In the last twenty years, we have witnessed a paradigm shift in the treatment and prognosis of acute myeloid leukemia (AML), thanks to the introduction of new efficient drugs or approaches to refine old therapies, such as Gemtuzumab Ozogamicin, CPX 3-5-1, hypomethylating agents, and Venetoclax, the optimization of conditioning regimens in allogeneic hematopoietic stem cell transplantation and the improvement of supportive care. However, the long-term survival of non-M3 and non-core binding factor-AML is still dismal. For this reason, the expectations for the recently developed immunotherapies, such as antibody-based therapy, checkpoint inhibitors, and chimeric antigen receptor strategies, successfully tested in other hematologic malignancies, were very high. The inherent characteristics of AML blasts hampered the development of these treatments, and the path of immunotherapy in AML has been bumpy. Herein, we provide a detailed review of potential antigenic targets, available data from pre-clinical and clinical trials, and future directions of immunotherapies in AML. In the last twenty years, the knowledge of mechanisms of leukemogenesis and molecular/genetic features of acute myeloid leukemia (AML) has rapidly progressed, allowing a precise characterization of the disease and several updates on AML classification ,2. In thThe introduction in clinical practice of new drugs, such as Gemtuzumab Ozogamicin, CPX 3-5-1, hypomethylating agents (HMA) and Venetoclax (VEN) ,7, the eThus, the recent and successful introduction of immunotherapies, such as antibody-based therapy, checkpoint inhibitors, and chimeric antigen receptor (CAR) strategies, in lymphoid malignancies has raised hopes for their possible use in AML as well.Furthermore, other immunologic therapies showed efficacy in AML, and some of them are established as gold standards in different settings. The most illustrative example is HSCT: after HSC engraftment, the donor immune system is capable of targeting the residual leukemic cells, giving life to the so-called “Graft-versus-Leukemia” (GvL) effect . Key chaFLT3+ AML cells, which causes an increase in CD8+CD107a+IFN-γ+ T cells and is capable of eradicating leukemia cells , morphologic leukemia-free state [MLFS] and partial response [PR]) of 22% and a composite complete response  rate of 19%. Infusion-related reactions (IRR)/CRS occurred in all thirty patients treated (grade ≥ 3 in 13.3%)  infusion), an overall response rate  .In 2021, the report of the dose expansion cohort was published. cCR rate was 20%, with an ORR of 24%. IRR/CRS were observed in 96% of patients (grade ≥ 3 in 4%). Grade ≥ 3 neutropenia and thrombocytopenia occurred in 2% and 6%, respectively . A trialThe phase 1 dose escalation study evaluating AMG330 as a continuous IV infusion in patients with R/R AML showed a cCR rate of 11.4%, with an 89% treatment discontinuation rate for disease progression, adverse events, and/or patient requests. Grade ≥ 3 CRS was observed in 31.4% . The stuIn 2019, the results of AMG673, a CD33 × CD3 BiTE genetically fused to the N-terminus of a single-chain IgG Fc region , phase I study were presented: an ORR of 22.2% with a cCR rate of 3.7% were observed. CRS occurred in 50% of patients (≥grade 3 in 13.3%) .CD123 was also targeted by a BiTE, Vibecotamab; the phase I dose-escalation study, enrolling 104 AML, 1 B-cell ALL, and 1 chronic myeloid leukemia (CML), showed, at the higher dose tested (0.75 µg/kg), a cCR rate of 13.7%; CRS occurred in 58.5% of patients (8% grade ≥ 3) . A phaseA combination with conventional therapy was tested with a second CD123 × CD3 BiTE: APVO436. This drug was at first evaluated as monotherapy in relapsed/refractory (R/R) AML or MDS. In the AML cohort, 2 of the 34 evaluable patients (5.8%) obtained CR. Overall, IRR/CRS were observed in 50% of patients (≥grade 3 in 13%) . An updaRecently, new antigen targets are being explored in ongoing clinical trials: Tepoditamab (MCLA-117), a BiTE targeting CD3, and CLEC12A, an antigen present on leukemic stem cells and leukemic blasts whose expression seems stable throughout diagnosis, treatment, and relapse , showed,Since the early 2010s, efforts have been made to redirect NK-cell activity against AML blasts. In 2013, Wiernick et al. observed that CD16 × 33 BiKE and ADAM17 inhibitors can overcome inhibitory signals mediated by class I major histocompatibility complex (MHC) recognition and CD16 clipping, resulting in potent NK cell killing of CD33+ AML targets . In the More recently, the role of CD16 × CD33 BiKE was investigated in pediatric CD33+ AML and ALL patients. NK cells from the patients profited from restoration of effector functions by BiKE treatment, which, thus, could constitute a promising new option for supporting maintenance therapy or “bridging” consolidation chemotherapy before HSCT . The onlCAR-T cells are constructs composed of an antigen recognition extracellular domain, a transmembrane domain, and an intracellular domain consisting of a co-stimulation domain and cluster of differentiation 3   Figure . The varThe first clinical trial published in 2015, testing a CD33-directed CAR-T, enrolled one R/R AML patient. A decrease in blasts was observed without a clinical response; furthermore, IRR occurred .In 2019, a CAR-T directed against NKG2D , a receptor that transmits activation signals in NK-cells and activates the immune system against NKG2D ligands, expressed in AML blasts and poorly expressed in healthy tissues , was tesIn 2021, a new trial evaluated a CD33-directed CAR-T in 3 patients; again, none of them met criteria for response to treatment. Two patients developed CRS .In the same year, one case of a CD38+ AML refractory to chemotherapy combined with an anti-CD38 monoclonal antibody (Daratumumab) successfully treated with CD38-directed CAR-T was reported . AlthougIn the 2022 ASH meeting, several encouraging new data were presented.2 and cyclophosphamide 500 mg/m2 days −5 to −3; cohort 2). No response was observed in cohort 1; among the 10 AML patients enrolled in cohort 2, the cCR rate was 20% with an ORR of 30%. Overall, CRS occurred in 70.8% of patients (≥grade 3 in 4.2%) [PRGN-3006, a CD33-directed CAR-T with IL-15 membrane bound, was tested in 20 R/R AML, 1 chronic myelomonocytic leukemia (CMML) with ≥5% blasts, and 3 high-risk MDS without (cohort 1) or with lymphodepletion (fludarabine 30 mg/min 4.2%) .UCART123v1.2, an universal, anti-CD123 allogeneic CAR-T product obtained by the ex vivo knockout of the genes of the T-cell receptor (TCR) alpha constant and CD52 to minimize the risk of GvHD and allow the use of anti-CD52-directed therapy as part of lymphodepletion, was tested in combination with Alemtuzumab (anti-CD52) in 16 patients: 1 patient (6.2%) achieved MLFS, and 1 patient had MRD-negative CR. CRS occurred in 93.7% of patients (≥grade 3 in 18.7%) .An anti-CD123 CAR-T was also tested in 12 R/R AML pediatric patients in a phase I dose escalation trial; the ORR was 16.7%, with one patient (8.3%) achieving CR. No CRS occurred .In 2020, the first case of successful CAR-T targeting CLL1 , also targeted by bispecific antibody-based strategies (see above), was reported in a patient with secondary AML . In 2022CLL1 was also successfully targeted in the pediatric population; in 2021, a phase I interim analysis showed an ORR of 81.8% with a cCR rate of 72.7% (50% achieving MRD negativity) . In 2022Recently, anti-CLL1 CAR-T with stimulatory domains was tested: 4 patients were treated with CD28/CD27-equipped anti-CLL1 CAR-T, while 3 were treated with 4-1-BB-equipped anti-CLL1 CAR-T. In the first group, 3/4 patients achieved MRD-negative CR, while in the second one, 1 pt achieved MRD-positive CR and one pt achieved MRD-negative CR; CRS was experienced by all patients without ≥grade 3 events . Other CLL1-directed CAR-T trials are currently ongoing .To date, early results from only one trial testing CAR-NK in R/R AML patients have been presented.8, 1.2 × 109, or 1.8 × 109 cells per round after the precondition with Fludarabine (30 mg/m2) and Cytoxan 300–500 mg/m2 for 3 days to 5 days, determined by tumor burden at baseline).An anti-CD33 CAR-NK was tested in 10 heavily pre-treated patients  in a phase I dose escalation trial , whose targeted CAR-T was able toefficiently kill CD7+ AML cells and CD7+ primary blasts of R/R-AML patients in vitro and significantly inhibit leukemia cell growth in a xenograft mouse model ;CD19 (NCT04257175), occasionally expressed in AML blasts  ;FLT3 and DNMT3A-mutated AML and already successfully targeted by CAR-T constructs in pre-clinical studies [CD44v6 (NCT04097301), overexpressed in  studies ;CD70 (NCT04662294), expressed on most leukemic blasts but with little or no expression in normal bone marrow samples, is already targeted by the human monoclonal antibody Casatuzumab ;FLT3 , whose targeted CAR-T showed potent inhibition of leukemia proliferation in xenograft models ;LILRB4  (NCT04803929), highly expressed in AML with monocytic differentiation and suspected to be involved in tumor escape, maintains an immunosuppressive milieu for tumor cells ;CD276 (NCT04692948), expressed in AML with monocytic differentiation as well, whose targeted CAR-T exhibited efficient antigen-dependent cytotoxicity in vitro and in xenograft models of AML . Ongoing clinical trials are exploring new targets, such as:The comprehensive knowledge of tumor microenvironment and mechanisms of therapy escape led to the development, since the early 2010s, of drugs targeting the broad landscape of cells and molecules around the tumor cells, first in solid tumors than in hematologic malignancies . In a siCD47 is a transmembrane protein broadly expressed in human cells and on many types of tumors, where it plays a pivotal role in immune escape; in fact, the binding to its ligand, SIRPα , triggers a “don’t eat me” signal to the macrophages, inhibiting phagocytosis . In the In AML, CD47 is overexpressed and correlates with a poor prognosis ,94. MajeA second T cell-dependent mechanism in vivo was documented by Tseng et al., who showed that treatment with an anti-CD47 antibody had a greater effect in wild-type mice with established tumors than in T cell-deficient nude mice with equivalent tumor burden .The first drug targeting SIRP/CD47 interaction was CC-90002, an anti-CD47 monoclonal antibody, whose results were presented in 2019: among the 24 R/R AML patients and 4 high-risk (HR) R/R MDS patients treated, no objective response was documented, with an 82% rate of treatment-emergent adverse events .TP53-mutated AML patients achieved cCR. The combination therapy was well tolerated, with a safety profile similar to AZA monotherapy [In the same year, the first results of another anti-CD47 antibody, Magrolimab, in combination with AZA were presented. 25 newly diagnosed (ND) AML patients ineligible for induction chemotherapy were enrolled; among the 16 patients evaluable for response, 11 obtained an objective response , and 5 presented stable disease. Interestingly, 5/6 otherapy .TP53-mutated AML, the ORR was 71%  [The 2021 update of the study confirmed the promising results, with a 65% ORR ; in the subset of 5% MLFS) . A phaseSurprisingly, the ENHANCE study, testing Magrolimab plus AZA in HR-MDS, was recently discontinued due to futility based on a planned analysis ; these rA trial testing Magrolimab plus Atezolizumab  combination therapy (NCT03922477) is ongoing.Lemzoparlimab is an anti-CD47 antibody endowed with a red blood cell (RBC) sparing property and unique binding epitope, potentially differentiating itself from other CD47 axis-targeting therapies. Preliminary results from the phase I study, presented in 2020, showed a 20% cCR rate (MLFS) with an acceptable safety profile (no patient developed a treatment-related serious adverse event) . The phaIn 2022, Evorpacept, a fusion protein containing two engineered high-affinity CD47 binding domains of SIRPα linked to an inactive Fc region of human immunoglobulin was designed to promote the phagocytosis of tumor cells and was tested in combination with VEN and AZA in R/R AML or ND HR AML. A blast decrease was observed in all patients, with an objective response in all ND AML patients and 44.4% of R/R AML patients. Among the 14 patients enrolled, 1 grade 3 CRS occurred . Programmed cell death protein 1 (PD1) is a cell surface receptor expressed on several types of T cells  and other immune cells  and has a fundamental role in countering positive signals through the TCR and CD28 by engaging its ligands PDL1 and/or PDL2 . Thus, sSeveral drugs targeting this pathway, blocking PD1  or PDL1 , have been successfully tested and approved by the FDA in solid cancers and hematologic malignancies ,111, altThe first data on Nivolumab in combination with AZA dates back to 2017; the early results, on 53 R/R AML patients, showed an ORR of 35% with a cCR rate of 14%. Furthermore, the median overall survival (5.7 months) was superior to historical survival with AZA-based salvage protocols from the same institution. ≥grade 3 immune-related adverse events (IrAE) were observed in 14% of patients and responded rapidly to steroids .TP53 mutation was more frequent in non-responders (p = 0.06). Six patients had seven grade 3–4 IrAEs with an overall acceptable safety profile [In the same year, the results of the therapy combination with Cytarabine and Idarubicin as first-line therapy in a cohort of ND AML and HR MDS (30 patients with AML 2 with HR MDS) were presented: the cCR rate was 72%. More than grade 3 IrAE occurred in 5 patients . The fin profile .Nivolumab was also tested as maintenance therapy. In 2018, it was tested in 14 patients with HR AML  in CR, with a 6 and 12 month CR duration of 79% and 71%, respectively. Five patents experienced ≥grade 3 IrAE . In 2022In a similar fashion, Pembrolizumab was tested in different clinical settings: the combination with AZA was investigated in R/R AML and older ND AML by Gojo et al., who presented the preliminary results at the 2019 ASH meeting. The R/R AML cohort achieved an ORR of 17.2% (cCR 13.8%), while the ND AML cohort achieved 58.8% (cCR 47%). Grade 3/4 IrAE was observed in 9 (24%) patients in the first cohort and 3 (14%) patients in the second one . The combination with Decitabine (DEC) was tested in a cohort of 10 R/R AML patients, of whom 3 obtained cCR. The toxicity profile of the combination therapy was consistent with that expected from DEC alone, with the exception of three patients who suffered IrAE, likely as a consequence of Pembrolizumab . The combination with chemotherapy was explored in R/R patients by Zeidner et al.: 37 patients were enrolled to receive Pembrolizumab after high-dose cytarabine in a phase II study. The ORR was 46%, with a cCR rate of 38%; the administration of Pembrolizumab was associated with 5 IrAE, and no severe toxicity or delay in hematologic recovery were observed . In a foFinally, Pembrolizumab was tested as maintenance therapy after autologous HSCT in non-favorable-risk AML patients in the first CR or beyond who were ineligible for alloSCT. Among the twenty patients enrolled, after a median follow-up of 80 months, 14 patients remain alive, with 10 in continuous remission. Treatment was well tolerated, with only 1 non-relapse death .In 2022 the results of a large randomized phase II trial evaluating older patients with AML receiving AZA with or without Darvalumab were published. Although the therapy combination did not raise safety concerns, it failed to achieve a better response  . Trials testing anti-PDL1 are currently ongoing ; no results are available at the moment.T cell immunoglobulin and mucin domain 3 (TIM3) is a transmembrane protein expressed on immune cells  that binds multiple ligands. One of the most known is Galectin-9 , which plays an important role in immune cell regulation. Zhu et al. showed that Gal-9, through the TIM3 link, was able to induce intracellular calcium flux, aggregation, and death of T helper cells, and administration of Gal-9 in vivo resulted in selective loss of interferon-gamma-producing cells . In the The detection of TIM3 in myeloid malignancies cells, in particular MDS  and AML The first anti-TIM3 antibody, Sabatolimab, was tested in combination with HMA in a cohort of ND or R/R AML patients. The combination Sabatolimab plus DEC achieved an ORR of 41% in ND AML patients and 25% in R/R AML patients, whereas Sabatolimab plus AZA, tested only in ND AML patients, obtained a 27% ORR. Three IrAE were observed in the DEC cohort vs. none in the AZA cohort . In the study update in 2021, in a cohort of 40 evaluable patients with ND AML, the ORR was 40.0%. The response rate was consistent in AML patients bearing an adverse-risk mutation . The combination was safe and well tolerated, with grade ≥ 3 adverse events similar to HMA alone . Trials evaluating new clinical settings, such as AML in MRD+ CR after HSCT (NCT04623216), and therapy combinations, such as Sabatolimab plus AZA/VEN , are currently ongoing.CD70 is a molecule expressed in activated T cells and dendritic cells that, by binding its ligand, CD27, a costimulatory receptor of the tumor necrosis factor superfamily, constitutively expressed on lymphocytes and hematologic stem cells, promotes lymphocyte expansion and cell survival ,138. FurThus, as also briefly discussed earlier, the CD27/70 pathway represents a good target to address AML; in fact, Riether et al., in in vitro studies, documented strong proliferation inhibition blocking CD27 and CD70 .Casatuzumab, an anti-CD70, has been tested in combination with AZA in ND AML patients ineligible for chemotherapy in a phase I/II trial. The ORR in patients treated at the phase II dose was 37.9% . Seven patients (18.4%) reported infusion-related reactions, including two with grade 3 events. There were 10 fatal treatment-emergent adverse events; none were considered drug-related .In 2021, the preliminary results of the therapy combination Cusatuzumab plus AZA/VEN in ND AML patients ineligible for chemotherapy were presented: among the evaluable 42 patients, the ORR was 92.9% . Apart from the IrAE (11.4% of patients with 2.3% at grade ≥ 3), the safety profile was consistent with that previously reported for the AZA/VEN regimen .CTLA4 (cytotoxic T-lymphocyte-associated protein 4) is a transmembrane protein member of the immunoglobulin gene superfamily expressed by both CD4+ and CD8+ T cells, which, interacting with its ligands, CD80 and CD86, inhibits immune response . InteresIpilimumab, an anti-CTLA4 antibody, was first tested as post-HSCT salvage therapy in various malignancies, including 2 AML patients; no effect was observed . In 2023A phase I trial investigating Ipilimumab in combination with donor lymphocyte infusion (DLI) for myeloid malignancies relapsed after HSCT is ongoing (NCT03912064).CD200 is a cell membrane glycoprotein of the immunoglobulin supergene family, present on both cells of myeloid/lymphoid origin. Its receptor, CD200R, is expressed on cells of the monocyte/myeloid lineage and some T-cell subsets [ subsets . CD200/C subsets ,148. On  subsets ,150,151. subsets ,153. A tLAG-3 (lymphocyte activation gene-3) is a type I transmembrane protein with four Ig-like domains structurally similar to CD4 expressed on activated T cells. The bind with its ligand, MHC II, leads to the inhibition of T-cell functions; in fact, its blockade has been demonstrated to reinvigorate exhausted T cells and strengthen anti-infection immunity [immunity . Multiplimmunity ,155,156.Immunotherapy is based on mechanisms of immune escape and responsiveness. Such mechanisms can be different in the pre-leukemic and transforming stages of diverse subsets of leukemia. Failure to receive immunotherapy can be triggered by diverse disease etiologies and by chemotherapy resistance and immune evasion. Furthermore, the complex and disease-specific microenvironment may contribute significantly to the differency in efficacy observed between AML and lymphoid disorders . In thisGiven the genetic and epigenetic heterogeneity of pediatric and adult AML, immunotherapy has still not provided a paradigm-shifting breakthrough. The accumulation of genetic mutations and alterations of pathways defining anti-tumor activity are also very different across subsets of leukemia, which might explain different levels of response. However, the safety profile has been good for most of the new drugs, and the therapy was well tolerated by patients, who were commonly elderly and subjected to a heavy previous burden of therapy. In addition, IrAEs were usually low-grade and easily treated with steroid therapy. Concerning the anti-leukemic activity, some compounds showed encouraging results, but, unfortunately, there are still only a few randomized comparative studies, which are warranted to clarify the actual benefits and to frame these strategies in the most appropriate clinical subsets.The ongoing trials evaluating drugs targeting new markers and addressing different patient subsets allow us to envision the possibility, for immunotherapies, of expanding both safe chemo-free strategies and first-line combination therapies in AML.


ADAMTS13 gene, which is present on chromosome 9. In both syndromes, TTP results from a severe deficiency of ADAMTS13, which is responsible for the impaired proteolytic processing of high-molecular-weight von Willebrand factor (HMW-VWF) multimers, which avidly interact with platelets and subendothelial collagen and promote tissue and multiorgan ischemia. Although the acute presentation of the occurring symptoms in acquired and hereditary TTPs is similar , their intensity, incidence, and precipitating factors are different, although, in both forms, a severe ADAMTS13 deficiency characterizes their physiopathology. This review is aimed at exploring the possible factors responsible for the different clinical and pathological features occurring in hereditary and immune-mediated TTPs.Thrombotic thrombocytopenic purpura (TTP) is a thrombotic microangiopathy caused by a hereditary or immune-mediated deficiency of the enzyme ADAMTS13 . TTPs are caused by the following pathophysiological mechanisms: (1) the presence of inhibitory autoantibodies against ADAMTS13; and (2) hereditary mutations of the  Thrombotic microangiopathies (TMAs) are a heterogeneous group of syndromes associated with the generation of disseminated microthrombi responsible for a clinical triad composed of microangiopathic hemolytic anemia (MAHA), thrombocytopenia, and variable ischemic end-organ injury . Such syThe present review is aimed at exploring the possible factors responsible for the different clinical and pathological features of TTP.ADAMTS13 gene, which is present on chromosome 9q34  ([Ca2+]i). The rise in [Ca2+]i takes place via second messenger-mediated processes, which, in turn, trigger the release from intracellular Ca2+ stores in the endoplasmic reticulum (ER), and this is followed by Ca2+ entry from the extracellular space. This mechanism of Ca2+ entry can involve ER Ca2+ depletion but also directly receptor-activated Ca2+ entry. Of interest, the influx of Ca2+ into ECs occurs via some channels that are not gated by voltage. Among these, several polymodal transient receptor potential cation/canonical channels (TRPCs) have been shown to mediate the endothelial Ca2+ influx in ECs [2+ influx, including ECs [All of the IgG subclasses of anti-ADAMTS13 antibodies were detected in patients with iTTP, with the IgG(4) isotype followed by IgG(1) and IgA antibodies dominating the anti-ADAMTS13 immune response . IgG(1) al cells . Howeveral cells . Likewisal cells ,68. Plasal cells . The posal cells . Of inteal cells . Likewisal cells . Howeveral cells . Notably release . The autx in ECs . Seven Tx in ECs . TRPC5 eding ECs . TRPC5 cding ECs . TRPC5 pding ECs , the latding ECs . Thus, Tding ECs . HoweverThe different pathogenetic effectors and varying clinical phenotypes described above in cTTP and iTTP determine the different therapeutic approaches for their treatments. Plasma infusion may be administered in both clinical forms, but it is predominantly used in cTTP while waiting for the final approval of recombinant ADAMTS13 by regulatory agencies . By contIn conclusion, the understanding of the immunological basis of iTTP, which accounts for the majority of TTP cases, has progressively increased in the last few years. Plenty of previous studies investigated the contribution of antibodies against different ADAMTS13 domains to the inhibitory potential in plasma and revealed how these autoantibodies may cause both accelerated clearance with depletion of the metalloprotease and inhibition of its activity A. Howeve


Toxoplasma gondii is an obligatory intracellular protozoan in the family Apicomplexa. It infects almost one-third of the world’s population and causes toxoplasmosis, a prevalent disease. The parasite’s egress from infected cells is a key step in the pathology caused by T. gondii. Moreover, T. gondii’s continuous infection relies heavily on its capacity to migrate from one cell to another. Many pathways are involved in T. gondii egress. Individual routes may be modified to respond to various environmental stimuli, and many paths can converge. Regardless of the stimuli, the relevance of Ca2+ as a second messenger in transducing these signals, and the convergence of various signaling pathways in the control of motility and, ultimately, egress, is well recognized. This review attempts to outline intra- and extra-parasitic regulators that mediate T. gondii egress, and provides insight into potential clinical interventions and research. Toxoplasma gondii is an intracellular parasite that can infect the nucleated cells (including immune cells) of almost all warm-blooded animals, including humans [T. gondii [g humans . It is e. gondii .T. gondii infection may not exhibit clear clinical signs, multiple investigations have identified a possible link between T. gondii chronic infection and various mental diseases, including schizophrenia, bipolar disorder [T. gondii replicates slowly in the form of bradyzoites in tissue cysts in central nervous system (CNS) and muscles of people with competent immunity. However, in immunocompromised people , pre-existing tissue cysts rupture, and the released bradyzoites may develop into tachyzoites, the cytolytic form of T. gondii, causing toxoplasmic encephalitis [Although patients with chronic disorder , and depdisorder . In chrophalitis  and chorphalitis . Crucialphalitis .T. gondii alternates between asexual reproduction in intermediate hosts and sexual reproduction in definitive hosts. Tachyzoites, the major pathogenic stage of T. gondii, actively invade nucleated cells, multiply within host cells via endodyogeny, and egress from infected cells. T. gondii’s lytic cycle starts with active parasite invasion into host cells, which relies on protein release from two secretory organelles: micronemes and rhoptries [T. gondii—unsporulated oocysts—are shed in faeces, and develop to the sporulated stage containing infective sporozoites. After consuming contaminated food or water, intermediate hosts become infected. After ingestion, oocysts develop into tachyzoites in the small intestine. Tachyzoites cause the acute stage of infection and evolve into tissue cyst bradyzoites. When the host’s immune system is suppressed, bradyzoites may either stay dormant for the duration of the host’s life or return to tachyzoites.The life cycle of hoptries . In catsT. gondii invasion, parasitophorous vacuole (PV) formation occurs through host cell membrane invagination (HCM) [T. gondii development, the PV membrane (PVM) acts as a molecular sieve, allowing tiny molecules to exchange passively. Proteins in dense granules (GRA) are discharged to construct the tubulovesicular network (TVN), which is a network of membranous tubules present throughout the PV, to maintain the nascent PV, which offers a safe niche for T. gondii proliferation [T. gondii tachyzoites remain inside the PV until they egress, restarting the lytic cycle by attacking surrounding host cells. Egress was formerly assumed to be a simple passive process in which a nutrient-depleted host cell popped from inside, owing to the rising mechanical stress of the rapidly enlarging PV. The parasitic vacuolar membrane, the host plasma membrane, host endomembranous organelles , and host cytoskeletal components must all be disrupted by the escaping parasites.During on (HCM) . For T. feration . T. gondT. gondii, which is characterized by damage to tissues and organs. Egress is seen as the initial phase. Exiting a parasite from a host cell is a damaging process that releases motile parasites. The gliding motility of extracellular tachyzoites is mediated through the interaction of microneme secretion and an actinomyosin motor. To initiate invasion, the parasite extrudes its conoid and adheres to the host cell at its apical end. This process is aided via microneme secretion. The parasite dwells in the parasitophorous vacuole formed via the invagination of the host membrane. During the invasion, the host mitochondria and ER are attracted to the parasitophorous vacuolar membrane. The parasites replicate until the egress signal causes egress, completing the lytic cycle. The genes that regulate egress are unclear, and the particular signals involved are unknown, although it is apparent that Ca2+ signaling is essential in mediating this process. Researchers realize that egress is a dynamic process in which the parasite employs various approaches, including intraparasitic egress signaling, and exogenous compounds [Host cell lytic cycle resulting from replication of tachyzoites leads to acute infection of ompounds ,12. ThesT. gondii must adjust quickly and effectively to rapidly changing conditions, such as those encountered during invasion and egress. Ca2+ is a versatile second messenger that may work across a wide spatiotemporal range and control a wide range of essential cellular responses. T. gondii tachyzoites, akin to other eukaryotic cells, keep cytosolic Ca2+ at a significantly lower concentration (about 100 nM) than the external environment  [2+ release into the cytosol from intracellular storage or Ca2+ influx activates downstream effectors and propagates the signal along several pathways. As sustained high cytosolic Ca2+ concentrations are hazardous, a mixture of buffers, exchangers, and pumps immediately eliminated the calcium ion. Cells have, thus, developed regulatory mechanisms to control Ca2+ release and uptake in response to environmental stimuli. The location, amplitude, and frequency of these Ca2+ transients lead to many downstream effects. The endoplasmic reticulum (ER), acidocalcisomes, plant-like vacuole (PLV), and mitochondria are the vital intracellular depots involved in Ca2+ homeostasis in T. gondii [2+. Moreover, Ca2+ mobilization signals cause inositol 1,4,5-triphosphate (IP3) or ryanodine to bind to their respective ER receptors, causing Ca2+ to be released into the cytosol. Despite pharmacological evidence that IP3 and cyclic ADPR (cADPR) increase Ca2+ release from intracellular storage, there is no genetic support for the existence of an IP3 or ryanodine receptor homologue in T. gondii [2+ ATPase (SERCA) catalyzes Ca2+ absorption into the ER in T. gondii and is sensitive to thapsigargin, a tumor-promoting sesquiterpene lactone and inhibitor of SERCA-type Ca2+ ATPases [2+ leakage from the ER and inhibition of its re-uptake contribute to an increase in cytosolic Ca2+ resulting from thapsigargin inhibition [2+ storage in T. gondii [+-ATPase (V-H+-ATPase) and a vacuolar H+-pyrophosphatase (V-H+-PPase), both of which have been reported and identified in T. gondii (TgVP1) [2+ ATPase (TgA1) was also studied and found in both the acidocalcisome and plasma membrane of T. gondii. This protein helps move Ca2+ out of the cytosol [2+/H+ exchanger is involved in Ca2+ efflux. This suggestion is based on experiments that measured change in the proton gradient after Ca2+ was added. The acidocalcisome probably has a molar concentration of Ca2+; however, its main role seems to be storing Ca2+ since most of it is bound to polyphosphate and can only be released when the pH is more fundamental [To survive, ference) . The coo. gondii ,15. The . gondii ,17. This ATPases ,19. Ca2+hibition ,21,22. A. gondii . Acidoca (TgVP1) . A Ca2+  cytosol ,26. Therdamental ,27.2+ is also stored in T. gondii in apicoplast. This organelle possesses Ca2+/H+ exchanger functions, and the addition of glycyl-l-phenylalanine-naphthylamide (GPN) to extracellular parasites results in its breakdown with a cathepsin C protease, a rise in osmolarity, and a swelling effect that causes Ca2+ to leak into the cytoplasm [2+ homeostasis [2+ uniporter (MCU) that is activated via the electrochemical gradient produced via ATP hydrolysis, which promotes Ca2+ influx [T. gondii intracellular stores are shown to play a role in activating virulence features required for the lytic cycle, Ca2+ entry may also play a role in replenishing intracellular depots and promoting downstream effects triggered via high cytosolic Ca2+. Depletion of the ER activates store-operated Ca2+ channels in the plasma membrane, resulting in Ca2+ influx from the extracellular environment, known as store-operated Ca2+ entry (SOCE) [2+channels (ORAI), ligand-operated channels, or second-messenger operated channels [T. gondii [2+ entry route helps a voltage-gated Ca2+ channel function (VGCC). T. gondii’s genome contains many orthologues to VGCC and sequences that resemble transient receptor potential (TRP) channels [2+ influx and intracellular Ca2+ release both result in an increase in cytosolic Ca2+, activation of a variety of downstream processes, and the eventual return of cytosolic Ca2+ concentration to baseline levels, which is mediated through Ca2+-binding proteins. Ca2+-binding domains, such as EF-hand, allow signal transmission via these proteins. Calmodulin (CaM) from T. gondii was identified at both the apical end of external parasites and under the membrane of internal parasites, while the CaM inhibitors calmidazolium and trifluoperazine inhibited invasion [2+ signaling is widespread and vital in controlling many cellular activities, including T. gondii invasion and egress [Caytoplasm . There ieostasis . Mammali+ influx . While Ty (SOCE) . Howeverchannels . SOCE ha. gondii . The exichannels ,34. Ca2+invasion ,36. Ca2+d egress .T. gondii active egress was originally linked to perforin-like protein 1 (TgPLP1), which plays a role in developing pores on PVM and HCM [2+ were recognized as lysosomes in mammalian fibroblasts and epithelial cells. It is widely known that Trypanosoma cruzi, the causative agent of Chagas disease, requires host lysosome recruitment for endocytic entrance into non-professional phagocytic cells [T. gondii can host lysosome fusion and acidification [T. gondii actively recruits host microtubules for the transport of host endo-lysosomes into the PV lumen in a time-dependent manner, with 73% of host cell lysosomes clustering around the PV two days after infection [Toxoplasma egress [ and HCM . Gene kn and HCM . TgPLP1  and HCM . TgPLP1  and HCM . Accordi and HCM , pH-depeic cells . Previoufication ,45, and nfection . The disa egress . In miceTgPLP1 and TLN4 are both in micronemes and have a calcium-dependent role in parasite egress ,47. HoweT. gondii. Pingret et al. found that calcium is concentrated in the PV compared to the host cell cytoplasm, and the frequency of calcium sequestration rises before parasite egress, using non ratiometric markers in the PV to quantify calcium levels [T. gondii intracellular growth, affects the timing of parasite egress via regulation of calcium homeostasis in the PV [GRA41 results in dysregulation of parasite calcium, which disrupts vacuolar morphology and alters the structure of the TVN. GRA41, unlike GRA1 [T. gondii; however, the absence of GRA22 does not influence the membrane region and intra-vacuolar network of PV [T. gondii egress. Following secretion, TgLCAT is a GRA-like protein that binds to the PV and parasite plasma membrane. T. gondii lacking TgLCAT shows delayed egress, whereas parasites over-expressing TgLCAT exit faster from infected cells [T. gondii synthesizes and releases TgLCAT from 7 h post-invasion until late in infection. In vitro, recombinant TgLCAT displays membrane lytic activity and dual enzymatic activity as a PLA2 and a cholesterol transacyltransferase, showing its potential to disrupt PVM. The upstream signal inducing TgLCAT expression is not yet clear; therefore, whether the accumulation of TgLCAT in PV lumen affects calcium flux through pore-forming on PVM is worth investigating.The main functions of GRAs include changing the PV membrane (PVM) for nutrient acquisition from the host cell into the PV , the form levels . Interesn the PV . Completike GRA1 , is a TVike GRA1 ,47, resuike GRA1 . A similrk of PV . GRA41 aed cells . Unlike ed cells , T. gondT. gondii cytoskeleton, and regulates parasite movement and egress. TgGRA8I is the first proline-rich protein found in the cytoskeleton of Toxoplasma. It links microtubules and actin filaments in the sub-pellicular region. Lack of the GRA8 gene alters the parasite morphology and the sub-pellicular cytoskeleton, and partially inhibits parasitic egress [Besides disrupting PV structure and altering calcium, some GRA functions as a component of the c egress .2+ as a messenger, have been found in plants, ciliates and certain protists, but are absent from fungi and animals [Plasmodium spp., CDPKs play essential roles at various critical physiological stages during parasite development, including merozoite invasion and asexual intraerythrocytic development in humans and sexual/pre-erythrocytic development in Anopheles mosquitoes [T. gondii, such as gliding motility, cell invasion, and egress [Toxoplasma, TgCDPK3, which is found on the parasite’s periphery, controls T. gondii egress through particularly phosphorylated serines 21 and 743 of TgMyoA, which drives the parasite’s gliding motility [Tgcdpk3-mutant parasites display motility defects.Calcium-dependent protein kinases (CDPKs), which are responders that use Ca animals . Due of squitoes . Currentd egress . TgCDPK1d egress . TgCDPK1motility . ExtraceT. gondii egress. After the invasion, T. gondii produces and secretes proteins critical for intracellular replication, including GRA41 for PV modification. T. gondii initiates TgLCAT production and releases TgLCAT into the PV lumen 7 h after the invasion. Accumulative TgLCAT uses its membrane lytic activity to create holes in the PVM, enabling calcium influx into the PV lumen from the host cell cytoplasm or calcium reserves in the host cell, such as mitochondria and ER. When calcium levels rise, micronemes release TgPLP1 and TLN4, which destroy PVM and HCM. However, damage to PVM and HCM by pore-forming proteins in microneme may not be sufficient for complete T. gondii egress. TgCDPKs phosphorate TgMyoA, which initiates parasite motility when calcium levels rise. T. gondii then completes its egress from infected cells. T. gondii egress occurs in three stages: (i) production and accumulation of TgLCAT; (ii) calcium influx from the host cell; and (iii) PVM and HCM damage, along with parasite motility.Collectively, we can provide a concise picture of how parasite proteins control TgEFP1 [TgEFP1 results in faster propagation in tissue culture, hypersensitivity to calcium ionophore-induced egress, and premature natural egress   and TgEFTgEFP1 . Pcr2 knl egress .T. gondii infection elicits host type I immune responses, characterized through the proliferation of specific CD8+ T cells and production of a variety of inflammatory cytokines, such as IL-12 and IFN-γ. These cytokines are vital for parasite control, intracellular replication, and activating host cells to remove parasites. Previous studies showed that externally triggered egress (ETE) is the main fate of T. gondii during acute infection in mice, which does not require reactive oxygen or nitrogen species, the mitochondrial permeability transition pore, or a variety of signal transduction mediators; instead, it depends on intracellular calcium and is highly sensitive to SB203580, which is an inhibitor of p38 MAPK and a related parasite-encoded kinase [d kinase .T. gondii invasion into murine astrocytes in an IGTP-dependent manner [T. gondii replication in astrocytes in certain cases. In HFFs, IFN-γ induces cell death and early egress of T. gondii, which limits intracellular replication of T. gondii in HFFs and can promote clearance of the parasite by immune cells. IFN-γ-induced egress does not depend on the deprivation of tryptophan or iron [T. gondii instead of egress [T. gondii egress was a direct consequence of PV disruption resulting from effectors involved in IFN-γ signaling, although the role of parasitic motility and intraparasitic calcium needs future investigation. T. gondii can infect and multiply intracellularly in “naive” cells that inflammatory cytokines have not triggered. It is currently unknown if IFN-γ post-treatment causes T. gondii egress.IFN-γ induces PV disruption shortly after t manner . IFN-γ c or iron . Howeverf egress . Cell liT. gondii tachyzoites from infected peritoneal macrophages and HFFs [T. gondii [T. gondii from host cells in vivo.One variable related to tachyzoite and bradyzoite interconversion was NO  . NO releand HFFs ,74. The . gondii . HoweverT. gondii-infected cells causes rapid egress of tachyzoites and lytic necrosis of the host cell, which is mediated via the release of intracellular calcium because of caspase activation early in the apoptotic cascade. More importantly, upon acting on infected cells via death receptor- or perforin-dependent pathways, T cells induce rapid egress of T. gondii capable of infecting neighboring cells, including the Ag-specific effector cells [T. gondii infection. Active macrophages or other immune cells may engulf and kill free tachyzoites because of this death receptor- and perforin-mediated parasite egress.Death receptor ligation and the perforin-dependent granule exocytosis are two pathways used by cytotoxic cells to induce apoptosis of target cells, such as pathogen-infected cells. A study showed that death receptor ligation in or cells . Death rT. gondii dissemination [T. gondii from infected HFF cells in a time-dependent manner. Blocking the host apoptosis pathway significantly decreased TNF-α– induced egress [TNF-α is a key inflammatory factor in controlling mination . An in vd egress . HoweverT. gondii egress. According to previous research using ionophores to artificially stimulate egress, increasing cytoplasmic Ca2+ stimulates microneme production, motility, and egress. Using a Ca2+ ionophore—A23187—and an ionophore-resistant mutant, the link between Ca2+ and egress was first proven [2+ from intracellular reserves, and the stimulation of extracellular parasite microneme secretion [2+ ionophores through increasing Ca2+ fluxes, and that the same intracellular reserves engaged in microneme production and invasion may also be involved in egress.Besides inflammatory factors, various exogenous compounds were used to investigate the processes of t proven . Ionophot proven . The mutt proven . Ethanolecretion . The appecretion . These f+ in egress was highlighted using an artificial permeabilization system that exposed intracellular tachyzoites to buffers of various ionic compositions [+ in the extracellular environment was postulated to activate a parasite PLC, which would then increase the parasite’s cytoplasmic Ca2+ and, eventually, events prompting egress. The ionophore nigericin, which selectively causes efflux of potassium from the cell, effectively causes egress of T. gondii within 30 min of treatment with that drug, which requires phospholipase C function and parasite motility [2+ signaling and motility, implying that host cell K+ loss occurs upstream of intraparasitic Ca2+ fluxes on the egress route. The intracellular environment’s high K+ content has been linked to regulating microneme production and motility, both controlled via Ca2+ release from intracellular reserves and essential for parasite egress from the host cell. Acidification of the parasitophorous vacuole has been shown to overcome the inhibition of high K+ levels in the intracellular environment, enhancing motility and microneme secretion [Other investigations have shown that permeabilization of the host cell happens immediately before natural egress. When membrane integrity is lost, ions and small molecules exchange between the intracellular and external environments, exposing parasites to these alterations. The significance of Kositions . On hostmotility . This woecretion . Perforiecretion ,39,50. TT. gondii egress [T. gondii-infected host cells stimulated motility and egress of intravacuolar parasites, invariably accompanied by a detectable Ca2+ flux within the host cell [2+ just before egress. Uninfected host cells and extracellular parasites do not display an increase in Ca2+, and only infected host cells show a drop in ATP when stimulated with DTT, reinforcing the response’s reliance on both the host and parasite, and suggesting that Ca2+ flow and NTPase activity may be connected. The NTPases are thought to cause the disrupted Ca2+ ATPases in the host ER, resulting in an increase in Ca2+ in the cytosol around the parasite. It is still unclear whether the mechanism causes NTPase activation in vivo, or if their lowering serves as a critical egress sensor.Using dithiothreitol (DTT), which is a reducing chemical that stimulates parasite nucleotide triphosphate-degrading enzymes (NTPs) released into the PV, is also shown to promote i egress ,85. Dithost cell . When th2+ in T. gondii and plants [The accumulation of the phytohormone abscisic acid (ABA), continually generated by the parasites during intracellular reproduction, was also examined as an egress route . The pard plants . The exaT. gondii. This mechanism causes inflammation and is intimately linked to the pathogenesis of toxoplasmosis. There are flaws in the existing in vitro models for egress. The HFF line is most commonly employed in both traditional cultures and in vitro investigations. This method is used mostly for convenience since these cell lines are simple to maintain in culture, and their flat shape makes them perfect for microscopy. However, this cell type is unlikely to be infected with Toxoplasma during a natural infection. One alternative would be to perform egress assays in a context that more accurately reflects what we observe in vivo studies, namely through selecting immune cell lines or nerve cells to replace HFFs, and performing these assays in inflammatory conditions that mimic the severe inflammatory immune response that is characteristic of T. gondii infection. Current approaches for examining egress depend heavily on activating intracellular calcium signaling in the parasite to induce egress of intracellular parasites. Interactions between CD8 T cells and infected cells, for example, may be a trigger for parasite egress. This approach is probably a means for the parasite to detect and react correctly when its environment is under attack. At least two mechanisms describe this event: signaling through Fas ligand and perforin attack. An effective way of examining egress in vitro would be via a biological method, rather than pharmacological stimulation of egress. Infected cells that have been altered to express the correct receptors, or selected for endogenous expression of these molecules, might be stimulated via the addition of recombinant ligands or activated immune cells that express these molecules. This induction tactic would be a better approach to studying egress because it would more accurately mimic the actual events that stimulate egress during toxoplasmosis. It would also induce a response that uses relevant signaling pathways and, presumably, elicits responses at a natural level. Egress has always been challenging to examine due to how little is understood about the process and its dynamic nature. A more refined approach might enable future research to find phenotypes related to genes with minimal influence. In the future, organoid tissues may be used in the egress study of T. gondii in more complex systems.Egress is a critical stage in the intracellular lytic cycle of


Therapeutic advances in acute myeloid leukemia (AML) are dependent on identifying and targeting the molecular aberrations that drive disease. The European LeukemiaNet (ELN) 2022 guidelines have improved the categorization of AML into distinct molecular subgroups. One of the most notable recent inclusions is a group of mutations highly specific for secondary AML. This review examines how each of the secondary-type mutations contributes to the genesis of leukemia while spotlighting potential therapeutic avenues. While we highlight the limitations of the ELN 2022 revision, we also emphasize current progress, recent breakthroughs, and novel therapeutic options tailored to each molecular subset. This review provides a background and foundation for rational molecular-based therapeutic approaches and combination strategies to inspire future clinical trial designs.The characterization of the molecular landscape and the advent of targeted therapies have defined a new era in the prognostication and treatment of acute myeloid leukemia. Recent revisions in the European LeukemiaNet 2022 guidelines have refined the molecular, cytogenetic, and treatment-related boundaries between myelodysplastic neoplasms (MDS) and AML. This review details the molecular mechanisms and cellular pathways of myeloid maturation aberrancies contributing to dysplasia and leukemogenesis, focusing on recent molecular categories introduced in ELN 2022. We provide insights into novel and rational therapeutic combination strategies that exploit mechanisms of leukemogenesis, highlighting the underpinnings of splicing factors, the cohesin complex, and chromatin remodeling. Areas of interest for future research are summarized, and we emphasize approaches designed to advance existing treatment strategies. SF3B1, SRSF2, U2AF1, ZRSR2, BCOR, EZH2, and STAG2. These seven mutations, along with several others, comprise a group known as the secondary-type mutations, aptly named for their ubiquity in secondary AML [Next-generation sequencing has transformed the landscape of acute myeloid leukemia. Over the last several decades, the identification of recurrent genetic abnormalities in AML has guided modern clinical approaches toward a molecular era. The recent 2022 revision to the European LeukemiaNet guidelines builds upon its predecessor ,2; in adNPM1 or rearrangements of the core-binding factor retain their favorable prognoses [Secondary AML defines a subset of the disease with notoriously adverse outcomes. Based on preceding myelodysplastic neoplasms (MDS), myeloproliferative neoplasms (MPNs), or therapy-related clonal aberrations, secondary AML is associated with lower remission rates and overall survival compared with de novo AML ,5,6. Whirognoses ,8. In coSF3B1, SRSF2, U2AF1, and ZRSR2, are found in approximately 65% of patients with MDS [BCOR, EZH2, or STAG2; 36.8% harbored mutations in any of the seven previously mentioned genes or ASXL1 or RUNX1 -rich snRNPs: U1, U2, U4, U5, and U6. Starting from the proximal end of a target splice region, U1 binds to the 5′ splice site. Next, the budding spliceosome locates the distal end of a splice region through a two-step process. First, splicing factor 1 (SF1) binds to the branch site, an area near the 3′ splice site . Second,The diversity of proteins created through splicing is staggering; it is directly related to the selection of splice sites. The spliceosome always recognizes some splice sites, known as constitutive splice sites. In contrast, the recognition of other splice sites depends on the recruitment of additional factors; therefore, they are only occasionally spliced. These are alternative splice sites, which create combinatorial effects on protein diversity ,17. In mSF3B1, SRSF2, U2AF1, and ZRSR2. The first three are components of the major spliceosome; they exist in a heterozygous state and usually have altered or gain-of-function effects—consistent with their role as oncoproteins. In contrast, ZRSR2 is a component of the minor spliceosome and commonly exhibits a loss-of-function effect, acting as a tumor suppressor [The four most commonly mutated spliceosome mutations in AML are ppressor . The splppressor . This fuppressor . Thus, wSF3B1 occurred in 2.6%  of patients with AML, with a median upfront variant allele frequency (VAF) of 46.1%, consistent with its known heterozygous presentation; additional aggregate analyses have confirmed SF3B1mut frequencies of 2–5% in AML [SF3B1 frequently guide the spliceosome to an alternative 3′ splice site. Consequently, the introduction of a premature stop codon in target gene expression due to SF3B1mut is a common occurrence, resulting in reduced protein expression due to aberrant splicing [Splicing factor 3B subunit 1, also known as SF3B1, is a protein component of the U2 snRNP; it binds to the branch site adjacent to the 3′ splice site. Therefore, the function of SF3B1 is to assist in correctly localizing the spliceosome to the 3′ splice site. In Project ERIS, mutations in % in AML . Mutatiosplicing .SF3B1mut in hematological malignancies can be best appreciated in MDS with ringed sideroblasts. The abnormal splice site degrades a mitochondrial iron exporter, ABCB7 [SF3B1 create terminal blocks in erythroid maturation but without ringed sideroblasts [SF3B1 mutations are frequently found with AML-driving genes: RUNX1mut or rearrangements of MECOM [SF3B1 mutations are often early events, this finding suggests that AML with SF3B1mut arises following the acquisition of additional adverse-risk mutations—highlighting the need for more effective therapeutic approaches.The impact of r, ABCB7 . Reductir, ABCB7 . Other mroblasts ,24. In Aof MECOM . As SF3BThe numerous inhibitors of SF3b are the most well-studied spliceosome inhibitors. Pladienolide analogs were the first splicing modulators to enter clinical trials. One of the earliest splicing modulators was E7107, which disrupts splicing by targeting SF3b and inhibiting U2 ,27. AttrFLT3-ITD) may be more sensitive to the pladienolide analogs. In AML with FLT3-ITD, investigators noted increased induction of cell cycle arrest and a shift in the splicing patterns toward a pro-apoptotic state due to reduced MCL1 following pladienolide treatment [Newer pre-clinical studies suggest that AML with internal tandem duplications in FMS-like tyrosine kinase  is a regulatory SR protein that promotes splicing events by binding to splicing enhancer segments . Mutatedse genes ,36. Due ogenesis .SRSF2 ubiquitously occur at proline 95, identified in 90.9%  of AML in Project ERIS—the reminder commonly appear at proline 94 or 96. A single allele mutation in SRSF2 at proline 95 disrupts RNA-binding specificity and is sufficient to induce myelodysplasia [SRSF2mut AML.Mutations in ysplasia . ConsequSRSF2mut AML. Investigators administered E7107 to mice with SRSF2P95H. Untreated mice developed bone marrow failure; treated mice had a significantly reduced leukemic burden [SRSF2mut AML than in wild-type genotypes [SRSF2P95H mice compared with wild-type mice [Both investigational drugs that interfere with SF3b were evaluated in c burden . Moreoveenotypes . Similarype mice . NeitherSRSF2mut PDX model showed a significant and dose-dependent response to CTX-712; additionally, the drug demonstrated anti-leukemic efficacy in PDX AML models without spliceosome mutations [The discovery that spliceosome proteins require phosphorylation for nuclear transport—a process regulated by the CDC-like kinase (CLK) family—paved the way for innovation . CTX-712utations . In a phutations . A phaseU2AF1 knockout show pancytopenia, a reduction in hematopoietic stem cells, and decreased survival [U2 small nuclear RNA axillary factor 1 (U2AF1) recognizes the 3′ splice site and recruits the U2 snRNP during splicing . Mutatiosurvival .U2AF1S34F predisposes to increased translation of interleukin 8, upregulating the inflammatory response and correlating with a higher incidence of relapsed or refractory AML in humans; blocking interleukin 8 led to tumor burden reduction in mice [U2AF1mut is responsible for the alternative splicing of IRAK4, which results in the retention of an exon and creates a long isoform: IRAK4-long (IRAK4-L).U2AF1 has additional roles aside from splicing. U2AF1 and its binding partner, U2AF2, bind to cytoplasmic RNA and act as translational repressors . U2AF1S3 in mice . ConsistU2AF1mut in AML induces the expression of a novel therapeutic target, IRAK4-L, which is inhibited with emavusertib. Single-agent emavusertib demonstrated a composite complete remission rate of 40.0% in AML with spliceosome mutations, and 57.1% of high-risk MDS patients had <5% marrow blasts [Investigators discovered that inhibition of IRAK4-L disrupts leukemic growth . Thus, tw blasts . EmavuseZRSR2 mutations differ from the remaining spliceosome mutations in two critical ways. First, ZRSR2 mutations primarily affect the minor spliceosome; SF3B1, SRSF2, and U2AF1 mutations tend to affect the major spliceosome. Second, mutations in ZRSR2 are scattered throughout the length of the gene, in contrast to the other three spliceosome mutations, which occur at hotspots. As ZRSR2 is located on the X chromosome, mutations are linked to male-predominant leukemia—particularly in blastic plasmacytoid dendritic cell neoplasm [ZRSR2mut were male, suggesting a similar male-dominant presentation in AML.The zinc finger (CCCH type), RNA-binding motif, and serine/arginine rich 2 (ZRSR2) gene encodes a key member of the minor spliceosome that recognizes 3′-intron splice sites . ZRSR2 mneoplasm . In the ZRSR2 results in reduced differentiation of hematopoietic precursors due to the aberrant retention of U12-type introns, suggesting that a competent minor spliceosome is necessary for myeloid differentiation [ZRSR2 leads to increased self-renewal of hematopoietic stem cells [ZRSR2 knockdown exhibited slower growth compared with wild-type controls, a pattern not exclusive to loss of ZRSR2; U2AF1mut AML demonstrated similar findings of reduced hematopoietic reconstitution [ZRSR2 appear to lead to constitutive stem cell self-renewal but do not appear to contribute to proliferative phenotypes.ZRSR2 is required for splice site recognition of U12-type introns, which are removed by the minor spliceosome. Knockdown of ntiation . Loss ofem cells . Additiotitution . TherefoZRSR2mut AML. Novel agents that reverse the retention of U12-type introns and restore the function of the minor spliceosome have yet to be widely identified or studied, representing an area of unmet clinical need and a growing area of research interest. However, several mis-spliced targets may be of interest—ZRSR2 mutations frequently result in the aberrant splicing of several E2F and MAP kinase signaling regulators, including the RAS pathway [ZRSR2mut AML will likely require the identification of compounds that restore the function of the minor spliceosome, induce synthetic lethality through inhibition of other targets, or modify downstream mediators dysregulated by aberrant U12-type intron splicing.There is relatively limited data on adopting a targeted approach to  pathway . BreakthBCOR) is a transcription factor that regulates stem cell function and hematopoiesis. To understand the role of BCOR in AML, we will first review the polycomb group proteins. The polycomb group proteins are responsible for remodeling chromatin; they are recognized for silencing HOX genes through chromatin structure alteration, a critical process for hematopoiesis. Two key complexes in the polycomb group family are polycomb repressive complex 1 (PRC1) and polycomb repressive complex 2 (PRC2). PRC1 catalyzes the ubiquitination of histones (a histone ubiquitin ligase), repressing target genes that control stem cell pluripotency [BCORmut AML, we will shift our focus to the interaction between PRC1 and PCR2.The BCL6 corepressor . In striking accordance with these observations, BCORmut AML appears to be sensitive to tyrosine kinase inhibition targeting pathways involved in stem cell pluripotency [BCORmut-mediated disease refractoriness in AML [tory AML , frequenKRASG12D . In Projipotency . Ongoings in AML .EZH2 are a double-edged sword: as its target genes are required for stem cell maintenance, both gain- and loss-of-function mutations result in characteristic hematopoietic perturbations. A detailed review of epigenetics in leukemogenesis can be found here [As we have reviewed the role of chromatin compaction with BCOR in PRC1, we will similarly unpack the function of enhancer of zeste homologue 2 (EZH2) in PRC2. EZH2 is the catalytic component of PRC2, a transcriptional corepressor with lysine methyltransferase activity that controls gene expression through histone modification . The addund here .EZH2 acts as an oncoprotein in gain-of-function mutations. Well-studied in the context of lymphomagenesis, EZH2 overexpression results in transcriptional suppression of genes responsible for differentiation . During EZH2mut will require more innovative approaches compared with its gain-of-function counterpart.In contrast, loss-of-function EZH2 mutations are more challenging to approach. As gain-of-function mutations act as oncoproteins, loss-of-function EZH2 mutations, conversely, act as tumor suppressors. In mouse models, loss-of-function EZH2 mutations activate bivalent promoters that accelerate AML induction . TherefoBCORmut AML, loss-of-function of EZH2 is associated with resistance to cytarabine [EZH2mut-mediated resistance to treatment. An additional therapeutic avenue involves potentially targeting a mutually exclusive mutation. For example, SRSF2mut and EZH2 loss-of-function mutations are mutually exclusive in MDS [EZH2 mutations, SRSF2 inhibition theoretically induces synthetic lethality. Further research is needed to characterize the dynamic landscape associated with EZH2 loss-of-function in AML.Similar to tarabine ; howevere in MDS . Thus, iAdditional sex combs-like 1 (ASXL1) is jointly responsible for deubiquitinating histones—a function it performs with its histone deubiquitinase partner, BRCA1-associated protein 1 (BAP1). BAP1 is part of the polycomb repressive deubiquitinase (PR-DUB) complex, which reverses the repressive ubiquitination signatures of polycomb repressive complex 1. Therefore, the PR-DUB complex promotes gene expression .HOX genes. Mutations in ASXL1 result in BAP1 hyperactivation. BAP1 gain-of-function creates a state of histone deubiquitination at HOX gene regions, leading to leukemic transformation [HOX genes through histone ubiquitination at these regions; however, hyperactive BAP1 reverses PRC1-mediated ubiquitination signatures and creates constitutive HOX gene expression, resulting in myeloid leukemogenesis [ASXL1mut AML [ASXL1 activates BAP1 . In turnormation . Under nogenesis . Therefo1mut AML .Additionally, mutated ASXL1 acquires a novel binding partner: bromodomain-containing protein 4 (BRD4) . BRD4 acASXL1mut AML by inhibiting the interaction between mutated ASXL1 and BRD4, suggesting BET inhibitors may have a role in ASXL1mut AML [ASXL1mut AML, which were shown to inhibit HOX gene expression [Indeed, small-molecule inhibitors that target bromodomain and extraterminal (BET) domain proteins—including BRD4—demonstrate antileukemic activity . BRD4 in1mut AML . Further1mut AML ,68. Othepression .STAG2, one of the four core units of the cohesin complex. The cohesin complex regulates chromatin structure; it is named because it mediates the cohesion of sister chromatids during replication and facilitates the transition from metaphase to anaphase during cell division [The cohesin subunit SA-2 is encoded by division ,70. WhilFLT3-ITD [STAG2 mutations in AML, however, have not focused on indirectly targeting common cooperating mutations but on leukemic clone elimination directly through synthetic lethality.STAG2, alongside the remaining three core cohesin partners—RAD21, SMC1A, and SMC3—promotes hematopoietic stem cell differentiation . InactivFLT3-ITD . EffortsSTAG2 knockout cell lines had homologous recombination deficiencies [SRSF2mut cells compared with wild-type cells [STAG2 knockout cells to PARP inhibition. Talazoparib showed moderate anti-leukemic activity as monotherapy in a molecularly unselected, heavily pretreated AML population [ciencies . Talazoppe cells . Therefopulation ; combinapulation ,78.STAG2mut AML. Cancer cells depend on the overlapping function of STAG1 in STAG2 knockout cell lines to maintain the integrity of chromatin cohesion; double knockout of STAG1 and STAG2 induces synthetic lethality [STAG2mut AML cells and was well tolerated by wild-type hematopoietic stem cells [STAG2mut AML.STAG1, a protein paralogous to STAG2, is an additional target for the induction of synthetic lethality in ethality . Recentlem cells . These fRUNX1 encodes the α subunit of the core-binding factor and is therefore responsible for binding transcription complexes to DNA [RUNX1 aberrations in AML: RUNX1-related chromosomal rearrangements and RUNX1 somatic mutations. In the most well-known RUNX1-related rearrangement in AML, RUNX1 fuses to a transcriptional corepressor (RUNX1T1), creating the t fusion protein, RUNX1::RUNX1T1, which we have reviewed elsewhere [RUNX1-related rearrangement in AML confers a favorable prognosis, somatic mutations do not [RUNX1mut in AML.Runt-related transcription factor 1 (RUNX1) mutations do not directly alter chromatin structure. However, they affect the binding of transcription factors to chromatin and indirectly influence gene expression through altered chromatin interactions . RUNX1 es to DNA . There alsewhere . While tg the t8;1 fusion RUNX1 mutations lack DNA binding activity; missense mutations similarly lack DNA binding activity and down-regulate wild-type RUNX1, creating a dominant-negative effect [Truncated e effect . Other de effect . These ae effect . Althouge effect . In addie effect .RUNX1 mutations in AML suggest sensitivity to ribosome disruptions .The effects of ruptions . Investihibitors . Homoharnockdown . AML blasistance ,86. A clThe addition of these seven recurrent genetic aberrations to the ELN 2022 adverse risk category has refined the prognostication of AML. This group of newly added mutations plays a dominant role in splicing and epigenetic regulation, with unique opportunities for targeted therapies in each molecular cohort. In addition, alternative therapeutic approaches likely exist in larger subgroups by exploiting the nature of the underlying epigenetic or splicing aberrations.For example, spliceosome mutations lead to alternative splicing of other splicing factors . This reZRSR2mut AML that underwent 19 cycles of consolidation with nivolumab following conventional 7 + 3; the patient achieved a complete response, and the overall survival was 26.3 months. Nevertheless, reports of immunotherapy-associated outcomes in AML with spliceosome mutations are limited.A phase II study of pembrolizumab and cytarabine in relapsed or refractory AML resulted in a composite complete remission rate of 25.0% in secondary AML ; similarHOXA9, resulting in the preferential lethality of spliceosome-mutated AML [Other approaches indirectly target splicing. RBM39, an RNA splicing factor, prolongs the survival of AML cells, suggesting a role in AML maintenance . E7820 iated AML . In a phated AML . Other tated AML ,92. The Mutational cooperation invariably complicates targeted approaches. Combination therapies with multiple targeted agents hold promise in the treatment of AML, and clinical trials should be designed to encourage the recruitment of specific molecular cohorts. Innovations in AML require mutation-driven treatment paradigms to bring new hope to the field as we begin a new chapter of the molecular era.


These functions are intricately regulated by a variety of Ca2+-dependent cellular processes, encompassing synaptic plasticity, neuro/gliotransmitter release, and gene expression. In our previous work, we developed ‘monster OptoSTIM1’ (monSTIM1), an improved OptoSTIM1 that selectively activates Ca2+-release–activated Ca2+ (CRAC) channels in the plasma membrane through blue light, allowing precise control over intracellular Ca2+ signaling and specific brain functions. However, the large size of the coding sequence of monSTIM1 poses a limitation for its widespread use, as it exceeds the packaging capacity of adeno-associated virus (AAV). To address this constraint, we have introduced monSTIM1 variants with reduced coding sequence sizes and established AAV-based systems for expressing them in neurons and glial cells in the mouse brain. Upon expression by AAVs, these monSTIM1 variants significantly increased the expression levels of cFos in neurons and astrocytes in the hippocampal CA1 region following non-invasive light illumination. The use of monSTIM1 variants offers a promising avenue for investigating the spatiotemporal roles of Ca2+-mediated cellular activities in various brain functions. Furthermore, this toolkit holds potential as a therapeutic strategy for addressing brain disorders associated with aberrant Ca2+ signaling.Calcium ions (CaThe online version contains supplementary material available at 10.1186/s13041-023-01061-7. In esponses –15.2+ signals and cellular functions in space and time, we and others have previously developed a series of molecular optogenetic technologies designed to control intracellular Ca2+ levels through light stimulation [2+-release–activated Ca2+) channels, which are widely expressed and highly selective for Ca2+ [2+ entry (SOCE), plays a key role in these technologies. Under natural conditions, STIM1, which senses Ca2+ levels in the ER lumen through domains in its N-terminus, resides in the endoplasmic reticulum (ER) membrane [2+, STIM1 oligomerizes and undergoes conformational changes in its C-terminal domains. It subsequently translocates to the plasma membrane, where it binds to and activates CRAC channels, thereby leading to an increase in intracellular Ca2+ levels.To directly examine causal relationships between Camulation . These tfor Ca2+ . STIM1  mutant significantly reduces the basal activity of OptoSTIM1 in the dark while simultaneously enhancing its sensitivity to blue light . This pr2+ modulators in the mouse brain [However, combining the size of the coding sequence of monSTIM1 with AAV cassette components, including ITRs , WPRE  and hGH (human growth hormone) polyadenylation signal results in a total sequence size of ~ 5.1 kb without a promoter, exceeding the packaging capacity (~ 5.0 kb) of adeno-associated viruses (AAVs) , CRY2 , and the cytosolic STIM1 fragment (a.a. 238–685)  [2+ channels  , which i2+ influx under light stimulation. Only Set 1 construct was able to induce a Ca2+ increase at a comparable level to monSTIM1, suggesting that the expression levels of CRY2-fused STIM1 proteins can influence both the basal Ca2+ level in the dark and the maximal Ca2+ level under light illumination.To determine whether the basal intensity of R-GECO1 could be decreased by reducing the expression level of CRY2-STIM1 variants, we employed an IRES2  to drive protein expression  [2+ influx, indicating that the N-terminal fusion of vhhGFP to CRY2 retained the property of GFP binding and light-dependent oligomerization of CRY2. In contrast, C-terminal fusion proteins (Sets 2 and 3) showed significantly lower levels of Ca2+ increase, suggesting the C-terminal fusion interfered with GFP binding and/or light-dependent oligomerization of CRY2. Notably, none of the candidates exhibited an increase in basal R-GECO1 fluorescence in the dark, indicating that the STIM1 fragment (a.a. 238–685) sufficiently inhibited CAD activity in the absence of light stimulation  . Therefo2+ levels. To obtain a more precise analysis of Ca2+ levels in both dark and light conditions, we performed Fura-2 imaging on cells expressing each monSTIM1 variant , exhibited comparable levels of basal Ca2+ to those observed in non-transfected cells, consistent with the trends observed using R-GECO1. The slightly elevated Ca2+ level observed in GFP-CRY2-STIM1(318–450) suggests that the N-terminal autoinhibitory domain of STIM1 was insufficient to completely suppress the activity of CAD.In the results described above, we utilized R-GECO1 signals as a surrogate measurement of relative Ca2+ signals in the brain, we introduced these proteins into cultured neurons, astrocytes, and microglia. In neurons expressing monSTIM1 variants, we observed rapid and sustained increases in intracellular Ca2+ levels upon repeated light exposure  . To further increase the available space for packaging the coding sequence, we additionally replaced WPRE and hGH polyadenylation signal in the AAV cassette with W3SL, a smaller component that enhances the expression of a transgene [2+-responsive immediate-early gene, in approximately 10% of STIM1-positive neurons  [2+ influx and subsequent activation of signaling downstream of Ca2+ influx in the brains of freely moving animals through non-invasive delivery of blue light. Therefore, these tools offer a strategy for circumventing the compounding effects associated with conventional in vivo optogenetic experiments, such as tissue damage, glial scar formation, inflammation and tissue heating, that arise from the long-term insertion of optic fibers [Here, we have developed AAV-compatible monSTIM1 variants that enable precise and selective modulation of intracellular Cafindings , these CptoCRAC) . ImportaptoCRAC) , the exac fibers –37.2+ levels in HeLa cells in the absence of blue light , indicating that the majority of these molecules are in an inactive state. However, it is important to note that the expression of EGFP-CRY2-STIM1(318–450) in the CA1 neurons under dark conditions did not lead to any significant increase in cFos expression , none of the monSTIM1 variants induced significant increases in basal Caion Fig. C, E, indactivity –40. Desp2+ [2+-specific events or controlling the function of non-excitable cells, such as astrocytes and microglia, in the brain. In contrast, monSTIM1 specifically induces Ca2+ influx in both neurons and glial cells owing to the ubiquitous expression of Ca2+-selective CRAC channels [2+-mediated neuronal and glial cell functions, as well as their interactions in various contexts.A number of opsin-based optogenetic tools, such as channelrhodopsin 2 (ChR2) variants, have been developed for controlling neuronal activity . While t2+ . Consequchannels . Therefo2+ activity in specific cell types within the brain, obviating the need for brain surgery. We anticipate that our optogenetic tools for controlling Ca2+ signals will provide a framework for developing potential therapeutic interventions for diseases associated with dysregulated Ca2+ signaling.AAVs have attracted significant attention for their ability to deliver genes to treat a wide range of human diseases . Recent NheI and SalI sites after excising EGFP. The LOV2(404–546) sequence in OptoCRAC [BsrGI and HindIII sites to generate the EGFP-LOV2 vector. The STIM1(336–486) sequence in GFP-monSTIM1 was PCR-amplified using STIM1(336–486)-F and STIM1(336–486)-R primers and ligated into the EGFP-LOV2 vector at HindIII and BamHI sites to generate the OptoCRAC expression plasmid. For construction of GFP-CRY2-STIM1(238–448), GFP-CRY2-STIM1(248–448), GFP-CRY2-STIM1(336–448) and GFP-CRY2-STIM1(342–448) expression plasmids, sequences encoding STIM1(238–448), STIM1(248–448), STIM1(336–448) and STIM1(342–448) fragments were PCR-amplified using STIM1(238–448)-F, STIM1(248–448)-F, STIM1(336–448)-F, STIM1(342–448)-F, and STIM1(238–448)-R primers and ligated into monSTIM1 at BspEI and BamHI sites. For construction of GFP-CRY2-STIM1(318–463) and GFP-CRY2-STIM1(318–450) expression plasmids, sequences encoding STIM1(318–463) and STIM1(318–450) fragments were PCR-amplified using STIM1(318–463)-F, STIM1(318–463)-R and STIM1(318–450)-R primers and ligated into monSTIM1 at BspEI and BamHI sites. For generation of the IRES2-CRY2-STIM1(238–448) expression plasmid, the IRES2 sequence was PCR-amplified using IRES2-F and IRES2-R primers and ligated into GFP-CRY2-STIM1(238–448) at NheI and BsrGI sites after excising EGFP. The sequence encoding EGFP was then inserted at NheI and NotI sites to generate the EGFP-IRES2-CRY2-STIM1(238–448) expression plasmid. The STIM1(238–448) fragment was replaced with other STIM1 variants at BspEI and BamHI sites. The CRY2 sequence in monSTIM1 was PCR-amplified using CRY2-F and CRY2-R primers and ligated into EGFP-N1 at AgeI and BsrGI sites after excising EGFP to generate the CRY2-N1 vector. The vhhGFP sequence in mCherry-CRY2-vhhGFP [NheI and AgeI sites to generate the vhhGFP-CRY2 expression plasmid. The CRY2 sequence from CRY2-N1 was digested with AgeI and BsrGI and ligated into EGFP-C1 (Clontech) after excision of EGFP to generate the CRY2-C1 vector. The vhhGFP sequence in mCherry-CRY2-vhhGFP was PCR-amplified using vhhGFP-F2 and vhhGFP-R2 primers and ligated into CRY2-C1 at BsrGI and XhoI sites to generate the CRY2-vhhGFP vector. For construction of virus vector for pAAV-CamKIIa-GFP-monSTIM1, an exchange enzyme site was designed by using NheI-BamHI oligomer and inserted into pAAV-CamKIIa-EGFP (addgene #50469). GFP-monSTIM1 was ligated into pAAV-CamKIIa-EGFP at the NheI and BamHI sites after excising EGFP. The W3SL sequence in pAAV-CW3SL-EGFP (Addgene plasmid #61463) was PCR-amplified using W3SL-F and W3SL-R primers and then ligated into pAAV-CamKIIa-GFP-monSTIM1 vector at the BamHI and RsrII sites after the excision of WPRE and hGH poly(A) signal to generate the pAAV-CamKIIa-GFP-monSTIM1-W3SL vector. For construction of virus vectors of pAAV-CamKIIa-FLAG-monSTIM1-W3SL and pAAV-CamKIIa-GFP-STIM1(318–450)-W3SL, FLAG-monSTIM1 or GFP-STIM1(318–450) was inserted into the pAAV-CamKIIa-GFP-monSTIM1-W3SL vector using NheI and BamHI sites after excising GFP-monSTIM1. The GfaABC1D promoter sequence was PCR-amplified using GfaABC1D-F and GfaABC1D-R primers and ligated into pAAV-CaMKIIα-FLAG-monSTIM1 at MluI and XbaI sites after excising CaMKIIα promoter sequence to generate pAAV-GfaABC1D-FLAG-monSTIM1. Oligonucleotides used in this research are listed in Additional file Expression plasmid for R-GECO1 (Addgene plasmid #32444) was obtained from Addgene. For construction of expression plasmids for HA-monSTIM1 and FLAG-monSTIM1, the monSTIM1 sequence from GFP-monSTIM1  was amplOptoCRAC  was PCR-Y2-STIM13–448 expr2 atmosphere. Hippocampal neurons were prepared from embryonic day 15–16 mice, and the hippocampi from collected embryos were dissected in Hank’s balanced salt solution . The collected hippocampus was dissociated by incubating with 0.05% trypsin for 5 min at 37 °C, filtered through a 0.4-μm filter, and then seeded onto a 24-well polymer-coverslip–bottom plate  coated with 50 μg/mL poly-D-lysine . Neurons were grown in Neurobasal medium Cat# 21103-049) supplemented with 2% B-27, 2% N-2 supplements, 2 mM GlutaMAX , and 1000 units/mL penicillin–streptomycin and maintained at 37 °C in a humidified 5% CO2 atmosphere. Primary cortical astrocytes were dissected by removing the adherent meninges from P0-P1 C57BL/6 mouse pups, followed by dissociation into a single-cell suspension by trituration through a Pasteur pipette. Dissociated cells were plated on a 60-mm dish coated with 50 μg/mL poly-D-lysine. Cells were grown in high-glucose DMEM (Gibco) containing L-glutamine, supplemented with 10% horse serum, 10% FBS and 1000 units/mL penicillin–streptomycin, and maintained at 37 °C in a humidified 5% CO2 atmosphere. Cells were transfected using Lipofectamine LTX  according to the manufacturer’s instructions.HeLa cells (ATCC) and BV2 cells (ATCC) were maintained in Dulbecco’s Modified Eagle’s Medium  supplemented with 10% fetal bovine serum  at 37 °C in a humidified 5% CO2 and 37 °C during imaging with the microscope-mounted Chamlide TC System . Ca2+ influx in cells co-expressing R-GECO1 was measured using genetically encoded red Ca2+ indicators with each of the monSTIM1 variants; 488 and 561 nm laser sources were used to excite cells at 30-s intervals. Captured images were analyzed using NIS-Elements AR microscope imaging software . Time-lapse images of Ca2+ influx were analyzed in defined regions of interest (ROI) in the cells, and changes in R-GECO1 intensity were quantified.For live-cell imaging, a Nikon A1R confocal microscope (Nikon Instruments), mounted onto a Nikon Eclipse Ti body and equipped with a CFI Plan Apochromat VC objective ) and digital zoom Nikon imaging software , was used. Cells were maintained at 5% COHeLa cells were loaded with Fura-2 AM , dissolved in dimethyl sulfoxide and diluted to 2 μM in DMEM, by incubating at room temperature for 30 min. The cells were then washed three times for 5 min at each step. Fura-2 imaging was performed using a LAMBDA DG-4 lamp (Sutter Instrument Company) and a × 40/0.75 NA CFI Plan Fluor objective, with intermittent excitation using 340 and 380 nm filtered fluorescent light. The emitted light was collected with a Nikon DS-Qi1 monochrome digital camera after passing through a 510-nm emission filter.Male C57BL/6 mice, 12–15 weeks old, were group-housed on a 12-h light/dark cycle with ad libitum access to food and water. Experimental protocols were approved by the Institutional Animal Care and Use Committee of IBS . Mice were randomly assigned to experimental groups.Surgical procedures were performed under IBS IACUC guidelines. Anesthesia in mice was induced with 5% isoflurane and maintained with 1–2% isoflurane during the stereotaxic surgery. After securing the skull in a stereotaxic apparatus (RWD), the skin was shaved and the scalp was sterilized with povidone-iodine, and a small craniotomy was performed. The following coordinates were used for microinjections into the CA1: AP, 2.0 mm, ML, ± 1.5 mm, DV, -1.4 mm. All microinjections were carried out using a glass capillary Picospritzer (Parker). Following each injection, the capillary was kept in place for an additional 10 min to allow for diffusion, and then slowly withdrawn. In vivo light-stimulation experiments were performed 3 weeks post injection using 473-nm light, delivered via a solid-state LED excitation system (Live Cell Instrument) . The dur+ and FLAG+ cells.Mice were anesthetized with mixture of Alfaxan (40 mg/kg) and xylazine (10 mg/kg) 90 min after dark or light-illumination conditions and transcardially perfused first with PBS and then with PBS containing 4% paraformaldehyde (PFA). Brains were extracted, postfixed overnight in 4% PFA at 4 ℃, and then sectioned to a thickness of 30 μm using a vibratome (Leica). For immunostaining, free-floating sections were washed in PBS, incubated for 1 h in blocking solution  and incubated overnight at 4℃ with primary antibodies (see below) in blocking solution. Sections were then washed in PBS, incubated for 2 h at room temperature with secondary antibodies (see below) in PBS. Finally, brain tissue sections were washed in PBS and slide-mounted with Vectashield antifade mounting medium containing DAPI  . For CA1 imaging, fluorescence images were obtained using a Leica Stellaris 8 confocal microscope with 20×, 40×, and 60 × objectives. For all brain regions, fluorescence images were acquired using a Zeiss Axio scan Z1 with 10 × objectives. Images were analyzed using ImageJ (NIH). More than three coronal brain sections were analyzed for each mouse sample for quantification of cFosThe following primary antibodies were used: rabbit anti-cFos , rat anti-FLAG , chicken anti-GFAP , chicken anti-GFP .The following secondary antibodies were used: Alexa Fluor 488-conjugated goat anti-rabbit , Alexa Fluor 594-conjugated goat anti-rat , Alexa Fluor 647-conjugated goat anti-chicken , Alexa Fluor 488-conjugated goat anti-chicken , and Alexa Fluor 594-conjugated goat anti-rabbit .Additional file 1: Figure S1. Correlation between protein expression level and basal RGECO1 intensity. A Schematic depiction of CRY2-fused STIM1 fragment constructs. B Summary data showing expression levels of monSTIM1 variants. Data presented as means ± SEM (one-way ANOVA followed by multiple comparison test); ns, not significant (p > 0.05). C Graphs showing correlation between expression level of CRY2-fused STIM1 fragment and the basal RGECO1 intensity. monSTIM1: n = 110; Set 1: n = 85; Set 2: n = 104; Set 3: n = 150; Set 4: n = 136; Set 5: n = 104; Set 6: n = 56 cells. Scattered plots were analyzed by simple linear regression. Figure S2. Measurement of relative Ca2+ levels by Fura-2 imaging in cells expressing monSTIM1 variants. A Fura-2 ratio (Emission 340 nm/380 nm) measured in the dark condition. B Fura-2 ratio measured after blue light illumination. EGFP-monSTIM1: n = 120; FLAG-monSTIM1: n = 150; EGFP-CRY2-STIM1(318–450): n = 201; EGFP-IRES2-CRY2-STIM1(238–448): n = 146; EGFP-STIM1 + vhhGFP-CRY2: n = 152; CIBN-STIM1 + CRY2: n = 144 cells. Data are presented as means ± SEM . Table S1. Oligos used in this study.Additional file 2: Movie S1. Reversible Ca2+ increase in HeLa cells expressing monSTIM1 variants by light illumination.Additional file 3: Movie S2. Light-induced Ca2+ increase in cultured hippocampal neurons expressing monSTIM1 variants.Additional file 4: Movie S3. Reversible Ca2+ increase in cultured astrocytes expressing monSTIM1 variants by light illumination.Additional file 5: Movie S4. Reversible Ca2+ increase in microglia (BV2) expressing monSTIM1 variants by light illumination.


FLT3 internal tandem duplication (ITD) mutations. These mutations are present in about 30% of adult AML cases and are associated with a poor prognosis [Fms-like tyrosine kinase 3 inhibitor(s) (FLT3i), including sorafenib, quizartinib, and FDA-approved midostaurin and gilteritinib, are commonly used to treat adult acute myelogenous leukemia (AML) with rognosis . Howeverrognosis , 3, whicrognosis .BMM or “niche”-mediated resistance may result from the influence of extrinsic cytokines or chemokines, such as CXC chemokine receptor 4 (CXCR4)/CXC motif ligand 12 (CXCL12) and endothelial (E)-selectin CD62E)/E-selectin ligands (E-selectin-L). Both CXCR4 and E-selectin ligands play crucial roles in directing leukocyte or hematopoietic stem cell (HSC) homing to BMM , 6. In a2E/E-seleWe hypothesized that concomitantly targeting CXCR4 and E-selectin could enhance the anti-leukemia efficacy and alleviate the BM niche-mediated resistance to FLT3-targeted therapy. If this hypothesis is validated, this concept could serve as a backbone for developing combinatorial strategies with FLT3i as a frontline therapy for FLT3-mutant AML.FLT3-mutated AML patients exhibited higher levels of CXCR4 and CD44  compared to those of newly diagnosed or complete remission patients  also demonstrated higher levels of CXCR4, E-selectin-L, and CD44 expression on their cell surface compared to FLT3i sensitive cells Ba/F3-FLT3-ITD cells  treatment in vel Fig. –S4. As evel Fig. .NRAS(G12D) cells  using the specific small molecule CXCR4 inhibitors plerixafor and E-selectin inhibitor GMI-1271, and then comparing them with the dual CXCR4/E-selectin antagonist GMI-1359. GMI-1359 was found to be more effective than single inhibitors plerixafor or GMI-1271 in reducing leukemia cells adhesion and migration to BM components in normoxia and in hypoxia Fig. , and triOf note, leukemia cell mobility in the BM increased profoundly in mice BM compared with control vehicle-treated mice after one dose of GMI-1359 in a time-dependent manner (from 20 min to 4 h after drug infusion), as determined by intravital 2-photon laser scanning microscopy of intravasation and cellular outflow through the BM capillary vasculature. GMI-1359 infusion resulted in a 66% increase in the average speed of cellular motility of AML cells within 20 min and a >100% increase within 3.5 h (5.4 µm/min) Fig. . The finFLT3-mutant AML blasts from a relapsed patient who had received sorafenib-targeted therapy), resulting in prolonged survival of the mice compared to vehicle controls or single-agent treatments   model (xenografted with 01) Fig. , and was01) Fig. . These fp < 0.05) and myelocytes  in the sorafenib-alone treatment group compared with in the vehicle group. However, a much higher increase in megakaryocytes  and myelocytes  was observed in the combination therapy compared to the vehicle group in the BM  of mouse normal hematopoiesis in the AML PDX model, with a significant increase in megakaryocytes and myelocytes in the BM, as evidenced by histological analysis (H&E staining) (Supplementary Fig.  BM Fig. . These rFLT3-mutant AML. This could potentially overcome secondary resistance to FLT3-targeted therapy, which may involve the upregulation of surface CXCR4/E-selectin-L in AML cells. Further clinical studies are needed to evaluate the safety and efficacy of this combination therapy in AML patients.We have previously reported in phase 2a clinical study that targeting CXCR4 with the antagonist BL-8040 achieved significant mobilization of blasts into circulation, accompanied by a decrease in BM blasts . These fFLT3-mutant AML.Myelosuppression represents a major dose-limiting toxicity in chemo or targeted therapies . Normal FLT3-mutant AML, and highlights the importance of further investigating the underlying mechanisms of action in both leukemia cells and normal hematopoietic cells.Indeed, further investigation into the effects of co-targeting CXCR4/E-selectin/FLT3 on normal hematopoiesis and chemokine/cytokine profiles in the BMM scenario would be crucial for better understanding the protective mechanisms of normal hematopoiesis by CXCR4/E-selectin inhibition during FLT3-targeted therapy. Future clinical trials could help shed more light on these mechanisms and provide insights into potential strategies to mitigate myelosuppression while improving the efficacy of targeted therapies for AML. Overall, this study provides important pre-clinical evidence for the potential of co-targeting CXCR4/E-selectin/FLT3 in FLT3-mutant AML patients. We here report for the first time that CXCR4/E-selectin-L are transcriptionally upregulated by FLT3 inhibition, which was associated with suppression of MAPK signaling, and nuclear translocation of phospho-ERK may be involved in CXCR4 transcriptional regulation (Supplementary Fig. FLT3-mutant AML.In summary, CXCR4/CXCL12 and E-selectin/E-selectin-L axes play critical roles in leukemia cell homing to the BMM niche and are closely associated with resistance to FLT3-targeted therapy in Supplementary video for observing cell mobilitySupplementary materials and methodsSupplementary figures