TT) system is essential to normal Ca2+ cycling and cardiac function. We explored the relationship between the progressive disruption of TTs and resulting Ca2+ cycling during the development of heart failure (HF). Confocal imaging was used to measure Ca2+ transients and 2‐D z‐stack images in left ventricular epicardial myocytes of intact hearts from spontaneously hypertensive rats (SHR) and Wistar‐Kyoto control rats. TT organization was measured as the organizational index (OI) derived from a fast Fourier transform of TT organization. We found little decrease in the synchrony of Ca2+ release with TT loss until TT remodeling was severe, suggesting a TT “reserve” characterized by a wide range of TT remodeling with little effect on synchrony of release but beyond which variability in release shows an accelerating sensitivity to TT loss. To explain this observation, we applied a computational model of spatially distributed Ca2+ signaling units to investigate the relationship between OI and excitation‐contraction coupling. Our model showed that release heterogeneity exhibits a nonlinear relationship on both the spatial distribution of release units and the separation between L‐type Ca2+ channels and ryanodine receptors. Our results demonstrate a unique relationship between the synchrony of Ca2+ release and TT organization in myocytes of intact rat ventricle that may contribute to both the compensated and decompensated phases of heart failure.A highly organized transverse‐tubule ( Signaling occurs when Ca2+ entry due to an LCC opening initiates a local rise in Ca2+ concentration which induces a coordinated release of the local RyR cluster. This signaling is mediated via Ca2+‐induced‐Ca2+ release and leads to local release events referred to as Ca2+ sparks  network of a ventricular myocyte is essential for conducting the action potential from the surface of the cell into the cell center  Ca2+ release. In order to investigate this relationship in situ, we compared TT organization and Ca2+ release characteristics in individual ventricular myocytes in intact hearts of spontaneously hypertensive rats (SHRs) and Wistar‐Kyoto rats (WKYs) between 4 and 22 months of age. All animals were treated in accordance with the protocol approved by the Institutional Animal Care and Use Committee at Northwestern University, as dictated by National Institute of Health guidelines.The purpose of this study was to examine the relationship between TT remodeling and changes in Ca2) at a stable pressure of 60–100 mmHg. Re‐circulation was initiated and the heart was stained with di‐4‐ANEPPs (3–5 μmol/L) and Fluo‐4AM and contraction was abolished with a combination of cytochalasin‐D (60 μmol/L) and belbbistatin (25 μmol/L). z‐stack images showing the organization of TTs within subepicardial left ventricular myocytes were acquired and two‐dimensional (2‐D) images were analyzed to obtain a value representative of the level of organization within individual cells. An organizational index (OI) value was determined using ImagJ software to select the entire intracellular area, excluding the cell membrane, then further analyzed using customized Matlab software to produce an output value representative of the level of organization within the cell, also taking disorganization – including axial tubules – and any background signal into account. Each cell image was converted to a fast Fourier transform which served as the basis for calculation of OI as the fraction of the organized TT signal compared to the total of organized and disorganized TTs. OI values were measured in a range of approximately 0.4–0.95, representing low to high levels of TT organization, respectively  was used to wash the heart for 20 min. Recirculation was restored and included cytochalasin‐D and blebbistatin. Basal pacing at a basic cycle length (BCL) of 700 msec was established via the placement of silver needle electrodes in the apex of the left ventricle. To allow for comparison of TT organization to intracellular Ca2+ transients, longitudinal linescan images for each myocyte were recorded for analysis after a 2‐D image for TT measurements was recorded during basal pacing and during rapid pacing. Measurements of F/F0 were obtained using a locally developed Matlab program for Ca2+ transient measurements in which F/F0 was measured for each pixel. In these experiments, the main parameter of interest was the time of rise to 50% of the peak of the transient (TR50), as a measure of rate of SR Ca2+ release  with no sudden loss of TT density indicating a threshold effect with disease progression that would indicate that some minimal TT opening size would somehow limit dye access to the TTs. As far as uniformity of Ca2+‐sensitive dye distribution is concerned, it is always quite uniform in normal cells and there is no reason to think that it distributes differently in diseased myocytes. This idea is supported by our observations  along the entire cell length, which was used to calculate the TR50 at each interval. The linescan images chosen for each cell were obtained at the fastest pacing in which transients had a reasonable signal‐to‐noise ratio . The overall range of cycle lengths used in this study was 150–400 msec depending on the severity of disease. For the analysis, two representative transients were averaged for each cell, with cycle lengths chosen to exclude high levels of background noise, spontaneous waves, and Ca2+ alternans. After adjusting the baseline, individual sarcomeres were culled if they had an unacceptably low signal‐to‐noise ratio, were highly divergent from the mean, or had double peaks. Subsequently, the program converts F versus T to F/F0 versus T. In order to avoid over‐smoothing of Ca2+ transients, the program incorporated a default low‐pass filter (50 Hz). The analysis was then completed and an output of various parameters was exported to an excel file upon verification that the program correctly identified the initiation and peak of the mean transient and the transient of an individual sarcomere. TR50 values were calculated for each of the two sequential transients chosen for an individual cell by calculating the difference between the initiation time of the transient and the time to 50% width for each sarcomere. The standard deviation of these values was recorded as the TR50 HI (heterogeneity index)  where nx denotes the longtitudinal direction, ny is the width of the cell, and nz is the height. The subcellular compartments in the model are: (1) The dyadic junction, with concentration cpn, where a few LCCs on the cell membrane are in close proximity to a cluster of ∼100 RyR channels attached to the junctional SR (JSR). In this model the distance between the LCC channels and the RyR cluster is denoted by the variable h, which will be varied to model various states of HF  The bulk myoplasm, with concentration cin, which characterizes the volume of space into which Ca2+ diffuses before being pumped back into the SR via SERCA . (4) The junctional SR, with concentration cjsrn, that is the portion of the SR network that is positioned close to the cell membrane; (5) The network SR (NSR), with concentration cnsrn, which represents the bulk SR network that is spatially distributed in the cell. In this study, our cardiac cell model will consist of 60 planes representing Z‐planes, where each plane contains an array of 20 × 20 regularly spaced compartments  clamp. Our AP clamp is taken to have the simple functional form  where a = 2/3.In this study, we consider the dynamics of Ca2+ release during normal EC coupling, there has been a great deal of interest in how TT remodeling affects Ca2+ transients, particularly SR Ca2+ release. Figure 2+ transient measurements were recorded along the length of the cell in subsequent panels. Figure 2+ from the cytoplasm. The white vertical line indicates the initiation time for each transient and the fact that there is virtually no deviation in fluorescence intensity along the line indicates the uniformity of release along the cell length. Since we would expect that any heterogeneities in SR Ca2+ release would be exaggerated during rapid pacing, we also repeated the pacing protocol at a fast rate in this heart  and WKY control rats (open circles). Since WKY rats are considered normal controls, these served to provide a basis for comparison with myocytes studied in the SHR hearts during the development of hypertensive disease. The inset at the left of Figure 50 HI versus OI, the TR50 HI (variability in Ca2+ release) was fitted to both linear and exponential relationships during both basal and rapid pacing in order to determine the nature of the reliance of SR Ca2+ release on TT organization at both slow and rapid heart rates. Note that the goodness of fit is indicated by both the r and r2 values shown on the summary graphs. We found that there was an overall increase in variability in SR Ca2+ release with decreasing OI during basal pacing  where nx denotes the coordinate along the longitudinal direction. In the same panel (right) we indicate the local Ca2+ concentration in the cytosol at two distinct sites in the cell, and in the top trace we show the cytosolic Ca2+ concentration averaged over the whole cell. In Figure q = 0.3. In this scenario we observed that the timing of Ca2+ release is substantially more heterogeneous. The global signal (top) appears more blunted as the rise time of the Ca2+ transient is increased due to asynchronous Ca2+ release. Consequently, the local cytosolic Ca2+ concentration exhibits heterogeneities due to the failure of Ca2+ release in a large fraction of dyadic junctions. In Figure 50HI as a function of q in order to quantify the relationship between release heterogeneity and the spatial distribution of Ca2+ signaling. To compute TR50HI we measured the time it takes for cin to reach half of its maximum. This quantity is computed along the center of the cell and the standard deviation was computed from that population of dyadic junctions. We then averaged this quantity over 100 independent simulations of the whole cell. These computations were performed for cycle lengths of 300 and 700 msec and show that TR50HI increases more rapidly with decreasing q at 300 msec. Our results indicate that TR50HI increases in a nonlinear fashion as q is decreased, which is consistent with our experimental observations shown in Figure To explore the relationship between t‐tubule organization and Ca2+ channels during HF. This is in contrast to our previous model where signaling was completely eliminated at a fraction of junctions. To simulate the spatial variability that is expected in a cardiac cell we picked the spacing h from a Gaussian distribution of the form P(h)=(1/2πσ2)exp[−(h−h¯)2/2σ2], where h¯ is the average spacing between channels, and σ denotes the standard deviation. Using this approach we modeled the progression to HF as an increase in the average spacing h¯. Under normal conditions we fixed h¯=10nm, and model HF progression by increasing h¯ to 30 nm. For simplicity we kept our standard deviation fixed at σ=2nm. In Figure 2+ concentration cin during four beats at BCL=300msec. In this case the spacing was fixed at h¯=30nm. In this simulation we kept q=1.0 so that under normal conditions subcellular Ca2+ release is synchronized and similar to that shown in Figure 2+ release was highly heterogeneous since the coupling fidelity between LCC and RyR is substantially reduced. To summarize this data, in Figure 50HI versus the average spacing h¯ at BCL=300msec. Here, we found that TR50HI remains flat for channel spacings in the range 10–20 nm but then increases substantially as the spacing is increased further. Thus, as in the previous model, Ca2+ release variability depends on channel spacing in a nonlinear manner.The spatial distribution of LCC and RyR channels under different stages of HF progression is not known in detail. Thus, we applied our model to investigate how release variability depends on the spatial arrangement of LCCs under a broad range of conditions. In this section we considered an alternative model for HF in which the spacing between LCC and RyR channels was varied in order to model the increased separation between Ca2+ release and t‐tubule organization we analyzed specifically the dependence of Ca2+ signaling on the average distance h¯ between LCC and RyR channels. To quantify this relationship we measured the probability that a spark is induced at a dyadic junction as function of the distance h¯. To compute this quantity we drove our cell with an AP clamp and computed the probability that a Ca2+ spark occurs in that junction during the 300 msec duration AP. To compute this probability we ran 1000 independent simulations and computed the number of times a spark occurred at a given junction. In Figure Ps as a function of the average spacing between channels h¯. Our result shows that Ps is a threshold function of h¯ with Ps∼1 for h¯<20nm and where Ps decreases to Ps∼0 when h¯ is increased above 20 nm. This result suggests that the signaling fidelity between LCCs and the nearby RyR cluster drops substantially once the spacing between channels is increased above a threshold. This nonlinear relationship indicates that TR50HI will be insensitive to h¯ below the threshold (∼20nm) since the number of sparks recruited in the cell remains fixed.In order to understand the mechanism for the nonlinear relationship between Ca2+ transient characteristics. This decreasing state of TT organization during the development of HF has an impact on EC coupling, and consequently can affect overall heart function. A diminished TT network has been characterized as a contributing factor to phenotypic changes seen during HF, such as Ca2+ transients that are slower and have a smaller amplitude; however, these changes have been recently attributed to TT remodeling, rather than detubulation  and perhaps as problematic, causing some cell regions to be passively stretched while others are contracting. Our results as well as others  but that when some critical degree of TT remodeling is achieved, the result is a progressively more severe fractionation of Ca2+ release along the cell length. Our simulation studies also demonstrate that the accelerating slowing in release occurs because of the 3‐D volume considerations that determine local [Ca2+] at the cytoplasmic side of the RyR as the distance to the closest TT and resident LCCs increases in a highly nonlinear fashion as OI decreases.Based on our findings, the progression in TT remodeling does not appear to significantly affect EC coupling until a critical extent of remodeling occurs at least during rapid pacing, where nonuniformities in SR Ca2+ cycling. For example, it is well known that mitochondrial Ca2+ handling is altered during disease development. In addition, given the importance of nuclear Ca2+ fluxes in normal cell function, it is possible that changes in nuclear Ca2+ handling might also affect EC coupling, especially during long‐term changes in protein regulation and resulting myocyte function, often referred to excitation‐transcription coupling.It is also important to acknowledge that there are other changes that occur during hypertrophic remodeling and HF development that could contribute to abnormal Ca2+ cycling in order to uncover the relationship between TT remodeling and EC‐coupling. To model the progression to HF we first varied the fraction of dyadic junctions where Ca2+ signaling occurs between LCC and RyR channels. In this case we found that TR50HI increased in a nonlinear fashion as the fraction of signaling junctions q was decreased. This result is consistent with our experimental findings which show that TR50HI increases with decreasing OI with an effect that is accelerated at smaller OI. Using an alternate approach we have also modeled HF as an increase in spacing between LCC and RyR channels. In particular, we find that the probability that an LCC opening induces a Ca2+ spark depends on the spacing h¯ in a nonlinear sigmoid fashion. This threshold relationship occurs because an increase in channel spacing leads to a larger dyadic junction volume, which causes a decrease in the amount of Ca2+ sensed by the cytosolic side of the RyRs. Since RyRs transition from the closed to the open state in a Ca2+‐dependent manner, the cooperative response of an array of RyR receptors is highly sensitive to the local Ca2+ concentration in the dyadic junction. Thus, an increase in dyadic junction volume leads directly to a loss of signaling fidelity between LCCs and RyRs. A consequence of this relationship is that variations of the average channel spacing in the range h¯<20nm will not influence the whole cell Ca2+ release since the function Ps is constant in this range. However, when the average junction spacing increased beyond h¯=20nm then Ca2+ signaling in a large population of junctions will fail. This result may explain our observation that Ca2+ release heterogeneity is small for large OI but increases in a nonlinear manner when the OI is decreased.In this study we applied a detailed computational model of Ca2+ signaling channels. Our numerical findings reveal that both these effects lead to a marked increase in release variability which depends nonlinearly on both the fraction of signaling sites and the spatial distance between sites. Thus, our experimental findings shown in Figure 2+ release heterogeneity is highly sensitive to the changes in cell architecture that are expected during progression to HF.In a cardiac cell we expect that HF progression will lead to multiple changes to the cell architecture and even ion channel distribution and kinetics. Thus, it is likely that HF involves both the loss in signaling within a population of channels, and with a reduction in signaling fidelity due to an increased separation between Ca2+ cycling within the cardiac myocyte. However, there may also be a threshold up to which TT remodeling may occur without resulting in the development of dyssynchronous Ca2+ release and disrupted CICR. Our numerical simulations reveal that this behavior can be due to both a reduced density of active signaling junctions, and also dependence of Ca2+ signaling on the spatial separation between ion channels. In particular, we find that the degree of remodeling has to be above a threshold such that signal transduction fails within a large population of dyadic junctions. It is the failure of signaling that causes the increased variability in Ca2+ cycling at the more advanced stages of TT remodeling. Finally, our results from these theoretical and experimental observations suggest the idea that there is a TT reserve, which allows TT loss without interfering with Ca2+ release. This feature of the TT – Ca2+ release relationship may serve as a compensatory mechanism to maintain efficient myocardial performance during the progressive changes in cell ultrastructure and function that occur during disease development. However, as TT remodeling reaches critical levels, the high sensitivity of Ca2+ release desynchronization to TT disruption is a likely contributor to diastolic and systolic dysfunction and an overall decline in myocardial performance. Thus, it may ultimately be the TT reserve that is critical for efficient EC coupling at the cellular level during disease progression by maintaining the synchronization of Ca2+ release throughout the cell.Our results demonstrate that the TT remodeling that takes place during the transition from hypertension to heart failure are responsible for the increased variability in CaNone declared.


Chronic urticaria is a spontaneous or inducible group of diseases characterized by the occurrence of wheals  for more than 6 weeks. These are rather frequent conditions that may severely affect patients’ quality of life and sometimes represent a challenge for doctors as well. The causes of chronic urticaria are still poorly defined, although there is growing evidence that different biologic systems including immunity, inflammation, and coagulation may take part in the pathomechanism eventually leading to mast cell and basophil degranulation and hence to wheal formation. This review will discuss the main findings that are (slowly) shedding light on the pathogenesis of this disorder. While there is little doubt that the release of histamine by mast cells and basophils represents the final stage in the pathomechanisms involved in this group of diseases, there is still some uncertainty about the factors activating these cells and eventually inducing their degranulation. Several lines of evidence indicate that different biologic systems like immunity, inflammation, and coagulation may contribute to a common mechanism leading to wheal generation. This short commentary will summarize the current knowledge about the mechanisms theoretically leading to histamine release in chronic urticaria.Chronic urticaria, defined as the recurrent occurrence of wheals with or without angioedema for longer than 6 weeks, can be classified into spontaneous or inducible based on the absence or presence of identifiable physical stimuli able to elicit the skin lesionsThe bulk of studies carried out during the last 30 years have shown that different biologic systems  may concur to produce the wheal and flare lesions as well as the angioedema that characterize chronic urticaria. For clarity, these biologic systems, which are largely linked with each other, along with the main cell lines involved in either their activation or their behavior as effector cells will be considered singularly in the following subsections.2; this led them to conclude that chronic urticaria is characterized by circulating histamine-releasing factors. Since then, a positive autologous serum skin test (ASST) has been considered as a marker of “autoreactivity”. Thisin vivo test is quite easy to perform and seems sufficiently specific for chronic urticaria, as normal subjects do not show any skin reactivity3, although it may score positive in inducible urticarias such as cold urticaria4 as well as in some other “allied” conditions, such as the multiple drug allergy syndrome and multiple hypersensitivity to non-steroidal anti-inflammatory drugs6. However, the ASST scores positive in only about one half of chronic urticaria patients8, leaving a large proportion of patients who do not show any autoreactivity despite their maybe severe and ongoing disease.More than 30 years ago, Grattan and co-workers demonstrated that the intradermal injection of autologous serum induces a wheal-and-flare reaction in a large proportion of patients with chronic urticaria12.Following the first observation on skin reactivity to autologous serum that remains a cornerstone in urticaria research, the second important step in the definition of the pathogenic mechanism was the demonstration of histamine-releasing autoantibodies about 20 years ago, suggesting an autoimmune origin. This was based on the detection of circulating and functionally active IgG autoantibodies directed against either the high-affinity IgE receptor (FcεRI) present on both mast cells and basophils (in most cases) or membrane-bound IgE (in a minority of patients)13. Furthermore, both types of autoantibodies have been detected with similar frequency in chronic urticaria patients and controls without chronic urticaria13. Nonetheless, in more recent times, the autoimmune pathogenesis hypothesis has received new, indirect but strong support from the detection of circulating autoreactive CD4+ T cells that proliferate in response to FcεRI in >50% of patients with chronic urticaria examined14.Although this was a fascinating explanation for the ongoing histamine release, several subsequent studies found that this mechanism is in fact viable in only a minority of patients15 and that basopenia is directly related to disease severity. Recent studies found that chronic urticaria blood basophils show a reduced surface expression of CRTH2 (the chemoattractant receptor–homologous molecule expressed on TH2 cells), the receptor for prostaglandin D2 (PGD2). It is therefore possible that decreased CRTH2 levels on basophils in patients with chronic urticaria are due toin vivo PGD2-mediated activation, suggesting the engagement of CRTH2 in patients with chronic urticaria16. Furthermore, basophils from chronic urticaria patients show different phenotypes  that seem independent of the presence of IgG autoantibodies to IgE or FcεRI and seem stable during active disease17, and their IgE receptor-mediated degranulation is enhanced during disease remission17.Although there is little doubt that the mast cell is the main final effector cell in chronic urticaria, basophils and their role in the disease have received much attention during the last decade. It is well known that active chronic urticaria is characterized by basopenia due to the elective recruitment of circulating basophils into the skin lesionsin vitro tests aiming to diagnose autoimmunity. The first test used was the time-consuming and cumbersome basophil histamine release assay (BHRA), first introduced in the 1960s and subsequently marketed as a more convenient testing kit18; on the other hand, the observation that chronic urticaria sera were able to induce the surface expression of basophil activation markers such as CD6319 led to the investigation of the diagnostic usefulness of the basophil activation test (BAT) measuring CD63 and CD203c surface expression by flow cytometry21. However, in view of the fact that no more that 50% of chronic urticaria patients have an autoimmune disease and of the intrinsic variability of basophil releasability, the clinical usefulness of these tests has been questioned22 and they have not reached the status of routine investigation for this disease.The finding that at least a proportion of chronic urticaria patients show autoimmune phenomena targeting mast cells and basophils has led to the investigation of the diagnostic accuracy of basophil-based23. This pathogenic mechanism could be classified as “auto-allergic” and autoimmune at the same time. However, the proportion of patients in whom such IgE autoantibodies can be detected is limited, and TPO-specific IgE autoantibodies are generally associated with the more common TPO IgG autoantibodies that characterize patients with Hashimoto’s thyroiditis which are found in a minority of patients. However, research in this field is very active, and it cannot be excluded that other “auto-allergens” will be detected in larger proportions of patients with chronic urticaria in the near future24.Another important step was the discovery of specific IgE for thyroid peroxidase (TPO) for dsDNA and ssDNA, which has recently provided a different mechanism potentially able to promote the survival, proliferation, and activation of mast cells25; patients with both chronic urticaria and MS were older, had a more severe disease, had higher levels of inflammation markers , and, interestingly, more frequently scored negative on the ASST than those without MS. In another Asian-based population study, chronic urticaria was more frequent among subjects with a prior diagnosis of hyperlipidemia26. Recently, chronic urticaria patients were found to show increased levels of a series of adipokines  but lower levels of adiponectin27. The findings with IL-6 are in keeping with a previous study28 showing an association between IL-6 and disease severity.It is increasingly clear that chronic urticaria is characterized by a systemic pro-inflammatory state. The studies showing increased levels of different single markers of inflammation  that have appeared in the medical literature over the years will not be reviewed in detail here. Recent studies showed the co-existence of chronic urticaria and metabolic syndrome (MS) in a Korean population29 has prompted investigation of the coagulation system in patients with chronic urticaria. Specific studies have shown that the coagulation cascade is activated in chronic urticaria and involves the extrinsic pathway first and the intrinsic pathway second32. The process seems to be triggered by the hyper-expression of tissue factor by activated eosinophils33 and parallels disease activity. IgG autoantibodies to the low-affinity IgE receptor FcεRII, which is present on the membrane of eosinophils, have been detected in quite a large proportion of patients (about 65%) with chronic urticaria34 and might be involved in the activation of such cells with subsequent release of major basic protein and other mediators causing mast cell degranulation. The activation of the coagulation cascade might potentially play a relevant pathogenic role if one considers that thrombin can markedly increase the vascular permeability and is a potent inducer of mast cell degranulation in experimental models. The activation of the coagulation cascade occurs in other skin disorders characterized by an increase of vascular permeability, such as angioedema due to C1-inhibitor deficiency and bullous pemphigoid36, but also in a wide spectrum of systemic diseases, such as disseminated intravascular coagulation37, deep venous thrombosis38, and endotoxemia39, all prothrombotic conditions that are not characterized by urticaria or edema. However, the activation of coagulation should not necessarily be considered a non-specific phenomenon but rather a common intermediate step occurring in the pathophysiology of different diseases. Thus, the involvement of coagulation is another piece of truth unable to explain the whole story.The observation that the autologous plasma skin test (APST) may score positive in some ASST-negative patients40. This fact suggests a pathomechanism that bypasses the IgE receptor mechanism. In subsequent studies, we showed that low-molecular-weight circulating factors (molecular weight about 30 kDa) may be involved in the activation of mast cells in chronic urticaria patients41, thus confirming very old data by Grattan and co-workers42.Quite recently, we were able to show that sera from patients with chronic urticaria induce significant activation of mast cells lacking the high-affinity IgE receptor, irrespective of the autoreactivity status or of the presence/absence of circulating autoantibodies43. It is easy to speculate that different pathogenic mechanisms might underlie the different patterns of response. Several potential markers of response to omalizumab treatment have been investigated so far. In one study looking at the potential use of a CD203c assay  as a biomarker of responsiveness to omalizumab, it was found that the lack of basophil CD203 upregulating activity was associated with a higher likelihood of response to the drug44. This observation seems to agree with the results of another study finding that a positive response to omalizumab was associated with a negative histamine release assay45. On the other hand, Gericke and co-workers found that a positive BHRA, which was associated with a positive ASST, was frequently observed in “slow responders” to omalizumab43. Another interesting observation is that omalizumab has been found to be effective not only in the spontaneous form but also in several inducible forms of chronic urticaria47. This suggests that the drug interferes with a common pathogenic mechanism underlying a proportion of both spontaneous and inducible urticarias. Notably, the fact that the symptomatic cold urticaria, a frequent chronic inducible urticaria, can be passively transmitted to normal subjects via the serum of affected individuals has been known for many years. Since the mechanism of action of omalizumab in urticaria is all but elucidated, the next few years will probably shed some light on these questions.Omalizumab was recently licensed for the treatment of chronic urticaria. All three phase III studies as well as the real-life studies carried out in large numbers of patients have demonstrated that this humanized monoclonal IgG antibody to IgE is highly effective in a large proportion of patients with chronic urticaria who don’t show any clinical response to second-generation antihistamines given at doses even higher than licensed ones. The pattern of response to this drug has indirectly led to the categorization of patients with severe disease into “fast responders” , “slow responders” , and “non-responders” (about 10–20% of cases)The pathogenesis of chronic urticaria is probably characterized by a multiplicity of mechanisms, including autoimmunity, auto-allergy, and coagulation, each of which may carry different weight in each patient. Much research is in progress, and the overall perception is that the solution to the puzzle is not too far away.


In the latter, voltage‐induced Ca2+ release (VICR) is regarded as the dominant mechanism facilitating contractions. Any role of the SOICR in the regulation of cytoplasmic Ca2+ concentration ([Ca2+]i) and its dynamics in skeletal muscle in vivo remains poorly understood. By means of in vivo single fiber Ca2+ microinjections combined with bioimaging techniques, we tested the hypothesis that the [Ca2+]i dynamics following Ca2+ injection would be amplified and fiber contraction facilitated by SOICR. The circulation‐intact spinotrapezius muscle of adult male Wistar rats (n = 34) was exteriorized and loaded with Fura‐2 AM to monitor [Ca2+]i dynamics. Groups of rats underwent the following treatments: (1) 0.02, 0.2, and 2.0 mmol/L Ca2+ injections, (2) 2.0 mmol/L Ca2+ with inhibition of ryanodine receptors (RyR) by dantrolene sodium (DAN), and (3) 2.0 mmol/L Ca2+ with inhibition of SR Ca2+ ATPase (SERCA) by cyclopiazonic acid (CPA). A quantity of 0.02 mmol/L Ca2+ injection yielded no detectable response, whereas peak evoked [Ca2+]i increased 9.9 ± 1.8% above baseline for 0.2 mmol/L and 23.8 ± 4.3% (P < 0.05) for 2.0 mmol/L Ca2+ injections. The peak [Ca2+]i in response to 2.0 mmol/L Ca2+ injection was largely abolished by DAN and CPA  supporting dependence of the [Ca2+]i dynamics on Ca2+ released by SOICR rather than injected Ca2+ itself. Thus, this investigation demonstrates the presence of a robust SR‐evoked SOICR operant in skeletal muscle in vivo.In contrast to cardiomyocytes, store overload‐induced calcium ion (Ca In skeletal muscle myocytes, contraction and relaxation are evoked by [Ca2+]i increase and decrease, respectively. The primary Ca2+ releasing systems from the sarcoplasmic reticulum (SR) are action potential‐induced calcium release  (Schneider 2+‐induced Ca2+ release (CICR) (Endo 2+ release (SOICR)  Endo  and stor2+ releasing channel on the SR membrane. In mammals, three RyR isoforms exist  with RyR1 being dominantly expressed in skeletal  is a Ca Fabiato . In mark Fabiato . Because2+release via the SR , or indirectly via the depletion of Ca2+ from the SR by inhibiting SERCA with cyclopiazonic acid (CPA).Because RyR regulation is extremely sensitive to the cytosolic and luminal environments, and changes thereof  were used in this study. Rats were maintained on a 12:12‐h light/dark cycle and received food and water ad libitum. All experiments were conducted under the guidelines established by the Physiological Society of Japan and were approved by the University of Electro‐Communications Institutional Animal Care and Use Committee. The rats were anesthetized using pentobarbital sodium (60 mg/kg i.p.), and supplemental doses of anesthesia were administered as needed. At the end of the experimental protocols, animals were killed by pentobarbital sodium overdose.Male Wistar rats  equilibrated with 95% N2‐5% CO2 and adjusted to pH 7.4, at 37°C. The fluorescent Ca2+ indicator Fura‐2 AM  was dissolved in dimethyl sulfoxide (DMSO) and Pluronic F‐127 and dispersed into KHB solution at a final concentration of 40 μmol/L. The muscles were incubated in Fura‐2 AM/KHB solution for 60 min on a 37°C hot plate . After incubation, muscles were rinsed with dye‐free KHB solution to remove nonloaded Fura‐2 AM.All experimental techniques, including the spinotrapezius muscle preparation, were performed, as described previously  was selected, and bright‐field images were captured. Thereafter, 340 and 380 nm wavelength excitation light was delivered using a Xenon lamp equipped with appropriate fluorescent filters, and pairs of fluorescence images were captured through the 510 nm emission wavelength filter for ratiometry . After ensuring that the spinotrapezius muscle was not grossly damaged and supported robust capillary blood flow, a sampling area . Subsequently, using a microinjector , a single muscle fiber was microinjected with 0.02, 0.2, and 2.0 mmol/L Ca2+ solution by microinjection at 35 psi  for 1 sec (∼0.78 × 10−3 μL)  and the Ca2+ solution (CaCl2・2H2O: 10 mmol/L). Criteria for successful injection included an unchanged [Ca2+]i in adjacent muscle fibers. Fluorescence images were captured by a high‐sensitivity CCD (charge‐coupled device) digital camera  using image‐capture software  for 60 sec after injection. The scan requirements were set at 300–800 msec/image. We confirmed that insertion of the capillary micropipette itself and injection of Ca2+‐free solution did not induce any [Ca2+]i change. The specific region of interest  for the Ca2+ measurement was set at a distance of 40 μm from the injection point. All experiments were performed with the rat's body temperature maintained at 37–38°C with the exteriorized muscles mounted on the 37°C glass hotplate.Capillary micropipettes were generated with a tip of 5 2+ release channels  and the pharmacological action was confirmed by constructing dose–response curves. Based upon these studies, following Fura‐2 incubation and rinse, DAN (100 μmol/L) or CPA (100 μmol/L) was applied for 5 min before the start of experiments. Subsequently, a single muscle fiber was microinjected with either of the 0.02, 0.2, and 2.0 mmol/L Ca2+ solution.Pharmacological agents DAN and CPA were purchased from Sigma‐Aldrich . DAN inhibits single ryanodine receptor (RyR) Ca2+ injection. The caffeine solution (100 μmol/L) was injected into each single muscle fiber both with and without DAN conditions.This protocol was the same as for the Caμm from the ROI. The resolution was 0.73 μm per pixel.The muscle fiber shortening  was calculated frame‐by‐frame using autologous fluorescent landmarks, located close to the injection point and thus ~40 P < 0.05 (two‐tailed).All experimental data are expressed as means ± SE. All statistical analyses were performed in Prism version 6.0 . Comparisons among different groups were analyzed by either one‐way or two‐way ANOVA followed by Tukey's multiple‐comparison test. The level of significance was set at 2+‐free and 0.02 mmol/L Ca2+ injections which caused no detectable [Ca2+]i change, 0.2 and 2.0 mmol/L injections elicited significant increases in [Ca2+]i, indicated by 9.9 ± 1.8% and 23.8 ± 4.3% increases in R/R0 within 1–2 sec after injection, respectively . With CPA‐induced SERCA blockade, the [Ca2+]i increase after injection was substantially blunted compared with the unblocked control condition .The functional concentrations of each inhibitor (DAN and CPA) were verified under in vivo conditions before Caion Fig. A. As evi2+]i increase above baseline  and substantial  fiber shortening as seen in Figure P < 0.03, vs. Ca2+ alone condition) and CPA  on shortening fiber length. Specifically, the mean shortening length was significantly reduced in both DAN (25.1 ± 6.6 μm) and CPA (14.4 ± 4.0 μm) compared with 2.0 mmol/L Ca2+ alone  Ca2+ itself modulates RyR channel activity  and inhibitory at extremely high Ca2+ (~1 mmol/L) (Meissner 2+ currents by disrupting communication between RyR1 and Ca(V)1 which provides the only treatment for malignant hyperthermia resulting from RyR1 mutations  . This realization tempers confidence in translating ex vivo findings to the in vivo environment. Notwithstanding this consideration, the present investigation has extended previous observations regarding [Ca2+]i control and represents the first investigation to provide evidence for SOICR function in vivo.CaMeissner . DAN inh2+ release from the SR is inhibited, either directly via the blockade of RyR (DAN), or indirectly via the depletion of Ca2+ from the SR (CPA). This hypothesis was supported by the almost complete abolition of [Ca2+]i increases by DAN and CPA applied separately.We hypothesized that SOICR function would be abolished when Ca2+ content exceeds some threshold level. SOICR has been observed for RYR1 in skeletal muscle myocytes as well as RyR3 in smooth muscle  this would acutely increase net SR Ca2+ stores. Consequently, local Ca2+ release following Ca2+ injection would be triggered by the SOICR system and contribute to the contractile behavior evident in Figure 2+ bound immediately to troponin and directly facilitated contraction.SOICR has been observed in RyR2 containing cardiomyocytes  has been shown to inhibit rather than facilitate RyR function  and the resultant free [Ca2+]i is expected to be reduced immediately by intramyocyte buffering mechanisms and dilution within the far larger cytosolic volume.The i and significant myocyte shortening; both of which were diminished substantially by RyR blockade or SERCA inhibition.In conclusion, in the absence of VICR, we demonstrated, for the first time, evidence for SR SOICR in in vivo single mammalian skeletal muscle myocytes. The SOICR demonstrated induced elevated [CaNone declared.


NPM1, FLT3ITD, DNMT3A, NRAS, TET2, RUNX1, MLLPTD, ASXL1, BCOR, PHF6, CEBPAbiallelic, IDH1, IDH2R140, and IDH2R170, with an emphasis on their relevance to the leukemic stem cell compartment. We have reviewed the available literature and TCGA AML databases (2013) to highlight the potential role of stem cell regulating factor mutations on outcome within newly defined AML molecular subgroups.Adult acute myeloid leukemia (AML) clinically is a disparate disease that requires intensive treatments ranging from chemotherapy alone to allogeneic hematopoietic cell transplantation . Historically, cytogenetic analysis has been a useful prognostic tool to classify patients into favorable, intermediate, and unfavorable prognostic risk groups. However, the intermediate-risk group, consisting predominantly of cytogenetically normal AML (CN-AML), itself exhibits diverse clinical outcomes and requires further characterization to allow for more optimal treatment decision-making. The recent advances in clinical genomics have led to the recategorization of CN-AML into favorable or unfavorable subgroups. The relapsing nature of AML is thought to be due to clonal heterogeneity that includes founder or driver mutations present in the leukemic stem cell population. In this article, we summarize the clinical outcomes of relevant molecular mutations and their cooccurrences in CN-AML, including Current AML induction therapies are successful in obtaining complete remission in approximately 75% of young (age < 60 years) de novo AML patients; however, most are destined to relapse. This clinical behavior suggested the presence of an underlying leukemic cell population responsible for the relapsing nature of AML despite the attainment of a complete remission through induction chemotherapy. The existence of leukemic stem cells in AML capable of recapitulating the disease was firmly established by transplant experiments utilizing immunocompromised mouse models two decades ago [ FLT3, NPM1, and CEBPA mutations has added a great deal to the prognostic stratification of CN-AML. Therefore, it is vital to build upon these advances by continuing to elucidate the biological characteristics and properties of leukemic stem cells and their regulating factors to assess their impact on AML treatment plans, the overarching question being what is the optimal consolidation strategy for each AML patient? Perhaps the incorporation of leukemia-stem cell mutations will add further clarity to which patients merit consolidation with allo-HCT and its attendant mortality and comorbidity and which AML patients can be safely managed with chemotherapy alone.Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults. Although there have been landmark targeted therapies developed in other hematologic malignancies, such as imatinib for chronic myeloid leukemia and ibrutinib in chronic lymphocytic leukemia, induction chemotherapy for AML has not changed significantly for several decades , 2. The ades ago , 4. To dHistorically, the French American British (FAB) classification system was used to subdivide AML into 8 subgroups (M0–M7) on a morphological basis . The adv (NPM1), fetal liver tyrosine kinase 3 (FLT3), and CCAAT/enhancer binding protein α (CEBPA) have been shown to have clinically significant prognostic value [ FLT3 internal tandem duplication (ITD) mutation (FLT3ITD) is present in nearly one-third of AML cases and has been associated with adverse clinical outcomes including increased relapse risk and decreased overall survival (OS) [ FLT3ITD presence in CN-AML identified a subgroup of patients with more adverse outcome, particularly patients with a high mutant allelic frequency [ FLT3ITD is a therapeutic target as well. Sorafenib, a tyrosine kinase inhibitor targeting FLT3ITD mutations, has been shown to increase event-free survival (EFS) and relapse-free survival (RFS) when added to both induction and consolidation therapies, although there was no OS benefit in the three years of follow-up in the newly diagnosed AML patients aged 60 years or younger [ NPM1 mutation has also been recently validated as a molecular marker of minimal residual disease (MRD) in NPM1 mutation positive patients and the presence of MRD was shown to be the only independent prognostic factor for death in multivariate analysis [The focus on improving our understanding of CN-AML prognosis and outcomes leads to the identification additional molecular markers of clinical significance. Mutations in nucleophosmin 1ic value . The FLTval (OS) . FLT3ITDrequency . In addi younger . NPM1 muanalysis .Consolidative chemotherapy is utilized to eliminate residual leukemia cells and/or leukemic stem cells (LSC) after induction chemotherapy to reduce the chance of relapse. Risk of a relapse after induction chemotherapy and consolidation chemotherapy increases with the increased MRD, a condition which can be assessed by immunophenotypical detection of leukemia cells , 13. LevAdvances in clinical genomics have identified an expanding array of recurrent molecular lesions in AML that will add layers of complexity to prognostic stratification needed to guide treatment and provide needed targets for new AML therapies. The evolving challenge is to incorporate these molecular abnormalities and their combinatorial effect on AML prognosis and in turn treatment strategies. The availability of this new AML data has created a requirement of a new classification system based on both cytogenetics and additional molecular lesions, which will be pivotal in establishing new clinical treatment guidelines. Furthermore, new classifications based on molecular abnormalities may help clinical trial design to develop targeted therapies to specific subgroups of AML patients. Recently a new AML classification system has been proposed by Papaemmanuil et al. . Here thThe proposed new AML classification system is based on a retrospective genomic analysis of 1540 AML patients in three prospective trials of the German-Austrian AML Study Group . Patient TP53 mutations, chromosomal aneuploidies, or both (13%); and (3) AML with IDH2R172 mutations (1%). Many of the mutations used to define the novel subgroups involve genes which have roles in stem cell functions. Of note, only 48% of patients were classifiable based on the current WHO guidelines, whereas 80% of patients could be allocated into this novel classification system. Only 8% of patients had either no detected driver mutations (4%) or ≥2 genomic subgroups (4%).In addition to cytogenetic analyses, 111 candidate driver genes were sequenced and 5234 somatic driver mutations were identified across 76 genes or genomic regions. Nearly all AML patients (96%) had at least one mutation and 86% patients were found to have two or more mutations. Statistical analysis of comutation patterns was utilized to define 11 mutually exclusive AML subtypes including three novel genetic subgroups that have not been described in the World Health Organization (WHO) classification in 2008 . These n NPM1-mutated AML, as the largest subgroup in this novel classification, had variable clinical outcomes influenced by the presence of concurrent mutations such as FLT3, DNMT3A, NRAS, IDH, PTPN11, or chromatin-spliceosome mutations. We focused on the clinical outcomes of the most significant single or concurrent molecular mutations based on this novel classification  is an enzyme that catalyzes the transfer of methyl groups to specific CpG structures in DNA and hence plays an essential role in DNA methylation and gene silencing regulatory processes [ DNMT3A is important in normal hematopoietic stem cell differentiation and self-renewal [ DNMT3A mutations were found in 22.1% of all AML and 33.7% of AML with intermediate-risk cytogenetic profile and were independently associated with a poor outcome regardless of age [ DNMT3A mutations tend to cooccur with FLT3, TET2, or IDH1 in AML  yields the worst prognosis and the consolidation with allo-HCT should be considered, although prospective study is needed to confirm these results.Patients withenotypes . DNMT3A ms of OS . Howeverms of OS . InsteadNRAS belongs to the RAS GTPase family of genes. It plays important roles in the regulation of proliferation, differentiation, and apoptosis in AML and is a fairly common mutation in AML ranging from 11% to 30% [ NRAS mutation consists of 19% of NPM1-mutated AML [ NRAS mutation has been reported to be insignificant for OS, EFS, and disease-free survival (DFS) [ NPM1 and DNMT3A in the presence of NRASG12/13 in AML patients were associated with a more favorable outcome [% to 30% . NRAS muated AML . The proal (DFS) . However outcome .TET2 (ten-eleven translocation) protein is an epigenetic modifier that converts methylcytosine to 5-hydroxymethylcytosine and plays a role in DNA methylation and myelopoiesis. Normal expression and function of TET2 are essential in maintaining the hematopoietic stem cell (HSC) pool and in controlling HSC differentiation [ TET2 (TET2−/−) or TET2 haplodeficiency (TET2+/−) impaired hematopoietic stem cell differentiation, held cells in a more immature state, and initiated aberrant hematopoiesis both in vitro and in vivo [ TET2 expression is tightly regulated by the master stem cell transcription factors Oct4 and Sox2 [ TET2 mutations are present in 5–25% of adult AML cases, with the highest frequency in the elderly [ TET2 mutations are significantly correlated with NPM1 (16%) in this most recent study [ MLLPTD [ IDH1/2 mutations [ TET2 mutation resulted in a lower complete remission rate, shorter EFS and DFS in patients with mutated NPM1 without FLT3ITD [ NPM1 without FLT3ITD [ NPM1 [ TET2 mutations were also reported to have no impact on the clinical outcomes of de novo AML [ NPM1 or CEBPA without FLT3ITD [ TET2 mutations has yet to be fully understood.ntiation . Studies in vivo –46. TET2and Sox2 . TET2 mu elderly , 48. TETnt study  and wereutations . TET2 mu FLT3ITD , and sho FLT3ITD , 50 and novo AML , CN-AML novo AML , 50, and FLT3ITD . The fulIDH1 and IDH2 (isocitrate dehydrogenases 1 and 2) are enzymes that catalyze the interconversion of isocitrate and alpha-ketoglutarate and appear to play an epigenetic role in histone and possibly DNA methylation. IDH1 or IDH2 mutations confer a hypermethylation phenotype in leukemia and inhibit hematopoietic stem cell differentiation [ TET2 loss-of-function mutations [ IDH1 mutation is in the arginine residue at position 132 (IDH1R132), occurring in 6–9% of adult AML, while IDH2 mutations occur in 9–19%, predominantly IDH2R140 in 8–12% [ IDH1 and IDH2 mutations are mutually exclusive in AML. Furthermore, IDH1 and IDH2R140 are strongly associated with NPM1 mutations [ IDH1 [ IDH2 [ NPM1 and FLT3ITD mutations. In patients with cooccurring DNMT3A and IDH2R140 mutations, the OS was significantly poorer than those with wild-type or a single mutation [ntiation . These putations . The mosin 8–12% , 52–54. utations . IDH1 [5s [ IDH1 , 54 and 1 [ IDH2  mutationmutation . IDH1 and IDH2 mutations constitute poor prognostic factors in cytogenetically normal AML with NPM1 mutation without FLT3ITD [ IDH1 or IDH2R140 had good prognoses for OS in AML patients with the NPM1 mutation without FLT3ITD [ NPM1 mutations was restricted to patients with cooccurring NPM1 and IDH1 or IDH2R140 mutations [An earlier study demonstrated that the FLT3ITD . In some FLT3ITD , 52, andutations .SRSF2, SF3B1, U2AF1, and ZRSR2), chromatin , and transcription (RUNX1). Functional proteins encoded by these genes have functions in hematopoietic stem cell self-renewal and differentiation. Using the European LeukemiaNet (ELN) guidelines, the majority (84%) of the patients in this new chromatin-spliceosome group would be classified as having intermediate prognostic risk. However, this new subgroup demonstrated resistance to induction chemotherapy and inferior long-term outcomes [This chromatin-spliceosome group is the second largest subgroup in this new classification . This isoutcomes  suggesti RUNX1  gene encodes the alpha subunit of the core binding factor involved in transcription and is required for definitive hematopoiesis [ RUNX1 protein also plays an essential role in mesenchymal stem cell proliferation and promotes cell survival in AML [ RUNX1 mutations are present in 5% to 18% of AML [ ASXL1 [ MLLPTD [ IDH1/IDH2 mutations [ NPM1 mutations [ RUNX1 mutations were found to be associated with resistance to chemotherapy, inferior DFS, EFS [ RUNX1 mutations were deemed to be an independent prognostic marker for shorter EFS in multivariable analysis [ RUNX1-mutated AML patients benefited from allo-HSC in terms of RFS [Theopoiesis . RUNX1 pl in AML , 58. RUN% of AML –62. They [ ASXL1 , MLLPTD utations , 62. RUNDFS, EFS , 61, 62,DFS, EFS –62. More MLL (Mixed Lineage Leukemia) gene, located on chromosome 11q23, is frequently involved in translocations that recur in AML and have been classified into an individual subgroup, AML with MLL fusion genes, t [ MLL partial tandem duplication (MLLPTD), the result of a tandem duplication of an internal portion of the MLL gene that spans either exons 2 to 6 or exons 2 to 8, is present in approximately 10% of CN-AML [ MLLPTD mutation has been identified as a poor prognostic factor for EFS [ MLLPTD regardless of the presence of FLT3ITD [The1) . MLL fus1) . A MLL pf CN-AML , 65. MLL for EFS , 66, 67  for EFS , 60, 67. FLT3ITD . ASXL1  gene is a human homologue of the Drosophila additional sex combs (Asx) gene, which is highly conserved across multiple species. The ASXL1 protein functions in both epigenetic activation and repression of gene transcription [ ASXL1 mutations are more common in the aberrant karyotypes, the elderly, and MDS-associated and secondary AML [ ASXL1 mutations are detected in cytogenetically normal AML [ NPM1 and ASXL1 mutations appear to be mutually exclusive [ ASXL1 mutations had worse outcomes when compared to those without these mutations [ ASXL1 mutations were associated with inferior complete remission, DFS, OS, and EFS [Thecription –70. Its cription , 72. ASXdary AML –77, whilrmal AML –77. NPM1xclusive –78. Seveutations , 60, 75. BCOR (BCL6 corepressor) gene is located on chromosome X and encodes a nuclear protein that is a key transcriptional regulator of hematopoiesis [ BCOR retains hematopoietic stem cells in a quiescent, undifferentiated state and loss-of-function BCOR mutations result in enhanced HSC cell proliferation and differentiation [ BCOR mutations occur in 3.8% of CN-AML, and DNMT3A mutations are detected in 43.5% of these patients. BCOR mutations tend to be associated with an inferior EFS and OS [Theopoiesis . Studiesntiation . BCOR muS and OS . PHF6 (plant homeodomain finger 6) gene, also located on chromosome X, plays a key role in chromatin remodeling [ PHF6 mutations are found in approximately 3% of adult AML and confer worse OS in intermediate-risk patients that are FLT3ITD negative [Themodeling . PHF6 munegative . CEBPA gene is located on chromosome 19 band q13.11 and encodes a 42 kDa size protein that is a member of the basic region leucine zipper transcription factor family [ CEBPA is crucial in maintaining adult hematopoietic stem cell in a quiescent state and CEBPA gene knockout in mice results in impaired HSC differentiation [ CEBPA regulates Sox4 expression which affects self-renewal of leukemic stem cells [ CEBPA mutations is reported to range from 7% to 22% in patients with AML [ CEBPA-mutated patients are biallelic-mutated , and the remaining one-third carry a single mutation . In a meta-analysis of 10 clinical studies covering 6219 patients, CEBPAbiallelic mutation was found to be associated with favorable clinical outcomes with regard to EFS and OS in patients with AML or CN-AML; conversely, no significant difference was found between CEBPAmonoallelic mutation and wild-type CEBPA in patients with AML or CN-AML [ CEBPAbiallelic mutations showed longer OS, longer relapse-free survival, and a lower cumulative incidence of relapse compared to those with CEBPAmonoallelic mutation. The ten-year OS rate for patients ≤60 years and with CEBPAbiallelic mutation was 81%. CEBPAbiallelic-mutated AML was associated with TET2 mutation in 34% of the patients, and the combination resulted in significantly worse OS, whereas it was associated with GATA2  mutation, found in 21% of CEBPAbiallelic-mutated AML, resulting in improved OS [Ther family . Normal ntiation . The CEBntiation . Mutatedem cells . The frewith AML  and 15% with AML , 88. Abor CN-AML . In a loroved OS .IDH2R172 is a distinct IDH2 mutation that occurs in AML with a frequency of 1–3% [ IDH2R140 that significantly correlates with NPM1 mutation, IDH2R172 is generally not associated with other molecular mutations. The gene expression and DNA methylation profiles of IDH2R172-mutated AML differ from those of other IDH mutations and lead to more severe aberrations in metabolic activity [ IDH2R172 mutation has been defined as a unique subgroup in the new classification scheme [ IDH2R172 mutation was associated with a higher relapse rate and lower OS that were comparable with those of the adverse-risk cytogenetics patients [ IDH2R172 mutation was associated with a favorable prognosis with regard to OS, similar to patients with NPM1-mutated AML [ of 1–3% , 52, 90.activity , 92. Thun scheme . In prevated AML . NPM1- or CEBPAbiallelic-mutated CN-AML to have favorable risk has significantly impacted the clinical management of these patient groups. The novel AML classification system proposed by Papaemmanuil et al. has been especially valuable in organizing the growing array of AML mutations in terms of additive effects on prognosis. As an example, the subgroup “AML with NPM1 mutation” is the largest subgroup and has a large number of comutations. The different comutation combinations do not have a strictly additive effect on clinical outcomes indicating further risk stratification in this group is necessary. Conversely, the subgroup “AML with mutated chromatin, RNA-splicing genes, or both” is more consistent as an adverse prognostic group, at least among the five genetic mutations we reviewed here: RUNX1, MLLPTD, ASXL1, BCOR, and PHF6. A central theme of this adverse-risk group is that the majority of these genes have roles in maintaining normal HSC quiescence by their epigenetic regulation and their mutations result in transformation of HSC into leukemic stem cells. These malignant stem cells, in turn, are thought to be the wellspring of leukemic cell expansion, likely directly responsible for the relapsing nature of AML. Of note, mutations in epigenetic modifiers or regulators such as DNMT3A, TET2, and IDH1/2 alter normal HSC quiescent state and prime HSC to a preleukemic state [The progress in AML risk stratification using next-generation sequencing technologies over the past decade has been truly remarkable. As an example, the identification ofic state . These e


Genomic integrity is constantly threatened by sources of DNA damage, internal and external alike. Among the most cytotoxic lesions is the DNA double-strand break (DSB) which arises from the cleavage of both strands of the double helix. Cells boast a considerable set of defences to both prevent and repair these breaks and drugs which derail these processes represent an important category of anticancer therapeutics. And yet, bizarrely, cells deploy this very machinery for the intentional and calculated disruption of genomic integrity, harnessing potentially destructive DSBs in delicate genetic transactions. Under tight spatiotemporal regulation, DSBs serve as a tool for genetic modification, widely used across cellular biology to generate diverse functionalities, ranging from the fundamental upkeep of DNA replication, transcription, and the chromatin landscape to the diversification of immunity and the germline. Growing evidence points to a role of aberrant DSB physiology in human disease and an understanding of these processes may both inform the design of new therapeutic strategies and reduce off-target effects of existing drugs. Here, we review the wide-ranging roles of physiological DSBs and the emerging network of their multilateral regulation to consider how the cell is able to harness DNA breaks as a critical biochemical tool.  Just as DNA breakage can devastate genomic integrity, it can also be deliberately and precisely exploited by cells in feats of genetic craftsmanship. Indeed, various integral cellular processes and genetic transactions are underpinned by the measured use of such potentially deleterious breaks.The genome is continuously exposed to a panoply of DNA damaging agents, both endogenous and exogenous, which pose a considerable threat to genomic integrity. These genotoxic insults result in diverse DNA lesions including mismatches, base adducts, pyrimidine dimers, intra- and interstrand cross links, DNA-protein adducts, and strand breaks . Among tDSBs are highly mutagenic and can induce potentially tumorigenic chromosomal translocations. If unrepaired, DSBs may also lead to cell death . It is tDespite the dangers inherent in such lesions, DSBs are precisely employed for the intentional but controlled disruption of genomic integrity in biological processes. The cellular roles of physiological DSBs can be broadly considered as one of two major functionalities: genetic recombination or manipulation of DNA topology. In the former role, DSBs can act as genomic “shufflers,” carefully but permanently recombining DNA segments for genomic diversification in lymphocytes , 7 and gHere, we review the wide-ranging biological functions of these various physiological DSBs and the multilayered regulation thereof, together constituting cellular “DSB physiology” . By consβ and δ chains, whereas only V and J segments are required for the Ig light chain and TCR α and γ chains [The role of physiological DSBs in the diversification of the adaptive immune response is well documented. V(D)J recombination describes the process whereby lymphoid cells recombine a repertoire of germline variable (V), diversity (D), and joining (J) exon gene segments in various permutations to generate enormous antigen-receptor diversity in immunoglobulins (Ig) and T cell receptors (TCRs). All three exon segments are used for assembling the variable region of the Ig heavy chain and the TCR γ chains . The proEach V, D, and J segment is flanked by a recombination signal sequence (RSS), comprising conserved heptamer-spacer-nonamer sequence elements , 22. Cleλ and μ combined with exonucleases generates compatible coding ends [Prior to joining, hairpin coding ends are nicked open through the endonuclease activity of the nuclear enzyme Artemis, which complexes with the serine/threonine kinase DNA-PKcs, which, in turn, activates Artemis by phosphorylation , 39. Howing ends , 51 whicing ends , 47, 52.An important point of control in the DSB formation step of V(D)J recombination is the cell cycle-dependent regulation of RAG expression. Cyclin A-CDK2 phosphorylation-mediated degradation of RAG-2 at the G1/S transition  exerts tH) germline transcription with its joining with DJH suggested an open chromatin state was required for recombination [β chain before α chain), as well as the process of allelic exclusion [Additional temporal control is exerted over RSS selection for DSB formation and only certain RSS sites are available for recombination, exhibiting cell- and stage-specific biases , 57. Thebination –64. RAG2bination , 66. Thixclusion . Aside f RAG-1 or RAG-2 genes are also observed in humans with an analogous form of autosomal recessive SCID known as Omenn syndrome [ RAG-1 and RAG-2 mutants exhibit lower efficiency of V(D)J recombination [Broadly, perturbations in V(D)J recombination can be from either an inability to form DSBs, an inability to repair them, or inaccurate pairing of RSSs. Although viable, Rag-1- or Rag-2-deficient mice are unable to carry out V(D)J recombination and lack mature B and T lymphocytes, presenting with severe combined immune deficiency (SCID) , 68. Missyndrome . Geneticbination . These mbination . DCLRE1C hypomorphism in humans has been found to underlie an Omenn syndrome phenotype associated with defective endonuclease activity [Impairment of the joining phase of V(D)J recombination can also cause immunodeficiency. While all mammalian NHEJ pathway mutants display sensitivity to ionising radiation and a lack of B and T lymphocytes, a spectrum of phenotypes is observed in mouse models. DNA-PKcs and Artemis mutants exhibit subtle defects in DSB repair  but dispactivity . Ku70 anactivity , 75 likeactivity . Mutantsactivity –79. ThesLymphomas have been found to exhibit a range of chromosomal aberrations as a result of erroneous DSB handling in V(D)J recombination. Oncogenic lesions have been found to include chromosomal translocations, insertions, deletions, and inversions and broadly result from errors in RSS pairing or joining –82. InacH) genes via DSB generation, followed by ligation of the remaining segments through DSB resolution [Class switch recombination (CSR) is another example of a DSB-dependent recombination event, occurring in mature B cells. In response to antigenic stimuli and costimulatory signals from other immune cells, B cells undergo CSR to alter their Ig constant heavy chain while retaining the same antigen specificity, in effect, generating an alternative Ig isotype with the same paratope but distinct effector functions . This ensolution .H gene are the site of DSB generation [Conserved motifs called switch (S) regions upstream of each Cneration , 89. AIDneration –93. The neration . BER proneration  followedneration , 95. AIDneration . Processneration , 97.As aforementioned, MMR is another pathway employed in CSR DSB generation and is thought to provide auxiliary support to BER-mediated mismatch excision . Indeed,H genes, the free end of the variable domain region is ligated to that of the new constant domain exon by the NHEJ machinery [After intrachromosomal deletion of the intervening unwanted Cachinery –110.Recombination underlying isotype switching of constant heavy chain genes is regulated by germline transcripts which modify switch region accessibility to the DNA handling machinery . TranscrAID deamination can only occur on single-stranded DNA (ssDNA)  and one Beyond its roles in lymphoid cells, DSB-induced recombination also acts in meiosis where it is essential for both the fidelity of chromosome segregation and diversification of the germline. Meiosis is a form of reductive cell division exclusive to gametogenesis and involves two successive divisions following replication, known as MI and MII . A hallm Spo11−/− mouse spermatocytes [ S. cerevisiae, between Spo11 and other meiotic proteins. These proteins include components of the Mre11-Rad50-Xrs2 (MRX) complex and multiple Spo11-accessory proteins including Mei4, Mer2, Rec102, Rec104, Rec114, and Ski8 which facilitate DSB formation [Meiotic recombination is initiated by DSB induction by a dimer of the topoisomerase-like transesterase Spo11 which acts via a covalently linked protein tyrosyl-DNA intermediate complex –127. Intatocytes . DSB forormation , 130. Thormation –133. Recormation  which plThe resulting DSBs then undergo endonucleolytic processing which releases Spo11 to allow repair . This inA multitude of interacting mechanisms operate in meiotic recombination to control DSB formation and resolution. This tight spatiotemporal regulation of DSB handling allows the intentional disruption of gamete DNA integrity for diversification, while ensuring coordinated cell cycling and, ultimately, generation of an intact haploid germline.DSB induction is mechanistically coupled to DNA replication  and is s S. cerevisiae to control Spo11 loading on DSB target sites [Posttranslational modifications on the Spo11-accessory protein Mer2 have been shown to contribute to the timely execution of DSB formation in meiotic recombination. Phosphorylation of Mer2 by the budding yeast cyclin-dependent kinase Cdc28 stimulates DSB formation via promotion of interactions both with itself and with other proteins involved in DSB generation including Mei4, Rec114, and Xrs2 . It is ket sites . It has et sites . As wellet sites , 159 whiet sites .Additional temporal control is afforded by meiosis-specific expression of Spo11 and accessory proteins Rec102, Rec104, Rec114, and Mei4, largely achieved via transcriptional regulation . In contMeiotic recombination and DSB formation exhibit nonrandom patterning across the genome, being more frequently localised to particular sites on chromosomes called “hotspots” , 164. WhIn mice, recombination hotspots have been found to be enriched for H3K4me3 histone modifications , 168. Th cis and in trans also exert an inhibitory effect from one DSB on further break formation, thus limiting DSB frequency [Negative feedback systems acting both inrequency . In addirequency , 175. Rerequency  and it hrequency . S. cerevisiae have shown that Rec114, Mer2, and Mei4 stably associate with chromosome axis association sites between loops, in a manner dependent on components of the axial element [DSB formation is also subject to spatial regulation via control of chromosome ultrastructure. During the leptotene stage of prophase I, DSB formation occurs alongside the development of the axial element, a proteinaceous axis comprising various proteins including cohesin . The sis element . Meiotic element . Such di element . Further element .To access the information stored in compacted nuclear DNA in such processes as transcription, replication, and repair requires the repeated tangling and untangling of the DNA double helix. Such molecular contortions introduce topological entanglements into the duplex which if left unchecked could compromise genomic stability , 181. ReWhile, in general, a Top II-induced DSB is resolved in the enzyme's catalytic cycle, it does possess a capacity for formation of abortive breaks  and can Supercoiling, entanglement, and knotting are frequent topological obstacles encountered in DNA physiology. Type II DNA topoisomerases, through DSB induction and repair, act to resolve such obstacles using a repertoire of supercoil introduction/relaxation, catenation/decatenation, and knotting/unknotting processes , 198. EuReplication fork arrest, such as due to impassable DNA damage, can give rise to DSBs , 201 whiTop II enzymes are essential in the elongation of replicating DNA chains and during segregation of newly replicated chromosomes. During semiconservative replication, the replisomal machinery at the advancing replication fork forces the intertwined DNA strands ahead of it to become overwound or positively supercoiled. These positive supercoils are redistributed behind the fork by the rotation of the replicative machinery around the helical axis of the parental duplex. This causes intertwinement of the newly replicated DNA double helices, forming precatenanes . Studies top1 top2 temperature-sensitive double mutants exhibit significant inhibition of rRNA synthesis and, to a lesser extent, mRNA synthesis at nonpermissive temperatures [Transcription of nascent mRNA presents topological obstacles akin to those seen during replication . Transcreratures . In addieratures , 219. Moeratures . Pharmaceratures . top2 yeast exhibits normal levels of transcription; however it has been shown in budding yeast that loss of Top2 produces a specific reduction in Pol II-dependent transcription of genes longer than around 3 kb, attributed to defective elongation due to Pol II stalling [As aforementioned,stalling . More restalling .β-estradiol-dependent transactivation, via the regulated formation of site-specific longer-lived DSBs in target gene promoters [Transcription has long been known to stimulate HR in a locus-specific manner –225 and romoters . Top IIbromoters . More re Fos, FosB, and Npas4 [ Fos and Npas4 is not affected by inhibition of DSB signalling following ATM blockade, which also suggests that nIEG induction is independent of the effects of a generic DDR to nonspecific DSBs [ Fos and Npas4 promoters also produced upregulation thereof in the absence of neuronal activity. Together these indicate a role of DSBs acting within target gene promoters in the regulation of nIEG transcription [Neuronal early response genes or “immediate early genes” (nIEGs) are a subset of activity-regulated genes induced with shortest latency following neuronal stimulation and typically encode transcription factors involved in transcriptional modulation for synaptic plasticity , 229. Trnd Npas4 . This innd Npas4 . Furtherfic DSBs . Similarcription . Fos and Npas4 mRNA accompanied by elevated γH2AX levels [γH2AX following stimulation with NMDA (N-methyl-D-aspartate) demonstrated preferential clustering of yH2AX elevations at actively transcribed genes and downstream sequences. Differential peak calling on NMDA-treated samples showed that yH2AX enrichment was confined to only 21 loci including the nIEGs Fos, FosB, Npas4, Egr1, Nr4a1, and Nr4a3 among other transcription factors and noncoding RNAs, pointing to the specific localisation of DSB induction following neuronal stimulation [γH2AX was also observed to increase following pharmacological or electrical stimulation of mouse acute hippocampal slices as well as in hippocampal lysates from mice exposed to a fear-conditioning paradigm [ Fos promoter following NMDA treatment demonstrated long-lived breaks, being repaired within 2 hours of stimulation. However, the significance of this extended lifespan is as yet unclear [Furthermore, electrical or pharmacological stimulation of cultured primary neurons revealed upregulation ofX levels , a knownX levels , suggestmulation . Interesparadigm . This coparadigm . These Dparadigm . Overall unclear . Top2b in cultured primary neurons reduces yH2AX enrichment at nIEG exons following NMDA stimulation, pointing towards a role of Top IIb in activity-induced DSB formation at nIEGs [ Top2b knockdown in cultured primary neurons inhibits induction of nIEGs following NMDA stimulation but could be rescued by Cas9-generated DSBs targeted to the nIEG promoter [Other experiments showed that short hairpin RNA-mediated knockdown ofat nIEGs . In ChIPat nIEGs . In addiat nIEGs . Linkingpromoter .While the mechanism whereby Top IIb-mediated DSBs could induce nIEG transcription is as yet unclear, one model proposed involves a functional interaction with the architectural protein CTCF . CTCF orAdditional mechanistic complexity derives from the need for concerted repair of activity-induced DSBs. Top IIb exhibits preferential binding and cleavage at the promoters of target nIEGs, cooccupying the site with the transcriptional activator ELK1 and histone deacetylase HDAC2, the latter repressing nIEG expression . Also fo (ATM), ataxia-telangiectasia-like disorder (MRE11), Nijmegen breakage syndrome (NBS1), and Alzheimer's disease [ Ercc1Δ/- mice, which have compromised nucleotide excision repair, interstrand crosslink repair, and DSB repair pathways, display age-associated neurodegeneration and progressive loss of motor function [Defective DSB repair and/or loss of DSB repair components is a feature of various neuropathologies and age-associated neurodegenerative disorders, including ataxia-telangiectasia disease –245. In function , 247.β (Aβ), aggregates of which are widely recognised as a key factor in AD pathogenesis, is found to increase DSB formation in primary neuronal cultures via dysregulation of synaptic transmission [β oligomers [Increased incidence of DNA strand breakage has been identified in the brains of Alzheimer's disease (AD) patients , 249, ansmission , 250. Exsmission , 252, isligomers . Moreoveligomers . TOP2B mutation associated with intellectual disability, autistic traits, microcephaly, and developmental retardation [ TDP2 have been linked with cases of intellectual disability, epilepsy, and ataxia [There is growing evidence suggesting that aberrant Top II activity may play a role in the aetiology of neurodevelopmental defects. Recent work involving clinical whole-exome sequencing identified a novelardation  and homod ataxia . Top IIbd ataxia , 14. Phad ataxia . Furtherd ataxia , 257.Together, these findings point towards the importance of DSB repair regulation in neuronal physiology and activity-induced DSB handling, but further studies will be required to elucidate more specific roles of activity-induced DSBs in neuropathology. cis-elements which are involved in long-range chromatin organisation [Top II is a highly abundant chromatin protein, constituting 1-2% of total chromosomal protein content in mitosis . Studiesnisation , 261. Honisation . top2 yeast [ Xenopus egg extracts [ Drosophila ATP-dependent chromatin remodelling complex CHRAC (chromatin-accessibility complex) [ Drosophila protein Barren [ S. pombe demonstrate an intergenic localisation of both Top I and Top II which strongly correlates with distributions of the chromodomain ATPase Hrp1 and the histone chaperone Nap1, both involved in nucleosome disassembly [DNA topology and chromatin structural changes are known to be intricately related . Early wp2 yeast  and compextracts  revealedcomplex) , and mamcomplex) . Furthern Barren , which in Barren . ChIP exassembly –271. Topassembly . While tassembly . Thus, i (TOP1MT) and Top IIIα (TOP3A) of the type I family and Top IIb (TOP2B) from the type II family of enzymes [ TOP2B−/− MEFs and TOP1MT−/−TOP2B−/− display wild-type levels of transcription and only the double-knockout elicits a stress response, suggesting nonoverlapping functions of the two molecules [Mitochondrial DNA (mtDNA) in humans is typically a closed circle of dsDNA . As in g enzymes . Althoug enzymes . Top1mt  enzymes . Howeverolecules . This piolecules . Drosophila. Indeed, loss of mitochondrial import of Top IIIa in Drosophila is associated with a reduction in mtDNA copy number, mitochondrial dysfunction, impaired fertility, and accelerated ageing [Top IIIa is a type IA topoisomerase which is found in both the nucleus and mitochondria and has been shown to play a crucial role in the maintenance of mtDNA genomic integrity ind ageing , 280. ThA recently proposed but unexplored theory suggests that mitochondrial Top II may be involved in ageing . With ag Trypanosoma and Leishmania species, which possess a characteristic dense granule of DNA called a kinetoplast within their single mitochondrion. The kinetoplast consists of an enormous network of catenated circular mtDNA (kDNA) comprising short minicircles and much larger maxicircles and thus represents a considerable topological challenge in replication and segregation [Further insight into the role of Top II-induced DSBs in mitochondria may be drawn from parallels in Kinetoplastids, a group of flagellated protozoa which includes the parasiticregation . The mitregation , 297 andregation , mendingregation , segregaregation , and theregation .DSB physiology is thus, at its very core, a molecular surgery, reliant on both calculated incisions and timely suturing thereof, all the while, under the intense scrutiny of numerous regulatory mechanisms. While the basic biochemical mechanisms of the processes discussed above are well established, their multilateral spatiotemporal regulation remains a field of active research.The roles of recombination defects in human disease and tumorigenesis are being increasingly appreciated and further studies will be needed to examine the nature of the dysregulation of DSB physiology in pathogenesis. New work may reveal novel pharmacological targets for human disease such as the possibility of targeting DSB signalling and repair proteins in neurodegenerative conditions such as Alzheimer's disease, to lessen aberrant DSB formation.Novel tissue-specific roles of topoisomerases in neurophysiology are only just being uncovered with future studies required to elucidate the function of DSBs therein to understand mechanisms of learning, memory, and cognition. In addition, future work is necessary to characterise the nature of mitochondrial DSB physiology and the as yet unexplored implications on mitochondrial metabolism, disease, and ageing.Furthermore, both topoisomerases and cancer DSB repair deficiencies are important areas of investigation in the development of anticancer therapeutics, and an understanding of the physiological roles of these targets in multiple disparate processes will inform the design of specific inhibitors with fewer off-target effects.This account illustrates the breadth of DSB physiology across cellular biology and adds a new dimension of complexity to the regulation of genomic transactions on a basic biochemical level. The emerging role of DSB-dependent changes in topology, not only in replication and basal transcription, but also in activity-induced transcriptional changes, and potential roles in the chromatin landscape and mitochondria paint a picture of a far more active role of DSBs in fundamental DNA metabolism than previously thought.


MCL1 transcripts. In concert, there was a decline in total Mcl-1 protein in CLL lymphocytes. Isogenic mouse embryonic fibroblasts lacking MCL1 had higher sensitivity to bendamustine alone or in combination compared to MCL1 proficient cells. Collectively, these data indicate that bendamustine and idelalisib combination therapy should be investigated for treating patients with CLL.Idelalisib is a targeted agent that potently inhibits PI3Kδ which is exclusively expressed in hematological cells. Bendamustine is a well-tolerated cytotoxic alkylating agent which has been extensively used for treatment of chronic lymphocytic leukemia (CLL). Both these agents are FDA-approved for CLL. To increase the potency of idelalisib and bendamustine, we tested their combination in primary CLL lymphocytes. While each compound alone produced a moderate response, combination at several concentrations resulted in synergistic cytotoxicity. Idelalisib enhanced the bendamustine-mediated DNA damage/repair response, indicated by the phosphorylation of ATM, Chk2, and p53. Each drug alone activated γH2AX but combination treatment further increased the expression of this DNA damage marker. Compared with the control, idelalisib treatment decreased global RNA synthesis, resulting in a decline of early-response and short-lived  Chronic lymphocytic leukemia (CLL) is a malignancy that is driven by active B-cell receptor (BCR) pathway. The primary BCR down-stream network includes two pivotal enzymes, Bruton's tyrosine kinase and phosphatidyl inositol three kinase (PI3K). Both BTK and PI3K isoform delta are selectively expressed in hematopoietic cells specifically normal and malignant B-cells such as CLL lymphocytes. This exclusive expression provided an impetus to design potent and selective inhibitors of these two proteins. Ibrutinib, a BTK poison and idelalisib a PI3K delta antagonist were tested in CLL and demonstrated on-target effect during preclinical experimentations and efficacy during clinical trials. Both drugs were FDA approved for patients with CLL.As described above, PI3K delta isoform is pivotal in the BCR axis  and is sIn the front-line setting, the idelalisib and rituximab combination was tested for older patients with CLL . SimilarFrom phase I, phase II in previously treated CLL, and phase II in newly-diagnosed CLL, a few points emerged. First, while the drug was well-tolerated during earlier studies, in treatment naïve patients, there was unacceptable toxicity profile. Second, responses were mostly partial and complete remissions were limited. Third, in both cohort of patients (previously untreated or treated), similar to other BCR pathway antagonists, there was egression of CLL cells from lymph node which remained in peripheral blood. Finally, there is a need to combine idelalisib with CLL-specific agents that may result in deeper responses without much untoward toxicity.Chlorambucil, cyclophosphamide, and bendamustine are three alkylating agents that have been used for decades for treatment of CLL. Of these three, regarding potency in the clinic, the cyclophosphamide is the strongest, followed by bendamustine, and chlorambucil. However, cyclophosphamide results in high untoward toxicity, a feature not favorable to combine with idelalisib. Bendamustine not only possesses alkylating agent properties but is also well-tolerated and FDA approved for patients with CLL along with established treatment recommendations. , 13.Previously, we have demonstrated utility of bendamustine in preclinical setting for primary CLL cells and shown synergistic or additive interactions with fludarabine . ImportaIn the current project, we tested that idelalisib-mediated suppression of BCR signaling would sensitize CLL cells to bendamustine, and this mechanism-based combination may lead to a synergistic interaction. We evaluated the cytotoxicity induced by idelalisib or bendamustine alone or in combination in primary CLL cells and the impact of this combination on DNA damage response and Bcl-2 family survival protein levels which are regulated by PI3K/Akt pathway. Our data demonstrate that this combination is synergistic, and we suggest a mechanism by which idelalisib increased the cytotoxicity of bendamustine.n = 9) revealed that single agent idelalisib induced moderate but statistically significant cytotoxicity  in a dose-dependent manner, ranging from 4% to 16% (p = 0.002 to < 0.0001 for each concentration). Whereas single agent bendamustine resulted in 6%-33% (p = 0.002 to < 0.0001 for each concentration) cell death. Combination of idelalisib and bendamustine at clinically relevant concentrations resulted in 13% to 49% of apoptosis (p = < 0.0001 for each concentration). At each drug concentration, compared to single agent apoptotic response, there was an increase in the level of cell death when both drugs were combined together.Treatment of primary CLL cells with idelalisib alone Figure , bendamuThis dose-response profile led us to investigate whether idelalisib and bendamustine combination could be synergistic combination. To test this, we plotted the apoptotic values obtained from Figure Bendamustine is an alkylating agent known to induce DNA damage response. γH2AX is a prominent marker of DNA damage response  and coulWe further evaluated the effects of these agents on other proteins involved in DNA damage/repair signaling pathways. Compared to the control, bendamustine alone increased phosphorylation of ATM(Ser1981) and Chk2(Thr68), an effect that was further enhanced by combination treatment. Additionally, bendamustine alone and in combination with idelalisib resulted in stabilization of p53 protein, marked by phosphorylation of p53(Ser15). Overall, these observations indicate that idelalisib enhanced the DNA-damage response elicited by bendamustine.n = 4; Figure MCL1 and BCL2 are the members of the BCL2 family antiapoptotic proteins, -1-methyl-2-bezimidazolyl} butyric acid hydrochloride. The nitrogen mustard group of bendamustine is similar to that in chlorambucil , 21. HowMCL1 gene expression in a p53-independent manner. -uridine , and the radioactive counts were measured by scintillation counter. [Primary CLL cells were either untreated or treated with idelalisib for the indicated time. For the last 30 min, the cells were incubated with [5,6-counter.  Each treBCL2 and MCL1 transcript levels, which were normalized to 18S ribosomal RNA as an endogenous control. [CLL lymphocytes were treated with DMSO or with idelalisib alone, bendamustine alone, or two drugs together for 24 hours. TaqMan real-time reverse transcription polymerase chain reaction assay was used to measure control. t-tests were performed using Prism-6 software . For combination treatment, fractional analysis was used to determine whether the combination led to less than, equal to, or more than the additive effect on inducing apoptosis. [Paired 2-tailed Student optosis.  CalcuSynP.M. designed the experiments, performed the experiments, analyzed the results, and wrote the manuscript. K.B. directed P.M. in the laboratory and reviewed the manuscript. Q.Y. assisted in experimental planning and reviewed the manuscript. M.J.K. and W.G.W. identified patients to obtain peripheral blood samples, provided clinical and patient-related input, and reviewed the manuscript. V.G. conceptualized and supervised the research, obtained funding, analyzed the data, and wrote and reviewed the manuscript.