However, how Ca2+ uptake by the mitochondrial Ca2+ uniporter (MCU) shapes receptor-evoked interorganellar Ca2+ signaling is unknown. Here, we used CRISPR/Cas9 gene knockout, subcellular Ca2+ imaging, and mathematical modeling to show that MCU is a universal regulator of intracellular Ca2+ signaling across mammalian cell types. MCU activity sustains cytosolic Ca2+ signaling by preventing Ca2+-dependent inactivation of store-operated Ca2+ release–activated Ca2+ channels and by inhibiting Ca2+ extrusion. Paradoxically, MCU knockout (MCU-KO) enhanced cytosolic Ca2+ responses to store depletion. Physiological agonist stimulation in MCU-KO cells led to enhanced frequency of cytosolic Ca2+ oscillations, endoplasmic reticulum Ca2+ refilling, nuclear translocation of nuclear factor for activated T cells transcription factors, and cell proliferation, without altering inositol-1,4,5-trisphosphate receptor activity. Our data show that MCU has dual counterbalancing functions at the cytosol–mitochondria interface, whereby the cell-specific MCU-dependent cytosolic Ca2+ clearance and buffering capacity of mitochondria reciprocally regulate interorganellar Ca2+ transfer and nuclear factor for activated T cells nuclear translocation during receptor-evoked signaling. These findings highlight the critical dual function of the MCU not only in the acute Ca2+ buffering by mitochondria but also in shaping endoplasmic reticulum and cytosolic Ca2+ signals that regulate cellular transcription and function.Mitochondrial Ca As such, mitochondrial Ca2+ uptake is thought to take place at specialized microdomains where cytosolic Ca2+ concentrations are high. Such microdomains include the mitochondria-associated membranes (MAMs) where the endoplasmic reticulum (ER) membrane is within 10 to 30 nm from the outer mitochondrial membrane and Ca2+ is transferred to mitochondria through closely apposed inositol-1,4,5-trisphosphate receptor (IP3R) channels within ER membranes   and posser Letm1 . The gat present , but seeembranes .3  undergo a conformational change and move to ER-PM junctional spaces where they form puncta and activate Orai channels mediating the highly Ca2+-selective, store-operated, Ca2+ release–activated Ca2+ (CRAC) current  transcription factors (Activation of plasma membrane (PM) receptors that couple to isoforms of phospholipase C by hormones, neurotransmitters, and growth factors causes the breakdown of membrane-associated phosphatidylinositol-4,5-bisphosphate into two second messengers: the membrane-bound diacylglycerol and the diffusible IP3 . Activat current , 22, 23.llations , 25, 26. factors , which a factors , 29.2+ enters through CRAC channels and accumulates on the cytosolic side of the PM, it mediates inhibition of CRAC channels through several feedback mechanisms. First, there is fast Ca2+-dependent inactivation (CDI) that involves Ca2+ binding to inhibitory sites within few nanometers of the mouth of CRAC channels  -mediated dissociation of STIM1 puncta and STIM1–Orai1 complexes  and antimycin A1, which inhibit mitochondrial Ca2+ uptake by altering mitochondrial respiration or depolarizing its membrane, to propose that mitochondria can buffer Ca2+ to alleviate slow CDI and sustain store-operated Ca2+ entry (SOCE) in Jurkat and primary T cells  . This stchannels  and was omplexes . This la T cells , 37. A mllations .2+ dynamics using an arsenal of cell lines and primary cells from different species and tissues. We utilized CRISPR/Cas9 to produce several clones of MCU knockout in cultured cells and used the Cre-LoxP system to generate populations of primary cells from tissue-specific MCU knockdown mice. We show that, despite promoting slow CDI of CRAC channels and enhancing cytosolic Ca2+ extrusion, MCU deletion leads to enhanced cytosolic Ca2+ in response to passive store depletion. MCU-KO also enhanced the frequency of cytosolic Ca2+ oscillations in response to agonist stimulation without affecting IP3R-mediated Ca2+ puffs. MCU-KO causes enhanced ER Ca2+ refilling and increased nuclear translocation of NFAT isoforms and increased B-lymphocyte proliferation. Mathematical modeling is consistent with MCU as an important regulator of interorganellar Ca2+ transfer that fine-tunes cytosolic Ca2+ signaling. Our data provide critical insights into the role of MCU and mitochondrial Ca2+ uptake in cellular signaling. Despite the critical function of MCU-mediated mitochondrial Ca2+ uptake in inhibiting CRAC channel slow CDI and limiting Ca2+ extrusion, MCU deletion leads to a paradoxical enhancement of cytosolic Ca2+ upon receptor stimulation. Thus, the inability of mitochondria to store its “allocated share” of Ca2+ in the absence of MCU offsets the effects on Ca2+ extrusion and CRAC channel activity. The net result of MCU-KO is enhanced ER Ca2+ refilling and increased cytosolic Ca2+ signaling. Our data show that MCU has dual counterbalancing functions at the cytosol–mitochondria interface. MCU activity can enhance near PM Ca2+ signaling to increase NFAT activity and can also inhibit NFAT activity through the “Ca2+ sink” effect of mitochondria. This reciprocal regulation by MCU fine-tunes interorganellar Ca2+ transfer and NFAT activation during receptor-evoked signaling.Here, we have investigated the role of MCU in regulating cytosolic, ER, and mitochondrial Ca2+ uptake in shaping intracellular Ca2+ signaling. To alleviate potential off-target effects of CRISPR/Cas9 KO, we studied two independent MCU-KO clones for each cell line . These clones were identified; validated for MCU-KO with genomic sequencing, Western blot, and mitochondrial Ca2+ measurements; and assayed in parallel to their corresponding parental controls. Absence of the MCU protein in knockout cells was documented by Western blot . Functional knockout of MCU was documented by simultaneous Ca2+ and mitochondrial membrane potential fluorescence measurements in a permeabilized cell system  using thapsigargin , which causes passive depletion of ER Ca2+ stores (recorded in Ca2+-free bath solutions) and activation of SOCE, which manifests upon addition of 2 mM Ca2+ to the extracellular bath . Surprising, the use of this protocol revealed that MCU-KO led to a significant increase in apparent SOCE in all cell lines, with the exception of MCU-KO HEK293 cells where SOCE was increased in some runs but not significantly different from that of the parental HEK293 cells when all cells were averaged . This notable difference between HEK293 cells and the other six cell lines will be addressed further below. Western blots showed no change in STIM1 protein levels upon MCU-KO in either HEK293 cells or Jurkat cells  or blockade of complex III and complex V of the electron transport chain with Antimycin A1 and oligomycin, led to inhibition of SOCE (2+ buffer (1.2 mM EGTA) but including metabolites that promote energized mitochondria  of CRAC channels to sustain SOCE.Previous reports showed that abrogation of the ability of mitochondria to buffer cytosolic Ca of SOCE . CRAC cuchondria . These fA–C, when added after SOCE was initiated, FCCP significantly inhibited SOCE in both parental cells and their MCU-KO counterparts. The inhibition of SOCE in parental Jurkat (B) and RBL-1 (C) cells was slower than in their MCU-KO counterparts and was preceded by a transient increase in Ca2+, presumably reflecting bigger mitochondrial Ca2+ stores in the parental cells. In HEK293 cells, the rate of SOCE inhibition by FCCP was similar between wildtype and MCU-KO cells (A), suggesting a bigger contribution of mitochondria as a reservoir for Ca2+ in Jurkat and RBL-1 cells compared with HEK293 cells. Addition of 1 μM ionomycin to HEK293 and Jurkat cells  to release any remaining Ca2+ from thapsigargin-independent stores (presumably mitochondria) showed a small mitochondrial Ca2+ release in wildtype Jurkat cells but not in wildtype HEK293 cells. We subsequently added 10 μM ionomycin (blue arrow) in the presence of 2 mM Ca2+ to confirm that maximal Fura2 signal is equal between MCU-KO and parental cells. Overall, these data argue that inhibition of SOCE by FCCP cannot be explained simply by lack of mitochondrial Ca2+ buffering through MCU. One recurring observation in our recordings is the enhanced rate of decline of SOCE steady-state plateaus in Jurkat and RBL-1 MCU-KO cells compared with parental cells (B and C), suggesting that mitochondrial Ca2+ buffering by MCU inhibits PM Ca2+ ATPase–mediated Ca2+ extrusion, as shown previously in Jurkat T cells  and RBL-1 parental cells (C and D).Therefore, we performed SOCE measurements in response to thapsigargin in parental HEK293, Jurkat, and RBL-1 cells and their MCU-KO counterparts and tested the effect of 5 μM of the protonophore trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP) on SOCE. As seen in l Jurkat B and RBLnd RBL-1 C cells wKO cells A, suggesal cells , B and C T cells . Indeed,h Jurkat , A and Bal cells , C and D2+ CRAC currents in 20 mM Ca2+ bath solutions and performed rapid switches to divalent-free bath solutions to record Na+ CRAC currents, which are larger in size. We measured whole-cell CRAC currents from Jurkat and RBL-1 cells under two different conditions. The first condition uses a pipette solution with a low buffering capacity (1 mM EGTA) that includes 30 μM IP3 to empty the stores and contains a cocktail of mitochondria-energizing metabolites (see Methods) that can reveal potential mitochondrial effects on CRAC channel slow CDI. The second condition uses a pipette solution with a very strong buffer (50 mM BAPTA) that would negate any potential effect of mitochondrial Ca2+ buffering on CRAC currents. Native Ca2+ and Na+ CRAC currents under both weak and strong buffer conditions in MCU-KO clones of both Jurkat and RBL-1 cells were statistically of similar magnitude compared with currents from their corresponding parental cells . Furthermore, when the pipette solution included the mitochondria-energizing cocktail, ICRAC slow CDI of parental Jurkat cells was inhibited on average by ∼90%, whereas ICRAC slow CDI in MCU-KO cells remained unchanged (D–F). CDI recordings with and without mitochondria-energizing cocktail in each individual wildtype Jurkat and MCU-KO cell are shown in We then sought to determine whether increased SOCE upon MCU-KO is due to increased net CRAC currents across the PM by using the whole-cell patch clamp technique. We chose to record from two widely studied cell lines that produce the largest native CRAC currents, namely, Jurkat and RBL-1 cells. We recorded both Caal cells . To accusolution . ICRAC w mM Ca2+ ; see Exp mM Ca2+  either w mM Ca2+ . Of inte mM Ca2+ , A–C. Funchanged , D–F. CD2+ uptake promotes Ca2+ extrusion and CRAC channel CDI, its net effect is an increase, rather than a decrease, in cytosolic Ca2+ levels. Our findings suggest that preventing mitochondria from buffering its allocated share of cytosolic Ca2+ could lead to accelerated ER Ca2+ refilling and enhanced frequency of agonist-evoked cytosolic Ca2+ oscillations, NFAT activation, and cell proliferation. These concepts are explored further below.Taken together, these data indicate that, although abrogation of MCU-mediated mitochondrial Ca2+ signaling triggered by physiological agonist stimulation. To induce cytosolic Ca2+ oscillations, Jurkat cells were stimulated with a relatively low concentration (0.125 μg/ml) of anti-CD3 antibody, which is commonly used to activate the T cell receptor and drive clonal expansion and interleukin 2 (IL-2) production. Under these conditions, essentially all Jurkat cells that responded displayed regenerative Ca2+ oscillations (A and B) with MCU-KO Jurkat cells displaying enhanced frequency of Ca2+ oscillations compared with the parental cells (A–C). Furthermore, Ca2+ oscillations in MCU-KO Jurkat cells were of longer duration compared with those of the parental cells (D and E).We determined the effect of MCU-KO on Callations , A and Bal cells , A–C. Fual cells , D and E2+, evoked regenerative Ca2+ oscillations in the majority of cells (F and I–K). Note that HEK293 cells were the only cell line studied where SOCE in response to thapsigargin was not significantly enhanced by MCU-KO. Yet, MCU-KO HEK293 cells showed a higher proportion of cells responding with a Ca2+ plateau at the expense of cells responding with repetitive Ca2+ oscillations compared with their parental counterparts (I–K). Furthermore, when oscillating cells are considered MCU-KO HEK293 cells showed a higher frequency of cytosolic Ca2+ oscillations than their parental counterparts (H), suggesting that MCU-KO leads to higher cytosolic Ca2+. It is not obvious why the cytosolic Ca2+ signal in response to thapsigargin is similar between MCU-KO HEK293 cells and their parental counterparts (despite clear differences in Ca2+ oscillations triggered by agonist). We reasoned that differences in mitochondrial and/or cytosolic volumes between these different cells might play a role. We performed high-resolution three-dimensional multispectral confocal microscopy to generate volumetric reconstructions of the cytosol and mitochondria of Jurkat, A20, RBL-1, and HEK293 cell lines (A–E). Our volumetric measurements showed that HEK293 cells have a higher surface area and cytosolic and cell volume compared with all other cell lines (A–G). However, we found no obvious correlation between the mitochondrial volume or the mitochondria/cell ratio and the differences in SOCE in response to thapsigargin between parental and MCU-KO cells (D and E). Of interest, side-by-side comparisons of mitochondrial Ca2+ extrusion between wildtype HEK293, Jurkat, A20, and RBL-1 cells using the permeabilized cell preparation showed that HEK293 cells have significantly enhanced mitochondrial Ca2+ extrusion compared with all other cell types (H–K). The enhanced mitochondrial Ca2+ extrusion in HEK293 cells suggests a moderate role of mitochondria in buffering cytosolic Ca2+ in these cells . This might contribute to the blunting of small differences in the Ca2+ signal between MCU-KO and parental HEK293 cells under the thapsigargin protocol. Additional studies are needed to resolve this issue.Stimulation of HEK293 with a low concentration of carbachol (CCh) (10 μM CCh), in the presence of 2 mM extracellular Caterparts , I–K. Futerparts H, suggesll lines , A–E. Oull lines , A–G. HoKO cells , D and Ell types , H–K. Th2+ stores in MCU-KO and wildtype HEK293 cells by transiently expressing the genetically encoded Ca2+ sensor R-CEPIAer. Treatment of cells with supramaximal concentration of carbachol (300 μM CCh) in Ca2+-free solution caused a significantly bigger drop of CEPIAer fluorescence in MCU-KO cells compared with parental cells (L), suggesting a higher ER Ca2+ content in MCU-KO cells compared with parental cells. These CEPIAer fluorescence measurements are represented as normalized data in Figure 6M. We did not resolve a difference in the rate of ER Ca2+ depletion between wildtype and MCU-KO HEK293 cells (N). However, the rate of ER Ca2+ refilling after CCh wash-off and addition of 2 mM Ca2+ to the bath was significantly faster in MCU-KO HEK293 cells (O), suggesting that MCU function limits cytosolic Ca2+ and ER Ca2+ content.We also performed measurements of ER Caal cells L, sugges93 cells N. Howeve93 cells O, sugges2+ oscillatory frequency of MCU-KO cells is due to increased IP3R activity, we used total internal reflection fluorescence (TIRF) microscopy to measure the elementary Ca2+ signals called Ca2+ puffs, which are mediated by IP3Rs. We loaded parental HEK293 and MCU-KO cells with a Ca2+ dye and caged IP3 . The amplitude distribution of puffs did not differ between HEK293 and MCU-KO cells with most puffs ranging from 0.2 to 0.7 peak amplitude (C and H). The mean rise (r) and fall (f) time of the Ca2+ puffs were also similar between HEK293 and MCU-KO cells (D and I), indicating that the fundamental biophysical properties of IP3R clusters were not different between these two groups of cells. Furthermore, there was no difference between both groups of cells in the proportion of cells that showed a globalized Ca2+ signal within 60 s (E and J). These data suggest that MCU knockout does not alter IP3R channel activity.To determine whether enhanced Caf ci-IP3 . Howevermplitude , C and HKO cells , D and Ihin 60 s , E and J3R activity is unaltered by MCU-KO, the simplest explanation for enhanced Ca2+ oscillations in MCU-KO cells is that MCU-KO causes extramitochondrial Ca2+ buildup as mitochondria are unable to buffer their allocated share of cytosolic Ca2+. Hence, cytosol-ER-mitochondrial Ca2+ transfer would be backed up in MCU-KO cells, leading to faster ER refilling and/or increased net cytosolic Ca2+. We tested whether the net increase in global cytosolic Ca2+ we observed in MCU-KO cells is associated with enhanced NFAT activity, which is controlled by Ca2+ microdomains near CRAC channels. NFAT activity was significantly enhanced in MCU-KO cells of both HEK293 (A–C) and Jurkat cells (D–F) compared with their respective controls. We determined the effect of MCU-KO on NFAT4 nuclear translocation in HEK293 cells using a reporter NFAT4-GFP construct coupled to fluorescence microscopy (A–C). In Jurkat cells, we determined the effect of MCU-KO on native NFAT1 nuclear translocation using an NFAT1-specific antibody and ImageStream (D–F). Stimulation with 10 μM CCh caused a significantly bigger nuclear translocation of NFAT4 in MCU-KO HEK293 cells when compared with parental controls (A–C). Using a multispectral imaging flow cytometer (ImageStream) we tracked native NFAT1 nuclear translocation in response to ER store depletion with 2 μM thapsigargin for 30 min in populations of tens of thousands of MCU-KO Jurkat cells and their parental counterparts. MCU-KO Jurkat cells showed a significantly more robust nuclear translocation of endogenous NFAT1 compared with the parental Jurkat cells (D–F). These data suggest that the global cytosolic Ca2+ increase triggered by deletion of MCU leads to sufficient Ca2+ buildup at the vicinity of CRAC channels to enhance NFAT nuclear translocation.Given IPh HEK293 , A–C andat cells , D–F comcroscopy , A–C. IngeStream , D–F. Stcontrols , A–C. Usat cells , D–F. Th2+ upon MCU-KO also occurs in primary cells and is not restricted to cell lines, we generated tissue-specific knockdown (KD) mice for CD4+ T cells and B cells (see + T cell–specific MCU-KD (A–G). The B cell–specific MCU-KD mice were generated using the noninducible MB1 Cre line (H–M). The LoxP-Cre system led to reduction in MCU protein expression by ∼60% to 80% .To determine if the increase in cytosolic Cac MCU-KD , A–G. ThCre line , H–M. Th+ T cells were isolated with negative selection from spleens of MCUflx/flx mice (MCUflx/flx CD4Cre(-)) and MCUflx/flx CD4CreERT2 mice (MCUflx/flx CD4Cre(+)), activated in vitro using anti-CD3/anti-CD28 conjugated beads and mouse interleukin-2 (IL-2) and treated with (Z)-4-Hydroxytamoxifen (4-OHT) to induce Cre activity for 96 h before experimentation. Control cells for both MCUflx/flx CD4Cre(+) and MCUflx/flx CD4Cre(-) conditions originated from the same mice but were not subjected to 4-OHT treatment. We verified that the administration of 4-OHT does not alter MCU protein levels in MCUflx/flx CD4Cre(-) mice (C). Stimulation of cells with 2 μM thapsigargin showed that only MCU-KD CD4+ T cells, namely, CD4+ T cells isolated from MCUflx/flx CD4Cre(+) mice and treated with 4-OHT, displayed significantly higher SOCE (E–G). Because the B cell–specific MB1Cre line is noninducible, primary B cells were isolated with negative selection from spleens of MCUflx/flx mice either with MB1Cre (MCUflx/flx MB1Cre(+)) or without (MCUflx/flx MB1Cre(-)) and immediately used for Ca2+ imaging to measure SOCE. Stimulation of B cells with 2 μM thapsigargin showed that B cells isolated from MCUflx/flx MB1Cre(+) showed significantly higher SOCE compared with B cells isolated from MCUflx/flx MB1Cre(-) mice (J and K).Primary CD4(-) mice C. Stimulher SOCE , E–G. Be(-) mice , J and K+ T cells from MCUflx/flx CD4Cre(+) mice precluded us from performing proliferation assays on these primary CD4+ T cells. We did, however, perform proliferation assays on B cells isolated from MCUflx/flx MB1Cre(+) and MCUflx/flx MB1Cre(-) mice. B cell proliferation induced by weak B cell receptor (BCR) stimulation can be enhanced by Ca2+-independent signals such as costimulation with anti-CD40 or stimulation of Toll-like receptors by lipopolysaccharides (LPSs) (via their BCR with anti-IgM (L), costimulated with anti-IgM + anti-CD40 (M) or stimulated with LPS alone (C), or stimulated with anti-CD40 alone as a control (D) and B cell proliferation was monitored using the CFSE dye (A). There were no differences in cell viability between different groups of cells and stimulatory conditions (B). Primary B cells from MCUflx/flx MB1Cre(+) mice (MCU-KD B cells) showed significantly enhanced proliferation in response to BCR stimulation with anti-IgM compared with control MCUflx/flx MB1Cre(-) B cells as documented by dilution of the CFSE dye with increased proportion of B cells that underwent a third cycle of division (L). As expected, this difference in B cell proliferation was not observed when cells were costimulated with anti-IgM + anti-CD40 (M) or when stimulated with LPS (C). Stimulation with anti-CD40 alone had marginal to no effect on B cell proliferation (D).The induction of MCU-KO with 4-OHT for 5 days in CD4s (LPSs) . Therefoanti-IgM L, costimnti-CD40 M or stimPS alone C, or sti control D and B cCFSE dye A. There nditions B. Primardivision L. As expnti-CD40 M or whenwith LPS C. Stimulferation D.2+ transport alone  can change the properties of cytosolic Ca2+ rise activated by passive store depletion with thapsigargin as well as Ca2+ oscillations activated by agonist stimulation. We took an existing model of Ca2+ oscillations  but•Model 3 is the same as model 2 but includes the mitochondrial membrane potential and metabolism.•3R model and includes models of the mitochondrial membrane potential and metabolism, although it does not include MAMs.Model 4 is the model of  with no •Model 5 extends model 3 to include MAMs, following .•3R model.Model 6 is the model of  with no First, we tested how mitochondrial Callations , 42, 43,3R, the mitochondrial potential, mitochondrial metabolism, and MAMs. Model 1 is considerably simpler than the others, whereas models 5 and 6 are the most complex.Our six models thus combine in various ways different models of the IP2+ rise, we found that MCU-KO and MCU-KO + NCLX-KO cause similar increases in cytosolic [Ca2+], whereas NCLX-KO leads to a slight decrease (A–E), in agreement with our experimental data with MCU and previous work with NCLX . These thapsigargin simulations could not be performed for models 1, 4, and 6 as discussed in more detail in These simulations were performed for models 2, 3, and 5 with three variations on model 5 representing different percentages of total IP2+ oscillations, the qualitative results from each model differ significantly. For each model, the oscillation period is plotted against the relative [IP3], which is a model proxy for agonist stimulation (F–L). In each case, increasing agonist concentration increases the oscillation frequency . Knockout of the MCU results in oscillations that, in some models, are slightly faster but in others are slightly slower. In some cases (models 2 and 4), the effect on oscillations changes as agonist stimulation increases. Furthermore, in all cases, for a small range of high agonist concentrations, MCU-KO can turn a plateau response into an oscillatory response (this is the region where the red “MCU-KO” curves overlap the black wildtype “WT” curves). It follows that mitochondrial Ca2+ transport does not have a simple, monotonic effect on the properties of Ca2+ oscillations. Instead, the effects of mitochondrial Ca2+ transport are critically dependent on the exact details of each mitochondrial Ca2+ flux, as well as other cellular parameters, such as the choice of IP3R model, the relative mitochondrial volume, or the proportion of the ER membrane that is closely associated with the mitochondrial membrane. In particular, it is not correct to say that “buffering” by the mitochondria will necessarily increase oscillation period; there is no clear relationship between mitochondrial Ca2+ transport and oscillation frequency.When considering agonist-evoked Camulation , F–L. InCRAC microdomain (IM model) whereby MCU activity prevents slow CDI of ICRAC. This model also considers that 10% of IP3Rs are within MAMs but does not include the equations for mitochondrial metabolism and membrane potential. The inclusion of the mitochondrial metabolism equations makes no qualitative difference to the results. Thus, the IM model is just model 2 with the inclusion of ICRAC CDI and MAMs with all other parameters being unchanged from model 2. We modeled CDI of ICRAC after  are required to explain the experimental data.In summary, our mathematical modeling predicts that all the experimental results presented here are consistent with the hypothesis that the MCU affects SOCE activated by thapsigargin and Ca2+ uptake and extrusion, mitochondria can shape receptor-activated intracellular Ca2+ signaling. Mitochondria rapidly take up Ca2+ during receptor-evoked Ca2+ signaling and then extrude it much more slowly  are required to explain our experimental data. Ishii et al. . The absence of mycoplasmal contamination was verified for all cell lines using a sensitive PCR-based detection kit (ABM: G238).Parental HEK293, Jurkat E6-1, RBL-1, DLD1, HCT116, and A20 cell lines were purchased directly from ATCC . HEK293, RBL-1, and HeLa cells were cultured in high-glucose (4.5 g/l) Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum and 1× Antibiotic–Antimycotic solution. Jurkat, A20, and DLD1 were maintained in RPMI 1640 with L-glutamine supplemented with 10% heat-inactivated fetal bovine serum and 1× Antibiotic–Antimycotic solution. HCT116 were cultured in McCoys 5A medium supplemented with 10% heat-inactivated fetal bovine serum and 1× Antibiotic–Antimycotic solution. All cell lines were stored in a heated (37 °C) humidified incubator under standard cell culture conditions -treated 25-mm glass coverslips 30 min prior to dye loading. Once attached, HEK293, HeLa, HCT116, and DLD1 cells were incubated in Dulbecco's modified Eagle's medium containing the ratiometric Ca2+ indicator Fura-2 AM (2 μM) for 30 min. RBL-1 cells were loaded with 4 μM Fura-2AM following the same procedure. Jurkat and A20 cells were incubated with 2 μM Fura-2 AM in Hepes-buffered salt solution  supplemented with 2 mM Ca2+ for 30 min at room temperature. Before imaging, all coverslips were transferred from a standard six-well plate into an Attofluor cell chamber and washed 3× with Ca2+-containing HBSS to remove excess Fura-2 AM. Using a fast shutter wheel (Sutter Instruments), cytosolic Ca2+ was measured by alternatively exciting Fura-2 with 340- and 380-nm wavelengths and recording the resulting 510-nm dye emission on a pixel-by-pixel basis through a 20× fluorescence objective paired with a Hamamatsu Flash 4 camera and processed using Leica Application Suite X software.Adherent and semiadherent cell lines were seeded onto round 25-mm glass coverslips 24 h prior to imaging at a concentration of 1.5 × 106 cells were incubated with a solution containing 1 μg of NFAT4-GFP and 3 μl Lipofectamine 2000 for 3 h. One day later, coverslips containing transfected cells were transferred to imaging chambers and washed with Ca2+-containing HBSS as described above. Using a Leica imaging system and a 40× oil immersion objective, NFAT4-GFP was excited at 488 nm, and emission spectra were selectively captured through a GFP filter cube. Real-time analysis of NFAT4-GFP nuclear translocation in response to 10 μM Cch stimulation was calculated using the equation:Measurement of NFAT4 nuclear translocation in HEK293 cells was achieved by transient overexpression of NFAT4-GFP (Addgene #21664) as previously described , 42, 43.2+ store depletion and refilling was done by transfecting parental and MCU-KO HEK293 cells with red R-CEPIA1er (Addgene: #58216) using Lipofectamine 24 h prior to imaging. Cells expressing R-CEPIA1er were then excited at 552 nm and relative ER Ca2+ measurements recorded through a 40× oil immersion objective. Immediately after identifying R-CEPIA1er-positive cells the bath solution was replaced with nominally Ca2+-free HBSS. One minute into the experiment cells were treated with CCh followed by washout and readdition of 2 mM Ca2+ to the bath. The resulting traces represent ±SEM of all cells from at least three independent experiments.Quantification of ER Ca2, 4.7 mM KCl, 1.26 mM CaCl2, 10 mM Hepes, 1 mM Na2HPO4 at pH 7.4). Cells were subsequently incubated with Cal520-AM  and 6-O-[methyl]-2,3-O-(1-methylethylidene)-D-myo-Inositol 1,4,5-tris[bis[(1-oxopropoxy)methyl]phosphate]  in imaging buffer with 0.01% bovine serum albumin in dark at room temperature. One hour later, cells were washed three times with imaging buffer and incubated in imaging buffer containing EGTA-AM . After 45 min incubation, the medium was replaced with fresh imaging buffer and incubated for an additional 30 min at room temperature to allow for de-esterification of loaded reagents  in a 35-mm dish for 2 days and imaged as described . Prior treagents .3, UV light from a 405-nm laser at 50% power was introduced to uniformly illuminate the field of view. Both the intensity of the UV flash (∼2.4 mW) and the duration (1000 ms) for uncaging IP3 were optimized to prevent spontaneous puff activity in the absence of loaded ci-IP3. TIRF images were captured using 4 × 4 pixel binning (433.333 nm/pixel) from equal field of views for WT-HEK293 and MCU-KO cells at a rate of ∼166 frames per second. After visualizing images with the cellSens [Ver.2.3] life science imaging software (Olympus), images were exported as vsi files. Images, 10 s before and 60 s after flash photolysis of ci-IP3, were captured.Following loading, the coverslip was mounted on chambers and imaged using an Olympus IX83 inverted TIRF microscopy equipped with oil immersion Olympus UPLAAPO60XOHR (NA = 1.5) objective. The cells were illuminated using a 488-nm laser to excite Cal-520, and the emitted fluorescence was collected through a band-pass filter by a Hamamatsu ORCA-Fusion CMOS camera. The angle of the excitation beam was adjusted to achieve TIRF with a penetration depth of ∼140 nm. Images were captured from a field of view by directly streaming into RAM. To photorelease IP3 were averaged to obtain a ratio image stack (F/Fo) and standard deviation for each pixel for recording up to 20 s following photolysis. The image stack was Gaussian filtered, and pixel that exceeded a critical value  was located. The “Detect-puffs” plug-in was utilized to detect the number of clusters (puff sites), number of events (number of puffs), amplitudes, and durations of localized Ca2+ signals from individual cells. All the puffs identified automatically by the algorithm were manually confirmed before analysis  or 50 mM BAPTA. Clampfit 10.3 software was used for data analysis. The solutions employed for patch clamp recordings are as follows.RBL-1 cells were seeded onto 30-mm round glass coverslips 12 h before recording; Jurkat cells were seeded onto Poly-L-lysine-coated coverslips 1 h before recording. Native Ita 1440A , 72, 73.4, 10 mM Hepes, 20 mM CaCl2, and 10 mM glucose .115 mM Na-methanesulfonate, 10 mM CsCl, 1.2 mM MgSOBath solution:155 mM Na-methanesulfonate, 10 mM HEDTA, 1 mM EDTA, and 10 mM Hepes .Divalent-free bath solution:2, and 10 mM Hepes, 0.03 mM IP3, 2 mM pyruvic acid, 2 mM malic acid, 1 mM NaH2PO4, 2 mM Mg-ATP (pH 7.2 adjusted with CsOH).135 mM Cs-methanesulfonate, 1 mM EGTA, 8 mM MgClPipette solution 1 (low buffer):2, and 10 mM Hepes (pH adjusted to 7.2 with CsOH).85 mM Cs-methanesulfonate, 50 mM Cs-1,2-bis-(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (Cs-BAPTA), 8 mM MgClPipette solution 2 (high buffer):2+ and in the presence of 2 μM thapsigargin for 3 to 5 min before beginning of recordings. The bath and pipette solutions were as follows:Recordings of CRAC channel store-independent slow CDI were performed according to . Cells w2+ bath solution (in mM): 155 NaCl, 4.5 KCl, 3 MgCl2, 10 D-glucose, and 5 Hepes (pH = 7.4 with NaOH).0 mM Ca2+ bath solution (in mM): 125 NaCl, 4.5 KCl, 20 CaCl2, 3 MgCl2, 10 D-glucose, and 5 Hepes (pH = 7.4 with NaOH).20 mM CaBath solutions:2, 0.66 CaCl2, 1.2 EGTA, 10 Hepes (pH = 7.2 with CsOH).Low buffer pipette solution (in mM): 140 Cs-methanesulfonate, 2 MgCl2, 0.66 CaCl2, 1.2 EGTA, 10 Hepes, 2.5 mM malic acid, 2.5 mM pyruvate acid, 1 mM NaH2PO4, 5 mM Mg-ATP, 0.5 mM Na-GTP (pH = 7.2 with CsOH).Low buffer + mitochondrial cocktail pipette solution (in mM): 140 Cs-methanesulfonate, 2 MgClPipette solutions:To determine the cytosolic volume of all cell lines pEGFP-N1 (Addgene# 6085-1) was transiently overexpressed. One day before cytosolic volume measurement all cell lines were electroporated using the Amaxa Nucleofector II. Cells were first resuspended in the transfection reagent supplemented with pEGFP-N1 (1 μg). Cell line–specific protocols Q-001, X-001, T-030, and L-013 were used to electroporate HEK293, Jurkat, RBL-1, and A20 cells, respectively. One day later, live cells were stained with MitoTracker Deep Red (200 nM) for 15 min at room temperature in the dark before imaging on a Leica DMI8 confocal microscope. Stained and washed cells were observed through a 40× oil immersion objective. Both GFP and MitoTracker signals were then collected from randomly selected fields of view by compiling Z-Stacks of the lower and upper bounds of all cells. MitoTracker and GFP channels were then used to reconstruct 3D surfaces using IMARIS Cell Biology software (Oxford Instruments). Once both cytosolic (GFP) and mitochondrial (MitoTracker) surfaces were reconstructed, volume measurements were recorded individually.6 cells per replicate. Cells were then centrifuged and media replaced with HBSS supplemented with 2 mM Ca2+ to eliminate the possibility of serum-mediated activation. Experimental cells were then stimulated with α-CD3 (0.125 μg/ml) or treated with Tg (2 μM) to induce cytosolic Ca2+ oscillations or evoke SOCE. Following stimulation, cells were immediately fixed and permeabilized using a transcription factor staining kit according to the manufacturer protocol (Tonbo Biosciences Cat: TNB-0607-KIT). After fixation and permeabilization, cells were spun down, the supernatant was removed, and stained with a primary conjugated NFAT1-FITC antibody  for 1 h at room temperature protected from light. Unbound NFAT1 antibody was then washed away and cells were resuspended in 40 μL of PBS. Nuclear staining was performed immediately before image acquisition by adding 10 μL of the nuclear stain Dapi (5×) to give a final concentration of (0.25 μg/ml).Jurkat cells were first counted and transferred to 1.5-ml Eppendorf tubes at a concentration of 1.2 × 10Images were acquired using a 10-color Amnis ImageStream X Mk II imaging cytometer. Channels 1 and 9 (430–480 nm) were used to obtain bright-field images, channel 2 was used for FITC (Ex:488 Em:480–560 nm), and Dapi through channel 7 (Ex:405 Em:430–505 nm). Prior to final image acquisition cells were gated to exclude debris and doublets and Ex laser power was adjusted to prevent pixel saturation. From each stained and unstained sample 3000 to 5000 in-focus single-cell events were collected. After running all samples single color compensation samples were run for FITC then Dapi. All data were analyzed using IDEAS software version 6.2 (Amnis Corp). During the analysis in-focus cells were first identified, then doublets were excluded, and finally cells positive for both DAPI and NFAT1 were selected for further analysis. The extent of nuclear NFAT1 was then determined using the nuclear translocation module. Raw nuclear similarity scores were then exported to Prism GraphPad resulting in the final figures.+ T cells by breeding MCUflx/flx mice  (T2 (B6(129X1)-Tg(Cd4-cre/ERT2)11Gnri/J) purchased from Jackson laboratory -Cd79atm1(cre)Reth/EhobJ) mice from Jackson laboratory with MCUfl/fl mice. From that cross MCUfl/wtMB1Cre+/- were then bred with MCUfl/fl mice to generate the experimental and control groups (We generated inducible MCU-KO CD4versity)  with CD4boratory . The resl groups . PCR-basfl/fl CD4Cre+ or MCUfl/fl MB1Cre+ mice and control counterparts were humanely sacrificed immediately prior to immune cell isolation. Superficial cervical and inguinal lymph nodes were first collected followed by the spleen. All lymphoid tissues were then disrupted by passing the tissue through a 70-μm mesh screen. The resulting single-cell suspension was than washed with 10 ml of cold RPMI and resuspended in 1 ml of Robosep buffer (STEMCELL technologies) prior to the negative selection of B or T cells. Using the corresponding negative selection kit primary mouse CD4+ T cells and B cells were isolated using the magnetic beads and antibody cocktail as recommended by the manufacturer.About 6 to 8-week-old MCU+ T cells were then cultured ex vivo to increase the efficiency of CREERT2 activity. Naïve primary CD4+ T cells were maintained in RPMI 1640 supplemented with 10% fetal bovine serum, 1× Antibiotic-Antimycotic, 1× Glutamax, mIL2 (30 U/ml), and Mouse T-Activator anti-CD3/CD28 Dynabeads (Thermo Fisher) at a concentration of one bead per cell. Experimental mice (MCUfl/fl CD4Cre+) and control (MCUfl/fl CD4Cre-) mice were both treated with 2 μM 4-OHT for 4 days. As a control, the second group of (MCUfl/fl CD4Cre-) mice was only administered vehicle (dimethyl sulfoxide). Cells were subsequently washed and beads removed.Isolated CD4via nucleofection as described. Forty-eight hours after transfection, CRISPR/Cas9-positive cells were selected by treating the culture medium with puromycin (2 μg/ml) (Gemini Bio Products). After 6 days of selection, cells were trypsinized and seeded at a concentration of 0.5 cells per well into a 96-well plate. Resulting colonies were then screened using the Guide-it Mutation Detection Kit . Knockout was further confirmed via Western blot, functional analysis, and genomic sequencing.To generate MCU-KO HEK293 and HCT116 cells, human MCU gRNA3 (hMCU gRNA1) was subcloned into the lentiCRISPR v2 backbone  and transfected into wildtype HEK293 cells An alternative method was used for all other cell lines to avoid any potential effects of constitutive Cas9 expression. The same two human gRNAs (hMCU gRNA1and 2) were used to generate all Jurkat and DLD1 MCU-KO clones. Two independent mouse-specific gRNAs (mMCU gRNA 1 and 2) were used to generate MCU-KO A20 cells, whereas a single gRNA (rMCU gRNA1) was used to generate MCU-KO RBL-1 cells. All gRNA sequences can be found in 2+ uptake were used for all experiments.gRNAs for all cell lines were cloned into one of two fluorescent vectors to allow for the identification of transiently transfected cells using fluorescence-activated cell sorting (pSpCas9(BB)-2A-GFP and pU6-(BbsI)_CBh-Cas9-T2A-mCherry: Addgene). One day after transfection with the corresponding gRNA single cells with high gRNA expression were sorted into the wells of a 96-well plate using a fluorescence-activated cell sorting Aria SORP high-performance cell sorter. After sorting, cells were maintained in complete medium until colonies began to form ∼2 to 3 weeks depending on the cell line. Visible colonies were collected and analyzed by Western blot to identify knockout clones. Positive clones were functionally analyzed, and those clones that showed no mitochondrial CaFurthermore, all clones were sequenced to validate gRNA efficiency. In HEK293 MCU-KO cells we observed two different genomic deletions  that resulted in a genomic frameshift and the formation of early stop codons. In Jurkat MCU-KO cells we also observed two different mutations. The first was a 7-nt insertion into exon 3, and the second caused multiple indels that resulted in a genetic frameshift and formation of an early stop codon in exon 3. In RBL-1 we observed two different deletions from exon 3 (16 and 400 nt) that both resulted in genomic frameshifts. Two different deletion events were also observed in A20 cells; the first was a single-nucleotide deletion and the second a 7-nt deletion from exon 1. We also observed two different deletions in HCT116, the first being a 16-nt deletion and the second a 19-nt deletion; both occurred in exon2 and resulted in frameshifts. In DLD1 cells, two unique 2-nt deletions were observed within exon 4 that both resulted in genomic frameshifts and the introduction of an early stop codon.via centrifugation prior to lysis in ice-cold RIPA buffer  supplemented with protease and phosphatase inhibitor  for 15 min on ice. Crude protein lysates were clarified by centrifugation  and the supernatants collected. Total protein concentration was determined using the Pierce BCA assay as described prior to loading a NuPAGE precast 4% to 12% Bis-Tris gel. After subjecting the samples to 120 V for 1.5 h proteins were transferred to a polyvinylidene difluoride membrane using a Bio-Rad Criterion blotter and 1× NuPAGE Transfer Buffer (Invitrogen). After 1 h of transferring at 95 V the membrane was removed and blocked with LI-COR Tris-buffered saline (TBS) buffer for 1 h at room temperature. Primary antibodies were added to blocked membranes overnight at 4 °C in a shaker. On the next day, PVDF membranes were then washed 3× with TBST and probed with the corresponding species-specific LI-COR secondary antibody for 1 h at room temperature. Then the membrane was washed 3× with TBST and immediately imaged using a LI-COR odyssey imaging system and fluorescence quantified using Image Studio lite software (LI-COR).Cells were collected from culture flasks or isolated from mice, washed with ice-cold phosphate-buffered saline, and pelleted 6), Jurkat (10 × 106), A20 (10 × 106), and RBL-1 (7 × 106) cells were washed with PBS immediately before resuspension in an intracellular medium consisting of 10 mM NaCl, 120 mM KCl, 1 mM KH2PO4, 20 mM Hepes-Tris, pH 7.2, and 2 μM of the SERCA pump inhibitor thapsigargin. Just before beginning the measurement 2 mM succinate was added to the intracellular medium. Cells were permeabilized with 40 μg/ml digitonin, and simultaneous measurements of mitochondrial membrane potential (mtΨ) and extramitochondrial Ca2+ were achieved by loading permeabilized cells with the ratiometric mtΨ dye JC-1 (800 nM) and the fluorescent Ca2+ indicator Fura2-FF (0.5 μM). Both dyes were excited, and emissions were recorded using a dual-wavelength excitation and emission fluorimeter .HEK293 (6 × 106) and Jurkat cells (4 × 106) parental and MCU-KO cell lines using the RNeasy Mini Kit (Qiagen). Isolated RNA was then analyzed using a NanoDrop spectrophotometer (Thermo Scientific), and 1 μg of DNAse I-treated (Thermo Fisher) RNA was reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Total cDNA (1 μl) was diluted 1:5 and then combined with target-specific primers, SYBR Green qPCR Master Mix (Applied Biosystems), and mqH2O resulting in a 10-μl total reaction. All targets were subjected to the same PCR protocol that began with an initial activation step for 2 min at 50 °C followed by a 95 °C for 2 min melt step. The initial melt steps were then followed by 40 cycles of 95 °C for 15 s, a 15-s annealing step at 54.3 °C, and target amplification at 72 °C for 30 s. Once these steps were complete, a standard melt curve was generated to ensure primer specificity. Analysis of target and control samples was carried out using the comparative Ct method. All samples were normalized to the average of the two reference genes, GAPDH  and NONO (Non-POU Domain Containing Octamer Binding). All samples were run in triplicate to ensure reproducibility.Total mRNA was isolated from HEK293 , a flux through the NCLX (JNCLX), and (following (x). The four different models of mitochondrial Ca2+ transport are as follows.To incorporate mitochondrial transport, the base model was supplemented by the addition of three terms each representing a mitochondrial Caollowing ) a backgon = 3 μM-1s-1, koff = 5/s, bt = 5 μM were chosen so as to give a physiological resting value of cm and to ensure that the buffering was close to linear. The variable cm denotes the mitochondrial [Ca2+].Model 1. The mitochondria are modeled as simple buffers. ThusMCU = 15 μM/s, K1 = 6 μM, K2 = 0.38 μM, VNCLX = 0.005 μM/s, L = 50, and kx = 0.01/s. The variable c denotes the cytosolic [Ca2+].Model 2. The mitochondrial fluxes are modeled following  but with3R is taken from . However, the IP3R model is the same as in model 3, and thus models 5 and 6 give significantly different outcomes. To maintain a physiological resting cm, VMCU was increased to 150 μM/s.Model 5. The mitochondrial fluxes are identical to those of model 3, but now MAMs are included, in the manner of   of J2+] depending on whether we are modeling the influx into the IM or into the cytosol. Finally, Jin was replaced by hicracJin, so that an increase in [Ca2+] at the IM causes inhibition of the CRAC channel, with time constant τicrac. Calcium moves from the IM to the cytosol at a rate proportional to the concentration difference of the two compartments, with rate constant 0.001/min. Other parameter values  were chosen to give qualitative agreement with Figure 2A of . Since model 1 contains no specific models of either the MCU or the NCLX, simulations of MCU or NCLX KO would be arbitrary and unreliable at best. Model 3 is a closed-cell model , which again is incompatible with KO experiments that change the total Ca2+ in the cell, making KO simulations impossible. Finally, model 6 is unable to simulate MCU/NCLX KO owing to resultant instabilities in the model. We do not yet understand the source of these instabilities, which remain a topic of current investigation.Model 1 models mitochondria as a simple Cap-values of <0.05, <0.01, <0.001, and <0.0001, respectively. Statistical significance or lack thereof are reported within the figure legends, and the type of statistical tests performed and resulting p-values from that test are recorded in All statistical analyses were performed using GraphPad Prism version 9. Statistically significant differences between groups were identified within the figures where ∗, ∗∗, ∗∗∗, and ∗∗∗∗ indicate Statistical analyses and raw data of all experiments herein, including images of unprocessed Westerns, are included in a separate file. All original cell lines reported herein are available from the lead investigator upon request and completion of an MTA.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Despite the success of the combination of venetoclax with the hypomethylating agents (HMA) decitabine or azacitidine in inducing remission in older, previously untreated patients with acute myeloid leukemia (AML), resistance - primary or secondary - still constitutes a significant roadblock in the quest to prolong the duration of response. Here we review the proposed and proven mechanisms of resistance to venetoclax monotherapy, HMA monotherapy, and the doublet of venetoclax and HMA for the treatment of AML. We approach the mechanisms of resistance to HMAs and venetoclax in the light of the agents’ mechanisms of action. We briefly describe potential therapeutic strategies to circumvent resistance to this promising combination, including alternative scheduling or the addition of other agents to the HMA and venetoclax backbone. Understanding the mechanisms of action and evolving resistance in AML remains a priority in order to maximize the benefit from novel drugs and combinations, identify new therapeutic targets, define potential prognostic markers, and avoid treatment failure. For decades, the cornerstone of treatment for primary AML has been induction therapy with cytarabine and an anthracycline followed by consolidation with high-dose cytarabine chemotherapy or hematopoietic stem cell transplant (HSCT). Most patients with AML respond to initial induction chemotherapy and achieve a complete remission (CR), with rates ranging between 40 and 90% in different studies-5. However, long-term survival rates remain significantly lower at about 40%-50% for adults younger than 60 years of age and less than 10% in adults age 60 years and older,6. The high rate of relapse in older patients has been attributed to several factors, including lower tolerance to chemotherapy, poor performance status, comorbid diseases, a higher incidence of secondary AML, a higher incidence of adverse karyotypes, and acquired drug resistance,8. In addition, 20% of patients treated with standard chemotherapy experience primary induction failure,10. Therefore, mechanisms of de novo and acquired resistance to chemotherapy remain central barriers to achieving long-term remission.Acute myeloid leukemia (AML) is a heterogeneous disease with diverse molecular profiles, clinical outcomes, and disease-specific mortality. In 2020, there will be an estimated 20,000 new cases of AML and 11,200 deaths due to the disease in the U.S.-14. For instance, the presence of the genetic abnormalities t or inv(16)/t is associated with a high rate of response to conventional combination chemotherapy and a favorable prognosis,11. Conversely, a complex karyotype or aneuploidy is associated with resistance to chemotherapy and a poor prognosis,11. Although the relatively high proportion of patients with cytogenetically normal AML limits the prognostic and clinical utility of these cytogenetically defined groups, recent advances in the understanding of the molecular landscape of AML have simultaneously expanded our prognostic ability and facilitated the development of agents targeting specific actionable alterations, including mutations of fms-like tyrosine kinase 3 (FLT3) and isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2)-17. Agents that target these abnormalities, including IDH inhibitors , FLT3 inhibitors , and others, have offered an opportunity for remission with AML harboring targetable mutations,18. Because targetable genetic mutations are found in only a minority of patients with AML, the parallel introduction of apoptosis-inducing therapies has shown promise in overcoming conventional chemotherapy resistance and can be used irrespective of the genetic signature,20.The biological basis for this therapy resistance has been extensively studied. Certain cytogenetic abnormalities and genetic mutations are predictive of clinical course, prognosis, and response to conventional chemotherapy or more novel targeted therapies. However, response rates to HMAs alone are low (10%-50%), with a median survival of about 6-10 months-25. The observation that antiapoptotic proteins, including BCL2, BCLXL, and MCL1, are frequently overexpressed in AML and are associated with resistance to chemotherapy, eventually led to the addition of venetoclax, an oral selective small molecule inhibitor of BCL2, to LDAC or HMA therapy, initially in older adults with primary or secondary AML who were ineligible for conventional chemotherapy. Rates of CR or CR with incomplete hematologic recovery (CRi) were 54% for the LDAC/venetoclax regimen and 67% for the HMA/venetoclax doublet, with a significant extension in overall survival (OS) to a median of 10-18 months. These findings were more recently confirmed by the results of the phase III randomized placebo-controlled trial showing superiority of the combination of azacitidine and venetoclax over single-agent azacitidine in a group of previously untreated patients with AML who were not eligible for standard induction chemotherapy. With a CR/CRi rate of 66% in the azacitidine/venetoclax group vs. 28% in the control group, a significant prolongation in median OS was achieved (14.7 months vs. 9.6 months respectively).The “hypomethylating agents” (HMAs) azacitidine (5-azacitidine) and decitabine (5-aza-2ʹ-deoxycytidine) are nucleoside derivatives that are incorporated into DNA, where they inhibit DNA methyltransferases (DNMT). HMAs have been primarily used to treat AML patients who are 60 years of age and older or those who are unable to withstand the rigors of conventional chemotherapy. In comparison to conventional combination chemotherapy, single-agent HMA or low-dose cytarabine (LDAC) regimens are usually better tolerated and are associated with lower rates of treatment-related mortality. Therefore, although the last few years have witnessed important advances in the treatment of AML, drug resistance continues to be an important clinical problem. As the armamentarium of therapeutic options continues to expand with agents, like the Nedd8 activating enzyme (NAE) inhibitors  and the mouse double minute 2 homolog protein (MDM2) inhibitor KRT-232, being added to the HMA/venetoclax backbone, understanding the mechanisms of action and evolving resistance remains crucial in order to maximize the benefit from emerging drugs and combinations, identify new potential therapeutic targets, and determine potential prognostic markers. Hence, we review the individual actions of the HMAs and venetoclax as well as their individual mechanisms of resistance, and we then focus on potential mechanisms of resistance to the HMA/venetoclax combination.Despite these promising results, about half of the responders to azacitidine/venetoclax relapse after a median duration of about 18 months-32. Whether used as single agents or, more recently, in combination with venetoclax, HMAs have shown clinical activity in patients with primary, secondary, and relapsed/refractory AML,28,33.The HMAs azacitidine and decitabine constitute the backbone of treatment of high-risk myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML). In both azacitidine and decitabine, a nitrogen atom replaces a carbon atom in position 5 of the pyrimidine ring . There is evidence to support a dual mechanism of action for these agents: (1) a direct cytotoxic effect at higher doses, where the formation of covalent DNMT-DNA adducts leads to DNA damage; and (2) DNA hypomethylation and epigenetic modulation at lower doses with subsequent cell differentiation and tumor suppression,37.HMAs are pyrimidine analogs of the nucleoside cytidine that showed promising cytostatic activity at higher doses in the 1960s. The role of DNA methylation, specifically cytosine methylation, in the control of gene expression and epigenetic regulation was identified in different eukaryotic cells in the 1970s-41. It was consequently thought that compounds capable of changing the patterns of cytosine methylation might impact cell differentiation and differential gene expression in various cell types that harbor the same genetic information. Azacitidine and decitabine were subsequently shown to alter differentiation in cultured mouse embryo cells-44. Therefore, the focus with the use of HMAs shifted from their cytostatic and cytotoxic effects seen at higher doses to their role as potent inhibitors of DNA methylation at lower doses and with longer exposure. Importantly, at these low doses, HMAs reportedly do not cause cell cycle arrest.One of the ways HMAs exhibit their action is through epigenetic mechanisms,47. They are then further phosphorylated to diphosphate and triphosphate derivatives (5-aza-CTP and 5-aza-dCTP) by pyrimidine monophosphate and diphosphate kinases. These cytosine derivatives, like their natural counterparts, are also subject to degradation by cytidine deaminase (CDA).Azacitidine and decitabine are prodrugs that require phosphorylation to monophosphate forms by uridine-cytidine kinase (UCK) and deoxycytidine kinase (DCK), respectively. The RNA-dependent effects of azacitidine are independent of the cell cycle and affect both messenger and transfer RNA-51. The remaining 10%-20% of azacitidine is converted to 5-aza-dCTP by ribonucleotide reductase and, like the 5-aza-dCTP that is the main metabolite of decitabine, is incorporated into DNA.The fates of 5-aza-CTP and 5-aza-dCTP are somewhat different. Approximately 80%-90% of azacitidine incorporates into RNA as 5-aza-CTP, resulting in alteration of RNA methylation and inhibition of protein synthesis-59. Thus, after HMA treatment, formation of DNA-protein cross-links and loss of DNMT activity are inextricably linked.Once 5-aza-cytosine (5-aza-C) is incorporated into DNA, the downstream effects are the same whether the altered base is derived from azacitidine or decitabine. When DNMTs act on normal cytosine, they form a transient covalent bond between a cysteine at the enzyme active site and C6 of cytosine. When these same enzymes act on 5-aza-C, the same covalent adduct is formed, but it is more stable and cannot be enzymatically reversed. As a result, cells are left without the enzymatic activity of the adducted DNMT enzyme molecule and with a new covalent DNA-protein crosslink for each DNMT molecule inhibited,60,61.According to current understanding, HMA-induced hypomethylation of promoters of tumor suppressor genes leads to the interruption of feedback loops between DNA methylation and histone methylation. This reverses the silenced chromatin state of the histones at the tumor suppressor genes, thus facilitating their expression, the resulting suppression of leukemogenesis, and the induction of cellular differentiation, indicating that one or more of the DNA damage-activated kinases ATM, ATR or DNA-PK have been activated. The irreversibly bound DNMT can also lead to cell cycle arrest at the transition from S to G2 through the activation of the ATM and ATR pathways. The events that occur downstream of cell cycle arrest and lead to removal of the DNA-DNMT protein cross-links are still being elucidated. Current understanding, enhanced by the recent demonstration of DNMT inhibitor/PARP inhibitor synergy, suggests that DNMT-DNA covalent complexes might be removed by base excision repair, although the glycolyase involved and how it is activated remain to be determined. Inhibition of DNMT1 may also indirectly affect DNA repair mechanisms,66. Vispé et al. showed that combining treatment with decitabine and DNMT1 siRNA uncouples DNA damage from DNA demethylation and again suggests a dual mechanism of action for HMAs.HMAs also trigger DNA damage response pathways. At high HMA concentrations, phosphorylation of histone H2A.x is observed,33,68-72. The initial observation that a variety of tumor suppressor genes are silenced as a consequence of promoter hypermethylation in MDS and AML prompted the investigation of HMAs as epigenetic drugs in these hematological diseases. When initially tested in phase I studies in AML at higher doses (1500-2500 mg/m2 of decitabine per course), HMAs caused prolonged and dose-limiting myelosuppression with overall response rates of 30%-60%,75. The mechanism of action at those higher doses was thought to reflect formation of DNA-protein cross-links, activation of the DNA damage response, and direct cytotoxicity. Lower doses of decitabine  were subsequently found to have an overall response rate of 49% in patients with high-risk MDS in phase II studies,77. Subsequent phase III studies confirmed the clinical effectiveness of decitabine in the treatment of patients with MDS, with an overall response rate of 73% with decitabine vs. 0% with supportive care in the initial phase III trial, and 54% with decitabine in a follow-up phase II trial in elderly patients with intermediate- or high-risk MDS. Additional randomized trials have also shown a survival advantage in patients receiving azacitidine, with a median survival of 18 months in the azacitidine arm vs. 11 months in the supportive care arm in the Cancer and Leukemia Group B (CALGB) 9221 trial, and 24.5 months vs. 15 months in patients with intermediate-2- or high-risk MDS in the AZA-001 trial.HMAs have been used for the treatment of myeloid malignancies for more than a decade. The role of HMAs is currently expanding across wider age groups for various therapeutic indications (including preemptive and maintenance therapies), in novel formulations , and in combination with multiple other agents 2/day for five days in phase I and II studies in the pediatric patient population with AML and acute lymphoblastic leukemia. Six out of the 14 patients with refractory AML achieved remission, but prolonged marrow suppression was a significant adverse event. Prolonged myelosuppression was also dose limiting in a study of 154 adult patients with refractory AML treated with 150-700 mg/m2/day for 1-7 days. After a hiatus of several decades, decitabine and azacitidine were studied again in patients with AML, but at the lower doses that proved effective in MDS. In the AZA-001 trial, analysis of outcomes in a subgroup of patients with 20%-30% bone marrow blasts, who were originally classified as RAEB-t  and subsequently considered as AML by the 2000 World Health Organization classification,84, indicated prolonged median overall survival with azacitidine (24.5 months) compared to conventional care regimens such as LDAC, conventional chemotherapy, or best supportive care (16.0 months). An analysis of three CALGB trials with azacitidine 75 mg/m2/day for seven days every 28 days by intravenous (CALGB 8421) or subcutaneous routes (CALGB 8921 and CALGB 9221) showed an overall response rates of 36%-48% and median survival of 19.3 months in the azacitidine group compared to 12.9 months for the observation group. Retrospective data from European registries showed median overall survival estimates of 9-10 months,87. A large phase III study, AZA-AML-001, randomized patients older than 65 years and with > 30% bone marrow blast counts to azacitidine (75 mg/m2/day subcutaneously for 7 days every 28 days) or conventional care regimens. Although there was no statistically significant difference in survival in the overall study population , survival was significantly prolonged in the subgroups with poor-risk cytogenetics or AML with myelodysplasia-related changes, leading to the European Medicines Agency approval of azacitidine for the treatment of AML in adults above 65 years of age. A phase III study (DACO-16) that randomized 485 patients age 65 years or older to decitabine (20 mg/m2/day intravenously for 5 days every 28 days) or conventional care regimens (supportive care or LDAC) likewise failed to show a significant improvement in the primary endpoint of overall survival  but showed a higher rate of CR/CRp (CR with incomplete platelet recovery) with decitabine , leading to approval of single-agent decitabine for the treatment of AML in the older adults in Europe. More recently, the U.S. Food and Drug Administration (FDA) in September 2020 approved once-daily oral azacitidine as maintenance therapy for the continued treatment of AML in adults who achieve their first CR/CRi with induction chemotherapy and are not candidates for further intensive consolidation therapy with HSCT,72.As indicated earlier in the Introduction, HMAs are also used to treat AML. Azacitidine monotherapy was first evaluated at 150-200 mg/mA common theme across the clinical trials of HMAs in AML is a consistent trend towards improved survival despite the low CR rates of < 20%. This led to a number of studies examining the impact of adding various agents to HMAs, ultimately resulting in the HMA/venetoclax regimen described in a subsequent section of this review.When HMAs are used as single agents in myeloid malignancies, rates of remission are low, and durations of response are often short. Resistance, whether primary or secondary, is a central challenge in the treatment of MDS and AML. Therefore, potential mechanisms of resistance to HMAs have been extensively investigated. In brief, for HMAs to be active, they must accumulate in cells, undergo phosphorylation to the nucleoside triphosphates, avoid metabolic inactivation, and get incorporated into DNA. Resistance has been observed at each of these steps.. Consistent with a role for hENT1 in HMA cellular uptake, the level of hENT1 mRNA expression is significantly higher in patients who respond to decitabine. Interestingly, the decreased activity of azacitidine in cells with low hENT activity can be overcome by the use of an elaic ester of azacitidine, CP-4200, with reduced dependence on membrane transporters.Resistance to HMAs can be mediated by membrane proteins that are involved in drug uptake. Human equilibrative nucleoside transporter-1 (hENT1) facilitates transport of nucleosides, including decitabine and azacitidine, in a bidirectional manner across the cell membrane,89. Both of these changes are thought to play a role in HMA resistance. Low expression of DCK diminishes the activating phosphorylation of decitabine, and high CDA results in rapid catabolism of decitabine and azacitidine to uridine counterparts that do not contribute to DNMT inhibition,92. Consistent with these observations, malignant cell lines engineered to overexpress CDA are decitabine resistant. Azacitidine and decitabine have poor oral availability, likely reflecting high CDA expression in gastrointestinal and liver cells,93,94.The balance between HMA phosphorylation and enzymatic inactivation also plays a role in response. Cell lines that are resistant to decitabine have not only low expression of hENT1 and hENT2 but also low expression of DCK and high expression of CDA and that DCK is significantly reduced in the bone marrow and peripheral blood specimens from MDS patients who relapsed after decitabine, highlighting the importance of metabolic activation in the action of HMAs. These findings, however, have been far from universal. It has also been reported that expression of DCK or CDA does not distinguish MDS cases that respond to decitabine from those that do not, but the ratio of CDA to DCK was elevated in nonresponding cases.In the clinical setting, it has been reported that lower expression of UCK1 is associated with blunted response to azacitidine. For example, DCK protein is diminished in MDS samples with decitabine resistance and upregulated in azacitidine-resistant cells. Conversely, UCK2 protein is upregulated in decitabine-resistant cells and diminished in azacitidine-resistant cells. These observations raise the possibility of tailoring therapy based on DCK and UCK levels or diminishing HMA resistance by alternating HMAs,97.Emerging evidence suggests that metabolism of pyrimidines, including HMAs, reflects an adaptive network of enzymes that detect nucleotide levels and respond in a compensatory manner [Figure 1],98,99.After HMAs are metabolically activated, they are incorporated into DNA in place of dCTP. The enzyme carbamoyl-phosphate synthetase (CAD) facilitates the synthesis of dCTP, which competes with aza-dCTP, for incorporation into DNA. Notably, CAD has been found to be upregulated in both cell lines and clinical MDS samples that are resistant to HMAs, although this has not been a universal finding-104, although this effect has not been universally observed. The variations in conclusions among studies have been attributed to an interaction between different mutations and disease heterogeneity, as suggested by the relationship between lower clonal burden of secondary mutations and higher response rate.A number of additional determinants of response to HMAs have also been identified. Mutations in genes regulating methylation, including TET2, IDH1/2, and DNMT3A, have been associated with improved responses to HMAs in myeloid malignancies. About 90% of azacitidine is incorporated into RNA, raising the possibility that alterations in RNA metabolism might contribute to azacitidine action. Consistent with this hypothesis, azacitidine response has been linked to NSUN1, an RNA:5-methylcytosine (m5C) methyltransferase that binds BRD4 and RNA-polymerase-II to form chromatin structures that are not sensitive to azacitidine. Bone marrow specimens from patients with azacitidine-resistant AML or MDS showed a significant increase in RNA:m5C and NSUN1-/BRD4-associated active chromatin as compared to specimens from patients with sensitive AML or MDS. This resistance mechanism is not applicable to decitabine, which contains 5-aza-cytosine bound to 2’-deoxyribose.As evidence of discordance between DNA methylation status and clinical response accumulated, the effects of HMAs on RNA were also explored,110. Within the intrinsic pathway of apoptosis, cell death is initiated by mitochondrial outer membrane permeabilization (MOMP) that facilitates the release of cytochrome c, formation of cytosolic apoptosome complexes, and subsequent caspase activation. The MOMP process is regulated by BCL2 family members, which dynamically modulate pro- and anti-apoptotic signaling,111. Among the pro-apoptotic signaling proteins are BAK and BAX, which permeabilize the mitochondrial outer membrane upon activation-112. These are in turn neutralized when they are bound and sequestered by the anti-apoptotic paralogs BCL2, BCLXL, MCL1, BCLW, BCLB, and BFL1-113. A third group of BCL2 family members called BH3-only proteins includes BID, BAD, BIK, BIM, PUMA, HRK, NOXA, and BMF, which act through competitive inhibition of the anti-apoptotic BCL2 family members, direct activation of BAK and BAX, or a combination of these two processes-114. Upregulation of anti-apoptotic signaling  has been implicated in the pathogenesis of cancer, particularly in hematopoietic malignancies, where the upregulation serves as both an essential hallmark of lymphoma- or leukemogenesis and a mechanism of drug resistance,116. BCL2 is, therefore, an important therapeutic target for pharmacological inhibition in leukemia and other malignancies-120.Apoptosis is a regulated cell death process in eukaryotes that is activated by either extrinsic or intrinsic stimuli. Apoptosis mediates well-conserved decomposition of cellular macrostructures through activation of caspases, cysteine proteinases that cleave next to aspartate residuesBH3 mimetics are small molecules that have been developed to overcome apoptosis resistance in various neoplasms. These agents were designed to specifically inhibit members of the anti-apoptotic pathway by mimicking the BH3 domain found in BH3-only proteins. Like these pro-apoptotic proteins, BH3-mimetics bind the BH3 binding groove of anti-apoptotic proteins, thereby inhibiting ability of anti-apoptotic family member to bind and neutralize BAX and/or BAK.L, and BCLW. Navitoclax showed efficacy in chronic lymphocytic leukemia (CLL) in early human studies. However, navitoclax also displayed dose-limiting thrombocytopenia,124 that was traced to involvement of BCLXL in platelet survival. To avoid this toxicity, venetoclax was engineered to specifically inhibit BCL2 only. Venetoclax sensitivity was shown to be BCL2-dependent, with decreased lethality in platelets and nanomolar potency in the BCL2-dependent disease CLL,126-128.The first BH3 mimetic to enter clinical trials was navitoclax (ABT-263), which was designed to specifically mimic BAD binding to BCL2, BCLX,119,129. More specifically, venetoclax has been shown to form a hydrogen bond at Asp103 in BCL2 and interact with the intercalating indole on a partner BCL2. Alternatively, it has also been suggested that venetoclax might also lead to cell death by destabilizing the proton gradient across the mitochondrial inner membrane, leading to acidification of the cytosol and metabolic crisis. Whatever the proximate action of venetoclax, the final outcome is MOMP and release of cytochrome c to the cytoplasm, thereby resulting in apoptosome formation as well as decreased ATP production due to diminished mitochondrial cytochrome c,132.As a BH3 mimetic, venetoclax is thought to act primarily by binding to BCL2, causing release of sequestered BAX and BAK, thereby leading to MOMP and apoptosis. High BCL2 protein in this disease, which results from deletion of genes encoding two microRNAs that target BCL2 mRNA,135, was taken as a sign that CLL might be dependent on BCL2 for survival and, therefore, sensitive to BCL2 inhibition.Biomarkers that predict response to venetoclax are not fully understood. Venetoclax was initially approved for the treatment of relapsed CLL with chromosome 17p loss,136. In secondary AML refractory to HMAs, an increase in BCL2 and/or BIM protein detected by immunohistochemistry in myeloblasts in pretreated bone marrow aspirates was strongly associated with venetoclax monotherapy response and overall survival. In a phase II, single-arm clinical study of venetoclax monotherapy at a dose of 800 mg daily in patients with relapsed/refractory AML or patients with previously untreated AML who were unfit for conventional chemotherapy, an objective response rate of 19% (6 of 32 patients) was observed, with 6% (2 of 32 patients) achieving CR. As described below, high expression of BCL2 relative to other anti-apoptotic BCL2 family members was associated with better outcomes.The correlation between high expression of BCL2 protein and sensitivity also appears to hold for other neoplasms, but this relationship can be lost when high levels of venetoclax-insensitive BCL2 paralogs such as MCL1 are expressed,139-142. De novo resistance was attributable to a lack of BCL2 dependence, either because BCL2 was not activated or the CLL depended upon other paralogs such as MCL1 or BCLXL-145. Selection of cells that are dependent on an alternative BCL2 family member, such as BCLXL or MCL1, was also a major mechanism of acquired resistance [Figure 2]-148.Mechanisms of resistance in early trials of venetoclax in CLL were well studiedL may play a major role in the pathogenesis of various types of leukemia and mediate venetoclax resistance if BCL2 overexpression is not the primary antiapoptotic driver. In venetoclax-resistant AML cell lines, concurrent inhibition of MCL1 can mitigate venetoclax resistance-151. Combining venetoclax with daunorubicin or cytarabine has been shown to reverse some of the BIM sequestration mediated by MCL1, also sensitizing AML to venetoclax. In T-cell acute lymphoblastic leukemia cells with both MCL1 and BCL2 overexpression, combination therapy with inhibitors of both MCL1 (S63845) and BCL2 (venetoclax) is highly efficacious.This mechanism is not limited to CLL. Overexpression of MCL1 and/or BCLX-141,153. Mutations in the transmembrane domain of BAX have also been shown to prevent mitochondrial anchoring of BAX, thereby preventing BAX from facilitating MOMP and inducing apoptosis. In contrast to the BCL2 Gly101Val mutation, which would be expected to affect only drugs that bind BCL2, BAX mutations would be expected to confer resistance to a wide variety of agents that activate the intrinsic apoptotic pathway. Another preclinical study using a genome-wide CRISPR/Cas9 screen in AML cell lines identified BAX, along with TP53 and PMAIP1, as genes whose inactivation results in venetoclax resistance.A number of additional mechanisms of venetoclax resistance have been described. One involves the acquisition of mutations within BCL2 itself, such as the Gly101Val mutation, which prevents or reduces venetoclax bindingCLPB as a determinant of venetoclax sensitivity. Expression of CLPB was shown to be significantly higher in AML cells, even greater in cells resistant to venetoclax, which resulted in tighter mitochondrial cristae. Conversely, CLPB loss was shown to restore venetoclax sensitivity. From a genome-wide CRISPR-Cas9 knock-out screen in a venetoclax-resistant MOLM-13 derivative, the top hits that restored venetoclax sensitivity  pointed to inhibition of mitochondrial translation as a potential mechanism of sensitizing resistant cells to venetoclax. Further studies with doxycycline and tedizolid, which pharmacologically inhibit mitochondrial translation, led to the suggestion that diminished mitochondrial protein synthesis leads to respiratory dysfunction, which activates an integrated stress response that overcomes the resistance, possibly by upregulating the pro-apoptotic BCL2 family members NOXA (PMAIP1) and PUMA (BBC3).A number of resistance mechanisms also involve alterations in mitochondrial metabolism. Amplifications of changes in energy metabolism as a consequence of mitochondrial macrostructure rearrangement also appear to contribute to venetoclax resistance. A genome-wide CRISPR-Cas9 knock-out screen in the AML cell line MOLM-13 identified the mitochondrial chaperonin TP53, SMARCA4, CELSR3, CCND1, and KMT2D alterations, and allele loss often correlated with super-enhancer remodeling at 18q21. Next-generation sequencing or whole exome sequencing of 29 of the 32 cases of AML enrolled in the phase II venetoclax monotherapy study suggested that SRSF2/ZRSR2 and IDH1/2 mutations may predict sensitivity to venetoclax therapy in AML,159. The effect of SRSF2 mutation on venetoclax sensitivity may be explained by the alternative splicing of genes involved in apoptotic pathways.Epigenetic changes also appear to contribute to venetoclax resistance. Mantle cell lymphomas that were resistant to single-agent venetoclax therapy exhibited In summary, mechanisms of resistance to venetoclax monotherapy are varied, from mutation of the drug binding pocket of BCL2 itself, to amplification of parallel anti-apoptotic signaling pathways, changes in energy metabolism, and epigenetic alterations.ex vivo revealed synergy, at low nanomolar concentrations, between azacitidine and venetoclax,162. Azacitidine treatment in AML cell lines was also shown to cause several pro-apoptotic changes, including a decrease in MCL1 protein levels,164 and an increase in NOXA through activation of the integrated stress response. Combination therapy with azacitidine and ABT-737, an inhibitor of BCL2, BCLXL, and BCLW, not only displayed synergistic killing of AML cells but also decreased tissue invasion of leukemic cells in vivo, thus dampening a potential niche microenvironment-based evasion mechanism of resistance in AML. Other preclinical studies have shown that the HMA/BCL2 inhibitor combination decreases oxidative phosphorylation and avoids genomic amplifications that reduce venetoclax monotherapy-induced energetic stress. The HMA/venetoclax combination was made even more attractive by the observation that the cytotoxic synergy occurs preferentially in neoplastic cells while sparing normal hematopoietic cells.Combination therapy is employed to increase treatment efficacy beyond the action of a single agent, avoid dose-dependent adverse events, and evade potential mechanisms of resistance. With these goals in mind, the combination of venetoclax and HMAs was explored as a potential drug combination in preclinical studies. Treament of clinical AML samples initially et al. evaluated the safety and efficacy of the combination of azacitidine or decitabine with venetoclax in previously untreated AML patients 65 years of age or older who were ineligible for conventional induction chemotherapy. The most common grade 3 and 4 adverse events included neutropenic fever, pneumonia, leukopenia, neutropenia, anemia, and thrombocytopenia. With a median time on study of ~9 months and a median duration of follow-up of ~15 months, the CR/CRi rate was 67% and the overall leukemia response rate, including patients with partial response and morphologically leukemia free state, was 83%. Median overall survival was not reached at the time of the initial report. Very similar results were observed in the phase III study of azacitidine and venetoclax in treatment-naïve AML patients ineligible for standard induction therapy. Based on the phase II and III HMA/venetoclax results, as well as a phase II study of LDAC that also showed an increased CR/CRi rate and relapse-free survival compared to LDAC alone, the combination of venetoclax with HMA or LDAC was approved by the U.S. FDA for the treatment of elderly or medically unfit patients with AML.Based on these preclinical findings, DiNardo  and the phase III azacitidine/venetoclax trial both indicate that 1/3 of patients with newly diagnosed AML fail to achieve a CR or CRi with this therapy,28 This high rate of treatment failure has prompted investigation into mechanisms of resistance to the combination.The phase II HMA/venetoclax trialet al. suggest that resistance to the azacitidine/venetoclax doublet is mediated by the presence of monocytic AML cells, which upregulate MCL1 to evade BCL2 blockade and relieve mitochondrial energetic stress. These monocytes, which lose BCL2 expression and preferentially rely on MCL1 for survival, show outgrowth in relapsed/refractory AML and have a distinct immunopathogenic signature (CD45-bright/SSChigh/CD117-/CD11b+/CD68+) that might allow early detection and therapeutic decision making. Additional analysis showed that AML with monocytic differentiation  had little or no response to the combination of azacitidine and venetoclax.Based on the mechanism of action of HMAs and venetoclax, it is logical that resistance might involve changes in BCL2 family members. Results of Pei vs. resistance. Through genomic analysis of AMLs treated on studies with venetoclax-based combinations, DiNardo et al. found that higher response rates and durable remissions were associated with NPM1 or IDH2 mutations. AMLs that did not respond to venetoclax-based combination therapy (primary resistance) or initially responded and then relapsed (adaptive or secondary resistance) had new or expanded clones with activating mutations of FLT3 or RAS. The variety of mutations occurring in different kinase pathways  and the increase in tumor heterogeneity following treatment indicate that resistance is likely driven by an evolving genetic diversity in the cell pool rather than a single dominant gatekeeper mutation. Genomic changes between diagnosis and relapse involved mutations in a variety of pathways, including kinase signaling such as FLT3-ITD, NRAS, and JAK1 mutations; alternative RNA splicing due to mutations in U2AF1, U2AF2, SRSF2, and ZRSR2; cancer-related transcription factors, including IKZF1, SETBP1, RUNX1, and STAT5A; tumor suppressors such as TP53 and WT1; and epigenetic modifiers such as BCOR and CREBBP. The observed increase in AML cells harboring a FLT3-ITD mutation following treatment may indicate a clonal selection in which a small pool of FLT3-ITD cells are able to selectively withstand treatment and repopulate the marrow. The addition of inhibitors able to target mutated FLT3 such as sorafenib or midostaurin to the HMA/venetoclax combination treatment may help to combat this particular form of resistance.Another approach to understanding resistance has involved genomic characterization of AMLs that exhibit sensitivity FLT3 mutation or even possibly TP53 mutation), others such as monocytic differentiation are more difficult to directly address pharmacologically. Accordingly, targeting a more general mechanism of resistance that is not usually dependent on specific genetic mutations may be of clinical utility. AML cells take advantage of stromal-dependent pro-survival signals to create a permissive niche within the bone marrow microenvironment via interactions between upregulated cell surface receptors, including very late antigen-4 (VLA-4), CD44, E-selectin ligand-1, and CD98, with adhesion molecules, including vascular cell adhesion molecule-1, fibronectin, hyaluronan, osteopontin, selectins, and integrins. This niche is favorable for the maintenance and progression of chemotherapy-resistant disease,171. This mechanism of resistance, although less specific to the topic of this review, may be potentially modified or targeted; treatment with anti-VLA-4 antibodies and cytarabine improves survival in AML mouse models relative to cytarabine alone. Additionally, patients with VLA-4-negative AML have a more favorable prognosis.While some of these mechanisms of resistance can potentially be targeted therapeutically  monitoring disease status; and (2) devising effective maintenance or salvage therapies. Multiple venetoclax-based combinations are currently being tested with the goal of circumventing drug resistance. These agents added to the HMA/venetoclax doublet include: a combination of cladribine and low-dose cytarabine, the NEDD8-actviating enzyme inhibitor pevonedistat, the anti-CD33 antibody/calicheamicin conjugate gemtuzumab ozogamicin, MDM2 inhibitors, and the anti-programmed death ligand-1 (PDL-1) antibody avelumabThe HMA/venetoclax combination represents are major advance for AML patients who previously fared poorly with conventional induction chemotherapy. As this combination is increasingly used, studies evaluating the causes of loss of response or relapse in clinical samples are needed to elucidate the patterns and mechanisms of resistance. It also remains crucial to explore modified dosing schedules for HMAs as a strategy to avoid acquired HMA resistance based on the results of recent studies with single-agent decitabine and azacitidine.


Ca2+ homeostasis is indispensable for neuronal functions and survival. The cytosolic Ca2+ concentration ([Ca2+]CYT) is regulated by channels, pumps, and exchangers on cellular membrane systems. Under physiological conditions, both endoplasmic reticulum (ER) and mitochondria function as intracellular Ca2+ buffers. Furthermore, efficient and effective Ca2+ flux is observed at the ER-mitochondria membrane contact site (ERMCS), an intracellular membrane juxtaposition, where Ca2+ is released from the ER followed by mitochondrial Ca2+ uptake in sequence. Hence, the ER intraluminal Ca2+ concentration ([Ca2+]ER), the mitochondrial matrix Ca2+ concentration ([Ca2+]MT), and the [Ca2+]CYT are related to each other. Ca2+ signaling dysregulation and Ca2+ dyshomeostasis are associated with Alzheimer’s disease (AD), an irreversible neurodegenerative disease. The present review summarizes the cellular and molecular mechanism underlying Ca2+ signaling regulation and Ca2+ homeostasis maintenance at ER and mitochondria levels, focusing on AD. Integrating the amyloid hypothesis and the calcium hypothesis of AD may further our understanding of pathogenesis in neurodegeneration, provide therapeutic targets for chronic neurodegenerative disease in the central nervous system.Although anything that changes spatiotemporally could be a signal, cells, particularly neurons, precisely manipulate calcium ion (Ca SimilarInsP3R3) . Functioe (CICR) . Seeminglarities .3Rs and RyRs share similar but not identical distributions in neurons  and oscillations or spikes (at the whole-cell level)  . Ca2+ waechanism . Furthered later . Under habortive .2+-release activity from the opened InsP3R, at least, is regulated by the InsP3, [Ca2+]CYT, and [Ca2+]ER. Under a modest concentration of InsP3, the opening of InsP3R is biphasically regulated by cytosolic Ca2+: the low [Ca2+]CYT (<1 μM) can activate InsP3R; in contrast, the high [Ca2+]CYT (>1–10 μM) can inhibit the channel  is applied secondary to the maximal concentration of InsP3 (10 μM), the cytosolic Ca2+ fails to inhibit the Ca2+ release from the liganded InsP3R; in turn, if the high [Ca2+]CYT (100 μM) is given before the InsP3 (10 μM), the cytosolic Ca2+ can entirely inhibit the Ca2+ release from the unliganded InsP3R , another primary function of mitochondria is buffering intracellular Ca2+ . Neuronanditions . The Ca2 neurons . Mitochoxhausted . It is cxhausted .2+ channels, such as NMDAR on the postsynaptic density, can accumulate the cytosolic Ca2+ and prevent the propagation of Ca2+ waves, a global Ca2+ signal  and the inner mitochondrial membrane (IMM). The OMM is permeable to ions attributed to the massive expression of voltage-dependent anion channels (VDAC)  . The not[Ca2+]MT . Among tpoptosis . Consistpoptosis .2+ into the mitochondria matrix across the electrochemical gradient (2+ with extremely high affinity (KD ≤ 2 nM)  on the IMM can rapidly accumulate Cagradient . MCU sel ≤ 2 nM) . MCU conpression . Acidic pression . The mitchondria .The ER has distributed the entire intracellular space from the nucleus to the plasma membrane intertwining all organelles, including mitochondria . The ER 2+ uptake can occur at the ERMCS, where a high concentration of Ca2+ transports from the ER lumen into the mitochondrial matrix , first described in 1907 , is a tyPathologically, the senile plaques  and the neurofibrillary tangles (NFT) , observed inside and outside neurons, respectively, are two of several neuropathological features related to AD .Based on the age of morbidity, Alzheimer’s disease is divided into two subtypes: the early-onset AD (EOAD), ranging from 30 years to 60 or 65 years; the late-onset AD (LOAD), defined with an onset age later than 60 or 65 years . At the amyloid precursor protein (APP), presenilin-1 (PSEN1), and presenilin-2 (PSEN2) genes are genetically associated with FAD  gene, residing in chromosome 19, fulfill a significant role in SAD  peptide is the predominant force of AD-related pathogenesis, including plaques, tangles, synapse loss, and neuronal death . Althoug2+ dyshomeostasis as one of the early causes of AD, is first proposed by Khachaturian based on limited direct evidence in the 1980s  on the plasma membrane and disrupt Ca2+ homeostasis by rapidly elevating intracellular Ca2+ concentration, leading to neuronal death in AD , exhausting the neuronal Ca2+ buffering capacity rapidly, subsequently leading to the neurotoxicity  is named by its specific agonist, N-methyl-D-aspartate (NMDA), which does not occur naturally. NMDARs belong to one ionotropic family of glutamate receptors located on the plasma membrane. NMDARs can integrate two extracellular chemical stimuli (glycine and glutamate) and one membrane electrical stimulus (the depolarization of the plasma membrane) into the Ca2+ signal   . The Aβ stage AD  , causing the opening of the permeability transition pore (PTP) and further depolarising the ΔΨ, which parallels with the level of neuronal death  acts with 100% efficiency and the [Ca2+]CYT level is 0.1 μM, the calculated upper limit value of the [Ca2+]ER is 2,400 μM    (Tu et aKO-MEFs) . The per control . SubsequI do not . Similar can not .2+]ER level in DKO-MEFs (190 μM) is approximately 2-fold higher than it is in wild-type control (87 μM); moreover, it is calculated that PSENs account for 80% of the ER endogenous Ca2+-leaking ability  probe  probe . The knoER level .2+ leak from ER and the decreased [Ca2+]ER level in the same DKO-MEFs model   significantly elevate the probability of InsP3R opening compared with wild-type control  also considerably enhance the InsP3R opening, which indicates that the γ-secretase activity of PSEN is not required for its influence on InsP3R opening  and profoundly decreases both Aβ accumulation and tau protein hyperphosphorylation in cortical and hippocampal neurons of transgenic mice  . In 1999utations . The und control . Interes opening . Suppresnic mice . These l for FAD  (Shillin for FAD .CYT) and RyR regulates the Ca2+ signal from ER (2+ concentration (10 nM < [Ca2+]CYT < 1 μM) and pathological high Ca2+ concentration ([Ca2+]CYT > 10 μM) are required for the cytosolic amino-terminal fragment residues 1–82 of presenilin-1 (PSEN1-NTFCYT1-82) and the cytosolic amino-terminal fragment residues 1–87 of presenilin-2 (PSEN2-NTFCYT1-87) to bind RyR, respectively , and long-term depression (LTD)  . For exagger LTD . For neugh ERMCS . Collect2+ signaling has been reported in neurodegenerative diseases, such as AD, Parkinson’s disease (PD), and Huntington’s disease (HD)  can elevate the [Ca2+]MT level, and reducing the Ca2+ signal from ER to mitochondria normalizes the [Ca2+]MT level and the mitochondrial function (2+ homeostasis (Dysregulation in Caase (HD) . The [CaD and HD . In Caenfunction . In neurfunction . Area-Gofunction , later tfunction . Moreoveeostasis .2+ signaling regulated by molecular machinery on cellular membrane systems and the Ca2+ dyshomeostasis linked to Aβ and presenilins. Connecting the amyloid hypothesis with the calcium hypothesis may further the understanding of Alzheimer’s disease pathogenesis. At ER and mitochondria levels, understanding the regulation of cellular Ca2+ signaling and the mechanism underlying neuronal Ca2+ dyshomeostasis in AD may provide therapeutic targets for chronic neuronal degeneration disease in the central nervous system.The present review summarizes the intracellular Ca


These events promote the efflux of cations and anions from guard cells. As a result, guard cells lose turgor leading to stomatal closure -induced stomatal closure in the epidermis took about 30 min (and in leaf 3 h). In contrast, recovery took 1–6 days, implying short-term and long-term effects on stomata  activate RbohD/F and stimulate reactive oxygen species (ROS) production during stomatal closure. However, the role of RBOHD in resistance against pathogens, particularly during pre-invasive stage is not clear. The elevated ROS, in turn, trigger a rise in nitric oxide (NO) and Ca2+. The interaction of these secondary messengers (ROS/NO/Ca2+) regulates the downstream components in guard cells. Both NO and Ca2+ (via Ca2+-dependent protein kinases) promote the ion efflux by activating K+ out, SLAC1, and SLAH3 channels and at the same time inhibit the K-influx channel  or elicitors (flg22) by their respective receptors. Upon binding to ABA or flg22, the receptor kinases , ABA, methyl jasmonate (MJ), and ethylene (ET) accumulate when microbes attack plants. The concerted action of these hormones causes stomatal closure and induces systemic resistance  or abiotic stress (drought) , upregulated the genes involved in SA signaling and PA catabolism, reinforcing plant defense responses  and maize leaves , synthesis of pathogenesis-related (PR) proteins, and programmed cell death (PCD)  and even PAs could induce systemic resistance  of the Council of Scientific and Industrial Research [No. 38 (1404)/15/EMR-II]. SG was supported by a Senior Research Fellowship from University Grant Commission, New Delhi. PB was supported partially by a University of Hyderabad BBL fellowship.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.