Interestingly, the gating ring’s intersubunit assembly interface harbors the RCK2 Ca2+-binding site, known as the Ca2+ bowl. The gating ring’s assembly interface is made in part by intersubunit coordination of a Ca2+ ion between the Ca2+ bowl and an RCK1 Asn residue, N449, and by apparent intersubunit electrostatic interactions between E955 in RCK2 and R786 and R790 in the RCK2 of the adjacent subunit. To understand the role of the intersubunit assembly interface in Ca2+ gating, we performed mutational analyses of these putative interacting residues in human BK channels. We found that N449, despite its role in Ca2+ coordination, does not set the channel’s Ca2+ sensitivity, whereas E955 is a determinant of Ca2+ sensitivity, likely through intersubunit electrostatic interactions. Our findings provide evidence that the intersubunit assembly interface contains molecular determinants of Ca2+-sensitivity in BK channels.The large-conductance calcium-activated K In the brain, BK channels regulate neuronal firing and neurotransmitter release7 and are involved in motor coordination8, rhythmic control of the circadian clock9, as well as frequency tuning of cochlear hair cells10. Mutations or dysregulation of neuronal BK channels can cause epilepsy and paroxysmal dyskinesia12. BK channels control the contractile tone of smooth muscle and are involved in the regulation of blood pressure13, bladder contractility14, and erectile function15. In non-excitable secretory epithelial cells, BK channels provide an essential pathway for resting K+ efflux16.Large-conductance, Ca2+- and voltage-sensing α subunits either alone or with regulatory β or γ subunits19. Each α subunit contains a transmembrane domain for voltage sensing and channel pore formation and a large cytosolic C-terminus composed of two RCK domains (RCK1 and RCK2) for Ca2+ and Mg2+ sensing26. BK channels possess many biophysical features that make them an ideal system for studying ion channel gating mechanisms27. The biophysical mechanism of BK channel activation by voltage and Ca2+ can be described by a well-established allosteric gating model, in which the channel pore opening is allosterically regulated by the movement of each voltage sensor and the binding of Ca2+ at each Ca2+ sensor on the four subunits in a largely independent manner28. X-ray crystallography of BKα C-termini in humans and zebrafish21 and cryo-electron microscopy (cryo-EM) of the entire BKα channel in Aplysia californica23 at near-atomic resolutions have revealed that the tandem RCK1 and RCK2 domains from four individual subunits are organized into a gating ring of eight RCK domains by tetramerization at the intersubunit assembly interface. In each subunit, the two RCK domains are assembled into a pseudo-dimeric bi-lobed architecture21 harboring a Ca2+-binding site in the RCK1 domain and another Ca2+-binding site, known as the Ca2+ bowl, formed by a string of Asp residues in the RCK2 domain30. Comparisons of the BK channel structures in the absence and presence of Ca2+ indicate that the gating ring expands in response to Ca2+ binding and propagates conformational changes to the channel pore22, suggesting a likely involvement of the interfaces between the RCK domains of adjacent subunits, known as the “intersubunit assembly interface”21, in BK channel activation by Ca2+.BK channels are composed of homotetramers of the pore-forming, Ca2+ gating, we perturbed the structure of the interface by eliminating intersubunit Ca2+ coordination mediated by N449 residues, and by disrupting intersubunit electrostatic interactions mediated by E955 residues, in human BK channels. We found that the Ca2+-coordinating N449 residue has little role in determining BK channel Ca2+ sensitivity. However, E955 plays an important role in determining BK channel Ca2+-sensing likely through intersubunit electrostatic interactions, by a mechanism that is mediated in part through the Ca2+ bowl.To probe the role of the intersubunit assembly interface in BK channel Ca2+-free gating ring21 and the zebrafish Ca2+-bound gating ring20, and cryo-EM structures of the full-length Aplysia californica BK channel in the presence and absence of Ca2+ 23. The intersubunit assembly interface is formed by the RCK1 domain’s α-helices C, D, and E  and the RCK2 domain’s α-helix H (αH2) in one subunit and the RCK2 domain’s Ca2+ bowl and α-helices C, D, and E  in the neighboring subunit  in the αD1 from one subunit provides additional coordination for the Ca2+ atom of the neighboring subunit’s Ca2+ bowl   located on αH2 of one subunit (on the same side as the human N449 residue) and two positively charged Arg residues  located on αC2 of the neighboring subunit (on the same side as the Ca2+ bowl)  is close (within 3.4 Å) to the two Arg residues (R786 and R790 in humans) to allow these residues form intersubunit electrostatic interactions , which is immediately next to the other Glu residue  in the amino acid sequence but with its side chain positioned approximately 10 Å from the other subunit’s two Arg residues (R786 and R790 in humans)  Fig. . On the ons Fig. . The abons) Fig. . These i2+ (V1/2 = 167.5 ± 2.4 mV for wild type [WT] vs 127.2 ± 1.4 mV for E955Q)  in the absence of Ca2+ at very negative voltages where the voltage sensors are forced into resting states. The relationship between PO and L can be described by the equation PO = L = L0exp(zLV/kT), in which L0 is L at 0 voltage and zL is the partial charge associated with channel opening. To examine a possible effect of E955Q on L, we measured the channel’s PO at very negative voltages  and found that it was increased by approximately 10-fold in E955Q channels compared with WT channels 27, we estimated that L0 is increased from 4.8 × 10−6 in WT to 7.2 × 10−5 in E955Q channels  curve towards the hyperpolarizing direction by 40 mV in the absence of Ca5Q) Fig. . Becauseels Fig. . By fittels Fig. . Assumina2+ Fig. .Figure 22+ concentrations ranging from 1.5 µM to 1 mM, we observed that the E955Q mutation greatly decreased the apparent Ca2+-sensitivity of BK channels. In the WT channels, the Ca2+-induced shifts in V1/2 (ΔV1/2 (−/+Ca) = V1/2(0 Ca) − V1/2 (+ Ca)) were 69 mV at 1.5 μM Ca2+ (V1/2 = 98.2 ± 4.0 mV), 149 mV at 7.5 µM Ca2+ (V1/2 = 18.7 ± 2.8 mV), 199 mV at 90 μM Ca2+ (V1/2 = −31.6 ± 2.8 mV), and 236 mV at 1 mM Ca2+ (V1/2 = −68.7 ± 2.2 mV)  values of E955Q mutant channels were reduced to 21 mV at 1.5 μM Ca2+ (V1/2 = 106.0 ± 8.0 mV), 64 mV at 7.5 µM Ca2+ (V1/2 = 62.9 ± 5.2 mV), 106 mV at 90 μM Ca2+ (V1/2 = 21.3 ± 2.6 mV), and 153 mV at 1 mM Ca2+ (V1/2 = −25.3 ± 4.1 mV), which were decreased by 70% (48 mV), 57% (85 mV), 47% (93 mV), and 35% (84 mV), respectively, compared with those of WT channels  values of the E956Q channel were 90 mV at 7.5 μM Ca2+ (V1/2 = 70.6 ± 6.3 mV) and 172 mV at 90 μM (V1/2 = −11.6 ± 6.2 mV), which were decreased by 40% (59 mV) and 13% (27 mV), respectively, compared with those of the WT channel  with a resultant ΔV1/2 (−/+Ca) value of 36 mV, which was 76% (113 mV) less than that of the WT channels and 45% (29 mV) less than that of the E955Q channels  value of 105 mV, which was similar to that of the E955Q channel  Fig. . Howeverels Fig. . To detenel Fig. . In the nel Fig. . The comels Fig. . At 90 μnel Fig. . These r2+ sensing, the equivalent residue  is located more than 20 Å from the nearest Ca2+ in the Ca2+ bowl site in the gating ring structures of Aplysia  and zebrafish (N449) were found to directly coordinate Ca2+ in the Ca2+ bowl , the G-V curve of the N449A channels was shifted to the depolarizing direction by approximately 30 mV (V1/2 = 193.2 ± 5.3 mV) in the absence of Ca2+  values of the N449A mutant channels were 67 mV at 1.5 µM Ca2+ (V1/2 = 126.5 ± 2.6 mV), 140 mV at 7.5 µM Ca2+ (V1/2 = 53.1 ± 2.0 mV), 204 mV at 90 µM Ca2+ (V1/2 = −10.3 ± 3.9 mV), and 233 mV at 1 mM Ca2+ (V1/2 = −39.8 ± 2.9 mV), all of which were similar to those of the WT channels  mutation or that lack RCK1 Ca2+ sites owing to the D362A/D367A mutation. Similar to those reported previously31, the 5D5N and D362A/D367A mutant channels were much less responsive to Ca2+ than the WT channels, as they encountered decreases in ΔV1/2 (−/+Ca) at 7.5 µM Ca2+ (ΔV1/2 (−/+Ca) = 49 and 66 mV) of 67% (100 mV) and 56% (83 mV), respectively, and at 90 µM Ca2+ (ΔV1/2 (−/+Ca) = 108 and 105 mV) of 46% (92 mV) and 47% (94 mV), respectively  in ΔV1/2 (−/+Ca)  in ΔV1/2 (−/+Ca) at 7.5 µM Ca2+ and a decrease of 53% (56 mV) in ΔV1/2 (−/+Ca) at 90 µM Ca2+  Fig. ; Fig. S1a2+ Fig. ; Fig. S11/2 (−/+Ca) values of channels with the single R786A or R790A mutations or the double R786A/R790A mutation were decreased by 25% (37 mV), 40% (60 mV), and 39% (59 mV), respectively, at 10 µM Ca2+ and by 27% (54 mV), 24% (48 mV), and 33% (67 mV), respectively, at 90 µM Ca2+  = 52 mV, 112 mV, and 148 mV at 1.5, 7.5, and 90 µM Ca2+, respectively) were either similar to  or slightly larger than  those of the R786A/R790A double mutant channel (ΔV1/2 (−/+Ca) = 46 mV, 90 mV, and 133 mV at 1.5, 7.5, and 90 µM Ca2+, respectively)  = 13 mV)  that could neutralize the charge but cause no other changes in the structure or physical and chemical properties of the side-chain. Compared with that of the WT channels, the ΔVa2+ Fig. . Thus, sly) Fig. ; Fig. S1mV) Fig. . These rion Fig.  had a smdid Fig.  could ha2+-binding molecules and proteins32, Ca2+ ions are preferentially coordinated by six to eight oxygen atoms. In the BK channel structures of both Aplysia and zebrafish22, the Ca2+ ion of the Ca2+ bowl is coordinated by seven oxygen atoms: one from the side-chain carboxylate of N449, four from the side-chain carboxylates of D895 and D897, and two from the backbone carbonyl oxygen atoms of Q889 and D892  approaches and coordinates the Ca2+ ion in the neighboring subunit’s Ca2+ bowl site23. Interestingly, we found that N449 does not contribute overall to the BK channel’s Ca2+ sensitivity, as N449A mutant channels and WT channels had similar ΔV1/2 (−/+Ca) values over a wide range of Ca2+ concentrations (1.5 µM to 1 mM). This finding argues against an obligatory functional role of N449 residues in Ca2+-binding affinity23. Consistent with our finding, in Ca2+-free structures  in human BK channels21 and D905 (2.3 Å) in Aplysia BK channels22. The ineffectiveness of the N449A mutation in altering Ca2+-sensitivity may therefore arise from an energetically greater contribution of other Ca2+-coordinating interactions and/or other intersubunit interactions at the interface that are stabilized in the Ca2+ bound state.In structures of Ca892 Fig. . Among tres Fig. , this As2+ ion, caused a decrease in the Ca2+ sensitivity, to an extent similar to that of the two RCK1-Ca2+ and Ca2+ bowl null mutants (D362A/D367A and 5D5N)  of human E955 is more than 40 Å from the RCK1-Ca2+ site of both the same and the neighboring subunits  to the Ca5N) Figs . We founite Fig. . In the its Fig. . Althoughem Fig. 20–23. Th2+ sensitivity and the function of the Ca2+ bowl remains unclear. Because the distances between E955 and R786 and R790 appear similar in the absence or presence of Ca2+ in reported structures . Cells were transfected with the designed plasmid(s) using Lipofectamine 2000 (Invitrogen) and used in electrophysiological assays within 16–72 hours.Expression constructs of BK channel mutants were made with the QuickChange site-directed mutagenesis kit (Stratagene) using the recombinant cDNA plasmid HF1-hSlo1, which encodes the c-Myc-tagged human BK channel α subunit (GenBank accession number AAB65837), as a template as describe previously+ currents were measured by patch-clamp recording in excised inside-out patches of HEK-293 cells with symmetric K+ solutions of 136 mM KMeSO3, 6 mM KCl, and 20 mM HEPES (pH 7.20). Data were acquired at room temperature using an EPC-10 patch clamp amplifier (HEKA). To obtain the desired concentration of free Ca2+, the internal solution was supplemented with a certain amount of CaCl2 buffered by 5 mM HEDTA or nitrilotriacetic acid. No chelator was used for 1 mM internal Ca2+. The free Ca2+ concentration was measured with a Ca2+-sensitive electrode (Orion Research Inc.). The steady state of channel activation was expressed as G/Gmax, calculated from the relative amplitude of the tail currents . The voltage of half maximal activation (V1/2) and the equivalent gating charge (z) were obtained by fitting the relationship of G/Gmax with voltage with a single Boltzmann function: G/Gmax = 1/(1 + exp-zF(V − V1/2)/RT).BK channel KAll data generated or analyzed during this study are included in this published article and its Supplementary Information.Supplementary Information


AML initiating cells (LICs), defined functionally as cells capable of initiating AML in immunocompromised mice3, are also challenging to maintain in culture, presenting a major obstacle to developing new therapies for AML that target the LIC. Co-culture with stromal cells6 can maintain and expand LICs; however these culture systems use multiple cytokines that may promote differentiation and loss of LICs ex vivo. Small molecules such as SR1 (an aryl-hydrocarbon receptor antagonist) and UM729 support AML viability without stromal co-culture, but also require multiple cytokines1. In this work, we demonstrate an alternative approach that enhances the viability of primary AML cells and supports ex vivo maintenance of LICs in cytokine-free medium. We show that inhibition of GSK-3, which activates Wnt signaling, markedly improves the viability of primary human AML cells in cytokine-free medium and further that combined inhibition of GSK-3 and the nutrient sensor mTORC1 maintains LICs capable of engrafting immunocompromised mice, identifying a signaling network that regulates the viability of LICs.Acute myeloid leukemia (AML) is an aggressive malignancy of the hematopoietic system that arises through clonal expansion of myeloid precursor cells that have arrested at an early stage of differentiation and ultimately causes death in over 50% of patients. Despite the aggressive growth characteristics of AML in vivo, AML blasts are difficult to culture once removed from the patient, suggesting that AML cells depend on signals from the microenvironment7 and Gsk3 knockout causes a myeloproliferative neoplasm suggestive of AML in mice8, we tested whether direct inhibition of GSK-3 would enhance the viability of primary AML cells ex vivo. AML cells in culture do not proliferate and typically die over a few days. Cells from AML patients were cultured in defined medium without cytokines in the presence of the GSK-3 inhibitor CHIR99021 (Chiron) or vehicle. After 5 days, cells cultured with vehicle alone were small with fragmented nuclei whereas cells cultured with Chiron were uniformly larger with intact, round nuclei . Knockdown  and consistent with redundant functions for GSK3A and GSK3B9  and measured cell viability; CR reduced viability significantly compared to Chiron alone   than control cells Fig. , consist18) Fig.  whereas 18) Fig. . Cells c18) Fig. .Fig. 2Co10 or AML patients. β-catenin is essential for long-term HSC self-renewal in response to Gsk3i10, but the parallel activation of mTORC1 drives HSCs to proliferate and enter more differentiated lineages, leading to HSC exhaustion, as also observed with Pten knockout in hematopoietic cells14. Similarly, activation of mTORC1 by GSK3i prevents AML cells from engrafting whereas LICs are maintained with GSK3i and parallel inhibition of mTORC1. Consistent with these observations, knockout of Gsk3a and Gsk3b in mouse bone marrow causes a severe myeloproliferative neoplasm with increased blasts and features of AML8. The mechanism downstream of GSK-3 again appeared to be through enhanced Wnt/β-catenin signaling, although other effectors may contribute. Taken together, these findings support a model in which AML cells in vivo are supported by signals from the microenvironment that inhibit GSK-3 and that activation of GSK-3 could serve as a strategy to treat AML.Data from this study and our previous work with nonmalignant HSCs suggest that inhibition of GSK-3 and mTORC1 is a general mechanism to maintain self-renewing hematopoietic cells from healthy donorsIn conclusion, we show that the signaling molecules GSK-3 and mTORC1 regulate the viability of primary human acute myeloid LICs. Combined inhibition of GSK-3 and mTORC1 maintains LICs under cytokine-free conditions, suggesting a novel approach to study signaling in primary AML cells, screen for new therapies for AML, and test the molecular response to targeted AML therapies in a patient-specific manner.6 cells/ml were cultured in cytokine-free medium in the presence of DMSO or PBS (vehicle control) or the indicated concentrations of Chiron, LiCl, and/or Rapamycin for 4–10 days. Viability was measured by trypan blue dye exclusion every other day for time course experiments and on day 4 for dose response experiments. Viable fraction was calculated as the number of live cells/(live cells+dead cells).De-identified cells from patients with AML were obtained from the Stem Cell and Xenograft Core (SCXC) at the University of Pennsylvania. In total 5 × 104, 105, and 106 AML cells were injected prior to culture and an equivalent volume of the remaining culture was injected after 4 days into 10-12 week NSG mice. After 10 weeks of transplantation, engraftment was measured by flow cytometry for hCD45+hCD33+ cells. Engraftment of 0.5% was considered as positive.Transplantations were performed through the SCXC. Non-obese diabetic severe combined immunodeficient IL-2Rγ null (NSG) mice were conditioned with busulphan or irradiation 24 h before intravenous or intrafemoral injection of cells. 10Detailed description of other methods is provided in supplement.Supplemental Data and Methods


We employ an ultrafast random access multi-photon (RAMP) microscope to simultaneously record action potentials and Ca2+ transients from multiple T-tubules in ventricular cardiomyocytes from a HF rat model of coronary ligation compared to sham-operated rats as a control. We confirmed that β-adrenergic stimulation increases the frequency of Ca2+ sparks, reduces Ca2+ transient variability, and hastens the decay of Ca2+ transients: all these effects are similarly exerted by β-adrenergic stimulation in control and HF cardiomyocytes. Conversely, β-adrenergic stimulation in HF cells accelerates a Ca2+ rise exclusively in the proximity of T-tubules that regularly conduct the action potential. The delayed Ca2+ rise found at T-tubules that fail to conduct the action potential is instead not affected by β-adrenergic signalling. Taken together, these findings indicate that HF cells globally respond to β-adrenergic stimulation, except at T-tubules that fail to conduct action potentials, where the blunted effect of the β-adrenergic signalling may be directly caused by the lack of electrical activity.Alterations of the β-adrenergic signalling, structural remodelling, and electrical failure of T-tubules are hallmarks of heart failure (HF). Here, we assess the effect of β-adrenoceptor activation on local Ca PKA phosphorylates several key proteins that modulate cardiac function: L-type Cas (LTCC) , ryanodis (LTCC) , troponis (LTCC) , and myos (LTCC) . In hears (LTCC) , the β-As (LTCC) ,8,9,10, s (LTCC) ,12, one s (LTCC) . Moreoves (LTCC) . Among ts (LTCC) . The twos (LTCC) . However2+ release [2+ release after acute β-AR stimulation in HF, in light of the new findings demonstrating the presence of T-tubular electrical defects that directly impair local Ca2+ release [Furthermore, beyond the alteration and loss of the T-tubular structure ,18 obser release . With th release .2+ release [−7 M isoproterenol show 5.6% ± 3% of AP− , in line with our previous findings in HF cells at basal conditions [2+ transients (in cyan) recorded from AP+ and AP− T-tubules in the absence  after isoproterenol is observed exclusively in the proximity of AP+, while Ca2+ transient TTP of AP− is not affected by isoproterenol treatment (2+ transients (CaT50) is significantly hastened in HF + ISO cardiomyocytes as compared with HF cells at basal condition at both AP+ and AP− sites. These results suggest that the Ca2+ transient detected at AP− tubules results from the Ca2+ signal propagating from neighbouring sites, and the velocity of propagation is not regulated by β-AR signalling. Contrarily, the increased phosphorylation of phospholamban by PKA activation is preserved, increasing the sarcoplasmic reticulum Ca2+ ATPase (SERCA) reuptake rate across the whole cardiomyocyte, at both AP+ and AP− sites.We previously demonstrated  that the release . Here, wnditions ,20. In F absence A, HF andreatment C. On the2+ transients both in time  and in space (among different sites). A coefficient of variability (CV) is calculated as σ/μ, where σ is the standard deviation and μ is the mean. The CV of Ca2+ release is calculated based on time (beat-to-beat CV) and space  and the corresponding graphs are reported in 2+ release; such variability is significantly reduced with isoproterenol application [2+ decay of failing cardiomyocytes is also more variable when compared to CTRL cells. In 2+ traces recorded in three different T-tubules of a HF and HF + ISO myocyte. It can be noticed that isoproterenol application reduces the variability of local Ca2+ release and reuptake in HF cells and, as reported in the columns  [2+ transients. Here, we confirmed that the Ca2+ sparks frequency during diastole is dramatically increased after isoproterenol treatment in control cardiomyocytes, their frequency being 1.8 ± 1.8 mHz and 32.9 ± 5.4 mHz, without and with isoproterenol treatment, respectively. In 2+ sparks (grey arrow) in HF (above) and in isoproterenol-treated HF cardiomyocytes (bottom). In HF, we previously reported a significantly augmented Ca2+ sparks frequency in isolated cardiomyocytes [2+ sparks in their vicinity. Here, the isoproterenol treatment of HF cardiomyocytes significantly enhances the frequency of Ca2+ sparks in AP+ (2+ leakage from the sarcoplasmic reticulum (SR) similarly to CTRL cells, irrespective of T-tubule excitability.To further evaluate β-AR’s role on RyR open probability (Po), we studied the Cait (CRU)  during amyocytes  as compas in AP+ C as comp2+ and voltage at a sub-cellular level, in order to disclose the effects of β-AR signalling on Ca2+ release at the local level in failing rat cardiomyocytes. As demonstrated by our investigations on Ca2+ sparks and Ca2+ transient variability, we find that acute stimulation of the β-AR system increases the open probability (Po) of RyR channels by a similar extent in HF cells and control cardiomyocytes. It is of note that the RAMP microscope has a sensitivity capable of detecting single Ca2+ sparks [3) [2+ sparks are not evoked by cellular permeabilization or specifically designed stimulation protocols, but occur spontaneously in intact cardiomyocytes during regular pacing at steady state. We confirmed that stimulation of β-AR signalling increases Ca2+ sparks frequency [2+ sparks rate is changed in HF compared to control cells. Previous work by Gomez et al. did not observe an increased frequency of Ca2+ sparks in failing rat cardiomyocytes [2+ leak has been found in intact ventricular myocytes of failing rabbits [2+ sparks are increased compared to the control in the absence of β-AR stimulation. The factors influencing RyR gating have been extensively reviewed [2+ sparks frequency is increased in HF cardiomyocytes similarly to isoproterenol-treated control cells. This finding suggests that β-AR signalling still has leeway to generate Ca2+ sparks in HF cells, using the same mechanisms of control cells . In addition, the microenvironment pertinent to failing T-tubules shows a Ca2+ sparks rate comparable to that of electrically coupled elements and displays a similar response to β-AR stimulation.In the present work, we employ an optical technique capable of simultaneously studying Ca+ sparks , by probarks [3) ,24. In orequency  in intacrequency . These mmyocytes , while a rabbits . In the reviewed ,30, the reviewed  and othereviewed ,33. PKA-reviewed . After β2+ transients. In fact, a homogeneous recruitment of RyR2 operated by β-AR signalling is observed both in space and in time and it is similar in failing and CTRL cardiomyocytes after isoproterenol challenge. Altogether, these observations allow inferring that, despite altered basal levels, the β-AR signalling on RyR2 is well preserved in HF cells.Moreover, we observe a synchronization of RyR2 recruitment upon β-AR challenge as demonstrated by the coefficient of variability of Ca2+ release have been previously observed in HF cells with structurally remodelled T-tubules [2+ release [2+ handling per se, here we focused on the microenvironment around preserved T-tubules. Employing a novel functional imaging technique [2+ rise in HF [2+ release previously observed. In HF cardiomyocytes, the AP failure prevents LTCC activation, because, even though β-AR signalling phosphorylates LTCC, this response does not translate into higher sarcolemmal Ca2+ influx or into the downstream SR Ca2+ release. In fact, shortening of the Ca2+ transient TTP is reduced by β-AR activation exclusively in the vicinity of electrically coupled T-tubules (AP+). The acceleration of Ca2+ transient TTP after β-AR stimulation in HF cells is lower than in control cardiomyocytes. This can be explained by the fact that HF cardiomyocytes show a lower density of LTCC but a higher level of basal Ca2+ channel phosphorylation [2+ transient decay at every location in HF cells. It means that β-AR–mediated phosphorylation of phospholamban is maintained in the cardiomyocyte, increasing the SERCA reuptake rate across the whole cell. Again, this is the hint that β-AR machinery is still capable of working in failing cardiomyocytes when the target substrates are present. Alterations of Ca-tubules ,36 and w release . Althougechnique , we haveechnique ,20 and technique . Such T-rylation , likely rylation  that mayIn conclusion, our work provides functional information about the subcellular responsiveness to β-AR signalling in HF that should be integrated in the complex scenario of the β-adrenergic molecular modifications found in this disease. Here, we provide evidence that a novel mechanism for the blunted β-AR signalling is present in HF and it does not directly involve receptors and downstream effectors, but it is caused by the absence of electrical activity in some T-tubules.t-test applied. Rats were sacrificed six to eight weeks after surgery and used for cell isolation. All animal procedures are performed conform the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes; experimental protocol is approved by the Italian Ministry of Health on the 6th of July 2015 (approved protocol number 647/2015-PR). Cells were loaded in extracellular buffer added with 10 μM blebbistatin, 4 μM cytochalasin D, and 500 μM CaCl2. First, 0.5 μg/mL of GFP-certified Fluoforte dissolved in DMSO were added to the cell suspension for 15 min. After washing, 2 μg/mL of di-4-AN(F)EPPTEA dissolved in ethanol was also added for 15 min and then cells were resuspended in fresh extracellular buffer containing 10 μM blebbistatin, 4 μM cytochalasin D and 1 mM CaCl2. Loaded preparations were used for experiments within 1 h. Although blebbistatin is known to be fairly safe [2+ transient at 40 μM [Ventricular myocytes were isolated from male Wistar Han rats  as previously described . Myocardrly safe , cytochaat 40 μM . For thi2+ detection, respectively. The measurements were performed during steady-state stimulation (0.34 Hz). The cells were field-stimulated using two parallel platinum wires (250 μm in diameter) placed at a distance of 6.3 mm. Square pulses of 10–20 V and duration of 3 ms were used to reach AP threshold. In a typical measurement, we probed 5–10 different sarcolemmal sites for ten subsequent trials. The length of the scanned lines ranged from 2 to 10 μm with an integration time per membrane pass of ~200 μs, leading to a temporal resolution of 0.4–2 ms. The large Stokes shift of fluorinated VSD is not sufficient to prevent spectral contamination between the two channels. For this reason, we optimized a simple un-mixing procedure under two hypotheses: negligible contamination of the green channel on the red one and constant VSD sensitivity across the emission spectrum.The basic design of our RAMP imaging system has been already described . Briefly2+ were manually identified trace by trace for the calculation of the coefficient of variation (CV), while the mean values of each probed site were determined after averaging 10 subsequent trials to increase accuracy. Spontaneous Ca2+ sparks were scored when a sudden increase of fluorescence intensity occurred with a ΔF/F0 two-fold above the trace noise not correlated to the electrical stimulus. Ca2+ spark frequency was normalized to time and excitation volume (sparks·pl−1·s−1), assuming the two-photon excitation volume as xy) and axial (z) intensity squared profiles [0 = 0.037 in agreement with our previous finding [2+ sparks, we calculated the systole as the CaT95 for each experimental group, the rest of the recording is considered diastole. Data are expressed and plotted as means ± SEM (Standard Error of Mean) obtained from a number of independent determinations on different myocytes. Number of cells and number of animals (N) are indicated in the figure legends for each set of measurements. Unpaired Student’s t-test is used for comparisons. A p-value of < 0.05 is considered statistically significant .Optical data were analysed with software written in LabVIEW 2010 . The amplitude and kinetics parameters of the Caprofiles . An aver finding . In orde


The pediatric AML and ALL cells differed by 139/34 gene expression/pathway activation biomarkers. For the adult 30 AML and 17 normal blood samples, we found 132/33 gene expression/pathway AML-specific features, of which only 7/2 were common for the adult and pediatric AML and, therefore, age-independent. At the pathway level, we found more differences than similarities between the adult and pediatric forms. These findings suggest that the adult and pediatric AMLs may require different treatment strategies.Acute lymphoblast leukemia (ALL) is characterized by overproduction of immature white blood cells in the bone marrow. ALL is most common in the childhood and has high (>80%) cure rate. In contrast, acute myeloid leukemia (AML) has far greater mortality rate than the ALL and is most commonly affecting older adults. However, AML is a leading cause of childhood cancer mortality. In this study, we compare gene expression and molecular pathway activation patterns in three normal blood, seven pediatric ALL and seven pediatric AML bone marrow samples. We identified 172/94 and 148/31 characteristic gene expression/pathway activation signatures, clearly distinguishing  Acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) are heterogenous diseases of hematopoietic stem cells and progenitor cells. AML is a cancer of the myeloid line of blood cells, characterized by the rapid growth of abnormal white blood cells that accumulate in the bone marrow and interfere with the production of normal blood cells [od cells . AML invod cells . AML is od cells . As an aod cells .Unlike AML, acute lymphoblastic leukemia (ALL) is most common in childhood, with a peak incidence at 1–6 years of age , 5. AML However, the genetic or gene expression factors responsible for the age-related manifestation features of AML and ALL remain uncertain. To date, gene expression analysis has been performed on a very limited number of AML/ALL cancer samples, especially for the pediatric onset. This may be due to relative rarity of AML/ALL and very limited access to pediatric cancer patient biopsies . On the In this study, we used microarray hybridization to compare the gene expression in the two groups of human pediatric AML and ALL biosamples. We compared pediatric onset-specific AML gene expression profiles with those characteristic of adult AML. To analyze the expression data, we used molecular pathway approach which was shown to reduce platform-specific bias in various assays . We idenThe bone marrow biopsy samples were collected and analyzed for seven pediatric acute lymphoblast leukemia (ALL) and seven pediatric acute myeloid leukemia (AML) samples. The mean ages were 7 and 6 years old (y.o.) for the patients in the AML and ALL groups, respectively. Leukemia samples were compared with the normal peripheral blood isolated from the three healthy donors, with the mean age in the group 12 y.o. .The total RNA preps were extracted, and gene expression was profiled with microarray hybridization. We used original customized microchip system developed using the CustomArray (USA) technology of direct electrochemical oligonucleotide synthesis on the array . Using tFor the adult leukemia samples, we took GEO dataset GSE37307containing 30 AML and 17 peripheral blood samples profiled using the Affymetrix hgu133a microarray hybridization platform. The gene expression data were quantile normalized and further compared with pediatric samples.p) is as follows: activator/repressor role (ARR), which equals 1 for an activator, −1 for a repressor, and shows intermediate values −0,5; 0,5 and 0 for the gene products having intermediate repressor, activator, or unknown roles, respectively. The nCNR value is the ratio of the expression level of a gene n in the sample under investigation to the average expression level in the control sampling. N is the number of individual gene products in the pathway p. The positive value of PAS indicates activation of a pathway, and the negative value - its repression in a biosample under investigation. The CNR and PAS values obtained for the normal and leukemia samples are shown on We processed the transcriptomic data under investigation to establish normalized cancer-to-normal (CNR) expression rates and to build pathway activation strength (PAS) profiles corresponding to intracellular signaling pathways for each sample. The analysis included 334 pathways  and 2016CNRn))/N . The funi) pediatric ALL and AML from normal samples and (ii) pediatric AML from the adult AML samples.The CNR and PAS data were analyzed in two ways to identify gene expression and PAS signatures that distinguish  values  for the Inositol-3-phosphate signaling pathway responsible for gene expression with the transcriptional factors CREB3, NFATC2 and MEF2D was found to be strongly upregulated in the AML samples in this study. Of note, AML cells also demonstrated several upregulated p53-related apoptosis-promoting pathways: the branch of Mitochondrial apoptosis pathway related to the activation of p53-dependent gene expression, the branch of P53 signaling pathway responsible for promotion of apoptosis, and the branch of TNF signaling pathway responsible for apoptosis. However, this enhanced upstream regulation of apoptosis was blocked by the strongly suppressed downstream branch of Mitochondrial apoptosis pathway responsible for the irreversible mechanisms such as the DNA fragmentation  genes from the list, four ubiquitin protein ligase genes , and four genes for proteasome subunit A (PSMA). There were also five genes for tubulins and associated proteins that were all significantly upregulated in the ALL compared to AML. The same trend was observed for the four tumor necrosis factor (TNF) superfamily genes and their receptors .To identify the differential molecular features in the AML and ALL samples, we compared seven ALL versus seven AML gene expression profiles. At the level of gene expression, we found 139 biomarkers with high AUC scores , Table 1G protein subunits , five genes for fibroblast growth factor (FGF) proteins and three glutamate metabotropic receptor (GRM) genes were upregulated in the AML versus ALL samples.In contrast, all five genes from the list for TNF signaling pathway, Ubiquitin-proteasome pathway  individual gene products and (ii) molecular pathways, may help to decode the mechanisms for greater curability of the ALL tumors and provide avenues for finding new molecular targets for treating AML in the future. In addition, these data suggest that using of proteasomal inhibitors like Bortezomib may be beneficial also for the treatment of the pediatric patients with the ALL, not only for the AML patients, as this is the case now in several clinical studies [e.g., [For the first time, our data clearly suggest, that the ALL cells are highly enriched in the proteasomal activities compared to the AML cells. In turn, the AML cells are enriched in GPCR (G protein-coupled receptor) signaling. Those molecular differences clearly seen at both levels of  was repressed instead  and acute lymphoblast leukemia (ALL) differ greatly in their behavior, mortality and curability. While ALL occurs primarily in the childhood, the incidence of AML increases with age. The factors that act in an age-dependent manner to promote AML are poorly understood. Although it is widely accepted that the cellular physiology, epigenetic regulation and gene expression of normal hematopoietic stem cells change with age, molecular grounds of such age-dependent cancer transformation remain largely unknown . In the CD40 pathway, and a branch of the Ras pathway), and one (a branch of the ATM Pathway governing control over the cell cycle checkpoints) was regulated oppositely, being upregulated in children and downregulated in the adults  completely matching molecular pathways, of which two were commonly regulated  and one pathway , for which association with the AML was previously unknown.In this study, we identified multiple gene expression and pathway activation markers specific for the AML Table , among tGPCR and TRAF-associated apoptosis marker pathways) were also new (Table For the pediatric acute lymphoblast leukemia (ALL), we found 97 gene expression markers and 47 characteristic molecular pathways, of which two , Moscow, Russia, were analyzed. All patients provided written informed consent to participate in this study. This study was approved by the local ethical committee at the CPHOI. The mean age of the ALL patients at the time of sampling was 5.7 years (range 1–14 years), and 7 years for the AML patients (range 1-15 years). The mean age for the healthy donors of peripheral blood at the time of sampling was 11.7 years (range 10–13 years). Both the tumors and normal tissues were evaluated by a pathologist to confirm the diagnosis and estimate the tumor cell numbers. All tumor samples used in this study contained at least 90% tumor cells. Mononuclear cells were extracted shortly after bone marrow or peripheral blood samples collection. Cells were obtained by a density gradient centrifugation method using DiacollTM (Ficoll-1077) . 2-3 ml of bone marrow or blood was dissolved in PBS up to 10 ml. Cell suspension was layered on 2.5 ml of Diacoll in 15 ml centrifuge tube and centrifuged at 400 RCF for 40 min. Buffy coat was removed and dissolved in PBS up to 14 ml followed by centrifugation at 800 RCF for 10 min. PBS wash procedure was performed twice. Pellet was dissolved in 0,5-1 ml of RNALater solution (Thermo Fisher Scientific). Cells were counted by Scepter™ 2.0 Handheld Automated Cell Counter (Merck Millipore), aliquoted and stored at −20°C till RNA extraction and microarray hybridization. For RNA extraction, cell suspensions with RNALater were centrifuged at 3000 RCF for 5 min. Pellets were dissolved in TRI Reagent (MRC), Direct-zol™ RNA MiniPrep (Zymo Research) was used for RNA extraction. RNA was quantified using Nanodrop (Thermo Fisher Scientific).For the adult leukemia samples, we took GEO dataset GSE37307 containing 30 AML and 17 peripheral blood samples profiled using the Affymetrix HG-U133a microarray hybridization platform.B3 microarray synthesizer  was used for forty nucleotides-long oligonucleotide probe synthesis on CustomArray ECD 4×2K/12K slides. Synthesis was performed according to the manufacturer's recommendations. Four replicates of total 2228 unique oligonucleotide probes specific to 2016 human gene transcripts were placed on each chip. Chip design was performed using Layout Designer software .Complete Whole Transcriptome Amplification WTA2 Kit (Sigma) was used for reverse transcription and library amplification. Manufacturers protocol was modified by adding to amplification reaction dNTP mix containing biotinylated dUTP, resulting to final proportion dTTP/biotin-dUTP as 5/1. Microarray hybridization was performed according to the CustomArray ElectraSense™ Hybridization and Detection protocol. Hybridization mix contained 2.5 ug of labeleled DNA library, 6X SSPE, 0.05% Tween-20, 20mM EDTA, 5x Denhardt solution, 100 ng/ul sonicated calf thymus gDNA, 0,05% SDS. Hybridization mix was incubated with chip overnight at 50°C. Hybridization efficiency was detected electro-chemically using CustomArray ElectraSense™ Detection Kit and ElectraSense™ 4×2K/12K Reader.Probe signals were geometrically averaged, thus obtaining expression value for each specific type of the probe. The whole dataset was next rounded down to integer values and normalized using the “DeSeq2” package's “estimateSizeFactors” function with respect to the sample type . The same geometrical averaging was performed for the GSE37307 dataset  using the correspondence table from the “hgu133a” Bioconductor package. Then quantile normalization  was perfhttp://www.sabiosciences.com/pathwaycentral.php) was used to determine structures of intracellular pathways, as described previously [n is the ratio of the expression levels of a gene n in the sample under investigation to the average expression in the control group of samples. In this study, we used normalized PAS scores (PAS2), where each initial PAS score obtained according to [The SABiosciences signaling pathways knowledge base (eviously . We appleviously  for funcrding to  was diviHierarchical clustering heatmaps with Euclidean distance and complete-linkage were generated using heatmap.2 function from “gplots” package . Pathway


The pathway involving the key components phosphatidyl inositol-3-kinase (PI3K), protein kinase B (PKB/AKT) and mammalian target of rapamycin (mTOR) plays a major role in regulation of proliferation and cell death of DLBCL3. PI3Ks play multiple roles, ranging from cell growth and proliferation to migration. Dysregulation of the PI3K pathway is associated with numerous cancers5. Here, we have focused on the contribution of the δ isoform of PI3K, which plays a central role in normal B-cell development and function. PI3Kδ signaling is overactive in many B-cell malignancies and shown to drive proliferation, survival, and trafficking to lymphoid tissue. The most effective single PI3K-based therapeutic agent is idelalisib, a p110δ-selective inhibitor. Idelalisib has achieved notable success in clinical trials for patients with relapsed chronic lymphocytic leukemia or indolent lymphoma7. However, treatment with idelalisib alone is insufficient due to induced resistance, clearly highlighting the urgent need to identify effective new targets for combination therapy.Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma. DLBCL represents a highly heterogeneous group of tumors with variable clinical features, molecular genetic alterations, therapeutic responsiveness levels, and prognosis9. Additionally, CXCR4 expression is a marker of DLBCL recurrence and associated with shorter survival10. Here, we established idelalisib-resistant ABC-DLBCL cells and addressed the functional consequences of CXCR4 expression.Stromal-derived factor (SDF-1 or CXCL12), a CXC chemokine, and its cognate seven transmembrane G-protein coupled receptor 4 (CXCR4) play significant roles in promoting migration and trafficking of malignant B cells in hematological malignancies. CXCR4 has been detected in several tissues, in particular hematopoietic cellsTo determine the molecular targets in primary resistance to idelalisib, we isolated surviving cells after exposing three DLBCL cell lines  , U2932-Idela(3 μM), and OCI-Ly10-Idela(3 μM)) were generated by exposing each parental cell line to progressively increasing concentrations of idelalisib for 4 weeks. The cell lines established were more resistant to idelalisib and displayed superior ability to form colonies (Supplementary Figure  μM, U29314. Primary refractory cells expressed high levels of CXCR4 on the cell surface and application of CXCR4 antagonists combined with idelalisib resulted in a profound synergistic effect of growth inhibition, compared to individual treatment. However, in acquired resistant cell lines, CXCR4 expression was gradually decreased during long-term idelalisib treatment and selection of resistant cells (Supplementary Figure 10. Idelalisib resistance in Waldenstrom’s Macroglobulinemia cells has been overcome through BCL-2 targeting15. However, in our experiments, BCL-2 expression was not increased (Supplementary Figure In the current study, we identified CXCR4 as a cause of primary resistance to idelalisib in ABC DLBCL via gene expression analysis, consistent with earlier studies showing that CXCR4 is associated with drug resistance of cancer cellsSupplementary tables and figuresSupplementary materials and method


However, the ways in which perturbation of specific molecular mechanisms alters the probability of ectopic beats is not understood. We present a multiscale model of cardiac tissue incorporating a biophysically detailed three-dimensional model of the ventricular myocyte. This model reproduces realistic Ca2+ waves and DADs driven by stochastic Ca2+ release channel (RyR) gating and is used to study mechanisms of DAD variability. In agreement with previous experimental and modeling studies, key factors influencing the distribution of DAD amplitude and timing include cytosolic and sarcoplasmic reticulum Ca2+ concentrations, inwardly rectifying potassium current (IK1) density, and gap junction conductance. The cardiac tissue model is used to investigate how random RyR gating gives rise to probabilistic triggered activity in a one-dimensional myocyte tissue model. A novel spatial-average filtering method for estimating the probability of extreme  stochastic events from a limited set of spontaneous Ca2+ release profiles is presented. These events occur when randomly organized clusters of cells exhibit synchronized, high amplitude Ca2+ release flux. It is shown how reduced IK1 density and gap junction coupling, as observed in heart failure, increase the probability of extreme DADs by multiple orders of magnitude. This method enables prediction of arrhythmia likelihood and its modulation by alterations of other cellular mechanisms.Ectopic heartbeats can trigger reentrant arrhythmias, leading to ventricular fibrillation and sudden cardiac death. Such events have been attributed to perturbed Ca Arrhythmias are electrical abnormalities of the heart that can degenerate into fibrillation, thus preventing normal heartbeats and leading to sudden cardiac death. The mechanisms leading to ventricular arrhythmias and the unexpected nature of sudden cardiac death are not fully understood. One hypothesis is that a group of cardiac myocytes, which generate contraction, spontaneously depolarize at precisely the same moment to excite the surrounding tissue. In individual myocytes, such misfires, known as delayed afterdepolarizations, are driven by random ion channel gating and thus stochastic in nature. While incidental afterdepolarizations in a large number of myocytes is highly improbable on any given beat, it may be feasible over a long time frame, thus explaining the unpredictability of arrhythmias. We developed a detailed model spanning the molecular, cellular, and tissue scales that realistically reproduces the mechanisms underlying this hypothesis. An efficient method is presented for estimating the probability of extremely rare delayed afterdepolarizations in tissue from a limited set of simulations. Furthermore, we demonstrate how altered tissue and ion channel properties in heart disease increase the risk of arrhythmia. This approach can be used generally to probe the effects of specific molecular mechanisms on the likelihood of rare delayed afterdepolarizations. Depolarization of the cell membrane during an action potential (AP) increases LCC open probability, generating a flux of Ca2+ ions into the dyad. The resulting local increases of dyad Ca2+ concentration ([Ca2+]d) increase RyR open probability, which when open allow flux of Ca2+ ions from the JSR into the dyad. This process, known as Ca2+-induced Ca2+ release (CICR), causes brief, spatially localized release events known as a Ca2+ sparks [2+ sparks cause a cell-wide rise in cytosolic [Ca2+] ([Ca2+]i), leading to myofilament activation and force generation. This process, known as excitation-contraction coupling (ECC), is central to the function of the myocyte ) is elevated during Ca2+ sparks and cell-wide Ca2+ release [2+ release sites have revealed that RyR clusters exhibit edge-to-edge spacing of less than 100 nm [2+ diffusion. Therefore Ca2+ diffusion between SM compartments was implemented to reflect Ca2+ transport across steep [Ca2+] gradients on the periphery of the release site during release [2+ in the SM is buffered by calmodulin and sarcolemmal binding sites. Ca2+ transport rates from the SM to the cytosol and between SM compartments were constrained to yield a realistic Ca2+ wave threshold (~100–150 μmol/[L cytosol]) )  in the b2+ transport , dyadic subspace , and SM  compartments. Spatial Ca2+ diffusion is modeled as transport between SM compartments of adjacent release sites in the 3D lattice , is represented by a set of ordinary differential equations describing local Caransport . [Ca2+]  lattice . Model e2+]i and network SR [Ca2+] ([Ca2+]NSR). The use of global compartments to represent these quantities eliminates two Ca2+ diffusion parameters and provides a three-fold reduction in the number of Ca2+ diffusion terms, thus substantially reducing model complexity and computational burden. While these simplifications result in over-estimation of the rise in [Ca2+]i and fall of [Ca2+]NSR at sites far away from an initiating Ca2+ wave, the model produces realistic spatiotemporal dynamics of spontaneous Ca2+ release and DADs.Global compartments are used to represent average cytosolic i by 50% d signal caused by the brief high-amplitude unitary LCC currents characteristic in this potential range.In order to reproduce protocols designed to measure ECC properties, membrane potential was stepped to varying test potentials for 200 ms from a holding potential of -80 mV. The dependence of normalized peak RyR and LCC Camentally   compared to under baseline conditions due to greater RyR Ca2+ sensitivity. The wave shape and velocity of 68 μm/s are similar to those observed in experimental studies i were controlled over time. β-adrenergic stimulation was applied and the cell was paced at 1 Hz. First, [Na+]i was fixed to 15 mM and the RyR opening rate was ramped from 1.5x to 5x that of baseline over t = 0 to 5 s. Intermittent Ca2+ waves, triggered by overload of JSR Ca2+, cause spontaneous [Ca2+]i transients, which began to occur  the next stimulus  . The fir+]i, . Upon Rythe LCCs . The modload see . Rather,2+ overload as a driver of DADs under pathophysiological conditions. Elevated RyR sensitivity and [Na+]i accumulation led to DADs of sufficient amplitude to trigger action potentials during the diastolic intervals. In addition, these results illustrate the interplay between [Ca2+]i and SR load dynamics during spontaneous Ca2+ release.These results demonstrate how the model reproduces SR Ca2+ load and spontaneous Ca2+ release was investigated. Simulations were run using initial conditions reflecting the cell state just prior to the moment when SR Ca2+ load reaches the Ca2+ wave threshold following an AP. Initial [Ca2+]i was set to 150 nM, similar to the level during the late decay phase of a cytosolic Ca2+ transient. 2+ load shown in 2+ load, DAD amplitude is large enough to trigger an AP, as shown in simulation (v) in 2+ load reduces the delay until the spontaneous release event, consistent with the observations of Wasserstrom et al. [2+ loads [The relationship between SR Cam et al. . The inc2+ loads .2+]SM at three time points in each simulation. The number of Ca2+ wave nucleation sites (Nnuc) was defined as the number of independently formed wave front epicenters and was estimated by inspection of the volume renderings  by enhancing the synchrony of RyR opening and number of nucleation sites.nderings . Nnuc gect heart , 10. The2+ wave dynamics and thus DAD amplitude and timing. To test this, five independent realizations were generated, each of which had identical initial conditions similar to simulation (i) from 2+ load was not reported [We hypothesized that stochastic gating of the RyRs drives variability in Careported . The are2+ release generates DADs by driving an inward current through NCX [2+]SM that is greater than [Ca2+]i because it can be localized near the release sites [2+ sparks occurring across the cell. This is consistent with a study showing that peak Ca2+ release flux is strongly correlated with the likelihood of ectopic activity [2+]i and JRyR are related to DAD amplitude. max) and the maximum of [Ca2+]i during the spontaneous [Ca2+]i transient (r2 = 0.950). There is an even stronger relationship between Vmax and the peak JRyR value (r2 = 0.998). This confirms that the inward NCX current is primarily driven by JRyR via the resulting rise in [Ca2+]SM.Spontaneous Caough NCX . Imagingough NCX  studies se sites , 20, 21.activity . An enseRyR reflects the combined Ca2+ release flux associated with all Ca2+ sparks occurring at any time. It was therefore expected that the variability in DADs was the result of spatio-temporal variations in Ca2+ wave dynamics. 2+ waves in each simulation. Waves emanate from nucleation sites of high Ca2+ spark activity. For example, in the simulation (iv) of 2+ sparks is visible at the left end of the cell, and by 900 ms a wave front of Ca2+ is seen propagating radially away from this cluster site. The random nature of nucleation site locations results in DAD variability across simulations. Simulations i and ii are both associated with the highest-amplitude DADs as well as the greatest number of nucleation sites, as shown in 2+]SM that drive inward INCX. The remaining three simulations have lower amplitudes due to fewer nucleation sites. Note that in simulation iv, two separate Ca2+ waves form over 100 ms apart, resulting in a prolonged low-amplitude DAD with two peaks. These results are consistent with the strong correlation between Vmax and maximum JRyR, which reflects the timing and pattern of Ca2+ wave formation.Note that J2+ loading and DAD amplitude and timing in ensemble simulations are investigated. 2+ load is varied. The dark lines indicate the median value of membrane voltage over time, and the shaded regions illustrate the range of the second and third quartiles (25th to 75th percentile). Consistent with findings from the individual cell simulations of 2+ load. The peaks of the upper and lower bounds are -92.4 and -90.6, -82.7 and -79.5, and -73.2 and -68.1 mV for initial SR [Ca2+] values of 0.32, 0.36, and 0.40 mM, respectively. This suggests that there is an increase in DAD amplitude variability at higher SR Ca2+ loads. Note also that the width of the shaded regions decreases as SR load increased, reflecting increased DAD synchrony.In this section, the statistical relationships between Ca2+]i plays a critical role in determining the SR Ca2+ wave threshold during pacing  are reflected in initial total cell Ca2+. The effect of 50% IK1 density reduction on the [Ca2+]i-dependence of Vmax was also analyzed. These results demonstrate two important conclusions.K1, the distributions of Vmax as a function of initial total cell Ca2+ are identical in both cases, as shown by overlap of the blue  and red  traces. This strongly suggests that DADs are driven by both [Ca2+]i and [Ca2+]SR and is consistent with the notion that total cell Ca2+ plays a major role in setting the DAD distribution, as observed in other models  is 5.8 fmol, but reducing IK1 density by 50% increases the probability to ~ 0.5. Thus IK1 density plays a critical role in modulating the Ca2+ load at which arrhythmia-triggering events occur.With increasing initial total cell Ca2+ load and that varying IK1 density shifts the Ca2+ load dependence. Simulations were performed to test whether the model could produce probabilistic triggered activity in a 1D fiber of myocytes during pacing, where electrotonic loading of any cell by adjacent cells becomes important. +]i set to 19 mM, as may be observed in heart failure [+] sensed by membrane channels during contraction may be higher than [Na+]i in the bulk cytosol due to local buildup of Na+ imported by NCX [K1 density and gap junction conductance were each reduced by 50% as observed in HF [+]i was set to 10 mM in the outer twenty-four cells near each end of the cable in order to prevent DAD generation. DADs, appearing as faint bands of depolarization between the paced beats, and reach Vmax values between approximately -70 and -60 mV. This is consistent with experimental observations of synchronized spontaneous Ca2+ release causing DADs in intact heart following rapid pacing i. At the highest [Ca2+]i, Vmax lies between -74.1 mV and -70.7 mV, a range of 3.4 mV. There is notably less variability than in individual cells at a similar Ca2+ load  compared to baseline. This is consistent with the increase in Vmax SD from 1.7 to 6.1 mV when IK1 density was decreased to 50% in isolated cells . For a fiber of a given length, the probability of this event depends upon both the mean Vmax and the likelihood of a deviation from the mean with sufficient amplitude to reach threshold. Under conditions where the mean Vmax is far below threshold, however, it is unclear whether it would be plausible to observe a sufficiently large fluctuation that causes a triggered beat. We sought to characterize the likelihood of such events by estimating the upper tail of the Vmax distribution.The DAD results presented thus far indicate that spatial fluctuations in Vmax was strongly correlated with Jmax in DAD simulations of isolated cells. In tissue, however, electrotonic coupling attenuates DAD amplitude by drawing current to neighboring cells via gap junctions. However, if spontaneous Ca2+ release occurs synchronously in a cluster of cells, the potential gradient between cells is smaller, thus reducing the gap junction current and increasing DAD amplitude. Therefore the Vmax of a given myocyte in tissue depends on the relative timing and amplitude of Ca2+ release in nearby cells.In max are caused by rare spatially clustered synchronized Ca2+ release events. However, estimating the probability of extremely rare events (e.g. 1 in 106) by direct simulation is computationally prohibitive using the full biophysical model since it would require potentially millions of simulations. Therefore, a method was developed for estimating the probability of such events using the output from a limited set of simulations.We hypothesize that extreme deviations in V2+ wave patterns and thus variable profiles of JRyR. Therefore JRyR is a stochastic process that is dependent on complex microscopic events, which are computationally intensive to sample. The method leverages the fact that JRyR has a relatively simple distribution by resampling JRyR profiles from a set of simulations that is sufficiently large to approximate the distribution of JRyR. Multiple realizations of release events  are generated using a resampling method in which cell positions along the cable are shuffled to produce independent, distinct realizations. The key to this approach is the fact that spontaneous Ca2+ release events are decoupled across the cells in the fiber . All values of μV fall within a narrow range from -77.0 mV to -79.1 mV, while the values of μJ in the two non-baseline conditions are less than half of the baseline value due to their lower Ca2+ loads.The filtering method was applied to simulations of DADs with baseline conditions, with 50% I50% ggap . In the max variability in the two pathological conditions is reflected in the parameters of the filtering model. It can be shown that the quantity SV = (σV/σJ)/W scales with the SD of Vfmax . While this distribution exhibits a very small bias of ~ -0.05 mV, it is not significantly different from the true distribution on the basis of a Kruskal-Wallis test after correcting for the bias by subtracting their means (p = 0.89). The shuffling method was then validated by resampling from the JRyRs in all 1,000 fibers to produce 1,000 96-cell fiber realizations and computing Vpeak with the filtering method. While the resulting distribution also exhibits a very small bias of ~ -0.08 mV, it is not significantly different from the true distribution after adjusting the means (p = 0.69) . Therefore, the method is accurate using a total of ~ 500 simulated cells. In summary, the method outlined here is a computationally efficient approach that permits the rapid estimation of the Vpeak distribution using output from a single 500-cell fiber simulation. = 0.69) . The upp = 0.69) . To valipeak—μV exceeds a given potential estimated from an ensemble of 106 realizations. Reducing IK1 by 50% shifts the distribution tail to greater amplitude DADs compared to the baseline model. The probability of observing a Vpeak 3 mV greater than μV (-77.1 mV) is 9.9 × 10−5 in the baseline model compared to 0.096 with 50% reduction in IK1, a nearly 1000-fold increase. The most extreme event observed with 50% IK1 was 6.1 mV greater than μV compared to 3.4 mV in baseline, corresponding to DAD amplitudes 41% and 21% above average, respectively. Reducing ggap by 50% further increases the likelihood of large-amplitude DADs, with the probability of a 3 mV DAD reaching 0.50, a 5000-fold increase over baseline, and the most extreme event observed at 7.5 mV, corresponding to a DAD amplitude 53% higher than average. These results demonstrate how changes ion channel expression or function can alter the probability of supra-threshold membrane potential fluctuations. In this case, reductions in IK1 and ggap considerably widen the distribution of Vpeak and increase the probability of occurrence of larger Vpeak values by multiple orders of magnitude.peak values were examined in each condition. max—μV. Because Vmax at any position is determined by an average of the Ca2+ release profiles over a window of cells, each rare depolarization occurs at a cluster of cells whose width corresponds to the window size. RyR profiles and the filter window centered on the Vpeak. In each case, the extreme event occurs at a cluster of cells within the window where JRyR tends to be greater and more synchronized than in the rest of the fiber. This result demonstrate that rare (occurring ~1 in 106 beats in a 500-cell fiber) proarrhythmic events result from spontaneous Ca2+ release exhibiting high (1) flux magnitude, (2) synchronization, and (3) spatial clustering. None of the 999,999 other simulations result in such an extreme event due to a lack of one or more of these properties. Note that because the window size is smaller in the case of 50% ggap, fewer cells need to fulfill these three criteria and thus the probability of extreme events is much higher.To illustrate the nature of these rare events, the realizations exhibiting the greatest V2+ diffusion between release sites and incorporated it into a tissue-scale model to study the mechanisms and statistical properties of DADs. Loading of SR Ca2+ is known to cause spontaneous Ca2+ release JSR-dependent regulation of the RyRs increases their sensitivity to cytosolic [Ca2+] JSR < 1 mM Jc [Ca2+] , 64, butR < 1 mM , 66 and K1 inhibition (in the absence of β-adrenergic stimulation) caused increased APD and cellular Ca2+ loading over time. This resulted in greater inward NCX current during the repolarization phase, thus further increasing APD and Ca2+ loading. This positive-feedback loop led to EADs and ultimately failure to repolarize. It is possible that the saturating behavior of LCC Ca2+-dependent activation in this model prevents sufficient LCC inhibition required for repolarization in the presence of elevated SR Ca2+. The LCC model also does not incorporate “global” [Ca2+]i sensing [2+]i. Future work is needed to understand the contributions of these mechanisms, as they may affect the conditions under which the model exhibits DADs during pacing.In our investigation, we found that pacing the baseline model at 1 Hz with 50% I sensing , which w2+]i and [Ca2+]SR. This was revealed by the apparent change in the threshold SR Ca2+ load for spontaneous Ca2+ wave formation during pacing  the probability of occurrence of potentially arrhythmogenic large-amplitude DADs. Note that the model does not include Ca2+-dependent regulation of IK1, as this mechanism has been controversial [2+]i at a fixed concentration upregulates IK1 via an increase in CaMKII activity, there remains insufficient data to constrain a model of this mechanism. Future models could be informed by further studying dynamic changes in CaMKII activity and IK1 in response to elevated [Ca2+]i. Reduction of gap junction conductance, another pathological feature of diseases such as HF   . Therefo unclear , 83–87.2+ wave dynamics and results in substantial DAD variability at the tissue scale, particularly in the pathological states tested where IK1 and ggap were reduced. Second, while one can imagine a case where a contiguous cluster of cells exhibit large synchronized spontaneous Ca2+ release, the probability distribution of such events has not been well characterized. In a 496-cell fiber with reduced IK1 and gap junction coupling, the largest DAD amplitude observed in an ensemble of 106 realizations had amplitude ~50% greater than the mean DAD amplitude. For such a fiber paced at 1 Hz and exhibiting a DAD after every beat, one could expect to observe such an event approximately once every 11 days. Thus extreme DADs, while quite rare, are still possible over relevant time frames. Further work is needed to estimate the probability of such events in whole heart, as 3D tissue is ~1–2 orders of magnitude less likely to exhibit triggered beats due to the increased electrotonic coupling i is ramped down to 15 mM over t = 35 to 40 s, resulting in sub-threshold DADs that occur at lower frequency. (b) APD of paced and DAD-triggered APs. APD decreases mainly as [Na+]i rises due to reduced inward NCX current. (c) [Ca2+]i and (d) average [Ca2+]JSR are shown.(a) DADs and triggered APs continue to occur after cessation of pacing after t = 30 s. [Na(EPS)Click here for additional data file.S4 Fig2+]i profiles were taken from the simulation represented by the red trace in K1 and 50% ggap. The absolute difference in peak [Ca2+]i was computed for each pair of adjacent (dC1) and 50th neighbor (dC50) cells. The absolute difference in the times of the peaks was also computed . Histograms and QQ plots of the differences in (a) time of peak and (b) peak [Ca2+]i. Both QQ plots exhibit linear trends, implying that the distributions are indeed alike. Therefore, the timing and amplitude of spontaneous Ca2+ release of adjacent cells do not differ substantially from those of distant cells.[Ca(EPS)Click here for additional data file.S1 Equations(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.S1 Movie2+ loads on Ca2+ wave dynamics, as shown in This illustrates the effect of varying SR Ca(M4V)Click here for additional data file.S2 Movie2+ wave dynamics due to stochastic Ca2+ spark activity, as shown in Each was started with identical initial conditions to illustrate the variability in Ca(M4V)Click here for additional data file.


Therefore, IB functions through a mechanism involving vulnerability to dysregulated calcium metabolism and the combination of fusing a lipophilic amine to a NOX inhibiting dye shows promise for further pre-clinical development for targeting high risk AML.Aberrant cytokine signaling initiated from mutant receptor tyrosine kinases (RTKs) provides critical growth and survival signals in high risk acute myeloid leukemia (AML). Inhibitors to FLT3 have already been tested in clinical trials, however, drug resistance limits clinical efficacy. Mutant receptor tyrosine kinases are mislocalized in the endoplasmic reticulum (ER) of AML and play an important role in the non-canonical activation of signal transducer and activator of transcription 5 (STAT5). Here, we have tested a potent new drug called imipramine blue (IB), which is a chimeric molecule with a dual mechanism of action. At 200–300 nM concentrations, IB is a potent inhibitor of STAT5 through liberation of endogenous phosphatase activity following NADPH oxidase (NOX) inhibition. However, at 75–150 nM concentrations, IB was highly effective at killing mutant FLT3-driven AML cells through a similar mechanism as thapsigargin (TG), involving increased cytosolic calcium. IB also potently inhibited survival of primary human FLT3/ITD FLT3/ITD mutations result in constitutive activation of key survival and growth pathways including STAT5, PI3K, and MAPK. Tyrosine kinase inhibitors (TKIs) that directly target FLT3  are currently being studied in clinical trials, however thus far they have produced modest responses, with TKI resistant mutations being a main cause of relapse3. Therefore, novel methods to target this high risk disease subset are warranted. Mislocalization of mutant type III receptor tyrosine kinases  occurs in leukemia5. In FLT3/ITD+ AML, a portion of mislocalized, underglycosylated RTK accumulates on the endoplasmic reticulum (ER) in a highly oxidized microenvironment where the RTK activates signal transducer and activator of transcription 5 (STAT5) through tyrosine phosphorylation, a phenomenon that does not occur with the mature fully glycosylated form, and there are high levels of localized reactive oxygen species (ROS) generated from ER-bound NADPH Oxidase 4 (NOX4)6. This non-canonical activation mechanism is leukemia-specific, thus opening the door to an improved therapeutic index for agents that are able to exploit this STAT5 activation mechanism.Acute myeloid leukemia (AML), the most common leukemia in adults and second most common in pediatrics, is a heterogeneous disease with a high relapse rate. FLT3/ITD mutated AML represents 30% of adult and 10–20% of pediatric AML and despite intensive therapy and allogeneic stem cell transplant the prognosis for these patients remains dismal7. NOX4 has been shown to be a STAT5 target gene, and its upregulation in FLT3/ITD+ AML mediates leukemic transformation through inactivation of protein tyrosine phosphatases (PTPs) such as DEP-1 by causing reversible cysteine oxidation at their catalytic site. Furthermore, NOX4 inhibitors have been shown to increase survival in murine models of FLT3/ITD+ AML8. STAT5 is also directly affected by ROS; in FLT3/ITD+ AML cell lines, hydrogen peroxide (H2O2) co-localizes to the endoplasmic reticulum and is required for STAT5 phosphorylation9. These important roles of ROS in FLT3/ITD+ AML raise the possibility the ROS may be a therapeutic target for this disease.ROS are highly reactive molecules containing oxygen. While typically produced by the mitochondria as a byproduct of cellular metabolism, in recent years ROS have been shown to play a key role in many aspects of cell signaling. ROS are elevated in AML and specifically AML with FLT3/ITD mutations10. IB has been shown to be effective in preclinical models of glioblastoma, Burkitt lymphoma, head and neck carcinoma, breast cancer, and chronic myeloid leukemia (CML)14. Given the prominent role of NOX4 and ROS in FLT3/ITD+ AML, we sought to determine if FLT3/ITD+ AML might be preferentially sensitive to ROS inhibition. However, we uncovered a new mechanism of action where AML cells are also highly sensitive to cytosolic calcium (Ca2+) and vulnerable to Ca2+ overload induced mitochondrial cell death16.Imipramine Blue (IB) is a triphenylmethane dye conjugated to the tricyclic antidepressant imipramine. Many effects of this compound have been discovered, however its most prominent effect is that of NOX inhibition+ cell lines MV4–11, MOLM-13, and MOLM-14 were a gift from Doug Graham (Emory University). MV4–11 cells were grown in IMDM supplemented with 10% FBS, and MOLM-13 and MOLM-14 cells were grown in RPMI supplemented with 10% FBS. FLT3/ITDneg cell line OCI-AML3 was purchased from DSMZ and grown in αMEM supplemented with 10% FBS. FLT3/ITDneg cell lines HL-60, HEL, and K562 (blast crisis CML) were gifts from Gang Huang  and William Tse (University of Louisville). HL-60 cells were grown in IMDM with 20% FBS and HEL and K562 cells were grown in RPMI + 10% FBS. MOLM-14 cells with acquired FLT3 point mutations17 F691L (gatekeeper mutation) and D835Y (tyrosine kinase domain mutation) were grown in RPMI + 10% FBS. 32D clone 3 cells transduced with wild-type c-KIT and c-KIT D814V mutation were grown in IMDM + 10% FBS supplemented with 5 ng/mL murine IL-3 (Gemini) and 32D KIT D814V cells were starved of IL-3 for at least 24 hours prior to experimentation.All cultured cell lines were grown in media containing 1% penicillin/streptomycin and found to be negative for mycoplasma contamination prior to use. The FLT3/ITD+ cells were then extracted utilizing the human UltraPure CD34 MicroBead Kit . Cells were expanded as described18 in Stemspan H3000 serum free media  supplemented with 100 ng/mL of human thrombopoietin, IL-6, FLT3 ligand, stem cell factor (Gemini) and 1 µM StemRegenin-1 .Umbilical cord blood units were obtained with IRB approval from the Carolinas Cord Blood Bank (Duke University). All methods were carried out in accordance with relevant guidelines and regulation. Informed consent was obtained for all subjects donating cord blood units to the bank. Mononuclear cells were extracted using Ficoll then counted via trypan blue to assess viability. CD3419. Samples were considered satisfactory quality for analysis if >50% viability was maintained after 5 days in culture.De-identified patient samples were obtained from the Children’s Oncology Group (COG) through the AML Biology Committee (AAML15B9-Q). Bone marrow samples containing 70% blasts or greater were from patients under 21 years of age at diagnosis prior to receiving any therapy. Samples were thawed and expanded in cytokines, SR-1, and UM-729  as describedMitochondrial complex I inhibitors rotenone and metformin (#R8875 and # D150959), NOX inhibitor diphenyleneiodonium , SERCA inhibitor thapsigargin (T9033), and STAT5 inhibitor pimozide (P1793) were purchased from Sigma. The lipophilic amine imipramine and the triphenylmethane dyes gentian violet and imipramine blue were obtained from Sigma. Imipramine blue was prepared at PCI Synthesis, Newburyport, MA. PCI is a cGMP facility which has prepared imipramine blue, and confirms the identity of imipramine blue by mass spectroscopy and NMR spectroscopy.Cells were lysed in RIPA buffer with protease and phosphatase inhibitors  for 30 minutes on ice followed by centrifugation for 10 minutes at 10,000 × g. Protein concentrations were determined using the Bio-Rad protein assay  and proteins were separated on an SDS-polyacrylamide gel followed by transfer to either a nitrocellulose  or PVDF  membrane. After blocking in 5% BSA for 1 hour, membranes were incubated in the appropriate antibody overnight and detection was by chemiluminescence  or by the Odyssey Clx imaging system (LI-COR Biosciences). Image Studio v4.0 software was used for densitometry quantification.20.For ROS detection, cells were washed twice with PBS then incubated for 30 minutes in 1 µM H2-DCF-DA  in PBS. After 2 more washes in PBS, hydrogen peroxide levels were detected by flow cytometry. For mitochondrial membrane potential, after 2 washes with PBS, cells were incubated with 5 µM JC-1  in PBS for 20 minutes, then washed 3 times with PBS prior to analysis by flow cytometryAfter 2 washes with serum-free RPMI medium, cells were incubated with serum-free RPMI with 5 µM Indo-1 AM  for 30 minutes. Cells were then washed 3 times with PBS. Flow cytometry was performed by ratiometric analysis of cytoplasmic calcium-bound vs unbound Indo-1 vs time. Analysis was performed for 30 seconds on untreated cells prior to the addition of treatment agents.The fluorescent properties of IB were utilized for localization analyses. IB is excited at 568 and emits fluorescence at >650. ER-tracker Green was purchased from Thermo-Fisher (E34250) and Golgi-ID and Lyso-ID were purchased from Enzo (ENZ-51028-K100 and ENZ-51034-0100). These dyes were chosen because they have no spectral overlap with IB. Hoechst stain provided in the Enzo kits was used for nuclear counterstain. Staining was performed according to manufacturer’s protocols. Cells were concurrently loaded with 150 nM IB. High resolution live-cell imaging was performed on the DeltaVision OMX . Post-acquisition data analysis was done using Fiji software (Fiji.sc).21.For apoptosis assay, after 2 washes in PBS, cells were resuspended in Annexin Buffer with Annexin V-PE  and DAPI, according to the manufacturers’ instructions. Cells were analyzed by flow cytometry. For cytotoxicity assay, after 48 hours of drug treatments, cells were analyzed by trypan blue exclusion assay as previously describedAll data were derived as a result of three or more independent experiments, unless stated otherwise. Student’s two tailed t-test was used to calculate p-values and values less than 0.05 were considered to be significant.3VO4), a protein tyrosine phosphatase (PTP) inhibitor restored pSTAT5 levels as measured by Western blot, implicating the liberation of PTPs by ROS inhibition as an effective method of pSTAT5 inhibition.  inhibitors  and NADPH Oxidase (NOX) inhibitors  for 4 hours, cellular levels of ROS as measured by hydrogen peroxide content in MV4–11 cells were effectively decreased. Western blot after 4 hour treatment demonstrated corresponding inhibition of pSTAT5 Fig. . Effectiion. Fig..Figure 1+ cell lines , a SERCA inhibitor which blocks Ca2+ reuptake by the ER and Golgi complex  and apoptosis. We then treated cell lines for 48 hours with TG and saw a similar sensitive and selective cytotoxicity for FLT3/ITD+ cell lines, confirming the role of cytoplasmic Ca2+  apoptosis . FLT3/ITDneg samples remained relatively unaffected  and more selective class III receptor tyrosine kinase inhibitors (TKIs) (e.g. quizartinib) have now reached early-phase clinical trials, however durable remissions with TKI monotherapy have been limited. Multiple mechanisms are responsible for acquired TKI resistance, with the most prominent being the acquisition or expansion of AML clones with the ITD as well as TKD point mutations27. Current translational and clinical efforts targeting FLT3/ITD expressing cells include the development of more robust TKIs (e.g. crenolanib) and utilization of TKIs in combination with chemotherapy backbones or following hematopoietic stem cell transplantation as a maintenance therapy28. Given the numerous proliferative and anti-apoptotic downstream pathways activated by FLT3/ITD , targeting of downstream lesions is an attractive alternative to direct inhibition of FLT3/ITD.AML with the FLT3/ITD mutation portends a poor prognosis. The mutation alone is insufficient to generate an acute leukemic transformation but induces myelo- and lymphoproliferative disease and FLT3/ITD has been validated as a therapeutic target+ AML through dual mechanisms of ROS/STAT5 inhibition as well as lysosomal Ca2+ release. Through the decreased production of NOX-derived ROS, IB potently inhibits the tyrosine phosphorylation and activation of STAT5 at doses of approximately 200–300 nM. Surprisingly, IB was highly selective against FLT3/ITD+ AML at 75–150 nM doses, which induced cytosolic Ca2+ release from lysosomes, resulting in mitochondrial outer membrane permeabilization, caspase activation, and intrinsic apoptosis29. The therapeutic strategy of cytosolic Ca2+ release was confirmed through SERCA inhibition with TG, which also resulted in potent and selective cell death of FLT3/ITD+ cells. Ca2+ release may occur via the inhibition of calcium re-uptake channels (e.g. SERCA inhibitors) or through the disruption of Ca2+-storing organelles 31. We also discovered a novel synergistic combination of IB with the STAT5 inhibitor pimozide which effectively targets FLT3/ITD+ AML while sparing cell lines without the mutation and sparing CD34+ cord blood cells. Importantly for IB monotherapy and the IB/pimozide combination, no drug resistance was observed for FLT3/ITD+ cell lines with acquired TKD mutations, indicating that IB and IB/pimozide may be able to overcome this substantial hurdle to FLT3/ITD+ AML treatment. IB also effectively induced cytotoxicity in FLT3/ITD+ primary patient samples, indicating this therapeutic strategy has potential in more heterogeneous leukemic cell populations. The dual-effects of IB cytotoxicity and the synergistic combination of IB/pimozide are attractive therapeutic strategies because of their selectivity for FLT3/ITD mutations, and there is likely less potential for development of drug resistance.The data presented here reveals that the NOX inhibitor IB induces a potent and selective cell death to FLT3/ITD32. Its downstream targets include several anti-apoptotic and pro-survival genes  that are known to be associated with oncogenic transformation33. STAT5 is downstream of many oncogenic tyrosine kinases including FLT3/ITD, BCR-ABL, JAK2V617F, KITD816V and others, making it an attractive target for anti-leukemic therapy34. Unfortunately, STAT5 inhibition as a therapeutic target has been difficult to achieve and there is lack of preclinical and clinical agents with the capability to directly target STAT5, although there are now some new compounds with advantages and some limitations37. Typically STAT5 inhibition occurs through the inhibition of upstream tyrosine kinase pathways and inhibitors to FLT3, JAKs, and BCR-ABL may lead to drug resistance.STAT5, a crucial transcription factor for hematopoiesis, is activated through tyrosine phosphorylation. Because of its broad roles in hematopoiesis and hematopoietic stem cells (HSCs), our lab and others have shown that deletion of STAT5 causes significant immune suppression, multilineage cytopenias, and stem cell dysfunction, thus far limiting the feasibility of STAT5 as a druggable target. STAT5 is classically activated through the canonical JAK-STAT pathway. However, STAT5 activation can be uncoupled from canonical JAK2-STAT5 activation39. FLT3/ITD+ AML has increased ROS through several mechanisms including the stabilization expression of the NOX subunit p22phox, increased expression of NOX4, and binding of Rac1-GTP to STAT540. One important role is the inactivation of PTPs through reversible oxidation of cysteines leading to the formation of disulfide bridges and inactivation of the enzyme41. As demonstrated here, IB treatment can restore PTP activity through inhibition of ROS leading to decreased tyrosine-phosphorylated STAT5 at nanoMolar doses. This may serve as an attractive STAT5 inhibitor not only for FLT3/ITD+ AML but more broadly for other leukemia and myeloproliferative neoplasms with constitutive STAT5 activation to be tested in future studies. Furthermore the combination of ROS inhibition with pimozide conferred a synergistic cell death at sub-therapeutic doses of each drug, demonstrating a potentially useful combination therapy.ROS was historically considered an unwanted by-product of cellular metabolism, however recent work has demonstrated several important roles of ROS as a cell signaling mediator and also roles in oncogenesis, including in AML2+ is also an important mediator of cell signaling. There exists an intimate interconnection of Ca2+-containing organelles including endoplasmic reticulum, lysosomes, Golgi, and mitochondria. The ER and mitochondria have multiple contact sites that allow transfer of Ca2+. Ca2+ release at high doses from the ER to mitochondria may trigger apoptosis through mitochondrial swelling, cytochrome C release, caspase cleavage and mitochondrial rupture. Importantly, lysosomes have recently been identified as an intracellular Ca2+ store42. Lysosomal Ca2+ efflux may also cause a robust release of Ca2+ from the ER, likely through the activation of IP3 and ryanodine receptors on the ER44. This may explain the robust Ca2+ release caused by IB, which is similar in intensity to that of the SERCA inhibitor thapsigargin. Peshakov et al. confirmed a selective cytotoxic and anti-proliferative effect of Ca2+ overload and caspase-induced apoptosis in AML cells using a combination of curcumin and carnosic acid, however the authors did not test AML cells with the FLT3/ITD mutation, so it is unknown if this strategy has greater effects on that subset of AML45.Like ROS, Ca+ AML cell death that remain to be determined. It is unclear at this time why IB would preferentially target the lysosome, however we hypothesize that this is due to “lysosomal sequestration”, in which hydrophobic amines such as imipramine accumulate in highly acidic environments (i.e. lysosomes) where they become protonated and are unable to diffuse out of the organelle46. This mechanism would not be specific to FLT3-ITD+ AML cells. If this is true, then IB must possess a unique property to induce lysosomal Ca2+ release as compared to its pro-drug imipramine, which did not induce a cytoplasmic Ca2+ release in our experiments. Another important issue is the question of IB’s selectivity for FLT3/ITD+ cells. One possibility is that mitochondria are more sensitive to Ca2+ overload compared to FLT3/ITDneg AML cells. In addition to STAT5 inhibition, pimozide, a neuroleptic agent, also induces multiple metabolic perturbations including blocking T-type calcium channels which may be responsible for synergy with IB. T-type calcium channels can alter cell cycle dynamics through the action potential generated by a calcium spike. When ER calcium release is accelerated by IB treatment, this hyperpolarization would normally be associated with relieved inactivation of T-type channels. However, pharmacologic inactivation of T-type channels with pimozide could prevent cell proliferation that would normally proceed through calcium mediated p53 inactivation, activation of p21, and release of CDK2 to promote S phase transition. Finally, a safe and efficacious in vivo therapeutic approach is needed. Ca2+ release as a therapeutic target is currently being tested by using a pro-drug of the SERCA inhibitor thapsigargin  in clinical trials for prostate cancer and hepatocellular carcinoma47. In this study, we have demonstrated that the imipramine/triphenylmethane conjugate agent imipramine blue possess potent and selective cytotoxic effects alone and in combination with pimozide for FLT3/ITD+ AML. The potential clinical utility of this combination remains to be determined. IB acts through a dual mechanism involving STAT5 inhibition and lysosomal Ca2+ release at nM concentrations. IB thus represents an exciting new agent and chimeric prototype drug with promise for future development in treating high risk subsets of AML.There are important implications regarding the underlying mechanism of IB-induced FLT3/ITDSupplemental Information