggested that this surface marker could be a promising new target for the eradication of LSCs. Second, AML cells positive for c-MPL, a thrombopoietin receptor regulating processes such as self-renewal and HSC proliferation, showed more chemoresistance than the c-MPL negative AML cell population in a mouse leukemic model. Moreover, the c-MPL+ cells had a higher self-renewal potential and were significantly better at initiating AML in vivo compared to the c-MPL- cell population. Although these results suggest that c-MPL could be a potential target to eradicate AML LSCs, more research is needed regarding unwanted toxicities since c-MPL is also a long-term HSCs marker.The surface markers in . Several of these factors are involved in drug resistance mechanisms, making them important therapeutic targets for elimination of AML LSCs.Besides the previously described surface markers, there are intracellular proteins such as transcription factors that are differentially expressed between LSCs and HSCs. The JAK family of intracellular non-receptor tyrosine kinases can be activated via extracellular cytokine or growth factor binding, resulting in the phosphorylation and activation of STAT proteins. This STAT protein family consists of transcription factors that interfere with proliferation, differentiation, and apoptosis. Previous research has shown that STAT3 and STAT5 are constitutively activated in AML leukemic blasts, which is not seen in HSCs. This may contribute to the uncontrolled proliferation of these blasts and resistance to chemotherapy-induced apoptosis. The role of JAK/STAT signaling in AML LSCs growth and survival was investigated several years later by evaluating the expression levels of JAK and STAT in AML patient samples at diagnosis and relapse. An ATP competitive inhibitor of JAK1/2 kinases named AZD1480 was used in both in vitro and in vivo experiments to analyze its effect on AML stem/progenitor cells. Inhibitor treatment of AML CD34+ cells in vitro showed decreased levels of JAK2 and STAT3/5 activity and reduced AML CD34+ cell proliferation and survival, but it did not affect normal CD34+ cells. Similar results were seen in NOD/SCID mice treated with AZD1480: the number of AML LSCs was reduced, but normal human HSC numbers were not affected. To further investigate the role of JAK1, JAK2, STAT3, and STAT5 in CD34+ AML cells, an RNA interference-mediated knockdown of these proteins was performed. JAK2, STAT3, and STAT5 knockdown resulted in a significant decrease of colony-forming cells, cell numbers, and survival, while this was not observed with downregulation of JAK1, indicating that inhibition of JAK2 is more effective in decreasing growth and survival of AML CD34+ cells than inhibition of JAK1.High CD123 expression is associated with elevated STAT5 activation by IL-3, suggesting that the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway plays a role in the fate of LSCs [39]. The. NF-κB is suggested to be involved in drug resistance, as NF-κB has anti-apoptotic activity and increased levels of NF-κB have been seen after chemotherapy and radiotherapy,97. Electrophoretic mobility shift assays on primary AML samples showed constitutively activated NF-κB in AML LSCs. In contrast, in normal human stem/progenitor cells, there was no NF-κB expression. Multiple studies have investigated the effect of NF-κB inhibitors on AML (stem) cells. For example, AML LSCs treated with MG-123, an NF-κB inhibitor, initiated cell death in vitro. Micheliolide (MCL), a natural sesquiterpene lactone, had cytotoxic effects on LSCs via the inhibition of NF-κB. In vitro MCL treatment initiated apoptosis in AML LSCs but not in normal HSCs. In vivo, treatment with DMAMCL, the pro-drug form of MCL, improved survival rates of NOD/SCID mice engrafted with human AML. A third NF-κB inhibitor, BMS-345541, conferred an altered expression of genes involved in a previously described 17-gene LSC score to primary AML patient samples,95. This gene signature contains 17 stemness-related genes differentially expressed between LSC-positive and LSC-negative AML samples. The NF-κB pathway is illustrated in .Nuclear factor-kappa B (NF-κB) is a proinflammatory transcription factor that plays an essential role in cellular processes such as proliferation, survival, stress responses, and inflammation. The Wnt/β-catenin pathway plays a role in the cellular processes of HSCs, including cell proliferation, differentiation, survival, and stem cell renewal. The transcriptional coactivator β-catenin plays a central role in this pathway; when the Wnt ligand binds to its cognate receptor Frizzled, β-catenin degradation is blocked, causing an accumulation of β-catenin and subsequent translation to the nucleus. There, β-catenin binds to nuclear transcription factors that belong to the T-cell factor/lymphoid enhancer factor (TCF/LEF) family, which recruits coactivators including the CREB-binding protein (CBP), resulting in the transcription of target genes involved in self-renewal and proliferation. A study evaluating the activity of Wnt/β-catenin signaling by transfecting AML and normal progenitors with a TCF/LEF reporter construct, found that in the majority of the AML samples, the TCF/LEF pathway was constitutively active, which was supported by other studies showing overexpression of β-catenin both in AML cell lines and patient samples,103. Moreover, the promoter regions of Wnt pathway inhibitor genes were frequently methylated in cell lines, and in 89% of AML patient samples with normal cytogenetics one or more of these inhibitor genes were methylated. Methylation of two Wnt pathway inhibitor genes named sFRP2 and sFRP5 was associated with elevated relapse risk, suggesting that enhanced Wnt activity has adverse outcomes in AML patients with normal karyotypes.The Wnt/β-catenin signaling pathway is a highly conserved signaling pathway involved in the development and tissue homeostasiset al., using an AML mouse model, showed that LSCs require β-catenin in order to maintain their self-renewal capacity. Several studies have shown the overexpression of β-catenin in LSCs; however, the expression of β-catenin in normal HSCs was also observed. Cobas et al. provided evidence that β-catenin did not show to be crucial for self-renewal of adult HSCs, since depletion of β-catenin in mice showed no impairment in hematopoiesis and lymphopoiesis. Targeting β-catenin is therefore suggested to be promising in the eradication of LSCs, while sparing HSCs , either due to a lack of efficacy or slow enrollment, but not because toxicities were seen. Despite this, many recent clinical trials are still recruiting and some clinical trials targeting LSC-related surface markers or pathways have even been completed with published results.Although some clinical trials are investigating the safety and efficacy of novel therapeutics as a monotherapy, most of the compounds are tested in combination with other drugs, such as chemotherapy and hypomethylating agents. A few clinical trials have been terminated . However, the multiple resistance mechanisms contained by LSCs are a major challenge in achieving an effective eradication of these cells. Therefore, many clinical trials in AML patients test the effectiveness of targeting LSCs using several therapies, both as a monotherapy and in combination with other drugs.The substantial number of recent studies investigating an LSC population in AML has led to the general acceptance of AML being hierarchically organized. The rare population of LSCs at the apex of this hierarchy plays a key role in the relapse development of the disease due to their drug-resistant mechanisms and self-renewal capacitypulation . In addipulation . The foc. For instance, inhibiting the CXCR4/SDF1 interaction involved in HSC/LSC homing releases LSCs from their chemoprotective niche, making them more sensitive to chemotherapy. This indicates that, besides specifically targeting LSCs, targeting the pathways involved in the BM/LSC interaction could also be a useful strategy to increase LSC eradication. However, this strategy has to be executed carefully, since releasing leukemic cells from the BM into the peripheral circulation could possibly increase the risk of these blasts infiltrating other organs. Future studies on AML should therefore focus on gaining better insight into the factors involved in LSC homing and the effectiveness and safety of targeting these factors.Accumulating evidence shows that the interaction between BM niche and LSCs plays a role in the survival and acquired drug resistance of AML LSCs,178. However, it is still not fully understood if T-ALL follows the same stem cell model as AML. The majority of B-ALL cells containing different immunophenotypes have the capacity to initiate ALL, indicating that B-ALL lacks a clear hierarchy as in AML. Hence, the term LSC as used in AML might not be correct to define the cells being able to cause relapse in ALL. For ALL, the term leukemia-initiating cells (LICs) is more preferred. Due to the dynamic phenotypes of LICs, their prospective purification has not been possible up to now. More research is needed to investigate ALL LIC plasticity and discriminating factors that make one population of cells more likely to result in relapse than the other. However, since this has shown to be highly challenging, the focus on interfering with the leukemic cells and BM interaction could be a more promising approach to eliminate ALL LICs in the near future. For all these novel LSC-targeted agents, it is difficult to assess whether specifically the LSC are really eliminated. Monitoring the LSC using flow cytometry during and after therapy might be an option,180 but is not standardly investigated yet.Compared to AML, research on the LSC population in ALL has been less successful with highly contradicting results. When comparing AML with T-ALL, pathophysiological similarities are seen within both diseases. In addition, it is possible that there are multiple populations of LSCs with different phenotypes present within one patient. Targeting only one marker may therefore not be effective, and more research focusing on simultaneously or subsequently targeting multiple markers is needed.Despite many different surfaces and other markers being promising targets in eradicating LSCs, there are some important considerations that need to be taken into account before applying anti-LSC therapeutics. First, it is extremely important that healthy cells, especially HSCs, are not targeted by the treatment. Since some surface markers and intracellular pathways involved in “stemness” are expressed in LSCs and HSCs, there is a concern that targeting LSCs can harm healthy stem cell populations. This might cause severe and dangerous side effects, so more research is needed to evaluate how many healthy cells are affected in patients treated with these therapies. Secondly, a major challenge is the heterogeneity of acute leukemia. For instance, in AML, heterogeneous phenotypes of LSCs have been identified between patients


Spermatogenesis associated serine rich 2 like (SPATS2L) was highly expressed in homoharringtonine (HHT) resistant acute myeloid leukemia (AML) cell lines. However, its role is little known in AML. The present study aimed to investigate the function of SPATS2L in AML pathogenesis and elucidate the underlying molecular mechanisms.Overall survival (OS), event-free survival (EFS), relapse-free survival (RFS) were used to evaluate the prognostic impact of SPATS2L for AML from TCGA database and ourcohort. ShRNA was used to knockdown the expression of SPATS2L. Apoptosis was assessed by flow cytometry. The changes of proteins were assessed by Western blot(WB). A xenotransplantation mice model was used to evaluate in vivo growth and survival. RNA sequencing was performed to elucidate the molecular mechanisms underlying the role of SPATS2L in AML.SPATS2L expression increased with increasing resistance indexes(RI) in HHT-resistant cell lines we had constructed. Higher SPATS2L expression was observed in intermediate/high-risk patients than in favorable patients. Meanwhile, decreased SPATS2L expression was observed in AML patients achieving complete remission (CR). Multivariate analysis showed high SPATS2L expression was an independent poor predictor of OS, EFS, RFS in AML. SPATS2L knock down (KD) suppressed cell growth, induced apoptosis, and suppressed key proteins of JAK/STAT pathway, such as JAK2, STAT3, STAT5 in AML cells. Inhibiting SPATS2L expression markedly enhanced the pro-apoptotic effects of traditional chemotherapeutics .High expression of SPATS2L is a poor prognostic factor in AML, and targeting SPATS2L may be a promising therapeutic strategy for AML patients.The online version contains supplementary material available at 10.1186/s12967-023-03968-0. Acute myeloid leukemia (AML) is the most common acute leukemia in adults and is characterized by the accumulation of immature myeloid blasts in the bone marrow or peripheral blood (PB) . DespiteSPATS2L  was one of the top 50 upregulated differentially expressed genes (DEGs) in HHT-resistant cell lines, which were established by our research group. Additionally, SPATS2L had prognostic significance for AML compared with other top 50 DEGs when analyzing online databases , Idarubicin (IDA), and HHT in AML cells. We hypothesized that SPATS2L was an independent prognostic factor in AML and might represent a novel therapeutic target for AML patients.AML cell lines THP-1, HL-60, OCI-AML3, NB4, Kasumi-1, KG-1, U937, THP-1-luc, were cultured in RPMI medium , MV4-11(MV4-11S), HHT resistance MV4-11 cell lines , MOLM13(MOLM13 S), HHT resistance MOLM13 cell lines  were cultured in IMDM medium . The medium was supplemented with 10% fetal calf serum (FCS), penicillin G (100 unit/mL), and streptomycin (100 μg/mL) at 37 °C in a 5% CO2 atmosphere. HEK-293 T cells were cultured in DMEM medium supplemented with 10% fetal bovine serum  at 37 °C with 5% CO2.A total of 228 adult de novo AML patients with detailed diagnosis and treatment information were included in this study from March 2010 to June 2016 according to standard French–American–British and World Health Organization criteria between 2003 and 2011. Patients with acute promyelocytic leukemia (APL) and those undergoing bone marrow transplantation were excluded. WHO classification, conventional cytogenetic banding assays, and molecular analyses were performed centrally at the Zhejiang Provincial Key Lab of Hematopoietic Malignancy, China, as previously described . We analMV4-11 and MOLM13 cells were treated with gradually increasing HHT concentrations (1 nmol/L–50 nmol/L). Cells that grew normally in an IMDM medium with 10% fetal bovine containing 10 nmol/L, 30nml/L, and 50 nmol/L HHT were preserved. These cells were named MV4-11 R10, MV4-11 R30, MV4-11 R50(MV4-11 R), MOLM13 R10, MOLM13 R30, and MOLM13 R50. Then, the IC50 value of those cells was calculated. The RI of those cells was calculated with the formula IC50 (Resistance)/IC50 (Sensitive).HHT (#61847) was purchased from MCE . Ara-c (# S1648), and IDA HCl (# S1228) were purchased from Selleck .™ IV 1st strand cDNA Synthesis Mix . Quantitative PCR was performed in triplicate using a SYBR Green PCR Master Mix kit  on an IQ5 real-time PCR instrument . The mRNA levels were normalized to GAPDH. The primers used were as follows.Total RNA was extracted using TRIzol reagent . First-strand complementary DNA synthesis was performed using the PrimeScriptSPATS2L forward:5’-AAGCAGCATCAAGGCAACAAA-3′,reverse: 5′-TTCTCGCAGCCATTCATGGG-3′,GAPDH forward: 5′-GGAGCGAGATCCCTCCAAAAT-3′;reverse 5′-GGCTGTTGTCATACTTCTCATGG-3′.6 cells were harvested and fixed in 4% paraformaldehyde for 15 min. Cells were then incubated with 0.3% Triton X-100 for 15 min and incubated with SPATS2L antibody at 4 ℃ overnight. Then cells were washed twice and incubated with a fluorescent secondary antibody for 1 h. After incubation with DAPI for 15 min, cells were transferred to a confocal 96-well plate and visualized using Nikon A1 Ti.To explore the localization of SPATS2L in AML cells, 1 × 105 for growth or 1 × 105 for drug sensitivity in each well. 20 μl MTS solutions  were added to each well, and the cells were incubated for 4 h at 37 °C. The plates were read at a wavelength of 490 nm using Varioskan Flash .Cells were seeded in 96-well plates at 0.5 × 10Cells transfected with control or SPATS2L KD lentiviral fluid were collected after transfection 96 h. For apoptosis analysis, cells were stained with PI and Annexin-V-APC for 15 min in a binding buffer . For the differentiation test, CD11b FITC  and CD14 FITC  were used. Cells were stained with CD11b or CD14 antibody for 30 min in 1xPBS. All the processed cells were analyzed using a NovoCyte D2060R flow cytometer . GFP-positive cells were sorted by Beckman moflo Astrios EQ.Cells were harvested, and 1 × RIPA buffer containing a protease inhibitor and phosphatase inhibitor cocktail  was added on ice for 30 min and centrifuged at 12,000 g for 15 min at 4 ℃ to pellet cell debris. Protein concentration was determined using a BCA reagent . After heating to 100 ℃ for 10 min, the protein samples were separated by SDS-PAGE and transferred to PVDF membranes . The membrane was blocked with 5% powder milk and probed with an antibody at 1:1000. After washing with 1 × TBST buffer, the membrane was incubated with HRP-conjugated secondary antibodies at 1:5000. The target proteins were visualized using an ECL kit  and imaged using a Bio-Rad imaging system. Antibodies against GAPDH #8884, STAT3 #4904, STAT5 #94205S, Phospho-STAT5 #9359, Phospho-BCL2 #2827S, Caspase3 #9662S, PARP #9532S were purchased from CST . Antibodies against SPATS2L 16938-1-AP, JAK2 17670-1AP, BCL2 26593-1-AP were purchased from ProteinTech . The antibody against CD45 (PE conjugated) was purchased from MULTI SCIENCES .https://portal.gdc.com). R packages were implemented by R  version 4.0.3. The mRNA expression profiles of HHT paternal strains (MV4-11 S) and HHT-resistant strains with different resistance indexes (RIs), MV4-11 R10, MV4-11R30, MV4-11R50, were obtained using high throughput sequencing (RNA-Seq). Sequence data were obtained from Illumina HiSeq X Ten platforms and uploaded to NCBI's SRA data (PRJNA664675). For RNA-Seq data, mRNA expression levels were calculated as RPKM (Reads Per Kilo-base per Million reads). The DE Seq (1.18.0) R package analyzed differentially expressed mRNAs. For SPATS2L KD RNA-seq, RNA was isolated from THP-1 control and THP-1 SPATS2L KD cells. The sequence was obtained from the Illumina HiSeq X Ten platform and uploaded to NCBI's SRA data (PRJNA707119). In the present study, the screening criteria for differential expression genes (DEGs) are fold-change  > 2 and P value  < 0.05.RNA-sequencing expression (level 3) profiles and corresponding clinical information for AML were downloaded from the TCGA dataset containing and package plasmids (psPAX2 and pMG2.G) were co-transfected into HEK-293 T cells in a 10 cm cell culture dish using Calcium Phosphate Cell Transfection Kit . Lentivirus particles were harvested at 48 h and directly added to 1 × 106 GFP positive cells were injected into mice through the tail vein. Each group had six mice. Bodyweight and CD45 expression of PB were measured every week. THP-1 luc cells transfected with SPATS2L control or KD lentiviral solution were injected into mice through the tail vein to evaluate the tumor burden more intuitively. The number of the two groups was five and six, respectively. Cell engraftment was assessed by intraperitoneal luciferin injection (100 mg/kg) and then by imaging using an IVIS Lumina LT system . The leukemic burden of mice was assessed by bioluminescence imaging every seven days after injection and analyzed using GraphPad Prism 5 [two-tailed Student's t-tests]. Mice were euthanized when they developed a bowed back, or their lower limbs were paralyzed. The survival of each group of mice was analyzed using Kaplan–Meier curve analysis by IBM SPSS Statistics 20 software.6–8 weeks old immunodeficient NCG(NOD/ShiLtJGptPrkdcem26Cd52Il2rgem26Cd22/Gpt) mice were raised in the Experimental Animal Center of Zhejiang Chinese Medicine University Laboratory Animal Research Center. All animal experiments were approved by the Ethics Committee of the Experimental Animal Center of Zhejiang Chinese Medicine University Laboratory Animal Research Center . THP-1 cells were transfected with the SPATS2L control or KD lentiviral solution. After 72 h, 1 × 10The primary endpoints of this study were OS, EFS, and RFS. OS was defined as the time from diagnosis until death due to any cause or last follow-up. EFS was defined as the time to diagnosis until its removal from the study due to incomplete remission, relapse, or death. RFS was defined as the time from CR to relapse or death as previously described. The surFor experimental analysis, unpaired two-tailed Student's t-test or ANOVA and Chi-square test were used by IBM SPSS Statistics 20 software or GraphPad Prism 5. P < 0.05 was considered statistically significant. P < 0.05 *, P < 0.01 **, P < 0.001 ***.g RI. Then, to investigate the mRNA expression profile changes associated with HHT resistance, we performed RNA sequencing between MV4-11 resistant and MV4-11 (MV4-11S) cells. A Venn diagram was generated, and 29 DEGs (belonging to the top 50 DEGs) were upregulated in the three MV4-11 resistant cell lines  with increasinhttp://ualcan.path.uab.edu/) and GEPIA (http://gepia.cancer-pku.cn/). Of them, SPATS2L had the strongest prognostic significance for OS (p < 0.0001) (Table S1). Interestingly, with the increase of RI, the mRNA and protein levels of SPATS2L were also increased in MV4-11 and MOLM13 resistance cells , EFS , and especially for RFS   for AML treatment was first proposed by our group and is the first-line therapy for non-elder AML patients in China , 24. Witdeno or relapsed/refractory AML [According to data gathered via the NCBI AceView tool, the function of SPATS2L is still unknown. In the present study, the expression level of SPATS2L decreased after CR, indicating SPATS2L may be a candidate therapeutic target for AML. It was confirmed by SPATS2L knockdown using shRNA. Growth inhibition and apoptosis promotion were observed after SPATS2 KD in AML cells. We also found AML cells with SPATS2L KD could induce differentiation. These results indicated SPATS2L could affect AML cell behavior in multiple aspects. By analyzing the RNA sequence, we discovered that high levels of SPATS2L may activate the JAK/STAT pathway. After silencing SPATS2L, the essential proteins in JAK/STAT signaling pathway were also decreased, leading to the inhibition of STAT3 and STAT5 phosphorylation. High expression and constitutive activation of JAK2, STAT3, and STAT5 in AML blasts have been confirmed , 29. JAKtory AML –32. The Three chemotherapy drugs, Ara-c, IDA, and HHT, did not affect the expression of SPATS2L, indicating that these three drugs may have a poor effect on SPATS2L high expression patients. These results also explained why AML patients with high SPATS2L expression had a poor prognosis. Furthermore, inhibition of SPATS2L expression enhanced the apoptosis-promoting effect of these three traditional chemotherapeutic drugs on THP-1 cells. Compared to other AML cell lines, THP-1 shows innate HHT resistance. These findings indicated that targeting SPATS2L may be a promising therapeutic option for treating Ara-c, IDA, and HHT resistant AML patients. Drugs targeting the SPATS2L or JAK/STAT pathways may reverse the chemotherapy resistance. Due to the lack of drugs targeting SPATS2L, we could not test the synergistic effect of SPATS2L targeting inhibitors and AML chemotherapy drugs.Here, we suggest SPATS2L is a promising marker for evaluating AML and an effective candidate target for AML treatment. Although the results are promising, the current study had several limitations. First, this study only clarified the prognostic significance and mechanism of SPATS2L in AML pathogenesis. We also found SPATS2L may partly induce drug resistance, and SPATS2L knockdown could reverse HHT resistance. But the underlying mechanism requires further investigation. Second, follow-up studies are needed to identify specific drugs targeting SPATS2L and reveal whether SPATS2L mediates AML chemotherapy resistance  and the in-depth drug resistance mechanism.Additional file 1: Figure S1. Protein localization and expression of SPATS2L in AML. Figure S2. The prognostic significance of SPATS2L for AML through quartile survival analysis. Figure S3. The changes of cell cycle and differentiation were analyzed by flow cytometry after SPATS2L KD in the AML cell line. Figure S4. The Global changes of gene expression between SPATS2L KD and SCR cells, and between SPATS2L high group and low group AML patients were detected by RNA sequencing. Figure S5. The top down-regulated pathways after SPATS2L KD were analyzed by KEGG enrichment analysis. Table S1. The fold change and prognostic significance of top up genes. Table S2. The top down-reguated pathways after SPATS2L KD in AML cells


CEBPA-double mutant (CEBPAN/C) AML. These mutations disrupt the structure of a major regulator of myelopoiesis. We identify the binding sites of mutated C/EBPα proteins in primary cells, we show that C/EBPα, AP-1 factors and RUNX1 colocalize and are required for AML maintenance, and we employ single cell experiments to link important network nodes to the specific differentiation trajectory from leukemic stem to blast cells. Taken together, our study provides an important resource which predicts the specific therapeutic vulnerabilities of this AML subtype in human cells.Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy caused by mutations in genes encoding transcriptional and epigenetic regulators together with signaling genes. It is characterized by a disturbance of differentiation and abnormal proliferation of hematopoietic progenitors. We have previously shown that each AML subtype establishes its own core gene regulatory network (GRN), consisting of transcription factors binding to their target genes and imposing a specific gene expression pattern that is required for AML maintenance. In this study, we integrate gene expression, open chromatin and ChIP data with promoter-capture Hi-C data to define a refined core GRN common to all patients with  N-t. N-tCEBPEBPAN/C) , 12.Fig.CEBPA mutated AML showed that homozygous C-terminal mutations promote the expansion of pre-leukemic HSCs but impair myeloid differentiation, whereas N-terminal mutations do not impact on proliferation and preserve myeloid commitment. The combination of both mutations accelerates leukemogenesis and explains the CEBPAN/C prevalence [CEBPAN AML [N/C patients is unknown.Mouse models of evalence  whereby evalence . A recenBPAN AML  but the CEBPAN/C AML patients by linking distal cis-regulatory elements occupied by specific TF families to their rightful promoter using promoter-capture Hi-C data from other AML subtypes [CEBPAC/N is based on distinct regulatory relationships. However, the C/EBPα binding pattern in patient cells remained unknown, the preliminary GRN was based on only three patients, considered only distal regulatory elements, and was not based on CEBPAN/C-specific Hi-C data. In this study, we defined the complete core GRN of CEBPAN/C AML by integrating multi-omics data from 10 patients. We identified CEBPAN/C AML-specific cis-regulatory elements and TF modules, highlight AP-1, RUNX1 and C/EBPα as crucial factors driving AML maintenance and show that mutant C/EBPα and AP-1 factors colocalize with RUNX1 in chromatin. Our study therefore provides important insights into the biology of the disease in humans.GRNs represent the regulatory connections between TFs and their target genes . GRNs insubtypes . This woDetailed methods can be found in the Kasumi-1 and HEK 293T cells were purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen. KO52 were obtained from the Japanese Collection of Research Bioresources Cell Bank. Kasumi-1 cells were cultured in RPMI-1640 (Sigma-Aldrich) supplemented with 10% fetal calf serum (FCS) (Gibco), 5% Penicillin-Streptomycin (Pen-Strep) (Sigma-Aldrich), 5% L-Glutamine (L-Glu) (Sigma-Aldrich). HEK 293T cells were cultured in DMEM supplemented with 10% FCS (Gibco), 5% Pen-Strep, 5% L-Glu. KO52 were cultured in Alpha MEM (Lonza) supplemented with 10% FCS (Gibco), 5% Pen-Strep, 5% L-Glu.C/EBPAN/C-8 cells were cultured at the concentration of 1 × 106 cell/mL in StemSpanTM Serum-Free Expansion Medium II (SFEMII) plus 10% StemSpanTM CD34+ Expansion Supplement and 175 nM UM171 (STEMCELL Technologies). All other patient cells were grown in SFEMII supplemented with 100 ng/mL thrombopoietin, 10 ng/mL FMS-like tyrosine kinase 3 ligand, 750 nM Stem Regenin 1 (SRI), 10 ng/mL Interleukin 3, 10 ng/mL human granulocyte/macrophage colony stimulating factor, 150 ng/mL Stem Cell Factor  and UM729 (STEMCELL Technologies). Patient CD34+ or CD117+ progenitors were isolated with CD34 MicroBead Kit, human or CD117 MicroBead Kit, human (Miltenyi-Biotech) as described in [The doxycycline inducible pCW57.1-dnC/EBP plasmid was generated using Gateway Gene Cloning (Thermo Fisher Scientific). Plasmids containing the dnC/EBP and dnFOS inserts were a gift from Charles Vinson, National Cancer Institute, Bethesda, USA . The pCWTotal RNA was extracted with NucleoSpin RNA (Machery-Nagel) and was retrotranscribed using SuperScriptTM II Reverse Transcriptase kit (Thermo Fisher) and Oligo(dT) primers (Promega). Real-time PCR and RNA-Seq was performed as described in .DHS mapping was performed as described in . ATAC liDouble protein–protein crosslinking followed by protein-DNA crosslinking with 0.25 M Di(N-succinimidyl) glutarate (DSG) (Sigma-Aldrich) for 45 min on rotation at RT was performed when immunoprecipitating chromatin with the c-FOS antibody, a single-crosslinking approach (protein-DNA crosslinking) in all other cases as described in .CHi-C in primary AML cells was performed as described in .Details of the bioinformatics analyses can be found in the CEBPAN/C and t AML clustered closely together [CEBPA is mutated in CEBPAN/C AML and is repressed by RUNX1/ETO in t AML [CEBPAN/C represents a specific epigenetic subclass of AML, we repeated these analyses with additional samples to include a total of 10 CEBPAN/C patients, and studied 7 t patients. CD34+ leukemic blasts were purified for RNA-seq and ATAC-or DNase-seq experiments  AML, we compared the open chromatin profiles (DNAse-Seq and ATAC-Seq data) of t and CEBPAN/C and also compared both AML subtypes to normal CD34+ peripheral blood stem cells (PBSCs)  AMLs [CEBPAN/C-specific peaks.In t the ETO gene is fused to the gene encoding RUNX1, creating the fusion protein RUNX1/ETO . The exptogether . We orig;21) AML , 25. To SCs) Fig.. IndividSCs) Fig.. Motif eSCs) Fig.. ExampleSCs) Fig., which hSCs) Fig., a transCEBP genes in both healthy PBSCs, CEBPAN/C and t leukemic blast cells    Fig. . To idenCEBPAC/N AML cell line model KO52 [RUNX1 itself was bound by all three factors sharing several binding sites  AMLs did not differ  dataset , arrows going from a node to a specific gene indicate the presence of a binding motif in the locus encoding that gene. The analysis highlights nodes and edges which are specific for CEBPAN/C AMLs and how different TFs and their genes are “wired”. The C/EBP and AP-1 families represent major nodes, with 341 and 353 connections to putative targets, respectively RNA-seq on LSCs and blasts from one ers Fig. . We theners Fig. . The anaers Fig. . In a psers Fig.  with theers Fig. . The Blaers Fig. .CEBPAN/C AML GRN on the cell clusters -inducible dominant negative C/EBP (dnC/EBP) [CEBPAN/C patient blasts and therefore used KO52 and the t cell line Kasumi-1   and FOS dnC/EBP)  peptidesdnC/EBP) . We coulAfter Dox treatment and FACS sorting for GFP positive cells, we performed ATAC-seq and RNA-seq on transduced cells. After induction of dnC/EBP expression for 72 h in KO52 cells, ATAC analyses identified 420 lost and 702 gained open chromatin regions Fig. . Gained CEBPE which is bound by FOS, C/EBPα and RUNX1  cell line models )CEBPAN/Cls  cells.RUNX1 has been shown to be essential for the survival of t AML . We therCEBPAN/C-specific DHSs and the colocalization of C/EBPα, RUNX1 and AP-1, in contrast to the enrichment of the AP-1 canonical motif in t and healthy blasts-specific sites. We showed previously that AP-1 activity is required for leukemic maintenance in FLT3-ITD and t AMLs [CEBPAN/C. C/EBP and AP-1 TFs form heterodimers [JUN, AP-1 family members were uniformly expressed throughout all LSC and blast subpopulations. JUN was downregulated in the blast compartment, following an expression pattern inversely correlated with CEBPA. During granulocyte differentiation C/EBPα heterodimerizes with JUN, impeding the formation of JUN:FOS complexes and preventing JUN from autoregulate its own promoter. Therefore, JUN downregulation promotes granulocytic differentiation [C/EBPα is required for myeloid differentiation and for maintaining the self-renew ability and the quiescent state of adult HSCs , 42. Theif in t8; and heal21) AMLs . Here werodimers –46, and rodimers . Our datntiation .CEBPAN/C-specific core GRN Highlights the AML-specific connections between TF-coding genes that are distinct from those found in PBSCs. The analysis of the TF modules  contains C/EBP proteins as a major node, (ii) that a large number of genes are bound by the mutant protein, including C/EBPAN/C-AML-specific cis-elements, (iii) that the latter are associated with downregulated myeloid differentiation genes and (iv) that eliminating this node using dnC/EBP affects the survival of the cells.Another important result described here is the finding that despite one essential member being mutated, the C/EBP family represents a highly connected node of the AN/C AML . The bZICEBPAN/C GRN, the RUNX TF family is highly connected. Targets of RUNX1 in the AML-specific GRN include, for example, the apoptosis regulator BCL2 which is upregulated in CEBPAN/C AML blasts, and which could be inhibited by Venetoclax [CEBPAN/C AML GRN, indicating that MAPK signaling plays an essential role in regulating growth. For FLT3-ITD and t AML this idea has been confirmed in vitro and in vivo [Within the netoclax . Similarnetoclax , the AP- in vivo , 21.CEBPAN/C AML growth. Expression of dnFOS had a strong effect on the chromatin landscape of KO52 cell, mainly causing loss of open chromatin sites. Interestingly, C/EBP:AP-1 composite motifs were enriched in hypersensitive sites lost upon dnC/EBP expression in KO52, but not in Kasumi-1 cells. This result suggests that the cooperation between these TFs is a specific feature of CEBPAN/C AMLs. Our gene expression analysis showed that loss of AP-1 binding in KO52 was accompanied by downregulation of signaling pathways, suggesting that these factors promote the survival of CEBPAN/C cells. Finally, we found that the expression of dnC/EBP or dnFOS induced growth arrest in both KO52 (this study) and Kasumi-1 cells (this study and [Inhibition of the RUNX TF family and the depletion of RUNX1 shows that it is important for tudy and ).CEBPAN/C AML-specific core GRN highlights subtype-specific connections between TFs and target genes that code for “druggable” gene products. It will be an important resource for researchers developing combination therapies for this particular AML subtype.In summary, the Supplementary MaterialSupplementary Dataset 1Supplementary Dataset 2Supplementary Dataset 3Supplementary Dataset 4Supplementary Dataset 5


Consortin (CNST) is a protein located on the trans-Golgi network that can target transmembrane proteins to the plasma membrane. Although CNST was discovered more than 10 years ago, there are still not enough studies on its function. During our search for possible new acute myeloid leukemia (AML) markers, we found that CNST was overexpressed in almost all patients with AML. By analyzing profiling data from public databases, we found that CNST expression inversely correlated with overall survival among AML patients. There was a great variation in CNST expression among different subtypes of AML, and the expression was the highest in the t subtype, which was probably due to the direct regulation of CNST transcription by RUNX1-RUNX1T1. In addition, we analyzed the expression of CNST in different cells of the hematopoietic system. We found that CNST was associated with the low differentiation degrees of hematopoietic cells and had the highest expression level in leukemia stem cells (LSCs). Finally, we analyzed the CNST-related gene network and found that the genes negatively correlated with CNST are involved in various immune-related pathways, which indicates that CNST is likely related to immune evasion, LSC niche retention, and assembly of stress granules. In conclusion, our study suggests that CNST has the potential to be a diagnostic and prognostic biomarker for AML. Acute myeloid leukemia (AML) is a malignant disease that occurs in the hematopoietic system. It is characterized by abnormal proliferation and differentiation of hematopoietic stem cells (HSCs) . The proConsortin (CNST) is a receptor located on the trans-Golgi network (TGN) and cytoplasmic transport vesicles , which iCNST is thought to be involved in pathways related to inherited non-syndromic hearing impairment (DFNB1) ; in miceIn this study, we investigated the expression of CNST in the blood system and AML, and its impact on the prognosis of AML by analyzing data from public databases. Moreover, the possible function of CNST in AML was analyzed.http://www.vizome.org/additional_figures_BeatAML.html, and the Gene Expression Omnibus (GEO) repository . The CaGSE13159  transcriWe used directly the already normalized transcriptome data. For unprocessed count data, we used the CPM method in edgeR to replace the count value with the CPM value. Differentially expressed genes between different groups were found using the limma (version 3.48.3)  package,For the downstream analysis of single-cell RNA-seq, we selected cells with at least 1000 UMIS  mapped to at least 200 unique genes. We also excluded cells with more than 20% of gene counts reflecting mitochondrial genes or ribosomal RNA. We normalized gene counts to a total of 10,000 for each cell. The type definitions for different cells used in the cell annotations were provided in the data.Single-cell transcriptome sequencing data were visualized using a combination of principal component analysis (PCA) and t-distributed stochastic neighbor embedding (t-SNE), The top 50 principal components were selected for downstream analysis. Specifically, PCA was performed using prcomp, which was followed by t-SNE visualization using the Rtsne package. The maximum number of iterations was 2000, and the random seed was the seed .High variable genes for clustering: HSC/hematopoietic progenitor cells (HPC): SPINK2, ZFAS1, NRIP1, GAS5, JUN, MEIS1, HLF, EGR1, CRHBP, NPR3; LSC: NPTX2, H1F0, EMP1, MEIS1, CALCRL, TPSD1, TPT1, CRHBP, CLNK, TSC22D1; leukemic progenitor cell (LPC): CDK6, HSP90AB1, SPINK2, EEF1B2, PCNP, TAPT1-AS1, HINT1, LRRC75A-AS1, DSE, PEBP1.Gene expression data plots in different tumors were built with the Gene Expression Profiling Interactive Analysis (GEPIA) web tool. The chromatin immunoprecipitation followed by sequencing (ChIP-seq) data were visualized using the WashU Epigenome Browser , and theKG-1a, THP-1, HL-60, HEL, and KO-52 cells were cultured in RPMI-1640 (M&C Gene Technology) containing 10% FBS (LONSA SCIENCE SRL), maintained at 37°C with 5% CO2. Kasumi-1 cells were cultured in RPMI-1640 containing 20% FBS.+ cells were enriched using the CD34 Positive Isolation Kit (Thermo). CD34-enriched AML cells were then incubated with CD34-FITC  and CD38-PE  monoclonal antibodies and sorted on a flow cytometer (BD FACSAria Fusion).CD34RNA extraction, reverse transcription, and quantitative real time polymerase chain reaction (qRT-PCR).Total RNA was extracted with RNAiso Plus (Takara). Reverse transcription was performed using the M5 Sprint qPCR RT kit (Mei5 Biotechnology). Afterward, RT-PCR was performed using THUNDERBIRD SYBR qPCR Mix (TOYOBO) on a LineGene 4840 Real-time PCR system (Bioer). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an endogenous control. GAPDH gene Forward primer: TGGCACCGTCAAGGCTGAGAA; Reverse primer: TGGTGAAGACGCCAGTGGACTC. CNST gene Forward primer: GCCACTTCGGGATGCTTCTGAG; Reverse primer: GCCACTTCGGGATGCTTCTGAG.Cells were lysed in sodium dodecyl sulfate (SDS) lysis buffer  supplemented with protease inhibitors and sonicated to shear DNA. Protein samples were separated by SDS-PAGE, transferred to the PVDF membrane (Millipore). Antibodies: CNST , GAPDH .t-test or Mann–Whitney U test was used to examine whether there was a difference between the two groups of data, and analysis of variance (ANOVA) or Kruskal–Wallis test was used to evaluate the relationship between a categorical variable and a continuous variable, as appropriate. For ANOVA test, multiple comparisons were performed using Tukey test. For Kruskal–Wallis test, multiple comparisons were performed using Kruskal–Wallis H test. The correlation between genes was estimated using the Pearson correlation coefficient. The prognostic effect of CNST expression was analyzed through Kaplan–Meier analysis using the log-rank test. The p value < 0.05 (two-tailed) was defined as statistically significant in all statistical analyses.All statistical analyses were performed using R4.1.1 and third-party R packages based on R. The Shapiro–Wilk test was used to determine whether the variables were normally distributed. Student’s p < 0.001; GSE13159, p < 0.001; GSE114868, p < 0.001; GSE15061, p < 0.001; p = 0.029; p = 0.002; GSE66525, p = 0.033)  .p < 0.001; To verify the ability of CNST as a diagnostic marker, we further examined whether CNST expression is associated with the demographic, molecular and biological characteristics of AML, including those based on age, morphology, cytogenetics, and genomic lesions. We analyzed five independent large clinical datasets and focused on bone marrow samples. In GSE10358 and GSE14468 datasets, the expression of CNST in young patients (aged < 60 years) was higher than that in the elderly group, but this trend was not found in GSE30285, TCGA, and Beat AML. To further explore whether CNST expression is related to age, we used the data of GSE11504 and GSE69408 to analyze the expression of CNST in bone marrow cells and HSCs from different ages of people. We found that there was little change in CNST expression in bone marrow cells and HSCs of different ages. CNST expression showed a significant variation according to the French–American–British classification, which subclassified AML into different categories based on the cytology as well as enzymatic profile , in all t-test, p < 0.001; p < 0.001; Patients with t  karyotype have been shown to have mutations in the RUNX1–RUNX1T1 fusion protein . Therefop < 0.001; p < 0.001; p < 0.001; p < 0.001; +CD38−, CD34+CD38+, and CD34− KG-1a cells and found that CNST expression was significantly higher in HSC-like (CD34+CD38−) KG-1a cells  enrichment analyses were performed on these DEGs, and pathways enriched in at least three datasets were selected. These included 325 pathways that positively correlated with CNST expression and 619 pathways that negatively correlated with CNST expression . Differentially expressed genes (DEGs) between CNSTpression . The DEGCNST is a protein located on the TGN that mediates the targeting of transmembrane proteins to the plasma membrane. At present, there is limited information about CNST, and the function of CNST is still poorly understood. Here, we investigated the expression patterns of CNST in AML and normal hematopoietic system and analyzed the possible role of CNST in AML.CNST was significantly elevated in AML compared with normal bone marrow and was associated with a poorer prognosis. In multiple different independent patient cohorts, CNST expression of AML patients with various subtypes showed higher expression than normal population, which led us to believe that compared with other AML makers such as CD33  and CD12To explore the phenomenon that CNST expression is generally elevated in AML, we analyzed other subgroups of AML. We found that the expression of CNST was higher in AML patients with M0–M2 subtypes that were poorly differentiated. This suggests that CNST may be associated with the low degree of differentiation of hematopoietic cells, which was confirmed by the expression pattern of CNST in the normal hematopoietic system. Thus, a question arises as to whether CNST is associated with poor differentiation levels in other tissues. However, the results indicated that CNST did not show this trend in other tissues; so, we considered CNST to be a hematopoietic system-specific marker of poor differentiation. The elevated CNST expression in LSCs also partially explains the higher CNST expression in relapsed AML patients . Next, wWe next considered whether the elevated expression of CNST was due to the activation of its reported function, namely sorting and cell surface targeting of transmembrane proteins through interactions with GGA adaptor proteins .However, GGA1 and GGA2, which are reported to interact directly with CNST, and Cx32, Cx43, Cx45, and other transmembrane proteins targeted to the cytoplasmic membrane by CNST did not show a similar expression trend to CNST in the hematopoietic system and AML. Some of them even showed a negative correlation with CNST, suggesting that the high expression of CNST in HSCs and AML is likely not due to more frequent Golgi-mediated vesicular trafficking but rather due to the activation of other pathways involved in CNST. This is consistent with reports in the literature that, in addition to GGA, CNST may also recruit other proteins to play its role  [1]. Ourhigh AML patients, various immune-related pathway genes showed low expression, suggesting that CNST may be involved in immune evasion (+ monocyte-like AML through the LILRB4/SHP-2/NF-κB/uPAR/ARG1 signaling pathway (For the possible role of CNST in AML, according to our analysis of the signaling network involved in CNST in AML, we propose the following three possibilities: first, CNST affects the drug resistance of AML by participating in stress granule assembly. SGs are membraneless ribonucleoprotein-based cellular compartments in the cytoplasm that are formed when translation initiation is impaired . SGs are evasion , which i evasion . Two mec pathway , which i pathway .Finally, CNST participates in LSC niche retention. HSC niche is a concept first proposed by Schofield , which dTaken together, our results suggest that CNST, a marker of poor differentiation of blood cells, may play multiple biological roles in AML. CNST may influence the prognosis of AML by participating in SG assembly, immune evasion, or LSC niche retention. The specific high expression of CNST in AML also indicates that targeting CNST and its related pathways is a potential therapeutic option. However, as it is a key protein in vesicle trafficking in cells, direct targeting of CNST may lead to serious side effects. Therefore, further in-depth study of the role of CNST in cells is needed to formulate more targeted treatment options.


To monitor sarcomere shortening and relaxation in cardiomyocytes in real-time, we generated a α-cardiac actinin (ACTN2)-copepod (cop) green fluorescent protein (GFP+)-human-induced pluripotent stem cell line by using the CRISPR-Cas9 and a homology directed recombination approach. The engineered human-induced pluripotent stem cells were differentiated in transgenic GECI-enhanced GFP+-cardiomyocytes and ACTN2-copGFP+-cardiomyocytes, allowing real-time imaging of [Ca2+]i transients and live recordings of the sarcomere shortening velocity of ACTN2-copGFP+-cardiomyocytes. We developed a video analysis software tool to quantify various parameters of sarcoplasmic Ca2+ fluctuations recorded during contraction of cardiomyocytes and to calculate the contraction velocity of cardiomyocytes in the presence and absence of different drugs affecting cardiac function. Our cellular and software tool not only proved the positive and negative inotropic and lusitropic effects of the tested cardioactive drugs but also quantified the expected effects precisely. Our platform will offer a human-relevant in vitro alternative for high-throughput drug screenings, as well as a model to explore the underlying mechanisms of cardiac diseases.Live-cell imaging techniques are essential for acquiring vital physiological and pathophysiological knowledge to understand and treat heart disease. For live-cell imaging of transient alterations of [Ca Live-cell imaging also delivers more physiologically relevant information in comparison to fixed cell microscopy.The classical non-live imaging procedures to monitor changes on the molecular level  are inefficient, static methods, executed on fixed cells. For more efficient (cost-effective and less time consuming) monitoring of the dynamic changes of molecular parameters, such as the cytoskeleton or of intracellular free Ca2+]i. Our novel video analysis software also allowed us to quantify functional parameters related to contraction and relaxation. We validated the applicability of our platform using Isoprenaline as a classic Adrenoreceptor agonist, the L-type agonist Bay-K8864, the L-Type blocker Nifedipine, and the muscarinic agonist Carbachol. We firmly believe that these sophisticated tools open new horizons to study the contractility of CMs under pathological conditions. This technology can be applied to screen pharmacological drugs and to study the mechanisms involved in the development of cardiac diseases.The clustered regularly interspaced short palindromic repeats (CRISPR) and associated (Cas)-9 technology efficiently facilitates genetic editing in human pluripotent stem cells, including hiPSCs and human embryonic stem cells (hESCs), thereby dramatically increasing the potential applications of hiPSCs as a disease model for monogenic, polygenic, and rare diseases, very often associated with gene mutations ). Here, 2. After reaching a confluence of 90%, cells were dissociated by trypsinization using the trypLE solution  and propagation of the cells was performed in StemMACS™ iPS-Brew XF media in the presence of 10 µM ROCK inhibitor. Differentiation of hiPSCs to CMs was performed using a combination of two differentiation protocols [N-(6-Methyl-2-benzothiazolyl)-2-[thio]-acetamide IWP2  and cells were cultured for further 48 h (day 4). The medium was replaced with basal RPMI1640/B-27 without insulin and was refreshed every 48 h. Spontaneous beating in cardiomyocytes was observed from day 9 onwards. Enrichment of the cardiomyocytes was performed by culturing of the cells with glycose-free Roswell Park Memorial Institute  containing 4 µM sodium DL-lactate for 6 d. The efficiency of the differentiation of hiPSCs to obtain a purity of CMs over 95% by flow cytometry and RT-qPCR and hiPSC-CMs was characterized using immunocytochemistry, electrophysiology, electron microscopy, and calcium transient measurements -3,4-dihydroxyfuran-2(5H)-one ascorbic acid , 1x Linoleic Acid-Oleic Acid-Albumin 100x , Non-Essential Amino Acid (NEAA) , 1x B27 media, and 1% Knockout serum replacement (KOSR) . The seeded hiPSC-CMs were cultured for 48–72 h to obtain the uniform beating pattern in the flasks prior to the experiments.Experiments were performed with the IMR90 hiPSCs . Cells were cultured on matrigel-coated petri dishes. Cells were cultured in StemMACS™ iPS-Brew XF media  supplemented with 50 U/mL penicillin and 50 U/mL streptomycin , at 37 °C and 5% COrotocols ,25 as derotocols . Brieflyurements . In all 2+]i in CMs, we generated genetically-encoded Ca2+-indicator (GECI)-enhanced green fluorescent protein (eGFP+)-hiPSCs (IMR90), as indicated in +-hiPSCs were then differentiated to GECI-eGFP+-CMs for further experiments.For live-cell imaging of transient alterations of the i during contraction of CMs. Expression of GECI in CMs was induced by adding 500 nM of -4-(dimethylamino)-1,5,10,11,12a-pentahydroxy-6-methyl-3,12-dioxo-4a,5,5a,6-tetrahydro-4H-tetracene-2-carboxamide;ethanol;hydrate;hydrochloride Doxycycline . The binding of Ca2+ to GECI results in a conformational change of CaM, thereby binding to target proteins, such as M13, and enhancing the fluorescence signal of eGFP (by de-protonation of the eGFP chromophore). In the absence of Ca2+, the eGFP chromophore is protonated and the fluorescence intensity is very poor. Plasmids and the gateway adapter sequences required for generation of GECI-eGFP+-hiPSCs are shown in In brief, first the +-hiPSC line using the CRISPR-Cas9 and the homology-directed recombination approach, as indicated in the To monitor CM sarcomeres contraction and relaxation in real-time, we generated an ACTN2-copGFP+-CMs for live-cell imaging.In brief, the ACTN2-gRNA was cloned into the pX330 plasmid and confirmed by DNA sequencing. Next, ACTN2 homology arms were cloned in the universal donor vector and confirmed by DNA sequencing. Then, the donor and gRNA plasmids were transfected in the hiPSCs and positive clones were selected using 2 µg/mL puromycin. The DNA sequences of the ACTN2-gRNA with the PAM sequence (underlined), the donor plasmid with the left and right 600 bp 5′ and 3′ homology arms , and the Gly-linker are shown in 2+]i during contraction of CMs, as well as the contractile sarcomere activity of the CMs. The software is based on the LabVIEW  application, where we have programmed the Vision Module to monitor and record fluorescence signals of microscopic live-cell imaging records. The software can be used to calculate the contraction/relaxation velocity of the CM sarcomeres, as well as changes in [Ca2+]i, based on video-image recordings. All videos were recorded using a frame rate of 50 fps. The velocity field was calculated for each frame, using the Horn and Schunck algorithm i occurred after 0.1 s and the (time to 90% of peak) T90 time was approximately 0.4 s. The video records of the [Ca2+]i fluctuation curves were automatically analyzed using the software Video Analyzer 1.9 . 2+]i waves of fluorescence in c-CMs during contraction in the absence (end concentration of Dimethylsulfoxid: 0.05%) and presence of Bay-K8864, Isoprenaline, Nifedipine, and Carbachol (end concentration of Dimethylsulfoxid: 0.05%). Temporal parameters of the [Ca2+]i fluorescence signals are shown in Imaging of eGFP fluorescence waves related to Ca for 6 h A. Repres0 represents the time point immediately before a rise in [Ca2+]i. Time-to-peak (TTP) represents the time required for the transient to reach a maximum max; this value was used as indicator for inotropic effects ethyl]benzene-1,2-diol;hydrochloride Isoprenaline  and subsequently with 1 µM of L-Type channel blocker dimethyl 2,6-dimethyl-4-(2-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate;hydrochloride Nifedipine  is shown in 2+-waves was significantly increased by Isoprenaline treatment; while, as expected, addition of Nifedipine not only reduced the velocity, but also the resting sarcoplasmic Ca2+ level . A representative video recording of the control (c-CMs) followed by addition with Isoprenaline (1 µM) and subsequently by addition of 1 µM of acetylcholine receptor agonist 2-carbamoyloxyethyl-trimethyl-azanium Carbachol  is shown in 2+-fluorescence waves, while Carbachol significantly reduced the velocity of the Ca2+ fluctuations as well as the Ca2+ fluorescence correlated with high [Ca2+]i. A representative video recording of the c-CMs treated with the L-type calcium channel agonist 1 µM of (±)-1,4-Dihydro-2,6-dimethyl-5-nitro4-(2-[trifluoromethyl]-phenyl)pyridine-3-carboxylic acid methyl ester Bay-K8864  is shown in 2+-fluorescence. As indicated in max over the values for c-CMs (= 100%), respectively. The addition of Carbachol or Nifedipine to the Isoprenaline-stimulated c-CMs resulted in a significant reduction of (∆F/∆T)max compared with the c-CM value, reducing these values from 100% to 59% and 19%, respectively. Isoprenaline alone resulted in the reduction of the T90 value, compared with that of the c-CM value, from 100% to approximately 78%, whereas Bay-K8864 induced a 20% increase in the T90 value over the c-CM value.T maximum A. T90 re effects A. Normalα-actinin (ACTN2)-copepod (cop) GFP+-hiPSCs was performed as described in Generation of the +-CMs . Liv2+ biosensor/GCaMP biosensor, which has been successfully applied in neurosciences for imaging of neuronal activities [2+ biosensor/GCaMP hiPSC-CMs is the real-time monitoring of transient fluctuations of [Ca2+] in a Tet-on inducible manner.The application of single fluorescent protein-based genetically encoded biosensors is based on allosteric modulation of the fluorescence of a single fluorescent protein . An examtivities . Moreovetivities ,33,34. H2+-imaging  over synthetic dyes, such as fluo-2, are outlined below. Clearly, in comparison to new technologies, the loading procedure of Ca2+ indicators, such as fluo-2, is invasive and time-consuming i. The binding of Ca2+ to troponin C, a protein of the troponin complex  initiates contraction of the sarcomeres and can be visualized as a shortening of the distance between Z-discs (systolic phase). Dissociation of Ca2+ from troponin C initiates the relaxation of CMs, which can be monitored by the increase in the Z–Z distance. (diastolic phase). To investigate the dynamic changes of the contractility of CMs, pending on the [Ca2+]i, we also generated a transgenic hiPSC cell line, allowing live-imaging of the transient changes in [Ca2+]i. The applicability of the transgenic hiPSC-CMs as a cellular platform for live imaging of beneficial or adverse functional effects of different drugs was tested using the sympathomimetic drug, Isoprenaline, and the parasympathomimetic drug, Carbachol, as well as an agonist Bay-K8864 and antagonist Nifedipine of the L-type channel. In parallel, we developed a 2-in-1, user friendly software VA1.9 to quantify the contraction and relaxation kinetics, as well as the kinetics of transient fluctuations of [Ca2+]i during contraction and relaxation phases of the CMs.The contractility of CMs is regulated by [Ca2+-influx via the L-type channel induces a Ca2+-release from the sarcoplasmic reticulum [2+-induced Ca2+-release mechanism). CMs from mouse ESCs showed prolonged APD [90 in ventricular human ESCs-derived CMs [90 period that correlates positively with the APD90. Moreover, we demonstrated that Bay-K8864 increased (∆F/∆T)max, indicative of an inotropic effect. In a comparative electrophysiological study in which several positive inotropic agents were tested on hiPSC-CMs, it has been shown that Isoprenaline slightly decreased APD90, whereas Bay-K8864-induced a prolongation of the APD90 [90 of hiPSC-CMs [max, and significantly shortened the T90 period, which correlates well with ADP90 values. Carbachol not only abolished the effect of Isoprenaline on the (∆F/∆T)max, and on the T90 value, but also significantly reduced the (∆F/∆T)max value, even below that of the c-CM value.Bay-K8864 is a potent inotropic compound that increases the action potential duration (APD) in adult animal ventricular heart muscles and Purkinje fibers . It is eeticulum  max values, again proving the inotropic effects of both drugs. As expected, Carbachol almost eliminated the effect of Isoprenaline on the basal values of c-CMs. Moreover, Isoprenaline induced a significant shortening of the contraction and relaxation phases, as observed by other authors in rat, mouse, and human engineered heart tissues [Our findings with the ACTN2-copGFP tissues . The lus tissues . Our dat