The efforts made in the last decade regarding the molecular landscape of acute myeloid leukemia (AML) have created the possibility of obtaining patients’ personalized treatment. Indeed, the improvement of accurate diagnosis and precise assessment of minimal residual disease (MRD) increased the number of new markers suitable for novel and targeted therapies. This progress was obtained thanks to the development of molecular techniques starting with real-time quantitative PCR (Rt-qPCR) passing through digital droplet PCR (ddPCR) and next-generation sequencing (NGS) up to the new attractive metabolomic approach. The objective of this surge in technological advances is a better delineation of AML clonal heterogeneity, monitoring patients without disease-specific mutation and designing customized post-remission strategies based on MRD assessment. In this context, metabolomics, which pertains to overall small molecules profiling, emerged as relevant access for risk stratification and targeted therapies improvement. In this review, we performed a detailed overview of the most popular modern methods used in hematological laboratories, pointing out their vital importance for MRD monitoring in order to improve overall survival, early detection of possible relapses and treatment efficacy. RUNX1-RUNX1T1 fusion gene [PML-RARA [CBF-β) and the smooth muscle myosin heavy chain (SMMHC), MYH11 [Acute myeloid leukemia (AML) is a clonal disorder that affects myeloid progenitor cells residing in the bone marrow (BM). This implies altered differentiation with subsequent abnormal proliferation and accumulation of inadequately matured myeloid cells ,2. AML aion gene  t15:17):17RUNX1-), MYH11 .NPM1, FLT-3, CEBPA, TP53, RUNX1 and ASXL1 are included to define the profile of prognostic-risk groups [A cytogenetic profile has allowed stratifying cases into groups that are favorable, intermediate and adverse-risk. Complete identification of genetic mutations has enhanced the previous classification, and it has been useful in determining personalized prognosis and risk stratification. Currently, k groups ,7.IDH-1, IDH-2, DNMT3A and TET2 emerged in this context [To date, diagnosis of AML according to the redesigned guidelines of the World Health Organization (WHO) must provide immunophenotypic, cytogenetic and molecular gene panel screening . Moreove context . These a context ,13.Although ongoing studies have shown improvement in the outcome of AML patients, the real prognosis remains poor. Approximately 70% of patients aged ≥65 died within one year of diagnosis, despite the newly available target treatment options . The molNPM1, FLT3-ITD, CEBPA, IDH1/2, KIT, RAS, RUNX1 and TP53 or gene overexpression (WT1). It is commonly used as a standardized method in hematology laboratories to obtain a diagnosis and to monitor the kinetics of MRD without inter-laboratory mismatch [The detection of leukemic cells moved in the last two decades from immune-phenotyping to polymerase chain reaction (PCR) and real-time quantitative PCR (RT-qPCR) . RT-qPCRmismatch ,19. Thismismatch  , a group of 24 international experts in the clinical and translational knowledge in MRD in AML, has established the RT-qPCR approach as “highly sensitive” and as the “gold standard” .PML-RARA, RUNX1/RUNX1T1, CBFB-MYH11 and mutated NPM1 need to be considered as a standard of care for AML patients, and it recommends performing that assessment at diagnosis and every 3 months for 24 months after treatment editing [ELN indicates that molecular evaluation of  editing . PML-RARA is the fusion gene responsible for acute promyelocytic leukemia (APL). Three main alternative transcripts  may arise according to the location of breakpoints [PML-RARA levels remain detectable [PML-RARA transcript [PML-RARA fusion transcripts [akpoints . RT-qPCRakpoints . Furthertectable . Monitortectable . The molanscript . This apnscripts . RUNX1/RUNX1T1 translocation or inv(16) and associated with a better prognosis compared to other types of AML [RUNX1/RUNX1T1 after consolidation therapy and during remission and in predicting relapse risk [RUNX1/RUNX1T1 or inv(16) reduction is useful for discriminating high-risk patients from low-risk patients. However, a recent study investigating the real contribution of measuring transcript kinetics of CBF-AML showed that the majority of relapses were not predicted by molecular monitoring and occurred in a very short period of time, suggesting that MRD monitoring may be poorly informative in the follow-up of CBF-AML patients [Core-binding factor acute myeloid leukemia (CBF-AML) is a subtype of AML characterized by s of AML ,5. RT-qPpse risk . Jourdanpse risk  illustrapatients . Moreovepatients . NPM1 has an incidence of mutation near to 30–40% of adult AML and is frequently associated with normal karyotype (50–60%). To date, more than 50 different mutations are described [NPM1 mutated AML patients) [escribed . Types Aatients) . Being oatients) . HoweverNPM1 transcripts in BM and PB. The recurrence of NPM1 mutation in BM after treatment and the not attainment of a 4-log reduction in PB requires closer monitoring every 4 weeks for at least 3 months [NPM1 mutation is classified as a favorable risk, its persistence after the second cycle of chemotherapy was associated with a higher relapse risk and reduction in OS independently of other prognostic factors [NPM1 qPCR analysis has proved to be essential as a marker of allogeneic stem cell transplantation in poor responders [ELN recommends the monitoring of 3 months . Indeed, factors . Finallysponders ,32. Nevertheless, RT-qPCR has some limitations: (1) more than 60% of AML patients cannot be analyzed by RT-qPCR because they lack common MRD molecular targets; (2) RT-qPCR needs a plasmid standard curve for each molecular target analysis, limiting its usage; (3) identification of the most clinically significant time-points and MRD thresholds; and (4) detection limit in monitoring AML patients with long term remission, where the level of fusion transcript is extremely low. These difficulties can interfere with risk assessment, and they have driven to development of droplet digital PCR (ddPCR), a recently designed technology that can reach high-precision absolute quantification . ras mutations in colon cancer patients, it has been used primarily to detect mutations in genes linked to cancer genesis [Digital polymerase chain reaction (dPCR), or more precisely called droplet digital polymerase chain reaction (ddPCR), is a superior adaptation of real-time quantitative polymerase chain reaction (RT-qPCR). Since its first use in 1999 to detect  genesis . Althoug genesis ,35,36. A genesis . Then, a genesis .The technical similarities and differences between dPCR and RT-qPCR are well defined, and the most important ones are listed as follows: (i) dPCR consists of thousands of individual PCR reactions divided during analysis while in RT-qPCR there is only one exponential amplification of the nucleic acids; (ii) dPCR measures the absolute number of the molecule of interest, while RT-qPCR determines the relative number using a standard curve; and (iii) dPCR has higher sensitivity and better accuracy compared to RT-qPCR ,39. The Nevertheless, the genetic complexity and molecular profile of AML complicates the decision to select a factor for determining MRD, which in turn is necessary for better risk stratification and therapy selection. In addition, the different approaches available for determining MRD have not been the subject of international standardization to date. Thus, monitoring patients with AML and early detection of possible relapse remains a major clinical challenge ,16. In tNPM1 mutations and the fusion genes  by using the dPCR technique and then evaluating them after induction and consolidation chemotherapy are suitable approaches for molecular MRD assessment [DNMT3A, NRAS, ASXL1, TET2, FLT3-ITD, FLT3-TKD, IDH1, IDH2, WT1, etc. A recent study evaluated the use of a newly developed double drop-off droplet dPCR (DDO-ddPCR) assay for mutation screening and AML disease monitoring using cell-free DNA [NPM1  and IDH2 (therapeutic target for enasidenib). In conclusion, this new dPCR assay offers rapid diagnosis and detection of somatic mutations in AML patients, crucial for a prompt and targeted therapeutic decision, and then facilitates disease monitoring. The cost–benefit compared to other modern diagnostic methods such as NGS is on the dPCR side in terms of price, availability and sensitivity. In general, proving the presence of sessment . There afree DNA . In the IDH1/IDH2 mutation as an appropriate marker for molecular monitoring in AML using dPCR [IDH1/IDH2 will be the focus of future scientific research. dPCR can also be used to quantitatively assess the WT1 molecular marker in AML patients with high accuracy [Several other studies have analyzed the ing dPCR ,45,46. Gaccuracy . Its impaccuracy .Finally, allogeneic hematopoietic stem cell transplantation (HSCT) as a curative method remains one of the greatest successful hopes over AML. Balsat et al. have shown that the presence of MRD prior to or after HSCT is associated with a negative outcome . CurrentThe scientific data so far place dPCR at the zenith of interest when it comes to hematological diseases, especially where we need long-term monitoring. Its reliability as a method with high sensitivity and precision, most importantly in low levels of the disease, has been confirmed in numerous studies. The fact that, unlike RT-qPCR, there is no need for a standard curve and reference material makes it attractive, easier to access and fast to use. However, the main disadvantage of dPCR in monitoring AML patients is that it requires a specially designed assay for each specific gene aberration. NGS or massively parallel sequencing is a revolutionary method of DNA and RNA sequencing. It is called parallel because it sequences millions of DNA fragments simultaneously. Sequencing may be limited to selected segments of certain genes or to the entire exome . The worIn the early years of its appearance, NGS platforms were used primarily for cancer research purposes. Recently, they are increasingly emerging as irreplaceable diagnostic tools in clinical settings. So far, there are several commercially available NGS myeloid panels. They target about 30 genes directly or indirectly involved in the pathophysiology of myeloid neoplasms. Depending on their function, these genes may belong to the group of transcription factors, epigenetic modifiers, signal molecules, etc. ASXL1, SRSF2, EZH2, IDH1 and IDH2 in PMF patients is associated with shorter leukemic-free survival and an increased risk of leukemic transformation [IDH2, U2AF1, EZH2, TP53, SH2B3 and SF3B1 genes are associated with a worse prognosis [The clinical use of NGS proved to be important in demystifying myeloid neoplasms that lack classical chromosomal or gene aberrations. For example, in Ph-MPNs, in addition to the presence of “driver” mutations, the application of NGS in a clinical setting reveals new mutations that facilitate risk stratification and treatment decision . NGS revormation ,56. Simirognosis . NGS enaNPM1 and FLT3-ITD genes, showed that NGS had assured MRD assessment and 95% concordance with RT-qPCR for mutated NPM1 [The idea of MRD assessment with NGS in AML patients has existed since the very beginning of NGS expansion in clinical settings. Scientific studies have shown that 96% of AML patients have at least one driver mutation, and 86% have at least two . By imprted NPM1 . RUNX1 gene evaluation with NGS is also a possible choice for MRD analysis in AML patients. In one study in this context, mutational burden <3.61% was associated with better event-free survival (EFS) and OS [DTA mutations  had a reduced risk of developing relapse, while patients with persistent mutations in other genes had an increased risk of developing relapse [In a study by Morita et al. using targeted sequencing of 295 genes in 131 AML patients, the lower cumulative incidence of relapse (CIR) and better overall survival (OS) were found among patients who had no residual mutations until 30 days after induction therapy . RUNX1 g relapse . PML-RARA, RUNX1-RUNX1T1 and CBFB-MYH11. The NGS method is particularly superior in detecting intra-chromosomal rearrangements compared to FISH, which can detect these changes with great sensitivity only on larger chromosomes [The fact that at least one leukemic mutation is present in a large number of AML patients permits us to believe that any of these mutations may be an appropriate marker for MRD, and NGS can provide an effective MRD assessment. Additionally, NGS can detect reciprocal gene rearrangements such as omosomes . Howeveromosomes . At thisThe future advent of genome-wide approaches in clinical practice could allow the identification of additional driver gene mutations and potential MRD markers suitable for prognosis and innovative therapeutic procedures .TET2, ASXL1, RUNX1, IDH, DNMT3A and others may be present [p53 mutations in 6 of 107 AML patients after cancer treatment [NGS offers precise gene sequencing, but what is the true impact of the discovered mutations on leukemogenesis? These questions remain unclear to clinicians and scientists; therefore, one of the imperfections of NGS is the inability to determine the impact of a particular mutation. Hence, the challenge of introducing it into routine clinical practice . Further present . Along treatment . Thus, tIDH1, IDH2 or FLT3-ITD/TKD genes, as they may represent a hot spot for target therapy. NGS as a newly introduced method will proceed through many more processes of intensive comparative analysis with existing methods of molecular diagnosis before being applied in clinical practice. A multidisciplinary approach must be taken to overcome technical, economic and organizational aspects.It will be of great importance in the future to create updated and extensive cancer databases that would include all mutations that can initiate the leukemogenic process. Moreover, NGS analysis in AML patients is of particular importance applied to the The qualitative and quantitative collection of cell metabolites including carbohydrates, vitamins and lipids featured in a specific condition or in a common metabolic reaction is defined as metabolome. It is a result of biochemical reactions catalyzed by the proteins of the proteome. Therefore, the metabolome can be considered as the interaction of genome and proteome information where microenvironmental factors can play an active role.The understanding of metabolome functions in the past resulted in strong contributions in biological research. Roughly, from the 1920s to the 1960s, the “golden age of biochemistry” clarifies most metabolic networks involved in organisms and that are responsible for nutrient use and energy production. Thus, the main processes such as glycolysis, tricarboxylic acid (TCA), urea cycles respiration, glycogen catabolism, oxidative phosphorylation and the supremacy of ATP in energy transfer reactions have been characterized.The latest scientific findings and the development of “omics” technologies open a modern concept of metabolism as a complex system operating in network with other biological systems. Metabolomics is the analytical and informatic technique used to study the metabolome and to define cell type, tissue, organ or organism metabolomic signatures in a specific condition . MetabolIn the last ten years, differential metabolomics approaches have been used to untangle AML and have paved the way to new information crucial for diagnosis, prognosis and target identification, although the complexity of the related technologies does make it easy to use in routine clinical practice. Indeed, this approach provides several skills that many laboratories do not manage: the equipment is very expensive, the laboratory staff needs optimal knowledge on biochemical pathways and the samples must be prepared with the optimized and reproducible method in order to avoid bias associated to technical procedures .By the Nuclear Magnetic Resonance (NMR)-based metabolism approach, significant serum metabolomic differences between different risk subgroups of AML were found. These studies enriched data concerning several metabolic pathways such as glycolysis, TCA cycle, proteins and lipoproteins biosynthesis, metabolism of fatty acids and cell membrane components . All theBeing an early and noninvasive area of interest in AML, metabolomics can improve the investigation of a new biomarker. Combining gas chromatography coupled with triple quadrupole tandem mass spectrometry and statistical analysis, Musharaf et al. studied differentiated metabolic alterations in the serum of patients with acute lymphoblastic leukemia (ALL), and AML Fatty acid palmitic, stearic and oleic acid metabolism emerged as deregulated in patients with acute leukemia, thus representing an essential metabolic pathway associated with disease progression . Metabolomic profiling by ultra-high performance liquid-chromatography mass spectrometry was used to study the metabolome of FLT3-ITD positive AML , which uRecently, a global untargeted metabolomics approach was used to ascertain sorafenib resistance in FLT3/ITD-mutated leukemic cells . AlteredCombining different types of “omics” represents a promising future goal in order to offer personalized therapeutic approaches even for AML patients with identifiable and targetable genomic lesions ,58,83. RAlthough there were enhancements in AML diagnosis, classification and remission achievement, more than 50% of patients relapsed due to the persistence of residual resistant clones. Considering this, the new era of the AML study approach passed through a precise and accurate evaluation of residual disease.The improvement of new techniques such as ddPCR and NGS, attempting MRD detection, completely modified the relevance of this issue; currently, MRD monitoring has become an objective methodology for setting up remission statuses. In this context, the “minimal” residual disease (MRD) term has been substituted with “measurable” residual disease to emphasize the crucial role of modern techniques in determining the threshold of remission and in activating preventive treatment of molecular relapse. In addition to these technologies, metabolome analysis of AML serum highlighted the possibility to identify significant metabolic signatures suitable for the detection of relapsed patients and of highly responding patients with risk stratification. For this issue, standardization of sample selection and processing will be necessary to improve the use of metabolomics in AML stratification. In the near hematological future, characterizing novel drugs for metabolomics targeting and improving personalized treatment approaches will be challenging.


Resistance of cancer patients to DNA damaging radiation therapy and chemotherapy remains a major problem in the clinic. The current review discusses the molecular mechanisms of therapy resistance in acute myeloid leukemia (AML) conferred by cooperative chemotherapy-induced DNA damage response (DDR) and mutational activation of PI3K/AKT signaling. In addition, strategies to overcome resistance are discussed, with particular focus on studies underpinning the vast potential of therapies combining standard chemotherapy AML regimens with small molecule inhibitors targeting key regulatory hubs at the interface of DDR and oncogenic signaling pathways. Resistance to conventional chemo- and radiotherapy is a mainstay of many cancer entities and a major obstacle in the clinic. Thus, understanding the molecular mechanisms of resistance in cancer patients is essential to define relevant druggable targets for therapeutic interventions and overcome therapy resistance.,2. Survival remains particularly poor for elderly/unfit and relapsed/refractory (R/R) AML patients, who rarely survive beyond two years-3.Acute myeloid leukemia (AML) patients exhibit a dismal overall survival (OS) rate (five-year OS 25%) due to primary resistance or relapse when treated with current standard AML chemotherapy regimens,4-8. Thus, inhibition of drug resistance conferred by aberrant mutational activation of DDR and signaling pathways holds great potential to improve therapy response and OS of AML patients.AML emerges through sequential acquisition of genetic aberrations comprising a few drivers that partially promote abnormal activity of DNA damage response (DDR) and “oncogenic” signaling pathways, which together confer DNA repair, survival, proliferation, and ultimately therapy resistance toward current AML therapies-21.The current review deals with chemotherapy resistance in AML with particular focus on studies highlighting the potential of targeting DDR and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathways to overcome resistance in AML patients. For detailed information on therapeutic strategies targeting DDR and PI3K/AKT signaling pathways in other types of cancers, we refer to recent comprehensive reviewsetc.) ,2,22. Following induction therapy, AML patients will proceed with consolidation therapy, comprising additional cycles of intensive chemotherapy or allogeneic stem cell transplantation, dependent on the individual AML patient’s age, fitness, and genetic risk stratification,2. Currently, only 40% of younger and fit AML patients are long-term survivors , as the majority relapse or exhibit primary resistance (R/R) toward current intensive therapeutic regimens,2. Of the remaining elderly/unfit (> 75 years) AML patients, who are not eligible for intensive standard AML chemotherapy, the vast majority do not survive two years (< 10%) due to limited clinical response to current non-curative standard regimens. These include best supportive care with or without hydroxurea, low-dose cytarabine , and hypomethylating agents , which also cause some degree of DNA damage,2,23. More recent studies have highlighted the substantial therapeutic potential of the anti-apoptotic BCL2 inhibitor venetoclax in combination with current standard AML regimens. More specifically, a seminal phase III trial combining azacytidine with venetoclax demonstrated significant improvement of response rates and survival of elderly/unfit AML patients as compared to single treatment with azacytidine alone . Consistently, preliminary studies combining the hypomethylating agent decitabine (DEC) or azacytidine with venetoclax for R/R AML patients have demonstrated improved response rates  and survival rates  as compared to single treatment with azacytidine or decitabine  and similar response rates when compared to treatment with conventional intensive AML salvage chemotherapy -27.Standard curative treatment of younger/fit AML patients (< 65-75 years) consists of intensive induction chemotherapy encompassing an antimetabolite  and a DNA damaging anthracycline  and WEE1 G2 checkpoint kinase (WEE1). Consistently, combinations of cytarabine and ATR inhibitors have demonstrated combinatorial therapeutic effect in some but not all studies on AML,37. HR and NHEJ are tightly orchestrated by the DDR master regulators ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase, catalytic subunit (DNA-PK), respectively, which through various DDR downstream substrates promote: (1) delay or block of cell cycle progression; (2) repair of DNA; or (3) TP53-mediated apoptosis if DNA is irreversibly damaged,11,38. Hence, given the high rate of relapse and primary resistance of AML patients, there is a compelling rationale to combine DNA damaging standard AML chemotherapy and HMAs, with inhibitors of the DDR master regulators ATR, ATM, DNA-PK, and their DDR downstream substrates, to boost therapeutic efficacy and overcome therapy resistance.Anthracyclines and to some extent HMA induce DNA double-strand breaks (DSB), leading to DDR activation and repair of DSB by homologous recombination (HR) and non-homologous end-joining (NHEJ), respectively,13,15,21.Mutational activation of the PI3K/AKT signaling pathway is a common theme in cancer and is generally associated with a poor response to DNA damaging cancer therapies,40-49.Recent studies, mapping the genomic landscape in large AML cohorts, have identified mutations in more than 60% of AML patients, which directly or through intermediaries confer aberrant “oncogenic” activation of the PI3K/AKT signaling pathway . The lat,50. AKT itself activates numerous downstream targets including mTOR complex 1 (mTORC1), glycogen synthase kinase 3 (GSK3), and forkhead box proteins (FOXO). Together, PI3K/AKT downstream signaling directs: (1) proliferation; (2) survival; (3) glucose metabolism; and (4) DNA repair,21,51,52. Notably, PI3K/AKT exerts these activities partially through: (1) activation of MDM2-promoted TP53 degradation; (2) regulation of DDR molecule activities (CHK1 and BRCA1); and (3) upregulation of the anti-apoptotic MCL1 and BCL2 proteins through the cyclic adenosine monophosphate response element-binding protein (CREBBP)-56. Because PI3K/AKT-dependent MCL1 expression can promote resistance to the recently approved BCL2 inhibitor venetoclax in patients with AML, simultaneous targeting of anti-apoptotic proteins such as MCL1, BCL2, or BCL2L1 (BCL-xL) and PI3K/AKT signaling might cooperatively boost apoptotic activity in AML cells. Indeed, this is corroborated by preclinical PDX trials of solid cancers and AML, highlighting the therapeutic benefit of PI3K/AKT inhibitors in combination with the BCL2 inhibitor venetoclax-59. Since the majority of patients with AML will either not respond or in time develop resistance to venetoclax/azacytidine treatment, there is a strong reason to explore the therapeutic efficacy of combination therapies including venetoclax and PI3K/AKT inhibitors alone or in combination with standard AML chemotherapy/HMAs.Upon activation, PI3Ks and their second messenger phosphatidylinositol 3-phosphate promote phosphoinositide-dependent protein kinase-1 (PDPK1)- and mTOR complex 2 (mTORC2)-dependent phosphorylation of AKT at its T308 and S473 residues, respectivelyIn conclusion, the frequent mutational activation of PI3K/AKT signaling in AML patients, as well as its prominent role in therapy resistance, provides a rationale for targeted inhibition of PI3K/AKT signaling to enhance the efficacy of standard AML therapies and ultimately overcome therapy resistance.,61. Studies have shown that AKT is directly phosphorylated  by the DDR master regulators DNA-PK and ATM upon therapeutic DNA damage,60,62. The latter marks the PI3K/AKT axis as a key regulatory hub, which enhances DNA repair and survival in response to DNA-PK or ATM cross-activation, through collateral regulation of DNA repair and inhibition of TP53-dependent apoptosis,53. Consistently, targeted inhibition of PI3K/AKT signaling can confer replicative stress and cell death of cancer cells, underpinning its role in collateral enhancement of the DDR-mediated resistance to DNA damaging therapies,63. Overall, these findings highlight the potential of therapies combining DDR and/or PI3K/AKT inhibitors with conventional therapies conferring replicative stress and DNA damage.There is compelling evidence of crosstalk activation and regulation between DDR and “oncogenic” signaling pathways including the PI3K/AKT signaling pathway . vs. wild-type KIT-67. In a recent study, Estruch et al. applied a genetically modified inv(16)/KITD816Y AML mouse model, mimicking AML patients with constitutive mutational KIT-dependent activation of the PI3K/AKT signaling pathway, to explore the therapeutic efficacy of standard AML chemotherapy in combination with DDR or PI3K/AKT inhibitors . Treatment of inv(16)/KITD816Y AML cells with chemotherapy markedly increased activation of AKT via DNA-PK-dependent phosphorylation. Complementary mechanistic experiments further demonstrated that chemotherapy in combination with either PI3K or DNA-PK inhibitors significantly decreased chemotherapy-induced activation of AKT signaling, leading to a significant increase of DNA damage and apoptosis of inv(16)/KITD816Y AML cells. Subsequent treatment, with a PI3K or a DNA-PK inhibitor in combination with standard AML chemotherapy, synergistically inhibited in vitro growth and survival of AML cells in clonogenic assays. Consistently, combination of standard AML chemotherapy and these inhibitors significantly improved overall survival of inv(16)/KITD816Y AML mice in preclinical trials compared to single treated or untreated mice. Intriguingly, treatment with the PI3K/AKT inhibitor alone abrogated PI3K signaling activity and exhibited some therapeutic activity in clonogenic assays and preclinical trials, whereas single treatment with the DNA-PK inhibitor elicited no therapeutic effect due to lack of DNA-PK activation in absence of chemotherapy-induced DNA damage.Mutational activation of the PI3K/AKT signaling pathway in AML patients is associated with poor clinical outcome when treated with intensive AML chemotherapy regimens including cytarabine and anthracyclines. The latter is illustrated by a significantly higher relapse rate of t and inv(16) AML patients harboring gain-of-function KIT mutations et al. recently demonstrated that simultaneous treatment with a FLT3 inhibitor  and standard AML chemotherapy markedly enhanced chemotherapy response in FLT3-ITD mutant AML patient-derived xenografts.In agreement with these findings, Ueno Together, these preclinical studies suggest that DNA repair and survival of AML cells mediated by therapy-induced activation of a DDR and its collateral enhancement of constitutive mutational PI3K/AKT activation represents a key resistance mechanism in AML patients treated with DNA damaging therapies. Both studies further suggest that AML patients, who have a high mutational PI3K/AKT signaling activity potentially will benefit from combinatorial treatment with chemotherapy and DDR inhibitors and/or direct PI3K/AKT inhibitors or, alternatively, inhibitors of upstream PI3K/AKT activators such as KIT, FLT3, JAK2, ASXL1, and NRAS/KRAS. Since both standard AML induction chemotherapy and HMAs induce DSBs, which boost PI3K/AKT signaling in a DDR-dependent manner, the majority of AML patients treated with curative or non-curative therapeutic regimen will potentially benefit from complementary treatment with relevant small molecule inhibitors.,70. Moreover, two recent studies depicted in ,71.Currently, DDR inhibitors remain to be approved for treatment of AML patients. However, several DDR inhibitors are currently being tested in clinical trials of solid cancers and AML, alone or in combination with DNA damaging and cytotoxic chemotherapy and radiation therapies, including CC-115 (DNA-PKi/MTORi), nedisertib/M3814 (DNA-PKi), VE-821 (ATMi/ATRi), and VX-984 (DNA-PKi),72. However, recent studies have demonstrated that simultaneous treatment with FLT3 inhibitors, which potentially block mutational PI3K/AKT downstream signaling, significantly improved therapeutic efficacy of: (1) HMAs in elderly/unfit FLT3 mutant AML patients (azacytidine/gilteritinib); and (2) standard AML chemotherapy in younger FLT3 mutant AML patients (cytarabine/anthracycline/midostaurin or cytarabine/anthracycline/quizartinib) .  0.0001) [76]. Con= 0.005) [77].In conclusion, clinical trials highlight the therapeutic potential of DDR and PI3K/AKT inhibitor combination therapies and advocate for future AML trials investigating the efficacy of dual or triple combination therapies including DDR and/or PI3K inhibitors in combination with either chemotherapy or HMAs.vs. sequential drug administration, and finally the length of treatment cycles. In this respect, recent experimental and clinical studies underscore that inhibitors of DDR and PI3K/AKT signaling pathways including FLT3 inhibitors should be administered simultaneously rather than sequentially with chemotherapy/HMAs to potentiate rather than complement therapeutic efficacy, as is the case with AML treatment regimens and ongoing trials combining standard AML chemotherapy with sequential inhibitor administration,75. Although simultaneous treatment of AML patients with chemotherapy/HMAs and complementary drugs frequently comes with enhanced toxicity, primarily in the form of prolonged cytopenia and associated neutropenic infections, recent AML studies have demonstrated manageable toxicity and overall clinical benefit of standard AML therapies in combination with simultaneous administration of novel drugs,78-80. However, future clinical trials are needed to define the optimal drug dosages for combination therapies encompassing a backbone of standard AML chemotherapy or HMAs and inhibitors of DDR and/or PI3K/AKT signaling pathways. Ideally, such clinical trials should be accompanied by exploratory biomarker analyses for guided treatment of responders and prevention of overtreatment of non-responders. Such biomarker assessment should include NGS-based genomics and complementary functional analyses such as drug screening and phosphoproteomics to predict AML patients responding to specific inhibitors of DDR and PI3K/AKT signaling pathways alone or in combination with chemotherapy/HMAs.Conceptually, cancer poly-therapies should combine drugs which: (1) exhibit additive or synergistic therapeutic activity; (2) target distinct cancer vulnerabilities to circumvent the development of resistance; and (3) can be administered at active therapeutic doses without surpassing clinically manageable levels of toxicity. Hence, the hallmark of cancer therapies combining novel drugs with standard therapies relates to whether their higher therapeutic efficacy outweighs the increase of toxicity and ultimately leads to a significant clinical benefit. The latter is particularly relevant in AML, considering the substantial toxicity of standard AML induction chemotherapies in younger AML patients as well as HMAs in elderly/unfit AML patients. Thus, the balance of benefit and toxicity is key to the design of clinical trials combining DDR and/or PI3K/AKT inhibitors with current AML therapies in terms of drug doses, duration of drug administration during treatment cycles, concomitant Oncogenic DDR and PI3K/AKT signaling activity is a common theme in various cancers, including AML, and is frequently associated with therapy resistance and poor clinical outcome. Hence, therapeutic targeting of DDR and PI3K/AKT signaling pathways alone or in combination with current standard AML chemotherapy and HMAs stands out as a potential strategy to overcome resistance and improve clinical outcome of AML patients. In a broader perspective, current findings highlight the vast potential of combining conventional DNA-damaging therapies with inhibition of signaling molecules at the interface of DDR and oncogenic signaling pathways to overcome therapy resistance and improve clinical outcome in patients with AML as well as other cancer entities.


However, the underlying mechanism is unclear. Recent establishment of the molecular identity of the mitochondrial Ca2+ uniporter (MCU) and associated proteins allows modification of mitochondrial Ca2+ transport in intact cells. We examined the consequences of deficiency of the accessory protein MICU2 in rat and human insulin-secreting cells and mouse islets.Transport of CaMicu2 in the INS-1 832/13 and EndoC-βH1 cell lines was performed; Micu2−/− mice were also studied. Insulin secretion and mechanistic analyses utilizing live confocal imaging to assess mitochondrial function and intracellular Ca2+ dynamics were performed.siRNA silencing of Micu2 abrogated GSIS in the INS-1 832/13 and EndoC-βH1 cells. The Micu2−/− mice also displayed attenuated GSIS. Mitochondrial Ca2+ uptake declined in MICU2-deficient INS-1 832/13 and EndoC-βH1 cells in response to high glucose and high K+. MICU2 silencing in INS-1 832/13 cells, presumably through its effects on mitochondrial Ca2+ uptake, perturbed mitochondrial function illustrated by absent mitochondrial membrane hyperpolarization and lowering of the ATP/ADP ratio in response to elevated glucose. Despite the loss of mitochondrial Ca2+ uptake, cytosolic Ca2+ was lower in siMICU2-treated INS-1 832/13 cells in response to high K+. It was hypothesized that Ca2+ accumulated in the submembrane compartment in MICU2-deficient cells, resulting in desensitization of voltage-dependent Ca2+ channels, lowering total cytosolic Ca2+. Upon high K+ stimulation, MICU2-silenced cells showed higher and prolonged increases in submembrane Ca2+ levels.Silencing of 2+ uptake. β cell mitochondria sequestered Ca2+ from the submembrane compartment, preventing desensitization of voltage-dependent Ca2+ channels and facilitating GSIS.MICU2 plays a critical role in β cell mitochondrial Ca  •MICU2 deficiency impairs glucose-stimulated insulin secretion.•MICU2 deficiency abrogates mitochondrial calcium uptake.•MICU2-deficient cells.Depolarization-evoked cytosolic calcium increases are lower in •Mitochondria clear calcium from the subplasma membrane region, maintaining calcium influx. INS-1 832/13 and HEK-293T cells were seeded onto poly-d-lysine-coated 8-well-chambered cover glasses  at a cell density of 70,000 cells/cm2. Twenty-four h after seeding, the cells were co-transfected with siRNA and 1 μg of plasmid encoding mito-case12 at approximately 50% cell confluency. The cells were further grown for 48–72 h before measurements. On the day of imaging, the cells were pre-incubated at 37 °C in 400 μL of buffer P containing 2.8 mM of glucose. After 1.5 h, the cells were imaged using a 488 nm laser and emission recorded at 505–530 nm on a Zeiss LSM510 inverted confocal fluorescence microscope using a 100x/1.45 objective. Images were recorded at a frequency of 0.25 Hz (scan time = 3.93 s).Single-cell mito was performed on a Zeiss LSM510 inverted confocal fluorescence microscope using a 100x/1.45 objective. Images were recorded at a frequency of 0.13 Hz (scan time = 7.86 s).EndoC-βH1 cells were seeded onto poly-2.102+]c and [Ca2+]mito in EndoC- βH1, the protocol was the same except that after 1.5 h of incubation, 2 μM of Rhod-2 AM (Abcam) and 2 μM of Fluo8 AM were loaded for 30 min at 37 °C in buffer P. Imaging was performed on a Zeiss LSM510 inverted confocal fluorescence microscope using a 40x/0.75 objective with images recorded at a frequency of 0.30 Hz (scan time = 3.36 s).For experiments measuring the effect of 36 mM of KCl on mem measurements were carried out using PeriCam-based genetically encoded inner plasma membrane-targeted Ca2+ probe mem-case12 (Evrogen cat# FP992.). Specifically, INS-1 832/13 and HEK-293T cells were seeded onto poly-d-lysine-coated 8-well-chambered cover glasses  at a cell density of 70,000 cells/cm2. After 24 h of seeding, the cells were co-transfected with siRNA and 1 μg of plasmid-encoding mem-case12 at approximately 50% cell confluency. The cells were then further grown for 48–72 h before measurements. On the day of imaging, the cells were pre-incubated at 37 °C in 400 μL of buffer P containing 2.8 mM of glucose. After 1.5 h, the cells were imaged at 488 nm excitation (8%) and emission recorded at 505–535 nm on a Zeiss LSM510 inverted confocal fluorescence microscope using a 100x/1.45 objective. Images were recorded at a frequency of 0.64 Hz (scan time = 1.57 s).Single-cell mem) to be determined. If mitochondrial Ca2+ sequestration was indeed facilitating net Ca2+ entry into the cell by removing Ca2+ from the subplasma membrane compartment, then it would be predicted that cells with defective mitochondrial Ca2+ transport would show enhanced subplasmalemmal Ca2+ elevation in response to depolarization by KCl. Indeed, the maximal [Ca2+]mem elevation was 20% greater in MICU2-deficient INS-1 832/13 cells than in control cells  cytosolic ATP production. Mitochondrial Ca2+ transport, however, being dependent upon Δψm, is abolished [2+]mem elevation, the Ca2+ concentration remained elevated longer at the membrane, with a delayed return to the basal state  were selected, after which the starvation medium was rapidly replaced with medium containing 36 mM of KCl and the recording started simultaneously. It was expected that MICU2-deficient cells would show enhanced subplasmalemmal Ca2+ elevation in response to depolarization by KCl, reflected by a stronger fluorescence signal intensity at the edge of the line scan. We found that, upon KCl-induced depolarization, Ca2+ accumulated at the subplasma membrane compartment in siMICU2-treated INS-1 832/13 cells  at the edge of the cell compared to its center (p = 0.0266). Furthermore, there was a 2.5-fold higher Ca2+ response at the submembrane compartment in MICU2-deficient INS-1 832/13 cells compared to controls  insulin secretion was significantly higher compared to that in their wild-type counterparts . Both strains of mice responded robustly to insulin, with decreased plasma glucose levels  [2+]mito on ATP production would be unlikely to reach the threshold to affect KATP channel activity, meaning using oligomycin was not necessary. Reminiscent of the findings in INS-1 832/13 cells, [Ca2+]mem elevation upon 50 mM of KCl stimulation increased by 35% in MICU2-silenced HEK293T cells compared to the control cells ethane-N,N,N′,N′-tetraacetic acid (BAPTA) mito and the cytosolic ATP/ADP ratio in response to glucose in mouse β cells mito, cytosolic ATP levels, and GSIS in clonal insulin-secreting cells mito affects mitochondrial metabolism and insulin secretion. However, our experiments could not resolve how Micu2 knockout mice maintained euglycemia despite reduced early insulin secretion. Insulin tolerance, albeit a rough estimate of insulin action in vivo, was unchanged. In fact, we previously reported that ITT is not sensitive enough to reflect changes in insulin sensitivity in the face of reduced early insulin secretion in vivo mito in β cells. However, the mechanisms by which MICU2 and [Ca2+]mito might exert control over GSIS were unexpected and add a new dimension to how mitochondrial Ca2+ transport might regulate β cell function and insulin secretion.Herein, using  studies ,9,61 sho in vivo . This phsitivity . A hyper2+ uptake exerts a profound stimulatory effect on the net flux of cations across the plasma membrane, either by enhancing entry or depressing efflux. Insulin-secreting cells have a high concentration of subplasma membrane mitochondria, with roughly 20% of the membrane inner surface associated with mitochondria exerting a strong Ca2+-buffering effect c is seen with elevated K+ as well as with high glucose and is thus not directly dependent on bioenergetics. Had this effect only been observed in response to glucose, then the previously postulated positive effect of mitochondrial Ca2+ uptake on mitochondrial dehydrogenases would have been the most plausible explanation [To rationalize a decreased Ca deficit . In any lanation .2+]c elevation following inhibited transport of mitochondrial Ca2+ was previously observed in cultured neurons c transients induced by Ca2+ entry through NMDA-selective glutamate receptors or voltage-dependent Ca2+ channels drastically reduced. In the present study, treatment with oligomycin + antimycin further confirmed a role of mitochondrial Ca2+ uptake in clearing subplasmalemmal Ca2+ in insulin-secreting cells, and the observations were similar to those in neurons c amplitude wave propagation via feedback regulation of plasma membrane channel activity in diverse cellular systems, including rat cortical astrocytes and Xenopus laevis oocytes mem between Micu2 silencing and treatment with oligomycin/antimycin, whereby the former caused an increase in the duration of the subplasmalemmal Ca2+ increase while the latter resulted in an increased magnitude of the response. This may be explained by the different mechanisms by which mitochondrial Ca2+ uptake is inhibited. When MICU2 is deficient, uptake at low Ca2+ is likely to increase as the protein plays an important role in suppressing MCU activity under these conditions [2+]c is elevated, the mitochondrial buffering capacity decreases. In contrast, oligomycin and antimycin prevent proton transport from the mitochondrial matrix to the mitochondrial intermembrane space, increasing the matrix pH, subsequently collapsing Δψm and inhibiting Ca2+ entry. These two methods affect the Ca2+ electrochemical gradient differently and as a result the Ca2+ uptake kinetics, potentially explaining the differential effects on [Ca2+]mem.It was hypothesized that mitochondria are able to deplete a subplasma membrane layer of elevated Ca2+ ,46. Volt removed ,69,70. Iecretion . Further oocytes ,75. The ted Ca2+ ,46 durinnditions ,13,16,17d, Fluo4 is unsuitable for measuring [Ca2+]mem, which can reach concentrations of 10–100 μM mem was in fact an underestimation. Moreover, the saturation problems were mitigated against the complementary findings using the lower-affinity mem-case12 probe (Kd = 1 μM) mem effect on MICU2-silenced Endo C-βH1 cells may lie in differences in the composition of voltage-gated Ca2+ channels between the two cell lines. Notably, the L-type Ca2+ channel current, which is largely responsible for Ca2+ entry upon high K+ stimulation due to the rapid inactivation of other Ca2+ channels [2+ current in Endo C-βH1 cells [2+ current in INS-1 832/13 cells [2+]mem levels attained in EndoC-βH1 cells in the line scan experiment may still be able to be cleared by MICU2-deficient mitochondria. Thus, any buildup of Ca2+ in the subplasmalemmal compartment may occur at a slower rate, evading detection in our experiments. This would also explain the smaller effect of MICU2 silencing on depolarization-evoked [Ca2+]c increases in EndoC-βH1.The lack of a c increases may also occur as a consequence of endoplasmic reticulum Ca2+ release. Indeed, there is evidence in β cells of a basal endoplasmic reticulum Ca2+ leak channeled toward the mitochondria, which stimulates mitochondrial bioenergetics, priming the mitochondria for an increase in glucose mito and the associated increase in [Ca2+]mem were more or less instantaneous upon exposure to KCl and plasma membrane depolarization as opposed to the endoplasmic reticulum Ca2+ leak, which seems to occur at a constant rate c alone c alone . However of GSIS . However of GSIS .52+ into mitochondria located at the vicinity of the voltage-dependent Ca2+ channels in the plasma membrane that coupled with the well-established bioenergetic effects accounts for the regulatory effect of mitochondrial Ca2+ uptake on insulin secretion. The recent molecular characterization of the mitochondrial Ca2+ uniporter holocomplex holds promise for further elucidating the complexities of β cell stimulus-secretion coupling in which the mitochondria are at center stage.In summary, we uncovered a crucial role of mitochondria in maintaining β cell excitability involving sequestering of CaMicu2 knockout mice. A.T., V.M., D.G.N., and H.M. provided critical insights to the project. N.V., A.H., A.T., V.M., D.G.N., and H.M. wrote the manuscript.N.V., A.H., D.G.N., and H.M. designed the study. N.V., A.H., A.B., A.M.B., H.B., Y.S., K.K., P.S., E.C., and M.F. performed the experiments. N.V., A.H., A.M.B., and M.F. analyzed the data. V.M. provided the All of the experimental animal protocols were approved by the local animal ethical committee of Lund/Malmö and complied with the guidelines of the Federation of European Laboratory Animal Science Association (FELASA).


However, the molecular targets of dystrophin deficiency-dependent Ca2+ overload and the correlation between Ca2+ overload and contractile DMD phenotypes in in vitro human models remain largely elusive. In this study, we utilized DMD patient-derived induced pluripotent stem cells (iPSCs) to differentiate myotubes using doxycycline-inducible MyoD overexpression, and searched for a target molecule that mediates dystrophin deficiency-dependent Ca2+ overload using commercially available chemicals and siRNAs. We found that several store-operated Ca2+ channel (SOC) inhibitors effectively prevented Ca2+ overload and identified that STIM1–Orai1 is a molecular target of SOCs. These findings were further confirmed by demonstrating that STIM1–Orai1 inhibitors, CM4620, AnCoA4, and GSK797A, prevented Ca2+ overload in dystrophic myotubes. Finally, we evaluated CM4620, AnCoA4, and GSK7975A activities using a previously reported model recapitulating a muscle fatigue-like decline in contractile performance in DMD. All three chemicals ameliorated the decline in contractile performance, indicating that modulating STIM1–Orai1-mediated Ca2+ overload is effective in rescuing contractile phenotypes. In conclusion, SOCs are major contributors to dystrophin deficiency-dependent Ca2+ overload through STIM1–Orai1 as molecular mediators. Modulating STIM1–Orai1 activity was effective in ameliorating the decline in contractile performance in DMD.Ca However, the association between dystrophin deficiency and pathogenesis progression remains unclear, and related treatment options targeting such mechanisms have not yet been developed.Duchenne muscular dystrophy (DMD) is a progressive muscular degeneration disease caused by the complete loss of dystrophin protein, eventually leading to ambulatory and respiratory deficiency, whose symptoms typically occur in early childhood . AlthougInduced pluripotent stem cell (iPSC) technology has markedly advanced the field of regenerative medicine, including cell therapies and drug development, as well as basic science, facilitating the understanding of intractable disease pathogenesis . It is i2+ overload has been widely accepted as a candidate regulating DMD pathogenesis SR) reduction, and SOCs normally provide Ca2+ to refill the SR and maintain homeostasis [2+ sensor in the SR and Orai1 localizes in the transverse tubule membrane and functions as a Ca2+ channel [2+ mobilization regulates skeletal muscle growth and fatigue as well as exacerbating Ca2+ entry in dystrophic muscles in mdx mice [2+ homeostasis during skeletal muscle development and function [2+ mobilization is important for muscle development and function [2+ mobilization contributes to Ca2+ overload as well as functional phenotypes in an in vitro human model remains unknown.Caogenesis . Howevered iPSCs . Muscle ntration . SOCs areostasis . STIM1 aeostasis . STIM1 l channel ,23,24. Omdx mice . In contfunction . In addifunction . Thus, Sfunction . Moreovefunction . However2+ overload in myotubes differentiated from DMD patient-derived iPSCs using small-scale chemical screening. siRNA-mediated gene knockdown identified STIM1 and Orai1 as potential targets responsible for SOC-mediated Ca2+ overload in skeletal muscle cells. In addition, this result was further confirmed using STIM1 and Orai1 inhibitors, which prevented Ca2+ overload in dystrophic myotubes. Finally, to demonstrate how modulation of Ca2+ overload is linked to the functional rescue of dystrophic phenotypes, we utilized a recently reported disease model to evaluate the contractile performance decline in DMD. Orai1 and STIM1 inhibitor administration successfully rescued the decline in contractile performance. Thus, this study indicated that modulating STIM1–Orai1-regulated Ca2+ overload improves functional phenotypes and their potential for use in therapeutic purposes.In this study, we identified that SOCs are potent regulators of dystrophin deficiency-mediated CaThis study was approved by the Ethics Committee of the Graduate School of Medicine, Kyoto University and Kyoto University Hospital , and Takeda Pharmaceutical Company Ltd.  and conducted according to the guidelines of the Declaration of Helsinki. All patient information was kept confidential, and written informed consent was obtained from all patients.The Δ44 DMD-iPSCs established from skin fibroblasts of a DMD patient  and its isogenic control line, ∆44 DMD-ctrl-iPSCs as described previously ,29, wereFor the preparation of laminin-coated plates, 1.5 or 8 mL of Easy iMatrix-511 silk  were added to each well of the six-well plates or a 10-cm dish, respectively, and incubated overnight at 4 °C. The coated plates were stored at 4 °C for up to 2 weeks and equilibrated at 24 °C for at least 30 min prior to use. StemFit AK02N  (750 µL or 3 mL) was added to wash the plates and aspirated. For the Matrigel-coated plates, all tubes, pipette tips, and reagents were pre-chilled in the fridge or on ice. Matrigel  was prepared at 1:100 dilution with pre-chilled media without any additives. An appropriate volume of diluted Matrigel was dispensed into each well of the 6- or 96-well plates, which were incubated overnight at 4 °C and stored at 4 °C for a few weeks until use. For the gel culture, 2 mL or 10 µL of 0.5 mg/mL collagen solution (Nippi) were dispensed into a well of 6-well plates (BD) or µPlate Angiogenesis 96 plate  and solidified in an incubator at 37 °C overnight. The plates were then coated with Matrigel.6 cells were resuspended in Opti-MEM . Doxycycline (Dox)-inducible MyoD1-expressing piggyBac vector, Tet-MyoD [piggyBac transposase vector PBaseII using NEPA 21 at 125 V for 5 ms . After selecting the successfully differentiated clones from puromycin-resistant colonies, the cells were established and used for experiments.iPSC lines stably expressing Tet-inducible MyoD1 were generated as described previously . BrieflyTet-MyoD , was co-4 cells/well in 6-well plates or 10-cm dishes for the first two days after plating. At 48 h after passaging, Y-27632 was removed and replaced with StemFit containing a suitable antibiotic. The medium was changed at least every other day.The iPSCs were cultured on laminin-coated plates in StemFit AK02N  containing 100 µg/mL G418 or 0.5 µg/mL puromycin, depending on the antibiotic resistance of each line. The cells were passaged every seven days using Accutase  and seed4 cells/cm2. After 24 and 48 h, the media were replaced with primate ES cell medium  and PECM+1 µg/mL Dox , respectively. The next day, the pre-differentiated iPSCs were treated with Accutase and re-plated into new Matrigel-coated microplates in 5% knockout serum replacement  in αMEM +Y-27632+1 µg/mL Dox. The medium was replaced every other day with fresh medium. To induce further maturation of myotubes, the cells were cultured in 2% horse serum /αMEM. The medium was replaced a few times per week.The cells were re-plated as described previously . Briefly4 cells/cm2. After 24 and 48 h, the media was replaced with PECM and PECM+0.3 µg/mL Dox, respectively. After an additional 48 h, the pre-differentiated iPSCs were treated with Accutase and re-plated into new Matrigel-coated microplates or hydrogels in 2% HS/αMEM supplemented with 200 µM 2-mercaptoethanol , 4.5 g/L glucose (Invitrogen), 10 µg/mL insulin , SB431542 (Wako) and 3 µM Y-27632. After two days, the medium was replaced with 2% HS/αMEM+1 µg/mL Dox and changed every other day. To induce further maturation of myotubes, doxycycline was removed from the media and the cells were cultured in 2% HS/αMEM for 3 weeks. The medium was replaced every other day.iPSCs were treated with Accutase and plated on Matrigel-coated plates in StemFit+Y at 3.0 × 10pmol Silencer Select Negative Control No.1 siRNA or Silencer Select STIM1L and STIM1S  by forward transfection with 1  article ) on day The cells were fixed with 2% paraformaldehyde (PFA)/PBS (Wako) and methanol (Wako), blocked with Blocking One  for 45 min, and subsequently incubated with primary antibodies diluted in 5% Blocking One/PBST (Wako) at 4 °C overnight. The cells were washed in PBS and incubated with secondary antibodies diluted in 5% Blocking One/PBST for 1 h at room temperature (24–26 °C). Then, 4′,6-diamidino-2-phenylindole  was used to counterstain the nuclei. The samples were visualized and photographed with a BZ-710X  or Opera Phenix System . The primary antibodies used for this study were: mouse anti-myosin heavy-chain (pan-MHC) monoclonal , mouse anti-dystrophin (Rod domain) monoclonal , rabbit anti-STIM1 monoclonal , and mouse anti-Orai1 monoclonal  antibodies. Alexa Fluor 488-conjugated anti-mouse/rabbit and Alexa Fluor 647-conjugated anti-mouse/rabbit antibodies  were used as secondary antibodies.RPLP) served as the reference gene for TaqMan assays, and TATA-binding protein (TBP) served as a reference gene for SYBR Green assays. The following TaqMan probes were used: MYH1 (Hs00428600), MYH2 (Hs00430042), MYH3 (Hs01074230), MYH4 (Hs00757977), MYH7 (Hs0110632), MYH8 (Hs00267293), IL-1β (Hs00174097), TNFα (Hs01113624), IL6 (Hs00985639), and RPLP0 (Hs99999902). The sequences for the primers used for SYBR Green are shown in the Total RNA was isolated using the RNeasy Mini Kit  according to the manufacturer’s protocol. The residual genomic DNA was digested and removed using DNase I (Qiagen). First-strand cDNA was generated from 100 ng of total RNA using a PrimeScript RT reagent kit . Quantitative PCR was performed using PowerUp SYBR Green (ThermoFisher) or TaqMan assay  and QuantStudio 7 Flex (Applied Biosystems). Ribosomal protein lateral stalk subunit P0  in the presence of a cOmplete Mini protease inhibitor cocktail (Roche). Protein concentrations were determined using a bicinchoninic acid (BCA) protein assay kit (Takara). For sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE), 4 × Laemmli sample buffer and any KD mini-protean TGX precast gel with Mini-Protean Tetra Cell  were used to load samples, except the DYS1 and RYR1 samples, which were loaded using 4 × LDS sample buffer, 10 × reducing agent and 3–8%NuPAGE, and Tris-acetate protein precast gels with Mini Gel Tank (ThermoFisher). Western blot analysis was performed using the iBind Flex Western Device (ThermoFisher), PVDF membrane (Bio-Rad), and the following primary antibodies: mouse anti-myosin heavy-chain (pan-MHC) monoclonal , mouse anti-dystrophin (Rod domain) monoclonal , and rabbit anti-TBP monoclonal  antibodies. A horseradish peroxidase (HRP)-conjugated rabbit anti-mouse antibody  was used as a secondary antibody, and ECL Select or Prime Western Blotting Detection Reagent (GE) were used for protein visualization.2+ mobilization assay was conducted as previously described [2+ indicator , in FluoroBrite Dulbecco’s modified Eagle’s medium  in the presence or absence of each chemical and dimethyl sulfoxide (DMSO) control and incubated at 37 °C for 1 h. The fluorescence was detected using an FDSS/µCELL system . Electrical stimulation was applied at 15 V with a 50-ms interval and 0.2 Hz mono phase for 1 min after a 5-s resting phase. The fluorescence was measured at 480 nm excitation and 540 nm emission using an LED excitation light source and an electron-multiplying charge-coupled device (EMCCD) camera. Measurements for 96 wells were performed under uniform conditions at 37 °C with simultaneous stimulation and detection.The Caescribed . Briefly2+ was determined according to a previously published protocol [2. Then, they were washed with Ca2+-free Ringer’s solution for 30 min in an incubator, and fresh Ca2+-free Ringer’s solution was added. The cells were excited at 340 and 380 nm, and the emission at 535 nm was captured using an EnVision (Perkin Elmer). A Ca2+ release cocktail containing 10 µM ionomycin (Wako), 30 µM CPA, and 100 µM EGTA in Ca2+-free Ringer’s solution was injected 5 s after the start of imaging. The total releasable Ca2+ content was determined by calculating the difference between the basal and peak fura-FF ratios.Total releasable Caprotocol ,35. Brie2. Six-well hydrogel or collagen gel plates with cells were cultured in an incubator equipped with a C-Dish connected to the C-Pace EP. An EFS was applied between 2 and 20 V with a 2-ms interval and 0.5 and 1 Hz. The movie was recorded for 270 frames at 27 f/s  and analyzed using the associated software.The cells were differentiated on Matrigel-coated 6-well collagen gel plates and stimulated by an EFS system with C-Pace EP and 6-well C-Dish  for differentiated myotube maturation and muscle cell training. A movie was recorded and analyzed using an SI8000 Motion Imaging System  equipped with an inverted fluorescent microscope  and a stage top incubator  to maintain the cells under humidified conditions at 37 °C and 5% COThe cultured cells were visualized under a BZ-X710 fluorescence microscope (Keyence) and an Opera Phenix High-Content Screening System (PerkinElmer). The images were acquired and analyzed using the associated software.t-test, Dunnett’s test, or one-way analysis of variance (ANOVA), followed by post-hoc Dunnett’s test using GraphPad Prism . Statistical significance was set at p < 0.05.All experiments were conducted independently at least in triplicate. Data are reported as the mean ± standard deviation (SD) and were analyzed using an unpaired 2+ overload condition was recapitulated from dystrophic myotubes using the standard method. In the standard replating method, both DMD∆44-ctrl and DMD∆44 iPSCs were plated on a 10-cm dish and treated with doxycycline to induce MyoD-overexpression-mediated myogenic differentiation before the cells were replated on day 3. On day 3, cells were plated in a 96-well plate in the presence of rock inhibitor and doxycycline in 5% KSR medium and assayed on day 9 of myogenic differentiation. An isogenic control of DMD∆44, whose dystrophin expression was restored [2+ mobilization assays using a Ca2+ indicator, and Ca2+ overload conditions were observed in DMD∆44 myotubes , Na+/Ca2+ exchanger, and SOC, we chemically screened the channel responsible for Ca2+ overload in the model using 10 µM solutions of commercially available compounds with different chemical activities and molecular targets (2+ overload (2+ uptake inhibitor) [2+ mobilization assay and confirmed the effects in a dose-dependent manner  , did nott manner F. Althou2+ overload in dystrophic myotubes. STIM1 and Orai1 are well-known SOCs, and the interaction between STIM1 and Orai1 regulates intracellular Ca2+ levels [2+ levels in SR, STIM1 relocates to the plasma membrane–SR junction to bind to Orai1, triggering Orai1 activation. Thus, the prevention of STIM1 or Orai1 binding reduces Orai1-induced Ca2+ influx into the cytosol. Therefore, we studied whether Orai1 and STIM1 inhibitors also inhibit Ca2+ overload. First, we checked the mRNA expression levels of Orai1 and STIM1 during myogenic differentiation and found that both mRNA expression levels increased -mediated muscle training model [2+ overload and contractile phenotypes remains unknown. To address this question, we evaluated the activities of CM4620, AnCoA4, and GSK7975A in this training model. In the DMSO control of DMD∆44-ctrl, the contractile performance was maintained for two weeks , intracellular space (~100 nM), and inside the SR (~0.4 mM) were precisely maintained under normal conditions [2+ overload is defined as a condition where the intracellular Ca2+ concentration is abnormally higher than normal, leading to abnormally activated Ca2+ signaling. In turn, it is expected that abnormally activated Ca2+ signaling activates calpain signaling or proteolysis, ultimately leading to muscle weakness and degeneration [2+ overload and muscle function in DMD has been well studied in animal models. Tutdibi et al. reported that ion channel blockers reduce Ca2+ entry into muscle fibers in mdx mice [2+ influx is sufficient to induce DMD in mdx mice [mdx mice are leaky in handling Ca2+ [2+ release channel stabilizer, improves muscle fiber function in mdx mice, and Rycal ARM210/S48168 has been used in a clinical stage program [2+ overload conditions have been recapitulated in vitro using human cells [2+ overload conditions lead to the functional phenotype in human models remains unclear. Thus, a disease model that recapitulates functional DMD phenotypes is required for functional analysis, evaluation of potential compounds, and molecular pathogenesis studies.Caogenesis . While inditions . Ca2+ ovneration . In contmdx mice . Milay emdx mice . Bellinging Ca2+ . Moreovean cells ,36, whet2+ overload and correlations between Ca2+ overload and functional activities using myotubes differentiated from patient-derived iPSCs using a recently reported method [2+ could originate from both intracellular and extracellular space. Inside the cells, the SR–mitochondria axis contributes to increased Ca2+ levels in DMD. Upon cellular stimulation, robust Ca2+ is released from the SR to the cytoplasm. Thus, if the released Ca2+ amount from the SR is abnormally increased, it could contribute to Ca2+ overload. On the contrary, mitochondrial Ca2+ uptake may also contribute to Ca2+ overload. Normally, the Ca2+ concentration in the mitochondria is ~100 nM. However, it would reach ~100 µM upon Ca2+ signaling activation through mitochondrial Ca2 uptake [2+. Dubinin et al. reported decreased mitochondrial Ca2+ uptake in mdx mice, indicating that mitochondria could also contribute to Ca2+ overload in DMD [2+ overload in our model remains unknown, it would be interesting to further investigate this in future studies. However, Michelucci et al. reported a correlation between reduced Ca2+ content in the SR and increased Ca2+ influx through SOCs [2+ content in dystrophic myotubes compared to that in control myotubes upon ionomycin application. While the molecular mechanism remains unclear, there are a few possible explanations. First, the responses of myotubes to ionomycin and EFS are different. Ionomycin is a calcium ionophore that facilitates Ca2+ transport across the plasma membrane, leading to Ca2+ release from intracellular stores [2+ depletion in the SR compared to ionomycin. Thus, it will be important for future studies to assess the details of SR Ca2+ homeostasis in dystrophic myotubes under EFS. Second, correlated with this result, Robin et al. reported that the total SR Ca2+ content was reduced in mdx fibers compared to that in control fibers [2+ influx and SR leak contribute to high cytosolic Ca2+ levels in DMD. Thus, a high SR leak in DMD might explain the lower total SR Ca2+ store content in dystrophic myotubes than that in control myotubes reported in this study. Abnormal Ca2+ homeostasis in dystrophic myotubes may increase Ca2+ influx from the extracellular space as well as Ca2+ fluxes from the SR upon EFS. If this is a specific phenotype of dystrophic myotubes, further investigation of how dystrophin regulates the Ca2+ content in the SR and SOC activities, along with the reduction of Ca2+ in the SR could be a key to identify the molecular function of dystrophin in DMD.This study aimed to reveal the molecular target of how dystrophin deficiency causes Cad method . Initial2 uptake ,49. Mitod in DMD ,50. Sincugh SOCs ,35. An ur stores . On the r stores , which ml fibers . They al2+ inhibitors/modulators, we identified that SOCs were the most attractive targets for regulating dystrophin deficiency-mediated Ca2+ overload. The role of SOCs in abnormal Ca2+ handling in DMD has been investigated, and targeting of SOCs to restore Ca2+ homeostasis for DMD therapies has been discussed previously [2+ channels, such as TRPC1, TRPV, and TRPM7, also contribute to SOC activity [2+ overload in DMD. One of the limitations of our study is that ruthenium red inhibits TRPV activity and mitochondrial Ca2+ uptake [2+ uptake, increasing the cytoplasmic Ca2+ levels. Thus, ruthenium red treatment could possibly decrease the cytoplasmic Ca2+ levels via TRPV, increase cytoplasmic Ca2+ levels via mitochondrial Ca2+ uptake, or cause no change. While it remains elusive what targets the primary activity of ruthenium red in our model, further investigations of mitochondrial Ca2+ uptake would be interesting in future studies.In this study, we focused on the extracellular space because we targeted the development of a channel blocker that does not require the compounds to pass through the cell membrane. Using commercially available Caeviously ,54. Whilactivity ,56. In t+ uptake ,58. In a+ uptake . On the 2+ overload contributes to disrupted Ca2+ homeostasis in mdx muscles [2+ mobilization induces a muscular dystrophy-like pathology in STIM1 transgenic, dominant-negative Orai1 mutant, and mdx mice [2+ overload without affecting myogenic differentiation [2+ homeostasis in different roles [Correlations between Orai1–STIM1 and DMD have been previously shown in vivo. Zhao et al. found that upregulated Orai1-induced Ca muscles . Wei-LaP muscles . Furthermdx mice . These dntiation . Both STnt roles ,59,60. H2+ leakage from the SR via RYR inside the cells, STIM1–Orai1 modulators target Ca2+ channels on the cell membrane and Ca2+ influx from the extracellular space. Rycal inhibits RYR1 S-nitrosylation-induced SR Ca2+ leakage [2+ influx contribute to DMD pathogenesis. In addition, Ca2+ leakage from the SR via RYR may also lead to hyperactivated STIM1–Orai1. Nevertheless, further investigation is necessary to determine the molecular pathology of Ca2+ overload in dystrophic myotubes in our model.Lastly, to assess the effects of modulating STIM1–Orai1 activities on the functional phenotype, we performed a functional analysis using a previously reported model . In this leakage , which c