All Ca2+ flux perturbations impact cell function and may contribute to various diseases, including cancer. Several modulators of Ca2+ signaling are attractive pharmacological targets due to their accessibility at the plasma membrane. Despite this, the number of specific inhibitors is still limited, and to date there are no anticancer drugs in the clinic that target Ca2+ signaling. Ca2+ dynamics are impacted, in part, by modifications of cellular metabolic pathways. Conversely, it is well established that Ca2+ regulates cellular bioenergetics by allosterically activating key metabolic enzymes and metabolite shuttles or indirectly by modulating signaling cascades. A coordinated interplay between Ca2+ and metabolism is essential in maintaining cellular homeostasis. In this review, we provide a snapshot of the reciprocal interaction between Ca2+ and metabolism and discuss the potential consequences of this interplay in cancer cells. We highlight the contribution of Ca2+ to the metabolic reprogramming observed in cancer. We also describe how the metabolic adaptation of cancer cells influences this crosstalk to regulate protumorigenic signaling pathways. We suggest that the dual targeting of these processes might provide unprecedented opportunities for anticancer strategies. Interestingly, promising evidence for the synergistic effects of antimetabolites and Ca2+-modulating agents is emerging.Calcium ion (Ca However, following stimulation by extracellular stimuli and intracellular signals, the activation of two receptors, the inositol 1,4,5-triphosphate receptor (IP3R) and ryanodine receptor (RYR), mobilizes Ca2+ from this store, creating cytosolic Ca2+ hotspots in close proximity to mitochondria (2+ uptake via voltage-dependent anion-selective channel protein (VDAC) and mitochondrial calcium uniporter protein (MCU) is a major regulator of several mitochondrial processes, including energy production and cell death  at different subcellular locations. In turn, many Ca2+ channels and transporters are regulated by redox modifications.In recent years, a growing number of studies have yielded significant insights into how Cain cells . This in2+ signaling with metabolic processes has come from the cardiovascular system and organ physiology, with well-known examples of Ca2+ coupling workload and energy metabolism in cardiomyocytes and skeletal muscle. Spikes in intracellular Ca2+ levels during contractile activity lead to changes in glucose metabolism by enhancing glucose uptake and transport     by modulgenesis) . Furtherlycogen) . The reltic mice . In thesr damage .2+ from the metabolic changes associated with muscle contraction or insulin sensitivity, and the interpretation of this interaction is often hindered by the causality dilemma. Although the development of sensitive methods has allowed for the recording of subtle, transient and localized intracellular Ca2+ peaks in response to metabolic changes, much remains to be understood regarding the reciprocal regulatory role of metabolism.Nevertheless, it is difficult to clearly separate the effects of Ca2+ signaling and metabolism are aberrant, and it is therefore difficult to define whether dysregulated Ca2+ homeostasis leads to metabolic reprogramming or vice versa. Moreover, since much of the research investigating Ca2+ signaling in cancer has focused on evaluating relevant functional processes  as a result of aberrant expression levels of channels, pumps and transporters, the contribution of this interplay to cancer pathogenesis has often been overlooked.The “chicken and egg” situation is particularly evident in cancer cells, where both Ca2+ transportome to control signaling and metabolism (2) levels to support mitochondrial respiration, cancer cells are highly glycolytic and, as such, demonstrate increased glucose consumption and lactate production, a phenomenon known as the Warburg effect . We discuss how Ca2+ flux between different cellular compartments affects these pivotal metabolic processes in the cell and highlight how metabolic enzymes and metabolites function as signaling molecules to influence Ca2+ homeostasis. We also describe how the cellular redox state defined by the metabolic features of cancer cells controls the activity of several Ca2+ transporters and pumps through posttranslational modifications.In this review, we examine the main findings regarding the interplay between Ca2+ and metabolism in cancer has only begun to be explored. Highlighting emerging findings on this crosstalk is important to provide further understanding of the mechanisms affecting cell function. This may yield great returns by opening up new areas of investigation and identifying new potential therapeutic opportunities.The connection between Ca2+ levels are maintained at low resting concentrations (approximately 100 nM) by the activity of the major Ca2+ extrusion system, the plasma membrane Ca2+-ATPase (PMCA), which exchanges extracellular protons (H+) for cytosolic Ca2+  without the need for Osurvival , 2015. Pll death . Recentlll death , furtherIn normal cells and under physiological conditions, cytosolic ATP derived from glycolysis saturates PMCA function. However, in the case of impaired mitochondrial metabolism, ATP derived from glycolysis becomes a critical regulator of PMCA activity. Acute metabolic stress inhibits the PMCA, but this effect can be attenuated by a metabolic switch from mitochondrial metabolism toward glycolysis, which restores the ATP supply to the PMCA .While the amount of ATP derived from glycolysis regulates PMCA activity, there is no significant evidence of regulatory feedback by the PMCA on metabolism. Indeed, silencing of PMCA4 in pancreatic ductal adenocarcinoma cells has only minimal effects on metabolic functions .2+ homeostasis are much more complex than simple changes in energy supply.The mechanisms regulating the interplay between glycolysis and Ca2+. ATP is provided to the PMCA by glycolytic enzymes located in close proximity to the plasma membrane  opening and cell death by preventing ER Ca2+ release through IP3R  at mitochondria-ER contact sites [called mitochondria-associated ER membranes (MAMs)] prevents mitochondrial Caugh IP3R . HK2 canugh IP3R . This asIP3R  has recently been identified as a potent regulator of SERCA pumps, which transfer Cadrolysis . G6P redll death . This ATmulation .2+ flux and activation of the downstream nuclear factor of activated T-cell (NFAT) signaling pathway, which is critical for anti-tumor T cell functions  . In cytounctions . Insuffiesponses . Althougesponses .2+ homeostasis by PEP has been described in two human colon carcinoma cell lines, in which increased cytosolic Ca2+ due to SERCA modulation stabilizes the master regulator of glycolytic metabolism, the Myc proto-oncogene protein, ultimately supporting cancer cell proliferation  and the glycolytic inhibitor 2-deoxy-d-glucose has recently shown significant anti-tumor effects in preclinical studies  and the hexosamine biosynthetic pathway (HBP).There is interesting evidence for the indirect regulation of Ca2+ levels and impairing cancer cell proliferation and migration , which maintains antioxidant capacity, and ribose, the backbone of nucleotides. Recent studies in human breast cancer cells have shown that the production of NADPH by hexose-6-phosphate dehydrogenase (H6PD) in the ER lumen is indispensable for the activity of SERCAs . Followiigration . Dysreguout mice .2+ homeostasis.These findings define, for the first time, a direct link between a metabolic pathway and ER stress, paving the way for further investigations into the ER-related metabolic pathways that sustain tumor growth by affecting Ca2+ signaling [such as calcium/calmodulin-dependent protein kinase type II (CaMK-II)], as well as Ca2+ pumps and channels , is influenced by O-linked-beta-N-acetylglucosamine (O-GlcNAc) posttranslational modification  generated by the activity of the respiratory electron transport chain (ETC) (2)] to O2 while pumping H+ into the intermembrane space (IMS) (+ gradient provides the energy to generate ATP through the F1F0 ATP synthase (or complex V)  . The ETCce (IMS) . Togethemplex V) , a procel matrix .2+ homeostasis  cycle, and fatty acid oxidation, produce the electron donors that fuel the ETC. Upon exposure to high extracellular glucose concentrations, glycolysis provides a significant amount of pyruvate substrate to the TCA, elevating mitochondrial respiration and driving hyperpolarization of the IMM . Conversein NCLX . Mild deein NCLX . In turnhibition . Thus, N2+ uptake by the transporters VDAC (at the OMM) and MCU (at the IMM) is a key process in cell bioenergetics and functionality. In response to cellular demand, Ca2+ can directly modulate cellular metabolism by activating mitochondrial dehydrogenase enzymes and regulating ETC function , Ca2+ regulates the dephosphorylation state, and hence activation, of pyruvate dehydrogenase (PDH) complex, thus feeding the TCA -activated protein kinase catalytic subunit alpha-2 (AMPK)-dependent pro-survival autophagy , ATP synthesis continued for several minutes after the mitochondrial Ca2+ signal returned to basal levels, suggesting the existence of a metabolic memory imposed by Ca2+ in cancer cells  for one aspartate (from the mitochondria)  . Since t+ levels . Thus, cHOS rate . While mHOS rate , as findHOS rate .2+. This shuttle system catalyzes the exchange of ATP-magnesium ions (Mg2+) and adenosine diphosphate (ADP)-Mg2+ for one phosphate across the IMM, thus determining the levels of adenine nucleotides  in the mitochondria. The N-terminal region of these carriers contains Ca2+-binding domains that respond to high Ca2+ levels  to the cytosol. Once located in the cytosol, NADH influences the activity and expression of sirtuins , which is rapidly converted into non-radical hydrogen peroxide (H2O2), the major source of oxidative stress in cells , decreasing SERCA activity and inhibiting Ca2+ extrusion from the plasma membrane  to prevent cell death by Ca2+ overload. mPTP Ca2+ desensitization and PMCA overexpression are escape mechanisms used by cancer cells to increase their resistance to death ; these enzymes generate Omembrane . Althouggression . Howevergression .2+ signaling are targeted by ROS-dependent oxidation. Oxidants generated by mitochondria can oxidize, among others, CaMK-II, thus enhancing its activity . RNS are formed by peroxidase- and nitric oxide synthase (NOS)-mediated enzymatic reactions, as well as via the interaction of nitric oxide (NO) with ROS .2+ responses by RNS, most of the effects are attributed to posttranslational modifications that generate nitrosylation changes in cysteine residues of Ca2+ transporters, channels and pumps, altering their activity  plays an important role in modulating mitochondrial ROS levels. Upon inhibition of TRP cation channel melastatin subfamily member 2 (TRPM2), decreased intracellular Ca2+ levels result in reduced expression of mitochondrial genes and mitochondrial dysfunction , keeping ADP at steady-state levels. This in turn sets the ΔΨm to lower levels and decreases O leakage . When th leakage . Thus, V2– , the most important antioxidant synthesized in cells. Interestingly, the existence of a Ca2+-sensitive GSH transporter in the ER has been recently postulated  has been called into question by evidence that mitochondrial metabolism is essential for cancer biology. Increased mitochondrial Capathways .2+ addiction of cancer cells with defective oxidative metabolism. In these cells, Ca2+ transfer to mitochondria sustains survival by mediating the compensatory metabolic switch toward reductive carboxylation. Thus, specific anti-metabolic drugs may be effective as combination therapy with drugs that target mitochondrial Ca2+ transfer, regardless the OXPHOS status of cancer cells.An interesting observation is the mitochondrial Ca2+ is complex. In addition to the ATP-dependent regulation of Ca2+ pumps, the physical association of glycolytic intermediates with Ca2+ transporters modulates Ca2+ homeostasis, while changes in the subcellular localization of glycolytic enzymes influence cell fate by impacting Ca2+ homeostasis  cells in culture would be valuable in organ systems as well.Clearly, many gaps remain in terms of our understanding of Ca2+ levels or manipulating Ca2+ dynamics to selectively kill cancer cells. Unfortunately, the limited number of specific inhibitors has prevented the development of efficient anticancer strategies targeting aberrant Ca2+ signaling. An alternative strategy is to target the dysfunctional metabolic pathways that also regulate Ca2+ signaling, such as glycolysis. For example, targeting the glycolytic enzymes PFKFB3 and PKM2 which are highly expressed in cancer cells compared to normal cells, would provide a novel means of targeting Ca2+ pumps specifically in cancer cells. Given this possibility and the therapeutic potential of a synergistic approach to, for instance, reduce tumor acidification or induce Ca2+-dependent apoptosis in cancer cells during glucose deprivation, it is crucial to further dissect how Ca2+ levels and metabolism integrate to regulate tumorigenesis and cancer progression.One of the greatest challenges is restoring physiological Ca2+ dynamics to manipulate the epigenome is a fascinating idea. New findings are emerging (2+ signaling regulate epigenetic-mediated transcriptional control in cancer cells.Finally, the possibility of targeting metabolic pathways and Caemerging , and it ARC conceived the review and wrote, reviewed, and edited the text. CD provided a first draft of the manuscript and designed the figures. DG reviewed the text. All authors performed literature searching and contributed to discussion of the content.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Senescence is a cellular program in response to stress and can prevent the expansion of pre-malignant and malignant cells by cell cycle arrest. However, long-term pro-tumorigenic effects have been described. This review aims to compile molecular mechanisms of cellular senescence and to discuss potential therapeutic implications in myeloid malignancies.Senescence is a cellular state that is involved in aging-associated diseases but may also prohibit the development of pre-cancerous lesions and tumor growth. Senescent cells are actively secreting chemo- and cytokines, and this senescence-associated secretory phenotype (SASP) can contribute to both early anti-tumorigenic and long-term pro-tumorigenic effects. Recently, complex mechanisms of cellular senescence and their influence on cellular processes have been defined in more detail and, therefore, facilitate translational development of targeted therapies. In this review, we aim to discuss major molecular pathways involved in cellular senescence and potential therapeutic strategies, with a specific focus on myeloid malignancies. The only senescent cells that can re-enter the cell cycle under certain conditions are senescent tumor cells [INK4a and p21WAF1, as well as reduced proapoptotic caspase activity, are considered typical features of senescent cells [The inability of somatic mammalian cells to undergo further replication was first described as cellular senescence in 1961 ,3. Telomterphase . In additerphase . In contor cells ,11,12,13nt cells ,16,17,18nt cells .A widely used method for histochemical labeling of senescent but not pre-senescent or immortalized cells in vitro and in vivo is the senescence-associated β-galactosidase  assay . β-GalacDNA damage response (DDR) is an important mechanism for the initiation of the senescence pathway. In the context of replicative senescence, progressive shortening of telomeres leads to telomere uncapping, which is sensed as double-stranded DNA breaks and thereby activates DDR . On the MDM2 is, in turn, induced by p53 as a negative feedback loop since MDM2 acts as a negative regulator of p53 by direct binding and degradation via the ubiquitin–proteasome complex [+-dependent protein deacetylase activity. By deacetylation of p53, SIRT1 counteracts PML-mediated senescence [WAF1 gene, which encodes the cyclin-dependent kinase 2 (CDK2) inhibitor p21WAF1. The inhibition of CDK2 by p21WAF1 leads to decreased phosphorylation of the retinoblastoma protein (RB).Depending on the trigger, different pathways are activated to induce senescence. As already described, p53 is activated by a variety of triggers, such as DDR, critical telomere shortening, or activation of oncogenic signaling pathways . The activity of p53 is mainly controlled by the E3-ubiquitin ligase Mouse double minute 2 homolog (MDM2). The transcription of  complex ,33. MDMX complex . When ac complex ,36. Like complex . Indepen complex . The PI3 complex ,38. The  complex . Similar complex . The act complex . Silent nescence . SIRT1 inescence ,43,44. TMAPK pathway is activated independently of the telomere function but typically in the presence of oxidative stress or oncogenic signaling. Oncogenic RAS induces premature senescence by sequential activation of MEK-extracellular signal-regulated kinase (ERK). Accumulated MAP kinase kinases, MKK3 and MKK6, activate p38MAPK that thereby is able to degrade BMI1, a member of the Polycomb group gene family and transcriptional repressor of p16INK4a [INK4a [INK4a forms complexes with the RB kinases CDK4 and CDK6, which leads to long-term inhibition and stabilization of the hypo-phosphorylated state of RB.In contrast to DDR and telomere dependent mechanisms, the ERK-p38p16INK4a . In addia [INK4a . UpregulAs previously discussed, the final link of both pathways is formed by RB . RB is aSenescent cells are usually able to express a set of molecules that define a so-called ‘Senescence Associated Secretory Phenotype’ (SASP). These factors include a variety of chemokines, pro-inflammatory cytokines, proteases, growth factors, and non-protein molecules . The pheAnother activator of the SASP is the transcription factor GATA4 . Under nMAPK activation, a stress-inducible kinase that acts as a DDR-independent regulator of the SASP [Regulation of SASP has also been linked to JAK2/STAT3 signaling . This pathe SASP .Importantly, NF-κB signaling represents a bottleneck in the development of the SASP. In addition to described upstream kinase cascades, the activation of the NF-κB system is usually connected with several pattern recognition receptor pathways, e.g., toll-like receptors (TLR) and inflammasomes . The traCDKN2B encoding p15INK4b that promote p16INK4a by suppressing CDK4 and CDK6 [Another pro-inflammatory transcription factor, to which most SASP regulators converge, is CCAAT/enhancer-binding protein β (C/EBPβ). In collaboration with NF-κB, C/EBPβ regulates many SASP components and inflammatory cytokines ,56,67. Dand CDK6 .Myeloid malignancies are clonal disorders of the hematopoietic stem or progenitor cells that are caused by genetic mutations or epigenetic alterations. These mutations lead to changes in proliferation, differentiation, or self-renewal. Myeloid malignancies include chronic stages, such as myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), and acute stages, such as acute myeloid leukemia (AML). Recently, preclinical studies and early clinical trials have highlighted several molecular targets that are effective in the treatment of myeloid malignancies either by additional senescence induction or by eradication of senescent cells .The specific CDK4/6 inhibitor palbociclib not only blocks the proliferation of cancer cells but also induces senescence by the concomitant occurrence of CDK inhibition and disruption of proteasomal homeostasis ,72. The ADP-ribosylation by poly (ADP-ribose) polymerase 1 (PARP1) is an important mechanism in DNA damage repair. In addition to inducing synthetic lethality associated with a deficit in homologous recombination, PARP inhibition leads to cellular senescence trigger by DDR after DNA damage accumulation . The oraTP53 are a frequent event in specific subtypes of solid tumors, they occur in less than 10% of AML patients and, if so, mostly associated with complex aberrant karyotype in therapy-related AML [MDM2 inhibition induces a senescence-like state and has been shown to attenuate SASP ,83. Up tated AML ,87. For ated AML ,89. ALRNated AML . By induated AML . The MDMated AML . Howeverated AML .Sirtinol, a specific inhibitor of SIRT1, induces senescence through increasing acetylation of p53 . SirtinoIn various cell types, including MLL-rearranged AML cells, SIRT1 was shown to be downregulated by miR-181a ,44,96. IDisrupted telomere homeostasis can result in genomic instability in hematopoietic cells, causing myeloid diseases, such as MDS and AML . IncreasThe aberrant expression of the Polycomb complex member and epigenetic regulator BMI1 is associated with several human malignancies. BMI1 is highly expressed in specific AML subgroups and correlates with therapy failure ,106. InhThe trimethylation of H3K9 leads to stable repression of S-phase-relevant genes. Histone lysine-specific demethylase 4C (KDM4C) is a 2-oxoglutarate-dependent Jumonji C family member that demethylates trimethylated lysine residues of H1.4K26 and di- and trimethylated lysine residues of H3K9 and H3K36 . KDM4C wINK4a associated senescence in bone marrow stromal cells that promote the survival of AML cells by the secretion of SASP factors, especially IL-6 and MIP-3α [The tumor microenvironment is crucial for the progression of various solid and hematologic malignancies. Via NOX2-derived superoxide production, AML cells are able to induce p16d MIP-3α . Leukemid MIP-3α . Howeverd MIP-3α . Here, ad MIP-3α ,128. Thed MIP-3α . Howeverd MIP-3α . Chronic myeloid leukemia (CML) can be effectively treated with tyrosine kinase inhibitors (TKIs), such as imatinib. However, in rare cases, patients develop resistance to TKI therapy, which occurs independently of mutations in the BCR-ABL tyrosine kinase domain. Hyaluronan, a component of the bone marrow microenvironment, inhibits senescence induction of imatinib treated CML cell lines since survival signals in CML cells are triggered not only by BCR-ABL but also by hyaluronan through the activation of the PI3K/AKT pathway ,132. BesThe senescent microenvironment not only has a negative impact on leukemia development but also affects normal hematopoiesis, which may explain the development of resistance, disease recurrence, and long-term side effects of cytostatic drugs ,135. TheNavitoclax (ABT-263) is the first orally bioavailable inhibitor of the BCL-2 protein family and binds with high affinity to the anti-apoptotic proteins BCL-XL, BCL-2, and BCL-W ,138,139.WAF1 [Expression and phosphorylation of focal adhesion kinase (FAK) at its SRC-dependent phosphorylation site Tyr577 was increased in senescent cells, thus showing a reduced apoptotic response to cytotoxic stress . FurtherWAF1 ,153. UnlWAF1 . TreatmeWAF1 ,158,159.WAF1 . FurtherWAF1 .Considerable progress has been made in understanding the molecular regulatory mechanisms of senescence and its biological function in tissue aging and tumorigenesis, which appears to be highly dependent on cell type. Molecular specification of senescence depends on the type of stimulus as well as the environmental conditions of the cell . Senesce


Skeletal muscle atrophy is one of the clinical symptoms of myotonic dystrophy type 1 (DM1). A decline in skeletal muscle regeneration is an important contributor to muscle atrophy. Skeletal muscle satellite cells (SSCs) drive skeletal muscle regeneration. Increased autophagy can reduce the proliferative capacity of SSCs, which plays an important role in the early regeneration of damaged skeletal muscle in DM1. Discovering new ways to restore SSC proliferation may aid in the identification of new therapeutic targets for the treatment of skeletal muscle atrophy in DM1. In the pathogenesis of DM1, muscleblind-like 1 (MBNL1) protein is generally considered to form nuclear RNA foci and disturb the RNA-splicing function. However, the role of MBNL1 in SSC proliferation in DM1 has not been reported. In this study, we obtained SSCs differentiated from normal DM1-04-induced pluripotent stem cells (iPSCs), DM1-03 iPSCs, and DM1-13-3 iPSCs edited by transcription activator-like (TAL) effector nucleases (TALENs) targeting CTG repeats, and primary SSCs to study the pathogenesis of DM1. DM1 SSC lines and primary SSCs showed decreased MBNL1 expression and elevated autophagy levels. However, DM1 SSCs edited by TALENs showed increased cytoplasmic distribution of MBNL1, reduced levels of autophagy, increased levels of phosphorylated mammalian target of rapamycin (mTOR), and improved proliferation rates. In addition, we confirmed that after MBNL1 overexpression, the proliferative capability of DM1 SSCs and the level of phosphorylated mTOR were enhanced, while the autophagy levels were decreased. Our data also demonstrated that the proliferative capability of DM1 SSCs was enhanced after autophagy was inhibited by overexpressing mTOR. Finally, treatment with rapamycin (an mTOR inhibitor) was shown to abolish the increased proliferation capability of DM1 SSCs due to MBNL1 overexpression. Taken together, these data suggest that MBNL1 reverses the proliferation defect of SSCs in DM1 by inhibiting autophagy via the mTOR pathway. The mutant DMPK gene is also transcribed into CUGn-containing RNA, which forms nuclear RNA foci and disturbs RNA-binding proteins, resulting in reduced levels of MBNL1 and increased levels of CUG-binding protein 1 (CUGBP1)3. Muscle atrophy is one of the core symptoms of DM14, with decreased skeletal muscle regeneration as an important contributor5. New methods of enhancing the impaired regeneration of skeletal muscle are essential to improve skeletal muscle atrophy and the quality of life of DM1 patients.Myotonic dystrophy type 1 (DM1) is a neuromuscular disease caused by expanded CTG repeats in the myotonic dystrophy protein kinase (DMPK) gene6. SSCs are often located between the basal lamina and the plasma membrane in muscle fibers7. SSCs are at rest under normal conditions, but when muscles are damaged, they become activated and proliferate to form new skeletal muscle fibers8. However, the SSC proliferation capacity of DM1 patients is impaired9. The basic mechanisms of this process are not fully understood. Therefore, seeking ways to increase the proliferation of SSCs could inform the treatment of muscular atrophy.Skeletal muscle satellite cells (SSCs) are the source of skeletal muscle regeneration. Increased SSC proliferation can promote the early regeneration of skeletal muscle10. In DM1, the function of the MBNL1 protein is dysregulated due to the binding of MBNL1 to CUG expansion (CUGexp) RNA, which forms intranuclear lesions11. Previous studies have also shown that MBNL1 may be involved in the regulation of muscle atrophy in Drosophila models of DM213. In addition, atrophic muscle fibers are found in MBNL1-knockout mice14, but whether this is caused by affecting SSC proliferation activity has not been investigated in DM1. One study showed that DM1 neural stem cells exhibit defects in proliferation and increased autophagy associated with changes in the mTOR pathway. Furthermore, loss of MBNL1 function leads to changes in the mTOR pathway, and gain of MBNL1 function rescues the disease phenotype, including autophagy-induced reduction. The study also suggested that autophagy induction may also be involved in the pathogenesis of DM115. Notably, mTOR is an atypical serine/threonine protein kinase that acts as an important regulator of skeletal muscle atrophy and regeneration16. Some studies have demonstrated that mTOR can promote the proliferation of primary SSCs17. Therefore, MBNL1 may regulate the proliferation of SSCs via the mTOR pathway.The MBNL1 protein is an important factor responsible for RNA splicing18. Autophagy plays a role in various pathophysiological events, including cell proliferation, metabolic abnormalities, and cell differentiation, death, and growth20. However, macrophagy (henceforth named autophagy), a protein-degradation system closely related to cell proliferation21, is also involved in repairing damaged skeletal muscle22 and reversing muscle atrophy24. Previous studies have reported that melatonin can promote proliferation of neural stem cells by repressing elevated autophagy under hyperglycemic conditions25. Suppressing autophagy can also enhance the proliferation of germ cells26. Notably, mTOR is a major regulator of cell autophagy and proliferation20. In addition, overexpression of TOR (the human homolog of mTOR) inhibited autophagy to ameliorate atrophy of indirect flight muscles in a Drosophila model of DM127. Furthermore, MBNL1 modulated the mTOR pathway15. Therefore, MBNL1 may promote proliferation of DM1 SSCs by affecting autophagy levels via the mTOR pathway.Autophagy, also known as “self-eating,” is a process involving the degradation of proteins and organelles to recycle substances in response to cellular stress28. Muscle biopsy is also an effective method to obtain SSCs to describe this pathogenesis29. However, it causes pain to the patient and yields a limited number of SSCs30. In contrast, patient-derived induced pluripotent stem cells (iPSCs) not only carry the patient’s disease-causing genetic information, but can also be robustly differentiated into SSCs; thus, iPSCs are important tools for research on the molecular and functional abnormalities of DM131. Currently, we can obtain iPSCs from reprogrammed somatic cells and establish patient-specific cell models to study the molecular and functional abnormalities of human diseases32. We obtained DM1-03 iPSCs derived from a DM1 patient and normal DM1-04 iPSCs from a healthy volunteer. DM1-03 iPSCs contain 2829–3575 CTG repeats33. We have also developed a method for the directional insertion of polyA signals (PASs) upstream of the DMPK CTG repeats in DM1-03 iPSCs, which eliminates nuclear RNA foci and reverses the phenotype of MBNL1. We named genetically edited DM1-13-3 iPSCs. Furthermore, all three types of iPSCs maintain their pluripotency34.Studies investigating the pathogenesis of DM1 usually rely on mouse models, Drosophila modelsHere, we use SSCs differentiated from DM1 iPSCs and DM1 primary SSCs to study the mechanism responsible for the disruption of SSC proliferation in DM1. We named the corresponding SSCs from DM1-04 iPSCs, DM1-03 iPSCs, and DM1-13-3 iPSCs DM1-04, DM1-03, and DM1-13-3. Together, we innovatively obtained SSCs differentiated from DM1 iPSCs and used them as cellular models to study the pathogenesis of DM1, although we were unable to determine the role of MBNL1 in DM1 SSCs. Therefore, this study mainly investigated whether MBNL1 reverses the proliferation defect of DM1 SSCs by inhibiting autophagy via the mTOR pathway.+MyoD+) in the DM1-03 group was lower than that in the DM1-04 group, while that in the DM1-13-3 group was higher than that in the DM1-03 group, but was lower than that in the DM1-04 group 10 DNA probe to detect nuclear RNA foci, and found that nuclear RNA foci were present in DM1-03 SSCs Fig. . HoweverTo investigate the role of MBNL1 in the proliferation of DM1 SSCs, we conducted adenoviral overexpression of MBNL1 in DM1-03 and DM1-13-3 SSCs. Significantly elevated protein and mRNA expression levels of MBNL1 in the DM1-03 and DM1-13-3 SSCs were detected by western blot analysis and qRT-PCR Fig. . To examWe next performed western blot analysis and GFP-mRFP-LC3 to detect the autophagy levels in the DM1-04, DM1-03, and DM1-13-3 groups. Western blot analysis was also conducted to detect the autophagy levels in primary SSCs from healthy volunteers and DM1 patients. The ratio of LC3II/LC3I in DM1-03 SSCs was significantly higher than that in DM1-04 SSCs, while that in DM1-13-3 SSCs was significantly lower than that in DM1-03 SSCs, but higher than that in DM1-04 SSCs Fig. . The levTo investigate the role of MBNL1 in the mTOR pathway and autophagy in DM1 SSCs, we performed western blot analysis and GFP-mRFP-LC3. According to the western blot results, the levels of p-mTOR/mTOR and P62 in DM1 SSCs were increased after MBNL1 overexpression, but were still lower than those in the DM1-04 group Fig. . The LC3The analysis of GFP-mRFP-LC3 showed that the GFP/mRFP per cell in DM1 SSCs was increased after MBNL1 overexpression, but was still lower than that in DM1-04 SSCs Fig. . In addiTo further explore the role of autophagy activation in DM1 SSC proliferation, we conducted western blot analysis, CCK-8 assays, and Ki67 immunofluorescence to evaluate the proliferation of DM1 SSCs after autophagy was suppressed by mTOR overexpression.The protein and mRNA expression levels of mTOR in DM1-03 Fig.  and DM1-Ki67 immunofluorescence showed that the proportion of Ki67-positive DM1 SSCs was enhanced after mTOR overexpression, but the proportion was still lower than that in DM1-04 SSCs Fig. . The proThese results suggest that inhibiting autophagy activation by mTOR overexpression promotes DM1 SSC proliferation, and this effect was more pronounced in the genome-modification group.To investigate whether MBNL1 promotes proliferation by inhibiting autophagy via the mTOR pathway in DM1 SSCs, we performed western blot analysis, CCK-8 assays, and Ki67 immunofluorescence to examine the proliferation of DM1-03+Ad-MBNL1 and DM1-13-3+Ad-MBNL1 SSCs after rapamycin treatment.According to the western blot results, the level of LC3II/LC3I in DM1-03+Ad-MBNL1 Fig.  was incrBoth OD 450 values and the proportion of Ki67-positive cells in DM1-03+Ad-MBNL1+rapamycin SSCs were significantly lower than those in DM1-03+Ad-MBNL1 SSCs Fig. , whereasThese data suggest that MBNL1 promotes proliferation by inhibiting autophagy via the mTOR pathway in DM1 SSCs.1. SSCs, which play an important role in muscle regeneration6, can be activated into a proliferating state, and eventually differentiate and fuse with each other, restoring skeletal muscle fiber regeneration ability36. In this study, MBNL1 was shown to enhance the proliferation of DM1-03 and DM1-13-3 SSCs by inhibiting autophagy activation via the mTOR pathway.DM1, the most common form of muscular dystrophy affecting adults, is characterized by muscle atrophy, weakness, and myotonia7, but constitutively express Pax7, regardless of their activation state. Here, the proportion of activated SSCs (Pax7+MyoD+) in the DM1-03 group was lower than that in DM1-04 and DM1-13-3 groups, indicating that DM1-03 SSCs exhibited proliferation defects. In addition, similar results were observed in DM1 SSCs with Ki67 immunofluorescence and CCK-8 assays, consistent with the previous study9. However, the proportion of activated SSCs (Pax7+MyoD+), Ki67-positive cells, and OD 450 values in the DM1-13-3 group were higher than those in the DM1-03 group, but lower than those in the DM1-04 group, indicating that TALEN genome modification targeting CTG repeats promoted the proliferation of DM1 SSCs, but could not restore the proliferation level of normal human SSCs (DM1-04). Although genome modification has been widely used to correct repeat expansion mutations including DM1, it is still a challenge38.SSCs express the muscle-specific transcription factor MyoD only when they are activated into a proliferating state10. In this study, total MBNL1 protein levels were decreased in DM1-03 SSCs and DM1 primary SSCs. However, these reduced levels were ameliorated in DM1-13-3 SSCs, which exhibited enhanced proliferation compared with DM1-03 SSCs. Previous studies have also shown that MBNL1 overexpression can abate the DM1 symptoms39. Both low expression and nuclear retention of MBNL1 play an adverse role in the pathogenesis of DM1; therefore, reducing or eliminating the nuclear retention of MBNL1 could be another effective treatment for DM139. In this study, we observed nuclear RNA foci in DM1-03 SSCs but not in DM1-13-3 SSCs via RNA fluorescence in situ hybridization. Cytoplasmic MBNL1 expression was elevated in DM1-13-3 SSCs, which exhibited enhanced proliferation compared with DM1-03 SSCs. One previous study demonstrated that, consistent with our findings, elimination of CUGexp RNA with RNA-targeting Cas9 can redistribute endogenous MBNL1 between the nucleus and cytoplasm in DM140. Another study showed that MBNL1 in the cytoplasm of DM1 neurons is in a ubiquitinated state and promotes neurite outgrowth, while MBNL1 in a deubiquitinated state is transported into the nucleus and damages neurite outgrowth41. Protein ubiquitination is an ATP-dependent process44. However, mitochondrial dysfunction and metabolic disorders were observed in DM1 cells46. ATP deficiency may contribute to the reduction in the level of ubiquitination of MBNL1 and affect its localization in DM1 cells. Taken together, the literature shows that TALEN genome modification not only eliminates toxic RNA and increases total and cytoplasmic MBNL1, but also enhances the proliferation ability of SSCs. Hence, MBNL1 may regulate the proliferation of DM1 SSCs. To further investigate the role of the MBNL1 protein in the proliferation of SSCs, MBNL1 was overexpressed in DM1 SSCs. As expected, MBNL1 overexpression significantly enhanced the proliferative capacity of DM1 SSCs, but was still lower than normal, which indicates that, while MBNL1 could promote the proliferation of DM1 SSCs, it may not be the only regulatory factor. A previous study demonstrated that CUGBP1, another RNA-binding protein whose expression is affected by repeat expansion mutations, can affect the expression of the cell-cycle inhibitor p21 in DM147.MBNL1 dysfunction is reportedly a key event in DM118. mTOR is an important regulatory target for autophagy49. Downstream cascade autophagy is activated when phosphorylated mTOR is reduced18. LC3 (LC3I) in the cytoplasm is converted into LC3II and recruited to the autophagosome membrane. Autophagosomes and lysosomes fuse to form autolysosomes, and the substances within them, including LC3II, are degraded by lysosomal hydrolases. P62/SQSTM1, an autophagy adaptor protein that can interact with LC3 through the LIR domain, is also degraded in the autophagy–lysosome pathway50. This autophagy–lysosomal-mediated protein-degradation process can provide sufficient amino acid substrates for cell survival and proliferation51. However, autophagy acts as a double-edged sword in various pathophysiological states52. Normal autophagy can maintain cell homeostasis, but disordered autophagy will be accompanied by pathological phenomena, including muscle atrophy53. One recent study showed that autophagy can degrade cyclin D1 (CCND1) and arrest the cell cycle in the G1 phase to inhibit the proliferation of hepatocellular carcinoma cells54. Another study showed that lower-than-normal autophagy levels exert protective effects; however, elevated autophagy levels exert adverse effects55. In this study, autophagy levels in DM1 SSCs were elevated, with increased levels of LC3II/LC3I and decreased levels of P62. Similar results were observed in DM1 primary SSCs. Consistent with our findings, previous studies have shown that autophagy levels are increased in DM127. Autophagosomes and autolysosomes detected by GFP-mRFP-LC3 significantly increased in DM1 SSCs, indicating activation of the autophagy–lysosomal pathway. Both TALEN genome modification and MBNL1 overexpression significantly reduced autophagic flux in DM1 SSCs. However, TALEN genome modification cannot fully restore autophagy levels in DM1 SSCs. Furthermore, MBNL1 increases the level of phosphorylated mTOR in DM1 SSCs. These data show that MBNL1 may promote the proliferation of SSCs by affecting autophagy via the mTOR pathway.Autophagy is a catabolic and material recycling process, providing raw materials for the intracellular anabolism of cells26. mTOR overexpression consistently inhibits autophagy levels and promotes the proliferation of DM1 SSCs. PCNA is a eukaryotic protein found in the nucleus of dividing cells, and is essential for DNA replication, excision, and mismatch repair56. The expression of proliferation indicator PCNA in DM1 SSCs increased after mTOR overexpression, indicating that inhibiting elevated autophagy levels may promote cell division. However, other studies have reported findings inconsistent with our observations57. These differences in outcomes might be because the studies were conducted on different diseases and with different experimental models, cell types, etc. Another critical reason for the observed differences is that autophagy acts as a double-edged sword in various pathophysiological states52. Furthermore, the expression of P62 in DM1 SSCs increased after mTOR overexpression, but was still lower than DM1-04 SSCs. Research reports that the level of P62 is reduced in spinal and bulbar muscular atrophy (SBMA)58, and the complex restoration of P62 requires autophagic degradation, upregulation of transcription, and amino acid availability59. In summary, inhibition of autophagy activation promoted the proliferation of DM1 SSCs. Targeting autophagy and balancing autophagic activity may be valuable strategies for DM1 treatment.Next, we investigated the relationship between autophagy and proliferation in DM1 SSCs. Previous studies have shown that inhibiting elevated autophagy levels can promote cell proliferation60. To investigate whether MBNL1 could enhance the proliferation of SSCs by suppressing autophagy via the mTOR pathway, we used rapamycin to target mTOR. Inhibition of the mTOR pathway with rapamycin abolished the enhancement of proliferative capacity mediated by MBNL1 overexpression in DM1 SSCs. Some studies have shown similar results17. Therefore, we conclude that MBNL1 can promote the proliferative capacity of DM1 SSCs by suppressing autophagy via the mTOR pathway.Rapamycin is an mTOR inhibitor61. We inserted the PolyA signal for intron 9 to eliminate nuclear RNA foci with the production of truncated DMPK protein61. DMPK is a serine/threonine protein kinase, but its biological significance is rarely reported. Its homologous family molecule Rho-associated kinase (ROCK), which controls cell proliferation by regulating cell division, may provide a reference for our research. When a cell divides, the formation of an actomyosin-based contraction loop is important; it can drive the division groove into the cell and then divide it into two daughter cells62. Both ROCKs and DMPK can control the contraction of actomyosin by phosphorylating myosin-regulatory light chain (MLC)64. Truncated DMPK protein may lose or have weakened functions, including the function of phosphorylation of MLC, which needs further research to verify. Another problem is how MBNL1 upregulates the mTOR pathway in DM1 SSCs. Calcineurin is a calcium-dependent serine/threonine phosphatase composed of the catalytic subunit (CnA) and regulatory subunit (CnB) that plays an important role in many pathophysiological events65. CnAβ1 is an isoform of CnA that can be upregulated by MBNL166, and promotes the proliferation of myoblasts67. Furthermore, CnAβ1 regulates proliferation by sensing metabolic cues to maintain homeostasis in stem cells68. It can also reduce catabolism while activating AKT to promote proliferation through the mTOR pathway70. Interestingly, DM1 cells have metabolic disorders46. Therefore, we propose the following hypothesis: MBNL1 may reduce autophagy–lysosomal-mediated catabolism and promote proliferation through the CnAβ1–mTOR pathway.However, there are several issues that need further study. In this study, TALEN genome modification failed to fully restore proliferative capacity in DM1 SSCs. The shortcomings of TALEN genome modification in stem cell therapy have been reflected in previous studiesTaken together, these data illustrate the proliferation defect of SSCs differentiated from DM1 patient-derived iPSCs, and suggest that genome modification can eliminate nuclear RNA foci and enhance the proliferation of DM1 SSCs. We did not study the roles of CUG RNA repeat expansion and other factors in proliferation and autophagy in detail, but we conclude that MBNL1 reverses the proliferation defect of DM1 SSCs by inhibiting autophagy via the mTOR pathway. We investigated the mechanisms of DM1 SSC proliferation defects from the perspective of targeted modification of DM1 pathogenic genes and protein molecular pathways. We believe that our findings can provide guidance for the clinical treatment of muscle atrophy in DM1 patients.34.The normal control DM1-04 iPSC line, the DM1-03 iPSC line, and the DM1-13-3 iPSC line used in this study were in line with the requirements of the University of Florida’s Institutional Review CommitteeTM-E8TM Kit  to culture iPSCs in six-well plates coated with Matrigel . When the monolayers reached 70% confluency, we digested the cells with trypsin at 37 °C for 7 min and plated the single-cell suspensions on a new Matrigel-coated six-well plate. We changed the TeSRTM-E8TM medium every day until the cells again reached 70% confluency, and then pretreated the iPSCs with 10 µM Rocki  in TeSRTM-E8TM for 2 h. After Rocki treatment, we discarded the medium and washed the iPSCs with calcium- and magnesium-free DPBS, digested the cells with trypsin at 37 °C for 7 min, and plated them at a density of ~3.0 × 105 iPSCs per well in a new Matrigel-coated six-well plate. When the cultures reached 20% confluency, we initiated differentiation using the sequential culture media reported in our previous study to regulate specific signaling pathways, particularly the Wnt and BMP signaling pathways44. On day 25, SSCs were harvested with 0.05% trypsin and replated in Matrigel-coated 24-well plates in Skeletal Muscle Cell Growth Medium . Paired box protein Pax7  levels were monitored by immunofluorescence.We used the TeSRThree DM1 patients  with muscular dystrophy symptoms admitted from the First Affiliated Hospital of Chongqing Medical University and three recruited healthy volunteers without underlying disease  were included in this study. Muscle biopsies were obtained with the informed consent of the donors. The experimental protocol in this study was approved by the Ethics Committee of the First Affiliated Hospital of Chongqing Medical University. The collected muscle biopsy  was placed in cold PBS supplemented with 1% PeSt (100 U/ml penicillin and 100 µg/ml streptomycin)  and immediately sent to the laboratory.71, slowly stirred, and digested at 37 °C for 20 min. The supernatant produced by the digestion was collected and put into the growth medium . To obtain enough cells, the undigested tissue was digested once more, and the supernatant was collected. The supernatant obtained from the two digestions was centrifuged at 350 rpm for 10 min, the supernatant was removed, and the cell pellet was resuspended with Ham’s F-10 and plated on a noncoated disk for 1 h to purify SSCs. Then, the supernatant containing purified SSCs was transferred to the coated dish to continue culturing.The skeletal muscle of the biopsy was dissected, and the fat tissue and connective tissue visible to the naked eye were removed, minced into small pieces, transferred to the digestion fluid described in a previous study72; usually, the ratio of Ki67-positive cells can be used to assess the proliferation ability of SSCs73. For staining of cultured SSCs, cells were fixed in 4% PFA for 10 min at room temperature. After the cells were washed in PBS, they were blocked in 10% donkey serum for 1 h at 37 °C and then incubated overnight at 4 °C with primary antibodies targeting Pax7 , MyoD , and Ki67 . After three washes in PBS, the cells were incubated with secondary antibodies conjugated with Alexa Fluor Plus 488 , Alexa Fluor 555 , and Alexa Fluor 488  for 1 h at 37 °C. Finally, the cells were washed three times in PBS and stained with DAPI . RNA fluorescence in situ hybridization was performed with a Cy5-labeled (CAG)10 DNA probe , as described previously33. Images were captured with a Nikon AL90 laser confocal scanning microscope  and analyzed with ImageJ software.Ki67 is a nuclear protein that is expressed in G1, S, G2, and early mitosis cells, but not in G0-phase cellsThe cultured cells were homogenized in radioimmunoprecipitation assay (RIPA) lysis buffer  containing phenylmethanesulfonyl fluoride  and phosphatase inhibitors . After the lysates were centrifuged for 15 min, the supernatant was collected for total protein detection. To extract nuclear and cytoplasmic proteins, we used the Nuclear and Cytoplasmic Protein Extraction Kit  according to the manufacturer’s instructions. The protein concentrations were measured using bicinchoninic acid (BCA) protein assay reagent , after which the proteins were loaded into gels and separated by SDS-polyacrylamide gel electrophoresis. After separation, the proteins were transferred to polyvinylidene difluoride (PVDF) membranes , which were blocked in TBST with 5% nonfat dry milk  or bovine serum albumin for 1 h at room temperature according to the manufacturer’s instructions for primary antibodies. After they were blocked, the membranes were incubated overnight at 4 °C with the following primary antibodies: anti-Pax7 , anti-PCNA , anti-LC3 , anti-P62 , anti-mTOR , anti-p-mTOR , anti-MBNL1 , and anti-tubulin . After incubation with primary antibodies, the membranes were washed with TBST and incubated with goat anti-mouse IgG (H+L), HRP-conjugate antibody , goat anti-rabbit IgG antibody , or goat anti-rat IgG antibody  for 1 h at room temperature. Images were captured with a Fusion FX5 image analysis system .74 , were used as control groups. SSCs cultured on coverslips were treated with Skeletal Muscle Cell Growth Medium containing adenovirus with the mRFP-GFP-LC3 vector  for 2 h; after incubation, the treatment medium was discarded and replaced with regular medium, and the cells were cultured for another 48 h. After adenovirus transduction, the cells were washed with PBS and fixed with 4% paraformaldehyde for 10 min at room temperature. After the cells were washed three times in PBS, they were stained with DAPI . Images were captured and analyzed with a Nikon AL90 laser confocal scanning microscope . Upon transfection of GFP-mRFP-LC3, in the case of autolysosomes, the fluorescence of GFP is quenched so that the observed fluorescence is more red than green. In the case of mRFP, there will always be the same signal strength. In the case of observing more autophagosomes, it would be a nonacidic environment, and therefore, the observed fluorescence would be more yellowish (GFP (GREEN)+mRFP (RED)). A ratio (GFP/mRFP)/cell can quantify what has been broken out of GFP and normalized to the number of cells. The yellow dots after the merging of the red and green fluorescent channels are autophagosomes, and the free red dots indicate autophagolysosomes. ImageJ software was used to count the green and red fluorescent dots and normalize them to the number of cells according to guidelines in previous studies76.DM1-03 and DM1-13-3 SSCs treated with recommended 10 nM Bafilomycin A110 PFU/ml, FulenGen, Guangzhou, China) or an empty carrier adenovirus  for 2 h. Then, the virus-containing medium was replaced with regular medium, and the cells were cultured for another 36 h.To overexpress MBNL1, DM1-03 and DM1-13-3 were cultured in Skeletal Muscle Cell Growth Medium containing an MBNL1-expressing adenovirus , as recommended in previous publications10 PFU/ml, FulenGen, Guangzhou, China) or Ad-control  for 2 h. Then, the virus-containing medium was replaced with regular medium, and the cells were cultured for another 36 h.To overexpress mTOR, DM1-03 and DM1-13-3 were cultured in Skeletal Muscle Cell Growth Medium containing an adenovirus-expressing mTOR . A Q5 Gradient Real-Time PCR Detection System  and a TB Green Premix Ex Taq II  were used to conduct real-time qRT-PCR under the following reaction conditions: initial denaturation at 95 °C for 30 s followed by 40 cycles of 5 s at 95 °C, 30 s at 60 °C. The primer sequences are listed in Table Total RNA from SSCs was extracted with TRIzol , and cDNA was synthesized using a PrimeScript3 cells/well) and cultured in Skeletal Muscle Cell Growth Medium at 37 °C in 5% CO2 for 24 h. Then, we added 10 µl of CCK-8 solution  to each well, incubated the cells for 1 h, and immediately measured the absorbance  at 450 nm with a microplate reader .SSCs were seeded in 96-well plates . P < 0.05 was considered to indicate statistical significance.SPSS 23.0 was used to analyze the data. ImageJ was used to count cells with positive immunofluorescence, and GraphPad Prism 7.0 was used to create the graphs. Student’s


Contemplating the actual leaders of entrepreneurial firms and socio demographic dissimilarity between leaders and their teams, this study adopts panel data on the entrepreneurial firms of the China’s Growth Enterprise Market and empirically examines the influence of chair power on research and development (R&D) intensity of entrepreneurial firms from the perspective of social identity. The results indicate that chair power positively affects entrepreneurial firms’ R&D intensity. The chair–team sociodemographic dissimilarity moderates the relationship in such a way that chair power is negatively related to entrepreneurial firms’ R&D intensity only when chair–team sociodemographic dissimilarity is high. The execution of robustness checks authenticates the veracity of the empirical results. Leaders, as key decision-makers of enterprises, influence enterprise innovation, such as research and development (R&D) intensity, through using their power . To drivMany studies have examined the impact of leader’s power on organizational decision-making and performance e.g., . HoweverCombining with existing studies, there are still the following deficiencies. First, previous studies focus on the CEO/CTO power in large established corporations. These studies pay less attention to the entrepreneurial firms and their leaders, the chairs. For Chinese entrepreneurial firms, the legal representative are the chairs, who are the actual leaders and the most important decision-makers . CompareMoreover, the examination of leader power on innovation activities of entrepreneurial firms would be incomplete without assessing the cross-level interaction between leaders and their teams. Previous studies suggest that the leaders’ personal characteristics in large established corporations play a significant role in affecting the relationship between leaders’ power and enterprise innovation e.g., . NeverthAgainst this backdrop, this study constructs the theoretical framework of chair power, chair–team sociodemographic dissimilarity, and R&D intensity of entrepreneurial firms and analyzes the interaction mechanism among them. It contributes to the ongoing scholarly on leader power and firm innovation in a few distinct ways. First, this study probes the relationship between chair power and R&D intensity of entrepreneurial firms. Extant studies focus on the CEO/CTO power and organizational innovation in large established corporations e.g., . ConsideIn what follows, section “Theoretical Background and Hypotheses” explains the theoretical background and assumptions. Section “Materials and Methods” describes the research methodology and design, including sample selection, data sources, variable measurement indicators, and data analysis. Section “Results” reports the empirical results. Section “Discussion and Conclusion” presents a discussion of the research findings and their implications and outlines avenues for future research.This study mainly builds on the theories of behavioral agency model, social hierarchy theory, and social identity theory. The behavioral agency model combines prospect theory and classic agency theory, first proposed by To better explain the functional realization mechanism of decision-makers’ innovative strategic choice and the influence of the cognitive interaction between leaders and their teams, this study further introduces the social hierarchy theory and social identity theory. On the one hand, social hierarchy theory emphasizes that the ranking of individual members in social dimensions is the essential features of social hierarchy . ChineseR&D intensity usually refers to the degree an enterprise creates new products and knowledge by increasing R&D investment . And it Powerful chairs can promote the formulation and implementation of innovation strategies with their own influence. First, a social hierarchy governs the ranking of individual members in social dimensions . In thisHypothesis 1: Chair power is positively related to the R&D intensity of entrepreneurial firm in China.The chair–team sociodemographic dissimilarity reflects that each member of the team is different from the chair in age, gender, and other detectable attributes. According to the social identity theory, individuals will scrutinize and evaluate themselves by comparing observable demographic characteristics with other individuals. When individuals find that they have similar characteristics with other individuals, differences between “in-group” and “out-group” will be formed, leading to in-group favoritism and out-group hostility . The croThe destruction of social process between leaders and their teams will further affect the development of task process. The cross-level sociodemographic dissimilarity of chairs reflects the diversity among members. Thus, it may provide different knowledge for R&D strategic decisions. Past research suggests that the informational diversity would be beneficial because of the wider range of task-relevant resources brought by dissimilar participants, whereas social category diversity would be detrimental to the effective functioning of teams due to the formation of “insiders” and “outsiders” within the teams e.g., . ConsequUnlike the production and business behavior, R&D investment is a high-risk activity that requires comprehensive and high-quality strategic formulation in the early stage. Because of the market position and development stage of entrepreneurial enterprises, the innovation elements and knowledge acquired by entrepreneurial enterprises are limited. The leaders and their teams are the strategic decision-making body of entrepreneurial firms. And when facing the complex R&D investment decision-making, the cross-level interaction and identification between the leaders and their teams are more needed . In thisHypothesis 2:The chair–team sociodemographic dissimilarity moderates the positive effect of chair power on R&D intensity of entrepreneurial firm such that the positive effect is lessened when chair–team sociodemographic dissimilarity is higher.t to t + 1) is used. This study focuses on public organizations. First, CGEM has low threshold and convenient financing, which is favored by many innovative entrepreneurial firms (Given that China’s Growth Enterprises Market (CGEM) was officially opened on October 23, 2009, the firms listed before December 31, 2011, in CGEM are selected. And the listed firms that existed before 2003 are removed. Taking 2012–2016 as the research period, a total of 80 firms and 320 firm–year combinations were obtained. Those firms whose board chairs are changed and their annual reports without R&D expenditure data are further excluded. This leads to the selection of an eligible sample of 75 firms and 300 firm–year combinations. We set 2016 as the latest year for the sample enterprises. On the one hand, this allows meeting the definition of entrepreneurial ventures . On the al firms . The pheal firms . Third, Much of the data are taken from the China Stock Market and Accounting Research (CSMAR) database. The missing values from the CSMAR database are sought using the WIND database and annual reports. The consistency of multisource data is checked. And the annual reports shall prevail in case of discrepancy. The data for demographic characteristics and power structure of chairs and their teams are hand-collected from multiple sources, including the CSMAR database, annual reports, and corporate websites.R&D intensity is measured as the ratio of R&D expenditures to total assets. Entrepreneurial firms often have very low and unstable sales, and whose total assets are stable relative to established firms . Their iExisting studies usually focus on the CEO/CTO in organizations. However, for Chinese listed company, the legal representative is the board chair, who is the actual leader and the most important decision-maker . Thus, cPower in top managers as proposed by In this study, chair power is operationalized as the composite measure including structural power , ownership power , expert power (chair title), and prestige power . Chair duality is operationalized as a binary variable taking the value of 1 if the chair also serves as a CEO and 0 otherwise. Chair shareholding is also operationalized as a binary variable taking the value of 1 if the chair holds shares of the firm and 0 otherwise. Chair founder status is operationalized as a binary variable with a value of 1 if the chair is the founder or cofounder in team members and 0 otherwise. Chair title is operationalized as a binary variable with a value of 1 if the chair has high-level technical title in the industry and 0 otherwise. Chair political connections as a binary variable taking the value of 1 if the chair has political connections with the governments and 0 otherwise. Similar to the study of - Pikt)2, where Pikt is the proportion of the chair i for k organization in t year share the same gender category with team members. Chair–team age dissimilarity is measured using the distance formula: xikt is the age of chair i for k organization in t year, xjkt is the age of executive j (excluding the chair) for t year in k company, and n is the number of team members. Following This variable is a composite of a chair’s dissimilarity to the rest of the team in terms of two externally observable sociodemographic attributes: age and gender. The sociodemographic attributes include age, gender, and ethnicity . Considen-1]1/2 , where x- Pikt)2, where Pikt is the proportion of the chair i for k organization in t year share the same educational specialty category with team members, and chair–team functional background dissimilarity, measured using the formula: (1- Pikt)2, where Pikt is the proportion of the chair i for k organization in t year, share the same functional background category with team members. Educational specialty is categorized as science, engineering, economics, management, literature or art, law, or others. Functional background is categorized as manufacturing, R&D, finance or accounting, public relations, legal, literature or art, management, or others. At the organizational level, the variables are firm size, measured as the natural logarithm of total assets; asset–liability ratio, measured by the ratio of total liabilities to total assets; percentage of stock, measured by shareholding ratio of top 10 shareholders; ROE, measured as the ratio of net profits to net assets; cash ratio, measured as the proportion of corporate monetary funds and marketable securities to current liabilities; and turnover, measured as the natural logarithm of organizational turnover. Industry and year dummies are included to eliminate the potential impact of unobservable time and industry heterogeneities . Hypothesis 1 is thereby confirmed. Model 4 reveals that chair–team socio demographic dissimilarity has an insignificant moderating effect on the relationship between chair power and R&D intensity of entrepreneurial firm . Hypothesis 2 is thereby not confirmed.All the hypotheses were tested. As shown in r = −0.0010, P < 0.05), thus supporting Hypothesis 2. In addition, other results remain qualitatively similarly.The above results indicate that chair–team sociodemographic dissimilarity insignificantly moderates the relationship between chair power and R&D intensity of entrepreneurial firm. The possible reason is that the leader in entrepreneurial firm exhibits more risk-seeking behaviors relative to the general managers , thus wer = 0.0061, P < 0.01). Chair–team sociodemographic dissimilarity negatively moderates the relationship between chair power and R&D intensity . And both coefficient value and significance level are improved. This indicates that the results are more prominent in non-state-owned enterprises.Several robustness checks were conducted to further verify the findings. First, corporate ownership is one of the important differences among Chinese companies . Consider = 0.0044, P < 0.1). Chair–team sociodemographic dissimilarity negatively moderates the relationship between chair power and R&D intensity .To further investigate the impact of chair power on R&D intensity of entrepreneurial enterprises, chair power was measured from the cross-level perspective. Chair duality is operationalized as a binary variable taking the value of 1 if the chair also serves as a CEO and 0 otherwise. Chair shareholding is also operationalized as a binary variable taking the value of 1 if the chair holds shares of the firm at a higher rate than other members and 0 otherwise. Chair founder status is operationalized as a binary variable with a value of 1 if the chair is the sole founder relative to other members and 0 otherwise. Chair title is operationalized as a binary variable with a value of 1 if the chair has high-level technical title in the industry while other members do not and 0 otherwise. Chair political connections as a binary variable taking the value of 1 if the chair has political connections with the governments while other members do not and 0 otherwise. Similar to the study of r = −0.0396, P > 0.1).One could speculate that team-level compositional diversity may influence the functioning of chair power in the way as chair–team dissimilarity. To assess this, the regressions were re-evaluated with chair–team sociodemographic dissimilarity replaced with team-level diversity. Model 9 of r = −0.0001, P > 0.1).To address the possibility that too much sociodemographic dissimilarity may has positive effects through generating social norms . We asser = 0.0067 vs. r = 0.0040). Considering that tech-intensive industries requires high R&D intensity, this finding further supports the core argument that empowering more directed at the chairs becomes particularly effective.A fifth test shows that the association between chair power and R&D intensity of entrepreneurial firm is actually stronger in technology-intensive industries than that in non-tech-intensive industries   is 1.85, and the P-value is 0.174. It indicates that there is no endogenous explanatory variable. Durbin–Wu–Hausman test results show that statistic χ2(1) is 2.09, and the P-value is 0.147. The result also indicates that chair power is not an endogenous explanatory variable. Moreover, GMM C statistic χ2(1) is 1.79, and the P-value is 0.180. Thus, the above test results show that there is no obvious endogeneity problem.This problem was addressed by two ways. On the one hand, following t + 1 is taken as the dependent variable, and the R&D intensity of entrepreneurial enterprise in period t is taken as the independent variable. The regression results show that the coefficient of R&D intensity is not significant . Similarly, chair power in period t is taken as the dependent variable, and the R&D intensity of entrepreneurial enterprise in period t is taken as the independent variable. The results remain unchanged , which further helps to exclude the possibility of reverse causality.On the other hand, drawing on the method of Unlike large established corporations, does chair power promote the R&D intensity of entrepreneurial firms? How does chair–team sociodemographic dissimilarity in entrepreneurial firms affect the above relationship? The discussion of these issues is conducive to extend the current understanding of the impact leaders’ power has on the enterprises innovation. By adopting panel data on the entrepreneurial firms of China’s Growth Enterprise Market, the results reveal a significant positive relationship between chair power and entrepreneurial firms’ R&D intensity. Furthermore, chair–team sociodemographic dissimilarity attenuates the positive effect of chair power on R&D intensity of entrepreneurial firms. By changing the measurement method of key variables, testing the influence of non-linear effects, and controlling other factors, the veracity of the above findings is authenticated.The conclusion of this study provides a systematic theoretical logic to explain how chairs’ power affects R&D intensity of entrepreneurial firms under the context of China. The theoretical contributions of this study are twofold. First, prior studies investigate the impact of CEO/CTO power on the innovativeness of large established corporations e.g., . By conspost hoc null findings regarding the effects of team-level compositional diversity, highlight that, at least in certain situations , the moderating effect of team composition on chair influence may derive mostly from the relative differences between the chair and her/his team. Thus, distinguishing chairs from their teams may prove fruitful in advancing our overall understanding of the joint influence of the chairs and their teams.Second, extant studies focus on the moderating effect of leaders’ personal characteristics on the relationship between leaders’ power and innovation in large established corporations e.g., . This stThis study has a number of implications for research. First, by investigating the relationship between chairs’ power and innovation activity of Chinese entrepreneurial firms, this study extends the current understanding of power that chairs have on the organizations they lead. As such, the findings of this study improve scholars’ understanding by providing empirical evidence on the level of risky R&D investment of Chinese entrepreneurial firms that powerful chairs are more likely to pursue. Second, this research also illustrates the link between powerful chairs and their cross-level sociodemographic dissimilarity that influences R&D intensity of Chinese entrepreneurial firms. These findings also hold practical implications that include monitoring of executives appointment and cross-level management of teams in entrepreneurial firms. The positive relationship between chairs’ power and R&D intensity suggests that entrepreneurial firms that seek investment in R&D should be open to the idea of endowing their chairs with greater power to foster their innovative spirit. Specifically, the appointment of the chair in entrepreneurial firm should follow the principle that the chair has political connections with the governments, belongs to the founder, and has high-level technical title in the industry. In addition, the chair should hold shares and serve as a CEO and other important positions in entrepreneurial firms. Further, the results of robustness checks also provide insights into the importance of corporate ownership and industry categories. Specifically, these findings suggest that the innovation-driven effects of chairs’ power are more prominent in non-state-owned entrepreneurial firms. And empowering more directed at the chairs in entrepreneurial firms of tech-intensive industries becomes particularly effective. Furthermore, the findings of this study also provide insights into the importance of chair–team social category dissimilarity. Specifically, when making appointment decisions such as external introduction, internal promotion, and intergenerational inheritance, we should pay attention to the composition of observable demographic characteristics of leaders and their teams in addition to considering the person-post matching. By reducing the chair–team sociodemographic dissimilarity, especially the age differences between chairs and their teams in Chinese entrepreneurial firms, it can exert the innovative spirit of powerful leaders to a greater extent.As with any research, this study has some limitations. First, power is regarded as a comprehensive and unified whole, ignoring the heterogeneity in different power dimensions. For example, managers’ power can be divided into power depth and power breadth according to the multiple power sources . The funThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by School of Economics and Management at University of Science and Technology Beijing. The patients/participants provided their written informed consent to participate in this study.All authors contributed to the article and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Arterial blood pressure increases during aging . Periphe2+ release events (Ca2+-sparks) has been identified to limit myogenic tone. This functional unit involves type 2 ryanodine receptors (RyR2s) [2+-sensitive K+ (BKCa) channels, causing spontaneous transient outward K+ currents (STOCs), CaV1.2 L-type Ca2+ channels, and sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA). Recent data imply a novel role of Ca2+-influx via CaV3.2 T-type channels to trigger Ca2+-sparks [2+-sparks triggered at physiological membrane potentials in mesenteric arteries. Cav3.2 apparently activate RyR2s from the cytosolic side [2+ transferred into the cells and a more restricted spatial range than Ca2+-sparks triggered by L-type CaV1.2 channels. Consistent with this mechanism, proper function of the T-type CaV3.2-RyR axis requires sufficiently high SR Ca2+ load, which is regulated via Ca2+-influx through L-type CaV1.2 channels [Others and we have focused on mechanisms controlling VSMC contraction/relaxation states to regulate vessel diameter. An indirect mechanism involving elementary Ca+-sparks ,6, contrlic side , possiblchannels .2+, T-type channels: Ni2+, TRP channels: Gd3+) and specific knockout mice . We found that CaV1.2 L-type channels contribute to the same extent to Ca2+-sparks generation in young and aged mice. RyR2 is the predominant RyR isoform responsible for Ca2+-sparks [V1.2-mediated Ca2+-sparks but also for additional pathways. We found that the CaV1.2-RyR2 axis works efficiently in old VSMCs, i.e increasing age does not impair the formation of Ca2+-sparks in old VSMCs [There is a large body of experimental knowledge on key control processes of VSMC contractile state. However, the studies were usually performed in young adult rodents, whereas hypertension is a typical cardiovascular risk factor in humans at advanced age. We therefore extended our studies to aged mice, using approaches of pharmacological blockade (L-type channels: Cdld VSMCs .V3.2-deficiency (KO) does not affect blood pressure in young mice [v3.2 channels limit myogenic tone in young, but surprisingly not in old mice mesenteric arteries [V3.2 channel expression/activity to trigger Ca2+ sparks are possible underlying mechanisms for these effects [V3.2-T-type channel Ca2+-influx pathway obviously exists only in young mice, but not in mice at advanced age.Although Caung mice , Cav3.2 arteries ,8. Decre effects . The newV3.2 in caveolae close to RyRs is essential for triggering Ca2+-sparks, we analysed age-related alterations in caveolae to explain the complete malfunction of the Cav3.2-RyR2 axis in old mice VSMCs. Treatment with methyl-ß-cyclodextrin, depleting cholesterol as an essential caveolae component from the plasma membrane, failed to inhibit Ca2+-sparks in VSMCs of old mice. Electron microscopy images revealed a decrease in caveolae density and alterations in caveolae structure in advanced age [V3.2-Ca2+ sparks signaling by putative caveolemmal T-type CaV3.2 channels, which are insufficiently close to RyRs for extracellular Ca2+-influx through T-type channels to trigger Ca2+-sparks. Of note, we confirmed our findings using novel Eps15 homology domain-containing protein (EHD2)-deficient mice with genetically impaired caveolae formation. In this animal model, CaV3.2-expression was also decreased. Together, reduced caveolae density could downregulate CaV3.2 T-type channels to impair the ability of T-type CaV3.2 to generate Ca2+-sparks in old VSMCs.Advanced age can alter the composition of lipid rafts and caveolae, which could affect a variety of signaling molecules contributing to the pathophysiology of cardiovascular diseases. Since localization of Canced age . Thus, d2+-sparks remaining after L-type channel block. This finding enabled us to study effects of the non-specific TRP channel blocker Gd3+ (100 µM) to shed a light on the putative role of an TRP channel(s) in VSM elementary Ca2+ release (Ca2+-sparks) to compensate for loss of CaV3.2 T-type channels. We found that caveolae are presumably not required for the gadolinium-sensitive Ca2+-influx pathway, since caveolae disruption by methyl-ß-cyclodextrin did not alter Ca2+-spark events in old VSMCs. Due to the low specificity of Gd3+, further work is required to ascertain which TRP cation channel(s) or pathways are responsible for generation of these Ca2+ sparks.In mice at advanced age, there was a fraction of Ca2+ signaling and myogenic tone regulation. The CaV3.2-RyR-axis is an effective pathway to generate Ca2+-sparks in young VSMCs, but plays little or no role in aged VSMCs. A Gd3+-sensitive Ca2+-influx pathway, putatively through nonselective TRP channels, compensates for loss of the CaV3.2-RyR-axis. Clarifying the exact identity of the receptor(s)/pathways and the nature of tight vs. loose coupling between Ca2+ influx-RyR2s pathways  will reveal potential novel targets for antihypertensive therapies in patients in the elderly (Our results revealed age-dependent differences in elementary Ca elderly .


HOXA genes is thought to involve CTCF-mediated chromatin loops and the opposing actions of the COMPASS and Polycomb epigenetic complexes. We investigated the role of these mechanisms at the HOXA cluster in AML cells with the common NPM1c mutation, which express both HOXA and HOXB genes. CTCF binding at the HOXA locus is conserved across primary AML samples, regardless of HOXA gene expression, and defines a continuous chromatin domain marked by COMPASS-associated histone H3 trimethylation in NPM1-mutant primary AML samples. Profiling of the three-dimensional chromatin architecture in primary AML samples with the NPM1c mutation identified chromatin loops between the HOXA cluster and loci in the SNX10 and SKAP2 genes, and an intergenic region located 1.4 Mbp upstream of the HOXA locus. Deletion of CTCF binding sites in the NPM1-mutant OCI-AML3 AML cell line reduced multiple long-range interactions, but resulted in CTCF-independent loops with sequences in SKAP2 that were marked by enhancer-associated histone modifications in primary AML samples. HOXA gene expression was maintained in CTCF binding site mutants, indicating that transcriptional activity at the HOXA locus in NPM1-mutant AML cells may be sustained through persistent interactions with SKAP2 enhancers, or by intrinsic factors within the HOXA gene cluster.Transcriptional regulation of the  HOX genes encode developmentally regulated transcription factors that are highly expressed in acute myeloid leukemia (AML) and are important drivers of malignant self-renewal in this disease. Previous studies have shown that expression of HOX family members in AML is nearly always restricted to specific genes in the HOXA and/or HOXB clusters (HOXC and HOXD genes are rarely expressed), and that expression patterns correlate with recurrent AML mutations [HOX expression is most closely associated with AMLs with MLL rearrangements, which exclusively express HOXA genes, and AMLs with the recurrent NPM1c mutation, which nearly always express both HOXA and HOXB genes [NPM1 mutations make the combined HOXA/HOXB expression pattern the most common HOX phenotype in AML patients. However, the regulatory mechanisms that drive this expression pattern are poorly understood.The utations . HOX expHOX gene regulation in model organisms have established that colinear expression of each HOX cluster is mediated by COMPASS/Trithorax and Polycomb group proteins, which promote gene activation and repression and perform methylation of histone H3 at lysine 4 (H3K4me3) and 27 (H3K27me3), respectively [HOX gene regulation in AML cells, and are best understood for the HOXA cluster in AMLs with MLL rearrangements. MLL1 (KMT2A) is a component of the COMPASS complex, and MLL fusion proteins bind to the HOXA locus in AML cells and recruit the non-COMPASS histone H3 methyltransferase DOT1L, which is required for HOXA activation and AML development in MLL-rearranged leukemia models [HOXA gene regulation in AML cells. Specifically, the HOXA and HOXB clusters contain multiple binding sites for the chromatin organizing factor CTCF, and chromatin conformation experiments suggest these events mediate local chromatin loops in AML cells with MLL rearrangements [HOXA cluster in MLL-rearranged AML cells resulted in altered chromatin structure and reduced HOXA gene expression [HOXA locus in ways that require specific CTCF binding events or their associated chromatin structures.Studies of ngements . In addipression . These sHOXA regulation in MLL-rearranged AML, this molecular subtype accounts for <5% of all AML patients and only 25% of AMLs that express HOXA genes [NPM1 mutations nearly always express HOXA genes, it is unclear whether HOXA expression in these cells shares similar regulatory factors and chromatin structures that appear to be critical for HOXA expression in MLL-rearranged AML cells. In this study, we investigated histone modifications and chromatin interactions at the HOXA locus in NPM1-mutated AML samples vs. other AML subtypes, and used a NPM1-mutant AML cell line model to determine whether CTCF at the HOXA locus is required to maintain HOXA expression and chromatin structure.While these mechanistic insights have provided valuable information about XA genes . Althoug6 cell/mL in MEM alpha with 20% FBS and 1% penicillin-streptomycin. NPM1c was verified in the OCI-AML3 line by targeted sequencing and in RNA-seq data from wild type and mutant clones. Kasumi-1 (received as a gift from T. Ley), IMS-M2 (received as a gift from L. Brunetti), and MOLM13 (received as a gift from J. Dipersio) cell lines were cultured in RPMI-1640 with 1% penicillin-streptomycin and FBS .Primary AML samples and normal hematopoietic cells were obtained from diagnostic AML and normal bone marrow aspirates, respectively, following informed consent using protocol 201011766) approved by the Human Research Protection Office at Washington University as described previously 11766 app. OCI-AMLHypothesis testing was performed using the indicated parametric statistics after verification of normality. Sample sizes for genomic studies using read count data were selected to provide >80% power to detect a fold-change of at least 2.ChIP-seq was performed using ChIPmentation  with the2, 0.045% NP40, 0.45% Tween-20), PCR, and gel electrophoresis; clones with evidence for deletions were sequenced, and clones with deletions were expanded for analysis.Deletions were generated using CRISPR/Cas9 with guide RNAs from the UCSC genome browser , 21 thatHOXA9 exons 1-2 with a GUSB control (IDT). RNA-seq libraries were generated using 300 ng of RNA with the KapaHyper stranded RNA library kit for Illumina (Roche) following the manufacturer’s instructions and then sequenced on a NovaSeq 6000 to obtain ~50 million 2 × 150 bp reads. Reads were trimmed with trimgalore, aligned with STAR [RNA extractions were performed on ~1 million cells with the Quick-RNA MicroPrep Kit . One hundred nanograms of RNA was used for cDNA synthesis , and qPCR was done in duplicate in a StepOnePlus PCR System (Applied Biosystems) for ith STAR , and traith STAR .Hi-C libraries were prepared as described  using 4–HOXA cluster have accessible chromatin in primary AML samples that coincide with CTCF binding sites in other human cell types [NPM1, t and t MLL rearrangements, and t creating the RUNX1-RUNX1T1 gene fusion, which displayed the expected HOXA and HOXB, HOXA only, and no HOX expression patterns, respectively (Table HOXA6 and HOXA7 (“CTCF binding site CBSA6/7”), and HOXA7 and HOXA9 (“CBSA7/9”), near the transcriptional start site of HOXA10 (“CBSA10”), and in the 5′ UTR of HOXA13 (“CBSA13”)  Fig. . All foupes Fig. .Fig. 1CTHOXA cluster that overlapped CBSA6/7 and CBSA7/9, and was conserved in the MLL-rearranged and NPM1-mutant AML samples, and normal CD34+ cells   Figs.  and S2A.s) Figs. . Adjacenes Figs. , S2A. Chype Fig. . Similares Figs.  and S2A.NPM1-mutant AML cells. To this end, we used the OCI-AML3 cell line with a canonical NPM1 insertion mutation and that expresses MEIS1 and both HOXA and HOXB genes [HOX expression phenotypes, including MLL-rearranged MOLM13 cells that expressed only HOXA genes, and the RUNX1-RUNX1T1-containing Kasumi-1 cell line, which had low HOX expression . The resulting mutations did not appreciably alter markers of cell maturation in the edited cells Fig. , and theHOXA9 expression analysis via RT-qPCR to assess whether loss of CTCF binding affected HOXA9 expression. Surprisingly, HOXA9 expression was essentially unchanged in all deletion lines, with little evidence for consistent reduction in expression in the mutant clones, and no trend toward decreased expression between wild-type, heterozygous, and homozygous single mutants , which contacted regions in the SKAP2, and SNX10 genes, and an intergenic region 1.4 Mbp upstream with no associated gene annotations. Similar chromatin loops were observed in Hi-C data generated from the MLL-rearranged MOLM13 cell line, and in previously reported data from normal human HSPCs [HOXA genes.Given the role of CTCF in regulating chromatin architecture, we used in situ Hi-C  to defines Table . Analysies Table . The HOXary Fig. , with cean HSPCs   and a sample with the RUNX1-RUNX1T1 gene fusion and low HOXA gene expression , and the region of the HOXA cluster that interacted with intron 1 of SKAP2 involved posterior HOXA genes in the NPM1c sample, but was restricted to the anterior HOXA1 gene in the RUNX1-RUNX1T1 sample  displayed clear H3K27ac signal in NPM1-mutant AML samples, as well as MLL-rearranged samples that also express HOXA genes  to determine whether loci that interact with nes Fig. . Loci neX10 Fig. , but H3Kcus Fig. . AlthougHOX transcription factors are drivers of self-renewal in AML cells and are highly expressed in AMLs with NPM1c mutations. In this study, we demonstrated that the chromatin organizing factor CTCF is bound equally to specific sites at the HOXA locus in NPM1-mutant primary AML samples compared with other AML subtypes, and define a dynamic chromatin domain in primary AML samples and normal hematopoietic cells. Targeted deletions in the NPM1-mutant OCI-AML3 cell line eliminated CTCF binding, but surprisingly did not disrupt HOXA gene expression. This was true when multiple binding sites were deleted individually, or in combination, and was supported by independent mutant clones that showed little evidence for consistent decreases in HOXA gene expression, or changes to the histone modifications we measured. However, loss of CTCF binding did result in clear alterations to the chromatin loops involving the posterior HOXA genes, including HOXA9 and HOXA10. Specifically, long-range loops were diminished, and were replaced by compensatory interactions with regions of the SKAP2 gene, which also appear to directly interact with active HOXA genes in primary AML samples with the NPM1c mutation. Some of these candidate enhancers have been reported in other studies [HOXA gene activation in normal and malignant myeloid cells. studies , but havHOXA cluster is not absolutely required for maintenance of HOXA expression in NPM1-mutated AML cells. Deletion of CTCF site CBSA7/9 was previously shown to affect HOXA expression in the MLL-rearranged MOLM13 cell line [HOX genes are regulated in MLL-rearranged vs. NPM1-mutant AML cells. Indeed, the HOX expression phenotype of these AML subtypes is strikingly different: MLL rearrangements are associated with only HOXA gene expression, whereas both HOXA and HOXB genes are expressed in NPM1-mutant AML cells. HOXA and HOXB genes are simultaneously downregulated during normal myeloid maturation [NPM1-mutant vs. MLL-rearranged AML cells. We also observed differences in HOXA cluster expression patterns in MOLM13 vs. OCI-AML3 cells, including higher expression of HOXA11, HOXA13, and the HOTTIP long noncoding RNA in OCI-AML3 cells. HOTTIP has an established role in HOXA gene regulation [HOXA genes in NPM1-mutant AML cells.The central observation in this study is that CTCF binding at the gulation , 35, andHOXA genes during development. Our analysis of Hi-C data from OCI-AML3 cells and NPM1-mutant primary AML samples identified long-range loops between the active posterior HOXA genes HOXA9-HOXA13 and sequences with enhancer-associated epigenetic marks in the SNX10 and SKAP2 genes. Additional loops were identified at other loci, but these did not possess active chromatin modifications in primary AML samples. AML cells without HOXA expression did not display the same interactions, and lacked active histone marks at the interacting loci, which provides evidence that these contacts may be functionally relevant for HOXA gene expression in AML. Interestingly, loss of CTCF within the HOXA cluster revealed the plasticity of these interactions, implying that multiple loci may serve as exchangeable HOXA enhancers, which is reminiscent of other well-studied, complex regulatory systems, and has been implicated in developmental HOXA gene regulation [Three-dimensional architecture is an important component of the regulatory control of gulation , 37.HOXA cluster in OCI-AML3 cells, even after elimination of key CTCF boundaries. In fact, there was a tendency for deletion mutants to show increased HOXA gene expression levels compared with wild-type cells, which is consistent with recent evidence suggesting that reduction of CTCF levels could enhance HOXA gene expression [HOXA cluster possesses intrinsic properties that maintain its active state. It is unclear whether this activity is caused by the compensatory interactions we observed, or if these chromatin loops are consequences of persistent gene expression that is mediated by autonomous, sequence-specific factors that recruit transcriptional machinery. The HOXA cluster contains many highly conserved noncoding elements beyond CTCF binding sites. Additional studies will be required to better understand whether these elements provide key signals that promote and maintain HOXA gene expression in NPM1-mutant AML cells.Our data showed that transcriptional activity and active histone modifications remained highly localized to the posterior pression . These oSupplemental Figures and Figure LegendsSupplemental Table S1Supplemental Table S2Supplemental Table S3Supplemental Table S4Supplemental Table S5