For decades, intensive chemotherapy (IC) has been considered the best therapeutic option for treating acute myeloid leukemia (AML), with no curative option available for patients who are not eligible for IC or who have had failed IC. Over the last few years, several new drugs have enriched the therapeutic arsenal of AML treatment for both fit and unfit patients, raising new opportunities but also new challenges. These include the already approved venetoclax, the IDH1/2 inhibitors enasidenib and ivosidenib, gemtuzumab ozogamicin, the liposomal daunorubicin/cytarabine formulation CPX-351, and oral azacitidine. Venetoclax, an anti BCL2-inhibitor, in combination with hypomethylating agents (HMAs), has markedly improved the management of unfit and elderly patients from the perspective of improved quality of life and better survival. Venetoclax is currently under investigation in combination with other old and new drugs in early phase trials. Recently developed drugs with different mechanisms of action and new technologies that have already been investigated in other settings (BiTE and CAR-T cells) are currently being explored in AML, and ongoing trials should determine promising agents, more synergic combinations, and better treatment strategies. Access to new drugs and inclusion in clinical trials should be strongly encouraged to provide scientific evidence and to define the future standard of treatment in AML. Acute myeloid leukemias (AMLs) comprise a heterogeneous group of conditions that are characterized by the uncontrolled proliferation of a transformed malignant hematopoietic myeloid cell, inevitably leading to bone marrow failure.Intensive chemotherapy (IC), consisting of anthracycline and cytarabine in induction treatment, high or intermediate doses of cytarabine as consolidation followed by allogeneic stem cell transplantation (HSCT) in high/intermediate-risk patients, has been considered as the gold standard in the treatment of AML for decades.However, with a median age at diagnosis of 68 years, a high percentage of AML patients are not eligible for such an intensive therapeutic program . MoreoveIn recent decades, the hypomethylating agents (HMAs) azacitidine and decitabine have become the preferred low-intensity options in AML treatment for unfit patients, allowing about one-third of treated patients to reach remission and transfusion independence, improving their overall survival (OS) and quality of life ,4,5. In Recent advances in the genetic characterization of AML and in understanding the molecular mechanisms at the basis of leukemogenesis has allowed for a better prognostic assessment of the disease and opened the way to the exploration of new tailored therapeutic strategies based on patients’ risk profiles. In parallel, owing to advances in drug manufacturing, bioengineering, and cellular therapy, more potent and selective agents and more advantageous formulations of old drugs are now available.This progress is driving AML treatment from a ‘one fits all’ strategy to a precision therapy dimension, providing a real paradigm shift in the treatment of leukemia patients.With this work, we aimed to write a narrative review of the recent advances in AML treatment and describe the landscape of future possibilities in this setting. The most relevant papers were chosen after an extensive PubMed query; oral communications were selected from international hematology congresses (ASH and EHA congress). The main keywords used in the search strategy were adult acute myeloid leukemia, new drugs, target therapy, and immunotherapy.The B-cell lymphoma 2 (BCL-2) family proteins finely regulate the intrinsic cellular apoptosis pathway . In normIn response to cell stress or damage, the pro-apoptotic members of the BCL-2 family, BID and BIM, sensitized by BH3-only proteins, interact with the effector proteins BAX and BAK to provoke mitochondrial membrane permeabilization and caspase release, and, consequently, cell apoptosis .TP53 plays a key role in the intrinsic apoptosis pathway: it is activated in stress conditions, for example, with DNA damage induced by chemotherapy, and triggers apoptosis mainly by promoting the transcription of BH3-only proteins .A better understanding of mechanisms at the basis of the cellular apoptosis pathway prompted the development of different classes of molecules with anti-leukemic activity.First identified in follicular lymphoma (FL), the anti-apoptotic protein BCL-2 prevents cell death by binding the pro-apoptotic BAX/BAK proteins and protecting the integrity of the outer mitochondrial membrane ,11,12.BCL-2 has been found to be overexpressed in AML cells and to confer resistance to conventional chemotherapy ,14.While the anti-sense oligonucleotide oblimersen and the first BH3 mimetic obatoclax showed limited efficacy in early clinical trials, the oral BCL-xL and BCL-2 inhibitor navitoclax showed significant anti-tumoral activity in preclinical AML cells and xenograft models ,16. NaviVenetoclax is an oral, highly selective, and potent BCL-2 inhibitor, first employed in CLL treatment ,20. By bAfter preclinical studies confirmed the pro-apoptotic activity of venetoclax in AML cell lines, it was administered as monotherapy in relapsed or refractory (R/R) AML patients, where it showed a tolerable profile but modest clinical efficacy, suggesting the need to explore its potential combinations with other agents, particularly HMAs ,24.Venetoclax is already used in clinical combination with HMAs in AML patients who are not eligible for IC, and many associations with other agents (IC and new drugs) are currently under exploration . Preclinical data has shown a synergistic effect of the combination of venetoclax with HMAs and the association of venetoclax with azacitidine/decitabine has been proven to be safe and able to produce a favorable response even in high-risk AML patients in early trials ,26.In the phase III study VIALE-A, azacitidine and venetoclax produced significantly better OS (14.7 versus 9.6 months), complete remission (CR) rates (37% vs. 18%), and composite remission rate  compared to azacytidine alone in AML patients who had not been previously treated and who were ineligible for IC .Based on the VIALE-A study results, venetoclax received full approval from the Food and Drug Administration (FDA) in October 2020 in combination with azacitidine, decitabine, or low-dose cytarabine (LDAC) for the treatment of newly diagnosed (ND) AML in patients 75 years or older or in patients with comorbidities precluding the use of IC. Similarly, the European Medical Agency (EMA) approved the use of venetoclax in combination with an HMA for IC-ineligible adult AML patients.In the R/R setting, no randomized controlled trials are available; however, retrospective data show interesting results with ORR about three times higher than the conventional salvage treatment with a single HMA, allowing in some cases to proceed to allogeneic stem cell transplantation (ASCT) ,29.In one study, a ten-day decitabine schedule was associated with venetoclax and compared to standard IC in 65 patients with R/R AML; the experimental arm showed a better ORR , minimal residual disease (MRD) negativity by flow cytometry (28% vs. 13%), longer event-free survival , and OS (6.8 vs. 4.7 months) .The combination of azacitidine or decitabine with venetoclax can also be effective in AML relapse following ASCT, as shown in a retrospective analysis of 32 R/R patients conducted by the German Cooperative Transplant Study Group that reported an ORR of 47%, of which 50% were CR . The phase III study VIALE-C compared venetoclax plus low dose Ara-C (LDAC) with placebo plus LDAC in ND AML patients who were ineligible for IC .Although no statistically significant differences were observed in OS (7.2 for the combination of LDAC and venetoclax versus 4.1 months in the control arm), and thus the primary endpoint of the study was not met, venetoclax provided a 25% reduction in the risk of death over the LDAC alone arm. A higher rate of CR/CRi was observed with venetoclax plus LDAC vs. LDAC alone: 48% vs. 13% with CR achieved in 27% vs. 7% of the patients, respectively. A post-hoc analysis with an additional 6 months of follow-up reported a median OS for the venetoclax and placebo arm of 8.4 and 4.1 months, respectively .Considering the improved outcome when added to the treatment of elderly or unfit AML patients, venetoclax is under evaluation for younger and fit patients in addition to different IC regimens.Thus far, no published data is available concerning the association of the standard IC backbone (7 + 3) to venetoclax. A phase Ib trial (NCT03709758) enrolling ND AML is ongoing and a phase III randomized placebo-controlled 7 + 3 and venetoclax in ND AML or MDS-EB will be recruiting soon (NCT04628026).In a single arm, phase II trial at the MD Anderson Cancer Center, CLIA (cladribine plus high-dose aracytin and idarubicin) was associated with venetoclax in ND AML, higher risk MDS, and mixed phenotype acute leukemia patients. Composite complete response (CCR) was achieved by 94% of patients, with 82% having undetectable MRD. At a median follow-up of 13.5 months, the median duration of response (DOR), EFS, and OS were not reached. The toxicity profile was excellent, with only two deaths in CR occurring in patients receiving a concomitant FLT3 inhibitor (FLT3i) .In a phase Ib dose escalation study, venetoclax was associated with a 5 + 2 regimen in 51 elderly AML patients who were ineligible for IC; the authors reported noteworthy CR/CRi rates (72% in the whole population with 94% in de novo and 43% in secondary AML) and a median OS of 11 months. An impressive marrow blast reduction ≥ 50% was observed in NPM1-, IDH2-, and SRSF2-mutant AML during the venetoclax monotherapy pre-phase .p = 0.03) and an EFS of 8 months compared to not reached in the two groups (p = < 0.001), respectively ..133].The IgG4 anti-CD47 magrolimab showed a satisfactory tolerability profile with limited antileukemic activity as monotherapy in R/R AML patients in a phase I trial . The observation that azacitidine upregulates the ‘eat me’ signal on leukemic cells, increasing magrolimab efficacy, provided the rationale for testing their association .In a recent phase Ib trial, the combination of magrolimab with azacitidine showed a 64% and 91% CCR in untreated AML and high risk MDS patients non eligible for IC, respectively. Interestingly, 74% of CCR was found in the TP53-mutated AML subgroup .A phase III study evaluating the combination of magrolimab with azacitidine (NCT04778397) and a phase I study evaluating the combination magrolimab, azacytidine, and venetoclax are currently ongoing. (NCT04435691).Bispecific antibodies are compounds capable of binding two different targets, usually expressed by different cells. Particularly, bispecific T-cell engagers (BiTEs) are agents whose action is triggered by the engagement of both antigen sites, determining T-cell activation with subsequent killing of the target cell and increased pro-inflammatory cytokine release, independent of the costimulatory signal ,138.This technology started to be investigated more than 50 years ago; however, its clinical development and application in a hematological setting is much more recent and is mainly related to lymphoproliferative disorders ,140,141.The first bispecific antibody approved for use in a hematological malignancy was blinatumomab, which granted the indication for treatment of relapsed or refractory (R/R) acute lymphoblastic leukemia (ALL) in 2014.The first BiTE designed and employed for AML treatment was AMG330, which targets the antigens CD33 on tumor cells and CD3 on T cells; preliminary data showed important in vitro antileukemic activity and particularly the capacity of avoiding key mechanisms for tumor resistance, such as CD33 downregulation . The preliminary results of a phase I trial with AMG330 for R/R AML showed an ORR of 19% with an acceptable toxicity profile characterized mostly by cytokine release syndrome  and an AE correlating with blast count at the baseline and dose level. This drug presents a short half-life of approximately two hours, needing continuous intravenous administration .A modified CD33/CD3 BiTE is AMG673, which presents a longer half-life than AMG330, permitting an intermittent schedule of intravenous administration. Efficacy data reported in the phase I trial showed a decrease in blast count in 44% of evaluable patients; however, 50% of patients experienced CRS  . Other CVibecotamab (XmAb14045) has been conceived to target CD3 and CD123 and was tested in a phase I study in 63 heavily pre-treated R/R AML patients.Efficacy was reported only for the two highest dose level studies in the trial (1.3 and 2.3 µg/kg weekly) with CR/CRi in three of thirteen AML patients, of which two could proceed to transplant.The main toxicities were CRS , fatigue, neutropenic fever, and peripheral edema .A different type of molecule called DART  was recently developed; it differs from BiTE due to the presence of variable domains of heavy and light chains which engage the target antigens on two separate polypeptides . This coThe first DART investigated in AML was flotetuzumab (MGD006 or S80880), which binds to CD3 and CD123; the phase I/II trial showed a CR/CRi rate of 27% and a median OS of 10.2 months, independent from cytogenetic risk category. Grade 3 CRS was observed in 8% of patients and mostly reversible and frequently treated with the early employment of tocilizumab. The recommended dose was 500 ng/kg/day by continuous intravenous administration .One of the most innovative approaches in modern oncohematologic immunotherapy is represented by chimeric antigen receptor T cells (CAR-Ts). This technology is based on engineered synthetic receptors permitting the regulation of T cell activity to recognize and eliminate neoplastic cells expressing a specific target antigen. The induced activity of CARs is independent from the presence of co-stimulatory proteins or MHC receptors . Despite its great impact in lymphoproliferative disorders, leading to the approval of several CAR-T compounds by EMA and FDA in the last years, the development of this immunotherapy for AML treatment has been slower and less successful, due to adverse microenvironmental conditions and the reduced number of target antigens selectively expressed by AML cells, determining suboptimal efficacy and increased toxicity .Two promising target antigens are CD33, which is highly expressed on both AML and healthy stem cells, and CLL-1 (or CLEC12A), a more selective agent for leukemic blasts. In the first phase I trial using dual CD33-CLL-1 CAR-T cells on 9 R/R AML patients, a 4-week evaluation reported an interesting MRD negative rate of 78%, using flow cytometry, with six patients proceeding to ASCT. CRS was common, but was mostly of grade 1–2 (75% of cases) and efficaciously managed with corticosteroid treatment . Another widely studied AML antigen is CD123; in a phase I trial using a CD123 CAR-T cell treatment, the data showed significant antileukemic activity with manageable CRS . CD38 CAIn a phase I/II trial with six patients, four of them achieved CR or CRi, with a median PFS of 6.4 months. CRS was highly manageable, mostly of grade 1–2 (83% of patients) .A further improvement of CAR-T technology is the universal CAR-T platform (UniCAR): this CAR is designed to recognize a peptide motif included in the second component of the molecule, called the targeting module, which confers specificity against the antigen of choice. Three AML patients were treated with UniCAR-T-CD123 with encouraging efficacy  and toxicity (two cases of grade 1 CRS) results .CPX-351 is a liposomal formulation with a 1:5 molecular ratio of daunorubicin and cytarabine that optimizes drug delivery, with preferential uptake by LSCs compared to normal HSCs ,158,159.In a phase III trial including 309 patients from 60 to 75 years old with ND high-risk AML, CPX-351 showed a significantly improved median OS and higher ORR compared to the classic 7 + 3 approach, despite a longer time to neutrophil and platelet count recovery . CPX-351Ongoing trials are exploring the role of CPX-351 beyond TR-AML  and its associations with other drugs .The well-known HMA azacitidine has been recently developed in an oral form, which is administered in an extended doses schedule of 14 or 21 days, that exercises anti tumoral activity through hypomethylating and immunomodulatory effects which are not yet fully understood ,163.The oral formulation of azacitidine allows a prolonged schedule of administration, which consequently extends the exposure of AML cells to the drug, sparing inconveniences related to injections and promoting a particular hypomethylating profile and weaker cytotoxic effects ,166,167.2 for 7 consecutive days) ..189].RAS mutations are found in approximately 15–40% of AML diagnoses, and the most frequent mutated RAS protein in myeloid malignancies is NRAS ,194,195.Once synthesized, RAS proteins undergo cytosolic modifications before being collocated to the membrane surface where they are effective. One of these modifications is prenylation, which requires an enzyme called farnesyltransferase (FT) .Tipifarnib (R115777) is an inhibitor of FT that has been tested in R/R AML, with reported clinical responses in 29% of patients (10 out on 34) with two CR ,198.In phase I studies, tipifarnib was shown to be safe in combination with IC and with bortezomib in patients with ND AML .To target the RAS pathway, a RAF inhibitor has also been developed (LY3009120) and has shown synergistic proprieties with cytarabine in RAS-mutated AML cell lines .Acting on the same signal transmission pathway, some MEK inhibitors have been developed and studied in early phase trials. Their clinical development is still at an immature stage and their role will likely be in combination with other agents. The MEK inhibitor binimetinib has been shown to be safe in RAS-mutated AML, with very limited activity as monotherapy and in combination with the ATP-competitive pan-AKT inhibitor GSK2141795 (targeting the PI3K/PTEN/AKT/mTOR pathway) ,202.Trametinib is another MEK inhibitor that showed activity in RAS-mutated AML in a phase I/II study .Cobimetinib, a third MEK inhibitor, is currently being studied in combination with venetoclax .Downstream in the same pathway, ERK activation was shown to confer resistance to TKI; the association of trametinib with midostaurin showed a synergistic effect and could emerge as a new exploitable strategy in FLT3-mutated AML patients ,206.IDHs belong to a group of enzymes that catalyze the oxidative decarboxylation of isocitrate to alfa-ketoglutarate (α-KG), generating NADPH. Mutations involving arginine in the active site of IDH1/2 (R132 in IDH1 and R140 or R172 in IDH2) result in mutant IDH1/2 proteins that have acquired the capability of converting α-ketoglutarate in 2-hydroxyglutarate (2-HG) ,208.2-HG acts as a competitive inhibitor of α-KG, interfering with α-KG-dependent enzymes, which play a role in several metabolic processes and DNA methylation, and produces a characteristic hypermethylated DNA phenotype  209,210210.IDH1 and IDH2 mutations are found in approximately 8% and 12% of AML cases, respectively, frequently co-occurring with FLT3/NPM1 mutations ,212.It should be noted that IDH mutations have been also described in other malignancies  ,214,215.The prognostic impact of IDH1/2 mutations in AML is still a matter of debate .Enasidenib (AG-221) and ivosidenib (AG-120), which are IDH2 and IDH1 inhibitors, respectively, target IDH-mutated proteins.Enasidenib was proven to be well tolerated and safe as a single agent and capable of inducing responses in R/R IDH2-mutated AML patients in a phase I/II study, with an ORR of 40.3% and a median DOR of 5.8 months . These rClinicalTrials.gov (accessed on 7 February 2022), NCT02577406), which compared enasidenib to conventional treatments  in R/R IDH2-mutated AML patients was announced to have missed the primary endpoint of OS in the enasidenib arm [Unfortunately, the phase III trial IDHENTIFY  .Resistance to ivosidenib has been found to be correlated with receptor tyrosine kinase pathway mutations in this population of R/R AML patients . Both in the ND and R/R settings, enasidenib and ivosidenib are under investigation in association with other agents.Compared with azacitidine alone, Dinardo et al. showed that the combination of enasidenib with azacitidine resulted in a better and greater-than-additive effect on ORR (74% versus 36%) and a better CRR (54% versus 12%) .The safety and clinical activity of the combination of ivosidenib with azacitidine was evaluated in a phase b study in patients with ND IDH1-mutated AML who were ineligible for IC.This association, beside good tolerance without dose-limiting toxicities, reported interesting and encouraging results concerning clinical efficacy, with an ORR of 78.3%, a CR rate of 61%, and a median time to first response of 1.8 months ; the median duration of CR, CR/CRh, and overall response were not reached .The association of both agents with standard IC in ND AML in a phase I study did not delay hematological recovery compared with historical data. Ivosidenib-treated and enasidenib-treated patients had encouraging 12-month survival probabilities of >75%, considering the high portion of secondary AML (~30%) and a majority of elderly patients (more than 60 years old) in the study population. Phase III studies are currently ongoing to further evaluate the association of IDH inhibitors with IC .In the phase Ib/II enaven-AML trial, enasidenib was associated with venetoclax in 11 RR/AML very high-risk (VHR) MDS patients with an encouraging ORR (55%) .Multiple combinations of enasidenib/ivosidenib with venetoclax (NCT04092179), decitabine (NCT05010772), decitabine and venetoclax (NCT04774393), and classical induction/consolidation therapy (NCT03839771) are currently under investigation along with the role of IDH inhibitors in maintenance therapy following ASCT in AML ; in IDH2-mutated MDS (NCT03744390); in association with CPX-351, fedratinib (NCT04955938), and ruxolitinib (NCT04281498); and in IDH1/2-mutated high risk myeloproliferative syndromes. The hedgehog signaling pathway plays an essential role in embryonal and adult hematopoiesis and its aberrant activation has been found to drive hematological malignancies, including AML ,226,227.In the phase II BRIGHT AML 1003 trial, the combination of glasdegib and LDAC improved CR rate (17 versus 2.3%) and median OS (8.8 versus 4.9 months) compared to LDAC alone in a population of AML and high-risk MDS patients .These results led to the FDA and EMA approval of glasdegib in combination with LDAC in AML patients aged more than 75 years who are ineligible for IC .Moreover, the long-term BRIGHT AML 1003 analysis showed a survival advantage of the association of glasdegib with LDAC versus LDAC alone, with a more pronounced benefit in secondary AML .Ongoing trials are currently investigating new associations of glasdegib with azacitidine and IC with cytarabine/daunorubicin .IRAK4 (interleukin-1 receptor-associated kinase 4) in association with IRAK1 can activate NF-κB pathway signaling, triggering cell survival. IRAK4-L RNA isoform expression, generated by alternating splicing promoted by mutant U2AF1 splicing factor, has been found to be associated with AML and is required for leukemic cell function .The inhibition of IRAK-4 through the IRAK-kinase inhibitor CA-4948 was shown to arrest leukemic growth in AML cells expressing IRAK4-L and to limit the spread of THP1 leukemic cells in a xenograft murine model .Currently, the oral IRAK-4 inhibitor CA-4948 is under investigation in monotherapy and in combination with azacitidine or venetoclax in R/R AML and R/R MDS settings (NCT04278768).The histone-lysine-N-methyltransferase 2A (KMT2A) gene and menin-1 gene are part of a complex that plays an essential role in regulating the expression of homeobox genes. Mutations in KMT2A are responsible for aberrant Hox gene expression leading to leukemogenesis ,233.Accounting for the 5–10% of AML diagnoses, DNMT3A-mutated cases constitute a very poor prognostic group of disease .The menin inhibitor MI-3454 was revealed to block proliferation and induce differentiation in AML cells and remission in AML KMT3A/NPM1 mutated murine models .Indeed, the menin inhibitor KO-539 is under investigation in the KOMET-001 phase I/II study in R/R AML patients .Until recent years, AML has been treated with IC followed by ASCT for fit patients with the intention to cure, and with HMA for patients ineligible for IC, mainly with an intent to improve quality of life. A significant portion of more fragile patients could also be oriented towards pure palliative care.The absence of valid alternative treatments outside clinical trials leaves little room for therapeutical strategy in frontline treatment and, in the R/R setting, if remission cannot be obtained with salvage chemotherapy to proceed to transplant, supportive care should be encouraged. Recent advances in the understanding of the molecular basis of leukemogenesis and the mechanisms sustaining LSC survival has allowed a better characterization of AML disease and a more precise prognostic stratification. With the therapeutical plan, these advances led to the development of several classes of agents with different mechanisms of action, opening opportunities for new hopes for patients and prompting new management possibilities for a disease treated for many years in a single way.Nowadays, IDH1-2 inhibitors, FLT3is, and GO are frequently incorporated in AML treatment according to a tailored approach; future challenges include assessing their role in new combinations and the development of new, more potent, and better tolerated next-generation molecules. New formulations of drugs  with particularly interesting pharmacokinetic and tolerability profiles are also currently available and widely used.Among recently approved drugs, the BCL-2 inhibitor venetoclax is one of the most interesting agents, even if its role in treating AML is still largely unexplored. We know that in combination with HMAs and other agents, is a valuable upfront treatment component for unfit and fit patients; however, new combinations carrying promising results in early phase trials are currently under investigation. Beyond induction, venetoclax may keep an interesting role in consolidation and maintenance; however, again, appropriate schedules and more advantageous combinations need to be established.Following the path that has been traced by venetoclax, new classes of drugs will likely enrich the therapeutical landscape for AML, from which every population of patients could benefit, opening the pathway for several different treatment strategies. In this race, agents harnessing the intrinsic apoptotic pathway and their associations, target therapies, and mAbs are progressing more rapidly; however, owing to the acquired knowledge in lymphoproliferative disease, BiTEs and CAR-T cells could also gain ground in the next years and may turn out to be key weapons against AML. Frail patients could particularly benefit from the synergic association of new molecules without excessive side effects, owing to different and non-overlapping toxicity profiles of new single agents, allowing for globally better outcomes in terms of treatment efficacy and quality of life. Moreover, a larger portion of patients with preserved performance status could proceed to transplant, with expected advantages in long-term survival, questioning the optimal employment of new drugs in the maintenance post-transplant setting. On the other hand, the incorporation of new agents in IC schedules represents a hope for deeper and long-lasting responses in fit patients, maybe questioning the role of transplant for some AML patients in the future.Moreover, several articulated sequential strategies combining different agents at different moments  of treatment could be imagined. Defining which is the best place for every given agent is another challenge to deal with.No less important, on the bases of molecular and cytogenetic disease characteristics together with a better understanding in molecular biology and the immunology of LSCs, specific disease-tailored treatment could emerge.Complex karyotype disease and very bad prognosis mutations (such as EVI1 and TP53) characterize a population that is still difficult to treat with very poor outcomes, constituting an unmet therapeutical need. More effective drugs for such poor-prognosis disease should be determined and new agents will perhaps represent interesting therapeutic options.Undoubtedly, there is an unmet necessity for curative therapy in AML; access to new drugs should be encouraged, and well-designed, large-scale trials are needed to assess the efficacy of new molecules and to trace patient-tailored strategy. Observational data will be essential to support and to confirm clinical trial results in the real world AML population and, moreover, to provide survival outcomes on a longer-term follow-up.In conclusion, important and exciting progress in recent years is offering new therapeutical possibilities, projecting AML treatment into a precision medicine dimension, which is disease and patient tailored and capable of improving life quality and life expectancy for most patients.


It is noted that a vast majority of this labeling of Ac46AzGal occurs intracellularly in a range of mammalian cells. We also provided evidence that this labeling is dependent on not only the enzymes of OGT responsible for O-GlcNAcylation but also the enzymes of GALT and GALE in the Leloir pathway. Notably, we discover that Ac46AzGal is not the direct substrate of OGT, and the labeling results may attribute to UDP-6AzGlc after epimerization of UDP-6AzGal via GALE. Together, these discoveries support the conclusion that Ac46AzGal as an analogue of galactose could metabolically label intracellular O-glycosylation modification, raising the possibility of characterization with impaired functions of the galactose metabolism in the Leloir pathway under certain conditions, such as galactosemias.Galactose is a naturally occurring monosaccharide used to build complex glycans that has not been targeted for labeling as a metabolic reporter. Here, we characterize the cellular modification of proteins by using Ac O-GlcNAc modification of serine and threonine residues of intracellular proteins is ubiquitous in eukaryotic cells . The proMetabolic chemical reporters (MCRs) of sugar metabolites with bio-orthogonal functional groups have become a strategy widely used to visualize and identify glycoproteins. This strategy involves synthesizing non-natural monosaccharide analogues with bio-orthogonal functional groups, such as alkyne or azide analogues . These svia the Leloir pathway. Moreover, defects in the breakdown of Gal-related glycans result in several lysosomal storage diseases. Metabolic oligosaccharide engineering provides a powerful tool to investigate the roles of various monosaccharides through the copper-catalyzed azide-alkyne cycloaddition (CuAAC) in combination with appropriate tags. However, few documents disclose the roles of Gal including its analogues in the glycosylation changes in development and disease progression due to the strict substrate specificity. Recently, Baskin’s group developed 6-alkynyl UDP-Gal for imaging N-glycans in developing zebrafish in vivo  and analyzed by Western blot using streptavidin-HRP for biotin labeling. The results of Western blot indicate that dose dependence of labeling is observed with a similar pattern and remarkable labeling at the concentration of 500 μM  for different lengths of time; then, performed cells were lysed and reacted with the Biotin-PEG4-Alkyne tag, and Western blot analysis was performed  for 16 h. The cells were then lysed and reacted with CuAAC. Western blot analysis results show that Ac46AzGal enables efficient labels in different cell lines with a diversity pattern and intensity of modified proteins  in the hexosamine biosynthetic pathway (HBP) and OGA  in the presence of Ac46AzGal for 16 h and then treated CuAAc with azide biotin-alkyne, and Western blot analysis was performed  vehicle for 12 h. The corresponding cell lysates were then incubated with H2O or PNGase-F at 37°C for 6 h, followed by CuAAC with Biotin-PEG4-Alkyne and Western blot analysis. The results demonstrate that no significant loss in the signal is observed, suggesting that Ac46AzGal is not incorporated into N-glycans  to provide further insight into the tolerance of biosynthetic enzymes and glycosyltransferases to Ac46AzGal. We demonstrate that Ac46AzGal is able to label intracellular proteins by treatment with a variety of mammalian cells. Notably, the pattern of this labeling is conducted by OGT but is not related to OGA, which further reveals the substrate promiscuity of OGT. However, substrate specificity of OGT is detected for UDP-6AzGal, and the results demonstrate that UDP-6AzGal cannot be recognized by OGT. Therefore, we infer that the protein labeling of 6AzGal intracellular is incorporated in the form of UDP-6AzGlc instead of UDP-6AzGal, and the two donors are interchangeable via the enzyme in the Leloir pathway. Moreover, the enzymes of galactose-1-phosphate uridylyltransferase (GALT) and UDP-galactose 4′-epimerase (GALE) are also confirmed to regulate the efficiency labeling of Ac46AzGal, suggesting that the enzymes in the Leloir pathway, at least partially, participate in the metabolic biosynthesis of Ac46AzGal. These results support the conclusion that galactose analogues can modify endocellular proteins, and is significant on intracellular protein labeling under certain conditions that involve deficiency of the galactose metabolism.In conclusion, galactose is a naturally occurring monosaccharide used to build complex N-/O-glycans on the surface of vertebrates and is metabolized predominantly


A DNA double-strand break (DSB) takes place in the context of chromatin, and there is increasing evidence for chromatin structure to play a functional role in DSB signaling and repair. Thus, there is an emerging need for quantitative microscopy methods that can directly measure chromatin network architecture and detect changes in this structural framework upon DSB induction within an intact nucleus. To address this demand, here we present the phasor approach to fluorescence lifetime imaging microscopy (FLIM) of Förster resonance energy transfer (FRET) between fluorescently labeled histones in the DSB inducible via AsiSI cell system (DIvA), which has sufficient spatial resolution to map nuclear-wide chromatin compaction at the level of nucleosome proximity with respect to multiple site-specific DSBs. We also demonstrate that when phasor histone FLIM-FRET is coupled with immunofluorescence, this technology has the unique advantage of enabling exploration of any heterogeneity that exists in chromatin structure at the spatially distinct and genetically induced DSBs. Inside the nucleus of a living cell, DNA is folded around histone proteins into nucleosomes and compacted into a multi-layered three-dimensional (3D) structure called chromatin . At any The chromatin “opening” and “compacting” events that follow DSB induction  are undeHere in this study, we demonstrate the capacity of the phasor histone FLIM-FRET assay to spatially map chromatin architecture with respect to DNA damage in the DSB inducible via AsiSI cell system (DIvA) . DIvA cePhasor histone FLIM-FRET microscopy coupled with IF maps nuclear-wide changes in chromatin compaction with respect to DSB induction in the DIvA cell system. To quantify the local versus global chromatin compaction status of nucleus architecture with respect to multiple site-specific DSBs, here we combine phasor histone FLIM-FRET analysis with IF of γH2AX in the DIvA cell system. FRET is an optical phenomenon that reports fluorescent protein–protein interaction on a scale of 1–10 nm, and in the context of chromatin labeled with donor–acceptor fluorescent histones , FRET reTo next employ histone FRET as a readout of chromatin network architecture with respect to sites of DSB induction in the DIvA cell system, we first confirmed via IF for γH2AX Alexa Fluorophore 647 (γH2AX-AF647) in DIvA cells fixed 2 h after 4-hydroxytamoxifen (4OHT) treatment that multiple DSBs do form across the genome . Then frTo generate a mask that enables histone FRET analysis of chromatin compaction inside versus outside of DSB foci , a thresIF-guided image analysis of phasor histone FLIM-FRET microscopy data acquired in DIvA cells quantifies chromatin network organization and enables exploration of DSB foci heterogeneity. To demonstrate the potential of phasor histone FLIM-FRET microscopy and IF in DIvA cells to enable both 1) a quantitative insight into the nuclear-wide spatial organization of compact chromatin with respect to DSBs and 2) exploration of heterogeneity in the local chromatin response at DSBs, here we performed two types of image analysis to acquired FLIM maps of histone FRET. The first type of analysis extracts the nuclear-wide localization of high FRET compact chromatin foci within a FLIM map, treats them as particles, and then quantifies their spatial distribution in terms of particle size. From application of this analysis to DIvA nuclei that were untreated versus treated with 4OHT , we findTo next investigate heterogeneity in the local chromatin response reported by histone FRET at DIvA DSB sites, we performed IF against not only γH2AX, which is expected to highlight the total population of DSBs present, but also different DNA repair proteins that highlight the DSB sub-population set to undergo repair by one of two dominant DSB repair pathways. In particular, we performed an IF-guided mask analysis of histone FRET maps acquired in DIvA nuclei co-expressing H2B-eGFP and H2B-mCherry, which were treated with 4OHT for 2 h  and fixeWe find from this analysis that both NHEJ and HR DSB sites are statistically in a more “open” chromatin state than their surrounding undamaged chromatin environment , which iIn this study, we applied phasor histone FLIM-FRET microscopy to the measurement of nuclear-wide chromatin compaction at the level of nucleosome proximity and demonstrated that this assay can quantify sub-micron changes in the spatial organization of this nanoscale feature upon DSB induction in the DIvA cell system. From coupling this technology with immunofluorescence against histone modifications that highlight DSB sites  and DNA repair proteins involved in DSB resolution , we also highlight the capacity of phasor histone FLIM-FRET to explore spatial heterogeneity in the local DSB chromatin structure as a function of DSB repair pathway choice—NHEJ versus HR. In doing so, we found that DIvA DSBs induce a global chromatin network compaction event that reduces the average spacing between nucleosomes and reorganizes them into larger clusters, in parallel with the local opening of chromatin at DSB sites—especially those marked for repair via HR. Interestingly, this result, which stems from multiple site-specific DSBs being induced at distinct locations throughout the DIvA nucleoplasm, is in strong agreement with our previous study that implemented phasor histone FLIM-FRET in HeLa cells exposed to NIR laser micro-irradiation, which induces multiple DSBs at a single nuclear location . Thus, cThe next question is the following: What biological function do these detected changes in chromatin structure serve for DSB resolution? In the context of DNA repair, there is already evidence obtained via super-resolution microscopy that a nanoscale reorganization in the chromatin structure regulates DNA repair protein access and retention at DSB sites . Along t2. DIvA cells were then plated 24 h before transfection onto 35 mm glass bottom dishes and transiently transfected with H2B-eGFP and H2B-mCherry via use of Lipofectamine 3000 according to the manufacturer’s protocol. Transiently transfected DIvA cells were then treated (or left untreated) with 300 nM of 4OHT for 2 h and then fixed with 4% paraformaldehyde for 15 min, permeabilized with 1 mg/ml Triton X-100 for 15 min at room temperature, and blocked with 1% bovine serum albumin for 30 min. Three rounds of washing with phosphate-buffered saline (PBS) were performed in between each of these fixation steps. For IF against γH2AX (S139) , 53BP1 , and BRCA1 , the fixed DIvA cells were incubated with primary antibody (1:200) overnight at 4°C and then secondary antibody labeled with Alexa Fluor 405 (AF405) or Alexa Fluor 647 (AF647) for 1 h at room temperature. The three rounds of washing step with PBS were also performed in between each of these IF steps. In general, PBS washing not only was critical for fixation and IF but also counteracted a 4OHT-induced shift in the fluorescence lifetime of H2B-eGFP that was unrelated to histone FRET  were grown in Dulbecco’s modified Eagle’s medium (Lonza) supplemented with 10% bovine growth serum (Gibco), 1x Pen-Strep (Lonza), and 1 μg/ml puromycin (Thermo Fisher Scientific) at 37°C in 5% COone FRET .All fixed cell microscopy measurements were performed on an Olympus FV3000 laser scanning microscope coupled to a 488 nm pulsed laser operated at 80 MHz and an ISS A320 FastFLIM box. A ×60 water immersion objective 1.2 NA was used for all experiments, and the cells were imaged at room temperature. Prior to acquisition of FLIM data in the donor channel (H2B-eGFP) for histone FRET analysis, multi-channel intensity images  were acquired from each selected DIvA nucleus to verify that the FRET acceptor (H2B-mCh) was present in excess of H2B-eGFP  and to record the localization of DSB breaks labeled with either H2AX (γH2AX-AF647) or 53BP1 (53BP1-AF405) and BRCA1 (BRCA1-AF647). This involved sequential imaging of a two-phase light path in the Olympus FluoView software. The first phase was set up to image H2B-eGFP and H2B-mCh via use of solid-state laser diodes operating at 488 and 561 nm, respectively, with the resulting signal being directed through a 405/488/561/6033 dichroic mirror to two internal GaAsP photomultiplier detectors set to collect 500–540 nm and 600–700 nm. The second phase was set up to image 53BP1-AF405 and BRCA1-AF647 or just γH2AX-AF647 via use of solid-state laser diodes operating at 405 and 633 nm, respectively, with the resulting signal being directed through a 405/488/561/633 dichroic mirror to two internal GaAsP photomultiplier detectors set to collect 420–460 nm and 600–700 nm. Then in each DIvA nucleus selected, a FLIM map of H2B-eGFP was imaged within the same field of view  using the ISS VistaVision software. This involved excitation of H2B-eGFP with an external pulsed 488 nm laser (80 MHz) and the resulting signal being directed through a 405/488/561/633 dichroic mirror to an external photomultiplier detector (H7422P-40 of Hamamatsu) that was fitted with a 520/50 nm bandwidth filter. The donor signal in each pixel was then subsequently processed by the ISS A320 FastFLIM box data acquisition card to report the fluorescence lifetime of H2B-eGFP. All FLIM data were pre-calibrated against fluorescein at pH 9 which has a single exponential lifetime of 4.04 ns.The fluorescence decay recorded in each pixel of an acquired FLIM image was quantified by the phasor approach to lifetime analysis . As descIn the context of the histone FRET experiments presented, the phasor coordinates (g and s) of the unquenched donor (H2B-eGFP) and background (cellular autofluorescence) were first determined independently in fixed DIvA cells transfected versus un-transfected with H2B-eGFP. This enabled definition of a baseline from which a FRET trajectory could be extrapolated and then used to determine the dynamic range of FRET efficiencies that describe chromatin network organization in the DIvA cell system . From suTo quantify the local chromatin structure of DSB foci versus the undamaged nuclear-wide chromatin architecture in DIvA nuclei, we applied an intensity threshold mask based on a DSB protein IF intensity image to FLIM maps pseudo-colored according to histone FRET (compact chromatin) versus no FRET (open chromatin). This involved 1) smoothing each DIvA nucleus’ IF image of DSB localization  with a 3 × 3 spatial median filter, 2) transforming this smoothed image into a binary mask based on an intensity threshold that was sufficiently harsh to reject non-specific IF staining but retain DSB foci, 3) applying the IF-guided mask to its associated FLIM map pseudo-colored according to histone FRET, and 4) quantification of the fraction of compact chromatin within  versus outside  the IF-guided mask.To quantify the size of compact chromatin foci detected within a FLIM map pseudo-colored according to histone FRET (compact chromatin) versus no FRET (open chromatin), a binary image of compact chromatin foci was exported from the software SimFCS to ImageJ, and then a particle analysis routine was applied that identified particles based on the following criteria: 1) particle size was from 0 to infinity, 2) all adjacent non-zero pixels were considered one particle, and 3) holes inside connected pixels were considered part of the identified particle. The area of identified particles was calculated as the number of pixels times the area of a single pixel.Statistical analysis was performed by using GraphPad Prism software. Figures were prepared by using Adobe Illustrator, Microsoft PowerPoint, SimFCS, and ImageJ.


Pimozide contributes important calcium channel blocker activity converging with IB on mitochondrial oxidative metabolism. Overall, these data support the concept that antioxidants are a double-edged sword. Rationally designed combination therapies have significant promise for further pre-clinical development and may ultimately lead to improved responses.Acute myeloid leukemia (AML) is a heterogeneous disease with a high relapse rate. Cytokine receptor targeted therapies are therapeutically attractive but are subject to resistance-conferring mutations. Likewise, targeting downstream signaling pathways has been difficult. Recent success in the development of synergistic combinations has provided new hope for refractory AML patients. While generally not efficacious as monotherapy, BH3 mimetics are very effective in combination with chemotherapy agents. With this in mind, we further explored novel BH3 mimetic drug combinations and showed that pimozide cooperates with mTOR inhibitors and BH3 mimetics in AML cells. The three-drug combination was able to reach cells that were not as responsive to single or double drug combinations. In Flt3-internal tandem duplication (ITD)-positive cells, we previously showed pimozide to be highly effective when combined with imipramine blue (IB). Here, we show that Flt3-ITD L, used primarily in mouse models (ABT-737), to formulations optimized for oral administration , to second-generation agents that are more precise in binding only Bcl-2  and sparing Bcl-XL and consequent effects on platelet survival and thrombocytopenia. Notably, all three forms of this BH3 mimetic are equally effective in vitro on culture leukemia cells.Acute myeloid leukemia (AML) is a devastating disease that has remained difficult to treat despite major advances in understanding the mechanisms of leukemogenesis. AML has classically been treated with combination chemotherapy that includes anthracyclines and cytarabine . Recepto+ AML and intermediate risk Flt3-tyrosine kinase domain (TKD) or Flt3-ITD+ Nucleophosmin (NPM1)+ AML has not been clearly established in relation to BH3 mimetic usage.Major advances in the metabolic understanding of common features of AML survival mechanisms have been made in recent years. A variety of mutations are observed and underlying these signaling changes may be a common dysregulation of the metabolic state and changes in mitochondria turnover (fusion and fission) ,7. High Signal transducer and activator of transcription 5 (STAT5) is persistently activated in the high/intermediate risk AML subsets expressing mutant Flt3 or Kit receptors. Our lab has previously demonstrated that STAT5 induces Bcl-2 expression which is essential to off-set the pro-apoptotic impact of other STAT5 regulated genes ,14, whicSingle agent BH3 mimetic usage leads to resistance and is increasingly recognized as a clinical problem. Since patient responses are transient with monotherapy, new approaches are needed. Combinations with 5-aza-cytidine was recently given accelerated approval by the FDA. Additional combinations with MEK and PI3K inhibitors have been reported ,23,24,25+ cell line MV4-11 representing homozygous Flt3-ITD mutation and MOLM-13 representing heterozygous Flt3-ITD mutation were obtained from William Tse . MV4-11 cells were grown in IMDM supplemented with 10% FBS, and MOLM-13 cells were grown in RPMI supplemented with 10% FBS. Additional Flt3-ITDneg cell lines included: OCI-AML3, which was grown in αMEM supplemented with 10% FBS, and HL-60, HEL, CCRF-288, THP-1, Kasumi-1, NB4, Nomo-1, and K562 (blast crisis CML), which were gifts from Gang Huang (Ohio State University) and William Tse. HL-60 cells were grown in IMDM with 20% FBS. HEL and K562 cells were grown in RPMI +10% FBS. All AML cell lines were tested for authenticity by short-tandem repeat (STR) analysis in the Emory Integrated Genomics Core Facility.All cultured cell lines were originally sourced from the American Type Culture Collection (ATCC) or DSMZ (OCI-AML3 cells) prior to being obtained from other laboratories. Cells were grown in media containing 1% penicillin/streptomycin and were negative for mycoplasma contamination prior to use. The Flt3-ITDSTAT5 inhibitor pimozide (P1793) was purchased from Sigma-Aldrich Inc. . Imipramine HCl, ABT-737, ABT-263, ABT-199 were purchased from Selleck Chemicals  and used interchangeably in these studies. Initial experiments began with ABT-737 and as additional more clinically relevant forms were available the studies moved to ABT-263 and finally to ABT-199. These BH3 mimetics have identical function in vitro on cultured AML cells. AZD 8055 was purchased from Selleck Chemicals . Imipramine blue and gentian violet were provided by the Jack Arbiser lab. Mibefradil (M5441) and fluoxetine (F132) were purchased from Sigma-Aldrich Inc.  as indicated. DPI was obtained from Sigma-Aldrich Inc.  (D2926).For the cytotoxicity assay, after 48 h of drug treatments, cells were analyzed by trypan blue exclusion assay as previously described . Briefly6 cells using Trizol reagent  according to the manufacturer’s protocol. cDNA was synthesized from 1.0 μg of RNA using a SuperScript III First Strand Synthesis System . qRT-PCR was performed using an iQ SYBR Green Supermix (Bio-Rad) and amplification was performed on a 7500 Real-time PCR instrument (Applied Biosystems). MV4-11 cells were treated with ABT-263 (100 nM) and analyzed 24 h later for caspase 3 using an antibody to total caspase 3. Caspase 3 cleavage was detected by a different pH species migrating in the micro-capillary electrophoresis system. Antibodies used to total caspase 3 were obtained from Cell Signaling  (9662s).Total cellular RNA was extracted from 1 × 10g. Protein concentrations were determined using the Bio-Rad protein assay  and proteins were separated on an SDS-polyacrylamide gel followed by transfer to either a nitrocellulose  or PVDF  membrane. After blocking in 5% BSA for 1 h, membranes were incubated in the appropriate antibody overnight and detection was by chemiluminescence  or by the Odyssey Clx imaging system . Image Studio v4.0 software was used for densitometry quantification. Antibodies for Western blot were obtained from the following: Anti-STAT5 (Phospho-STAT5 Y694) antibody , Phospho-Akt (Ser473) (D9E), Phospho-Akt (Thr308) (D25E6) , Phospho-DRP1 (Ser637) (D3A4) , DRP1 Antibody , p38alpha MAPK antibody and Phospho-p38alpha MAPK . Primary antibody concentrations were used according to the manufacturers’ suggestions. The secondary antibody combinations used were either IRDye 680RD (925-68070)/IRDye 800CW (925-32210) or IRDye 680RD (925-68071)/IRDye 800CW (925-32211). These goat anti-rabbit  antibodies were used at 1:10,000 to 1:20,000 dilution.Cells were lysed in RIPA buffer with protease and phosphatase inhibitors  for 30 min on ice followed by centrifugation for 10 min at 10,000× t-test was used to calculate p-values and values less than 0.05 were considered to be significant. Throughout all figures, the following nomenclature was used to indicate the level of statistical significance: * p < 0.05; ** p < 0.01; *** p < 0.001. Synergy was tested using the SynergyFinder web-based tool using the ZIP method and the following parameters: readout: inhibition; baseline correction: yes. . The tool tests the significance of combined drug interactions and requires dose–response data with three data points to calculate a synergy score. For ABT–AZD combinations, only one concentration (250 nM AZD) was used experimentally. In order to be able to calculate synergy, intermediate concentrations were modeled with an assumption that cell death remained at the untreated level. Using this parameter, an estimated minimum SynergyFinder score was calculated. If experimental cell death increased above baseline on any intermediate drug concentration, then synergy would increase from that calculated. This method assumes that intermediate concentrations of drug do not have opposite effects from higher concentrations.All data were derived as a result of three or more independent experiments, unless stated otherwise. Student’s two tailed L, was used to treat a panel of lines at nM concentrations. MV4-11 cells showed the most sensitivity in the low nM range as compared with a range of other sensitive cell lines of varying FAB and cytogenetic composition  plus BH3 mimetic (ABT-263) was most selective for killing MV4-11 and MOLM-13 cells, but had more moderate selectivity for HEL and Nomo-1  and mTOR inhibitor (AZD 8055) was synergistic in MV4-11 and MOLM-13 cells but not in the control Kasumi cells which lack Flt3-ITD expression . Since Af pSTAT5 A,B. Sincd Nomo-1 C. In conGiven that the properties of high-risk AML differ from lower risk subsets, there is still a need to develop agents that target a wider range of subsets. Triple drug combinations are being developed and have been found to be more efficacious in some instances. Therefore, we tested BH3 mimetic (ABT-263), mTOR inhibitor (AZD 8055), and pimozide alone or in combination on several AML cell lines . Pimozid+ AML cells [50 from the μM to the nM range.We previously showed that imipramine blue (IB) was able to sensitively and selectively induce apoptosis in Flt3-ITDML cells . To exam+ AML cells, we explored the mechanism of action further. A range of genes known to be expressed during the unfolded protein response (UPR) were examined following vehicle or IB treatment   C. Finallgin (TG) D. Altoge+ vs. Flt3-ITD WT cells , MV4-11 cells were treated with or without NAC treatment A. NAC as+ cell lines used in this study and has been effectively targeted by combinations of conventional chemotherapy drugs and BH3 mimetics [Acute myeloid leukemia is characterized by poor prognosis and a high relapse rate despite initial remission. Since lack of durable remission is a clinical problem, detection of minimal residual disease in AML patients is an important clinical approach to early detection of relapsing clones. Multi-kinase  and more selective class III receptor tyrosine kinase inhibitors (TKIs)  are being actively investigated in clinical studies. Despite the recent flow of new agents entering trials, TKI monotherapy has not been a magic bullet. Currently targeting Flt3-ITD expressing populations and development of newer generation TKIs  has been a major objective. The approval of tyrosine kinase inhibitors has been limited to their use as single agents. In contrast, TKIs combined with chemotherapy or following hematopoietic stem cell transplantation  offer thmimetics . This mumimetics .L inhibition alone. Although Bcl-XL inhibition is obviously avoided in patients to bypass thrombocytopenia, we used early-generation and later-generation drugs in this study and found that for in vitro mechanistic and pre-clinical evaluation, they were functionally equivalent. We recently showed that imipramine blue and pimozide are highly synergistic in Flt3-ITD+ AML cells [+ AML may also be promising for clinical development. Imipramine blue induces calcium release from the ER/lysosomes and can inhibit tyrosine phosphorylation of STAT5. The calcium/reactive oxygen species/STAT5 signaling axis is a potential therapeutic target for high risk AML [+ cells, they have increased STAT5/mTOR activation, yet they are metabolically wired differently from AML cells. Therefore, targeting activated tyrosine kinase driven STAT5 is not perfectly straight-forward. There are other factors that need to be considered in regard to drug therapy and the induction of apoptosis. Further work in this area is needed to improve upon TKI treatments.In this study, double and triple combinations based on a BH3 mimetic backbone are shown to overcome some of the mechanisms of survival in the face of Bcl-2/Bcl-XML cells . Thereforisk AML . There aActivated Akt leads to increased amino acid and glucose uptake to support rapidly dividing cells. Synergistic combination approaches have been tested in a variety of cell types, including leukemia cells. Although rapalogs have been used for many years, they have not been replaced by newer mTORC1/2 inhibitors in the clinic. The triple combinations were the most effective and thus warranted further development in patients not characterized by homozygous Flt3 mutations. Instead, they were best suited for intermediate/low risk AML patients and thus provide a broader range of options. Multi-drug combinations in chemotherapy have been standard of care for many decades. Therefore, combining targeted agents is also a rational approach that is gaining acceptance, especially as wider understanding of the genetic make-up of individual patient is available to guide clinical decision-making. By leveraging substantial interaction among these agents, there are now therapeutic opportunities. Phase I and Phase II clinical studies are ongoing with BH3 mimetics, which already show promise in IDH-mutated relapsed refractory AML patients . Profiling of oxidative phosphorylation and associated metabolic rewiring may lead to discrete combinations with improved efficacy to be used in a patient-by-patient case. Furthermore, beyond our in vitro studies shown here, targeting STAT5 activation with pimozide promises to also target leukemia stem cell self-renewal in vivo, which might further reduce the relapse rate.+ AML cells, as supported by Surprisingly, in this study, we found that imipramine blue and pimozide synergize potently through mechanisms that have not been described previously for these drugs in AML. This phospho-STAT5-independent Drp1/p38/ROS signaling cascade is engaged through IB. Although we showed that ROS are ultimately responsible, we did not use pharmacologic targeting of Drp1 or p38 MAPK because of potential side effects that would make interpretation difficult. The combination therapy and is quite effective at low nM concentrations. Notably, the gentian violet moiety of IB has previously been shown to target mitochondrial apoptosis [Drug combinations are a very important approach in modern cancer biology and finding the best combinations for high-risk diseases such as AML is a high priority. We approached new ways to exploit mitochondrial ROS sensitivity in AML cells by using several agents targeting either the BH3-domain containing proteins, mTOR, or calcium regulation. While seemingly disparate, all of these agents converge on mitochondrial function , and we have shown dependence on MitoSox for their combined actions. In summary, the agents described here provide proof-of-principle for combinatorial treatments that effectively overcome resistance and induces selective mitochondrial cell death in AML cells independent of previously described inhibition of phospho-STAT5 by imipramine blue and pimozide. Tailored treatment regimens based on two- or three-drug combinations suggest that precision medicine for high-risk AML might be achieved in the absence of traditional chemo-radiotherapy.


Drosophila glutamatergic neurons augmented inositol trisphosphate receptor (IP3R)-dependent release of endoplasmic reticulum (ER) Ca2+, which in turn potentiated mitochondrial Ca2+ uptake and ATP production. Perturbations that induced chronic depolarization, including the expression of neurodegeneration-related transgenes, led to the diversion of released ER Ca2+ into lysosomes and an attendant shortening of animal lifespan. Thus, genetic disruption of PLC-β–IP3R signaling or lysosomal Ca2+ uptake restored longevity in animals with chronically depolarized glutamatergic neurons. Our findings point to aberrant Ca2+ signaling between the ER and lysosomes as a mechanism by which hyperexcitable glutamatergic neurons shorten animal lifespan.We demonstrate that depolarization of  Drosophila glutamatergic neurons. We show that depolarization increased phospholipase-Cβ (PLC-β) activity by promoting the association of the enzyme with its phosphoinositide substrate. Augmented PLC-β activity led to greater release of endoplasmic reticulum Ca2+ via the inositol trisphosphate receptor (IP3R), increased mitochondrial Ca2+ uptake, and promoted ATP synthesis. Perturbations that decoupled membrane potential from this mode of ATP synthesis led to untrammeled PLC-β–IP3R activation and a dramatic shortening of Drosophila lifespan. Upon investigating the underlying mechanisms, we found that increased sequestration of Ca2+ into endolysosomes was an intermediary in the regulation of lifespan by IP3Rs. Manipulations that either lowered PLC-β/IP3R abundance or attenuated endolysosomal Ca2+ overload restored animal longevity. Collectively, our findings demonstrate that depolarization-dependent regulation of PLC-β–IP3R signaling is required for modulation of the ATP/ADP ratio in healthy glutamatergic neurons, whereas hyperactivation of this axis in chronically depolarized glutamatergic neurons shortens animal lifespan by promoting endolysosomal Ca2+ overload.Mitochondrial ATP production is a well-known regulator of neuronal excitability. The reciprocal influence of plasma-membrane potential on ATP production, however, remains poorly understood. Here, we describe a mechanism by which depolarized neurons elevate the somatic ATP/ADP ratio in  In nel matrix . Approxiactivity , 6. Ther2+] and ATP production remains poorly understood. In an attempt to fill some of the gaps in knowledge, we examined the effects of PM potential on mitochondrial ATP production and Ca2+ homeostasis in Drosophila neurons. Owing to recent reports of neuronal hyperexcitability being a driver of diminished longevity in organisms ranging from Caenorhabditis elegans to humans  exerts reciprocal influence on mitochondrial /[ADP] balance in Drosophila neurons depends on mitochondrial oxidative phosphorylation (OXPHOS).We examined the effects of depolarization on cellular bioenergetics and Cal brains A. In neul brains , we founA and B) . These fA and B) , 15. Des2+ release. While depolarization alone did not elicit changes in the somatic [ATP]/[ADP] ratio , subsequent application of muscarine—an agonist of PLC-β–coupled metabotropic acetylcholine receptors (mAChRs) . This effect of muscarine was absent in electrically polarized neurons . Given the requirement for matrix [Ca2+] in mitochondrial ATP production  —signific neurons C. Oligomoduction , 18, we uscarine D and E. 2+ in the interplay between depolarization and mitochondrial [Ca2+], we examined changes in cytosolic [Ca2+] by expressing the GCaMP5G–tdTomato cytosolic Ca2+ sensor . Neither depolarization nor muscarine alone significantly elevated somatic [Ca2+] . Amplitudes of Ca2+ transients evoked by thapsigargin-mediated ER store depletion, however, were indistinguishable in depolarized or normally polarized cell bodies , which indicated that the larger Ca2+ transients evoked by muscarine in depolarized neurons did not reflect an increase in ER Ca2+ content.To assess the involvement of cytosolic Ca+ sensor . As with2+ transients? Stimulation of mAchR results in the release of ER Ca2+ via IP3Rs  cells . S. S2+ tramembrane A, they cmembrane A, howevea) cells , which r ouabain  , to form nanoclusters at the PM  . Shorter GFP lifetimes in cells expressing RFP-PH and GFP-PH indicate enhanced clustering. Consistent with the effects of high [K+] . Next, we asked whether depolarization influences the extent of association between PIP2 and PLC-β. To this end, we examined FLIM-FRET between PLC-β1-GFP and RFP-PH  , 24   upon itprRNAi expression is shown in SI Appendix, Fig. S3A] or deletion of PLC-β (norpA) restored the lifespan of flies expressing ATP-αDN . The additional finding that IP3R knockdown in isolation did not extend lifespan in controls  implicated the PLC-β–IP3R pathway in the early lethality apparent in ATP-αDN–expressing animals. In agreement, overexpression of IP3R in glutamatergic neurons was sufficient to shorten animal lifespan  , which aATP-αDN) B. Eitherlifespan C.3R signaling, and lifespan in fly models of tauopathy and amyotrophic lateral sclerosis (ALS). Using whole-cell patch-clamp recordings, we first confirmed that glutamatergic neurons expressing an ALS-causing variant of vapb (vapbP58S) or human tau  . Although the expression of vapbWT also shortened animal lifespan, effects of this transgene were mild compared to those evoked by vapbP58S . Concomitant knockdown of either IP3R or mAchR  or the deletion of PLC-β prevented the premature demise induced by vapbP58S or tau . Age-dependent decline in ambulation in animals expressing vapbP58S was also ameliorated by IP3R knockdown  D and E. nockdown I.IP3R knockdown, we first determined whether coexpression of UAS-itprRNAi could dilute GAL4-induced gene expression. Abundance of neutral protein  was not altered by the coexpression of UAS-itprRNAi . Similarly, coexpression of itprRNAi changed neither the abundance nor the distribution of ectopic human tau in glutamatergic neurons (IP3R complimentary DNA (cDNA) prevented itprRNAi from restoring the lifespan of vapbP58S- or tau-expressing animals . Lethality induced by concomitant expression of both vapbP58S and tau was also mitigated by IP3R knockdown , which indicates that both transgenes exert their effects via IP3R. Together, these data point to a critical role for the mAchR–PLC-β–IP3R pathway  gene dosage, which robustly reduces RyR-mediated ER Ca2+ release . Furthermore, animals expressing both tau and an RNA interference (RNAi) line against RyR (RyRRNAi) exhibited significantly shorter lifespan than did the animals expressing RyRRNAi alone . This result was in contrast to the finding that animals expressing both tau and itprRNAi lived even longer than did the flies expressing itprRNAi alone , and thus, demonstrated the inability of RyR knockdown to suppress tau-induced lethality. Finally, increased RyR dosage, which promotes the release of Ca2+ through these channels . Taken together, these data point to a role for IP3R, but not RyR, in the regulation of fly longevity.Do our findings suggest a general role for ER Ca release , 30, didchannels , 19, did3Rs have well-established roles in interorganellar Ca2+ transfer between the ER and organelles such as the mitochondria and endolysosomes –363Rs hauscarine B. Since d vapbWT B. Theref2+ uptake on lifespan, we knocked down the MCU, which participates in Ca2+ transfer into the matrix  . In contrast, MCU knockdown further worsened the survival of flies expressing vapbP58S , while lifespan of tau-expressing animals was not altered by this manipulation . These data indicate that although IP3R overexpression shortens lifespan via mitochondrial Ca2+ overload, the PLC-β–IP3R pathway contributes to the lifespan of vapbP58S- or tau-expressing animals via a distinct mechanism.To determine the consequences of altered mitochondrial Caig. S3A) , 38. Con3R-dependent transfer of Ca2+ into endolysosomes explain the effects of vapbP58S or tau? Endolysosomal [Ca2+] can be inferred from cytosolic transients triggered by the lysoosmolytic agent glycyl-l-phenylalanine-2-naphthylamide (GPN) . Inhibition of the vacuolar ATPase using Bafilomycin A1 (BafA1) also depletes endolysosomal Ca2+, albeit via an unknown leak mechanism , which indicates overlap between the Ca2+ stores targeted by BafA1 and GPN. Together, our data support the prevailing view that GPN induces lysosomal rupture and the acute release of Ca2+ stored in those vesicles.Could IPde (GPN) A  , we founde (GPN) C. ArguinGPN) . Conversely, GPN-induced Ca2+ transients were augmented by IP3R overexpression . These data are consistent with a requirement for IP3Rs in endolysosomal Ca2+ sequestration.GPN-induced CaS or tau D and E.  neurons D. Concom neurons D and E. pression F. These 2+] in neurons overexpressing IP3R. However, knockdown of trpml using either one of two independent RNAi lines  mitigated IP3R-induced endolysosomal Ca2+ overload . Conversely, overexpression of trpml augmented vesicular [Ca2+] . As was the case with knockdown of IP3R, knockdown of trpml did not significantly alter the abundance of ectopic tau . Thus, diminished perturbagen abundance did not underlie the observed restoration of vesicular [Ca2+]. Since IP3R-dependent augmentation of the GPN responses was not influenced by coexpression of a neutral transgene (luciferase) , we also ruled out the involvement of GAL4 dilution.We reasoned that concomitant knockdown of the endolysosomal cation channel, TRPML , 45, 46,overload A. KnockdvapbP58S B and C b2+] and lifespan, knockdown of trpml restored longevity in animals that overexpressed IP3R, tau, or vapbP58S in glutamatergic neurons . Although the effect of trpml overexpression on lifespan was countered by trpml knockdown, concomitant knockdown of IP3R did not confer similar benefits in those animals . Thus, trpml is epistatic to IP3R in the sense that increased TRPML abundance bypassed the beneficial effects of IP3R knockdown. In summary, TRPML is an intermediary in the effects of IP3R on endolysosomal [Ca2+] and lifespan.Pointing to the correlation between endolysosomal , were recorded by measuring CFP and cpCitrine emissions (2+] was established by recording cells for 1 min before addition of muscarine (1 mM). To depolarize the neurons, the bath was replaced with high [K+] HL-3 , which as per the Goldman–Hodgkin–Katz equation, would depolarize the membrane potential of fly neurons to ∼ −20 mV from the empirically determined values of −75 mV  was then added 2 min after depolarization, and PercevalHR signals were recorded for 3 min. We added 2-DG (10 mM), rotenone (100 μM), oligomycin A (10 μM), and FCCP (100 μM) as needed, and signals were recorded. Amplitudes of the emission ratio represented the cytosolic [ATP]/[ADP] ratio , we show both the raw data (thin lines) and smoothed values (thick lines). We used the smoothing function available in Prism 8, which averaged four neighbors on either side of each value, and a second order smoothing polynomial. For quantification purposes, we used custom R code to calculate the maximum change or the integrated change  in the ratio following the application of muscarine. A total of >80 cells from a minimum of five independently conducted experiments for each condition were used for the calculations.Culture media was replaced with HL-3. PercevalHR signals were recorded by measuring the ratio of fluorescence emissions at 525 nm sequentially excited at 487.5 nm and 407.8 nm. An A1R laser confocal microscope with 40× objective (Nikon) was used for measurement. Background emission signals were measured from a cell-free ROI. Baselines were established for 1 min before addition of muscarine (1 mM). For cells subjected to depolarization, the bath was replaced with high [KP] ratio . Data we+] HL-3. Muscarine (1 mM) was then added 2 min after depolarization and GCaMP5G-tdTomato signals were recorded for 3 min. Total ER [Ca2+] was quantified as the change in cytosolic Ca2+ signals following store depletion with the SERCA inhibitor, thapsigargin, as described . Emission signals at 525 nm and 595 nm were recorded. Backgrounds were measured from a cell-free ROI. Baselines were established for 1 min before addition of muscarine (1 mM). For cells subjected to depolarization, the bath was replaced with high , were recorded by detecting intensities of emission at 510 nm after excitation at 340 and 380 nm using a Nikon TiE wide-field fluorescence imaging system (Nikon). The background subtracted emission ratio (ΔF340/ΔF380) was measured and calculated by NIS Elements imaging software (Nikon). Cells were loaded with 10 μM fura-2 (Invitrogen) for 30 min in culture medium at 37 °C. Cells were then washed and bathed in a bath solution containing 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM Hepes, 10 mM glucose, and 30 mM sucrose; pH 7.4. Baseline fura-2 signals were recorded for 1 min, before replacing the bath with Ca2+-free bath solution. To depolarize PM with extracellular K+, we changed the concentrations of K+ and Na+ in the bath solution from 5 mM KCl/140 mM NaCl to 100 mM KCl/45 mM NaCl (Ca2+-free). To depolarize PM using ouabain, 50 μM ouabain or 0.2 μL dimethylsulfoxide  was added into the bath 30 s after Ca2+-free bath exchange. Histamine was added 1.5 min after depolarization. The fura-2 signals were recorded for 3 min after histamine application. Amplitude of the ΔF340/ΔF380 signal represented IP3R-dependent ER Ca2+ release. A total of >80 cells from seven independently conducted experiments for each condition were used for the calculations.Fura-2 signals, which represent cytosolic free [CaDrosophila neurons were identified and selected for whole-cell patch clamp. Before measurement of resting membrane potential, the bath solution was replaced with room temperature HL-3 . The pipette solution used contained the following: 109 mM K-gluconate, 10 mM NaCl, 1.7 mM MgCl2, 0.085 mM CaCl2, 0.94 mM EGTA, 2 mM ATP, and 8.5 mM Hepes; pH 7.2. Recording pipettes were pulled from micropipette glass (Sutter Instruments) to 8 to 10 M-Ω on a PC-10 puller (Narishige). Clamping was performed with an EPC10 (HEKA Instruments) amplifier. Commands were made from the PatchMaster program . G-Ω seal was achieved under voltage-clamping mode with V membrane holding at −70 mV to prevent cell excitation after the membrane was broken. Cells with resistance higher than 1 G-Ω were used for measuring resting membrane potential. Currents were clamped at zero, and voltage was continuously recorded at 10 kHz for a minimum of 3 min per cell.Approximately 3 h after plating, GFP-expressing dissociated primary Newly eclosed adult flies were collected and transferred to vials containing standard fly food . Flies were kept at room temperature (∼21 °C) and transferred to new vials twice a week. Dead flies at the bottom of the old vials were counted after each transfer until all the animals in a cohort died.Drosophila activity monitor . Individual flies were enclosed in a hollow 65 mm glass rod plugged with food (5% sucrose and 2.5% agar) on one end and cotton on the other. The vials are bisected by a central infrared beam, which reports the number of times each fly crosses the infrared beam per minute. Flies were placed in the monitor at least 2 h before data acquisition. The recordings lasted for 1.5 d, after which the flies were removed from the vials and maintained on standard fly food until the next recording.Adult flies were reared at 25 °C in an incubator with a 12 h light-dark cycle. Locomotor activity was recorded using a 32-sample 4OH. FRET pairs were transfected using standardized protocols to optimize for equal expression of respective proteins. Cells were imaged using a 60× Plan-Apo/1.4NA oil emersion lens mounted on a wide-field Nikon Eclipse microscope. GFP was sinusoidally excited by a modulating 3 watt 497 nm light-emitting diode at 40 MHz, and fluorescence lifetime was measured using a Lambert Instrument (Roden) FLIM unit mounted on the Nikon Eclipse microscope. A total of >60 cells were imaged and lifetime (phase) values were pooled and averaged.N2a cells coexpressing PLC-β1-GFP or GFP-tagged PH-PLC-δ with either empty vector pC1 or RFP-tagged PH-PLC-δ were washed with PBS, fixed in 4% paraformaldehyde, and quenched with 50 mM NHAdult heads or dissected larval brains were harvested and homogenized in 2× Laemmli sample buffer (Bio-Rad). Extracts were then loaded onto 4 to 20% Tris-glycine gels (Bio-Rad) for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After transfer to nitrocellulose membranes, blots were blocked in Odyssey blocking buffer (LI-COR Biosciences). Blots were then incubated with primary and secondary antibodies diluted in Odyssey blocking buffer. Blots were incubated with primary antibodies overnight at 4 °C and with secondary antibodies for 2 h at room temperature. Blots were imaged using the Odyssey imaging platform (LI-COR Biosciences). Quantification of band intensities was done using ImageJ (NIH). Primary antibodies used were mouse monoclonal anti-tau , rabbit anti-actin , mouse anti–α-tubulin , and rabbit anti-GFP . Secondary antibodies used were goat IRDye 680LT anti-rabbit and goat IRDye 800CW anti-mouse (LI-COR Biosciences) at 1:20,000 and 1:15,000, respectively.Cultured neurons were fixed in 4% PFA in PBS for 15 min at room temperature. The cells were then washed with PBS containing 0.1% triton-X100 and incubated with primary antibodies  for 1 h before wash and incubation with secondary antibodies  for another hour. Cells were then washed one more time before being mounted in Vectashield containing DAPI (Vector laboratories). Cells were imaged on Nikon A1 confocal microscopy using a 60× oil objective.3 in volume and divided the total number of vesicles per field with the number of cells in that field.Full growth media over the cultured neurons was replaced with media containing LysoTracker Red DND-99  for 30 min at room temperature. The cells were then washed 3× with PBS and fixed in 4% PFA in PBS for 15 min at room temperature. The cells were then washed with PBS containing 0.1% triton-X100 and mounted in Vectashield containing DAPI (Vector laboratories). To examine GPN-induced lysosomal rupture, we applied GPN to the media containing LysoTracker for the final 15 min before wash and fixation (see schematic in UAS-Luc under the control of heat shock–inducible promoter (hs-GAL4) were heat shocked in a 37 °C water bath for 1 h on 3 alternate days. The day after the third heat shock, RNA was extracted from whole-fly extracts using RNeasy mini kit (Qiagen) by following the manufacturer’s instructions. Using the high-capacity cDNA reverse transcription kit (Applied Biosystems), 1 μg of total RNA was reverse-transcribed. Real-time qPCR was performed using SYBR Green JumpStart Taq ReadyMix (Sigma) by following the manufacturer’s instructions. The primers used were as follows:rp49 (control):F: 5′-CTAAGCTGTCGCACAAATGG-3′R: 5′-GTTGTGCACCAGGAACTTCT-3′itpr:F: 5′-CTTAATCCTGAAATGCATGTCGG-3′R: 5′-GGGTATCTCGCTCCAAAGG-3′trpml.:F: 5′-TGACGGCCGACTGGAATTC-3′R: 5′-GGTATCCCATTGGTCCACC-3′mAchR:F: 5′-CAAACAGCAGTGACGAAAACAC-3′R: 5′-CATGTAGACACTCTCCGCGT-3′MCU:F: 5′-CCACTGGAAGAGAAAAAACTGG-3′R: 5′-ATCCCAAGAGTATTCCCACCA-3′Flies expressing the relevant RNAi transgenes or https://github.com/kvenkatachalam-lab/Wong-and-Karagas-et-al.-2021-PNAS-paper). Statistical significance was defined as a P < 0.05. P values were shown on the figures as asterisks: *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001. Lifespan (Kaplan–Meier) curves were generated using Prism 8. We used the log-rank (Mantel–Cox) test to determine P values.We used either a parametric or a nonparametric test of statistical significance on the basis of whether the data were normally distributed. Multiple comparisons were made by ANOVA. R, Excel (Microsoft), and Prism 8 (GraphPad) were used for statistical analyses. Custom R code used for quantifying data are available on GitHub (Supplementary File


All these results indicate that SQSBD is effective in the treatment of AML, and quercetin and licochalcone A are the major candidate compounds for AML treatment.Traditional Chinese Medicine (TCM) has been shown to be efficacious in treating leukemia for thousands of years. It has been shown that Shen Qi Sha Bai Decoction (SQSBD) has been extensively used in the treatment of acute myeloid leukemia (AML). However, the mechanism of SQSBD in treating AML remains unclear. In this study, we employed network pharmacology to analyze the potential active components and elucidate molecular mechanism of SQSBD in treating AML. A total of 268 active components were identified from SQSBD, among which 9 key components  were hit by the 6 hub targets  associated with leukemia. Molecular docking showed that two core active components, quercetin and licochalcone A, exhibited the highest component-like properties (DL), and could bind well to CDK1 and MAPK1 protein. The experimental validation of these two components showed that quercetin inhibited cell growth through CDK1 dephosphorylation-mediated cell cycle arrest at G2/M phase in human AML U937 and HL60 cells, and licochalcone A induced cell differentiation in these leukemia cells  Acute myeloid leukemia (AML) is a hematologic malignant disease that is characterized by the disruption of myeloid differentiation and an abnormal proliferation of myeloid precursor cells . The incTraditional Chinese medicine (TCM) treating AML has a history of more than 2000 years in China . For exaRecently, network pharmacology has been developed based on system biology, network biology and polypharmacology . This neAML cell lines HL-60 and U937 were purchased from American Type Culture Collection . Cells were cultured in RPMI-1640 medium supplemented with 10% FBS. CDK1 Rabbit pAb (310007), Phospho-CDK1 (Tyr15) Rabbit pAb (310063), ERK1/2 Rabbit pAb (343830) were purchased from Zen-bio ; GAPDH (2118) and p-ERK1/2 Rabbit mAb (4376S) antibodies were purchased from Cell Signaling Technology . Licochalcone A (A0558), Quercetin (A0083) were purchased from Must BioTechnology . Cyclin A (751), Cyclin B1(752) were from Santa Cruz Biotechnology . Secondary Goat-anti Mouse (0741802) and Goat-anti Rabbit (0741516) antibodies were purchased from Kirkegaard and Perry Laboratories .Cell cycle analysis was performed on the harvested cell pellets fixed in Cell cycle staining fluid containing 1% Triton X-100, 38 mM Na citrate in ddH2O, and PI solution (1 mg/ml) containing RNase A (10 mg/ml). The mixture was analyzed by a flow cytometer after dark stain for 30 min. Data analysis used Flowjo software .Cells were collected and resuspended in PBS containing PE-labeled anti-F4/80 , APC-labeled anti-CD11b , or CD133/1(AC-133)-PE , dark shade for 30 min, washed twice in PBS, Samples were analyzed on a FACSCaliburTM flow cytometer  Data analysis used Flowjo software.The protein samples ranging from 15 to 30 μg were separated using SDS-PAGE and transferred to PVDF membranes . Membranes were blocked with 5% fat-free dry milk in TBS buffer  and incubated with antibodies at 4°C for 24 h. Protein bands were detected by incubating with horseradish peroxidase-conjugated antibodies for 1–2 h. Signal was detected by Western ECL Substrate .https://old.tcmsp-e.com/tcmsp.php) with the criteria OB ≥ 30% and DL ≥ 0.18. The structural information, 2D molecular structure, “PubChem CID,” and “Canonical SMILES” of the active components were obtained from PubChem2. Targets of the active components in SQSBD were also obtained from the TCMSP database. Component-target network was constructed by Cytoscape 3.8.2 software.Oral administration is the most common form in clinical treatment, Oral bioavailability (OB), and drug likeness (DL) are the main factors that affect the activity of the drug during oral administration. Therefore, by using “Hedyotis Diffusae Herba,” “Scutellariae Barbatae Herba,” “Lobeliae Chinensis Herba,” “Codonopsis Radix,” “Licorice,” “Hedysarum Multijugum Maxim,” “Scutellariae Radix,” “Dioscoreaebulbiferae,” “Indigo Naturalis,” “Glehniae Radix,” “Asparagi Radix,” “Agrimonia Eupatoria,” as keywords, we filtered the active components of SQSBD in the Traditional Chinese Medicine System Pharmacology (https://www.ncbi.nlm.nih.gov/geo/). It contains the expression profiles of 9 healthy subjects and 30 AML subjects. Genes with |log2FC| >  1.0 and adjusted p values  < 0.05 were selected as DEGs. Volcano plots and heat map were constructed by using the ggplot2 package in R4.1.0 software.MRNA expression datasets with accession number GSE37307 was downloaded from Gene Expression Omnibus (GEO) database  values (DMD > 57), core network was filtered by degree centrality (DC) values (DC > 60), and betweenness centrality (BC) values (BC > 645) .p < 0.05.The top 10 GO terms were presented as chord, and the top 10 KEGG signaling pathways were presented as cnetplot (Gene Ontology (GO) annotation is used to define and describe the functions of gene products from three aspects, namely, biological processes (BPs), cell components (CCs), and molecular functions (MFs). The Kyoto Encyclopedia of Genes and Genomes (KEGG) is a database that integrates genome, chemistry, and system function information. In order to obtain more accurate genes functional enrichment information, GO and KEGG functional enrichment analysis results were obtained by using the clusterProfiler software package of the R4.1.0 platform, and the screening criterion was adjusted cnetplot .https://hgserver1.amc.nl/cgi-bin/r2/main.cgi) and results were presented as boxplot.Gene expression level between healthy people and AML, was calculated using R2 database  is a public online database including a large number of tumors and normal samples. R4.1.0 software was used to analyze gene coexpression.Cancer Genome Atlas (https://pubchem.ncbi.nlm.nih.gov) in SDF format and then converted into MOL2 files using a freely available Chem3D software of Chem Office 2014. The 3D structure of protein was downloaded from Protein Data Bank (https://www.rcsb.org/). Before molecular docking, the AutoDock 4.2.6 software was used to remove ligands, structure water molecules, and add polar hydrogen atoms and charges to the protein crystal structures. The Discovery Studio software and Pymol software were used to evaluate the interaction between the targets and the active components  to determine the relative abundance of protein expression. All data values are represented as mean ± SD. The comparisons were performed using Student’s Based on TCMSP, SQSBD was derived from 12 herbal medicines including Hedyotis Diffusae Herba, Scutellariae Barbatae Herba, Lobeliae Chinensis Herba, Codonopsis Radix, licorice, Hedysarum Multijugum Maxim, Scutellariae Radix, Dioscoreae bulbiferae, Indigo Naturalis, Glehniae Radix, Asparagi Radix, and Agrimonia Eupatoria. A total of 268 active components were identified in SQSBD by using the criteria of OB 30% and DL 0.18. Detailed information of these active components is listed in https://www.ncbi.nlm.nih.gov/) for RNA sequencing data from bone marrow samples in 30 AML patients and 9 healthy subjects . Differential expression analysis of RNA sequencing data GSE37307 in NCBI database showed that 1,505 differential expression genes   . The topBy using GO enrichment analysis, we found that BPs of the common targets are mainly associated to cellular response to oxidative stress and chemical stress, etc. . The maiAccording to the KEGG pathway analysis, the critical pathways  were mainly enriched in pathways in cancer . We screWe performed the protein–protein interaction (PPI) network analysis to evaluate AML targets by using Bisogenet software, and the results are shown in We further compared 8 core active targets of SQSBD and 26 targets related to AML signaling pathways, and 6 hub targets  were obtained, these targets may play a crucial role in the treatment of AML with SQSBD, because they were involved in PPI core network, enriched in KEGG signaling pathways, and regulated by active components of SQSBD .We used R2 databases to detect mRNA expression of hub targets in 1 dataset of health people and 18 datasets of AML patients, a total of 4,112 samples. The expression of hub targets in different AML samples is stable , and wasGSEA analysis was used to conduct hub gene ontology term and pathway enrichment analysis of high- and low-expression samples. As shown in Based on these 6 hub targets, we constructed AML-related gene targets-components network, and found that CDK1, JUN, and MAPK1 are the hub targets most enriched signal pathways regulated by 9 components of SQSBD . With thTo confirm further the interaction between active components of SQSBD and hub targets, we used 6 hub targets and 9 pivotal active components as receptors and ligands to perform the molecular docking analysis in AutoDock vina software. For each pair of receptors and ligands, we could get 20 binding result of modes in the form of affinity, and the first mode is used as the best mode. The Discovery Studio software and Pymol software were used to evaluate the interaction between the targets and the active components, and draw 3D and 2D structure diagrams of the combined position. The −5.0 kcal/mol binding energy and 2 hydrogen bond were used as a threshold to determine whether the binding of the receptors and ligands is good or not. As shown in via the dephosphorylation of CDK1, and licochalcone A may induce cell differentiation via phosphorylation of MAPK1.To further verify the reliability of above molecular docking analysis, we selected top two active components of SQSBD  as the ligands, and 3 hub targets  as the receptors, respectively. As indicated in To further confirm whether quercetin could target CDK1, we determined the effects of quercetin on cell cycle progression in AML cells. As shown in Exposure of HL-60 and U937 cells to licochalcone A at various concentrations increased the percentage of CD11b+ cells but decreased the expression of another cell differentiation marker CD133 in a dose-dependent manner . WesternAccumulating evidence suggests that multiple target therapeutics is a promising strategy for drug discovery and exhibits higher efficiency and lower toxicity than monotherapies. Traditional Chinese medicines (TCMs) are characterized by a complex system of multiple components, multiple targets and multi-action pathways . HoweverAccording to the active components identified based on TCMSP, we performed target fishing using the TCMSP and BATMEN-TCM, obtained a total of 264 potential targets of active components from SQSBD . We alsoIncreasing evidence reveals that molecular targets or cell signaling pathways play crucial roles in the treatment of AML through the regulation of cell cycle progression, apoptosis, cell differentiation, etc. . To explBy using TCMSP and BATMEN-TCM databases, we elucidated the chemical composition and potential mechanism of SQSBD against AML . A totalRecent evidence reveals that MAPK1 signaling is involved in the regulation of cell differentiation . It has It has been reported that quercetin inhibited cell proliferation through dephosphorylation of CDK1 mediating cell cycle arrest at G2/M phase . SimilarIn this study, we obtained 9 pivotal active components of SQSBD in treating AML, namely, licochalcone A, quercetin, luteolin, kaempferol, wogonin, oxoxylin A, formononetin, naringenin, and baicalein. By constructing a compound–target, we screened out 6 hub targets, namely, CDK1, HSPB1, JUN, STAT3, PCNA, and MAPK1. Molecular docking showed that two core active components, quercetin and licochalcone A, exhibited the highest ingredient-like properties (DL), and could bind well to CDK1 and MAPK1 protein. The experimental validation of these two components showed that quercetin inhibited cell growth through CDK1 dephosphorylation-mediated cell cycle arrest at G2/M phase in human AML U937 and HL60 cells, and licochalcone A induced cell differentiation in these leukemia cells via activation of MAPK1 and upregulation of CD11b. All these results indicate that SQSBD is effective in the treatment of AML, and quercetin and licochalcone A are the major candidate compounds for AML treatment.