Ca2+ release units (CRUs) are highly organized junctions between the terminal cisternae of the sarcoplasmic reticulum (SR) and the transverse tubule (T-tubule). CRUs provide the structural framework for rapid elevations in myoplasmic Ca2+ during excitation–contraction (EC) coupling, the process whereby depolarization of the T-tubule membrane triggers SR Ca2+ release through ryanodine receptor-1 (RyR1) channels. Under conditions of local or global depletion of SR Ca2+ stores, store-operated Ca2+ entry (SOCE) provides an additional source of Ca2+ that originates from the extracellular space. In addition to Ca2+, skeletal muscle also requires ATP to both produce force and to replenish SR Ca2+ stores. Mitochondria are the principal intracellular organelles responsible for ATP production via aerobic respiration. This review provides a broad overview of the literature supporting a role for impaired Ca2+ handling, dysfunctional Ca2+-dependent production of reactive oxygen/nitrogen species (ROS/RNS), and structural/functional alterations in CRUs and mitochondria in the loss of muscle mass, reduction in muscle contractility, and increase in muscle damage in sarcopenia and a wide range of muscle disorders including muscular dystrophy, rhabdomyolysis, central core disease, and disuse atrophy. Understanding the impact of these processes on normal muscle function will provide important insights into potential therapeutic targets designed to prevent or reverse muscle dysfunction during aging and disease.Skeletal muscle contraction relies on both high-fidelity calcium (Ca Ca2+ signaling occurs when the free cytosolic Ca2+ concentration, maintained around 100 nM under resting conditions, rises rapidly upon Ca2+ release from intracellular stores and/or Ca2+ entry from the extracellular space, due to the combination of the opening of Ca2+-permeable channels and a steep concentration gradient (~4 orders of magnitude).Calcium , and a continuous system of internal membranes of sarcoplasmic reticulum (SR). The SR is composed of two distinct functional and morphological compartments in direct continuity with each other: (1) the SR terminal cisternae and (2) longitudinal or free SR [2+ release unit (CRU), or “triad”, is composed of a central T-tubule flanked by adjacent junctions with two SR terminal cisternae [2+ concentration. In skeletal muscle, EC coupling involves a unique physical or mechanical interaction between two different types of Ca2+ channels: voltage-gated L-type Ca2+ channels (CaV1.1) or dihydropiridine receptors (DHPRs), which function as voltage sensors in the T-tubule membrane, and type-1 ryanodine receptor (RyR1) Ca2+ release channels in the SR [2+ release channels [2+ from the SR into the myoplasm provides the chemical signal used to drive muscle contraction. The high amount of free (~300–500 µM) and bound Ca2+ within the SR [2+ ATPases (SERCA). CASQ1, a highly acidic protein resident in the SR terminal cisternae, has two major roles during EC coupling: (1) to bind/buffer a large amount of Ca2+ needed for release and activation of muscle contraction [2+ depletion [2+ pump densely packed in the free SR [2+ reuptake is the primary mechanism for Ca2+ clearance in skeletal muscle following release during EC coupling, allowing muscle relaxation [2+ increase during a single twitch contraction  is cleared from the myoplasm through SERCA pumps. As a result, SERCA-mediated Ca2+ reuptake is responsible for muscle relaxation and recovery of SR Ca2+ to levels needed for subsequent cycles of EC coupling.Muscle contraction and relaxation are regulated by rapid changes of myoplasmic Cah other:  the SR tisternae   pathway was unequivocally identified in both skeletal myotubes [2+ influx pathway activated by depletion of intracellular Ca2+ stores, is among the most important Ca2+ influx pathways in non-excitable cells [2+ sensor [2+ permeable channel of the plasma membrane [2+ depletion also serves as the primary molecular machinery of SOCE in skeletal muscle [2+ stores, needed to sustain Ca2+ release and force generation during repetitive, high-frequency stimulation [2+ entry units” (CEUs) [DHPR/RyR1-mediated EC coupling and muscle contraction has long been known to persist in the absence of extracellular Ca2+  and extrl muscle ,27,28,29myotubes  and adulmyotubes . SOCE, ale cells ,33,34. S+ sensor ,36,37, amembrane ,39,40. Cl muscle . Althougmulation ,44,45,46mulation ,48. Toge” (CEUs) . CEUs ar” (CEUs) ,47 toget” (CEUs) ,51,52,532+ stores during repetitive stimulation. Interestingly, we found that a key prerequisite for exercise-induced assembly of CEUs and increased SOCE function involves elongation of the T-tubule (containing Orai1) and association with stacks of SR membranes (containing STIM1) within the I band of the sarcomere. Assembly of CEUs following exercise provides a greater surface area for increased STIM1/Orai1 coupling needed to enhance Ca2+ influx during sustained muscle activity [Exercise-induced assembly of additional CEUs, which requires remodeling of SR and of T-tubules at the I band, likely reflects the increased need to replenish intracellular SR Caactivity ,45,46.Besides being important in the physiology of adult muscle fibers, SOCE is also critical for the correct development and maturation of skeletal muscle tissue ,55. This2+, muscle contraction also requires energy from ATP [2) in a process referred to as aerobic respiration [2, through a series to reactions to molecular O2 as the final electron acceptor, produces an electrochemical proton gradient across the inner mitochondrial membrane that is used to drive ATP generation by the F1/F0 ATP synthase. Under normal conditions, only ~1% of electrons that flow through the electron transport chain “slip” to molecular O2 to produce superoxide anions (O2•−). Either spontaneously or in a reaction catalyzed by superoxide dismutases (SODs), O2•− is converted to hydrogen peroxide (H2O2). Together, O2•−, H2O2, and hydroxyl radical (HO•) are collectively referred to as reactive oxygen species (ROS). Besides mitochondria, other cellular sources of ROS include extra-mitochondrial enzymes such as NADPH oxidases or xanthine oxidase. While for many years ROS produced as a byproduct of the mitochondrial metabolism were only considered pathogenic, recent evidence indicates that low physiological levels of mitochondrial ROS play an important role in cellular redox signaling pathways that regulate multiple cellular functions [In addition to Cafrom ATP . ATP hydpiration ,67,68. Tunctions ,70. Howe2+ homeostasiswas proposed in early studies that showed robust Ca2+ uptake by purified rat kidney mitochondria [2+ within the mitochondrial matrix is provided by the large negative electrochemical gradient (>−180 mV) across the mitochondrial inner membrane generated by electron transfer chain dependent H+ pumping during oxidative phosphorylation. Nonetheless, the affinity of mitochondria for Ca2+ uptake is relatively low, with a Ca2+ concentration required for half-maximal Ca2+ transport of ~30 µM [2+ transient (1–3 µM) during activation of skeletal muscle contraction [2+ uptake in skeletal muscle. However, with the development of mitochondria-targeted Ca2+ sensitive probes [2+ uptake during cytoplasmic Ca2+ oscillations was confirmed in multiple cell types  including skeletal muscle fibers [2+ promotes the opening of the mitochondrial Ca2+ uniporter (MCU), a 40-kDa protein that functions as a highly Ca2+-selective channel within the mitochondrial inner membrane, thus enabling Ca2+ flux down the high electrochemical gradient from the cytoplasm to the mitochondrial matrix [2+ within the mitochondrial matrix stimulates the activity of several enzymes of the tricarboxylic acid (TCA) cycle and electron transport chain including pyruvate dehydrogenase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, and the ATP synthetic activity of the F1/F0 ATPase [2+ transient [2+-mediated regulation of mitochondrial TCA and electron transport chain activity. The essential role of Ca2+ in regulating mitochondrial function in skeletal muscle is further highlighted by the severe clinical muscle phenotypes  observed in patients with loss-of-function mutations in MICU1, a modulatory subunit of the MCU complex that acts as a gatekeeper for mitochondrial Ca2+ entry [The hypothesis that mitochondria regulate intracellular Cachondria . The drif ~30 µM . This cotraction ,74. These probes , mitochoe fibers ,77. The l matrix ,79. The dient >−1 mV acrosransient  and are 2+ entry ,83,84.2+ ions when the peak myoplasmic Ca2+ level is an order of magnitude lower than the concentration for half-maximal Ca2+ transport by MCU, is still debated. This apparent paradox is explained by a “Ca2+ microdomain” that results from the close positioning of mitochondria relative to sites of Ca2+ release . The concept of a Ca2+ microdomain suggests that mitochondrial Ca2+ uptake is driven by a high local concentration of Ca2+ around mitochondria that is much higher than that observed in the bulk myoplasm [How mitochondria in skeletal muscle are able to take up Camyoplasm . In skelmyoplasm ,87,88,89myoplasm . 2+ release. As a result of this structural linkage, the average minimal distance between the RyR1 foot (site of Ca2+ release during EC coupling) and the outer membrane of the adjacent mitochondrion is only ~130 nm [2+ signaling SR-mitochondrial “nanodomain”  mitochondrial positioning closely follows maturation of the EC coupling system, changing from predominantly longitudinal to transversal positioning during postnatal development; (b) in adult muscle fibers, most mitochondria are located on the Z-line side of the triad, closely associated with the terminal SR cisternae via small (~8–10 nm) electron dense bridges termed “tethers” . The tet ~130 nm , thus crodomain” . The CRU via MCU   across the membrane followed by pathological myoplasmic Ca2+ overload that triggers an array of intracellular mechanisms that lead to myofiber degeneration and death including: dysregulation of cytosolicCa2+ homeostasis, mitochondrial Ca2+ overload/damage, increased mitochondrial ROS production and oxidative stress, and activation of the Ca2+-dependent proteases [2+ handling and increased oxidative stress that underlie the dystrophic phenotype are far from being fully understood. Currently, two main pathomechanisms are hypothesized to be responsible for abnormalities in myoplasmic Ca2+ levels in DMD: (1) excessive transmembrane Ca2+ influx and (2) enhanced SR Ca2+ leak through oxidized RyR1 Ca2+ release channels  comprise a heterogeneous group of muscle diseases characterized by weakness, muscle wasting, and progressive muscle degeneration that can ultimately lead to an impairment of mobility and premature death. The most common MD is the Duchenne muscular dystrophy (DMD), a currently incurable inherited X-linked recessive muscle disorder that affects 1 in 3500 male births. DMD is caused by loss-of-function mutations in the gene encoding dystrophin, a 427 kDa structural protein located at the cytoplasmic face of the sarcolemma . Dystroproteases ,96  or the DGC (in -/-Sgcb mice) is associated with hypernitrosylation of specific cysteine residues in RyR1, which lead to FKBP12 dissociation, destabilization of the RyR1 channel closed state, and increased RyR1-dependent SR Ca2+ leak [2+ leads to mitochondrial Ca2+ overload and uncontrolled ROS/RNS production that further oxidizes/nitrosylates RyR1 in a destructive feed-forward cycle of Ca2+ leak and ROS/RNS production (mdx mice following treatment with N-actetylcysteine (NAC), a potent antioxidant [2+ signaling and excessive mitochondrial ROS production was shown to be a key pathomechanism not only in MDs, but even in other muscle diseases such as malignant hyperthermia (MH) and central core disease (CCD)  see . In factR1-Y522S  and CASQR1-Y522S  mice.2+ release by reducing the opening probability of destabilized RyR1 channels, thus interrupting the destructive feed-forward cycle of Ca2+ leak and ROS/RNS production and ameliorating the dystrophic phenotype. However, it remains unclear whether or not increased ROS/RNS levels modify proteins that mediate SOCE to augment Ca2+ entry and signaling. Likewise, it is unknown if inhibiting Ca2+ entry reduces oxidative stress. Future studies using mouse models to specifically inhibit SOCE in skeletal muscle will be needed to answer this important questionand, thus, validate SOCE as a potential therapeutic target to treat this incurable disease.A reduction in oxidative stress normalizes SR Ca2+ handling and increased oxidative stress are pathomechanisms of the myopathic phenotype of two overlapping muscle disorders: MH susceptibility and CCD. MH is a potentially lethal inherited pharmacogenetic disorder characterized by a life-threatening hyperthermic reaction in susceptible individuals exposed to volatile/halogenated anesthetics  and/or succinylcholine, compounds commonly used during surgical procedures [cores [Aberrant Caocedures . CCD, onocedures ,109. An s [cores ,111, amos [cores . In addis [cores ,114,115.RYR1 gene [RYR1 gene with MH  indicates that these disorders result, at least in part, from defective RyR1 function and Ca2+ regulation in skeletal muscle. MH-related mutations in RyR1 destabilize the SR Ca2+ release channel closed state, resulting in an increased susceptibility to opening in response to activators, SR Ca2+ leak, and mitochondrial Ca2+ uptake and subsequent ROS/RNS production [2+ leak. This destructive feed-forward mechanisms of increased RyR1 Ca2+ leak and oxidative stress enhances RyR1 sensitivity to activation in MH that eventually leads to mitochondrial damage and the development of central cores in CCD [MH and CCD are inexorably linked to one another as most cases for both conditions are linked to mutations in the YR1 gene ,117. Assoduction . In turns in CCD   reduces mitochondrial damage and the development of cores in RYR1-Y522S mice, but not MHS [2+ homeostasis in muscle are also involved in MH and CCD pathophysiology remains poorly understood. The reason might lie in the fact that for many years it was assumed that sustained elevations in myoplasmic Ca2+ are solely the consequence of increased RyR1-mediated SR Ca2+ leak/release. However, excessive RyR1 Ca2+ leak can lead to local SR depletion that activates SOCE to further enhance resting Ca2+ levels    and when2+ release function due to oxidative stress-dependent modification in RyR1, likely due to altered mitochondrial function. In line with this idea, Marks and colleagues reported an age-dependent increase in RyR1 oxidation/nitrosylation, FKBP12 dissociation, and SR Ca2+ leak [2+ leak, peak electrically evoked Ca2+ release and muscle specific force production were reduced in muscle from aged mice [A third mechanism proposed to contribute to an age-dependent reduction in intrinsic muscle specific force generation involves altered SR Caa2+ leak . As a reged mice . 2+ leak, a reduction in STIM1/ORAI1-mediated SOCE activity is also proposed tocontribute to age-dependent reduction in muscle specific force productionandincreasedsusceptibility to fatigue in aged muscle. In support of this idea, muscles from 2-year-old mice exhibit reduced SOCE and increased susceptibility to fatigue during high-frequency stimulation [extensor digitorum longus muscles from 2-year-old mice exhibited an accelerated decline in force generation during high-frequency stimulation compared to that of muscles from 4-month-old mice [2+ entry in aged muscle, removal of Ca2+ from the extracellular medium during repetitive high-frequency stimulation reduced contractility of muscles from young, but not aged, mice. These data are in line with findings of Thornton and colleagues who reported an inability of aged muscle to recover Ca2+ ions from the extracellular space via SOCE [Beside impaired EC coupling due to DHPR/RyR1 uncoupling, reduced CRUs and CRU–mitochondrial association, altered mitochondria structure/function, and increased nitrosylation-dependent RyR1 Camulation ,144. We old mice . Consistvia SOCE .2+ refilling during prolonged muscle activity, and thus, lead to reduced Ca2+ availability within the SR to support force production during sustained activity. This idea is consistent with the fact that pharmacological and/or genetic inhibition of SOCE in skeletal muscle of young mice results in a reduction in force generation and increased susceptibility to fatigue [An age-dependent reduction in SOCE activity could lead to impaired SR Ca fatigue ,63,143.  fatigue  and (2)  fatigue . Clearly2+-dependent signaling pathways involved in the dynamic process of protein degradation [2+ alloserstically activates calpainand caspase-12 to activate caspase-3, which cleaves cytoskeletal proteins to disrupt myofilaments and facilitate protein degradation. In this regard, dysregulation in myoplasmic Ca2+ levels promotes atrophy in various muscle disuse animal models and pathological conditions [2+ was observed in soleus muscle fibers following hindlimb unloading and reloading in rats [2+ level through activation of Ca2+ release via inositol 1,4,5 trisphosphate receptors, that was proposed to activate downstream caspase-mediated proteolytic pathways [2+ activation of calpain-mediated downstream proteolytic pathways in muscle atrophy during cancer cachexia.Skeletal muscle atrophy reflects the loss of muscle mass as a result of muscle disuse  and certain pathological conditions  . Muscle radation  .A potential mechanism for mitochondrial dysfunction in muscle atrophy is that enhanced mitochondrial Capathways . In cont atrophy . Electriomplex I . Consistomplex I  reported2+ homeostasis and increased oxidative stress in the loss of muscle mass, increase in muscle damage, and reduction in muscle contractility that are characteristic of sarcopenia, disuse atrophy, and a wide range of muscle disorders including DMD, CCD, and heat/exercise-induced rhabdomyolysis. Nevertheless, precise molecular mechanisms are yet to be fully understood and a considerable number of unresolved issues and open questions remain to be addressed. Over the past several years, substantial progress was made in elucidating exciting new insights into the role of impaired Ca2+ handling and Ca2+-dependent mitochondrial ROS/RNS production in the decline of muscle function during aging, muscle atrophy, and the pathogenesis of several genetically inherited muscle disorders. These advances identify several signaling pathways and molecular mechanisms that represent potential new targets for the development of more effective therapies to treat a wide range of debilitating human myopathies.This review provides a comprehensive overview of the literature supporting a role for dysfunctional Ca


Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are bone marrow cancers that have a poor prognosis. They have traditionally been treated with chemotherapy with limited success, and stem cell transplant, which many patients are too frail to receive. Perturbations in the immune system play a role in the development of MDS and AML. Recently, there has been progress in understanding how the immune system can be harnessed to treat these cancers. Effective immune therapies for MDS and AML have been developed at a rapid pace, including novel immune checkpoint inhibitor antibodies, bispecific T-cell-engaging antibodies, antibody drug conjugates, vaccine therapies, and cellular therapeutics including chimeric antigen receptor T-cells and natural killer cells. This review article highlights advances in this field for the practicing clinician to aid in understanding new developments for treating patients with MDS and AML.Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are hematologic malignancies arising from the bone marrow. Despite recent advances in treating these diseases, patients with higher-risk MDS and AML continue to have a poor prognosis with limited survival. It has long been recognized that there is an immune component to the pathogenesis of MDS and AML, but until recently, immune therapies have played a limited role in treating these diseases. Immune suppressive therapy exhibits durable clinical responses in selected patients with MDS, but the question of which patients are most suitable for this treatment remains unclear. Over the past decade, there has been remarkable progress in identifying genomic features of MDS and AML, which has led to an improved discernment of the molecular pathogenesis of these diseases. An improved understanding of immune and inflammatory molecular mechanisms of MDS and AML have also recently revealed novel therapeutic targets. Emerging treatments for MDS and AML include monoclonal antibodies such as immune checkpoint inhibitors, bispecific T-cell-engaging antibodies, antibody drug conjugates, vaccine therapies, and cellular therapeutics including chimeric antigen receptor T-cells and NK cells. In this review, we provide an overview of the current understanding of immune dysregulation in MDS and AML and an update on novel immune therapies for these bone marrow malignancies. Myelodysplastic syndromes (MDS) are a clonal malignancy of the bone marrow, which presents in a heterogeneous manner with peripheral blood cytopenias and a potential for progression to acute myeloid leukemia (AML). The World Health Organization (WHO) classifies MDS into eight discrete categories according to blood and bone marrow morphology, the number of cell lineages involved, cytogenetic abnormalities, and bone marrow blast percentage, with up to 19% myeloblasts in the blood and/or marrow representing MDS with excess blasts and a level of 20% or higher defining AML (doi: 10.1182/blood-2016-03-643544) . Py. Py12]. Recent studies implicate dysregulated innate immune signaling in MDS, including overexpression of TLRs, IL-8 and its receptor CXCR2, and IL-1 downstream effector signaling pathways  11,12,2,212,29. Genomic interrogation studies have revealed further mechanistic details of inflammasome signaling in MDS, including downstream effectors of TLR signaling ,29. DeleInnate lymphoid cell (ILC) dysfunction has been observed in human malignancies including AML ,43. ILCsAML blasts evade immune surveillance by disrupting the immune microenvironment through multiple mechanisms, including downregulation of human leukocyte antigen class I and II presentation and upregulation of the immune checkpoint inhibitors programmed death ligand-1 (PD-L1), T-cells immunoglobulin-mucin 3 (TIM-3), and C-lectin-like inhibitory receptor-4 (CTLA-4) . AML blaDysregulation of the adaptive immune response is also a well-described feature of the MDS bone marrow in a pattern characteristic of autoimmune states . The MDSThe recognition of immune perturbations in MDS patients led to clinical investigations into the use of immunosuppression therapy (IST) in MDS, with more than 20 studies published to date ,58. Initp = 0.0009). A multivariate analysis was performed to assess predictors for response, including all of the variables identified in single institution studies, and none of them demonstrated statistical significance. Among predictors for transfusion independence after IST, only a bone marrow cellularity of less than 20% was a predictor for red cell transfusion independence  . TT. TT70]. Immune checkpoint inhibitors have revolutionized the treatment landscape of solid tumor oncology over the last decade. Their basic principle is based on targeting immune checkpoints that cause the tumor cell’s escape from immune surveillance, including (i) CTLA-4 inhibitors, which prevent CTLA-4 on the tumor cell from binding to B7 on the T-cell/antigen-presenting cell that causes downregulation of T-cell activity and (ii) PD-1/PD-L1 inhibitors that prevent T-cell inactivation that is a result of programmed death-1 (PD-1) (T-cell) binding with programmed death –ligand 1 (PD-L1) (on tumor cell). T-cell immunoglobulin mucin-3 (TIM-3) and lymphocyte activation gene 3 (LAG-3) are more recent immune checkpoint targets being studied. TIM-3 is a glycoprotein on the surface of T-cells and innate immune cells, which downregulates Th1 immune responses. LAG-3 is a transmembrane receptor on activated T-cells, T-regs, and NK cells that suppresses T-cell and NK cell responses. Amongst hematologic malignancies, overexpression of PD-1 ligands by Reed–Sternberg cells in classic Hodgkin lymphoma formed the basis of study and eventually approval of these drugs for R/R disease. These agents are currently under study in MDS and AML either alone or in various combinations with conventional MDS/AML therapies. Increased expression of B7 on antigen-presenting cells correlates with inferior outcomes in AML ,79. ThisIncreased expression of PD-1 on MDS and AML cells has been documented, especially in patients treated with hypomethylating agents ,82. HoweAnother trial studied nivolumab in high-risk AML patients in remission but ineligible for allogeneic HSCT, with high risk defined as secondary AML in first CR, high-risk cytogenetics, FLT-3 ITD mutation, presence of minimal residual disease, or second CR or more . This waFc-optimized monoclonal antibodies are engineered to enhance recruitment of FcγRIIIa-positive effector cells in an effort to improve antibody-dependent cellular cytotoxicity (ADCC). In addition to targeting the AML antigens, Fc-optimized antibodies increase NK cell activation to enhance ADCC. This is in contrast to antibody drug conjugates, which are combined with a cytotoxic agent but do not augment the immune response . Fc-optiAlthough NK cell activity is impaired in AML ,48,49,87Bispecific T-cell engagers (BiTE) are a promising new area of research in MDS and AML. BiTEs are derivatives of two single-chain antibody fragments (scFv) from two different antibodies, each targeting components of the effector T-cells (the epsilon subunit of T-cells—CD3ε) to an associated cell surface antigen on malignant cells, thus facilitating close approximation and promoting T-cell-mediated killing of malignant cells . EffectiAMG330 and AMG673  are two BiTE cell constructs developed with specificity for CD33 and CD3 epsilon. In a recent human phase 1 dose escalation trial, AMG330 showed antitumor activity in R/R AML, with 11.4% patients achieving CR/CRi. However, a majority of patients (87%) discontinued treatment due to progression of disease, while 73% experienced serious adverse events, including cytokine release syndrome (CRS) and cytopenias related to treatment . A recenp < 0.001). AMV564, another BiTE construct with specificity to CD33 on target cells and CD3 on effector T-cells is currently undergoing phase 1 dose escalation study in R/R AML patients that have undergone at least 1–2 lines of prior induction regimens, with preliminary data showing a 49% reduction in bone marrow blasts . C. C103]. wo weeks . The ORRwo weeks .2 on days 1, 4, and 7, plus DA [p = 0.25). Event-free survival (EFS) was 17.1% (10.8–27.1) at two years in the control group, versus 40.8% (32.8–50.8) in the GO-treated group , and OS was 41.9% (33.1–53.1) versus 53.2% (44.6–63.5), respectively , and RFS 22.7% (14.5–35.7) versus 50.3% (41.0–61.6), respectively  [p < 0.001). Subgroup analyses stratified for EFS and OS by age, sex, ECOG, CD33 expression, and cytogenetic risk categories demonstrated benefit for all patients except those with AML with adverse cytogenetics [GO was then studied in the de novo AML setting in the SWOG S0106 study, in which patients received induction therapy with daunorubicin and ara C (DA) with and without GO . This st plus DA . Complet 0.0003) . A subsegenetics .p = 0.01). The survival benefit was particularly clear in those with favorable cytogenetics , p = 0.0005), but also observed in intermediate-risk patients , p = 0.007). Patients with adverse karyotype did not benefit overall or within any trial [The efficacy of GO in frontline AML was confirmed in a meta-analysis of five trials, which included a total of 3325 patients . Remissiny trial .p = 0.005), and the 1-year OS rate was 24.3% with GO and 9.7% with BSC. The OS benefit with GO was consistent across most subgroups and was especially apparent in patients with high CD33 expression, in those with favorable/intermediate cytogenetic risk profile, and in women. Complete remission occurred in 30 of 111 (27%) GO-treated patients. The rates of serious AEs were similar in the two groups, and no excess mortality from AEs was observed with GO [GO was also studied as monotherapy versus best supportive care (BSC) as first-line therapy in 237 older patients with AML unsuitable for intensive chemotherapy . Those w with GO . Based oVadastuximab talirine (VT) is a CD33-directed antibody conjugated to pyrrolobenzodiazepine (PBD) dimers. A phase 1 study assessed safety, tolerability, and activity of vadastuximab talirine with hypomethylating agents . No doseThe ADC IMGN779 comprises a humanized anti-CD33 mAb conjugated via a cleavable disulfide linker to DGN462, a DNA-alkylating agent. IMGN779 was found to be highly active in vitro against primary patient AML cells, with cells harboring FLT3-ITD mutations being more sensitive to IMGN779 inhibition compared with FLT3 WT AML samples . IMGN779AVE9633 is an anti-CD33-maytansine conjugate ADC studied in three phase 1 trials, enrolling a total of 54 patients with R/R AML . PatientThe IL-3 receptor α chain CD123 is expressed on a majority of AML blasts and has been found to be expressed more in leukemic cells relative to normal hematopoietic stem cells, making it an attractive target for immune-modulating therapies. Several CD123-targeting ADCs have been developed. IMGN632 is a humanized anti-CD123 antibody G4723A linked to a recently reported DNA mono-alkylating payload of the indolinobenzodiazepine pseudodimer (IGN) class of cytotoxic compounds . A phaseCD45 is widely expressed on AML blasts and is thought to play a role in AML development and maintenance . This moCD30 is a member of the tumor necrosis factor receptor superfamily. In addition to being expressed in other hematologic malignancies, CD30 is present on the surface of myeloblasts in patients with MDS and AML . BrentuxChimeric antigen receptor (CAR) T-cells are patient-derived T-cells transduced with viral vectors which express a chimeric antigen receptor for specific recognition of a tumor antigen, independent of major histocompatibility complex (MHC). CAR T-cells are composed of a single variable chain of an antibody domain linked to the signal transduction domain of the CD3 T-cell receptor . CAR T-cThe use of natural killer group 2D (NKG2D) CAR T-cells is promising due to the significance of NKG2D as a highly conserved ligand upregulated in the setting of malignant transformation in AML . In 2018CD123, while not specific to MDS, has been shown as a marker of disease progression due to upregulation during MDS pathogenesis. CD123 is also a potential immune cellular therapy target for AML, but the non-specific nature may result in off-target effects, which are especially dangerous due to expression in vascular endothelium, associated with the risk of capillary leak . A high Recently, FLT3-targeted CAR T-cells have been developed, including FLT3-CAR-R2, a construct modified with a rituximab-responsive mimotope off-switch between the hinge and scFv to mediate depletion . This trAnother promising target for AML lies with the investigation of C-type lectin-like molecule-1 (CLL1) as a target highly expressed on AML stem cells and blasts but not on healthy HSCs. The first human use of anti-CLL1 CAR T-cells in a pediatric patient with relapsed AML was reported in 2020 . The patFinally, a recent study investigated the use of CAR T-cells targeting CD70, which is expressed on most leukemic blasts with absent or low-level expression in normal marrow . The invNatural killer (NK) cells are type 1 ILCs that function as mediators of immunosurveillance and the elimination of malignant cells using activating and inhibiting receptors . Once acNK cell-based therapy is generally well-tolerated and has the potential to induce complete remission in patients with AML. NK cell therapy is based on early studies involving the infusion of NK cells in conjunction with hematopoietic stem cell transplants, where it was shown to have a protective effect against AML relapse . ChallenVaccine-based therapy is a rapidly evolving field of cancer immunotherapy designed to manipulate the immune system to recognize and eliminate tumor-specific cells through T-cell activation. Two common antigens include tumor-associated antigens (TAAs) and tumor-specific antigens (TSAs). TSAs are tumor- and patient-specific, so less likely to evoke autoimmunity compared to TAAs, which are self-antigens that are often over-expressed by tumor cells . The WilNY-ESO-1 is an antigen expressed in various tumors, which elicits robust humoral and cellular immune responses and is another candidate TAA under investigation in vaccine trials for MDS and AML. A recent pilot study employed an HLA unrestricted NY-ESO-1 vaccine combined with decitabine in a study of nine high-risk MDS patients to induce an antigen-specific cytotoxic response against the malignant myeloid compartment in MDS . Among sThere has been remarkable progress over the past decade in understanding mechanisms of immune dysregulation in the pathogenesis of MDS and AML, as well as a refinement in knowledge of how immune effector cells exert their actions against tumor antigen targets. It is now well recognized that both innate and adaptive immunity are perturbed in the MDS and AML bone marrow microenvironment, and a variety of immune targets have emerged. A considerable advantage of immune therapies over cytotoxic chemotherapy is the potential for sustained immune surveillance and ongoing effector cell activity, which can lead to durable responses to treatment. IST in the form of ATG with cyclosporine continues to have a role in the treatment of selected patients with MDS, particularly patients with low and intermediate-1 disease and those with a hypocellular bone marrow. Immune checkpoint inhibitors administered as single agents have modest clinical activity in MDS and AML. However, upregulation of PD-L1 has been shown to induce resistance to AML therapy ,143, and


Interestingly, the duration of the cardiac action potentials lengthened in the presence of both isoproterenol and carbamylcholine. To evaluate cardiac contractility, the Goldfish heart was perfused with the Ca2+ indicator Rhod-2 and ventricular epicardial Ca2+ transients were measured using Pulsed Local Field Fluorescence Microscopy. Following isoproterenol perfusion, the amplitude of the Ca2+ transient significantly increased, the half duration of the Ca2+ transient shortened, and there was an observable increase in the velocity of the rise time and fall time of the Ca2+ transient, all of which are compatible with the shortening of the action potential induced by isoproterenol perfusion. On the other hand, carbamylcholine perfusion significantly reduced the amplitude of the Ca2+ transient and increased the half duration of the Ca2+ transient. These results are interesting because the effect of carbamylcholine is opposite to what happens in classically used models, such as mouse hearts, and the autonomic regulation of the Goldfish heart is strikingly similar to what has been observed in larger mammalian models resembling humans.Autonomic regulation plays a central role in cardiac contractility and excitability in numerous vertebrate species. However, the role of autonomic regulation is less understood in fish physiology. Here, we used Goldfish as a model to explore the role of autonomic regulation. A transmural electrocardiogram recording showed perfusion of the Goldfish heart with isoproterenol increased the spontaneous heart rate, while perfusion with carbamylcholine decreased the spontaneous heart rate. Cardiac action potentials obtained  In nearly all vertebrate species, direct input from the autonomic nervous system tightly controls cardiac contractility and excitability . Althoug2+ handling proteins such as the L-type Ca2+ Channel  from postganglionic cholinergic intracardiac neurons. The ACh subsequently binds to and stimulates muscarinic (M2) receptors. Activation of M2 receptors stimulates a ibits AC . This ins system .2+ transient kinetics and dynamics of adult Goldfish closely parallel those of humans, even more so than mice and Zebrafish models  or 5 μm carbamylcholine (parasympathetic agonist), was enough to stimulate the sympathetic branch or parasympathetic branch, respectively. Interestingly, our results indicate stimulation of the Goldfish autonomic nervous system by these commonly used agonists resulted in a corresponding change in cardiac dromotropism, chronotropism, inotropism, and lusitropism in a similar manner observed in humans.Previous literature suggests few fish models exhibit autonomic control in a similar manner to larger mammals; however, it is not clear how autonomically driven AP kinetics impact cardiac contractility in the fish intact heart specifically. To explore how stimulation of either autonomic nervous system branch would alter cardiac contractility and excitability, we performed experiments in which we perfused the Goldfish intact heart with either a sympathetic or parasympathetic agonist. Ventricular APs, electrocardiograms, and CaOur animal facilities are Association for Assessment and Accreditation of Laboratory Animal Care accredited and Office of Laboratory Animal Welfare certified and fully comply with all regulations, policies, and standards that protect animal welfare. Animal use in our studies were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals  and the Institutional Animal Care and Use Committee guidelines of the University of California Merced (Protocol # 2008–201). The animals were bought from Toledo Goldfish, United States.–1 tricaine methanesulfonate for 2–5 min. To assure the Goldfish were completely anesthetized prior to decapitation, the tail was held with a small, curved tweezer, and gently moved. Once the Goldfish were completely anesthetized, they were decapitated, and the intact heart was removed from the chest cavity.Adult Goldfish were anesthetized by immersion in ice-cold water containing 0.16 mg ml2 1.8 mm, MgCl2 0.5 mm, HEPES 10 mm, and glucose 5.5 mm. The Ca2+ dye Rhod-2 AM was perfused into the heart with a Harvard pump for 30–45 min. The temperature of the bath containing the heart was set to 28°C. The temperature was controlled with the aid of a Peltier unit positioned at the bottom of the recording chamber and measured with a linearized semiconductor temperature sensor.Goldfish hearts were dissected, and the bulbous arteriosus was cannulated onto a gauge 27 needle and perfused in a Langendorff system at a rate of 60 μl/min driven by gravity. Multiple solutions were perfused through the bulbus arteriosus with the aid of a self-designed μ-manifold. Goldfish hearts were perfused with a fish ringer solution containing: NaCl 137 mm, KCl 5.4 mm, CaCl2+ transient recordings. To determine if the sympathetic response to isoproterenol could be reversed, the Goldfish heart was perfused with fish ringer solution for a prolonged amount of time. Indeed, the effects of isoproterenol could be completely reversed if the Goldfish heart were continuously perfused with fish ringer solution for 20 min. In contrast, to elicit a parasympathetic response the Goldfish heart was perfused with fish ringer solution containing 5 μm carbamylcholine for 10 min prior to the start of any AP and Ca2+ transient recording. The effects of carbamylcholine could be completely reversed after continuously perfusing the heart with fish ringer solution for 60 min. Recordings obtained prior to perfusion with isoproterenol or carbamylcholine were considered as control, and recordings obtained following isoproterenol or carbamylcholine perfusion were considered as experimental.The Goldfish heart was perfused with fish ringer solution containing 4 mm blebbistatin, prior to obtaining any electrophysiological recordings to suppress cardiac motion. In order to elicit a sympathetic response, the Goldfish heart was perfused with fish ringer solution containing 100 nm isoproterenol for 10 min before the start of any AP and Ca2+ transients were recorded (N = 8 hearts) using Pulsed Local Field Fluorescence Microscopy  was prepared with 20 μl of 20% pluronic in 1 ml fish ringer solution.Cay PLFFM; . The PLFN = 4 hearts) were obtained using sharp glass microelectrodes filled with 3 M KCl connected to a high input impedance differential amplifier . Glass microelectrodes were fabricated with a micropipette puller  and had a resistance of 10–20 MΩ  were performed by placing one Ag–AgCl micropellet inside the left ventricle and a second pellet outside the left ventricle  of the heart, it is impossible to perform the recordings in the same precise location between different hearts. Thus, the data are presented as the measured times with their standard error (SEM). To assess electrical changes, AP traces were first normalized then evaluated at their respective repolarization times. Specifically, the time it takes for the AP to reach 30, 50%, or 90% repolarization, referred to here as APD30, APD50, or APD90, respectively.In whole heart experiments, there are two main causes of variance. First, no two animals have entirely identical hearts, regardless of the species. Second, although we are measuring Ca2+ transients were normalized between zero (minimum fluorescence) and one (maximum fluorescence) in order to evaluate the kinetics of the recorded Ca2+ transients. The kinetic parameters of the Ca2+ transients evaluated were the rise time , half duration (duration of the Ca2+ transient at 50% of the maximum amplitude), and fall time . The AP parameters and Ca2+ transient kinetics obtained for the control and the experimental groups were evaluated and normalized to their respective control values for each heart used. After this normalization, data were compiled, and statistical analysis was performed.Each wave of the electrocardiogram  was measured using its corresponding half duration. The recorded Can; dot cloud) recorded for different measurements (n) on different hearts (N) with the mean ± SEM (solid lines). To determine if the agonists produced a significant effect, the distribution of the data before and after administration was compared using a two-sample Kolmogorov–Smirnov test . The difference was significant if the value of p < 0.01.The data are presented as multiple measurements , fall time (FT), and half duration (HD) were also evaluated to better understand how stimulation of the cholinergic pathway affected Ca2+ handling kinetics in the Goldfish myocardium ; a major current found in fish atrial myocytes responsible for the repolarization of the membrane potential , although the contribution of this current is still poorly elucidated in fish ventricular myocytes .MB and AE designed and performed the research, analyzed data, and wrote the paper. All authors contributed to the article and approved the submitted version.The study was supported by NIH (R01 HL-084487 to AE) and NIH (1R01HL152296 to AE).The authors declare the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The t translocation is the most common chromosomal abnormality in human acute myeloid leukemia (AML) subtype 2 (M2), which forms the AML/ETO fusion gene. However, AML/ETO alone does not necessarily cause leukemia. Other factors are thought to contribute to the disease. Calcitonin receptor-like (CALCRL), a G-protein-coupled neuropeptide receptor, is involved in various biological processes, such as colony formation and drug resistance. CALCRL expression in AML patients with and without AML/ETO and the prognostic significance of CALCRL expression in AML patients was further evaluated. Next, we detected the CALCRL expression level in 67 AML/ETO+ AML patients and 16 patients with nonmalignant hematological diseases using qRT-PCR and identified its prognostic relevance.  First, The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases were used to determine any differences in CALCRL had a longer median survival time. In AML/ETO+ AML patients, higher mRNA levels of CALCRL were observed before treatment, which decreased after the complete remission that followed multiple chemotherapy sessions. Clinical features indicated that more patients in the CALCRLhigh group also had c-kit mutations compared with patients in other groups. Overall survival (OS) was longer in patients with lower levels of CALCRL expression, especially in patients with c-kit mutations or with more blast cells in bone marrow (BM). In addition, a longer OS was observed in the CALCRLlow group after hematopoietic stem cell transplantation (HSCT).  Individuals in the group expressing low levels of + AML patients. This preliminary study indicates that CALCRL could serve as a suitable prognostic factor in AML/ETO However, due to the leukemic clonal diversity, the recurrence rate is high. All these implied that additional factors must participate in the development of AML in those patients [The AML/ETO fusion transcription factor is one of the most common chromosomal abnormalities detected in human acute myeloid leukemia (AML) subtype 2 (M2). However, AML/ETO alone does not necessarily cause leukemia –3. Moreopatients , 5.CALCRL) was one of several genes whose expression decreased when H22954 was highly expressed. CALCRL is a G protein-coupled receptor engaged in regulating the concentration of calcium ions in cells. It inhibits cell proliferation and angiogenesis [CALCRL expression and the clinical features and prognosis of AML patients have only rarely been addressed. We conducted the present study to address these issues. We used The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets and also enrolled 67 newly diagnosed AML/ETO+ AML patients in this study. We found that the mRNA expression level of CALCRL is correlated with the diagnosis, survival, and prognosis of AML/ETO+ AML patients. This may provide new ideas for the diagnosis and treatment of AML.Our previous studies showed that H22954, a novel long noncoding RNA, may play an important role in the pathogenesis of AML . To undeogenesis . CALCRL ogenesis , 9. Howehttps://portal.gdc.cancer.gov/) following the guidelines and policies of our hospital. Kaplan–Meier (KM) survival analysis with the log-rank test was also used to compare the survival between the AML patients with and without AML/ETO in the GEO dataset (GSE61804). For KM curves, P values and hazard ratios (HRs) with a 95% confidence interval (CI) were generated by log-rank tests.Clinical information of AML patients was obtained from TCGA dataset  [In total, 67 newly diagnosed AML/ETOy (MICM)  at The Fμl cDNA, 1 μl 20x buffer, 2 μl MgCl2, 4 μl 5x Q-Solution, 0.8 mM dNTPs, 2 μM primers, 1 μM probes, and 1 U HotStar  [ABL served as the internal control, and the relative expression level of CALCRL was calculated using the gene relative quantitative method. The primers for CALCRL were CALCRL-F, 5′-GCAGCAGCTACCTAGCTTGAA-3′; CALCRL-R, 5′-TTCACGCCTTCTTCCGACTC-3′; and CALCRL-probe, 5′-FAM-ACTGCAGTGGCCAACAACCAGGCCT-TAMRA-3′. The primers for ABL were ABL-F, 5′-CGCTGACCATCAATAAGGA-3′; ABL-R, 5′-CACTCAGACCCTGAGGCTCAA-3′; and ABL-probe, 5′-FAM-TGCTTCTGATGGCAAGCTCTACGTCTCCT-TAMRA-3′.Bone marrow mononuclear cells (MNCs) were collected from the experimental group and the control group. Total RNA (500 ng) was directly reverse-transcribed into cDNA. PCR primers and TaqMan probes were designed using Primer Express 2.0 software with published sequence data from the NCBI database. Amplification reactions contained 2 Germany) . ABL serThe induction chemotherapy regimen used to treat the patients was as follows: IA (IDA + Ara-C) and DA (DNR + Ara-C). Whenever there was no remission after induction chemotherapy, the induction chemotherapy regimen was changed. Postremission treatment included consolidation chemotherapy and hematopoietic stem cell transplantation. In addition to this induction regimen, the consolidation chemotherapy regimen also included medium- and high-dose cytarabine (HD-Ara-C/MD-Ara-C), single-drug chemotherapy, and other treatments. All of the patients were followed up until December 2018 or death via consultation of hospitalization medical records, telephone calls, and other methods.t-test, and the mean values of multiple samples were analyzed by variance analysis. The survival curve was drawn using GraphPad Prism 8.2.1 software. Survival probabilities were estimated using the KM method and the log-rank test. P value < 0.05 was considered to be indicative of statistical significance.The data were analyzed using SPSS20. The mean values of the two groups were compared by P < 0.05 in H22954-overexpressing cells  in AML.To understand the mechanism underlying the antitumor activity of H22954, we compared gene expression profiles in the stable K562 cells that did and did not overexpress H22954 through microarray analysis and found 1613 differentially expressed genes that had an expression fold change of <0.5 and CALCRL gene expression. As shown in CALCRL expression was associated with inferior OS  (+ AML patients and 309 AML/ETO− AML patients. Lower CALCRL mRNA levels were observed in the presence than in the absence of AML/ETO (The full TCGA cohort contained 106 AML samples. All of the samples were split into high- and low-expression groups based upon median  < 0.05) . In the  AML/ETO .CALCRL expression levels and the clinical features or prognosis of AML patients, 67 primary AML/ETO+ AML patients were enrolled, including 40 men and 27 women, with a median age of 35  years. Additionally, 16 patients (8 men and 8 women) with nonmalignant hematologic disease  were set as the control group, who had a median age of 31  years. CALCRL mRNA expression was significantly higher in the 67 patients with primary AML (AML/ETO+) than in the nonmalignant hematologic group (P < 0.05) (To determine the relationship between  < 0.05) .CALCRL expression was lower in CR patients (P < 0.05) (CALCRL mRNA levels were observed in bone marrow mononuclear cells (MNCs) in the CR group than in the NR group (P < 0.05) .  < 0.05) . After t < 0.05) .+AML patients was divided into two expression groups based on the median value: 34 patients were included in the CALCRLlow group, and 33 patients were included in the CALCRLhigh group. Basic clinical features were similar between the two groups, including white blood cell counts, neutrophils, hemoglobin, platelets, and the percentage of blasts in the bone marrow. No significant correlations were detected between the mRNA expression of CALCRL and the risk stratification of leukemia  in the CALCRLhigh group than in the CALCRLlow group (14/27) (P < 0.05)  .CALCRL and OS (P < 0.05) in AML/ETO+ AML patients  (high) and a BMlow group. In the BMlow patients, the survival time was similar regardless of the level of CALCRL mRNA expression. However, in the BMhigh group, the OS was shorter in those with a higher level of CALCRL mRNA expression (P < 0.05)  . CALCRL  < 0.05) .low group and 25 patients were in the CALCRLhigh group. The OS of patients in the CALCRLlow group was significantly longer than that in the CALCRLhigh group (P < 0.05) following HSCT . Of these, 27 patients belonged to the CALCRLing HSCT .CALCRL was shown to be closely related to the OS in AML.AML is a malignant hematological disease that is affected by genetic and epigenetic factors . VariousCALCRL is a G protein-coupled neuropeptide receptor involved in blood pressure regulation, cell proliferation, apoptosis regulation, vascular biology, and inflammation –15. In s+ AML patients were enrolled in the present study, taking the advance of the AML1/ETO fusion gene as the biomarker of AML in these patients. It was deemed unethical to aspirate bone marrow from healthy controls, so nonmalignant hematologic patients were chosen as controls. Our investigation showed that there were higher mRNA levels of CALCRL in pretreated AML/ETO+ AML patients than in control samples and these levels decreased when patients got CR. More AML/ETO+ AML patients with high levels of CALCRL expression also had c-kit mutations than patients in other groups.To determine the relationship between CALCRL expression and the clinical features and prognosis of AML, AML/ETOCALCRL expression more readily achieved CR after the first course of chemotherapy and there was a negative correlation between the mRNA expression of CALCRL and patient survival time. This correlation was especially strong in BMhigh patients. Patients with lower CALCRL mRNA expression may reap greater benefits from HSCT than other patients would.Survival and prognostic analysis showed that patients with less CALCRL played an important role in stemness and chemotherapy resistance in AML. Knockdown of CALCRL expressed in the leukemic stem cell (LSC) subpopulation decreased the LSC frequency and sensitized it to chemotherapeutic agents, facilitating to eradicate the relapse-initiated cells (RICs) in AML [CALCRL expression may be related to tumor burden and prognosis in AML/ETO+ AML patients as well. Importantly, antibodies interfering with CALCRL signaling have recently been approved for the preventive treatment of migraine headaches [Some studies suggested that median survival times were longer among patients expressing low levels of CALCRL, both in patients without t8 ; 21)  in AML , 20, 21.) in AML . Consisteadaches –25. All + AML patients. Our experimental results need to be confirmed with larger patient cohorts.Our study indicates that CALCRL could serve as a suitable prognostic factor for designing the chemotherapy regimen and evaluating the risk of HSCT in AML/ETO


We find that trains of Ca2+ spikes are advantageous compared to a constant rise in Ca2+ concentration as being more efficient and much less prone to noisy fluctuations.We investigate the messenger role of calcium ions implicated in the regulation of wave-like bending dynamics of flagella. The emphasis is on microtubules of flagellar axoneme serving as nonlinear transmission lines for bell-shaped spikes of calcium ions. The calcium sensitive proteins, such as calmodulin, exhibit activation dependence on the spike train frequency and amplitude. Here, we analyze a Ca Cilia and flagella are characterized by different patterns of movement but are identical in structure and composition. In the following, for the sake of brevity, we will mostly use the term flagella. Flagella are long thin appendages of many living cells, whose oscillatory bending waves enable cells to be propelled through visco-elastic fluids, or to drive fluid flows across the surface of the cell. Motile flagella are capable of complex, subtly coordinated movements and can play versatile roles in fertilization and embryonic developments . SpecifiThe axoneme emanates from the flagellum cell body. The part inside the cell that anchors axoneme is called basal body, The microtubule (MT) cytoskeleton of flagella consists of triplet MTs in the basal body and microtubule doublets (MTDs) in the axoneme. The axoneme contains 9 MTDs and the central pair (CP) of single MTs, all in a parallel cylindrical arrangement. The MTDs and CP are connected by the radial spokes (RSs), while neighboring MTDs are linked with sets of nexin-linkers, see Chlamydomonas axoneme by mass spectroscopy-based proteomics. Among these proteins just MTs and dynein motors play the executive roles for flagellar bending motions. These motions are driven by ATP-induced conformational changes of dynein motors. Other accessory proteins constitute a complex toolkit for subtle support and aid in flagellar dynamics. It is known that axonemes are reinforced by a network of tektin protofilaments that together with nexin-linkers maintain the nine-fold integrity of axoneme and enhance its mechanical robustness. Some accessory complexes are indicated in More than 600 different proteins were detected from the Microtubules are long hollow cylinders a typical2+ sensor while trimers are associated with calmodulin (CaM) LC4 as a Ca2+ sensor. The ODAs provide much of the power required for flagellar movement. The ODAs repeat once every 24 nm and are very homogenous in the structure of the axoneme.Axonemal dyneins are structured in two complexes of inner and outer dynein arms  complex present in RS1. For example, MTD sliding in Chlamydomonas flagella is regulated by phosphorylation/dephosphorylation of the IC138 complex of I1-IDA. The phosphorylation and dephosphorylation of this protein inhibits and restores wild-type MTD sliding, respectively.The IDAs are much more complex and contain at least 12 different DHCs ,6. These2+ signaling. The I1 acts as a brake to slow and locally regulate MTDs sliding driven by ODAs and other IDAs. Its 1β-DHC contributes to force production while 1α-DHC is the motor domain that resists or limits MTD’s sliding. Below we elaborate on the decoding mechanism of calcium oscillations involved in the activation of I1 dyneins. This represents the main focus of this article.It should be strongly stressed here that effective functioning of I1-IDA is provided in terms of CaThe RSs act as spacers to position MTDs in a circle around a central pair of MTs. These RSs are large complexes comprising at least 23 different subunits including a diverse group of structural and signaling proteins. They represent two important regulatory hubs aimed at tuning of flagellar motility. The first spoke RS1 in association with I1 dynein is positioned near the proximal end of the 96 nm axonemal repeat . The secThe MTDs are not contractile but, instead, slide relative to one another under the action of forces and torques, produced by dynein motors. The variability of sliding velocity is the basis of flagellar bend formation. Very notably the different IDAs and ODAs undergo MTDs translocation at different speeds.Bending of the axoneme originates from the imbalance of dynein motors on the opposite sides of the bending plane. For instance, switching of dynein activity between MTD7 and MTD3 b is thou2+ signals indirectly activate dynein sliding, initiating the beat, and conversely, the beat tunes the dyneins. When activated, dyneins on one side of the axoneme win the tug-of-war, leading to relative motion between MTDs. Passive nexin-linkers and RSs, along with the basal body of axoneme, constrain sliding and convert it into bending [During their power stroke dyneins produce force that tends to slide attached MTDs with respect to each other, thus regulating the beat pattern of the axoneme. Accordingly, the flagellar beat is enabled by alternating episodes of activation of sets of dynein. It is concluded that these beats are highly organized processes such that Ca bending .2+ to play a pivotal role in cell biology results from the ease with which cells have to shape Ca2+ signals in the dimensions of space, time, amplitude, and frequency [Calcium, being the most abundant metal on the earth and the fifth most abundant element in the human body, was adopted as a cellular regulator at an early evolutionary stage. Calcium acts as an intracellular second messenger in living processes ranging from cell movement, important here for secretion and contraction, up to gene expression and even cell apoptosis. The ability of a simple ion such as Carequency ,10.2+ ion can accumulate 8–12 oxygen atoms in the primary coordination sphere. The range of Ca-O distances in complexes is 0.23–0.28 nm. The abilities of Ca2+ and negative phosphate ions to trigger changes in the shape and charge of different proteins are two universal tools of cellular signaling transduction. Thus, Ca2+ binds to thousands of different proteins with over a million-fold range of affinities (nM to mM). These binding events can influence protein’s change in localization, association, and function. Interestingly, cells spend a significant part of their totally consumed energy to maintain changes in Ca2+ concentration keeping the difference between their intracellular (0.1 μM free) and extracellular (mM) concentration [The advantage of calcium is its specific coordination chemistry. This is determined by its valence, the ionic radius of 0.99 Å and its radius of hydration of 4.5 Å. A Cantration .2+ varies between 2.0 and 2.6 mM, subdivided into three components: (a) ionized, (b) bound to small inorganic molecules, and (c) complexed with organic molecules. The concentration of ionized Ca2+ forms is of the order 1 mM. The total Ca2+ concentration in the cytosol is also on the order of mM. But there, the concentration of free ionized fraction is about 104-fold lower than the bound and complexed components. To this end, cells are endowed with specific organelles which contain sites that ligate Ca2+ with significant affinities. The first group of such ligand peptides are sequestered inside mitochondria, the endoplasmic reticulum and the sarcoplasmic reticulum , as well as in the Golgi apparatus. The second class of such proteins involves cytoskeletal structures: microtubules, actin filaments, and intermediate filaments. All these organelles buffer free Ca2+ within the nM range without modifying its total content in the cell. A very important fact for this article is that ERs and SRs are missing in mature flagella, including sperm cells. However, there are the ER remnants in the membranous forms with similar Ca2+ buffering properties to those of the ER.In extracellular fluids, the concentration of Ca2+ concentration in sperm cells flagella is played by mitochondria. Besides energy homeostasis, mitochondria play a vital role in maintaining ion homeostasis in sperm cells [It is known that axonemes in mammalian sperm cells are reinforced by a network of tectin-dense fiber axoneme complexes that together with nexin-linkers maintain the nine-fold geometry and enhance the mechanical robustness of the axoneme. A significant role in controlling Carm cells .2+ ionic channels enables mitochondria to support the calcium induced calcium release (CICR) process which underlies the pulsatile localized calcium waves along the flagella. The mammalian sperm mid-piece contains 50–57 mitochondria with diameters in the 0.7–3 μm range, which represents about 10% of the cell volume. It was demonstrated in [2+ wave activities in Xenopus leaves oocytes strengthened by oxidizable substrates that energize mitochondria, thus increasing the Ca2+ wave amplitude and velocity. This clearly describes the basic role of mitochondria in intracellular Ca2+ signaling. Therefore, mitochondria may be safely considered as the guardians of the gate between life and death since they also play an important role in cell apoptosis.Within the cylinder-shaped sheet, adjacent mitochondria associate both end-to-end and along their lateral surfaces. This arrangement sets a concentrated array of mitochondria closely to the flagellum membrane from outer side, rated in  that Ca22+ storage organelles are contained in mature mammalian sperm cells due to the presence of inosital triphosphate receptors (IP3Rs) in the acrosomal part of the cell head and over the sperm neck and mid-piece of the flagellum.There is also clear evidence that other Ca2+ concentration which is a crucial regulator of the modulation of flagellar movement. These modulations include: (1) changes in flagellar waveforms; (2) reversal of the direction of flagellar bending; (3) arrest of bending; (4) changes in the beat frequency and amplitude [Flagella change their motility in response to Camplitude .2+ signaling there should exist four functional segments as follows:2+ signal through Cat-sper channels;In the case of the human sperm, the signaling is triggered by a stimulus (progesterone or nitrogen monoxide -NO) that generates Ca2+ into flagellum and sperm head. Since flagella are thin cylinders with a very large surface-to-volume ratio [2+ fluxes are efficiently injected into the cytosol.It activates the ON mechanism that feeds Came ratio , these C2+ ions function as messengers, which stimulate several axonemal proteins, primarily CaM, calaxin and IC138 of IDA-I1, to perform the control of dyneins and to modulate flagellary beats [The pulses of Cary beats .2+ from the cytoplasm to internal Ca2+ stores and buffers, as well as out of flagella, in order to restore the resting state [Eventually the OFF mechanism, composed of pumps and ionic exchangers, removes Cang state .For successful Ca2+ entry and release channels introduce Ca2+ into flagellar cytoplasm. However, since these channels (for example Cat Sper) have short open times, they only introduce brief pulses of Ca2+ that form a small puff around the mouth of each channel [When activated, both Ca channel ,20.2+ signals are the building-blocks by which recruitment of the complex Ca2+ signals is constructed. This includes the localized “Ca2+ clouds” drifting along MTDs within the scope of our model [These elementary Caur model ,21.2+ waves instead of global increases of Ca2+ concentration in controlling flagellary movement are manifold. These signals can have a rapid action considered to have a much higher fidelity of information transfer than simple tonic changes in Ca2+ concentration since they are much less prone to noisy fluctuations.The advantages of such pulse-like oscillating Ca2+ binding protein CaM. It bears structural Ca2+-binding motifs known as “EF-hands”. The affinity of Ca2+ for CaM is 2+ increase seen with each incoming localized spike. One of the best-known enzymes that uses CaM to help it “count” Ca2+ spikes is CaM dependent protein kinase C, which activates dynein motors ODAs and IDAs via phosphorylation [Chlamydomonas and teleost fish sperm CaM affects the symmetry of flagellar beating.The main sensor for calcium signaling is a very ubiquitous Carylation . As a corylation . In sea 2+ is important for motility maintenance via phosphorylation of flagellar proteins involved in dynein complexes [Protein kinase C (PKC), which is activated via diacyglycerol in the presence of Caomplexes ,24.2+ sensor is neuronal calaxin, which directly acts on an ODA and regulates specific flagellar movement during sperm chemotaxis. Calaxin is essential for generation and propagation of Ca2+-induced asymmetric flagellar bending [2+ concentration, which regulates calaxin activity in catalyzing ODAs speeds. Immunoelectron microscopy and biochemical analysis showed that calaxin interacts with the ODA heavy chain in a Ca2+-dependent manner. Knockout of calaxin caused reduced motility in sperm flagella. In some cases, ODAs on the doublets 3, 5, and 8 were lost, suggesting that calaxin might play a role in stabilizing the association of ODAs with MTDs within axoneme.The second important Ca bending ,25. The 2+ ions stored in ER or SR organelles. These additional Ca2+ ions diffuse to nearby ER channels where they cause the release of yet more calcium. This self-sustaining cycle is primarily modulated by the increased levels of inositol  triphosphate (IP3) within the cytosol which forms calcium channels by the receptor proteins IP3Rs. These intracellular channels respond to Ca2+ concentration in a bell-shaped fashion; that is IP3R is inactive at low nM concentration of Ca2+ and active at mid-μM concentration, then inactivated again by high concentrations that are in the mM range of Ca2+. Thus, the speed of CICR waves is determined by the diffusion of Ca2+ ions combined with diffusion of IP3Rs. The diffusion constant of Ca2+ ions is of the order of 3Rs is much greater, namely Calcium waves were first seen at the calcium tsunami, which crosses a fertilizing medaka fish egg . These ws. Jaffe  presente300 μm2s .3Rs is higher, the speed of Ca2+ waves is further increased. We already stressed that ER and SR are missing in mature flagella. However, the presence of IP3Rs in the sperm cell’s head, neck, and mid piece . Moreover, the accumulated Ca2+ ions form bell-shaped “Ca2+ clouds” that propagate along MTs much faster than it could be achieved by simple three-dimensional diffusion. The estimated speeds of Ca2+ waves within the scope of our model compared with some prior experimental results are given in We earlier established ,34 and f2+ ions implicated in the modulation of flagellar bending.The differences within our model are the consequences of different estimates of capacitance and resistance of elementary electric building blocks of MTD viewed as nonlinear electric transmission line. These differences are caused by different post-translational modifications of CTTs which change the capacitance of these building blocks. Nonetheless, fairly satisfactory agreement of our results with experimental data indicates that our concept offers a very promising mechanism capable to explaining an efficient signaling pathway of Ca2+ repetitive spikes regulates sensitive proteins in a way such that strengthening of appropriate stimulus increases spikes frequency [2+ concentration with oscillatory spatio-temporal evolution from the flagellum to the sperm head. In the presence of 0.5 nM speract as agonist, the frequency of Ca2+ pulses in Sea urchin sperm is of the order of 1 Hz, while increased agonist concentration of 0.1 μM generates a frequency of about 5 Hz [2+ pulsatile train. This amplitude usually can be changed with the external stimulus [2+ pulsatile signal into an appropriate cellular response [2+ signals [2+ repetitive signals can be more efficient for protein phosphorylation if compared to constant Ca2+ signals of equal average concentration.Prior experimental evidence has demonstrated that signaling by intracellular Carequency . For exaout 5 Hz . Signal stimulus . Experimresponse . Further signals ,39. It a2+/CaM-dependent protein kinase II (PKII), and it is inactivated by dephosphorylation [n Ca2+ ions to the E-F hands domains of the associated CaM molecule. Thus, the Ca2+/CaM-dependent PKII exerts the phosphorylation of IC138, the vital ingredient of the IDA-I1 motor protein. This can be considered a rapid “post-translational” modification accompanied by the conformation of target proteins. As an example, the phosphorylation and dephosphorylation of IC138 inhibits and restores wild-type MTDs sliding, respectively in Chlamydomonas flagella.Here, we consider the activation-inactivation cycle of an axonemal target motor protein, for example an IDA-I1 which plays the fundamental role in the regulation of MTDs sliding. Its activation is triggered by phosphorylation through Carylation . The sti2+/CaM, denoted by The fraction of activated PKII by Ca2+ concentration within a single spike is denoted by 2+ binding and release in CaM, respectively, while Here, 2+ clouds” propagating along microtubule doublets of the flagellar axoneme. The scenario of such trains of spikes is elaborated in [Next, we implement our concept of bell-shaped spikes, so called “Carated in ,21. Simirated in . Interesrated in ,43,44.The train of spikes arising from our model obtained in  is prese2+ concentration within a single spike is taken to be our bell-shaped “Ca2+ cloud”, [2+ cloud”, t stands for the time variable, and 2+ concentration within each spike. We should fix the position of a specific dynein motor with accompanying regulatory proteins  involved in the motor’s activations. Hence, we can take 2+ concentration now readsEquation (1) can be solved if the local Ca cloud”, :(3)C by substituting2+/CaM activated PKII. The bound number of Ca2+ ions in CaM  is The parameter 2+ peak concentration and PKII half saturationWe can solve Equation (6) numerically for an increasing set of parameter values The initial condition is: 2+ binding to CaM is much faster than it’s unbinding  . It implThis implies the following solution of Equation (1):2+ pulse has a greater speed, the segment corresponding to a “constant” This expression shows that Thus, by inserting 2+ concentration within the spike progressing along the axoneme as follows:The effective phosphorylation rate of IC138 is the function of Ca2+ cloud”, Equation (3), and insert it into Equations (10) and (11), yieldingAgain, we use here the spike as the bell-shaped “CaIntroducing the new scaled dimensionless time into Equation (12) by substitutionIn order to obtain numerical solutions of Equation (14) we should choose a set of appropriate parameters taking into account that the activation rate of the phosphorylation of IC138 We also adopt the reasonable physiological values of concentrations given as: 2+ activation of the target protein LC138 has a rapidly rising phase reached by a peak spike of Ca2+ concentrations of the order of 2+ concentration level. We stress that elevated global cytoplasmic Ca2+ concentrations can be very toxic so, instead of a sustained manner, Ca2+ signals in flagellum are commonly generated in terms of pulsatile forms. Since the spikes of bell-shaped “Ca2+ clouds” (Equation (3)) drifting along the axoneme are fast compared to the rate of phosphorylation of the target IC138 protein, it is reasonable to conclude that the cumulative effect of several spikes is responsible for an efficient activation of the I1-IDA motor.Generally, Ca2+ clouds”, Equation (14) needs to be solved analytically but superposition of these spikes is analytically intractable. Therefore, we consider it semi-quantitatively by approximating the “Ca2+ clouds” with a piece-wise constant function having the equal profile areaIn order to analyze the cumulative activation outcome of a series of “Cariod see . The expThe corresponding duty ratio is2+ clouds”. In that respect, the average IC138 activity for a single We assume that the target protein IC138, in order to be phosphorylated, integrates the incoming spikes of “CaIf we consider a spike train with We now use the analytical result for cumulative activation of the target protein for the spike train containing the greater number of piecewise functions of Equation (18) evaluated by  as:(22)〈2+ spike repetition The dimensionless frequency Now, we fix the duty ratio to be 2+ spike trains from experimental assays with sea urchin sperm [As a special example, we can apply this concept to the flagellar Cain sperm ,47,48.The estimated parameter values in this case are as follows:If we fix the frequency Otherwise, setting The comparison of Eventually, on the basis of Equation (22) the cumulative activation of the LC138 complex in sea urchin sperm readsOn the basis of Equation (21) for the activation by a single bell-shaped spike, and using result of Equation (29) we estimate that approximately eight spikes are needed to achieve cumulative activation of the LC138 complex, a vital ingredient of the I1 inner dynein arm.In this paper we provided a description of the structure and bending dynamics of flagella. Special emphasis was placed on the microtubules within the axoneme and the associated dynein motor proteins. We revisited the accumulated knowledge about 15 different axonemal dyneins that are distinct in both their composition and location within the axoneme. These dyneins are implicated in complex mechanisms of flagellar beating.2+ ions in the regulation of flagellar dynamics. For example, in sperm cells the intracellular Ca2+ level, pH, and ATP are the key regulatory elements of bending motility. The first two factors are controlled by ion channels. The principal Ca2+ channel is CatSper, which is activated by progesterone and flagellar alkalinity. The most important Ca2+ sensors in flagella are EF-hand proteins, primarily calmodulin and neuronal calaxin whose roles in Ca2+ signaling are very crucial.Specific attention has been dedicated to the unique signaling role of Ca2+ cloud”. Calcium ions injected into the flagellum by ionic channels , through a so-called calcium induced calcium influx, and further elevated by CICR release mechanism, accumulate closely around microtubules in the axoneme. These “Ca2+ clouds” are not static but instead, move with the speeds of the order of hundreds of micrometers per second, see 2+ influx elevates their amplitude and speed. Such oscillations of concentration of free intracellular Ca2+ are a basic control mechanism for beating of flagella.We have paid significant attention to the concept of the bell-shaped “Ca2+ signaling in sea urchin sperm. The obtained numerical estimation of dynein I1 activation has an important physiological relevance as demonstrated in Equation (29). Our analysis indicates that trains of Ca2+ spikes are advantageous compared to a constant rise in Ca2+ concentration as being more efficient and much less prone to noisy fluctuations. We expect that our results shed more light for deeper understanding of subtle calcium control mechanisms implicated in flagellar dynamics.The main part of this article, which is our original and novel contribution, elucidates the decoding of these spike trains in the context of the phosphorylation cycle mediated by calmodulin and protein kinase. It appears that the decoding process is dependent on the amplitude and the frequency of these spikes within the train. We investigated the case where these spikes activate the target protein IC138 more efficiently if the frequency is higher. In that respect the oscillation frequency should be greater than the inactivation (dephosphorylation) rate of the IC138 target. As an illustrative example, we applied the concept of cumulative activation of IC138 phosphorylation to the case of Ca


Meningioma-1 (MN1) occur in a subset of acute myeloid leukemias (AML) and result in high expression of MN1, either as a full-length protein, or as a fusion protein that includes most of the N-terminus of MN1. High levels of MN1 correlate with poor prognosis. When overexpressed in murine hematopoietic progenitors, MN1 causes an aggressive AML characterized by an aberrant myeloid precursor-like gene expression program that shares features of KMT2A-rearranged (KMT2A-r) leukemia, including high levels of Hoxa and Meis1 gene expression. Compounds that target a critical KMT2A–Menin interaction have proven effective in KMT2A-r leukemia. Here, we demonstrate that Menin (Men1) is also critical for the self-renewal of MN1-driven AML through the maintenance of a distinct gene expression program. Genetic inactivation of Men1 led to a decrease in the number of functional leukemia-initiating cells. Pharmacologic inhibition of the KMT2A–Menin interaction decreased colony-forming activity, induced differentiation programs in MN1-driven murine leukemia and decreased leukemic burden in a human AML xenograft carrying an MN1-ETV6 translocation. Collectively, these results nominate Menin inhibition as a promising therapeutic strategy in MN1-driven leukemia.Translocations of  Acute myeloid leukemia (AML) is a heterogenous disease characterized by the clonal proliferation of myeloid cells arrested at an early stage of differentiation. Genome-wide sequencing efforts have allowed the identification of a large number of molecular abnormalities , 2, but MN1 gene is involved in translocations with the genes encoding the transcription factors ETV6  were maintained in RMPI-1640  supplemented with 20% fetal bovine serum (FBS) , 50 U/ML Penicillin–Streptomycin (P/S)  and 20 ng/mL human IL-6 and 40 ng/mL human GM-CSF . MV4;11 [KMT2A-AFF1], Molm14 [KMT2A-MLLT3], THP1 [KMT2A-MLLT3], OCI-AML3 [NPM1c], and KASUMI-1 cells [RUNX1-RUNX1T1] were maintained in RMPI-1640 supplemented with 10% FBS and 50 U/ml P/S. 293T cells were maintained in DMEM supplemented with 10% FBS and 50 U/ml P/S.Human cell lines were purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen  or the American Type Culture Collection . UCSD-AML1 )), p = 3.3e-21 .Leukemia-initiating cells (LIC) are defined as cells capable of giving rise to human AML in immunocompromised mice  but consCre Fig. . Given tvel Fig. . However-21 Fig. .Men1−/− MN1-driven AML isolated from moribund animals in more detail. In vitro, Men1−/− MN1-driven AML cells presented a profound impairment in both colony formation and cell growth  into secondary irradiated recipients. At D21 post transplantation, we found a significant decrease in peripheral blood leukemic burden in the Men1−/− versus Men1wt group (p < 0.0001) , with a failure to rearrange both alleles in one of the animals . This resulted in a dramatic improvement in survival compared to Men1wt control .We next analyzed wth Fig. , S2A. Anwth Fig. . To asse01) Fig. . Furtherals Fig. . This reMen1 in MN1-driven AML. RNA sequencing was performed on Men1−/− and Men1wt cells isolated from moribund animals after brief in vitro culture.We next investigated the transcriptional consequences of deletion of Men1−/− versus Men1wt MN1 driven leukemic cells revealed downregulation of 276 genes (Log2 Fold change (FC) < −2, padj < 0.05) and upregulation 645 of genes   in 05) Fig. . Gene Ser et al.  . As previously reported for KMT2A-r cells, VTP50469 also caused upregulation of the differentiation marker Cd11b  translocation. In AML cells with this rearrangement, most or all of the coding sequence of MN1 translocates to the C-terminal of ETV6, resulting in very high expression of MN1 either as a full-length protein or as an MN1-ETV6 fusion protein [RUNX1-RUNXT1] cells, a cell line characterized by low HOXA9 and MEIS1 expression . In a second independent experiment, animals were transplanted and treatment was initiated as detailed above, but VTP-50469 or control chow were continued for the remainder of the experiment. Sustained VTP-50469 treatment induced a modest but significant increase of survival in addition to lower leukemic burden . In secondary recipients, the decrease in LIC resulted in an almost complete exhaustion of Men1−/− MN1-driven leukemia cells in vivo. Furthermore, GSEA revealed that a set of genes that we had previously found to be downregulated upon loss of Kmt2a in MN1-driven leukemia was enriched in Men1−/− versus Men1wt MN1-driven AML cells. Menin-dependent genes in MN1-AML also contained a core transcriptional program that was found to be dependent on Menin in other subtypes of AML, including those with KMT2A rearrangements and NPM1c [Men1 and loss of Kmt2a have similar effects on gene expression suggests that such effect is mediated through the Menin/Kmt2a interaction. Indeed, pharmacologic inhibition of the Menin/Kmt2a interaction decreased colony formation and leukemia burden in models of murine and human MN1-driven AML in vivo and in vitro, respectively.Using a murine conditional knockout model, we found that loss of nd NPM1c . The obsKMT2A-rearranged leukemias, the transcriptional consequences of Men1 deletion/Menin/KMT2A inhibition mirror inactivation of the fusion, not inactivation of the KMT2A wild type allele [KMT2A allele is largely dispensable for KMT2A-fusion mediated leukemogenesis [KMT2A-fusion leukemias acts via disruption of the fusion complex, not the KMT2A complex. In contrast, Menin/KMT2A inhibition is effective in disrupting KMT2A dependent transcription in leukemia models that exhibit a dependency on KMT2A by preventing recruitment of the Menin/wild type KMT2A complex to a subset of target genes [Kmt2a and inactivation of Men1 supports that the effect of Menin/Kmt2a inhibition in MN1-driven leukemias is a result of disrupting the Kmt2a complex. Overall, our data confirms that inhibition of the Menin/KMT2A interaction is a therapeutic strategy in the absence of KMT2A-fusion proteins in selected AML subtypes.Menin interacts with the N-terminus of both wild type KMT2A  and KMT2e allele . The unmogenesis , 49. Thuet genes , 45. Intet genes . The strKMT2A-rearranged leukemias are critically dependent on Menin [HOXA9 and MEIS1 expression [Men1 only causes minor cytopenias [Men1−/− HSCs were at a disadvantage compared to Men1wt HSCs in repopulating secondary recipients after bone marrow ablation. Thus, this raises the concern that targeting the Menin/KMT2A interaction in leukemias that depend on wild type KMT2A could also negatively impact normal hematopoiesis. Prolonged treatment of non-leukemic mice with inhibitors of the Menin/Kmt2a interaction did not reveal any significant effect on normal hematopoiesis [The observation that on Menin  and moreon Menin  has promon Menin . Severalpression –53. Furtpression , 54. In topenias  and doesopoiesis , 54. Simopoiesis , 54. ThiWhile the exact contribution of MN1 to leukemogenesis remains unclear, high levels of expression of MN1 consistently correlate with poor prognosis; whether it is found in the absence of any karyotypic anomalies –8 or wheSupplemental MaterialSupplementary table 1 revisedSupplementary table 2


This study presents an algorithm to estimate the relative biological effectiveness (RBE) for cell survival under hypoxic conditions using the repair-misrepair-fixation model. The study finds that the RBE values are in the range of 1.0–3.0 for the linear energy transfer equal to 1.1 to 22.6 keV/μm under aerobic condition (21% O60Co γ-rays (linear energy transfer (LET) = 2.4 keV/μm) as the reference radiation, the RBE for DSB induction and enzymatic DSB under aerobic condition (21% O2) are in the range 1.0–1.5 and 1.0–1.6 along the track depth, respectively. In accord with RBE obtained from experimental data, RMF model-derived RBE values for cell survival are in the range of 1.0–3.0. The oxygen enhancement ratio (OER) for cell survival (10%) decreases from 3.0 to 2.5 as LET increases from 1.1 to 22.6 keV/μm. The RBE values for severe hypoxia (0.1% O2) are in the range of 1.1–4.4 as LET increases, indicating greater contributions of direct effects for protons. Compared with photon therapy, the overall effect of 62 MeV proton beams results in greater cell death and is further intensified under hypoxic conditions.This study uses the yields of double-strand breaks (DSBs) to determine the relative biological effectiveness (RBE) of proton beams, using cell survival as a biological endpoint. DSB induction is determined when cells locate at different depths (6 positions) along the track of 62 MeV proton beams. The DNA damage yields are estimated using Monte Carlo Damage Simulation (MCDS) software. The repair outcomes are estimated using Monte Carlo excision repair (MCER) simulations. The RBE for cell survival at different oxygen concentrations is calculated using the repair-misrepair-fixation (RMF) model. Using  Proton therapy (PT) has been used in radiation therapy (RT) because its dose is specific to the target and surrounding healthy tissues are only slightly affected . The dosAn RT treatment plan relies on relative biological effectiveness (RBE) to determine the doses for PT. Currently, the RBE for PT is 1.1, but many studies have demonstrated that this value does not correctly reflect the effectiveness of PT, compared with photon therapy ,5,6,7. R60Co γ-rays, protons, and helium ions are comparable to the levels of DSB induction; hence it is important to evaluate the yields of enzymatic DSB when RBE is evaluated [In addition to DSB induction, misrepair of DSBs also affects the total DSB yields. Here, the yields for DSB induction are produced immediately (seconds to minutes) after the time that cells are irradiated by ionizing radiations . Multiplvaluated ,23,24.2, which attacks DNA and leads to the formation of DNA damage. The DNA damage is “fixed” and irreversible, which is termed the oxygen fixation hypothesis [2 modifies the pathway and the final chemical products [Furthermore, oxygen also plays a vital role in the RBE. DNA damage induction is mainly caused by direct and indirect actions. For low LET radiations, about two-thirds of DNA damage is induced via indirect actions, mostly by hydroxyl radicals (OH) . These rpothesis . The preproducts . Normal products . The levproducts ,27. Moreproducts ,29,30,31In RT, mathematical models are able to theoretically predict the RBE for cell survival and provide essential biological information for treatment plans. These models include the local effect model ,33, the Although measured RBE values for cell survival under hypoxia conditions are available ,48, to oAll energy spectra for depths P1 to P6 see  are in a56Fe ions [Monte Carlo simulation is a mathematical technique that applies to stochastic events. It is based on repeated random sampling from numerical simulations, and the results depend on the average outcomes over a large number of runs . The MCD6Fe ions ,51,52.+), two or more strand breaks on opposite strands that do not constitute DSB (2SSB), DSBs with additional break(s) on a strand within 10 base pairs (DSB+) and more than one DSB within 10 base pairs (DSB++).MCDS is not a Monte Carlo radiation transport code and cannot simulate the stochastic events generated by ionizing radiations as track structure simulations ,54,55. T−4; polymerase error for LPBER and NER = 1.0−6; probability of incorrect insertion opposite a damaged base = 0.75; probability of incorrect insertion opposite a lost base = 0.75. The values for the parameters mentioned above were suggested by previous studies [The MCER code simulates the probability of the repair outcomes in the BER and NER pathways for DNA damage in cells that are irradiated with electrons, protons and helium ions  and has  studies ,21.i lesions, and iY is the yield of the total number of non-DSB clusters per Gy per gigabase pair (per Gy per Gbp), which is composed of i lesions, for proton ions with energy E.The yield for DSB conversion for proton ions is calculated by the formula below. Enzymatic is defined as the conversion probability for repair pathways for total DNA damage, which is composed of An RMF model  was devej defines the least number of lesions per DSB that cannot be rejoined, i lesions. The DNA damage yields were converted into the unit per Gy per Gbp using the factor 6 Gbp per cell for a typical mammalian cell [d and is defined as [The parameter ian cell . Assuminfined as :(5)Z¯F=0The diameter for this study was set as 8 μm. The size of nuclear diameter (~8 μm) was the typical size of V79 Chinese hamster cells used in many experiments . The yiej had a value of 9 to achieve the best fit for the measured survival data of AG01522 cells and U87 cells [j = 8 is used in moderate hypoxia (2% O2). When oxygen concentration decreased to 0.1%, the parameter j had a value of 7 to achieve the best fit to the measured OER data in ractions . For thi87 cells  for a LE2) or in moderate hypoxia (2% O2) [−3 and −5, which were used in a previous study [2) [−3 and −5. All calculations for RBE for cell survival in a normal oxygen concentration (21% O (2% O2)  used valus study . For sevtudy [2) , the calΣ, because DSB induction is linearly proportional to the absorbed dose, D, up to a hundred Gy under aerobic condition (21% O2) [2) [The RBE is defined as the ratio of the dose of low LET reference radiation to the dose of any other radiation that is required to achieve an equal biological effect . The RBE(21% O2)  and seve O2) [2) , as:(6)RP and γ, respectively, denote protons and γ-rays. The DSB yield for 60Co γ-rays is the reference for all reported RBE values.Subscripts S denotes the fraction of cells that survive at dose D and the curve-fitting parameters are α and β. Using Equation (6) and the RBE definition in Equation (5), the formula for RBE for cell survival [60Co γ-rays and proton exposure, respectively, and Cell survival is described using a LQ equation :(7)S=e−αsurvival  becomes:2) to the yield under severe hypoxia (0.1% O2). To calculate the OER for cell survival (Equation (8)), where the doses, aD (aerobic condition) and hD (severe hypoxia) (0.1% O2) are calculated using Equations (9) and (10), respectively, as shown below:2) and severe hypoxia (0.1% O2).The effect of oxygen on cells is quantified in terms of the OER, which is the ratio of the hypoxic dose to the aerated dose that is required to achieve the same biological outcome . OER is +, 2SSB, DSB, DSB+, and DSB were within 0.2%. As the depth and LET increased, the yield for simpler damage (BD and SSB) decreased whereas the yield for complex damage  increased. In total damage, the portion of BD decreased from 68 to 64%, while the portion of simple SSB only slightly increased from 29 to 30%, indicating that the percentage of complex DNA damage increased as the depth increased. That is attributed to the fact that protons are low-LET radiations, which suggests that the damage was distributed spatially sparsely and the majority of the damage was of the simple types such as BD and SSB [+, and DSB++ increased, as shown in P6 (Bragg peak).The limits of the standard deviations for all subtypes of DNA damage  as oxygen concentration decreased to 0.1%. Furthermore, the RMF model-derived RBE values for cell survival are compared with the experimental data in The yield for DSB induction for depths P1 to P6 increased as the LET increased but decreased as oxygen concentration decreased. The RBE values for DSB induction at 21% OIn Furthermore, repairing efficacy by different repair pathways was studied and all repair outcomes from SP BER, LP BER, SP BER/NER, and LP BER/NER for positions P1 and P6 were listed in In summation, for all repair pathways, the probability of correct repair decreases from 0.983–0.896 (P1) to 0.967–0.818 (P6). Furthermore, the probabilities of mutation and DSB formation increase from 0.008–0.065 (P1) to 0.017–0.110 (P6) and from 0.008–0.038 (P1) to 0.016–0.072 (P6), respectively. The limits of the standard deviations for all probabilities reported in The results show that the yield for DSB induction for depth P1 and P6 are 8.28 per Gbp per Gy to 12.4 per Gbp per Gy, respectively. The yield of the maximum enzymatic DSB for depth P1 and P6 was, respectively, 5.72 per Gbp per Gy and 9.66 per Gbp per Gy, which accounts for 69–78% of the yields for DSB induction. The RBE for DSB induction was in the range of 1.0–1.5 , which w2) is about 40–45% of the value at normal oxygen concentration . The effects of oxygen on DSB induction and cell survival are shown in 2 versus LET. The OER decline for cell survival is more obvious than that for DSB induction. That is, the OER for cell survival decreased from 3.0 to 2.5 as LET increased by 22.6 keV/μm while the OER for DSB induction was in the range of 3.0–2.9. The experimental data showed a decrease from 2.9 to 2.4 as LET increased from 9.9 keV/μm to 21.2 keV/μm [2 keV/μm  for heli2 keV/μm . Figure This study calculates the RBE for DSB induction and cell survival at various oxygen concentrations. The RBE values are compared with experimental data and the results show that the RBE for cell survival is greatly affected by LET (position of the track) and oxygen concentration. The effects of LET and oxygen on RBE are discussed in the following sections.+, and DSB++ for a 0.3 MeV proton beam are respectively 4.8, 3.6, and 1.3% and total DSB yield is 22.8 per Gbp per Gy [+, and DSB++ by MCDS (this work) are 4.0, 2.2, and 1.4%, and the total DSB yield is 22.2 per Gbp per Gy, which is comparable to the DNA profiles obtained by track structure simulations. However, other study reported that the measured DSB yields (γ-H2AX foci) are in the range of 5–16 DSBs per Gy per cell (0.8–2.7 per cell per Gbp) for a wide range of LET (1 ~ 270 keV/µm) [The MCDS-derived RBE for DSB induction and the RBE value using the RMF model for cell survival has been documented in several studies ,69, inclp per Gy . The per keV/µm) , which i keV/µm) . This is keV/µm) ,74.+, 2SSB, DSB, DSB+, and DSB++ increase, indicating that the composition of DNA profiles from P1 to P6 becomes more complex. Other studies using the Geant4-DNA Monte Carlo code also show similar trends for the SSB and DSB yields [B yields ,76. For B yields . The yiej  [j = 9 for RBE for cell survival of protons at all positions (LET < = 22.6 keV/μm) at normal oxygen concentrations and a value for j = 7 at severe hypoxia (0.1% O2). These results suggest that a very complex DSB, such as DSB with eight lesions, can be rejoined under normal oxygen concentration. Our results also show that the parameter j does not depend on the LET for low-LET radiations (LET < = 22.6 keV/μm), but its value decreases for lower oxygen concentrations , which  keV/μm) , but theions see c. For alα has been shown to be linearly proportional to LET by experiments [The LQ model parameter eriments . This reeriments  (see Apperiments .In 2) increases by 7% and the RBE for cell survival using the RMF model increases from 1.0–3.0 to 1.0–3.7as oxygen concentration decreases from 21% to 2% and to 1.1–4.4 as oxygen further decreases to 0.1% , then the dose would increase to 3.3 Gy for cells treated with60Co γ-rays Gy (under 21% O2) and further increase to 9.9 Gy  is 2.6 (60Co γ-rays) under 0.1% O2 is around 3.8, which is 16% less than the value, 4.4, derived by the RMF model. These higher RBE values for PT under hypoxic conditions represent the difficulty to efficiently and successfully kill tumor cells by photon therapy since the oxygen level is significantly lower in tumor cells [Because the trend of RBE for DSB induction increases as oxygen concentration decreases , the incs = 3.0, ) for cel) is 2.6 c and it or cells .2) are comparable to those under normal oxygen concentration (21% O2) and higher RBE values are predicted for cells irradiated at P5 and P6 (Bragg Peak) under severe hypoxia (0.1% O2). In contrast, the RBE value for positions P1–P2 (plateau region) only increases slightly. Experimental data also showed that the RBE values of therapeutic proton beams (LET < 5 keV/μm) under hypoxic conditions are about the same (1.0 ~ 1.2) as those under normal oxygen concentration [2) [2. Under hypoxic conditions, the yields of DNA damage induction reduce to one-third of the yields under aerobic conditions for low-LET radiations; however, for higher-LET radiations, the yields of DNA damage induction reduce less because the direct action contributes more in the DNA damage induction. Moreover, the enzymatic DSB yields also reduce due to the reduction in the total damage when oxygen concentration decreases. The overall effects on cells irradiated by proton ions under hypoxic conditions lead to a higher RBE for DSB induction and cell survival.In ntration ,48. For tion [2) ,86; thistion [2) . The preThe study by Lomax et al. (2013)  shows thAlternatively, instead of modifying the microenvironment in the tumors, a larger irradiated volume containing a wider SOBP with higher-LET radiations may be applied in RT. This proposal is in line with the idea of “LET-painting” , which sj, This paper presents an algorithm to estimate the RBE for cell survival under hypoxic conditions using the RMF model. This algorithm uses the yields of DSB induction and three parameters (