Background: Acute myeloid leukemia (AML) is a common malignancy in adults. A vast variety of environmental and lifestyle factors play a role in AML incidence. This study aimed to assess the factors related to AML.Study design: A case-control study.Methods: This case-control study was performed on 137 AML cases during 2018-2021 at Beheshti Hospital in Hamadan, Iran, and 137 gender/age-matched controls. A questionnaire including 12 items was used to obtain information about lifestyle and environmental factors. A univariate and multiple variate logistic regression was used to estimate the odds ratios (OR), and a 95% confidence interval (CI) was used to investigate the relationship between the studied variables and the incidence of AML.Results: Based on findings, 62 (45.3%) out of the 137 leukemic cases were male and 75 (4.7%) were females. A statistically significant increased risk for AML was found with regard to prior usage of cytotoxic agents , family history of malignancies , exposure to electrical power , and history of mental diseases . It was found that the AML incidence had no association with age, gender, radiation therapy, cigarette smoking, prior chemotherapy, congenital disorders, exposure to chemical agents, history of infectious mononucleosis, exercise, and blood transfusion (P>0.05).Conclusion: The current results suggested that cytotoxic agents, family history of malignancy, mental disorders, and exposure to electrical power could play a role in AML incidence. Rapid growth of abnormal myeloid progenitor cells in the bone marrow (BM) and peripheral blood (PB) is the most important sign of AML. According to reports by the World Health Organization, the incidence of AML is related to several chromosomal abnormalities that have similar clinical manifestations but different morphologic, immunophenotypic, and cytogenetic subtypes. Moreover, altered expression of a large number of genes, including cytokine, ion channel, as well as CD markers may play a role in the pathogenesis and prognosis of AML.Acute myeloid leukemia (AML) is the most common hematological malignancy in adults. Furthermore, it has been shown that the process of aging can be associated with an increased risk of AML that is likely due to the gradual deterioration of the immune system due to immunosenescence. It seems that lifestyle and educational intervention play a key role in the promotion of community health. Studies have also highlighted the impact of chemotherapeutic agents on AML incidence. For example, it has been found that older patients with AML have a history of prior chemotherapy exposure. This may be a result of BM niche alteration that leads to deregulation of balance within the marrow cavity as well as abnormal hematopoiesis.Besides the chromosomal abnormalities, epidemiologic studies demonstrated that several factors, including exposure to ionizing radiation, personal and family medical histories, exposure to chemical or biological agents, such as solvents and benzene, smoking habits, and lifestyle factors are associated with AML and acute lymphoid leukemia (ALL) incidence. However, the majority of epidemiologic studies that have demonstrated relationships of AML incidence with potential risk factors, such as exposure to ionizing radiation and chemicals, including hydrocarbons and pesticides, cigarette smoking, infections, and family history of malignancies, have shown that some of these factors are simply modifiable. In addition, it seems that these potentially etiologic exposures have an impact on clinical outcomes. Therefore, understanding relevant risk factors may have particular significance in the prevention and reduction of AML occurrence. Moreover, it should be mentioned that this may influence clinical decisions.The specific genetic abnormalities that cause AML are not usually modifiable; nevertheless, as the disease occurs sometimes years after the exposure, this risk factor is not usually considered. However, since leukemia develops sometimes years after environmental exposure, current knowledge of some risk factors is insufficient.It is noteworthy that since the development of leukemia takes place sometimes years after environmental exposure, current knowledge of some risk factors is insufficient. For example, in utero exposure to ionizing, radiation is considered an established cause of pediatric de novo AML Furthermore, few studies have been conducted to examine the possibility of synergistic interactions of genes and environmental factors with AML both during childhood and adulthood. It seems that the knowledge of AML etiology can be enhanced by exploring the potential effect of several environmental and lifestyle factors, namely diabetes, exposure to alcohol, and pesticides as well as maternal factors, including age at pregnancy and birth weight on genetic abnormalities. In spite of the high incidence rate of AML due to environmental risk factors, few studies in Iran explain the majority of risk factors. In the present case-control study, an extensive epidemiological analysis of AML was conducted based on personal characteristics, lifestyle, and other environmental risk factors, including occupations and specific exposures to the chemical agents.This matched case-control study was conducted on patients with AML who referred to Beheshti Hospital of Hamadan University of Medical Sciences, Hamadan, Iran. For the purposes of the study, 137 patients were selected from 391 newly diagnosed , relapsed, or refractory AML patients who were hospitalized during 2018-2021 . In addi The control group matched the case group by age (±5 years) and had resided in Hamadan during the last 20 years. If the control group members refused participation after enrolment or were not eligible, additional subjects were randomly selected. Control group members were also screened to make sure that they were not relatives of the case group members. Data collection from both case and control groups began in February 2018 and finished in April 2021. It should be noted that the matching criteria included gender and age. Moreover, inclusion criteria for cases were diagnosis of AML and residence location at the time of the study. The inclusion criteria for controls were no previous history of invasive cancers or hematological malignancies and residence in Hamadan at the time of the study. The exclusion criteria for cases and controls were unwillingness to participate in the study, other resident location at the time of the study, and lack of access to patients. Informed consent was obtained from each subject in this study before data collection. Detailed information on the variables under study was collected through a full questionnaire that was given to each participant of the study. The questionnaire included items about age, gender, and a large number of potential risk factors, such as personal and family medical histories  exposure, and lifestyle, including smoking (>100 cigarettes in a year) and exercise, environmental exposures, such as living on a farm, living within 100 meters of high voltage electrical power transmission lines for at least five years, and exposures to chemicals , physical or biological agents .P-value of less than 0.05 was considered statistically significant. All statistical analyses were performed using SPSS software (version 22) and STATA (version 14.2).The demographic data were presented as frequency and percentage while the quantitative data were expressed as mean ±SD. Relevant data were collected in Stata software (version 14.2) and the risk factors were determined in two ways. In the first method, the conditional logistic regression of univariate analysis was used with the assumption of individual matching while in the second method, the multiple conditional logistic regression analysis was employed. The odds ratios and a 95% confidence interval were used to investigate the relationship between the studied variables and predisposition to AML. The chi-square and t-tests which showed good results in all models were used to compare the case and control groups to determine the association between the studied variables and the risk of AML. It should be mentioned that a P=0.055) and almost homogeneity was seen in the distribution. As shown in the receiver operating characteristic diagram, no significant difference was observed between the goodness of fit criteria of the two models (P=0.08) and gender (P=0.45).A total of 137 patients with AML and 137 age/gender-matched healthy individuals as the controls group were included in the study analysis. There was no significant difference between the mean ages of both groups (o models . In totaP=0.05) and increased risk of AML  and history of mental diseases (P=0.001) were also associated with an increased risk of AML . Family history of hematological malignancies or solid tumor (k of AML . In thisP=0.002). People with exposure to electrical power had 3.22 times higher odds for AML occurrence, compared to others . In addition, the risk of AML was shown to be 1.76 times higher for patients with prior chemotherapy, although no statistical significance was observed (P=0.101), . Furthermore, it should be noted that no association was found between smoking and the risk of AML incidence (P=0.209). However, the univariate analysis of cigarette smoking showed that people with a history of smoking had 1.5 times higher odds for AML incidence in comparison to others.As seen in P=0.001)  .P=0.085). Moreover, the results of the multivariate analysis did not show a significant relationship between exposure to electrical power and AML incidence in conditional logistic regression (P=0.080)  .; hence, it can be said that the etiology of AML is complex. Several epidemiologic studies worldwide have demonstrated that AML incidence is associated with a number of possible risk factors, including infectious and environmental factors, such as ionizing radiation, cigarette smoking, history of malignancies, and drug use. In this case-control study, the association between environmental risk factors and the development of AML has been evaluated. In the present study, results of the univariate and multivariate analysis indicated that AML can be related to a family history of malignancies, exposure to electrical power, and mental disorders.The AML and other hematological malignancies may be the result of a combination of genetic abnormalities and environmental risk factors This study was performed on 137 cases and the same number of healthy controls, and it was found that family history of malignancies may be associated with AML incidence. Although only a small number of cases and controls had a family history of malignancies in this study, a significant association was observed between family history of hematological malignancies and solid tumors with the incidence of AML. which is inconsistent with the findings of the present study. Other studies have demonstrated that the family history of cancer has a relative risk for incidence of AML and different neoplasms which is in line with the findings of this study. However, it should be mentioned that this finding was not replicated in a case-control study carried out in United States.In their case-control study, Wong et al. reported only the weak and non-significant effect of family history of malignancies on AML . Since for the expression of a trait each allele is inherited from one of the parents, it seems that the acquisition of abnormal genes, as well as the incidence of cancer, is higher in populations with high rates of consanguineous marriage. Therefore, consanguineous marriage should be re-examined in populations where leukemia and other tumors seem more common.These findings suggest that studies should determine whether AML patients with a family history of malignancies have received benefit healthcare from an earlier age or not as healthcare utilization can be useful in cancer prevention. Moreover, it is obvious that the development and progression of cancer usually occur due to the abnormal expression of some genes, such as tumor suppressor. However, there are many studies that have found the relationship between exposure to electrical power and a large number of disorders, including cancer, genetic damage, reproductive disorders, immune dysfunction, kidney damage, and hypersensitivity. Since the progress of science and technology leads to the wide use of high voltage electrical power transmission lines, electromagnetic wave exposure has become a public health problem that requires public awareness.In the present study, the analysis showed a significant relationship between exposure to electrical power and risk of AML incidence. In contrast, in a case-control study performed by Wong et al., living within 100 m of high voltage electrical power transmission lines was not found to be associated with the risk of AML incidence. No study has been conducted to explore the direct impact of mental disorders, including anxiety and depression, on AML incidence. Accordingly, it is unclear why mental diseases are a risk factor for the development of AML. Ding et al. in their case-control study demonstrated that anxiety and depression have poor prognostic value for survival in AML patients; hence, mental diseases which are associated with anxiety and depression might play a role in AML incidence via an unfavorable effect on the quality of life.In the present study, an increased risk of AML was observed in approximately half of the cases who had a history of mental diseases, such as sadness, anxiety, depression, fear, and stress. It has been shown that the stress from the family is the most important source of stress associated with adolescent mental health; this is in line with the findings of the present study. However, many case-control studies have indicated that the increased risk of AML is associated with heavy smoking. In addition, findings of a large cohort study performed by Ma et al. demonstrated that smokers who smoked more than a pack per day had a statistically increased risk of AML.Generally, smoking has been recognized as a risk factor for AML but in contrast to previous studies, we did not find any significant association between smoking and AML incidence. Results of a case-control study carried out by Bjork et al. indicated no significant association between smoking and AML incidence. However, inconsistencies in definitions and measurements of this variable in different studies make it difficult to interpret the full impact of smoking on AML.Our non-significant results in this regard may stem from a lack of AML classification or exact duration of smoking in our cases. Accordingly, Wong et al. in a case-control study found that the number of daily consumed cigarettes had a non-significant association with AML, whereas the duration of smoking seemed to be positively associated with the risk of developing AML. It should be mentioned that the sample size of the present study was small (n=25); therefore, there is a need for further clinical studies with larger sample sizes. The leukemogenic effect of chemotherapy could be due to the fact that its purpose may be BM progenitor cells. Many models are available to estimate the risks of AML after chemotherapy; however, to our knowledge, no tools exist to reliably recognize what patients are at risk. Nevertheless, the use of BM and PB cytogenetic assessment and a more careful clinical follow-up is an important prognostic tool for awareness of the risk of AML.In this study, no clear relationship was found between a history of prior chemotherapy and AML. Despite this lack of association in the present research, the incidence of AML after chemotherapy is a well-established fact. However, Ojha et al. found an increased risk for AML due to radiotherapy secondary to benign diseases . These inconsistent results may be in part due to differences in the methods used for the assessment of radiation exposures (exposure to three or more rays) or the age of the studied population. Although there are heterogeneous results for the relationship between exposure to radiation and leukemia in previous studies, this risk appears to be associated with therapeutic radiation effects on DNA repair enzymes, such as topoisomerase II. In this regard, Leone et al. have shown that alkylators combined with radiotherapy could promote survival of misrepaired cells giving rise to the leukemic clone.In the present study, no significant association was observed between prior radiation therapy and AML. In line with our results, those of several case-control studies have not shown the increased risk of leukemia in children who were exposed to X-rays during the postnatal period ; however, in this study, no significant association was observed. Despite our negative results and similar findings of a case-control study performed by Poynter et al. on pesticides and agricultural chemicals exposure in Minnesota, it seems that any chemical exposure during any time period of life in houses or workplaces could be a potential risk factor for cancers. Therefore, these public health problems should be considered and public awareness should be raised more widely.Potential exposures to a variety of chemicals, such as paints, hydrocarbons, including benzene, chemical fertilizers, detergents, and pesticides, might have been potentially hazardous for AML incidence. Prior case-control studies have identified an association between chemical exposure, especially exposure to benzene, and increased risk of AML. Future studies should focus on age-stratified approaches to reach results with greater sensitivity.In this study, no significant associations were observed regarding the effects of congenital disorders, such as Down syndrome, Kostmann syndrome, and aplastic anemia on AML. There is small speculation about the possible effects of congenital disorders on AML. Although adults with Down syndrome show a decreased incidence rate of cancer, compared to healthy individuals, children with Down syndrome are at an increased risk of leukemia, especially AML, we believe that exercise can improve immune system function and increase the ability to fight diseases.Besides, when we evaluated the relationship between exercises at least twice a week with AML, we did not observe a significant association. Since several quasi-experimental studies have indicated that physical activity and aerobic exercise interventions have been helpful for the reduction of fatigue and improvement of the quality of life of leukemic patients. The current study showed evidence of EB infection in a very small number of AML patients and no significant association was found between them. The findings are inconsistent with those of previous studies that indicated EBV infection as a possible risk factor for the progression and poor clinical outcome of chronic lymphoblastic leukemia(CLL), ALL, and AML. Since we do not have enough evidence to conclude that EBV is associated with the pathogenesis of AML, it is suggested that further studies be conducted with larger sample sizes of patients to investigate whether there is a relationship between EBV and AML.The EBV is a ubiquitous human infection that has been associated with a variety of malignancies, such as Burkitt’s lymphoma (BL), Hodgkin lymphoma (HL), nasopharyngeal carcinoma (NPC), gastric cancer, and acute leukemia. However, a slightly higher risk of leukemia  was reported in individuals with a history of blood transfusion in a study performed by Ross et al. . Since the relationship between the history of blood transfusion and leukemia is a controversial issue, further studies should be performed on specific leukemia subtypes are required. There were also several limitations in the present research; first of all, this study was conducted in a single center. Moreover, there was a lack of identification of several cytogenetic groups. Furthermore, regarding the history of mental diseases, only anxiety and depression were included; however, it is better to include other mental disorders as well. Besides, the duration of the study was relatively short. Therefore, it is suggested that the results be validated in a long-duration study in the future.In addition, our study showed no association between a history of blood transfusion and AML incidence. Similar to our results, in the two US population‐based case‐control studies, the increased risk of hematological malignancies was not associated with blood transfusion In summary, it was found several potential risk factors of AML  include prior use of cytotoxic agents, family history of hematologic malignancy or solid tumors, and history of mental diseases. An unexpected finding in this study was the lack of associations of cigarette smoking and prior radiation therapy with increased risk of AML. Identification of AML risk factors may be helpful to improve therapeutic measures and public health protocols. However, the limitations of the study should be taken into consideration. First, because of the large number of risk factors in AML subtypes, only some of them were evaluated in this study. Second, the number of subjects in this study was limited; hence, the results need to be interpreted with caution. Finally, the findings need to be replicated in future studies to reach definitive results. The authors wish to thank all their colleagues at Hamadan University of Medical Sciences. All authors approved the manuscript and have no declaration regarding commercial or non-commercial intentions. This study was funded by Research and Technology Deputy, Hamadan University of Medical Sciences (No. 9709065171). H.R. conceived the manuscript and revised it. M.A provided clinical data and information. M.M.B, I.O., and S.G performed the statistical analysis, wrote the manuscript, and prepared tables and figures. All procedures were approved by the institutional Ethics Committee of Research Deputy of Hamadan University (IR.UMSHA.REC.1397.558). Accordingly, informed consent was obtained from all the participants in the study.Acute myeloid leukemia may be the result of a variety of environmental risk factors.Family history of malignancies, environmental exposure, and history of mental diseases may be a potential risk for acute myeloid leukemia.Identification of these risk factors may be helpful in the management of patients as well as improvement of public health protocols.


Aberrant Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling is implicated in the pathogenesis of acute myeloid leukemia (AML), a highly heterogeneous hematopoietic malignancy. The management of AML is complex and despite impressive efforts into better understanding its underlying molecular mechanisms, survival rates in the elderly have not shown a substantial improvement over the past decades. This is particularly due to the heterogeneity of AML and the need for personalized approaches. Due to the crucial role of the deregulated JAK-STAT signaling in AML, selective targeting of the JAK-STAT pathway, particularly constitutively activated STAT3 and STAT5 and their associated upstream JAKs, is of great interest. This strategy has shown promising results in vitro and in vivo with several compounds having reached clinical trials. Here, we summarize recent FDA approvals and current potential clinically relevant inhibitors for AML patients targeting JAK and STAT proteins. This review underlines the need for detailed cytogenetic analysis and additional assessment of JAK-STAT pathway activation. It highlights the ongoing development of new JAK-STAT inhibitors with better disease specificity, which opens up new avenues for improved disease management. KIT, FLT3, NPM1, CEBPA), epigenetic modulators  or as chromosomal rearrangements that generate fusion transcripts of genes encoding for transcription factors. These include, for example, RUNX1-RUNX1T1, PML-RARA or MLLT3-MLL [Acute myeloid leukemia (AML) is a heterogeneous malignant disorder characterized by abnormal proliferation and differentiation of hematopoietic stem and progenitor cells (HSPCs). This clonal expansion leads to an accumulation of immature myeloid precursors in the hematopoietic tissue and peripheral blood, which occurs at the expense of normal hematopoiesis and results in anemia, leukopenia or thrombocytopenia ,2. The gLLT3-MLL ,19,20,21STAT3β/α mRNA expression ratio positively correlates with overall survival, representing a promising prognostic marker in AML patients [The Janus kinase (JAK) family of nonreceptor tyrosine kinases comprises pivotal mediators of growth factor and cytokine signaling, activating i.a. downstream signal transducer and activator of transcription (STAT) factors ,23,24. Spatients . DespiteIn mammals, four JAK proteins ) and seven STAT proteins  have been identified. The binding of a ligand to its receptor is a critical step in precisely determining the context-dependent effect of downstream signaling by distinct JAK-STAT combinations with a high degree of specificity. The JAK-STAT signaling pathway links the receptor binding of extracellular ligands like cytokines, hormones and growth factors via a ubiquitous intracellular signaling cascade with the transcriptional machinery in the nucleus. It is tightly regulated under physiological conditions coordinating a wide range of fundamental biological mechanisms, including cell proliferation, differentiation, apoptosis and inflammation. Dysregulation has been identified in severe pathological circumstances and is commonly associated with constitutively active STATs promoting, among others, intense inflammatory conditions or tumorigenesis .MYC, BCL-2, and D-type cyclins, which are engaged by any STAT protein in various cell types [The highly conserved JAKs, JAK1, JAK2 and TYK2, are ubiquitously expressed, whereas JAK3 is predominantly expressed in hematopoietic cells, in particular lymphoid and myeloid cells . Among tll types ,42.+) leukemias), the platelet-derived growth factor receptor (PDGFR) and epidermal growth factor receptor (EGFR) [Based on the ligand, the receptor and their tissue specificity, distinct combinations of STATs and JAKs are activated. This canonical signaling is based on JAK-mediated STAT tyrosine-phosphorylation. Additionally, several non-canonical pathways have been described, including cascades building on the kinase-independent action of JAKs and unphosphorylated STATs, or the formation of higher-order or multifactorial STAT transcriptional complexes, which can also affect cytokine signaling and immune cell function ,43. The r (EGFR) ,44,45. Cr (EGFR) .During canonical STAT signaling, STATs act mostly as homodimers, but they are also found as heterodimers or higher-order tetramers . STAT1 aThe JAK–STAT signaling cascade is subject to constant multiple levels of activating and inactivating processes, which offer new possibilities for increased therapeutic exploitation. Pathway inactivation can be accomplished by basic cellular mechanisms including posttranslational modifications, microRNAs, RNA-binding proteins , the ubiThe complex network between genetic factors and extrinsically driven signaling pathways is crucial for tumorigenesis and other severe diseases. JAK-STAT signaling in the hematopoietic system tightly orchestrates survival and self-renewal of hematopoietic stem cells (HSC) and hematopoiesis, as well as proliferation. Thus, aberrant JAK-STAT signaling has been associated with the pathogenesis of inflammatory diseases and immunodeficiencies and also with hematopoietic malignancies ,81. SincJAK2V617F, a somatic point mutation within the autoinhibitory pseudokinase (JH2) domain, resulting in constitutive activation of the JH1 domain and thus increased activation of JAK2 and downstream STATs [JAK2V617F appears in the majority of secondary AML cases resulting from myeloproliferative neoplasm (MPN), where it acts as a main driver mutation. Mutated JAK2 is only present in <5% of de novo AML patients [Constitutive activation of STATs has often been found in cancer cells as a result of deregulated signaling of the upstream JAK. A classic example is am STATs . JAK2V61patients ,24.FLT3) [FLT3 mutations, especially internal tandem duplication mutations (ITDs) of the extracellular juxtamembrane (JM) domain, are the most frequent genetic alterations in AML patients with a normal karyotype [In de novo AML, constitutive activation of STATs can be achieved by auto- and paracrine factors such as IL-6, decreased expression of negative regulators such as SOCS1 or activating mutations in other (non-JAK) upstream kinases such as FMS-like tyrosine kinase 3 (FLT3) . FLT3 muaryotype ,87. FLT3aryotype . The FLTaryotype .FLT3 mutations frequently co-occur with alterations in the NPM1 gene, leading to a significantly worse prognosis [MYC and BCL2 expression in a bromodomain-containing protein 4 (BRD4)-dependent manner [FLT3-ITDs also combine with mutations of DNMT3A, which seems to lead to the worst prognosis for AML patients [PTEN promoter in AML cells [rognosis . Mutatedrognosis , as wellt manner . Howevert manner . FLT3-ITpatients ,93. DNMTpatients  and mutapatients . IncreasML cells .CEBPA or CEBPA/CSF3R mutations are sensitive to the inhibition of the JAK-STAT pathway [RUNX1–RUNX1T1 [KITDD816V) is frequently detected in AML patients [Another way in which the JAK-STAT pathway contributes to the pathogenesis of AML is the reliance of leukemic cells on its signaling. AML cells carrying  pathway . Similar–RUNX1T1 . Furtherpatients ,100 and patients . These dJAK2V617F mutation [Inhibition of JAK-STAT signaling via JAK1/2 inhibitor Ruxolitinib is a successful approach in the treatment of MPNs. In rare cases MPNs evolve to secondary AML (sAML), which provided the first rationale for investigating Ruxolitinib as a treatment for sAML ,106,107.mutation . TherefoJAK2V617F mutation [Ruxolitinib has been approved for the treatment of intermediate and high-risk MPNs that are resistant or intolerant to hydroxyurea. In MPN, inhibition of JAK1/2 as a monotherapy efficiently reduces symptoms . Unfortumutation . This comutation ,127,128.mutation . In partmutation .CEBPA- and CSF3R-mutated AML [Among many combinatorial approaches being tested in preclinical settings, a combination of Ruxolitinib with Lysine Specific Demethylase 1 (LSD1) inhibitor recently demonstrated a synergistic survival improvement in mouse models of ated AML . LSD1 deated AML . The effated AML .FLT3-mutated AML cells are sensitive to Lestaurtinib in vitro upon treatment with chemotherapeutic agents [FLT3-ITD AML [FLT3-mutated AML. Although a transient response appeared in treated patients, overall, no clinical efficacy was observed in this therapeutic setting [Lestaurtinib is a multikinase inhibitor initially developed as a tropomyosin receptor kinase A (TrkA) inhibitor, which also potently inhibits JAK2 and FLT3 . FLT3-muc agents ,134. The setting .FLT3-ITD+ AML cell lines, but combined inhibition of JAK2 and FLT3 also overcomes the resistance towards FLT3 inhibitors , which is often associated with overactivation of the JAK-STAT pathway [FLT3 and JAK2. The therapeutic effect was attributed to efficient IRAK1 inhibition rendering this kinase a novel target in AML [FLT3 mutations [Pacritinib is a multikinase inhibitor with high specificity towards JAK2 over other JAKs, but in addition it inhibits interleukin receptor-associated kinase (IRAK), Colony stimulating factor 1 receptor (CSF1R) and FLT3 . It is c pathway . Interest in AML . Indeed,utations .Fedratinib is another small molecule targeting JAK2 and FLT3, which has recently been approved for the treatment of MPN. In addition, Fedratinib inhibits BRD4 . PreclinFLT3 mutated AML suggests that Momelotinib-mediated combinatorial inhibition of JAK1/2, FLT3 and downregulation of MYC will be more effective than currently used FLT3 tyrosine kinase inhibitors (TKIs) [Momelotinib is a JAK1/2 inhibitor that has reached phase III clinical trials for MPNs ,143. Alts (TKIs) .FLT3 mutated AML. Although occurring with significantly lower incidences, many other mutations in AML drive constitutive activation of the JAK-STAT pathway [Inhibition of JAKs in combination with chemotherapy or epigenetic modulator inhibitors seems to be a promising approach in the treatment of different subtypes of AML. In contrast to MPNs, where the specific JAK2 inhibitors sparing other kinases are discussed as the best treatment option , AML pat pathway . TherefoA substantial proportion of AML patients harbor mutations that cause hyperactivation of JAK-STAT signaling ; therefoMYC, cyclin D1, BIRC5 (Survivin) and BCL2. In line, upregulation of STAT3 is associated with protection from apoptosis [STAT3 has been shown to play an important role during myeloid homeostasis and differentiation as well as proliferation ,162. Duepoptosis . Furtherpoptosis  and signpoptosis . STAT3 spoptosis ,167 or cpoptosis . Based oA small molecule targeting the SH2 domain of STAT3, OPB-51602, has entered clinical trials for the treatment of hematological malignancies. It was shown to efficiently repress proliferation by blocking the phosphorylation sites tyrosine705 and serine727 of STAT3 in various human cancer cell lines . More reThe small molecule STAT3 inhibitor, C188-9, was demonstrated to inhibit G-CSF-induced STAT3 activation thereby efficiently inducing apoptosis in primary pediatric AML samples and AML cell lines . The comBBI608, also known as Napabucasin, is a small molecule inhibitor blocking STAT3-mediated gene transcription, whose antitumorigenic properties have been demonstrated in phase Ib/II and phase II trials in solid cancers as a monotherapy and in combination with standard treatment ,174. RecAn emerging strategy to target proteins with small-molecule degraders are proteolysis targeting chimeras (PROTACs), which rely on hijacking the cellular degradation machinery, the proteasome. By using a heterobifunctional degrader, the protein of interest is linked to an E3 ligase, which subsequently leads to polyubiquitination and degradation of the target . Due to An attractive strategy that emerged with the fast progress of next-generation sequencing techniques involves antisense oligonucleotide (ASO) inhibitors, which are designed based on the genetic sequence of the target gene. One promising STAT3 ASO, AZD9150, showed efficiency when systemically administered in preclinical trials for lymphoma and lung cancer (NSCLC) patients . In vitrSTAT3 gene silencing. To deliver the inhibitor to Toll-like Receptor (TLR)-9 positive antigen-presenting immune cells, the inhibitor molecule was linked to a TLR9 ligand and a CpG [As a driver of immune evasion in cancer it is conceivable that inhibition of STAT3 enhances anti-tumor immune responses. Thus, Hossain et al. investigated small interfering RNAs (siRNAs) conjugated to cytosine guanine dinucleotides (CpG) that target nd a CpG . Adminisnd a CpG . NeverthBCR-ABL and FLT3-ITD, which contribute to its constitutive activation [FLT3-ITD, which is one of the most commonly detected cytogenetic abnormalities, several upstream inhibitors targeting FLT3 kinase achieved promising clinical outcomes [FLT3 mutated AML [FLT3 mutated AML patients, the occurrence of therapy resistance is an ongoing challenge. Thus, using several approaches to target a disrupted signaling pathway might be an efficient strategy to eradicate leukemic cells and prevent therapy resistance.STAT5 plays a pivotal role especially in AML patients who harbor fusion-oncogenes such as tivation . For AMLoutcomes . Thus, tated AML ,180. AltBCR-ABL positive K562 (CML) and FLT3-ITD positive MV-4-11 (AML) human cell lines revealed effective suppression of STAT5 phosphorylation and downregulation of target genes such as Cyclin D1, Cyclin D2, C-MYC and MCL-1. Further compounds, SF-1-087 and SF-1-088, were identified, which also showed high selectivity for STAT5 over STAT3 and STAT1.In 2012, Page et al. performed an in vitro screen focusing on salicylic- acid-containing inhibitors potentially serving as phosphotyrosine mimicry to the SH2 domain of STATs . Two of FLT3-ITD-driven AML cells. In this preclinical setting, AC-4-130 exhibited anti-leukemic activity in vitro and in vivo in a xenograft model based on MV-4-11 cells by selectively targeting STAT5 over STAT1 and STAT3. Furthermore, it showed enhanced efficiency in combination with Ruxolitinib [As a novel SH2 inhibitor of STAT5, AC-4-130 was demonstrated to efficiently diminish STAT5 activity in human AML cells lines and primary olitinib .To summarize, various strategies for the inhibition of deregulated JAK-STAT signaling have demonstrated promising results in vitro and in mouse models with some compounds reaching clinical trials for AML. However, the heterogenicity behind AML development and progression might be an explanation for the diverse treatment outcomes. To address this, a better understanding of the complex biology and the underlying mechanisms involved in JAKs and STATs modulating AML progression is needed. For example, a recent preclinical study identified strong dependency of rarely occurring MLL-AF10-driven AML on JAK1 . Thus, p


The better understanding of the genomic landscape in acute myeloid leukaemia (AML) has progressively paved the way for precision medicine in AML. There is a growing number of drugs with novel mechanisms of action and unique side-effect profiles. This review examines the impact of evolving novel therapies on survival in AML and the challenges that ensue.  The last few years have witnessed a therapeutic renaissance in the field of acute myeloid leukemia (AML), spearheaded by the FDA’s approval of 10 new therapies since 2017. While treatment for AML remains a therapeutic challenge, new therapeutic options have begun to create an emerging precision medicine framework in our approach to managing this condition. Along with the growing number of drugs with novel mechanisms of action is the arrival of an array of new adverse event profiles that are often uniquely linked to a particular class of drugs. This review examines the impact of evolving novel therapies on survival in AML and some of the novel toxicities associated with their introduction. We also discuss emerging strategies used to augment and further improve efficacy and the role of measurable residual disease (MRD) as a means for the assessment of clinical outcomes. We propose that the current evidence points to a future of AML management that will be more personalized in terms of treatment selection, safety planning, monitoring for disease response, and the re-evaluation of the mechanisms of adaptive resistance at relapse.FLT3 mutations are one of the most common and prognostically important genetic alterations that occur in AML. FLT3 mutations are present in approximately 30% of all patients with AML, with internal tandem duplication (ITD) accounting for approximately 25% of all AML cases and FLT3 mutation in the tyrosine kinase domain (FLT3-TKD) accounting for approximately 7–10% of all cases. Both mutations lead to the constitutive activation of the receptor and its downstream signalling pathways, including PI3K/AKT/mTOR, RAS/MAPK, and STAT5 . FLT3 muTargeted therapy with FLT3 inhibitors has significantly improved outcomes in this subgroup. First-generation tyrosine kinase inhibitors (TKIs), such as midostaurin, sorafenib, and lestaurtinib, have demonstrated limited anti-leukemic activity as monotherapies. Midostaurin was shown to improve 4-year OS from 44% to 51% when combined with intensive chemotherapy in patients with untreated FLT3-mutated AML, as demonstrated in a landmark randomized phase 3 study (RATIFY) . A recenNew data concerning the role of other FLT3 inhibitors for the first-line treatment of patients fit for intensive chemotherapy are emerging. The Australasian Leukaemia and Lymphoma Group (ALLG) conducted a randomized, placebo-controlled phase 2 trial of sorafenib in combination with intensive induction and consolidation chemotherapy followed by 12 months of sorafenib maintenance. Several themes emerged, including the high rate of clinical response in both the sorafenib and placebo arms (91% vs. 94%); the higher rate of early FLT3-ITD MRD clearance, as assessed by next-generation sequencing (NGS) in the sorafenib arm (43% vs. 32%); the high rate of alloHSCT in both arms (~60%); and the high frequency of FLT3-ITD-negative relapse (~60%) . AlthougIn the frontline treatment setting, gilteritinib in combination with chemotherapy has also shown favorable anti-leukemic responses, with a composite CR of ~80% and a 70% mutational clearance of FLT3-ITD found in a Phase I study . Based oDespite the improved OS and EFS found in the RATIFY trial, only 59% of patients in the midostaurin arm achieved CR and almost half of these patients relapsed after achieving remission. AlloHSCT remains beneficial for sustaining long-term remission . A post p = 0.07) [There is a need for increased awareness of how to anticipate, mitigate, and manage complications associated with the increasing use of FLT3 inhibitors. Severe adverse events associated with the use of multikinase FLT3 inhibitors include midostaurin-related grade 3 to 5 pneumonitis or radiographic pulmonary opacities, which were reported in 8% of patients in the phase RATIFY trial . Among o = 0.07) .FLT3-ITD-negative at the time of disease relapse or progression [In relapses after first-line midostaurin in combination with intensive chemotherapy, different modes of treatment failure have also been noted. A recent study by Schmalbrock et al. provided novel insights into clonal evolution and resistance mechanisms in the RATIFY study. Almost half the midostaurin-treated patients (46%) were gression . In othegression . p = 0.007) in the ADMIRAL study [In relapsed refractory (R/R) AML, second-generation TKIs including quizartinib, crenolanib, and gilteritinib have shown favorable single-agent clinical efficacy, related in part to their enhanced FLT3 target affinity. Gilteritinib was shown to be superior to salvage chemotherapy in the R/R setting by improving the median OS from 5.6 to 9.3 months  of 76.4%, including a significant proportion (62.5%) of patients with prior FLT3 TKI exposure, in contrast to 12% in the ADMIRAL study . This suFollowing the attainment of remission, FLT3 TKIs have been actively explored as maintenance therapies for patients with FLT3-mutant AML. However, the impact of maintenance therapy on survival is yet to be ascertained. The beneficial effects of sorafenib maintenance after alloHSCT have been demonstrated based on two randomized trials , albeit most patients were not exposed to FLT3 TKIs prior to alloHSCT ,17. A syIsocitrate dehydrogenases (IDHs) are NADP-dependent enzymes involved in cellular energy generation in the Krebs cycle. IDH catalyzes the oxidative decarboxylation of isocitrate to  enzymes . This reIvosidenib and enasidenib, selective inhibitors of mutant IDH1 and IDH2, respectively, have demonstrated encouraging clinical activity for use as monotherapies in the R/R and, more recently, in the frontline setting. In patients with R/R IDH1/2-mutant AML, ivosidenib and enasidenib have induced therapeutic responses in a considerable proportion of patients with an ORR ~40%, CR rates ~20%, and median time to CR of approximately 3 months ,23. Morep= 0.23) [p < 0.01; CR 53% vs. 12%, p < 0.01) and durations  [Despite the promising results of early-phase studies, comparative randomized trials with IDH inhibitors have delivered mixed results. The phase III IDHENTIFY trial was planned as the main study to obtain full registration for the IDH2 inhibitor enasidenib. However, this was to no avail, as the study did not demonstrate a superior OS as anticipated. IDHENTIFY was an open-label trial for patients aged ≥60 years who had received 2–3 prior AML therapies. Investigators preselected patients to one of four conventional care regimens (CCR) in the control arm, including either azacitidine, intermediate-dose cytarabine, low-dose cytarabine (LDAC), or best supportive care. Despite the improved ORR found in the Enasidenib arm (40.5% vs. 9.9%), the primary endpoint of OS was not significantly different between the Enasidenib and CCR arms: 6.5 months vs. 6.2 months  . The stu= 0.23) . Further = 0.05) . Again,  = 0.05) . This reAmong older patients unfit for intensive chemotherapy, the combination of azacitidine and ivosidenib in a phase Ib study demonstrated an ORR of 78.3%, including CR in 60.9% and mutation clearance in 43.4% . The medAlthough ivosidenib and enasidenib responses are clinically durable, the complex genetic heterogeneity of AML suggests that combination with other non-targeted therapies may augment remission and overcome primary resistance . PromisiThe emerging role of IDH inhibitors in AML has also led to improved insights into the mechanisms of therapeutic resistance to these agents. An analysis of 179 patients with R/R AML treated with ivosidenib suggested that primary resistance to ivosidenib was related to receptor tyrosine kinase (RTK) pathway mutations such as NRAS and PTPN11 . It was BCL2 signaling is thought to be a critical survival pathway in IDH1/IDH2-mutant AML based on preclinical studies, providing a rationale for targeted BCL2 inhibition in IDH-mutant AML . NotablyAs IDH inhibitors restore myeloid differentiation, they can induce myeloid maturation and proliferation, resulting in IDH inhibitor “differentiation syndrome”. This is reported in ~15% patients and may occur with hyperleucocytosis ,25. OfteTP53 mutation accounts for approximately 5–20% of cases. It is associated with a complex and/or monosomal karyotype with very poor survival outcomes after conventional treatment options, with reported CR rates of 20% using hypomethylating agents and a median OS of only 6–12 months [TP53 functions as a tumor suppressor gene and the majority of TP53 mutations are missense in nature and associated with dominant negative function [In AML, the 2 months ,40. TP53function .TP53 mutations in myeloid malignancies drive clonal selection through a dominant-negative effect without gain-of-function activities, several novel small molecules to reactivate missense-mutant p53 protein have been investigated to restore the wild-type function of p53  of azacitidine and eprenatopopt reported an ORR of 73%, with 50% achieving CR in the MDS cohort (n = 40) and an ORR of 64%, with 36% achieving CR in the oligoblastic AML cohort (n = 11) . The medApart from p53 reactivation, targeting immune evasion is another therapeutic strategy in TP53 mutated AML. CD47  is a key macrophage immune checkpoint and serves as an anti-phagocytic or “do not eat me” signal . CD47 isKMT2A gene is located on Ch 11q23.3, with >100 fusion partners identified to cause leukemogenesis. Fusion genes induce aberrant HOXA and MEIS expressions secondary to the recruitment of chromatin-associated complexes, including the enzymes menin and DOT1L, resulting in stem cell-like gene expression signatures ,50. RecuNovel drug discoveries including DOT1L inhibitors, bromodomain inhibitors, and menin inhibitors have demonstrated preclinical efficacy with ongoing validation in prospective clinical studies ,52,53. Tp = 0.0004) and CR (15% vs. 2.3%) [The Aberrant Hedgehog (Hh) signalling pathway affects the proliferation of leukaemia stem cells, and its upregulation results in chemo-resistance in AML cell lines . This pas. 2.3%) . This sts. 2.3%) . More roImmunotherapy, including alloHSCT and donor lymphocyte infusion, has been used in the treatment of AML for many years, with T cells considered to be the major contributor to the success of this therapy . HoweverADCs consist of three main components: a monoclonal antibody (mAb) that recognizes an antigen target on tumor cells; a cytotoxic molecule, often referred to as a payload, and a chemical linker that connects the mAb and payload . They arp = 0.009) [CD33 is a transmembrane receptor expressed by committed myeloid cells, including the majority of leukemic blasts but not normal hematopoietic stem cells . Hence, = 0.009) . Overall= 0.009) . The add= 0.009) ,67. HoweBispecific antibodies are recombinant protein constructs that engage T cells through CD3 and bind to tumor-associated antigen, typically with a higher affinity. In AML, potential therapeutic targets are lineage-restricted antigens including CD33, CD123, CLL-1, and FLT3, for which early phase clinical trials are underway. CD123 (also known as interleukin 3 receptor and LSCs . It is aand LSCs ,69,70. Tand LSCs . It is oand LSCs . This isand LSCs ,73. The observed response of patients with PIF/ER AML to CD123-targeted immunotherapy is in accordance with the finding that an immune-infiltrated (IFN-otherapy . In compotherapy . Stepwisotherapy . In summotherapy ,75. Several potential biomarkers used to predict responders in CD123-targeted immunotherapy have been identified, including a higher CD123 receptor density and higher CD123 mRNA using gene expression profiling in patients with PIF/ER . Future T-cell-directed therapy using bispecific antibodies is a promising immunologic approach in the treatment of AML. However, the most suitable and appropriate epitopes for limiting on-target off-tumor toxicity in AML are yet to be identified. One of the proposed ways to optimize the success of bispecific antibodies in AML is to test it earlier in the treatment sequence—for instance, in the first salvage or in the MRD setting—to minimize T-cell exhaustion, as preserved T-cell function is critical for the activity of bispecific antibodies . To imprUnlike the successes of CAR-T cell therapy in B cell malignancies, the progress of the use of CAR-T cells in the treatment of AML has been hindered, mainly due to the lack of suitable targetable antigens and the poorly tolerated consequences of complete myeloid progeny ablation ,77. MostThe powerful diagnostic technologies created in recent years as part of the therapeutic revolution have transformed the paradigm of AML treatment, with increasing focus now placed on precision medicine approaches. Targeting the disease heterogeneity of AML has provided a more rational therapeutic approach compared to the ‘one-size-fits-all’ approach using conventional chemotherapy. It is likely that, in the long term, targeted agents will be used in combination with chemotherapy to eliminate measurable residual disease and for maintenance treatment. The potential areas of focus for future research in AML therapy are summarized in


Myelodysplastic Syndromes (MDSs) affect the elderly and can progress to Acute Myeloid Leukemia (AML). Epigenetic alterations including DNA methylation and chromatin modification may contribute to the initiation and progression of these malignancies. DNA hypomethylating agents such as decitabine and azacitidine are used as therapeutic treatments and have shown to promote expression of genes involved in tumor suppression, apoptosis, and immune response. Another anti-cancer drug, the proteasome inhibitor bortezomib, is used as a chemotherapeutic treatment for multiple myeloma (MM). Phase III clinical trials of decitabine and azacitidine used alone and in combination with other chemotherapeutics demonstrated their capacity to treat hematological malignancies and prolong the survival of MDS and AML patients. Although phase III clinical trials examining bortezomib’s role in MDS and AML patients are limited, its underlying mechanisms in MM highlight its potential as a chemotherapeutic for such malignancies. Further research is needed to better understand how the epigenetic mechanisms mediated by these chemotherapeutic agents and their targeted gene networks are associated with the development and progression of MDS into AML. This review discusses the mechanisms by which decitabine, azacitidine, and bortezomib alter epigenetic programs and their results from phase III clinical trials. Myelodysplastic syndrome (MDS) is a hematological malignancy characterized by aberrant hematopoiesis and most commonly affects the elderly . From 20The last decade of research has characterized various epigenetic and genetic abnormalities linked to both MDS and AML, resulting in impaired genetic machinery and hematopoietic stem cell function, all of which are more common in the aged population ,7. More Decitabine was first synthesized in 1964  as a cyt2 intravenously for 5 days . T. T32]. TThis proteasome inhibitory action is exemplified in bortezomib’s effect on multiple myeloma (MM) cells. MM cells overproduce immunoglobulin and cytokine proteins to help maintain contact with bone marrow stroma and facilitate their survival; this adhesion-related survival mechanism is known as cell adhesion-mediated drug resistance and is a quintessential characteristic of MM cells. However, this overproduction can result in inappropriate accumulation of misfolded and functional proteins in the ER and cytosol; therefore, MM cells rely heavily on protein degradation systems to compensate for increased ER activity and stress ,35. LossXL, A1, cIAP, XIAP, FLIP)  C 35]. B. BXL, A1P, FLIP) C 42]. I. IXL, A1P, FLIP) C 124]. . XL, A1,Bortezomib has also been shown to downregulate the expression of several proteins involved in the protective cellular response to genotoxic stress . GenotoxMore recently, researchers have uncovered connections between bortezomib and its effect on the epigenome, specifically RNA interference mechanisms. A study looking to uncover new molecular mechanisms of bortezomib found 719 genes and 28 miRNAs downregulated, and 319 genes and 61 miRNAs upregulated, in neuroblastoma cells treated with the drug . MicroRNThe mechanism by which bortezomib activates CEBPD remains unclear. CEBPD is involved in regulating hematopoietic tissue homeostasis, as several members of the CEBP family are expressed during myeloid development . As a tuCell culture studies have found bortezomib can induce apoptosis in both hematological cancers and solid tumor malignancies , includiBortezomib has shown promise in vivo as well; in xenotransplant mouse models of human AML, mice receiving bortezomib and volasertib displayed prolonged survival and better disease control than mice receiving volasertib alone . Bortezo2 of bortezomib per day on days 1, 4, 8, 11, 22, 25, 29, and 32 on cycles 1–4 and days 1, 8, 22, 29 during cycles 5–9, alongside melphalan 9 mg/m2 and prednisone 60 mg/m2 per day of days 1–4 each cycle), or MP alone [Most recent phase III clinical trials involving bortezomib and hematological malignancies examine the drug’s effect in multiple myeloma patients, specifically when used in tandem with other chemotherapeutics, steroids, and antibody treatments. Of the several phase III clinical trials underway, only a few currently have results published and/or reported. One phase III trial, completed and published in February 2013, sought to compare the effect of Velcade (bortezomib), melphalan and prednisone in patients with previously untreated multiple myeloma; this trial was particularly interested in exploring secondary malignancy risk in MM patients post-treatment. Patients received nine 6-week cycles of VMP  induce global hypomethylation? Or do they target specific gene promoters unique to the development and progression of MDS, hematological malignancies, and other cancers? A 2011 study looking for such consistencies found that decitabine and azacitidine induced non-random demethylation patterns at specific loci in human colon cancer (HCT116) and leukemic cell lines (HL60) . AdditioINK4b, E-cadherin and MYOD1 [It is already known that decitabine demethylates tumor-suppressor genes such as p15nd MYOD1 ,51, makind MYOD1 ,70, it mnd MYOD1 ; DNMT3A,nd MYOD1 ) and mutnd MYOD1 . IdentifAlthough mutations in epigenetic factors like DNMT1/3a and TET2 are common in hematological malignancies, there are mixed findings when it comes to whether such genetic alterations are principal contributors to secondary resistances ,164,165.Recent success with cedazuridine, an oral formulation of decitabine, points to more accessible HMA treatments in the future . Within Xenopus laevis oocyte revealed the azanucleoside to be transported by all seven human nucleoside transporters (hNTs)  with hCNT3 with the highest affinity for direct uptake of azacitidine [The molecular mechanism of nucleoside transporters in cell membranes responsible for the uptake of natural and synthetic nucleotides is a cardinal area of focus for future research. Cellular uptake is regulated by the following two transporter families: human equilibrative nucleoside transporters (hENTs) and human concentrative nucleoside transporters (hCNTs). Radiolabeled azacitidine in citidine . Furthercitidine . As suchcitidine ,169. Thecitidine .This information can be used to deduce nucleoside drug potency levels and response since specific nucleoside transporters can serve as biomarkers for effective drug entry and subsequent action . This wi2 daily for 7 days. Further treatment cycles can be administered upon monitoring hematological response, and the additional cycles can be repeated every 4 weeks with a maximum increase to 100 mg/m2. The oral formulation CC-486 was approved by the FDA as a maintenance therapy for newly diagnosed AML patients who responded to front-line chemotherapy due to the results of the QUAZAR AML-001 study. Oral azacitidine (CC-486) in combination with the best supportive care is currently being evaluated for efficacy and safety over the injectable azacitidine  in a phase 2 pilot study. An oral formulation is clinically desired due to the opportunity of delivering at lower systemic doses over a more prolonged schedule. Moreover, the route of oral administration will alleviate the injection site reactions associated with vidaza.Azacitidine is an FDA approved treatment for patients with MDS, CMML, and AML with up to 30% blasts, and it is a preferred treatment option for patients who are ineligible for standard induction chemotherapy . ClinicaCurrently, there are no effective treatments to suppress leukemic transformation and increase survival outcomes in patients with higher-risk MDS. Allogenic haemopoietic stem-cell transplantation offer the only potentially curative treatment plan for this group of patients. As such, DNMTis such as azacitidine offer an alternative treatment that provides significant overall survival benefits for those with MDS. In a randomized controlled phase III (CALGB) trial, azacitidine treatment yielded a significantly higher response rate, improved quality of life, reduced risk of leukemic transformation, and increased survival compared to that of supportive care for high-risk MDS patients . Conventp < 0.001) for the azacitidine-placebo control group for previously untreated patients with AML [Individual azacitidine treatment has proven efficacious, but current research is focused on combinations of histone deacetylase inhibitor (HDACi) drugs with azacitidine to increase the overall safety and effectiveness of treatments. HDACs act on specific histone protein tails and selectively alter gene transcription by changing chromatin status to regulate gene transcription . The epiwith AML . Furtherwith AML . For inswith AML . As thesMoving to proteasome inhibitors, recent clinical trials have already explored bortezomib’s apoptotic induction abilities in various types of cancer, including hematological malignancies ,142,143.atgs), specifically Atg3, are downregulated in conditions like MDS; after overexpressing Atg3 in leukemic cell lines, researchers found malignant cells more sensitive to bortezomib-induced autophagy [Further understanding of bortezomib’s specific mechanism of action can help clarify its potential role in possibly slowing MDS/AML disease development and progression. Bortezomib’s proteasome binding abilities have been well recorded and its relationship to the NF-kB pathway has been thoroughly explored. Overactivation related mutations in NF-kB signaling molecules have been identified in the pathogenesis of MM ; overactutophagy . This hiutophagy ,117.Fas receptor; however, when the disease progressed to AML the FAS gene was epigenetically repressed in 60% of patients [FAS gene silencing [Researchers are still working to define mechanisms by which the drug influences transcript levels of cellular stress response genes and other genes related to malignancy. A study looking at transcription signatures in MDS and AML identified a relationship between NF-kB pathway inhibition and TNF receptor 6 (CD95/Fas) expression, an important component in initiating apoptosis . Low-rispatients . Interesilencing ; given bilencing ,149.Recent research is also seeking to define concrete relationships between bortezomib and the epigenome, as the drug has been implicated in RNA interference pathways and DNA methylation regulation. Researchers have continued to explore bortezomib’s influence over the transcription of genes associated with DNA damage repair and other genotoxic stress responses, specifically in cases where malignant cells display resistance to the drug. A recent study found MM cells resistant to bortezomib showed significant downregulation of the SENP2 gene; this gene encodes for a serine-protease heavily involved (de)sumoylation . SumoylaGiven its role in the pathogenesis of hematological malignancies like AML, and its responsiveness to bortezomib, CEBPD could also be a reasonable target for increasing bortezomib’s effectiveness in vivo. As mentioned above, CEBPD is methylated and silenced in AML patients. Therefore, hypomethylating agents such as 5-Aza-deoxycytidine could offer a novel approach to bolster bortezomib’s effect in hematological malignancies, in combination with pre-existing treatments . Given tThese findings are promising as researchers look to identify possible targets for eliminating bortezomib resistance in patients who have acquired or intrinsic resistance to the drug, and as more proteasome inhibitors are developed in order to overcome such resistances. Since the examination of proteasome inhibition as cancer treatments, researchers have also become interested in other aspects of protein ubiquitination as it relates to cancer development. Recent research has focused on deubiquitinating enzymes (DUBs) and how they can be selectively inhibited to create effective chemotherapeutics . These pCollectively, decitabine, azacitidine and bortezomib can remodel the epigenome of cancer cells to prevent their proliferation and are currently used as chemotherapeutic treatments in the clinic. However, their specific mechanisms of action remain under investigation, and they are being used as a basic research tool to elucidate new epigenetic programs associated with MDS and its potential to transition into AML.


MLL-rearrangement, NPM1 mutation, and NUP98 fusion genes. Potent menin-MLL inhibitors have demonstrated promising anti-leukemic effects in preclinical studies. The downstream signaling molecule SYK represents an additional target. However, the TP53 mutation continues to remain a challenge. While the p53 stabilizer APR-246 in combination with azacitidine failed to show superiority compared to azacitidine monotherapy in a phase 3 trial, next-generation p53 stabilizers are now under development. Among a number of non-canonical approaches to TP53-mutated AML, the anti-CD47 antibody magrolimab in combination with azacitidine showed promising results in a phase 1b trial. Further, the efficacy was somewhat better in patients with the TP53 mutation. Although clinical evidence has not been accumulated sufficiently, targeting activating KIT mutations and RAS pathway-related molecules can be a future therapeutic strategy.We describe recent updates of existing molecular-targeting agents and emerging novel gene-specific strategies. FLT3 and IDH inhibitors are being tested in combination with conventional chemotherapy for both medically fit patients and patients who are ineligible for intensive therapy. FLT3 inhibitors combined with non-cytotoxic agents, such as BCL-2 inhibitors, have potential therapeutic applicability. The menin-MLL complex pathway is an emerging therapeutic target. The pathway accounts for the leukemogenesis in AML with  FLT3-mutated AML. For patients with iso-citrate dehydrogenase (IDH)-1 or -2 mutation, specific IDH inhibitors ivosidenib and enasidenib are generally well-tolerated and expects complete remission (CR) rates of 30–40% as monotherapy . De. DeDNMT3. 22.4%) . The nex. 22.4%) .TP53 mutation) who were ineligible for intensive therapy in a phase 1b study. Although the immune escape mechanism through CD47 would not be specific for TP53-mutant AML, the CR/CRh rates were somewhat higher (67%) in TP53-mutated AML in this study  on macrophages and dendritic cells and results in inhibition of phagocytosis . Increasis study  96]..TP53 mutKIT mutations are associated with worse prognosis in CBF-AML [KIT gene, commonly on the exon 8 and 17, are frequently found in gastrointestinal stromal tumor (GIST) and systemic mastocytosis, as well as CBF-AML. A number of tyrosine kinase inhibitors (TKIs) targeting KIT such as imatinib, sunitinib sorafenib, regorafenib, dasatinib, nilotinib, ponatinib, and midostaurin are suggested to be a potential therapeutic agent for KIT-mutated tumors [KIT A-loop mutations  render resistance to imatinib and sunitinib in patients with GIST, some agents  remain effective for such resistant cases [KIT-mutated AML [KIT protein, also known as CD117, is expressed in 70% of AML as well as normal hematopoietic progenitor cells. KIT mutations are seen in less than 10% of all subsets of AML and in approximately 30% of the core-binding factor (CBF) AML ,98,99. K CBF-AML ,100. KIT CBF-AML ,102. Actd tumors . A part nt cases ,105. Altated AML ,107.KIT D816V-mutated CBF-AML which relapsed after the first-line decitabine monotherapy [KIT-mutated CBF-AML.Kampa-Schittenhelm and colleagues reported 77-years-old patient with otherapy . Upon caFLT3-ITD plus FLT3-TKD  which render resistance to FLT3 inhibition [Recently, a heat shock protein (HSP)-90 inhibitor pimitespib (TAS-116) showed significant improvement of PFS in patients with GIST resistant to imatinib, sunitinib, and regorafenib in the phase 3 CHAPTER-GIST-301 trial . HSP-90 hibition . HSP90 iRAS genes  are one of most popular proto-oncogenes and frequently mutated in human cancer, affecting the mitogen-activated protein kinase (MAPK), phosphatidylinositol-3-kinase (PI3K), and Ras-like  small GTPase  signaling pathways. Activating mutations of NRAS and KRAS are found in approximately 15–25% of patients with AML [RAS genes commonly co-mutated with RAS-regulating genes  and/or signaling receptor genes that rely on RAS-involving pathways  [FLT3-mutated AML. McMahon and colleagues sequentially analyzed clinical samples from patients with FLT3-mutated AML which progressed on gilteritinib treatment [NRAS and/or KRAS mutations after progression on FLT3 inhibition. Almost complete substation of RAS-mutated subclones for the original RAS-wild FLT3-mutated clone was observed in a few cases. In addition, leukemic cell lines with both FLT3 and RAS mutations showed resistance to gilteritinib monotherapy, which was canceled by co-administration of a MEK inhibitor trametinib.RAS proteins have GTPase activity responding to growth factor receptor activation and play an essential role in cell proliferation ,113. RASwith AML . RAS genand KIT) ,114. Recreatment . Among fNRAS-driven AML cell line relapsed after genetic suppression on NRAS (NRI-AML) was devoid of MAPK and PI3K signaling but dependent on the cyclin-dependent kinase (CDK) 5-mediated activation of RalGEF signaling. In in vivo experiments using PDX mouse model, a CDK inhibitor dinaciclib which inhibits CDK5-mediated RalGEF signaling induced leukemic cell apoptosis and prevented evolution of NRI-AML, suggesting that RalGEF signaling is involved in escaping mechanism of RAS-mutated AML and can be a potential therapeutic target.Preclinical experiments have shown that inhibition of both MAPK and PI3K pathway did not cause significant leukemic cell death but led to static effects instead ,117. SimG12C-specific inhibitor sotorasib demonstrated 7–32% of overall response rates in advanced solid tumors  with KRAS G12C mutation in a phase 1 study [KRAS inhibition have not been proved in hematologic malignancies neither clinically nor preclinically. However, given that activating RAS mutations are associated with worse prognosis and drug resistance in AML, KRAS inhibition might be included in future possibility.The KRAS 1 study  and now MLL rearrangements, NPM1 mutations, and NUP98 fusion genes. SYK inhibitors can be another strategy for AML with menin-related genomic alterations. For AML with TP53 mutation, p53 stabilizers and anti-CD47 antibodies can be a candidate for gene-specific therapeutic agents. Although clinical evidence has not been sufficient yet, anti-KIT strategy such as HSP-90 inhibitors and RAS-pathway interference might be a future approach for AML treatment. Mutations of FLT3, MLL, and TP53 are considered to be unfavorable prognostic factors so far. However, the development of targeted therapies with or without conventional therapy may improve or perhaps reverse the current situation. Aberrant oncogenic mechanisms depending on specific genomic abnormalities can be an ideal therapeutic target, though preventing or overcoming treatment resistance remains a challenge. Determining genomic features and following case-specific molecular-targeted strategy should be future therapeutic strategy in AML. Results from current developments and ongoing trials are anticipated.Molecular-targeted therapies, especially for specific genomic abnormalities, have had significant impacts on AML treatment. FLT3 inhibitors and IDH inhibitors have proved its efficacy in clinical trials and now available in practice. In addition, menin-MLL inhibitors have shown anti-leukemic effects in preclinical studies of AML with menin-related genomic alterations such as


Immunotherapy may be an attractive treatment option to increase survival, and to reduce treatment-related side effects, for children with acute myeloid leukemia (AML). While immunotherapies have shown successes in many cancer types, the development and subsequent clinical implementation have proven difficult in pediatric AML. To expedite the development of immunotherapy, it will be crucial to understand which pediatric AML patients are likely to respond to immunotherapies. Emerging research in solid malignancies has shown that the number and phenotype of immune cells in the tumor microenvironment is predictive of response to several types of immunotherapies. Such a predictive model may also be applicable for AML and, thus, knowledge on the immune cells infiltrating the bone marrow environment is needed. Here, we discuss the current state of knowledge on these infiltrating immune cells in pediatric AML, as well as ongoing immunotherapy trials, and provide suggestions concerning the way forward.Immunotherapeutic agents may be an attractive option to further improve outcomes and to reduce treatment-related toxicity for pediatric AML. While improvements in outcome have been observed with immunotherapy in many cancer types, immunotherapy development and implementation into patient care for both adult and pediatric AML has been hampered by an incomplete understanding of the bone marrow environment and a paucity of tumor-specific antigens. Since only a minority of patients respond in most immunotherapy trials across different cancer types, it will be crucial to understand which children with AML are likely to respond to or may benefit from immunotherapies. Immune cell profiling efforts hold promise to answer this question, as illustrated by the development of predictive scores in solid cancers. Such information on the number and phenotype of immune cells during current treatment regimens will be pivotal to generate hypotheses on how and when to intervene with immunotherapy in pediatric AML. In this review, we discuss the current understanding of the number and phenotype of immune cells in the bone marrow in pediatric AML, ongoing immunotherapy trials and how comprehensive immune profiling efforts may pave the way for successful clinical trials . Acute myeloid leukemia (AML) is a heterogeneous blood cancer characterized by both aberrant proliferation and arrested differentiation of immature myeloid cells in the bone marrow . Due to To date, therapeutic options that make use of T-cell-mediated effects to eliminate residual leukemic cells, such as allo-SCT, have shown to evoke anti-AML immunity and support the use of immunotherapy in pediatric AML ,5,6,7. HUnfortunately, clinical translation from basic immunobiology to effective immunotherapy appears to be relatively slow for both adult and pediatric AML in comparison to other cancer types . For insThe results in these trials and, consequently, the relatively slow implementation of immunotherapy in patient care can be explained in part by an incomplete understanding of the microenvironment in which the leukemic cells reside. For AML, the alleged tumor microenvironment (TME) is the bone marrow and includes, next to tumor cells, immune and other normal hematopoietic, stromal, and endothelial cells . During Extensive characterization of immune cells in the TME is crucial to tackle these challenges and to design successful immunotherapy trials ,35,36,37Immune cells and immune-related factors such as soluble molecules play a major role in cancer development and progression . For exaAs only a minority of patients respond in most immunotherapy trials, many studies have focused on elucidating factors that impact the response to immunotherapy. For instance, studies in both solid and hematological cancers have shown that patients with an immune-infiltrated or ‘hot’ TME have a relatively high probability of response to ICIs ,25,26,27Immunotherapy studies in adult AML have shown similar observations ,27,28. Fn = 273) had a median combined proportion of T- and NK-cells of nearly 25% out of all living cells in the bone marrow at diagnosis [n = 1585) in the same study [n = 102) showed a slightly higher immune cell abundance in comparison to children with AML [Since the prognostic and predictive potential of immune cells in the bone marrow of AML is increasingly recognized, we summarized the available information on immune cell abundance and phenotype in the TME for children with AML at diagnosis. To date, few studies have examined these parameters on a protein level. However, recent microarray- and RNA-sequencing based studies have provided valuable information ,45. Thesme study . Only adwith AML . This mawith AML .n = 28) revealed a significantly lower fraction of T-cells out of all mononuclear cells in the bone marrow (4%) [In contrast, a small study that used flow cytometry in children with AML (row (4%) . Phenotyrow (4%) . These crow (4%) . Howeverrow (4%) . Compararow (4%) .n = 151) and pediatric AML , T-cells in infant AML presented relatively low levels of clonal expansion in comparison to pediatric and adult AML [In addition, clonotype diversity of T- and BCRs can inform whether patients can generate an effective antigen-specific anti-tumor immune response, for instance after immunotherapy . High leTaken together, microarray- and RNA-sequencing based studies have provided valuable insights into the immune cell abundance in the bone marrow of children with AML. Apart from adults with AML ≥ 60 years, children with AML appeared to have similar levels of T- and NK-cell abundance in the bone marrow at diagnosis in comparison to adults with AML. Moreover, the high proportions of T- and NK-cells in the bone marrow of children with AML in these studies look promising and anticipate that children with AML may benefit from immunotherapy in the future. Studies that evaluated the immune cell abundance and phenotype on the protein level at diagnosis are scarce in children with AML. Although the microarray- and RNA-sequencing based methods in the discussed studies have been validated and were found to robustly estimate immune cell levels as measured by protein-based assays such as flow cytometry, functional studies are needed to deepen our understanding of the phenotype of immune cells in the bone marrow in children with AML.Unfortunately, the abundance and phenotype of immune cells in the TME of relapsed and refractory pediatric AML has not been elucidated yet. In addition, no trials on immunotherapy drugs for relapsed and refractory pediatric AML have been published to date. For adults, most data on the immune cell abundance in relapsed and refractory AML stem from the studies that measured these parameters before treatment in immunotherapy trials ,30,31. FOver the years, the use of immunotherapy during and after conventional therapy has gained interest in the field of AML due to the relatively low leukemic burden in these settings . AccordiTo date, few clinical trials have assessed the role of immunotherapy as maintenance therapy for children with AML ,61,62. Tn = 22) [n = 72) reported equal results in terms of T-cell activation after induction therapy in responders, while non-responders showed relatively high levels of dysfunction in comparison to the pretreatment setting [For adult AML, these data are also scarce. As mentioned above, one study revealed that the RNA-sequencing based immune cell abundance was significantly higher in complete remission in comparison to diagnosis in adults with AML (n = 22) . Furthern = 22) . Another setting . However setting . For exa setting ,66,67.Collectively, the use of immunotherapy during and/or after conventional treatment has the potential to support the eradication of MRD and consequently prevent relapsed disease. However, data on the immune cell abundance and phenotype is limited for both adult and pediatric AML. Since studies across a spectrum of cancers have observed plasticity of immune cell numbers and their phenotypes before- and after conventional treatment, delineating changes in bone marrow immune cell abundance and phenotype for pediatric AML will likely be important for the selection of suitable treatment time points for immunotherapy intervention ,70,71,72Next to the fact that leukemic cells compete with immune cells for the bone marrow niche occupancy, emerging research suggests that certain genetic alterations in the AML cells may as well impact the immune cell abundance in the TME ,73,74. FOver the last decade, the field of immuno-oncology has focused heavily on understanding the immune cells in the TME. However, the immune system is coordinated across tissues and communication with the peripheral blood (PB) is essential for immune function in the TME . RecentlIn pediatric AML, only one study that used flow cytometry examined the presence of immune cells in the PB . This stStudies that have assessed the immune reconstitution in the PB after allo-SCT might also be informative of whether immunotherapy is likely to be effective in this setting. For instance, T-cell reconstitution was shown to be excellent within one to two months after unrelated cord blood donor allo-SCT in pediatric AML in the absence of anti-thymocyte globulin as part of the conditioning regimen ,82,83,84Taken together, the research community has only just begun to recognize the role of immune cells in the PB for achieving responses to immunotherapy. However, results show that the immune response at the time of diagnosis and upon treatment should encompass immune cells in both the TME and the PB.For antigen-directed immunotherapies such as CAR T-cells and bispecific antibodies, the expression of the target antigen on AML blasts is crucial. An ideal target should be strongly expressed on the surface of AML blasts but preferably not or lowly expressed on normal cells. A recent review has summarized the current knowledge on different classes of target antigens in AML, and we refer to that article for a broad overview of this topic .n = 36) using RNA-sequencing and flow cytometry and identified distinct expression patterns data in comparison to what has been reported in adult AML [n = 139 and n = 316, respectively) reported no differences in expression levels [The identification of target antigens has been challenging in both pediatric and adult AML. In addition, target antigen selection for pediatric AML has been largely based on studies from adult patients due to a lack of studies in the pediatric population . Of intedult AML . In partn levels .Moreover, Willier et al. identified CD33 and CLEC12A (CLL1) as a promising combination of targets for immunotherapy covering about 60% of pediatric AML patients . Dual taA complete overview of currently ongoing and recruiting clinical immunotherapy trials for adult AML is presented elsewhere, whereas we focus on immunotherapy trials for pediatric AML in this review ,19,87 F; Table 2® for colorectal cancer and the tumor inflammation score for multiple cancer types [Immune profiling is the overarching term used for quantification and/or characterization of the immune response. Studies in adults with cancer showed that immune profiling data have substantial predictive value for immunotherapy and conventional treatments, illustrated by the development of the Immunoscoreer types ,25,94,95er types . Therefoer types . In thiser types . HoweverAccordingly, flow or mass cytometry approaches on bone marrow aspirates and PB or immunohistochemistry on bone biopsies could be used to assess and characterize the immune infiltration of specific immune cell subsets. Importantly, these analyses should include functional and exhaustion markers, such as PD-1 and CTLA4, to identify dysfunctional or exhausted immune cells that may benefit from ICIs. For this purpose, single-cell RNA sequencing could be used to dissect the heterogeneity and dynamics of immune cell subsets in detail . FurtherThe available evidence about the immune response in the TME and the PB is limited in pediatric AML. Nonetheless, current data suggest that the T- and NK-cell abundance in pediatric AML is comparable to adult AML at diagnosis ,45. As aAs the level of immune infiltration was shown to be inversely associated with the leukemic burden, there is an increased interest in the use of immunotherapy for the eradication of MRD ,57,58,59In conclusion, to allow children with AML to benefit from immunotherapy, major immune profiling efforts are needed. These efforts should focus on the systemic and TME immune profile during conventional therapy, as well as in clinical immunotherapy trials. Moreover, future immunotherapy trials should enrich for potential responders to prevent trials with unnecessarily low response rates in a disease with a limited number of patients. These and immunological data-guided preclinical efforts will improve our understanding of why children with AML develop a certain immune profile and which patients are likely to respond to immunotherapy. Importantly, in a rare disease such as pediatric AML, international collaboration will be key to expedite the development and implementation of immunotherapy into patient care.