Our results indicated that SRGS, which regulates the immunological microenvironment, is a reliable predictor of the clinical outcome and immunotherapeutic response in AML.In malignancies, cellular senescence is critical for carcinogenesis, development, and immunological regulation. Patients with acute myeloid leukemia (AML) have not investigated a reliable cellular senescence-associated profile and its significance in outcomes and therapeutic response. Cellular senescence-related genes were acquired from the CellAge database, while AML data were obtained from the GEO and TCGA databases. The TCGA-AML group served as a training set to construct a prognostic risk score signature, while the GSE71014 set was used as a testing set to validate the accuracy of the signature. Through exploring the expression profiles of cellular senescence-related genes (SRGs) in AML patients, we used Lasso and Cox regression analysis to establish the SRG-based signature (SRGS), which was validated as an independent prognostic predictor for AML patients via clinical correlation. Survival analysis showed that AML patients in the low-risk score group had a longer survival time. Tumor immune infiltration and functional enrichment analysis demonstrated that AML patients with low-risk scores had higher immune infiltration and active immune-related pathways. Meanwhile, drug sensitivity analysis and the TIDE algorithm showed that the low-risk score group was more susceptible to chemotherapy and immunotherapy. Cell line analysis  Acute myeloid leukemia (AML) is a variable disease that arises from the malignant clonal expansion of myeloid progenitor cells, which are mainly located in the bone marrow and peripheral blood . It is tCellular senescence is an essential aspect of aging  and a reIn order to thoroughly analyze the relationships between cellular senescence and AML patients’ prognosis, we developed a new signature according to cellular senescence-related genes (SRGs). We then investigated the potential value of serving as prognostic and immunotherapy response biomarkers. According to the SRG-related signature (SRGS), the interactions between risk groupings and immunological checkpoints, as well as immune cell infiltration, were then carefully investigated. Additional investigation of the processes revealed that cellular senescence in AML influenced the TME through SASP. In a word, this work gave new insights into the potential regulatory mechanisms related to cellular senescence and relevant immunotherapeutic treatments for AML.https://portal.gdc.cancer.gov) and the Gene Expression Omnibus . The TCGA-AML cohort . A total of 279 SRGs  is developed for assessing the immune evasion mechanisms. Hence, TIDE is often used as another robust biomarker for predicting immunotherapy response. While a higher TIDE score usually suggests a lower response rate for tumor cells to immunotherapy. The tumor mutation burden (TMB) for each AML patient was measured by the nonsynonymous mutation numbers per mega-base. The pRRophetic package (50) of cytarabine and YM155 in each TCGA-AML sample. In addition, the Pearson correlation analysis was carried out to explore the positive relationships between the prognostic SRGS and sensitivity to cytarabine or YM155.Tumor Immune Dysfunction and Exclusion (TIDE) , http:// package  in R sof2 in a humid environment. Cells were grown in RPMI-1640 media supplemented with 10% fetal bovine serum, 100 IU/mL penicillin, and 100 mg/mL streptomycin, respectively. Cytarabine and YM155 were acquired from Selleck Chemicals (United States). Using the Cell Counting Kit-8 (CCK-8) test , the viability of cells was determined. Twenty thousand cells per well were seeded in a 96-well plate and incubated at 37°C in a humidified cell incubator containing 5% CO2. After 48 h of exposure to the chemical at the prescribed concentrations, the CCK-8 reagent was added, and incubation continued for an additional 2 h. Optical density (OD) was determined using the Tecan Spark TM10M at 450 nm . For Western Blot analysis, cells were centrifuged at 1000 rpm for 5 min after 48 h of exposure to the drug at various doses. This was followed by a 0.5-hour resuspension in protease and phosphatase inhibitor-containing RIPA lysis buffer . Before being centrifuged at 12000 rpm at 4°C for 15 min, the lysates were subjected to vortex and sonication in a bath of cold water for 5 min on high with 30-second intervals and 1 min off. After determining the protein concentration, the protein was combined with loading buffer and boiled for 10 min at 100°C, followed by electrophoretic separation on a 10% SDS-PAGE and transferred to PVDF membranes. The membrane was then treated with 5% skim milk powder for 2 h at room temperature to eliminate nonspecific interaction, followed by overnight incubation with antibodies against GAPDH , BAX , and BCL-2  at 4°C, and subsequent incubations in secondary antibodies , prior to visualization with enhanced chemiluminescence (ECL) western . For the detection of mRNA levels, we used SYBR green I dye from Takara  and the ABI 7500 real-time PCR machine . Sequences of primers were designed in PrimerBank (http://pga.mgh.harvard.edu/primerbank/). We used the following primers: KDM5B (Primer Bank ID: 57242795c1), SMURF2 (Primer Bank ID: 56550041c1), MAP4K1 (Primer Bank ID: 110611904c1), G6PD (Primer Bank ID: 108773794c2), CDK18 (Primer Bank ID: 262527294c1), SOCS1 (Primer Bank ID: 4507232c1), ETS2 (Primer Bank ID: 372466581c3), AKR1B1 (Primer Bank ID: 24497579c2), and GAPDH (Primer Bank ID: 378404907c2).AML cell line (U937) was obtained from the American Type Culture Collection . All cells were grown in an incubator at 37°C and 5% COp < 0.05 was considered statistically significant.All the data analysis and the graph generation in this study were carried out in R version 3.5.1, SPSS version 25.0, and GraphPad Prism version 8.0. Unpaired Student’s t-test was applied for comparisons of two groups to analyze the statistical significance. The Kaplan-Meier survival analyses for the OS of AML patients were performed using the R package named “survminer”. Receiver operating characteristic (ROC) curves for survival evaluated the predictive efficacy of the SRGS . UnivariA total of 278 SRGs were compared between AML samples from TCGA and normal samples from GTEx to characterize their expression in AML. We identified a total of 227 DESRGs . Next, aTo construct an SRGS for the survival prediction in AML, the 11 OS-associated SRGs were analyzed using LASSO Cox analysis. An 8-gene SRGS was then built . A scoreTo further validate the predictive value of the constructed SRGS, an external AML dataset named GSE71014 from the GEO database was enrolled. Kaplan-Meier survival analysis in p > 0.05; Univariate and multivariate Cox analyses for the variables in clinic and SRGS were carried out to determine the independence of SRGS’s prognostic value for OS of AML patients. As shown in The evaluation above confirmed the predictive value of the constructed SRGS, prompting us to further explore the possible mechanism. Gene set variation analysis (GSVA) was done to elucidate the biological functions of various risk groups in the tumorigenicity and progression of AML . AML samSome upregulated SASPs possess immunosuppressive properties . ConsequPrevious research has shown the significance of immune checkpoint genes in regulating immune infiltration . To furtTo completely characterize the immunological aspects of AML, samples from the TCGA-AML cohort were evaluated. Based on the SRGS scores and hierarchical clustering method, all AML samples were grouped cleanly into two groups . The fea+ T lymphocytes that may recognize tumor cells and elicit an antitumor immune response. For this reason, we hypothesized that TMB mamightperate as a non-negligible predictive factor of antitumor immunotherapy responsiveness. Initially, we discovered that individuals with higher SRGS scores in the TCGA-AML cohort had significantly lower TMB  could improve the clinical outcomes of AML patients, but responses vary. Hence, accurate predictive biomarkers for AML are urgently needed. Given the association between SRGS, immune infiltration, and checkpoints, we analyzed the correlation between the proposed SRGS and the recognized immunotherapy predictor TIDE . We disctarabine  and YM15tarabine . Lower Starabine , widely tarabine .To further confirm the results of drug-relevant analysis, we investigated the expression of SRGs in the proposed signature in U937 cells treated with cytarabine or YM155. Firstly, we tested cell viability following exposure to cytarabine or YM155 of AML cell lines. As shown in Senescence is a complicated process, which involves cell-autonomous and paracrine effects, and is found to have a substantial influence on the microenvironment . There iIn this study, we explored the expression patterns of SRGs, as well as their predictive values, effects on the TME, and drug sensitivity in AML. In detail, we constructed a novel prediction model named SRGS. Then, the predictive value of the SRGS was well validated in the TCGA-AML set and an external public GEO dataset. We also explored the features of the TME in AML patients with different SRGS scores, which included immune cell distribution and the activities of the inflammatory response. Significantly, we recognized distinct SASP influencing TME remodeling as possible immune evasion and tumor growth pathways. In addition, we discovered that the SRGS was an independent predictor for AML patients when combined with immune checkpoints or TMB.This is one of the first studies to analyze SRGs’ expression patterns and discover their prognostic values via utilizing the TCGA and GEO datasets. Six highly upregulated and two considerably downregulated genes were found and included in the SRGS proposed in this work. Intriguingly, these hallmark genes have been identified as regulators of cellular senescence in a variety of malignancies and play crucial roles in tumor formation . The ectAs the significance of cellular senescence in cancer is mainly unexplored, it is essential to get a deeper understanding of the relationships among cancer, senescence, and the immunological milieu. To yet, however, the effect of cellular senescence on the tumor immune infiltration has been inadequately investigated, as well as whether or not this might influence the therapeutic response to ICIs. By undertaking a comprehensive assessment, we showed that SRGs might have significant impacts on the composition and location of the tumor immune cell infiltration. Moreover, we found that the SRGS score was negatively correlated with the infiltration levels of activated mast cells, resting mast cells, follicular T helper cells, cancer-associated fibroblasts, NK cells, and regulatory T cells. In contrast, it was positively correlated with the infiltration levels of M2 macrophages, B cells, monocytes, and myeloid dendritic cells in AML. This data revealed that individuals with higher SRGS scores might have a tumor microenvironment that is immunosuppressive, preventing the immune system from eliminating tumor cells. Then, to further investigate the processes of immunological remodeling caused by the rising number of senescent cells, we discovered that SASP changes might influence TME establishment, which leads to immune evasion and promotes tumor progression. The group with higher SRGS scores demonstrated increases in inflammatory regulators, such as IL-16 and CXCLs; growth factors, including ANG, AREG, EREG, FGF7, and HGF; receptors, including ICAMs, PLAUR, and TNFRSF11B; and proteases, including CTSB and SERPINE1. These variables may influence the recruitment of immune cells and promote tumor growth . In addiin vitro and in vivo (Consequently, our results have clear clinical value. On the one hand, individuals with low SRGS scores had considerably longer longevity, indicating that high-risk AML patients should get more frequent monitoring and appropriate treatments. In contrast, given that only a subset of patients may get long-term advantages from ICIs, we want more precise biomarkers with therapeutic usefulness. The created SRGS may be used as a prognosis tool as well as a guide for customized immunotherapy. In addition, small compounds targeting SRGs have been discovered and have exhibited anticancer potential  in vivo . And the in vivo .in vivo and in vitro studies of the mechanisms through which SRGs remodel the TME in AML are necessary. In addition, further research is required to demonstrate how the aging TME contributes to AML development. The early assessment of the processes behind the connection between SRGs and a worse response to ICIs needs to be clarified by utilizing fundamental investigations.Despite the fact that our research indicated the advantages of immunotherapy and the prognosis for AML, it still had significant drawbacks. First, the eight-gene risk model was created and verified using a publicly available dataset; hence, external validation in multicenter cohorts is required. Second, prospective clinical studies are required to confirm the relevance of our study findings to AML patients undergoing immunotherapy. Third, In conclusion, our work discovered and validated an SRGS with independent prognostic value for AML patients. Notably, the SRGS was strongly related to the immune cell infiltration levels and was implicated in the control of the immunological milieu in AML by SASP. At the end of this study, we characterized the complex interaction between the SRGS and immune checkpoint genes in AML. Meanwhile, we suggested the potential usage of the SRGS in combination with specific checkpoints as the predictive biomarkers of ICI response, which enabled a more accurate selection for AML patients who might benefit from ICI immunotherapy. Consequently, identifying SRGs influencing tumor immune responses and further investigating their regulatory mechanisms should aid in the risk classification and present intriguing targets which could enhance the immunotherapeutic response of AML.


The diversity and mosaic architecture of phage genomes present challenges for whole-genome phylogenies and comparative genomics. There are no universally conserved core genes, ∼70% of phage genes are of unknown function, and phage genomes are replete with small (<500 bp) open reading frames. Assembling sequence-related genes into “phamilies” (“phams”) based on amino acid sequence similarity simplifies comparative phage genomics and facilitates representations of phage genome mosaicism. With the rapid and substantial increase in the numbers of sequenced phage genomes, computationally efficient pham assembly is needed, together with strategies for including newly sequenced phage genomes. Here, we describe the Python package PhaMMseqs, which uses MMseqs2 for pham assembly, and we evaluate the key parameters for optimal pham assembly of sequence- and functionally related proteins. PhaMMseqs runs efficiently with only modest hardware requirements and integrates with the pdm_utils package for simple genome entry and export of datasets for evolutionary analyses and phage genome map construction. Bacteriophages (phages) are an enormous, genetically diverse population whose evolution likely spans billions of years . A hallmAssortment of phage genes into phamilies (phams) was initially described using Phamerator, which assembled phams (using amino acid sequences) with specific BLASTP and CLUSTALW parameters .Here, we describe PhaMMseqs, a new pipeline for Pham assembly, using MMseqs2. Phage gene sequences are first translated into amino acid sequences, and PhaMMseqs uses MMseqs2 to first derive sequence profiles, followed by profile-sequence clustering to merge phams containing more remote homologs; Clustal Omega (MMseqs2 (https://pypi.org/project/phammseqs/) or GitHub (https://github.com/chg60/PhaMMseqs.git). MMseqs2 and Clustal Omega must be installed separately, using a package manager, such as Anaconda or compiled manually; PhaMMseqs installation and usage instructions are available in the GitHub repository. PhaMMseqs has also been incorporated into the pdm_utils package  and Needleman–Wunsch global amino acid sequence alignments were computed using the PARASAIL library . Alignme Methods in Supplementary Materials and A gene present within a pham that should not be (i.e. a nonhomolog) is considered a false positive. To identify false positives, pairwise global alignments were first computed within each pham. A sequence was counted as a false positive if either of 2 conditions were met: (1) the gene’s length is less than 60% of the length of the longest pham member or (2) the gene’s best pairwise alignment within the pham is not significantly better than an alignment between unrelated gene pairs . The former identifies any gene whose homology is evidently nonglobal in scope, while the latter finds genes with dubious homology to the rest of the pham . Any such genes identified by BLASTP but not present in the pham were counted as false negatives.A gene expected to be present in a pham but which is absent is considered a false negative. To identify false-negative pham members, pairwise global alignments were computed within each pham, and the gene with the highest average similarity to all other genes was selected as the pham representative. BLASTP was used to identify genes in the complete dataset that are likely globally homologous to the pham representative (https://toolkit.tuebingen.mpg.de/tools/hhpred), with the following 4 structural/domain databases selected for search: PDB_mmCIF70, PDB_mmCIF30, UniProt-SwissProt-viral70, and NCBI_ConservedDomains(CD). Top hits were manually evaluated for agreement with the annotated functions.For every pham, we identified the set of unique functional annotations ascribed to members of the pham and counted how many times each was used. Counts were manually adjusted to account for labeling inconsistencies  and typographic errors within individual genome annotations. The most common function in each pham was used as its consensus function. For validation of consensus functions, pham MSAs were submitted to the Max Planck Institute’s HHpred server  based on shared amino acid sequences, and the primary function of PhaMMseqs is to perform pham assembly, which it does by interfacing with the sequence–sequence and profile–sequence clustering pipelines of MMseqs2. The program workflow  proceedserformed . The firE-value (-e), sensitivity , the number of cluster steps (–cluster-steps/–num-iterations), and the clustering algorithm itself (–cluster-mode). For each of these parameters, the value that PhaMMseqs uses by default was carefully optimized to maximize the sensitivity in grouping remote global homologs while avoiding domain-chaining , coverage (-c), chaining  and othechaining , retrievchaining .Parameter training was done in 2 stages by a pipeline we created to run PhaMMseqs across ranges of values for each parameter and evaluate the resultant phams for likely false positives and false negatives . In the Of the 2,025 possible combinations of values given in E-value threshold and number of cluster steps appear not to significantly alter runtime, number of phams, or the number of false negatives  into 17,248 phams with a mean size of 10.53 genes in ∼3 min on a 2020 MacBook Pro with an 8-core M1 processor. The largest pham contains 946 genes, and there are 7,644 (44.3%) phams with only 1 gene (orphams). To test the scalability of the optimized MMseqs2 parameters, we downloaded all 4,380 complete phage annotations available in RefSeq (accessed 2021 August 29). The RefSeq . There are several features of phage genomes that can present challenges to pham assembly including the abundance of small genes, the presence of inteins within genes, and intragene mosaicism leading to multidomain proteins with complex evolutionary histories. Thus, even when optimized, pham assembly is expected to be imperfect, reflected by false-positive inclusion or false-negative rejection of genes into phamilies. In a few instances, we have observed some “pham-instability,” where a single set of clearly homologous genes are split into 2 phams. This is rarely observed with repeated assembles on the same hardware but can emerge with identical runs on different hardware . We estimate such phams are rare (<0.1%) and it is unclear which if any software innovations may help to eliminate these. Nonetheless, PhaMMseqs provides a rapid and efficient method for pham assembly with broad utility in phage genomics.jkac233_Supplementary_DataClick here for additional data file.


C9ORF72 gene is the most prevalent cause of amyotrophic lateral sclerosis (ALS). To better understand neuronal dysfunction during ALS progression, we studied motor neuron (MN) cultures derived from iPSC lines generated from C9ORF72 (C9) expansion carriers and unaffected controls. C9 and control MN cultures showed comparable mRNA levels for MN markers SMI-32, HB9 and ISL1 and similar MN yields (> 50% TUJ1/SMI-32 double-positive MNs). Using whole-cell patch clamp we showed that C9-MNs have normal membrane capacitance, resistance and resting potential. However, immature (day 40) C9-MNs exhibited a hyperexcitable phenotype concurrent with increased release of calcium (Ca2+) from internal stores, but with no changes to NaV and KV currents. Interestingly, this was a transient phenotype. By day 47, maturing C9-MNs demonstrated normal electrophysiological activity, displaying only subtle alterations on mitochondrial Ca2+ release. Together, these findings suggest the potential importance of a developmental component to C9ORF72-related ALS.A large hexanucleotide repeat expansion in the  The C9ORF72 hexanucleotide repeat expansion (G4C2) accounts for 5–7% of sporadic MND and frontotemporal dementia (FTD), and 40% and 25% of familial ALS and FTD, respectively3. ALS causes the selective death of motor neurons (MNs) in the motor cortex, brainstem and spinal cord4. With electrophysiological activity central to neuronal function, understanding electrophysiological dysfunction in MNs carrying the C9ORF72 expansion (C9-MNs) may provide important information on disease aetiology.Amyotrophic lateral sclerosis (ALS) is the most common form of adult-onset motor neuron disease (MND) and leads to paralysis and death, typically within 2–3 years of diagnosis, with only 10–20% of patients surviving ≥ 5 yearsSOD1 ALS8, as well as in a high proportion of sporadic and SOD1 ALS patients11. By contrast, variation in MN excitability caused by C9ORF72 expansions is still an emerging and complex phenotype studied in mouse models and induced pluripotent stem cell (iPSC)-derived neurons, with a range of findings reported. C9 mouse models are limited by their inability to fully recapitulate the disease phenotypes seen in patients14 and neuronal excitability phenotypes are therefore not well established. iPSC-derived C9-MNs also show varied neuronal excitability phenotypes with groups reporting either hyper- or hypo- excitability18. Wainger et al.15 reported hyperexcitable spontaneous activity in C9-MNs using multielectrode arrays; Sareen et al.16 showed hypoexcitability, studying evoked action potential (AP) firing over a longer time course, whereas Devlin et al.17 observed a transition from hyper- to hypo-excitable behaviour over time. Hypoexcitability was defined as a reduction in the proportion of neurons able to fire repetitive APs. The same study also reported a decrease in NaV and KV currents over time during the switch from the hyper- to hypo-excitability phenotype. In contrast to the above ‘mixed’ culture models which also contained glial cells, Selvaraj et al.18 used a ‘pure’ MN culture and did not observe any evoked excitability changes between C9 lines and control lines (CTRL). The authors of that study proposed a ‘glial-mediated non-cell autonomous mechanism’ accounting for the previously observed excitability changes18.Neuronal hyperexcitability is often reported in embryonic and early postnatal rodent models of C9-MNs at early (day 40) and late (day 47) stages of maturity. We demonstrate hyperexcitability is paralleled by increased intracellular Ca2+ release without changes to NaV and KV currents in C9 cultures at day 40. The hyperexcitability phenotype is lost by day 47 and coincides with a normalization of intracellular Ca2+ release, with parallel dysfunction in mitochondrial Ca2+ emerging. Our work highlights a developmental component to ALS by showing an early disease-relevant disruption of neurophysiology which may impact on the pathophysiology of neurons later in life.Previous studies used various methods for measuring neuronal excitability and resulted in a lack of understanding of the proposed mechanisms underlying the changes in excitability. In this study, we used whole-cell patch clamp electrophysiology to evoke APs and compare the excitability of a physiologically relevant neuronal/glial mixed population of iPSC-derived C9ORF72 expansion  and three CTRL  lines were previously generated by Sendai Virus reprogramming19  through modulation of after-hyperpolarisations or after-depolarizations23.Ca2+ release from intracellular stores using the Fura-2AM ratiometric dye. When MNs were activated with ionomycin, we observed a typical biphasic Ca2+ response at both day 40 and 47 24. A significant change in delta Ca2+ release compared to baseline was detected in young neurons at day 40  of iPSC-derived MN maturation. This effect was, however, transient, with the induced AP firing frequency in the CTRL MNs increasing from day 40 to 47 resulting in equivalent AP frequency in both CTRL and C9-MNs at day 47. These changes in excitability may result from different rates of neurophysiological maturation, with C9-MNs reaching greater maturity at day 40 compared to CTRL MNs, a genotype-specific difference lost by day 47.This study focused on iPSC-derived MNs harbouring a C9 neuronal excitability. The data here confirm that hyperexcitability exists as an early stage phenotype, as also suggested by Devlin et al.17. Since Wainger et al.15 and Devlin et al.17 observed hyperexcitability at a similar timepoint to us  our hyperexcitability data at day 40 confirm the idea of an early disease-associated hyperexcitability phenotype. Devlin et al.17 noted hypoexcitability at 7–10 weeks post plating and Sareen et al.16 at 66–79 days of differentiation. That we observe the loss of a hyperexcitable phenotype in C9-MNs compared to CTRL MNs at day 47, illustrates a rapid change in excitability in only 7 days in vitro. This loss of phenotype could present an intermediate stage of disease progression between the hyper- and hypo-excitable states. Of note, the mutant C9ORF72 iPSC lines used in this study differed from those used by Wainger et al.15, Sareen et al.16 and Devlin et al.17 in their studies, thereby demonstrating the hyperexcitability phenotype in young neurons is robust and not simply the result of studying a particular set of iPSC lines.These data also provide a rationale for differing results in the field regarding V and KV channels have been implicated in excitability changes in ALS patients and C9ORF72 iPSC models26. However, in contrast, we found no change in fast inactivating NaV or delayed rectifier KV current. This does not rule out a contribution for Na+ or K+ current dysfunction as A-type K+ channels and the Na+/K+ pump could play a role. Our observations from day 40 to 47 show an increase in both NaV and KV current densities, indicating ongoing maturation of neuronal cultures in our study.Both Na2+ dyshomeostasis has strong links to ALS pathogenesis28. MNs are particularly vulnerable to Ca2+ dyshomeostasis due to high Ca2+ influxes during neurotransmission as well as their low overall Ca2+ buffering capability. Recent studies have shown AMPA receptors have increased Ca2+ permeability in C9-MNs which may contribute to excitotoxicity via increased [Ca2+]i and subsequent activated cellular stress pathways29. Using Fura-2AM, Dafinca et al.28 showed iPSC-derived C9 neurons to have increased internal Ca2+ release in response to ionomycin. In addition, the same study found that upon chemical neuronal depolarisation (KCl 50 mM), live cell imaging revealed increased Ca2+ release and delayed Ca2+ clearance from the cytosol in C9 vs CTRL MNs. We show that the dyshomeostasis in internal Ca2+ release in response to ionomycin coincided with the presence of evoked hyperexcitability. One potential explanation may be that the effect of increased intracellular calcium during the period of neuronal maturation may impact expression of ion channel genes over time contributing to hyperexcitability.Ca2+ release from intracellular organelles, we observed a change in stimulated mitochondrial Ca2+ release at day 47 in C9-MNs. Therefore, the loss of hyperexcitability at day 47 coincides with the loss of increased general intracellular organelle Ca2+ release and with the appearance of mitochondrial Ca2+ dysfunction. Mitochondria are important for ATP production as well as for Ca2+ homeostasis, free radical formation, metabolite generation and apoptosis30. Mitochondrial dysfunction has been previously implicated in ALS, particularly in SOD1 models31. However, a variety of aberrations, including reduced ATP production as well as disrupted architecture, are also observed in different ALS mutations as reviewed by Smith et al.32. Ca2+ overload is one mechanism proposed in ALS mitochondrial dysfunction and neuronal cell death pathway activation. Mitochondria rely on cross-talk with the ER for maintaining balanced Ca2+ concentration and Ca2+ signalling33. A shift in Ca2+ storage from the ER to mitochondria could lead to reduced Ca2+ in the ER, but increased Ca2+ in the mitochondria. Too little Ca2+ in the ER could result in protein misfolding, whereas too much Ca2+ in the mitochondria leads to opening of the ‘mitochondrial permeability transition pore’ and subsequent necrosis or apoptosis34. The shift from increased general intracellular organelle calcium release at day 40 to increased Ca2+ release specifically from the mitochondria at day 47 in C9-MNs compared to CTRL MNs could suggest the possibility of mitochondria Ca2+ overload later in the process of cellular degeneration.Finally, in addition to perturbed Ca30. In that paper the authors’ showed astrocytes are affected by the presence of the C9ORF72 mutation which then contributes towards MN dysfunction. It should, therefore, be noted that the cultures used in our study include a mixed population of neurons and astrocytes as a result of the differentiation protocol used.The presence of astrocytes in iPSC-MN cultures and their impact on the biology of MNs carrying ALS mutations is an area of debate as highlighted by Zhao and colleaguesC9ORF72 neurons. Here, we report transient disease-related hyperexcitability in a mixed ‘MN-Astrocyte culture’ which is directly affected by neuronal maturity. Our work associates the hyperexcitability phenotype to a temporally-linked dyshomeostasis in Ca2+ release, followed by a normalization of excitability and emergence of mitochondrial Ca2+ dysfunction at later stages. Together, these data provide a Ca2+ based neurophysiological phenotype which could be manipulated for therapeutic benefit in pre-symptomatic stages of disease.Overall, our study seeks to clarify the conflicting reports surrounding altered excitability in C9ORF72 expansion and control individuals19, then differentiated into MNs using the Du et al20 protocol with minor modifications. At day 28 the media was changed to basal media supplemented with 10 ng/ml IGF, 10 ng/ml CNTF, 10 ng/ml BDNF and 0.1 mM ascorbic acid.iPSC lines were previously generated from patients carrying the Coverslips were fixed in 4% PFA, permeabilized with 0.1% triton (PBS-T) followed by 1 h blocking in PBS-T with 5% donkey serum and 2% bovine serum albumin. Primary antibodies  were applied overnight at 4 °C. Secondary antibodies (Alexa 1:1000) were incubated for 1 h at room temperature. Cells were counterstained with 1 µg/ml DAPI then mounted in Vectashield.6 iPSC-derived MNs were dissociated using Trypsin–EDTA and fixed in 4% PFA on ice for 10 min. Cells were blocked for 10 min in 1 M glycine and solution PB . Blocked samples were split for staining. One sample used for the IgG control, one for unstained control and one for primary antibodies . Antibodies were incubated for 1 h at 4 °C. Alexa secondary antibodies (1:2000) were incubated for 15 min on ice. Primary stained samples were compared against their IgG control for positive cells and analysed using FlowJo v10.4 Software (BD Life Sciences).2.5 × 106 cells were used per RNA sample. Trizol reagent and chloroform were used along with the RNase Easy Mini Columns (Qiagen) with DNase I treatment. cDNA was produced using SuperScript VILO MasterMix, using 1 μg of RNA. Fast SYBR green was used for the qPCR reaction. Primer sequences in Supplementary Table 1.25 × 105 cells were plated per coverslip and selected for electrophysiology based on morphology. Neurons were selected for patch clamping both within and outside of neuronal clusters. Due to the enrichment of the culture for MNs the cells predominantly recorded were MNs. Although without live, cell-type specific labelling it cannot be excluded that some recordings may be from interneurons, the spread of passive properties data does not suggest a separation in cell types. Cells with Ra greater than 40 MΩ were rejected and recordings with a change in Ra or cell capacitance greater than 10% over the duration of the recording were omitted. Any cell lines post stained with less than 30% MNs were excluded.103GTP, adjusted to pH of 7.3 using KOH and osmolarity 290 mOsm. aCSF included (mM): 167 NaCl, 2.4 KCl, 1 MgCl2, 10 Glucose, 10 Hepes and 2 CaCl2 adjusted to a final pH of 7.4 and osmolarity of 300 mOsm.Internal solution (mM): 140 Potassium Gluconate, 6 NaCl, 1 EGTA, 10 Hepes, 4 MgATP, 0.4 NaV and KV currents were recorded in voltage clamp  during a series of 10 mV voltage steps, 100 ms in duration, from a holding potential of − 70 mV. Leak subtraction was applied with 4 sub-sweeps and a settling time of 250 ms.Induced APs were recorded in current clamp by injecting 500 ms current steps in 5 pA increments from − 10 pA. Membrane potential was maintained around − 70 mV by injecting a hyperpolarizing bias current. Na5 cells per well on 96-well plates. Stimulation by ionomycin and recordings using Fura-2 AM were performed as described by Wallings et al.35.Neurons were plated at a density of 10For the above techniques, data from multiple lines and differentiations have been pooled to allow more robust analysis with a higher number of cells and greater statistical power. Patch clamp electrophysiology is a low-throughput technique, and a much higher number of differentiations and patched neurons would be required to present separate data for individual lines. Key figures can be found in the supplementary data, displayed with each line separately for data transparency. Authors can confirm that all relevant data are included in the paper and/ or its supplementary information files.Supplementary Information 1.Supplementary Information 2.


Acute myeloid leukemia (AML) arises from the cells of myeloid lineage and is the most frequent leukemia type in adulthood accounting for about 80% of all cases. The most common treatment strategy for the treatment of AML includes chemotherapy, in rare cases radiotherapy and stem cell and bone marrow transplantation are considered. Immune checkpoint proteins involve in the negative regulation of immune cells, leading to an escape from immune surveillance, in turn, causing failure of tumor cell elimination. Immune checkpoint inhibitors (ICIs) target the negative regulation of the immune cells and support the immune system in terms of anti-tumor immunity. Bone marrow microenvironment (BMM) bears various blood cell lineages and the interactions between these lineages and the noncellular components of BMM are considered important for AML development and progression. Administration of ICIs for the AML treatment may be a promising option by regulating BMM. In this review, we summarize the current treatment options in AML treatment and discuss the possible application of ICIs in AML treatment from the perspective of the regulation of BMM. Acute myeloid leukemia (AML) stems from the myeloid cell lineage and is defined as the presence of immature myeloid precursors (blast cells) in bone marrow or peripheral blood . AlthougIn AML, nonfunctional abnormally proliferated blast cells dominate the bone marrow and thus impair normal hematopoiesis that may result in pancytopenia which will further demonstrate itself with manifestations such as anemia, clotting disorders, and immunosuppression, where the latter increases vulnerability to infections –6. In soDiagnosis of AML is made by the presence of 20% blast count in peripheral blood or bone marrow aspirate. Subtypes of the disease is assessed by flow cytometry to define the subtype of the disease, while chromosomal alterations are investigated using cytogenetic approaches, morphological changes in cells can be observed by bone marrow smears, and oncogenic mutations can be detected by genomic sequencing , 13.Treatment strategies in AML depend on prior toxic exposure, precursor myelodysplasia, karyotypic and molecular abnormalities and patient-specific factors, including assessment of comorbid conditions, age, risk status, or disease situation such as relapsed or refractory. National Comprehensive Cancer Network (NCCN) Clinical Practice Guidelines in Oncology offer annually updated recommendations for the diagnosis and treatment of AML in adults, based on the reviews of recently published clinical trials which have led to significant improvements in treatment. Although details of treatment strategies are not a focus of this review, we will summarize the current therapeutic opportunities to provide a general perspective based on the NHHC 2022 guidelines , 15.The European LeukemiaNet (ELN) risk stratification and the National Comprehensive Cancer Network (NCCN) guidelines classify AML patients into three groups that are associated with specific prognoses and may guide medical decision-making: favorable, intermediate, and poor . The claCurrently, the main treatment for most types of AML is cytotoxic chemotherapy that consists of two phases: remission induction and post-remission consolidation treatments. Although patients are managed according to the same general therapeutic principles, chemotherapy regimens may vary depending on whether the patient is a candidate for intensive or non-intensive therapeutic regimens. In patients eligible for high intensity induction chemotherapy, the “7+3 regimen” of cytarabine plus anthracycline is commonly used . Other aAntibody–Drug Conjugates (ADC), monoclonal antibodies that are linked to cytotoxic agents are novel treatment options in AML . The antBesides the abovementioned therapeutic interventions, other immunotherapeutic strategies in AML include immune checkpoint blockade, bispecific T cell engagers (BiTE), chimeric antigen receptor T cells (CAR-T) and tissue infiltrating lymphocytes (TIL) are under investigation , 22. As Immune checkpoints are receptor-based signal cascades that lead to negative regulation of immune cells, enabling escape from immune surveillance that eventually results in failure of tumor cell elimination favoring tumor progression. Immune checkpoint blockade exerts its’ anti-cancer effect by promoting the immune response through administration of monoclonal antibodies that target immune checkpoint proteins present on immune cells or tumor cells. Inhibition of immune checkpoints such as cytotoxic T lymphocyte antigen 4 (CTLA-4), programmed death-1 (PD-1), and programmed death-ligand 1 (PD-L1) enhances immune responses by inhibiting negative signaling receptors and supporting immune activation, where, in turn, elimination of the tumor promotes cancer regression. Currently, three different classes of Immune Checkpoint Inhibitors (ICIs); PD-1 inhibitors , PD-L1 inhibitors , and one CTLA-4 inhibitor (ipilimumab) have been approved by the US Food and Drug Administration (FDA) for the treatment of various malignancies while others targeting T cell immunoglobulin and mucin domain 3 (TIM3) and lymphocyte activating-3 (LAG-3) are still under investigation –32. All Although ICIs are already being used in the treatment of various malignancies, studies on AML are still ongoing. There are many completed and ongoing experimental studies and clinical trials in distinct phases evaluating ICIs in treatment of AML either as monotherapy or part of a combinational therapy with other agents including chemotherapeutics, HMAs or other immunotherapies. Experimental studies and clinical trials regarding ICIs, either in combination with other therapeutic interventions or alone are summarized below.Importance of IC pathways in immune evasion of AML as well as their blockade with specific agents in AML treatment has been underlined in several experimental studies which involved AML cell lines and murine models.in-vitro  that impair activity of cytotoxic T cells and NK cells . The assd format . Combinain-vitro  in AML treatment . In anotin-vitro . In anotin-vitro .CD47 is a macrophage ICP that is particularly involved in myeloid malignancies and has been identified as a leukemic stem cell marker in AML. Blockade of CD47-SIRPα pathway has been shown to increase several therapeutics in pre-clinical studies . Similarin-vivo study revealed the connection between PD-1H and epigenetic modifications as well as their role in immune evasion in AML where DNA methyl transferase inhibition by 5-aza-2’-deoxycytidine (Decitabine) increased T cell infiltration that potentiated the anti-leukemic effect of the PD-1H blockade and significantly prolonged survival  curve analysis suggested that CTLA-4 and LAG-3 co-positivity can be used as a diagnostic criteria for the disease  is a novel co-inhibitory molecule that promotes immune evasion in solid tumors, and an survival . VISTA hsurvival . Moreove disease .It should be noted that even if the ICIs are promising in the cancer treatment, the broadly distributed immune-related adverse events (irAEs) may not be tolerable in some cases. To overcome this, some experimental studies focus on restricted immune checkpoint blockade such as α-PD-1 × α-CD3 × α-CD33, a bifunctional checkpoint inhibitory T cell–engaging antibody (CiTE) that directs T-cells to CD33 on AML cells with locally restricted immune checkpoint blockade . By the HMAs have been approved by FDA, and they are being used as epigenetic modifiers for the treatment of myelodysplastic syndromes (MDS) and acute AML patients, who are not eligible for induction chemotherapy , 66. It In a phase 2 clinical trial, nivolumab was administered with azacitidine to a high-risk population of relapsed or refractory (R/R) AML patients. Among 70 patients, the response rate to therapy was 33%, with 22% being in complete remission or incomplete hematologic recovery. Grade 3/4 irAEs were reported in 11% of patients, the most frequent one being pneumonitis. For all 70 patients, the median survival was 6.3 months, while for 32 salvage-1 patients , it was 10.5 months. This finding indicates a promising response rate for the combination therapy, as also stated by the authors. A greater response rate was recorded in patients with higher CD3+CD8+ T cell infiltration pre-therapy. Thus, it was reported that pre-therapy T cell infiltration can be considered an inflamed tumor marker and a biomarker that can be used in deciding which patient group would benefit from ICI-based treatments . In anotA phase 1b/2 study reported that azacytidine leads to PD-1 and PD-L1 upregulation in AML which causes drug resistance that may be overcome by including the PD-1 inhibitor nivolumab. In a study, azacitidine was combined with nivolumab and administered to 35 relapsed AML patients. Out of 35 patients evaluated, the preliminary data from this study showed that 6 (18%) were in complete remission (CR) or CR with incomplete count recovery (CRi) and 5 (15%) were in hematologic improvement. A decrease in blast count greater than 50% was observed in 14 patients (26%), and the median overall survival was reported as 9.3 months . Patients with CR/CRi, higher levels of pre-treatment CD3+ and CD8+T-cell infiltration were detected in bone marrow aspirates . In concIn another multi-centered, randomized, international phase 2 clinical trial, azacitidine was administered to high-risk MDS or AML patients in combination with the anti-PD-L1 antibody durvalumab or as a single agent. In this study with 129 AML patients older than 65 years old who were not eligible for chemotherapy; a comparison between the azacitidine and Durvalumab combination therapy and azacitidine as a single agent therapy showed no statistically significant difference in total response rate (31.3% vs. 35.4%) or complete remission rate (17.2% vs. 21.5%). The overall survival rate was 13.0 and 14.4 months, respectively, with no unexpected side effects. Although this study portrays an important role regarding its comparatively larger sample size, it is worth considering that more than half of the patients did not continue with the study regimens, which might be taken into account in interpreting the results .The resistance mechanisms and biomarkers playing role in processes that play role in treatment response are not yet fully explained, but a study from Herbrich et al. puts forth a possible explanation. In their study, Herbrich et al. evaluated the bone marrow and peripheral blood samples taken from nine relapsed or refractory AML patients who received azacitidine and anti-PD-L1 antibody avelumab using single-cell mass cytometry. Out of nine evaluable patients, four had an initial decrease in blast count, and seven showed a fast progression subsequently. Authors reported that in AML bone marrow, CD4+ and CD8+ T cells had a significantly lower proportion of naïve T cells at baseline, along with a smaller ratio of terminally differentiated CD8+ cells. Contrarily, the largest portion of T-cells in AML bone marrow consisted of the effector memory CD4 and CD8 cells. In these patients, a high PD-L2 protein expression was observed in AML cells, and PD-L1 expression was low in the samples taken at both baseline and during therapy. PD-L2 was also frequently expressed in the newly formed clones which were not present at baseline. These findings may indicate a possible explanation for the different response rates to PD-1 and PD-L1 inhibition observed during AML treatment. These findings also indicate that the immune cell distribution is significantly affected in AML patients’ bone marrow. The T cell distribution ratio and the different checkpoints that are expressed on AML cells, such as PD-L2, may pose a key in the consideration of the approach and response of the treatment , 76.th day. The patients who responded to the treatment continued to receive pembrolizumab for two years. The overall response rate was 46%, the composite complete remission was 38%, and the median overall survival was 11.1 months. For refractory or early relapsed patients, and for patients who received the treatment as the first salvage, the median overall survival was 13.2 and 11.3 months, respectively, which was considered promising by the authors. Grade 3 and higher irAEs were reported to be rare and self-limiting, with 14% which is promising when treatment feasibility is considered  to patients with advanced hematologic malignities in a phase 1 clinical trial, where pidilizumab was administered to 17 patients (8 being diagnosed with AML) in doses between 0.2 and 0.6 mg/kg. Complete remission was observed in one patient, while clinical benefit is reported in 33%. Although serious adverse events were reported in 4 patients, who were all diagnosed with AML and passed away later, the study reported that none of these were related to the treatment but to fulminate-resistant leukemia and that the dose aforementioned can be considered safe , 78. Curnsidered . In anotnsidered . These fnsidered . The oveWhen ICIs’ role in maintenance is considered, preclinical studies indicate that ICIs can prevent leukemic cells’ evasion of the immune system and, thus, overcome tumor persistence. Another phase 2 study investigating efficacy of nivolumab on 14 high-risk AML patients in complete remission who were not eligible for allogeneic HSCT indicated that by the end of the year, 71% of patients were in complete remission, indicating the drug’s safety and feasibility in high-risk AML .In brief, numerous recent clinical studies involving ICI as a single agent or combined with other treatments demonstrated promising results regarding clinical efficacy and safety profile. However, it is early to draw distinct conclusions about ICIs’ use in AML and further research is needed. It should be noted that currently, there is no ICI approved by the FDA in the treatment of AML, and the clinical trials regarding ICI in AML treatment are still at the early stages with results revealing modest efficacy, especially for monotherapy the refractory settings .As mentioned earlier, chemotherapy in AML is divided into two phases; induction therapy and consolidation therapies which both vary according to the patient’s age, presence of co-morbidities and genetic alterations. Induction therapy aims to eliminate the blasts in the peripheral blood and to restore normal hematopoiesis while consolidation therapy is administered to remove residual leukemic cells . In clinBone marrow is an extraordinary tissue where various cells from lineages reside. BMM is a substantial gatekeeper in maintenance of the blood cell formation and is a complex structure which is composed of cellular and noncellular elements . The celvia secreting matrix metalloproteinases (MMPs)  , 123. MMs (MMPs) . Moreoves (MMPs) . Angioges (MMPs) . Lipolyss (MMPs) . Sympaths (MMPs) . Along ws (MMPs) , or overs (MMPs) . Similars (MMPs) . AML cels (MMPs) . Likewiss (MMPs) .Recently, upregulated ICP expressions including PD-1, TIM-3, LAG-3 in addition to expansion of myeloid-derived suppressor cells and increased Treg frequency in the BMM of AML patients were reported, which highlights the importance of IC blockade as a novel therapeutic strategy in the treatment of the disease .in vivo murine AML models as well as promoting chemosensitivity via mobilizing AML cells to circulation in clinical studies /CXCR4 (C-X-C chemokine receptor 4) axis is the most studied pathway in AML treatment; as reported, inhibition of this pathway leads to mobilization of leukemic cells, sensitizes them to chemotherapy and promotes apoptosis –138. The studies –141. Ano studies . Wnt/β-cd exvivo . Anotherd exvivo .Targeting adhesion molecules which support the leukemic cells is another approach in AML treatment. Being the receptor of vascular cell adhesion molecule (VCAM-1), integrin α4β1 (very late antigen 4 – VLA 4) plays role in the adhesion of leukemic myeloblasts to BMM . HumanizThe endothelial cell adhesion molecule E-selectin is another important component of the vascular niche that regulates the balance between HSC renewal and commitment. However, the inflammatory mediators secreted by AML blasts increase the expression of endothelial niche E-selectin, which, in turn, promotes their survival and chemoresistance through AKT/NF-κB/mTOR signaling pathways . In an AAML blasts modulate TME to enable disease progression, provide protection against therapeutic interventions and contribute to recurrence . In termPD-1/PD-L1 axis is the most studied IC pathway in AML , and PD-Bone marrow endothelial cells are an important part of the BMM; by secreting growth factors along with certain cytokines and forming physical contact with hematopoietic progenitors, they take part in the regulation of hematopoiesis . In canc+/CD8+ cells co-expressing TIM3 or LAG3 was detected, especially in patients who had multiply relapsed AML. Secreted by LSCs, Gal-9 promotes apoptosis of Th1 effector cells and CTLs expressing TIM-3 that eventually leads to T cell exhaustion and immune evasion , immunomodulatory B cells that exert immunomodulatory effects mainly ogenesis . Recentlogenesis . AccordiML model . Other IML model .lo/neg immature myeloid cells that consist of three major groups: monocytic MDSCs  that resemble monocytes in terms of their phenotypes and morphologies, polymorphonuclear MDSCs  that are similar to neutrophils and early-stage MDSC  (Myeloid-derived suppressor cells (MDSCs) are a heterogenous group of CD11b+ CD33+ HLA-DRA-DRneg) –176. AllA-DRneg) . VISTA hA-DRneg) . PreviouA-DRneg) .Polarization of TAMs to anti-inflammatory M2 phenotype has been well documented in AML, which hampers anti-tumor immunity and promotes cancer progression , 180. A via MHC molecules, leukemia-associated antigens (LAAs), or NK cell activating ligands  are thought to be differentiated from Sca1+ CD45− CD31− or LepR+ CD45− CD31− MSCs , 197. Th100-fold . Recentl100-fold . Howevervia BMM modulation, it should be noted that our current knowledge regarding ICIs mainly relies on studies with solid tumors, and more data involving larger patient cohorts are needed to determine whether they will be integrated into therapeutic routines in hematological malignancies, and the impact of tumor immune microenvironment on the success of ICIs require more investigation.Today it is widely known that the structure and the function of BMM is altered to facilitate AML progression, dissemination and escape immune surveillance . ManipulDrafting manuscript: BA, CP, ZD, NND-C and EG. Preparing the figures: BA and NND-C. Editing and revising the manuscript: GY-D. All authors contributed to the article and approved the submitted version.


Extracellular matrix (ECM) proteins that form the extracellular scaffolding supporting cardiac contractile activity are thought to play an integral role in the modulation of EC-coupling. At baseline, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) show poor utilisation of SR Ca2+ stores, leading to inefficient EC-coupling, like developing or human CMs in cardiac diseases such as heart failure. We hypothesised that integrin ligand–receptor interactions between ECM proteins and CMs recruit the SR to Ca2+ cycling during EC-coupling. hiPSC-CM monolayers were cultured on fibronectin-coated glass before 24 h treatment with fibril-forming peptides containing the integrin-binding tripeptide sequence arginine–glycine–aspartic acid (2 mM). Micropipette application of 40 mM caffeine in standard or Na+/Ca2+-free Tyrode’s solutions was used to assess the Ca2+ removal mechanisms. Microelectrode recordings were conducted to analyse action potentials in current-clamp. Confocal images of labelled hiPSC-CMs were analysed to investigate hiPSC-CM morphology and ultrastructural arrangements in Ca2+ release units. This study demonstrates that peptides containing the integrin-binding sequence arginine–glycine–aspartic acid (1) abbreviate hiPSC-CM Ca2+ transient and action potential duration, (2) increase co-localisation between L-type Ca2+ channels and ryanodine receptors involved in EC-coupling, and (3) increase the rate of SR-mediated Ca2+ cycling. We conclude that integrin-binding peptides induce recruitment of the SR for Ca2+ cycling in EC-coupling through functional and structural improvements and demonstrate the importance of the ECM in modulating cardiomyocyte function in physiology.Engagement of the sarcoplasmic reticulum (SR) Ca Human cardiomyocytes (CMs) are characterised by a finely tuned ultrastructure sensitive to external dynamic factors, in a phenomenon known as myocardial plasticity ,2. Use o2+ is a fundamental component of excitation–contraction (EC) coupling. To adapt to the increased postnatal and adult requirements, immature CMs engage the sarcoplasmic reticulum (SR) as the primary regulator of Ca2+ cycling [2+ cycling is proportionally more dependent on sarcolemmal influx compared to release of Ca2+ from SR stores [2+ cycling, bring the electrophysiological features of hiPSC-CMs closer to that of the physiological, adult CM [2+ cycling efficiency and SR recruitment are largely unknown.Regulation of cytoplasmic Ca cycling . In stemR stores . Our groadult CM . However2+ stores for EC-coupling.Human CMs respond to changes in the ECM via integrins; heterodimeric transmembrane receptors that function mechanically by connecting the cytoskeleton to the ECM and biochemically by activating intracellular signalling cascades . The intWe show that integrin ligands modulate EC-coupling through functional regulation of SR and structural regulation of SR-sarcolemma junctions. The integrin ligand–receptor interactions provide a mechanism for transduction of mechano- and biochemical reception into dynamic changes in CM contractility. Biocompatible integrin ligands present a novel mechanism for modulating hiPSC-CM maturation and CM function in disease.2+ cycling by integrin ligands, preliminary studies cultured CMs on glass dishes with various combinations of ECM proteins  for 24 h before optical recording at 1 Hz field-stimulation, identifying no change in Ca2+ cycling parameters (2+ transient modulation. Negative control RGES (Arg-Gly-Glu-Ser) had no effect on Ca2+ transient parameters. Soluble RGDS and GRGDSP shortened TTP  with no change in T80 , (2+ transient time to peak (TTP) (p = 0.0016) and time from peak to 80% back to baseline (T80) (p = 0.0039), indicating a role in regulating both Ca2+ release into the cytoplasm and Ca2+ removal, required for the initiation and cessation of CM contraction, respectively. Collagen, a common ECM protein, did not mediate any changes in Ca2+ transient morphology (The notion that flanking sequences determine RGD potency in abbreviating Casp-Ser), . More sprphology , indicatp < 0.0001), with no change in GRGDS-mediated abbreviation in T80 (p = 0.601), indicating that GRGDS-induced abbreviation of Ca2+ transients is independent of cell detachment. A confounding factor to the use of soluble GRGDS was that, while the hiPSC-CMs were initially cultured as a confluent monolayer , GRGDS c2+ sensitive dye to changes in Ca2+ availability [p = 0.691) and, unlike GRGDS, did not cause cell detachment from the glass substrate (p = 0.484), indicating that it is a suitable alternative to soluble integrin ligands for investigating integrin ligand–receptor interactions.The detachment of GRGDS-treated hiPSC-CMs from the substrate prevented us from attributing GRGDS-induced changes in signal amplitude of our non-ratiometric Calability ,11,12,13lability . The fib2+ transients (p = 0.0016) and T80 (p = 0.0055), in the absence of changes in Ca2+ transient amplitude. The weaker efficacy of ff_RGD compared to GRGDS may be, at least in part, accounted for by the differences in availability of RGD—in ff_RGD, the integrin-binding RGD tripeptide is likely to have a lower exposure to hiPSC-CMs than in the soluble GRGDS solution.Optical recordings of ff_RGD-treated hiPSC-CMs field-stimulated at 1 Hz showed that ff_RGD, like GRGDS following pretreatment with blebbistatin, abbreviated the Caansients A by redup = 0.0018) was accompanied by an abbreviation in APD (p = 0.0029). There was no change in the maximum diastolic membrane potential (p = 0.602) or AP amplitude.The lack of cell detachment following ff_RGD treatment afforded us the opportunity to analyse microelectrode recordings of APs in current-clamp in control and ff_RGD-treated hiPSC-CM monolayers D. An inc2+ transients in hiPSC-CM monolayers without changing the cell density afforded us the ability to assess the contributions of Ca2+ transporters to Ca2+ extrusion [2+ in both the standard and Na+/Ca2+-free Tyrode’s solution (2+ transient decay rate (p = 0.0446), attributed to a 63% increase in SR Ca2+ uptake rate (p = 0.0049). Ff_RGD had no effect on Na+−Ca2+ exchanger (NCX) (p = 0.941) or the slow Ca2+ removal mechanisms (p = 0.856). The SR:NCX ratio is an important indicator of Ca2+ cycling maturity [p = 0.376) but increased SR fractional release (p = 0.0426). These results demonstrate that ff_RGD increases SR contribution to Ca2+-induced Ca2+ release (CICR) and Ca2+ removal required for cessation of contractile activity.The ability of ff_RGD to abbreviate Caxtrusion . Locallysolution A,B. In ksolution C, ff_RGDmaturity . The SR:p = 0.0021), closer to that of the adult human CM. By labelling α-actinin (p = 0.0039), a key determinant of contractile activity. Other key changes include a reduction in hiPSC-CM cross-sectional area and volume ) that bears the Ca2+ release channels (ryanodine receptor (RyR2)) and the sarcolemmal L-type voltage-gated Ca2+ channels (LTCCs). Following immunofluorescence labelling of the 1.2α subunit of LTCCs and RyR2 in control (p = 0.0363). There was no difference in the Mander’s coefficient of RyR2 overlapped with a background of LTCC pixels (M2) (p = 0.1143). Pearson’s correlation coefficient between LTCC and RyR2 increased following ff_RGD treatment (p = 0.001). These findings indicate that integrin ligand–receptor interactions mediate co-localisation of RyR2 with sarcolemmal LTCCs responsible for CICR, in keeping with abbreviation in Ca2+ transient TTP —the sites at which Ca2+ sparks are detected [p = 0.0002).The junctions between the jSR and sarcolemma function as Cadetected . In adul2+ rise, as well as increasing the rate of Ca2+ decay by increasing the contribution of the SR to Ca2+ cycling. The EC-coupling changes are largely mediated by integrin-induced actin polymerisation. Fibril-forming integrin ligand ff_RGD induce changes in CM ultrastructure, including an increased co-localisation between LTCCs and RyR2, key proteins in CM Ca2+ cycling.Our data demonstrate that integrin ligands abbreviate the poorly orchestrated CICR in hiPSC-CMs, increasing the rate of CaIntegrins are cell surface receptors integral to the processes of cellular adhesion, mechanosensing and signalling. Mammals have been found to express over 18 α and 8 β integrin subunits, which heterodimerise to form 24 receptors . A major2+ transient properties when the collagen gel was present. Also, there was no change in the GRGDS-mediated abbreviation of the Ca2+ transient when it was applied in a collagen gel. This indicates that the abbreviation of Ca2+ transients by soluble RGD-containing compounds is independent of FAC formation.It has been reported that integrin-mediated regulation of cell function is through connections to the actin cytoskeleton and signalling via recruitment of focal adhesion molecules to form the focal adhesion complex (FAC) ,24,25. LThe current study used the fibril-forming amino acid sequence of RGDSGAITIGC, referred to as ff_RGD, to mimic fibronectin. The amino acid sequence of ff_RGD was built around the naturally occurring sequence NSGAITIG observed in the adenovirus fibre shaft at residues 385–392, identified to be the key amyloidogenic region of NSGAITIGC that led to self-assembly into amyloid fibril peptide nanostructures ,28. Stud2+ transient peak amplitude and slower SERCA and NCX Ca2+ transport [2+ availability is due to an initial rise in Ca2+ at the sarcolemma, which spreads inward [2+ transient TTP without changing the amplitude, allowing us to rule out the changes in Ca2+ availability during peak contraction as the trigger for changes in kinetics.There are two main differences when comparing the EC-coupling kinetics of healthy adult human CMs to hiPSC-CMs: hiPSC-CMs display slower rise in reaching Caransport ,16. In hs inward , an inef2+ transient amplitude, an indicator of contractile force, is largely determined by stretch force exerted on CMs [The Cad on CMs . hiPSC-Cd on CMs , much lod on CMs . There w2+ cycling [2+ removal from the cytoplasm and SR fractional release in the absence of a concomitant change in SR Ca2+ content indicates that RyR2 is more responsive to Ca2+ with a shorter opening time. The shift in SR Ca2+ uptake dependence mirrors our previous observations that co-culturing human CMs with human cardiac fibroblasts, the main modulators of ECM turnover, induces more efficient CM Ca2+ cycling compared to CM monocultures [The ratio of SR uptake to NCX activity is often used as a surrogate marker for the maturity of CM Ca cycling . In heal cycling . Ff_RGD cultures .2+ cycling, we demonstrate increases in spontaneous beating rate. hiPSC-CMs have previously been observed to exhibit a negative force–frequency relationship when cultured in monolayer [2+ handling mechanisms [2+ cycling machinery and CM ultrastructure induced by integrin binding motifs within the ECM, as demonstrated in this study. Alongside these improvements in Caonolayer , thoughtchanisms . Since wAPD in adult CMs varies depending on the subtype but lies between 100–300 ms . Our stu2+ extrusion. This reduces the inward current and is expected to shorten the APD. However, the APD is determined by a myriad of other transporters, and this reduced inward current produced by NCX could be compensated. Future studies should delineate the relationship between AP morphology and Ca2+ cycling in hiPSC-CMs.This study demonstrates that hiPSC-CMs treated with integrin ligands are less reliant on NCX-mediated CaAlthough rapid stimulation of hiPSC-CMs has been shown to drive maturation , we show2+ cycling properties. Blebbistatin-mediated inhibition of mechanical activation attenuated GRGDS-induced reduction in the Ca2+ transient TTP. However, the lack of effect of blebbistatin on T80 and the finding that modulation of Ca2+ transients is influenced by the sequence of amino acids flanking the RGD-motif suggest involvement of other mechanisms in a multifactorial signalling cascade.We hypothesised that changes in the integrin-linked cytoskeleton mediated the changes in hiPSC-CM Ca2+ release is the apposition of the SR to the sarcolemma, which forms the CRUs [A key determinant of SR Cathe CRUs ,42. CRUsthe CRUs . We idenp = 0.0004, We also investigated the co-localisation within CRUs—the size of the junctional gap between the SR and sarcolemma membrane. This space has previously been reported to be enriched with proteins that are recruited during formation of the CRU. The co-localisation is thought to be restricted by the size of the RyR2 cytoplasmic domain or foot , which a2+ cycling, integrin ligand–receptor interactions drive key changes in hiPSC-CM morphology. By labelling α-actinin (p = 0.0039). This is closer to the 2.0–2.2 μm widely considered to be the sarcomere length in the adult CM under load [In our study, we show that together with the improvement in Ca-actinin D,E, we ider load ,48.The average size of a human ventricular CM is 100–150 by 20–35 μm, significantly larger than neonatal or hiPSC-CMs. hiPSC-CMs show circular, triangular, or multi-angle morphology, unlike the elongated anisotropic shape that is typical of adult human CMs. During development, interactions with the ECM are crucial for cardiac morphogenesis ; at a si2+ cycling in CMs. Integrins are a complex family of receptors important in a myriad of cellular processes. Future studies should investigate the integrins that play a significant role in modulation of Ca2+ cycling and the genomic changes that occur downstream of integrin ligand–receptor interactions. In doing so, we can improve our understanding of the CM maturation process during development and the physiological changes that occur in cardiac disease. Our study presents important observations centred on the role that integrins play in modulating CaCommercially available hiPSC-CMs  were resuspended and plated according to the manufacturer’s instructions. An experiment was a single biological replicate formed from a single batch of hiPSC-CMs, with the technical replicates indicated in the figure legends.2.Cell resuspension occurred after 1 mL of a CM suspension was thawed, and the cellular content was calculated based on manufacturer-provided plating efficiency and viability measurements. Cells were re-suspended in commercially available plating medium . All procedures were carried out under sterile conditions in a BioMAT 2 class II microbial safety cabinet. Cells were maintained in an incubator  at 37 °C and 5% CO2, before washing with phosphate-buffered saline (PBS) and changed over to maintenance medium . Culture medium was replaced every two days, and the cells were used in experiments between days 10 and 14 after being thawed—at day 40–46 of differentiation.HiPSC-CMs were plated onto 35 mm dishes with a 14 mm glass-bottomed well in the centre . Wells were pre-coated with a fibronectin solution for 1 h at 37 °C unless otherwise specified. Sixty thousand cells were used to achieve a confluent monolayer. Cells could adhere for two days, incubated at 37 °C and 5% CO2+ transient recordings were carried out in standard Tyrode’s solution  at 37 °C. The cells were field stimulated at 1 Hz using a 20 ± 10 V pulse of 5 ms duration, and recordings were captured using a NeuroCMOS camera  at 0.5 kHz with a temporal bin of 2  using a 40x oil-immersion objective. Recordings were made after the cells had reached steady state under field stimulation. The first four transients of each recording were signal-averaged before the following parameters were calculated using custom in-house software for MATLAB R2019b : normalised fluorescent amplitude , time to peak , and time to 80% decay . Firstly, hiPSC-CMs were loaded with fluo4-AM (5 µM)  at 37 °C for 20 min. Fluo-4 was excited using a 470 nm wavelength light-emitting diode (LED) and emitted fluorescence collected through a 530 ± 35 nm long-pass filter. All Ca2+ content and Ca2+ removal mechanisms. A solution of 40 mM caffeine in standard Tyrode’s was loaded into borosilicate glass micropipettes mounted on a 3-axis micromanipulator. The pipette was lowered into the bath, and the tip positioned above the cells at the far end of the field of view, upstream of the flow of bath solution. The cells were paced at 1 Hz before stimulation was ceased, and caffeine was applied simultaneously by applying positive pressure with a syringe pump, triggering a Ca2+ transient approximately 1 s following the last field stimulated Ca2+ transient. Caffeine flow was maintained until the end of the recording , and caffeine was applied as described above and elsewhere [High concentrations of caffeine were locally applied to hiPSC-CMs to assess SR Caecording . To asselsewhere ,35.2+ removal mechanisms were assessed by fitting a monoexponential curve to the declining phase of the Ca2+ transient. The rate of decline (k) was calculated as 1/tau (s−1). As set out by Bassani et al., the rate of decline of the field-stimulated transient (Ktwitch) is the sum of KSR, KNCX, and Kslow [twitch and the decline phase of the caffeine elicited Ca2+ transient was taken as kSR. The difference between the caffeine-elicited Ca2+ transients elicited in standard Tyrode’s solution and Na+/Ca2+-free Tyrode’s solution is attributed to kNCX. Kslow is equal to that of the decline phase of the caffeine-elicited Ca2+ transient in Na+/Ca2+-free Tyrode’s solution.Cand Kslow . As such2+ content measured as the caffeine induced Ca2+ transient amplitude. Fractional release of SR Ca2+ stores was measured as twitch Ca2+ transient amplitude divided by caffeine induced Ca2+ transient amplitude × 100.SR CaAPs were recorded from a confluent hiPSC-CM monolayer using sharp borosilicate glass microelectrodes . Pipettes with 30–60 M Ω resistance were prepared using a Sutter Instruments P-97 Pipette puller. Pipettes were filled with 2 M KCl, 0.1 mM EGTA, 5 mM HEPES at pH = 7.2. bath solution was Tyrode’s . Spontaneous action potentials were recorded in current clamp using a MultiClamp 700B amplifier . Aps were recorded from a confluent hiPSC-CM monolayer using sharp borosilicate glass microelectrodes . Pipettes with 30–60 M Ω resistance were prepared using a Sutter Instruments P-97 Pipette puller. Pipettes were filled with 2 M KCl, 0.1 mM EGTA, 5 mM HEPES at pH = 7.2. bath solution was Tyrode’s . Spontaneous action potentials were recorded in current clamp using a MultiClamp 700B amplifier .2O. Fibronectin solution was diluted to a final working concentration of 5 µg/mL in sterile PBS. Matrigel was used at a final concentration of 300 µg/mL. MapTrix was used at a final concentration of 5 µg/mL. Dishes were coated with all ECM substrates by incubating at 37 °C for at least two hours before cell seeding.hiPSC-CMs were prepared and seeded in monolayers as described above on dishes coated with four different ECM derivatives: gelatin , fibronectin , Matrigel , and MAPTrix . A 0.1% gelatin solution was prepared from a 2% stock solution by diluting in dH2O. HiPSC-CM medium was removed, and the cells were washed with sterile PBS before medium with GRGDS (2 mM) was applied and compared to cultures without GRGDS as control. The cells were incubated for 24 h at 37 °C before electrophysiological assessment.The RGD-containing peptide used was GRGDS (Gly-Arg-Gly-Asp-Ser) . Peptide powder was made up to a stock solution of 50 mM in sterile dH2O, and 1 N NaOH. All solutions were kept on ice to prevent premature polymerisation. Gel polymerisation was activated by neutralising the collagen solution with 1 N NaOH to achieve an optimal pH of 7.0, assessed using universal indicator pH strips . For a GRGDS-collagen solution, GRGDS peptide in dH2O was used in place of dH2O alone.A type I collagen gel of 3 mg/mL was made from bovine collagen I  according to the manufacturer’s instructions. A 5 mg/mL stock solution of collagen was diluted in a combination of sterile 10X PBS, sterile dHFor the application of the gel, hiPSC-CMs were washed in sterile PBS as before, and suction was used to clear the dish of as much liquid as possible. Approximately 100 µL of gel solution or phosphate-buffered saline control was pipetted onto the cells to cover the recessed glass coverslip in the dish. The cells and gel or control solution were incubated at 37 °C for 1 h to allow for gel polymerisation and attachment. After this time, serum-free medium was added and incubated again at 37 °C for 24 h before electrophysiological assessment.3+-RGDSGAITIGC-CONH2 (ff_RGD) . The synthetic lyophilised peptide powders were dissolved in serum-free media  at a concentration of 2 mM. The gel or control were mixed by vortexing and sonicated for 1 min. Gel or control were then incubated at 37 °C for 3 h to allow for self-assembly before 80 μL of the ff_RGD-containing gel or serum-free control was added above the hiPSC-CM monolayer and incubated for 24 h.The fibril-forming synthetic peptide studied was NH2 incubator for 20 min. In order to assess the fluorescence of the dishes, images were collected using a 10× objective on a Zeiss AxioObserver Confocal microscope equipped with a motorised stage. Analysis of the images was conducted offline using Fiji software.The LIVE/DEAD viability/cytotoxicity assay was used to detect the % of living cells in ff_RGD-treated and control CM monolayers. A stock solution of 2 µM calcein AM and 4 µM ethidium homodimer-1 was prepared. A total of 20 µL of 2 mM ethidium homodimer-1 and 5 µL of 4 mM calcein AM were added to 10 mL of serum free medium. The stock solution was then vortexed to ensure thorough mixing. Glass-bottomed dishes with spontaneously beating hiPSC-CM cultures were treated with ff_RGD (2 mM) or serum free for 24 h control was filled with 200 µL of stock solution and incubated at 37 °C in a 5% COHiPSC-CMs were attached to 1 × 1 cm Aclar film squares treated with 10 µg/mL Matrigel in serum-free medium and cultured in maintenance medium. Self-assembling integrin ligand ff_RGD or control was formed as described above and 2 mM of the gel or control was added above the hiPSC-CM monolayer and incubated for 24 h. Samples were washed in PBS before fixing in 2.5% glutaraldehyde and 2% paraformaldehyde in 100 mM sodium cacodylate buffer. After washing in sodium cacodylate buffer, samples were subsequently stained in 1% osmium tetroxide and 1.5% potassium ferrocyanide, 1% tannic acid, 1% osmium tetroxide and 1% uranyl acetate, with washings in water after each staining step. After staining, samples were dehydrated in an ethanol ascending series  followed by further dehydration in pure acetone. Increasing concentrations of TAAB 812 hard resin  mixed with acetone were used for infiltration. To finish, samples were embedded in pure resin and cured at 60 °C for 36 h.Resin blocks were sectioned with a diamond knife at 80 nm on a Leica Ultracut UCT, and sections were imaged on an FEI Tecnai12 BioTwin operated at 80 kV. Images were collected in dm3 format  and analysed in ImageJ or IMOD ,54,55.Confocal microscopy was carried out at the Facility for Imaging by Light Microscopy . A Zeiss LSM780 confocal laser scanning microscope with 7 laser lines  and 34 detectors (34 Channel GaAsP Detection System) was used for all imaging. During all experiments, laser intensity was optimised to enhance resolution but minimise photobleaching and saturation. Z-stacks of images were acquired if cell volume was required for analysis.The average sarcomere length was measured in ImageJ by separating the α-actinin channel and viewing the profile plot of a line drawn perpendicularly across the α-actinin bands. Each peak on the graph corresponds to the increased fluorescence caused by an α-actinin band; thus, the distances between the 5 peaks were measured and averaged to give sarcomere length .Analysis of the average CM area was performed using WGA-stained images acquired using a 20× objective. A region of interest was drawn around the border of individual CMs, and the area was then measured digitally using ImageJ. The areas of 4 CMs were measured per dish. Three dishes were analysed per condition.WGA-stained images were used to calculate the aspect ratio of the cell and nucleus. The major and minor axes were measured digitally using ImageJ, and the aspect ratio was calculated as the major axis divided by the minor axis. Z-stacks of hiPSC-CMs stained with WGA were used to calculate cell volume. Individual images were acquired at 0.73 µm intervals. The areas of the lowest and highest stack images were measured, as well as the middle image in the series. These areas were averaged and multiplied by the stack height to give the cell volume.Brightfield images of hiPSC-CMs were obtained using a 40× objective mounted on a Zeiss Axio Observer Inverted Widefield Microscope at 20 frames/s. The samples were kept in 37 °C standard Tyrode’s. All the acquired images were processed in ImageJ (NIH).Cells were plated either on glass-bottomed dishes as described or on glass coverslips and maintained for 10 days. The culture medium was then removed, and the cells were washed in PBS  before fixing in 4% PFA for 20 min. Preparations were washed in PBS three times for 5 min each and used immediately or stored at 4 °C in PBS. PFA-fixed preparations were subsequently permeabilised in 0.2% Triton X-100  in PBS for 3 min followed by three 5 min washes in PBS.Preparations were incubated in blocking solution containing 3% BSA  in PBS for 1 h at room temperature. The primary antibodies mouse IgG1 anti-α sarcomeric actinin were diluted 1/500 in PBS. In the co-localisation immunostaining, preparations were stained with mouse monoclonal antibody IgG1 anti-RyR2 in a 1/500 dilution in PBS and guinea pig polyclonal antibody  anti-1.2 CaV in 1/250 dilution in PBS.Cells were washed three times in PBS and incubated with secondary antibodies for 1.5 h at room temperature. Cells were then washed three times and mounted with a DAPI-containing mounting solution .WGA staining was carried out to label the cell membranes. Cells on dishes were washed three times in PBS for 5 min each time and then fixed with 3.7% PFA for 15 min. The samples were washed three times for 5 min each to ensure that any remaining fixative was removed. According to the manufacturer’s protocol, the samples were incubated with 5 μg/mL WGA solution  for 10 min at room temperature . After the specimens were washed in PBS, the nuclei were counterstained with Vectashield mounting medium containing Hoechst, and the samples were mounted and kept refrigerated without light exposure until they were imaged using an inverted Zeiss LSM-780 confocal microscope.To measure co-localisation between LTCCs and RyRs on ImageJ, the merged images from the monolayers were split in separate channels, and brightness/contrast was adjusted to ‘Auto’ for each channel then merged again to create a Z-project. Using the Z-stack composite, the borders from individual cells were identified using the WGA membrane staining. The option ‘clear outside’ was selected to isolate individual cells from the preparations. Once channels were split again, the ones from RyRs and DHPRs were inputted in JACoP plugin for co-localisation on ImageJ . Pearsont-test was used. For comparisons of >2 sample groups, a one-way within-subject ANOVA  was used. Post hoc tests used with ANOVA were either Tukey’s test  or Dunnett’s test . For non-parametric distribution, a statistical test was performed using Mann–Whitney (2 sample groups) or Kruskal–Wallis (>2 sample groups). The data are expressed as means ± standard error of mean (SEM). * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.Cell parameter data were calculated manually and blindly by two operators not involved in the data analysis. Before statistical tests, all data were subjected to the D’Agostino and Pearson omnibus normality test. For parametric distribution, a two-way paired 2+ channels and ryanodine receptors and improving cell alignment. These findings are critical in understanding the mechanisms involved in myocardial plasticity and demonstrate the functional importance of the ECM in modulating CM function in physiology.The role of extracellular signals involved in the modulation of EC-coupling is multifaceted. This study demonstrates that integrin-binding peptides improve hiPSC-CM function through increased functional recruitment of the SR with improved CICR, and alterations in CM ultrastructure, increasing co-localisation between L-type Ca


More importantly, changes in the intracellular Ca2+ level can modulate signaling pathways that control a broad range of physiological as well as pathological cellular events, including those important to cellular excitability, cell cycle, gene-transcription, contraction, cancer progression, etc. Not only intracellular Ca2+ level but the distribution of Ca2+ in the intracellular compartments is also a highly regulated process. For this Ca2+ homeostasis, numerous Ca2+ chelating, storage, and transport mechanisms are required. There are also specialized proteins that are responsible for buffering and transport of Ca2+. T-type Ca2+ channels (TTCCs) are one of those specialized proteins which play a key role in the signal transduction of many excitable and non-excitable cell types. TTCCs are low-voltage activated channels that belong to the family of voltage-gated Ca2+ channels. Over decades, multiple kinases and phosphatases have been shown to modulate the activity of TTCCs, thus playing an indirect role in maintaining cellular physiology. In this review, we provide information on the kinase and phosphatase modulation of TTCC isoforms Cav3.1, Cav3.2, and Cav3.3, which are mostly described for roles unrelated to cellular excitability. We also describe possible potential modulations that are yet to be explored. For example, both mitogen-activated protein kinase and citron kinase show affinity for different TTCC isoforms; however, the effect of such interaction on TTCC current/kinetics has not been studied yet.Calcium (Ca Action potentials and subthreshold depolarizing impulses cause Ca2+ influx through VGCCs, which are found in many different cell types  pyrimidine) [2+, Src kinase, and TTCCs in neuritogenesis; however, the underlying pathway remains elusive. Whether Src phosphorylates TTCCs or whether Ca2+ influx through TTCCs is responsible for the activation of Src needs further investigation.Studies regarding the regulation of TTCCs by Src kinase are minimal, and only indirect evidence exists. Gasotransmitter hydrogen sulphide (Hscorbate . Src kinimidine) . OverallPhosphatase and Tensin Homolog deleted on Chromosome 10 (PTEN) is a dual phosphatase with both protein and lipid phosphatase activities . PTEN co2+ can regulate cellular fates such as proliferation, apoptosis, and autophagy linked to cell cycle and even pathologies like cancer and neuronal disorders. Several cellular proteins, including receptors, pumps, and Ca2+ sensing proteins, work together to maintain Ca2+ concentrations inside the cell and cellular compartments. TTCCs specialized membrane proteins that play a crucial role in the signal transduction of many excitable and non-excitable cell types. Scientific literature shows that the activity of TTCCs is regulated by several kinases and phosphatases, leading to the enhancement or inhibition of TTCC currents. A general scheme of TTCC modulation by kinases and phosphatases is shown in Ca2+ influx and thus contribute to pathology. An example of this kind exists for L-type Ca2+ channels where phosphorylation of L-type Ca2+ channels by CaMKII during heart failure contributes to the pathology, and inhibiting CaMKII is protective [In conclusion, there is still an opportunity to explore different regions of TTCCs for their modulation by biological molecules. With the advent of techniques like affinity capture-mass spectroscopy and anti-tag immunoprecipitation, researchers have revealed some new binding interactions as well. However, the functional significance of such interactions is still to be explored. TTCC modulation by kinases and phosphatases could be important in pathology, where the modulation can lead to altered Caotective . Much re


Venetoclax has proven to be a promising therapy for newly diagnosed, relapsed and refractory AML patients ineligible for induction chemotherapy. Current ongoing clinical trials are evaluating its effectivity as frontline therapy for all acute myeloid leukemia (AML) patients. However, response rates vary wildly, depending on patient characteristics and mutational profiles. This review elaborates on the efficacy and safety of venetoclax compared to conventional chemotherapy for treatment of AML patients, comparing the response rates, overall survival and adverse events. Moreover, it gives an overview of genetic and epigenetic AML cell characteristics that give enhanced or decreased response to venetoclax and offers insights into the pathogenesis of venetoclax sensitivity and resistance. Additionally, it suggests possible treatment combinations predicted to be successful based on identified mechanisms influencing venetoclax sensitivity of AML cells.Venetoclax is a BCL-2 inhibitor that effectively improves clinical outcomes in newly diagnosed, relapsed and refractory acute myeloid leukemia (AML) patients, with complete response rates (with and without complete blood count recovery) ranging between 34–90% and 21–33%, respectively. Here, we aim to give an overview of the efficacy of venetoclax-based therapy for AML patients, as compared to standard chemotherapy, and on factors and mechanisms involved in venetoclax sensitivity and resistance in AML (stem) cells, with the aim to obtain a perspective of response biomarkers and combination therapies that could enhance the sensitivity of AML cells to venetoclax. The presence of molecular aberrancies can predict responses to venetoclax, with a higher response in NPM1-, IDH1/2-, TET2- and relapsed or refractory RUNX1-mutated AML. Decreased sensitivity to venetoclax was observed in patients harboring FLT3-ITD, TP53, K/NRAS or PTPN11 mutations. Moreover, resistance to venetoclax was observed in AML with a monocytic phenotype and patients pre-treated with hypomethylating agents. Resistance to venetoclax can arise due to mutations in BCL-2 or pro-apoptotic proteins, an increased dependency on MCL-1, and usage of additional/alternative sources for energy metabolism, such as glycolysis and fatty acid metabolism. Clinical studies are testing combination therapies that may circumvent resistance, including venetoclax combined with FLT3- and MCL-1 inhibitors, to enhance venetoclax-induced cell death. Other treatments that can potentially synergize with venetoclax, including MEK1/2 and mitochondrial complex inhibitors, need to be evaluated in a clinical setting. Acute myeloid leukemia (AML) is a rapidly progressing heterogeneous disease caused by the accumulation of malignant myeloid hematopoietic precursor cells in the bone marrow and, in some cases, in the peripheral blood. AML often affects the elderly, with a median age at diagnosis of 68 years . The proCurrently, standard treatment of newly diagnosed AML patients consists of intensive induction chemotherapy with cytarabine and an anthracycline, more commonly known as the “7 + 3” regimen , followeFor patients considered ineligible for induction chemotherapy, treatment with hypomethylating agents (HMA), such as azacitidine or decitabine, or low-dose cytarabine, (LDAC) has recently been the standard of care . LDAC moIn 2018, venetoclax received accelerated approval by the FDA for use in AML patients. As a BH3 mimetic, venetoclax is a selective and oral inhibitor that binds to BCL-2 , an impoAML relapse is thought to be initiated by LSCs that have self-renewal abilities but may be cell cycle quiescent ,23,24. TSince its approval as a treatment for chronic lymphocytic leukemia (CLL), venetoclax treatment has primarily been investigated in AML patients unfit for or refractory to standard treatment regimens. Elderly AML patients, unfit for standard treatment with induction chemotherapy, showed a 67% CR/CR with incomplete blood count recovery (CRi) rate with a median OS of 17.5 months when treated with venetoclax and a HMA . For comUnfortunately, upfront resistance to venetoclax as well as relapse following CR still occurs . The mecMost clinical studies of venetoclax-based therapy for AML patients were performed in patients aged > 60 years or unfit for conventional treatment with induction chemotherapy. In these clinical trials, venetoclax was given together with HMA, azacitidine or decitabine, or LDAC. The AML patients included had different stages of disease, some newly diagnosed, while others had relapsed or refractory disease. In newly diagnosed elderly AML patients treated with venetoclax-based therapy, CR/CRi was reached in 34–90% with a median OS ranging from 8.4 to 17.5 months and progression-free survival (PFS) ranging from 4.7 to 22.4 months  31,36,3,42,43,44The efficacy of venetoclax was also analyzed in relapsed or refractory AML after standard chemotherapy, HMAs or a HSC transplantation . AlthougThe intensive FLAG-IDA regimen  in combination with venetoclax proved feasible and showed high response rates in both newly diagnosed and relapsed or refractory AML patients (median age of 46 years) . CR was As compared to conventional therapies such as azacitidine, LDAC and intensive chemotherapy the treatment with venetoclax-based combination therapy increased the number of adverse events (AE) ≥ grade 3, such as (febrile) neutropenia, anemia and pneumonia ,38,42,52FLT3-ITD, RUNX1, ASXl1 and TP53 mutations, or a monosomal karyotype have been classified as AML with adverse risk, while NPM1 or biallelic CEBPA mutations are classified as favorable risk, based on treatment with standard induction chemotherapy . AML molAML patients who received venetoclax-based therapy showed increased response rates and OS when harboring NPM1, IDH1/2, TET2 or RUNX1 mutations as compared to the other cases. NPM1 mutations are present in 20–30% of AML cases. In newly diagnosed NPM1-mutated AML patients treated with venetoclax combined with HMA or LDAC CR/CRi, rates up to 91% were observed, which were higher than the overall cohort  31,37,3,40,41,54The mechanism behind the increased sensitivity to venetoclax in NPM1-mutated AML patients remains unclear. NPM1 functions in ribosome biogenesis, genomic stability, p53-dependent stress response and activation of growth-suppressive pathways ,58,59. UIsocitrate dehydrogenases 1/2 (IDH1/2) mutations are observed in 20% of AML patients. IDH produces NADPH from NADP+ by catalyzing the oxidative decarboxylation of isocitrate to alpha-ketoglutarate (αKG) . MutatedSince IDH mutations result in inhibition of TET enzymes, the response of venetoclax-based therapy was also studied in TET2-mutated AML patients ,48,70. TMutations in RUNX1 often lead to loss of function of the RUNX1 protein and create HSCs with aberrant differentiation abilities and resistance to genomic stress . ApproxiAML harboring FMS-like tyrosine kinase 3 (FLT3) and TP53 mutations are classified as an adverse risk group . FLT3 isCo-occurrence of FLT3-ITD mutations with ELN favorable risk mutations, such as mutations in NPM1, generally decreases treatment outcomes . ResponsRelapse after venetoclax-based therapy was associated with clonal expansion of AML cells containing the FLT3-ITD mutation, suggesting that FLT3-ITD-positive cells had a survival advantage during venetoclax treatment . ResistaAML patients with RAS or PTPN11 mutations, although not classified in the ELN classification system , showed Since venetoclax inhibits BCL-2, a key member of the apoptosis pathway, additional mutations or epigenetic and transcriptional changes affecting apoptosis may influence treatment response. TP53 is one of the most frequently mutated genes in all types of human cancer . The adAn effective combination strategy for AML patients that acquired more dependency on MCL-1 for survival than on BCL-2 is the combination of venetoclax with an MCL-1 inhibitor ,120. MaiThe increased dependency on MCL-1 is caused by activation of the MAPK signaling pathway, which stabilizes the MCL-1 protein and prevents its degradation ,111. InhSince OXPHOS plays an important role in the activity of venetoclax, research is focusing on inhibition of the mitochondrial complexes, by for example tedizolid, metformin and AXL inhibitors, thereby potentially increasing sensitivity of AML cells to venetoclax ,140,141.Lastly, ongoing research is investigating novel possible combination therapies to enhance the response to venetoclax in AML cells by targeting specific pathways. Activation of the CXCL12-CXCR4 pathway by the adhesion molecule CD44 increases the level of embryonic stem cell transcription factors causing resistance to venetoclax . TherefoThe addition of venetoclax to induction chemotherapy may be beneficial for a particular subgroup of AML patients. It seems likely that most suitable patients can be identified by their mutational profile, with high sensitivity to venetoclax in NPM1-, IDH1/2-, TET2- and relapsed or refractory RUNX1-mutated AML patients. AML patients harboring FLT3, TP53, RAS or PTPN11 mutations, monocytic AML, or AML cases pre-treated with HMAs show reduced sensitivity to venetoclax-based therapies. Reduced sensitivity to venetoclax is caused by an increased dependency on other anti-apoptotic proteins, such as MCL-1, the ability to increase OXPHOS or the ability to use alternative sources for energy metabolism than OXPHOS, and aberrant levels or mutations in BCL-2 and BAX. Future research is investigating new ways to overcome the reduced sensitivity to venetoclax of AML cells with novel combination therapies such as MCL-1, FLT3, MEK1/2 and mitochondrial complex inhibitors, which need to be evaluated in a clinical setting.