AML is a genetically heterogeneous disease with a median age of diagnosis between 60 and 70 years. Thus, many AML patients are not eligible for intensive chemotherapy. Often, the disease is accompanied by a poor prognosis due to high-risk genetic features or due to antecedent hematologic disorders . Therefore, AML treatment remains a challenge; even after intensive chemotherapy and allogeneic stem cell transplantation , AML relapses are regularly observed. Thus, new concepts of AML therapy, considering tailored treatment approaches after comprehensive molecular diagnostic or implementing new immunotherapeutic strategies, are urgently needed. This review provides a detailed overview of recent developments and current promising concepts to improve the treatment and the outcome of AML patients.FLT3 mutations), or are restricted to such defined AML subgroups, such as AML-MRC (AML with myeloid-related changes) or therapy-related AML (CPX-351). With this review, we aim to present a comprehensive overview of current AML therapy according to the evolved spectrum of recently approved treatment strategies. We address several aspects of combined epigenetic therapy with the BCL-2 inhibitor venetoclax and provide insight into mechanisms of resistance towards venetoclax-based regimens, and how primary or secondary resistance might be circumvented. Furthermore, a detailed overview on the current status of AML immunotherapy, describing promising concepts, is provided. This review focuses on clinically important aspects of current and future concepts of AML treatment, but will also present the molecular background of distinct targeted therapies, to understand the development and challenges of clinical trials ongoing in AML patients.Treatment of acute myeloid leukemia (AML) has improved in recent years and several new therapeutic options have been approved. Most of them include mutation-specific approaches (e.g., gilteritinib for AML patients with activating  Acute myeloid leukemia (AML) is a heterogenous disease with a broad spectrum of cytogenetic and molecular aberrations contributing to the definition of distinct AML subgroups. Treatment options for patients suffering from AML are continuously expanding and targeted therapies are available for distinct molecularly defined subgroups. Nevertheless, AML treatment remains challenging; in particular, patients with high-risk AML not eligible for intensive treatment or allogeneic hematopoietic stem cell transplantation  are characterized by an unfavorable outcome.The occurrence of AML relapse is attributed to the persistence and clonal evolution of leukemic stem cells (LSCs). To date, the approval of AML-targeted therapy is mostly restricted to elderly AML patients or relapsed or refractory AML (r/r AML), while only a minority of patients who are refractory to chemotherapy subsequently undergo potential curative alloHSCT. Thus, current strategies of AML precision medicine aim to target the LSC compartment, to allow longer remission and to provide the chance of further consolidation treatment. This review provides a comprehensive survey of both current concepts of AML therapy and promising approaches currently being investigated in clinical trials or awaiting approval by the Food and Drug administration (FDA) or European Medicines Agency (EMA). We discuss neither present treatment strategies of acute promyelocytic leukemia (APL) nor recent advances of conventional chemotherapy, including liposomal formulation of CPX-351 (Vyxeos).Furthermore, we address potential mechanisms of resistance observed in venetoclax-based AML regimens and provide insights into the recent progress to overcome resistance by addressing different cellular targets. We further describe current development of immunotherapy in AML in detail and present an overview of targeted therapies and immunotherapeutic approaches that are characterized by their potential to effectively address LSCs of AML.FLT3) occurring in about 30% of newly diagnosed AML play a pivotal role in diagnostic algorithms, prognostic stratification, and first line treatment of AML patients. Besides the detection of the most frequent and almost patient-specific FLT3-ITD  that can be found in approximately 25% of all AML patients, activating mutations located in the FLT3-TKD (FLT3-tyrosine kinase domain) are found in about 7% of patients at diagnosis [FLT3-TKD mutations as a recommendation for alloHSCT since they are not associated with a poor prognosis in general [FLT3-TKD mutations completing intensive induction and consolidation chemotherapy should receive maintenance treatment with midostaurin for 48 weeks [Activating mutations of the FMS-like tyrosine kinase 3  mutations and is affected by the mutant-to-wild-type allelic ratio (AR) of FLT3-ITD, according to the ELN 2017 guidelines. The discrimination between FLT3-ITD “low” vs. “high” in terms of the allelic ratio is defined by a cutoff of 0.5 . In contrast to the NCCN recommendations, ELN 2017 classification stratifies AML patients with lowNPM1mut/FLT3-ITD as favorable while only those patients with highNPM1wt/FLT3-ITD are attributed to the adverse prognostic subgroup [In context of a normal karyotype, prognostic stratification of lowFLT3-ITD lacking a concomitant NPM1 mutation are difficult to monitor for minimal residual disease (MRD) and several studies demonstrate a clinical benefit of alloHSCT in first remission of AML patients harboring FLT3-ITD independently of the AR of FLT3-ITD [This prognostic stratification is still under debate and especially patients with FLT3-ITD ,8. FLT3 mutations undergoing intensive chemotherapy  should receive midostaurin for 14 days after each chemotherapy course followed by midostaurin maintenance unless subsequent alloHSCT is indicated [In general, all patients with activating ndicated .FLT3-ITD AR in presence of a NPM1 mutation on ELN classification and allocation to conventional consolidation or upfront alloHSCT, the occurrence of FLT3-TKD mutation does not implicate alloHSCT in first CR of AML. As described above, patients harboring FLT3-TKD mutations are recommended to undergo maintenance therapy with midostaurin for 12 cycles of 4 weeks each. Besides the impact of FLT3-ITD comprising almost unique tandem duplications of the FLT3 gene, many efforts were made to subclassify FLT3-ITDs and to understand potential differences in terms of biology and possibly prognosis of distinct FLT3-ITD subtypes. Approximately 30% of FLT3-ITD are localized within the tyrosine kinase domain 1 (TKD1) of FLT3 being associated with an inferior outcome following intensive AML treatment [FLT3-ITD located in the TKD1 region towards midostaurin that was caused by aberrant upregulation of the anti-apoptotic protein MCL-1 (myeloid cell leukemia-1) [Due to the molecular individuality of reatment ,10. Breikemia-1) . FLT3-ITD subtype on the treatment effect of midostaurin during intensive chemotherapy of AML patients. The prognostically relevant heterogeneity of FLT3-ITD and the poor outcome of FLT3-ITD localization in the TKD1 region have been confirmed by Rücker and co-workers [FLT3-ITDs” located in the juxtamembrane domain (JMD). Furthermore, the authors demonstrate an improved outcome for patients undergoing alloHSCT following induction chemotherapy in combination with midostaurin.Recent data obtained from the RATIFY study revealed a significantly different impact of -workers . This coFLT3-ITD subgroup analysis should be implemented in diagnostic algorithms at diagnosis; (2) midostaurin maintenance therapy needs to be evaluated critically in case of FLT3-ITD located within the TKD1 domain; and (3) when lacking reliable MRD markers in AML patients with a prognostically relevant FLT3-ITD subtype, alloHSCT should be considered in first CR.Considering these results, we postulate several clinical consequences: (1) the FLT3-ITD or FLT3-TKD mutations, undergoing conventional induction and consolidation chemotherapy without alloHSCT. All patients randomized to midostaurin treatment at diagnosis within the RATIFY trial were recommended to receive maintenance treatment with midostaurin unless they underwent alloHSCT. Thus, there was no second randomization investigating the impact of midostaurin maintenance alone [Maintenance therapy with midostaurin has been approved by EMA and reflects the standard of care in AML patients harboring either ce alone . FLT3 inhibitor maintenance therapy after alloHSCT. The placebo-controlled SORMAIN trial could demonstrate a significantly improved survival of AML patients who received sorafenib following alloHSCT [Several clinical trials addressed the question of alloHSCT . SimilaralloHSCT . ImportaalloHSCT . While sFLT3-ITD or FLT3-TKD mutations at primary diagnosis, confirmation of the FLT3 mutational status is required, because clonal evolution can confer the loss of activating mutations at relapse [FLT3 mutations is observed in half of patients at AML relapse or disease progression [In case of AML relapse, with detection of either  relapse ,17. Aftegression .FLT3 tyrosine kinase inhibitors (TKI) as quizartinib or gilteritinib compared with intensive salvage chemotherapy regimens. Notably, quizartinib can effectively address FLT3-ITD, but not FLT3-TKD mutations, and the inhibition of KIT by its “off target” activity contribute to prolonged cytopenia [FLT3 mutations has been shown to achieve second remission with less toxicity compared to intensive salvage chemotherapy regimens and currently offers the best choice of “bridging to transplant” in this challenging situation [Recent phase 3 clinical trials investigated the clinical outcome of AML patients at relapse following treatment with such ytopenia . The appituation . Moreoveituation .FLT3-directed TKI has been approved for first-line treatment of AML patients with activating FLT3 mutations who are not eligible for intensive chemotherapy. The current standard of care for these patients is the combined treatment with hypomethylating agents (HMA) plus venetoclax [FLT3 mutations presented by Aldoss and co-workers revealed an excellent composite CR (CRc = CR/CRi) rate of 94% for treatment-naïve AML patients undergoing combined treatment with HMA plus venetoclax. Even in r/r AML patients harboring either FLT3-ITD or FLT3-TKD mutations HMA plus venetoclax was able to achieve a CRc rate of 42% [So far, no netoclax . A retroe of 42% .FLT3 mutations [Preclinical studies could demonstrate synergistic effects between venetoclax and quizartinib in murine AML models . Recentlutations . Similarutations .FLT3 mutations might provide not only feasible but also highly effective targeted therapy combinations that have the potential of remission induction also in younger patients who are eligible for alloHSCT. Thus, current treatment approaches for AML patients with activating IDH1 and IDH2 mutations in AML is approximately 8% and 12%, respectively [IDH1 and IDH2 heterozygous missense mutations occur at conserved single arginine residues in the enzyme active site [IDH1 and R140 or R172 in IDH2 are affected, respectively. The neomorphic enzyme activity acquired by mutated IDH (mIDH) and consequently aberrant 2-HG production leading to an increase in DNA hypermethylation and a myeloid differentiation block [Presence of isocitrate dehydrogenase (IDH) mutations in AML blasts was described shortly after its discovery in glioblastoma ,28. The ectively . The impectively . Both DNectively . IDH1 anive site . Specifion block . IDH mutations on outcome is still conflicting and co-mutations, such as NPM1 or DNMT3A, appear to be important [IDH2 (R172K) has been associated with a favorable prognosis [IDH1/2 mutations. Impact of mportant . Mutatedrognosis . Larger IDH-inhibitors, enasidenib  for mIDH2 and ivosidenib  for mIDH1 were recently approved by the FDA for r/r AML based on two single arm studies [IDH inhibitors has yet received European approval. Two  studies ,37. Ivos studies . In contmIDH2 enzyme that inhibits the conversion of alpha-ketoglutarate (α-KG) to 2-HG thereby achieving a significant reduction in plasma 2-HG levels [Enasidenib is a selective allosteric inhibitor of G levels . Within G levels . OverallG levels . mIDH1 inhibitor also shows strong 2-HG inhibition and reinstatement of myeloid differentiation [Ivosidenib, a selective ntiation . FDA appntiation . The CRcmIDH1 AML the drug was also approved as first line monotherapy in patients ineligible for intensive therapy. Median age of these 33 patients was 77 years and 76% had secondary AML whereas most of them already received prior therapy with HMAs. CRc rate of 42.5% (CR rate of 30%) was attained with a median OS of 12.6 months [Based on the results of the original ivosidenib monotherapy trial by DiNardo and co-workers that included 33 patients with newly diagnosed/treatment naïve 6 months . IDH1/2 inhibitors show good tolerability but clinicians should be aware of the development of differentiation syndrome (DS). This has been reported in about 12–15% of patients receiving IDH inhibitor monotherapy and most frequently results in non-specific syndromes, such as fever, dyspnea, hypotension, pulmonary infiltrates, and hypoxia [IDH inhibitor and application of steroids (dexamethasone 10 mg bid) [Concerning adverse events of both  hypoxia .Treatmen mg bid) .Several mechanisms of resistance have been identified whereas primary and secondary causes are described. Co-occurring mutations in the RAS pathway, leading to both primary and secondary therapeutic resistance, and it has been demonstrated that AML patients harboring such mutations are less likely to achieve a response to IDH inhibition . Newly acquired mutations within the receptor tyrosine kinase genes and mutations restoring 2-HG levels contribute to secondary resistance . mIDH1/mIDH2 inhibitors represents an important cornerstone on the way to personalized treatment in AML patients. Since early relapses occur, clinical investigations are warranted to overcome mechanisms of resistance . In conclusion, the development of those oral, small molecule As an important mechanism in different types of cancer, aberrant hypermethylation of specific promotor regions contributes to elementary alterations in gene function, and cell regulation . Today, Currently, due to the pharmacological profile of AZA or DEC, respectively, treatment with HMAs requires subcutaneous or intravenous application over 5 to 7 days in 28-day cycles. In the phase III, multicenter and placebo-controlled QUAZAR AML-001 trial  a novel oral formulation of AZA (CC-486) as maintenance therapy for patients with de novo AML in first remission after induction chemotherapy which are ineligible for subsequent alloHSCT has been investigated. In detail, a significant prolongation of OS  and PFS  with comparable adverse event profiles to injectable AZA could be demonstrated. AML relapse was observed in 60% of patients in the AZA group and in 77% in the placebo cohort, respectively . For oral AZA, a more sustained epigenetic activity over the treatment course by a prolonged exposure time over 14 days is hypothesized. Notably, pharmacokinetic analysis demonstrated significant differences compared to parenteral AZA in terms of metabolization, while CC-486 is not considered as bioequivalent . In the De Lima and co-workers investigated CC-486 in a phase II trial as maintenance treatment in the situation of CR following alloHSCT . They coFurthermore, HMA in combination with donor lymphocyte infusions (DLI), in case of molecular relapse following alloHSCT, is a therapeutic option, which was investigated in the phase II RELAZA trial . AnotherNPM1-mutations are associated with excellent survival and response rates while TP53 or FLT3-ITD mutations are predictors for resistance towards HMA plus venetoclax [Recently, the BCL-2 inhibitor venetoclax has received FDA and EMA approvals for previously untreated elderly and unfit patients in combination with HMA or LDAC representing a remarkable improvement in a hard-to-treat patient cohort ,56. The netoclax . The use of venetoclax in various treatment scenarios is under intensive investigation. Application of HMA plus venetoclax in the situation of r/r AML has not been analyzed systematically in clinical trials so far. In a phase II study, including 32 patients with r/r AML who received monotherapy venetoclax, only a moderate ORR with 19% was seen . HoweverHMA plus venetoclax is also considered in relapsed AML following alloHSCT. A retrospective analysis of 32 patients demonstrated satisfying response rates when HMA/venetoclax is applied as salvage treatment or early at molecular relapse ,66. UsinImprovement of response by adding venetoclax to intensive induction chemotherapy regimens is subject of current research. Results of phase I and II trials for the combination of FLAG-IDA  with venetoclax show convincing results regarding efficacy (ORR 70–97%), deep response (96% of de novo AML achieved MRD negative CR) accompanied by an acceptable safety profile . AdditioFLT3 inhibitors plus venetoclax, which mainly seems to be caused by downregulation of MCL-1 und BCL(x)L [FLT3 inhibitor plus HMA and FLT3 inhibitor plus venetoclax with ORRs of 65–80% and 85%, respectively [FLT3 inhibitors plus venetoclax combinations are ongoing: a phase I trial testing a combination of venetoclax and the second-generation TKI gilteritinib (NCT03625505) or a phase Ib/II trial for venetoclax and quizartinib, both in patients with FLT3 mutated r/r AML (NCT03735875). A Phase Ib study is combining gemtuzumab ozogamicin with venetoclax in patients with CD33 positive r/r AML (NCT04070768).Preclinical studies indicate synergistic effects between  BCL(x)L ,69,70. Eectively ,72. SeveDespite high efficacy of HMA/venetoclax combinations, one-third of patients fail to respond. Several cellular mechanisms and molecules causing drug resistance have been identified; however, there is an urgent need of a deeper understanding to predict and overcome resistance . The folHMAs are acting as pyrimidine analogs of the nucleoside cytidine. Its incorporation into DNA leads to an irreversible link between the cytidine analogue and the DNA methyltransferase (DNMT), which causes inhibition of the latter. HMAs show differential cellular effects depending on its dosage. High dose levels result in short term cytotoxic effects by direct DNA damage through DNMT-DNA adducts while at lower dose hypomethylating and epigenetic effects lead to improved cell differentiation and tumor suppression ,74,75,76A crucial step is the uptake of HMA into the cell through the human equilibrated nucleoside transporter-1 (hENT1) . ElevateTET2 mutations as well as alterations of IDH1/2, SF3B1, or DNMT3A represent positive predictors for response towards HMAs in MDS and AML [ASXL1, PTPN11, CDKN2A, ETV6, and FLT3-ITD were associated with shorter OS and lower ORR [As HMAs are applicated as prodrugs, requirement of a stepwise phosphorylation to active cytosine derivatives by di- and triphosphatases is dependent on deoxycytidine kinases (DCK). Low DCK expression has been attributed to DEC resistance in AML cell lines ,79. Cyto and AML ,82,83,84ower ORR ,86. In aower ORR . BCL-2 protein family comprises multiple regulator proteins, which directly affect intrinsic apoptosis pathway at the mitochondrion. In detail, anti-apoptotic proteins like BCL-2, MCL-1, or BCL(x)L can bind and sequester the effectors BAK and BAX and prevent their function of oligomerization and induction of mitochondrial outer membrane permeabilization (MOMP). Consequently, subsequent release of cytochrome c and caspase 8 activation leading to cell death [BID, BAD, BIM, PUMA, NOXA), representing competitive inhibitors of anti-apoptotic proteins leading to release and activation of the effectors BAK and BAX [The ll death ,89. This and BAX . BCL-2, which in turn leads to release of BAK and BAX subsequently inducing apoptosis. Overexpression of BCL-2 in several lymphoid malignancies yielded to implementation of venetoclax in different disease entities, such as chronic lymphocytic leukemia. Myeloid blasts are not necessarily dependent on BCL-2 expression and leukemogenesis is driven by different apoptosis suppressors like MCL-1, BCL(x)L, BCL(w) [Usually, a strict balance between the anti- and pro-apoptotic effectors controls cell regulation. Venetoclax is mimicking BH3-only proteins through competitive binding of the anti-apoptotic protein , BCL(w) ,92,93. BCL2A1 as a potential key mechanism for venetoclax resistance. Hereby, BCL2A1 overexpression is described to be even more frequent than BCL-2 overexpression and seems to be predominantly in transformed, secondary AML or in the subset of M4/M5 according to FAB (French-American-British) classification. Knockdown experiments of BCL2A1 induced strong apoptosis in cell line models occurring almost exclusive in AML blasts, while sparing normal hematopoietic stem cells. This emphasizes BCL2A1 as a potentially attractive and important target for AML therapy [In the context of AML, a comprehensive experimental analysis of Zhang and colleagues revealed high expression levels of  therapy .MAPK and AKT, underlies different molecular patterns mediating primary or secondary resistance [TP53, BAX, BAK, NOXA (PMAIP1), TFDP1 have been identified as conferring resistance towards venetoclax [TP53 was mediating alterations in expression of BCL-2 family members and thereby had an impact on mitochondrial homeostasis and cellular metabolism. An increase of mitochondrial stress turns cells into resistance towards different anti-cancer drugs like FLT3 inhibitors—even besides venetoclax. This underlines the tight link of mitochondrial stress  and expression of anti-apoptotic proteins. The observation of distinct regulation of cellular pathways in venetoclax resistant cells, e.g., activation of sistance ,95. In anetoclax . AdditioKRAS, PTPN11, and FLT3-ITD are known to confer primary or secondary drug resistance. In KRAS mutated cell line models harboring an activating gain of function mutation G12D, consecutive overexpression of BCL2A1 and MCL-1 as well as reduced levels of BCL-2 and BAX have been described, which is crucial for venetoclax resistance [Mutations in genes like sistance ,96. PTPN11) encodes for the non-receptor protein tyrosine phosphatase SHP2 (SH2 containing protein tyrosine phosphatase-2), which positively mediates downstream signaling from receptor tyrosine kinases like FLT3 or KIT [E76K, A72D) in the PTPN11 gene convey activation of downstream pathways, such as RAS, JAK-STAT, and MAPK. Thereby effects of MCL-1, pMCL-1, and BCL(x)L are intensified and venetoclax sensitivity is reduced [FLT3-ITD and SHP2 activates STAT5 signaling, which strongly induces anti-apoptotic signaling  and promotes hematopoietic cell cycle progression and survival [FLT3-ITD mutations [Protein tyrosine phosphatase non receptor type 11  is elevated in venetoclax resistant AML cells, knockdown of CLBP was leading to significant decrease of IC50 for venetoclax. Even in context of TP53 depletion, knockdown of CLBP was able to rescue TP53-mediated venetoclax resistance highlighting the potential of targeting CLBP and other mitochondrial structures [Cell metabolism and mitochondrial structures are known as important targets. In a genome-wide CRISPR/Cas9 screen in human AML cells, Chen and colleagues demonstrated the association of the depletion of genes necessary for mitochondrial organization and integrity with an increased sensitivity towards venetoclax. In detail, considering that expression of the mitochondrial structure protein ructures . Pharmacructures . HMA/venetoclax combination effectively and selectively targets cellular energy metabolism, especially oxidative phosphorylation (OXPHOS) in LSCs. This mechanism is hypothesized a major contributor to high clinical effectiveness and durable responses of this drug combination. However, through activation of fatty acid oxidation (FAO), LSCs can maintain or reinforce OXPHOS, which in turn decreases venetoclax sensitivity. Thus, targeting and suppression of FAO might contribute to conserve HMA/venetoclax properties .BCL-2 inhibition mainly rely on targeting MCL-1 [MCL-1 prior to or subsequent BCL-2 inhibition requires adequate MCL-1 inhibition to induce effective apoptosis. Thus, abrogated binding of apoptosis sensitizers BIM, BAD, NOXA, BIM, and PUMA on MCL-1 has been identified to initiate apoptosis [MCL-1 inhibitors such as A1012477 and S63845 are under preclinical and clinical investigation demonstrating high synergistic efficiency with concurrent BCL-2 inhibition [MCl-1 inhibitor VU661013 provided additional evidence for rescuing venetoclax resistance in patient-derived xenografts [MCL-1 inhibitor AZD5991 a phase I trial in combination with venetoclax in r/r hematological malignancies has been launched recently (NCT03218683). Detailed knowledge about the applicability and safety profile as well the role of MCL-1 in physiological, non-hematologic tissues should be acquired in further clinical trials [Current pharmacological strategies to overcome resistance towards ng MCL-1 . Overexppoptosis . Severalhibition ,113. A pnografts . For thel trials ,115.FLT3-ITD or PTPN11 mutations, consecutive MCL-1 and BCL(x)L activation causes reduced venetoclax sensitivity. Thus, addressing MCL-1 in FLT3-ITD mutated cells seems to be of particular interest to improve drug sensitivity [FLT3-ITD induced resistance are described above. In PTPN11 mutations, ABT-263 (navitoclax) and ABT-737 as dual inhibitors of BCL-2 and BCL(x)L and the MCL-1 inhibitor AZD5991 were able to overcome venetoclax resistance. An important side effect of dual inhibitors was severe thrombocytopenia leading to discontinuation of early clinical trials [In the presence of sitivity ,116 Currl trials ,118,119.CDK9 inhibitor alvocidib and venetoclax through affecting the balance of intrinsic apoptosis effectors, such as downregulation of MCl-1 and upregulation of BIM or NOXA [MAPK pathway, representing another key downstream pathway that upregulates MCL-1 expression. Combination of cobimetinib with venetoclax was synergistic in 7 out 11 investigated cell line models including those that were resistant towards single agent treatment [Cyclin dependent kinase 9 (CDK9) has emerged as a potential target in cancer . Bogenbe or NOXA . A compareatment . MDM2 antagonist idasanutlin can lead to effective reconstitution of TP53 thereby enhancing mitochondrial apoptosis. Several preclinical and early clinical phase I/II studies investigating the combination of idasanutlin with venetoclax, in vivo and in vitro. The superior effect compared to single agent treatment might partially be explained by enhanced MCL-1 inhibition [As described previously, the hibition .SMO plays a key role in SHH pathway activation and its functional state is predominantly affected by the interaction of SHH ligands and the SMO suppressors PTCH-1 and PTCH-2 [The balance between proliferation and differentiation of stem cells is minutely regulated and several critical pathways including the sonic hedgehog pathway (SHH) are involved in these processes. The SHH pathway is characterized by a high complexity and its activation confers cell cycle entrance and inhibition of apoptosis. The transmembrane protein d PTCH-2 .GLI1) being associated with resistance to chemotherapy and inferior OS. Furthermore, it has been shown that sensitivity towards either chemotherapy or HMAs like AZA is enhanced by concomitant treatment with SMO inhibitors [LSCs in patients with r/r AML are characterized by overexpression of SHH pathway proteins . Current clinical trials address the question of other combinations with glasdegib including HMA or venetoclax.FLT3 inhibitors or the BCL-2 inhibitor venetoclax, are either directly addressing a constitutively active receptor tyrosine kinase identified by mutational analysis, or inhibiting an important anti-apoptotic protein in defined clinical setting, the biology of AML with MLL- (KMT2A) rearrangement (KMT2Ar) is almost unique and therapeutic strategies are more complex [While targeted therapies, such as  complex .KMT2Ar can be diagnosed in about 5% of adult AML patients and is associated with higher rates of treatment failure or AML relapse. Therefore, those patients are considered as poor prognosis and alloHSCT is recommended in first remission. In addition, approximately 5% of patients harbor partial tandem duplications within the KMT2A gene (KMT2A-PTD) demonstrating a similar biology as described for KMT2Ar-dependent AML as described below [AML with ed below ,129.KMT2A as well as menin can induce expression of homeobox (HOX) genes representing the master regulators guarding self-renewal and differentiation of hematopoietic stem cells (HSCs). In detail, the histone H3 lysine 4 methyltransferase KMT2A exerts its multiple functions by epigenetic mechanisms. In case of KMT2Ar fusion proteins, such co-factors as MEIS1 contribute to an aberrant gene expression program resulting in AML development, e.g., by induction of a differentiation block [The multifunctional scaffold protein menin is critically involved in the regulation of gene expression in a tissue-specific manner. Menin directly interacts with transcription factors, chromosomal regulators, and gene promotors resulting in enhanced gene expression of regulated target genes. The large protein on block .KMT2Ar or NPM1mut in phase 2 expansion cohorts. The KOMET-001 trial investigated the menin inhibitor KO-539 in adult AML patients presenting first results at the Annual meeting of the American Society of Hematology (ASH) in 2020. The well-tolerated KO-539 showed no relevant interaction with clinically important CYP3A4 inhibitor and 8 of 12 patients enrolled into this trial were evaluable. Beside a patient with KMT2Ar developing tumor lysis syndrome at lowest KO-539 dose level, clinical activity could also be demonstrated in NPM1mut patients including one patient achieving MRD-negative CR after being refractory to conventional chemotherapy [KMT2Ar and FLT3-ITD previously demonstrating resistance to chemotherapy and gilteritinib achieved cytogenetic CR with SNDX-5613 single treatment [Preliminary clinical data exist for two menin inhibitors recently evaluated in AML patients with either otherapy . The AUGreatment .NPM1 accumulates in AML blasts in about 30% of patients due to NPM1 frameshift mutations (NPM1mut). Subsequent disruption of physiological shuttling of the NPM1 protein shares important biologically features with KMT2Ar-positive patients. In detail, NPM1mut can confer a similar gene expression pattern as described for KMT2Ar AML and maintenance of HOX gene expression in NPM1mut AML cells also depend on both wild-type KMT2A and menin. NPM1mut often co-occur with activating FLT3 mutations leading to an impaired prognosis especially in case of a high ITD/wild-type ratio (highFLT3-ITD). In consideration that MEIS1 can also induce FLT3 gene expression, combined treatment of highNPM1mut/FLT3-ITD AML patients with menin inhibitors and FLT3 inhibitors might represent a promising approach [Notably, the nuclear chaperone protein approach ,133,134.TP53 encoding the transcription factor p53 plays a crucial role in many human cancers and mutations of TP53 occur in a broad percentage of AML patients depending on the history of AML . Beside the presence of TP53 mutations, the functional inactivation of p53 is observed in most AML cases. MDM2 represents one of the most important negative regulators of p53 and overexpression of MDM2 or inactivation of ARF (p14) are the main mechanisms of functional inactivation of p53 in TP53-unmutated AML patients. Thus, inhibition of MDM2 reflects a promising therapeutic strategy in those AML patients lacking TP53 mutations. MDM2 inhibitors can affect the ARF-MDM-p53 axis enabling functional reactivation of unmutated p53 [The tumor suppressor gene ated p53 ,136.TP53. Regularly observed adverse events were mainly gastrointestinal and myelosuppression. The second-generation MDM2 inhibitor idasanutlin has been investigated in a large phase II clinical trial. In 76 r/r AML patients undergoing combined treatment with intermediate-dosed cytarabine and idasanutlin, a CRc rate of 29% was demonstrated with a median duration of response of 6.4 months. The MDM2 expression level could be elucidated as a promising biomarker for response prediction to MDM2 inhibitor treatment in this study. While CR was achieved in only one of 25 patients with TP53 mutations, in the subgroup for TP53 wild type AML patients CR could be achieved in 31% (22 of 71 patients). The initiated phase 3 study (MIRRORS) had to be stopped early due to failure of achieving study endpoints [Inhibitors of MDM2—so called “nutlins”—have already been investigated in clinical trials including AML patients with wild-type ndpoints ,138.TP53 mutations or selection of subclones harboring MDM2 mutations disrupting its interaction with p53. Two clinical trials investigating the combination of idasanutlin and venetoclax are ongoing and preliminary data are available for one of these studies. The phase 1 part of one of these trials could determine the RP2D for idasanutlin and venetoclax and demonstrated an overall CR rate of 29% [Beside combination treatment with either HMA or cytarabine, combined targeted therapy approaches are of special interest. Thus, combination with venetoclax has the potential to delay development of resistance to MDM2 inhibitors that is regularly observed by the emergence of e of 29% .The compound ALRN-6924 as a first-in-class dual inhibitor of MDM2 and MDM4 is able to overcome resistance to MDM2 inhibitors resulting from MDM4 overexpression. ALRN-6924 is currently investigated in early-phase clinical trials with r/r AML and MDS patients .MEK inhibitor trametinib showed response in a few patients including one sustained CR. Current trial concepts consider combination therapy with DEC or chemotherapy [The MDM2 inhibitor AMG-232 is structurally different from nutlin derivatives and has recently been evaluated, but failed to achieve clinical activity at least as single treatment in a phase 1 dose-escalation study. In contrast, combined treatment of AMG-232 with the otherapy .TP53 or CDK9 may overcome resistance to conventional AML therapy or improve outcome of AML patients who are not eligible for intensive chemotherapy regimens.Beyond the scope of immunotherapeutic strategies that we will discuss separately, there are several additional and promising approaches of AML targeted therapy. Addressing distinct signaling molecules like mutant CD33-directed antibody-drug conjugate gemtuzumab ozogamicin (GO) has been approved based on the results of the ALFA0701 study. This phase III clinical trial investigated the impact of additional GO treatment in patients with newly diagnosed AML undergoing induction chemotherapy, with the 7 + 3 regimen containing cytarabine 200 mg per square meter as continuous infusion (days 1 to 7) and daunorubicin, 60 mg per square meter, for three consecutive days (days 1 to 3). Consolidation chemotherapy also contained combination treatment with cytarabine and daunorubicin. Due to a high rate of adverse events  in previous studies, GO was administered at lower doses (3 mg per square meter at day 1 and day 4 with a maximum dose of 5 mg) [After a complex history, the of 5 mg) .p = 0.1216) [The final results of the ALFA0701 trial confirmed a significantly improved event-free survival (EFS) for those patients treated with GO. The OS benefit was restricted to AML patients with favorable and intermediate subtype according to ELN classification. In detail, OS for both ELN subgroups was 45.6 months for the GO-arm compared to 26.9 months for the control group without achieving statistical significance  .CD33 gene (rs12459419) has been associated with an improved EFS and OS in pediatric AML patients undergoing chemotherapy combined with GO. This polymorphism caused a truncated CD33 protein, lacking part of the extracellular epitope recognized by GO. In contrast, the impact of this CD33 SNP was not observed in adult AML patients receiving GO treatment within two large MRC trials. In addition, this analysis confirmed the lack of association of CD33 expression level and response to GO treatment [Furthermore, a frequent single nucleotide polymorphism (SNP) of the reatment ,151.Several open questions, in terms of dosing and the clinical benefits of GO remain, including the impact of GO during consolidation chemotherapy on the survival of AML patients. Results of different clinical trials have been contradictory and several studies were not able to demonstrate a survival benefit for the addition of GO to consolidation chemotherapy ,153.trans retinoic acid (ATRA), with or without GO in AML patients with NPM1 mutations. A clinical benefit could be demonstrated for women, patients up to the age of 70 years and for patients without FLT3-ITD as demonstrated by EFS and CIR (cumulative incidence of relapse) subgroup analysis. Particularly, a companion study of the AMLSG-09-09 trial revealed a significantly enhanced reduction of NPM1 MRD level in the GO group resulting in a significant decrease of CIR. Thus, the addition of GO during consolidation chemotherapy improves both the molecular response and the outcome of ELN favorable and intermediate risk AML patients [In contrast, the recently published AMLSG-09-09 phase III trial investigated consolidation treatment with high-dose cytarabine and all-patients ,155. CD19-expressing acute lymphoblastic leukemia, demonstrates high remission rates including patients with MRD negativity [In contrast to antibody drug conjugates, such as GO, bispecific T cell engager (BiTE) molecules represent promising treatment approaches for AML patients. One of the most extensively studied BiTE blinatumomab being already approved for r/r and gativity ,157.Immunotherapy of AML can either consider neoantigens that arise from AML-specific mutations specific for the individual patient or addresses AML-associated antigens. Due to the high complexity and HLA-dependence of patient-specific neoepitopes that can be presented to the immune system after processing of neoantigens, this approach has not been implemented into clinical trials yet. In contrast, many AML-associated proteins are expressed on the cell surface or intracellularly provide the rationale for promising treatment strategies .In general, toxicity  or tumor lysis syndrome)) of such immunotherapeutic approaches does not only result from the interaction between BiTE or antibody construct and the target cell (“on-target on tumor activity”). Side effects can arise from so-called “on-target off tumor toxicity” due to the expression of the target antigen on other cell types beyond the hematopoietic system. With respect to detection of treatment-relevant AML antigens on distinct blood cells, expression levels of target antigens vary between LSCs and HSCs or may be restricted to either of these compartments .Currently, several clinical trials aim to investigate the impact of different BiTE molecules directed against AML surface proteins. Here, we will describe those BiTE strategies that have already been published or are characterized by relevant preliminary data. A common challenge of many BiTE approaches is their short half-life resulting in the necessity of continuous infusion of the drug as known for blinatumomab. Thus, a recent strategy is the development of half-life extended BiTE molecules that allow a short application of the drug by reducing its renal clearance. In the following, we give an overview about currently investigated BiTE strategies in AML patients that are also summarized in CD33-directed BiTE compounds are AMG 330 and the half-life-extended AMG 673. Results of a phase I AMG 330 trial including 55 r/r AML patients analyzed several dose steps of AMG 330 while AMG 330 needs to be dosed by continuous infusion. In detail, CRS of at least grade 3 was observed in 13% of patients and severity of CRS was associated with both AMG 330 dose-level and disease burden. CRc was documented in five patients, including one patient achieving CRmol [The most comprehensively investigated ng CRmol .CD33xCD3 BiTE AMG 673 is administered by short infusion and it has been investigated in r/r AML. In detail, 30 patients with r/r AML were treated in ten dose-dependent cohorts and received between one and six cycles with AMG 673 demonstrating CRS in 50% (15 of 30) patients including 13% of patients with CRS grade ≥ 3. A total of 27 patients were evaluable for response assessment whereas a reduction in AML blasts of at least 50% could be demonstrated in 6 patients. In addition, one patient allocated to a higher AMG 673 dose level achieved CRi [The half-life extended eved CRi . CD33 on AML cells include the bispecific antibody GEM 333, the tandem diabody AMV564 and the CD33xCD3 bispecific antibody JNJ-67571244, respectively, and are currently investigated in phase I clinical trials.Further immunotherapeutic strategies addressing CD123 on LSCs as compared to HSCs appears to be very promising for the treatment of AML patients.The high expression of First results of a phase I clinical trial analyzing flotetuzumab first presented at the ASH meeting 2020 have been published recently including a total of 88 patients r/r AML patients. Patients with primary refractory AML or early relapse within 6 months were eligible for this study. In 30 patients fulfilling these criteria and treated with the recommended phase 2 dose, the observed CRc rate was 30.0%. Flotetuzumab was well tolerated including only 1 of 30 (3.3%) patient presenting a CRS ≥ grade 3 .CD123 expression level [The bispecific antibody vibecotamab (XmAb14045) has been investigated in a phase I clinical trial including 106 patients while most patients had r/r AML. Vibecotamab was evaluated at three dose levels and applicated weekly in a 28-day schedule. While no tumor lysis syndrome was observed, CRS was documented in 62 of 106 patients and almost completely restricted to grade 1 or 2. In 51 patients receiving at least 0.75 µg/kg body weight, ORR was 14% (7 patients), including 5 patients with CRc. A low AML burden and distinct T cell subsets have been elucidated as potential biomarkers while there was no association with the on level .FLT3 (CD135) is not only constitutively activated in about 30% of AML patients, but also regularly expressed on bulk AML cells and LSCs as compared to HSCs. The BiTE construct AMG 427 is currently investigated in patients with r/r AML in a phase 1 clinical trial [The receptor tyrosine kinase al trial .FLT3-ITD (approximately 25% of AML patients) is mainly bound to the endoplasmic reticulum and Golgi apparatus, it is a matter of debate whether a lower surface expression of FLT3-ITD might be associated with a reduced efficacy of treatment strategies addressing FLT3 in those AML patients harboring FLT3-ITD [Considering that FLT3-ITD .CLL-1 (CLEC12A) is part of the C-type lectin superfamily and cannot only be detected on several cell types of innate immunity, but also on LSCs in AML patients. Since CLL-1 is not expressed on HSCs, it represents a promising target not only for antibody-based immunotherapeutic approaches. The bi-specific antibody MCLA-117 is evaluated in a phase 1 clinical trial including r/r AML patients. Preliminary results analyzing 50 patients revealed CRS and pyrexia as the most common adverse events while grade ≥ 3 adverse events were rare. Response assessment including all allocated dose levels of MCLA-117 demonstrates 26 evaluable patients with blast reduction of at least 50% in four patients [The surface protein patients .TIM-3) can be detected on LSCs; therefore, it appears as a promising target molecule for AML treatment. TIM-3 also represents a surface protein expressed on several subsets of immune cells with pleiotropic functions (comprehensively reviewed by Wang and colleagues). Briefly, TIM-3 can be detected not only on CD4+ or CD8+ T cells, but also on natural killer (NK) cells and macrophages. Several ligands  are able to bind TIM-3 with distinct functions dependent on the cellular context. Its interaction with TIM-3 actively regulates the functional state of such important cells as T cells or NK cells that can contribute to immunotolerance against AML cells [In contrast to HSCs, the expression of the T cell immunoglobulin and mucin protein 3  or with HMAs. Recently published results of the phase I clinical trial (NCT03066648) could demonstrate an ORR of 41.2% with a 6-months duration of response (DOR) rate of 85.1% for the combination of sabatolimab and HMA in patients with newly diagnosed AML. Sabatolimab was well tolerated in patients with AML, MDS and CMML [The monoclonal antibody sabatolimab (MBG453) represents one of the best-investigated treatment approaches directed against and CMML .Antibody-based strategies aiming at the activation of T cells have been approved for several solid tumors and contribute to an improved survival of patients, e.g., for lung cancer or melanoma. In AML, several ongoing clinical trials are investigating different treatment strategies considering classical checkpoint inhibitors, such as pembrolizumab, nivolumab, or a combination of nivolumab and ipilimumab. There is a broad spectrum of clinical settings being addressed by current clinical trials with checkpoint inhibitors ranging from AML patients with r/r AML to those who are in CR but characterized by a high risk of AML relapse including e.g., following alloHSCT ,173.Beside treatment approaches containing only one or two checkpoint inhibitors, several clinical trials are focusing on combination therapy with either conventional chemotherapy, epigenetic treatment with HMAs, or targeted therapy . One rationale of combination of conventional chemotherapy  with checkpoint inhibitors is the observation of an enhanced susceptibility towards cytotoxic T cells following treatment with cytarabine. In contrast, HMA treatment can up-regulate PD-1 expression on T cells suggesting a potential clinical benefit for the combination with checkpoint inhibitors ,175.The development of CAR-T cell treatment has significantly improved our therapeutic armamentarium for pretreated patients with diffuse large B cell lymphoma (DLBCL), mantle cell lymphoma (MCL), or acute B-lymphoblastic leukemia, and will also be implemented in treatment algorithms for follicular lymphoma and multiple myeloma .A major challenge of CAR-T cell approaches in AML patients is the expression of several target proteins on healthy hematopoiesis resulting in prolonged cytopenia being associated with an even more increased risk of severe infectious complications or the necessity of subsequent alloHSCT. Another important biological aspect concerning CAR-T cell therapy in AML is the potentially reduced T cell number and function following conventional chemotherapy.CD33, CD123, CLL-1, CD44v6, and NKG2D as target molecules expressed on AML, while conditioning therapy prior to CAR-T cell application is similar to lymphodepletion regularly administered in lymphoma patients [Current clinical trials with CAR-T cells in AML patients address patients .CLL-1 and CD33). Preliminary results of the corresponding clinical trial describe MRD negative CR following cCAR-T cell treatment in r/r AML patients [Efforts are ongoing to improve the specificity of CAR-T cells with respect to its “on-target but off-tumor” effects on HSCs and to reduce the toxicity profile . Two promising approaches to achieve those goals consider bispecific CAR-T cells. In detail, compound CAR (cCAR)-T cells contain two independent and functionally active CAR molecules  being recognized by bispecific and split CAR (BissCAR) T cells has been developed by He and co-workers. This CAR-T cell approach has a high potential to enhance the specificity by reducing its toxicity against HSCs [Another concept considering two target domains . In a recently published clinical trial, 52 therapy-naïve AML patients with adverse prognostic markers, including patients with TP53 mutations underwent magrolimab treatment in combination with AZA. Here, a high CRc rate of 50% including half of these patients with MRD negativity could be achieved in this study cohort resulting in a median OS of about 13 months. In patients with high-risk MDS, a phase 3 study is ongoing investigating the clinical impact of additional treatment with magrolimab [The grolimab ,183.CD47-SIRPalpha axis is considered as a promising treatment approach not only for AML patient.Furthermore, this immunotherapeutic approach has the potential to eliminate LSC effectively. Thus, this special checkpoint inhibitor targeting the CD70 represents an important ligand of the TNF superfamily receptor CD27, and can be detected on most AML blasts, while both surface molecules are not detectable in healthy bone marrow cells. The CD70-CD27 axis can promote AML cell growth and contributes to the blockage of cell differentiation in AML. The soluble form of CD27 (sCD27) can be detected in sera of AML patients displaying a negative prognostic marker in case of higher sCD27 levels. The expression of CD70 on LSCs in AML patients supports the hypothesis that the interaction between CD70 and CD27 plays a key role in LSC maintenance [The surface protein ntenance .CD70 can be targeted by the monoclonal antibody cusatuzumab that is characterized by an enhanced activity in terms of antibody-dependent cellular cytotoxicity (ADCC). In a recently published phase I study, cusatuzumab has been evaluated in a dose escalation cohort of 12 elderly treatment-naïve AML patients while all patients received a single dose of cusatuzumab followed by AZA treatment. The combination therapy of AZA and cusatuzumab was well tolerated at all dose levels. Importantly, 8 of 12 patients enrolled into this study achieved CRc including 4 patients with MRD negativity. In addition, the authors can demonstrate a significant reduction of LSCs following treatment with the CD70-targeting antibody cusatuzumab [atuzumab .CD70 represents an attractive target molecule for the improvement of AML therapy. Besides testing further treatment combinations , CD70-directed CAR-T cell approaches are under development [In consideration of the vulnerability of LSCs towards cusatuzumab, elopment ,187. CD34+CD38- AML fraction regularly expressing such important surface proteins, such as CD123, TIM-3, CLL-1, CD70, CD44v6, or GPR56. Some of these proteins are exclusively expressed on LSCs or display a higher expression level as compared to normal CD34+CD38- HSCs representing potential targets for immunotherapy approaches  in AML treatment [LSCs play a pivotal role in AML development, response to treatment and are responsible for the evolution of AML relapse following persistence of detectable MRD. LSCs can be characterized by a distinct immune phenotype within the reatment .TIM-3 on both LSCs and on a broad range of immune cells  makes TIM-3 an attractive target for AML therapy [The eradication of residual AML cells by addressing persistent LSCs by immunotherapy is also under investigation by means of reactivation of AML-directed T cells implementing checkpoint inhibitors in different clinical settings as described above. The expression of  therapy .Importantly, similar to clonal heterogeneity in “bulk AML” potentially responsible for persistent MRD and treatment failure by clonal selection under conventional chemotherapy or following targeted therapy  distinct LSC populations are hypothesized in AML patients. Such a diversity on the level of AML LSCs contributes to a high complexity of addressing LSCs effectively ,190,191.Transcriptome profiling of LSCs revealed a LSC signature that can be attributed to leukemia-initiating capacity and so-called “stemness”. Moreover, LSCs are predominantly regulated by epigenetic mechanisms rather than by driver mutations as detected in “bulk AML” cells resulting in a characteristic LSC expression signature resulting from a unique chromatin and epigenetic landscape. Together with a high plasticity and heterogeneity, LSCs can develop a dynamic resistance to chemotherapy or other treatment approaches ,192.SHH pathway). Thus, implementing targeted therapies addressing such pathways can not only reduce resistance to chemotherapy but also contribute to a deeper remission and potential improvement of survival in AML patients [Beside clonal selection of different AML clones or distinct LSC subtypes due to heterogeneous mutational patterns, in terms of clinically relevant molecular aberrations LSCs in AML are characterized by a series of unique molecular features. In detail, precise regulation of cell cycle entrance, differentiation or apoptosis is at least in part maintained by highly conserved signaling pathways  expression indicating higher IRS pathway activity and potential target for AML therapy [Another critical mechanism of LSC protection is represented by the integrated stress response (ISR) pathway responsible for balancing LSCs between apoptosis and survival under distinct critical conditions. Thus, the ISR pathway can rescue LSC within the hypoxic bone marrow niche by inhibition of reactive oxygen species (ROS) production. The  therapy ,196.In addition to these promising targets that can be addressed in LSCs in a more specific manner, as compared to HSCs, several additional mechanisms of LSC maintenance have been investigated in pre-clinical models or even in early-phase clinical trials. Cytogenetic, especially molecular genetic analysis of AML cells, at diagnosis and at relapse, is indispensable for risk stratification with respect to alloHSCT consolidation treatment, and to implement target therapies up-front.MCL-1) have the potential to overcome resistance in this clinical setting.Treatment algorithms for elderly AML patients not eligible for intensive chemotherapy have been changed and the addition of venetoclax to epigenetic therapy has improved survival of elderly AML patients tremendously. Understanding the molecular mechanisms of primary or acquired resistance to venetoclax-based regimens in AML is of importance to improve second-line strategies for those patients not responding adequately to this treatment. Several candidates that are responsible for resistance to venetoclax were identified and molecularly defined strategies  or the detection of individual phospho-proteome signatures able to reveal aberrant activation of signaling pathways able to mediate venetoclax resistance  can contribute to improve future AML therapy.In consideration of the limited prognosis of elderly AML patients, future strategies might not only implement ex vivo characterization of primary AML cells. A comprehensive analysis considering resistance-conferring mutations  or indirectly activating the innate immune system  are currently developed as single treatments or in combination with epigenetic therapy.While immunotherapy-based approaches have been implemented in the treatment of many entities, so far, gemtuzumab ozogamicin is the only antibody approved for AML therapy. Potential immunotherapeutic strategies either directly addressing AML surface proteins (e.g., Future diagnostic approaches need to implement a much broader immunophenotype characterization in order to provide an optimal selection of immunotherapy for the individual AML patient.The molecular and immunological characterization of LSCs provide substantial chances to improve response to AML therapy beyond controlling “bulk” AML cells and to achieve deeper remissions prior to alloHSCT to further increase survival of AML patients. Thus, both molecular and immunological characterization of AML cells should not be restricted to the whole population of AML cells, but instead focus on the prognostically relevant LSCs. Our vision for the future is a combined diagnostic approach, considering genetic and proteomic diagnostics that allow an integrated characterization for targeted therapy and the prediction of treatment resistance in AML.Taken together, the armamentarium to treat AML is still growing, and the landscape of ongoing clinical trials contains promising new treatment approaches.


Gentianaceae family, has antispasmodic and relaxant effects. However, the vasorelaxant effects of GPS on aortic rings and the molecular mechanisms involved in these effects are not yet clear. Therefore, we investigated whether GPS inhibits phenylephrine- (PE-) or KCl-induced contractions in isolated rat thoracic aortic rings. The present study found that GPS produced a dose-dependent relaxation in aortic rings precontracted with PE or KCl and significantly reduced CaCl2-, narciclasine- (Rho-kinase activator-), and phorbol-12,13-diacetate- (PKC activator-) induced vasocontractions. Pretreatment with NG-Nitroarginine methyl ester hydrochloride , methylene blue (sGC inhibitor), indomethacin (COX inhibitor), 4-aminopyridine (KV channel inhibitor), and glibenclamide (KATP channel inhibitor) had no influence on the vasorelaxant effect of GPS, while BaCl2 (Kir channel inhibitor), tetraethylammonium chloride (KCa channel inhibitor), ruthenium red (RYR inhibitor), and heparin (IP3R inhibitor) significantly reduced GPS-induced vasorelaxation. Moreover, GPS pretreatment remarkably inhibited the influx of Ca2+ in vascular smooth muscle cells stimulated using KCl or PE-containing CaCl2 solution. Western blot analysis confirmed that GPS treatment inhibited PE-induced increases in the protein levels of p-Akt, p-myosin light chain (MLC), and p-myosin-binding subunit of myosin phosphatase 1 (MYPT1) in the aortic rings. Additionally, the vasorelaxation activity of GPS was attenuated upon pretreatment with LY294002 (PI3K/Akt inhibitor), Y27632 (Rho-kinase inhibitor), and verapamil (L-type Ca2+ channel inhibitor). These findings demonstrate that GPS exhibits endothelium-independent vasorelaxant effects through inhibition of voltage-dependent, receptor-operated, and inositol triphosphate receptor (IP3R)/ryanodine receptor- (RYR-) mediated Ca2+ channels as well as the PI3K/Akt/Rho-kinase signaling pathway.Gentiopicroside (GPS), a main active secoiridoid glucoside derived from the roots of perennial herbs in the  Hypertension, which is associated with vasoconstriction and vascular remodeling, is a serious threat to global public health by causing cardiovascular diseases, such as atherosclerosis, myocardial infarction, and vascular hypertrophy –4. The i2+]in) is the primary regulator of tension in VSM. Ca2+ is a critical factor in excitation-contraction coupling in VSM, an increase in [Ca2+]in results in vasoconstriction and a decrease in [Ca2+]in results in vasodilation [2)], changing the resting membrane potential (K+ channels), the influx of extracellular Ca2+ through receptor-operated calcium channel (ROCC) and voltage-dependent calcium channel , and the release of intracellular Ca2+ from sarcoplasmic reticulum  for 30 min, the rings were treated with GPS  for 20 min, and subsequently, CaCl2  was added cumulatively to obtain concentration-response curves.After the aortic rings without endothelium were incubated with Ca2.The rat thoracic aorta vascular smooth muscle A7r5 cell line was purchased from Shanghai Cell Bank . The cells were cultured in high-glucose Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution  and incubated at 37°C with 5% CO4 cells/well) in chamber slides were loaded with 10 μΜ Fluo-4/AM  in Ca2+-free Krebs solution for 30 min at 37°C with 5% CO2 in the dark, as described previously  or 10 μM ruthenium red [ryanodine receptor (RYR) inhibitor] for 30 min , and then, the rings precontracted with PE (1 μM) were treated with GPS  for 20 min, followed by recording of the concentration-response curves.Aortic rings without endothelium were firstly contracted with 60 mM KCl in normal Krebs solution to assure rich Ca2. PE was added to the medium for 30 min, followed by GPS (160 μΜ) for 20 min. Aortic rings were snap frozen with liquid nitrogen; then, the protein was extracted using radioimmunoprecipitation assay (RIPA) lysis buffer mixed with 1% phenylmethanesulfonyl fluoride (PMSF). According to the standard WB procedure, the membranes were incubated with primary antibodies against GAPDH, Akt, p-Akt, MLC, p-MLC, MYPT1, and p-MYPT1  overnight at 4°C, followed by incubation with the corresponding secondary antibody for 1 h. The protein bands were visualized using an ECL reagent (EMD Millipore) and analyzed using the Tanon 5200 image acquisition system .Isolated aortic rings without endothelium were transferred to DMEM and incubated at 37°C with 5% COEmax, and the half-maximal effective concentration was presented as EC50. Data were analyzed by one-way and two-way analysis of variance (ANOVA), followed by Bonferroni's as a posttest, using the SPSS software . Differences with p < 0.05 were considered statistically significant.All data are expressed as mean ± SEM. Data were plotted using the GraphPad Prism software , with sigmoidal curve fitting performed by nonlinear regression using the Prism software. The maximal relaxation or contraction response was presented as μΜ). The results indicated that GPS has no direct vasoconstriction and vasorelaxation effects on aortic rings that sustained resting tension. However, GPS (5-320 μΜ) produced a dose-dependent relaxation in the aortic rings with or without endothelium that were precontracted with PE or KCl  values of the relaxant effects of GPS for endothelium-intact and endothelium-denuded aortic rings were 76.56 ± 3.62 and 78.81 ± 3.06 μΜ, respectively, in case of PE-induced contraction, and 72.68 ± 4.18 and 75.19 ± 4.22 μΜ, respectively, in case of KCl-induced contraction. No significant difference was observed in GPS relaxing PE- or KCl-induced contractions between endothelium-intact and endothelium-denuded aortic rings . These results showed that the vasodilation of GPS on aortic rings precontracted with PE or KCl was endothelium-independent.As shown in  Figures . The hal2, which are the main endothelium-dependent relaxing factors. Pretreatment with L-NAME (NOS inhibitor), indomethacin (COX inhibitor), and methylene blue (sGC inhibitor) had no interfering effect on GPS-  induced vasorelaxation  in aortic rings precontracted with PE when compared to control group (Emax: 78.58 ± 2.13%) (p > 0.05), which further confirmed that GPS-induced vasorelaxation was endothelium-independent (It has been reported  that NOSependent .+ (KATP) channel inhibitor] and 4-AP [voltage-dependent K+ (KV) channel inhibitor] had no effect on GPS-  induced vasorelaxation  in aortic rings without endothelium that were precontracted with PE, compared to the control group (Emax: 77.56 ± 3.03%) (p > 0.05). In contrast, pretreatment with TEA [Ca2+-activated K+ (KCa) channel inhibitor] or BaCl2 [inward rectifier K+ (Kir) channel inhibitor] significantly reduced the vasorelaxation by GPS with Emax of 25.68 ± 3.71% or 20.58 ± 3.35%, compared to the control group (p < 0.05). These results indicated that the activation of K+ channels may be related to the vasodilation of GPS.As shown in + and Ca2+-free Krebs solutions, the cumulative addition of CaCl2  induced concentration-dependent contractions in the aortic rings without endothelium, while GPS  treatment significantly inhibited these CaCl2-induced contractions   concentration dependently and significantly reduced the fluorescence intensity of A7r5 cells stimulated with CaCl2 or KCl (2+ influx might be involved in the underlying mechanism of GPS in the promotion of vasorelaxation.In the high K < 0.05) . To furt2 or KCl . These rμM) significantly decreased PE-, narciclasine- (Rho-kinase activator) or phorbol-12,13-diacetate- (PKC activator) induced contractions of aortic rings in Ca2+-free solution, compared to the control group  or heparin (IP3R antagonist) significantly reduced the vasodilatory effects of GPS on aortic rings precontracted with PE in Ca2+-free solution   . These r2+ channel in fluorescence intensity induced by KCl and CaCl2 in A7r5 cells, which indicated that GPS inhibited extracellular calcium influx, resulting in vasodilation. PE-induced contractions in Ca2+-free solution are ascribed to intracellular Ca2+ release via activation of PKC/Rho-kinase- and IP3R/RYR-mediated Ca2+ sensitization [3R inhibitor (heparin) in aortic rings without Ca2+. These results indicated that GPS inhibited the upregulation of [Ca2+]in via blockade of both extracellular Ca2+ influx in the cell membrane and intracellular Ca2+ release through IP3R/RYR-mediated Ca2+ channels in the sarcoplasmic reticulum by inhibition of VDCC and ROCC, and this inhibitory effect may be closely related to Rho-kinase- and PKC-induced phosphorylation of MLC [Kdilation . The foutization , 31, 35.n of MLC , 36.2+ channel and activation of Rho-kinase [2+ binds to calmodulin in the cytoplasm, activates MLC kinase, phosphorylate MLC, and finally causes vasoconstriction [2+ channel inhibitor (verapamil), PI3K/Akt inhibitor (LY294002), and Rho-kinase inhibitor (Y27632) were used to confirm that the vasodilatory effect of GPS on aortic rings involves regulation of the PI3K/Akt/Rho-kinase signaling pathway, as evidenced by the fact that vasodilatory effects of GPS were significantly attenuated compared to the control group upon pretreatment with these three inhibitors. Therefore, we suggested that GPS inhibited PI3K/Akt/Rho-kinase signaling pathway leading to vasodilation of aortic rings.It is well documented that activation of the PI3K/Akt pathway significantly enhances the contraction of VSM through stimulation of the L-type Cao-kinase , 38. As triction , 39. On triction –42. Our + channels and inhibition of Ca2+ channels by suppressing the activation of the PI3K/Akt/Rho-kinase signaling pathway.In summary, the present study demonstrates for the first time that GPS has evident vasodilation effects that are endothelium-independent, and the underlying mechanisms of action for these effects involve the activation of K


Acute myeloid leukemias (AML) results from the accumulation of genetic and epigenetic alterations, often in the context of an aging hematopoietic environment. The development of high-throughput sequencing—and more recently, of single-cell technologies—has shed light on the intratumoral diversity of leukemic cells. Taking AML as a model disease, we review the multiple sources of heterogeneity of leukemic cells and discuss the definition of a leukemic clone. After introducing the two dimensions contributing to clonal diversity, namely, richness (number of leukemic clones) and evenness (distribution of clone sizes), we discuss the mechanisms at the origin of clonal emergence and the causes of clonal dynamics including neutral drift, and cell-intrinsic and -extrinsic influences on clonal fitness. After reviewing the prognostic role of leukemic diversity on patients’ outcome, we discuss how a better understanding of AML as an evolutionary process could lead to the design of novel therapeutic strategies for this disease. Acute myeloid leukemias (AML) results from the accumulation of genetic and epigenetic alterations, often in the context of an aging hematopoietic environment. The development of high-throughput sequencing—and more recently, of single-cell technologies—has shed light on the intratumoral diversity of leukemic cells. Taking AML as a model disease, we review the multiple sources of genetic, epigenetic, and functional heterogeneity of leukemic cells and discuss the definition of a leukemic clone extending its definition beyond genetics. After introducing the two dimensions contributing to clonal diversity, namely, richness (number of leukemic clones) and evenness (distribution of clone sizes), we discuss the mechanisms at the origin of clonal emergence  and the causes of clonal dynamics. We discuss the possible role of neutral drift, but also of cell-intrinsic and -extrinsic influences on clonal fitness. After reviewing available data on the prognostic role of genetic and epigenetic diversity of leukemic cells on patients’ outcome, we discuss how a better understanding of AML as an evolutionary process could lead to the design of novel therapeutic strategies in this disease.  For several decades, leukemias as other cancers have been envisaged as evolutionary processes ,2. From In the cancer field, the term ’clone’ has been used to group cells sharing traits inherited from a common ancestral cell. Cells within a clone are considered to have the same fitness, i.e., the same competitive advantage in a given environment, compared to other clones and to normal cells. However, cells are characterized by multiple biological traits, some of which are highly context-dependent , raisingOwing to a long tradition of genetic reductionism in the field of cancer biology, and technological limitations to study other traits, clonal identity has long been based on genetic alterations . With agTET2, DNMT3A), or on the other hand, branched architectures where multiple mutations in the same oncogenic pathways occurred in different cells. Oncogenes with at least a partially overlapping function in AML include receptor tyrosine kinase and RAS-MAPK genes  . Ga. Ga59]. TP53 inactivation. Mutation rate can also transiently increase as an adaptive phenomenon in the context of therapeutic selection pressure .,65.64,65pressure . Finallypressure . Determipressure ,78. In Apressure . PDX exppressure ,24. Studpressure . Similarpressure , includipressure . In solipressure ,84. A repressure . The potTET2 [DNMT3A [With equal richness, clonal evolution can follow different paths, leading to differences in relative clonal abundance. A wide number of biological phenomena can account for these differences in clonal evenness. First, although clonal evolution is often conceived as a Darwinian process of evolution by natural selection, it is important to note that neutral evolution, which is widespread in the evolution of organisms, can also occur in cancer. Indeed, recent data suggest that neutral evolution is frequent after initial transformation in some solid cancers ,73 and mTET2  and DNMT [DNMT3A  are earl [DNMT3A .Clonal evolution can also be a deterministic process underlined by natural selection. In this case, clonal evenness will largely depend on the fitness of each clone. If a clone acquires a fitness advantage, it will expand and outcompete other clones. Interpretation of fitness is always relative and should account for the fitness of normal hematopoietic cells, which may decline with age, potentially explaining the age-dependence of some subsets of acute leukemias . RegardiIDH mutant clones may exemplify such ‘public good’ games in AML [TP53 and PPM1D mutations in therapy-related AML [NPM1 loss, genetic distance between diagnosis and relapse is more important, with only preleukemic drivers persisting at relapse. This pattern of evolution, often associated with a longer time to relapse, should lead research to consider the post-treatment leukemia as second AML rather than a true relapse [Furthermore, fitness is often envisaged as a cell-intrinsic trait of cancer cells. However, fitness of a clone may depend on its relative abundance because its expansion depends on a signal, notably, a diffusible factor  available in a limited amount (density-dependent fitness) secreted by the clone itself or by another clone . The pots in AML . Fitnesss in AML . In solis in AML ,100. Cons in AML . A p53-ds in AML . Leukemis in AML ,104, whis in AML ,107,108 s in AML ,104. Leus in AML ,112,113.s in AML . PDX exps in AML  suggestis in AML ,116,117 s in AML ,119; theated AML ,19,120. ated AML ,82. In a relapse ,121,122, relapse ,124 and/ relapse . Clonal  relapse ,45.IDH2 and DNMT3A mutations [Even when considering a fixed environment and neglecting density-dependent phenomena, inferring the fitness of clones harboring several drivers based on data for each individual driver  is nutations ,126,127.utations ), progreutations . This phutations , and couutations ,70. The utations ,25,70.Clones are often conceived primarily as competitors, but they can also cooperate ,133,134.Studying leukemia through the prism of multitask evolution theory helps to understand inter- and intraleukemic heterogeneity. Leukemic cells must perform several tasks to survive, but theoretically, no cell can be optimal for all tasks because of limited biomass or interference between them ,57,135. Evolution of cancer cells represents a challenge for cancer treatment. A better understanding of the underlying processes that drive clonal evolution can provide new therapeutic opportunities.Clonal diversity has been associated with poorer outcomes in AML. Clonal richness, defined on the number of cytogenetic clones, has been associated with poorer prognosis, especially in AML patients with a complex karyotype ,13. The FLT3 internal tandem duplications also portends greater risk of treatment failure [Clone size distribution and specific clonal architectures have been associated with prognosis in AML. In two independent cohorts, the presence of clonal dominance inferred from bulk sequencing was shown to predict shorter survival independently of the number of clones . Interes failure . ImportaThe link between clonal architecture and prognosis remains unclear in AML and has been mainly reported in studies where clonal architectures were inferred from bulk samples. Future studies defining clonal architecture based on multiple traits at the single-cell level might refine this association with outcome. Clonal architecture could play a direct, causal role on AML prognosis. Alternatively, clonal architecture could be a proxy of an underlying process itself, causing chemoresistance or relapse. Determinist mechanisms linking intraleukemic heterogeneity and chemoresistance remain elusive. Sequential driver acquisition could lead to additive fitness, ultimately resulting in an aggressive chemoresistant clone. However, lack of correlation between the accumulation of drivers in individual clones and clonal dominance in single-cell genotyping studies challenges this hypothesis . FurtherOn the other hand, clonal architecture might simply reflect some underlying mechanisms responsible for both clonal heterogeneity and chemoresistance. Mutation rate, number of generations, and effective population size can impact clonal architecture  and, thuIDH mutant clones [NRAS/FLT3-ITD double-mutated clones have been reported under treatment with the FLT3 inhibitor gilteritinib, whereas these mutations are usually mutually exclusive in the absence of this drug. Drug withdrawal (“drug holiday”) often induces regression of these addicted clones [In the past decade, the development of new drugs targeting genetic ,147,148 t clones ,150. Cont clones . Single-t clones ,152. Intd clones . This ild clones ,154,155.d clones . One of d clones ,158.Predicting drug sensitivity only through molecular profiling has been disappointing so far in oncology . It is tOther emerging strategies take advantage of selective pressures that have shaped the clonal architecture of AML at diagnosis. Clonal interference might point out a strong selective pressure on the involved oncogenic pathway, thus, representing a targetable Achille’s heel . One cruIntraleukemic diversity can occur at various scales, from local to global, and take various shapes that can usefully be described through richness (the number of clones) and evenness (the relative size of the clones). Multiple factors contribute to clonal emergence, including mutation rate, evolutionary time, and effective population size. Clonal expansion can occur due to a fitness advantage or by random drift in a small effective population. Clonal coexistence can be caused by competition or, instead, cooperation. It may simply reflect a task repartition with even fitness advantage. Single-cell multiomics will be necessary to determine a multimodal clonal identity. A clonal architecture reconstruction solely based on genetics may provide a biased view of the true diversity of leukemic cells. For instance, during therapy, diversity may remain high at the genetic level but converge to a single, potentially targetable phenotype, as recently observed in solid cancers . High-th