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Acute myeloid leukemia (AML) has become a worldwide malignant cancer. We intended to investigate the critical roles and mechanism underlying homo sapiens circular RNA 0003602 (hsa_circ_0003602) in AML progression, especially in tumor cell proliferation, migration, invasion, and apoptosis. Real-time PCR was applied to identify the differential expression of hsa_circ_0003602 and miR-502-5p in AML bone marrow tissues and cell lines. In addition, western blot analysis was employed to determine the levels insulin-like growth factor 1 receptor (IGF1R) protein. The biological behaviors were assessed by CCK-8 cell viability assay, flow cytometry assay for apoptosis detection, and Transwell migration and invasion assay. The relationships between target miRNA and downstream mRNA were investigated by bioinformatics, luciferase reporter assay, and biotin-labeled RNA pull-down assay. Hsa_circ_0003602 was upregulated and predicted poor survival in AML. Knockdown of hsa_circ_0003602 in AML cell lines induced the inhibition of proliferation, migration, and invasion and caused apoptosis. Hsa_circ_0003602 sequestered miR-502-5p by functioning as a competitive endogenous RNA (ceRNA), thereby regulating IGF1R expression. Hsa_circ_0003602 acted as a tumor promoter in AML via miR-502-5p/IGF1R axis. Our study provides evidence that hsa_circ_0003602, miR-502-5p, and IGF1R might form a regulatory axis to affect the carcinogenicity of AML cells and provide potential targets for the treatment of AML., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Despite recent approval of targeted drugs for acute myeloid leukemia (AML) therapy, chemotherapy with cytosine arabinoside and anthracyclines remains an important pillar of treatment. Both primary and secondary resistance are frequent and associated with poor survival, yet the underlying molecular mechanisms are incompletely understood. In previous work, we identified genes deregulated between diagnosis and relapse of AML, corresponding to therapy naïve and resistant states, respectively. Among them was MTSS1, whose downregulation is known to enhance aggressiveness of solid tumors. Here we show that low MTSS1 expression at diagnosis was associated with a poor prognosis in AML. MTSS1 expression was regulated by promoter methylation, and reduced by cytosine arabinoside and the anthracycline daunorubicin. Experimental downregulation of MTSS1 affected the expression of numerous genes. It induced the DNA damage response kinase WEE1, and rendered human AML cell lines more resistant to cytosine arabinoside, daunorubicin, and other anti-cancer drugs. Mtss1 knockdown in murine MLL-AF9-driven AML substantially decreased disease latency, and increased leukemic burden and ex vivo chemotherapy resistance. In summary, low MTSS1 expression represents a novel factor contributing to disease aggressiveness, therapy resistance, and poor outcome in AML., (© 2021. The Author(s).)

Despite recent advances in therapies including immunotherapy, patients with acute myeloid leukaemia (AML) still experience relatively poor survival rates. The Inhibition of Apoptosis (IAP) family member, survivin, also known by its gene and protein name, Baculoviral IAP Repeat Containing 5 (BIRC5), remains one of the most frequently expressed antigens across AML subtypes. To better understand its potential to act as a target for immunotherapy and a biomarker for AML survival, we examined the protein and pathways that BIRC5 interacts with using the Kyoto Encyclopedia of Genes and Genomes (KEGG), search tool for recurring instances of neighbouring genes (STRING), WEB-based Gene Set Analysis Toolkit, Bloodspot and performed a comprehensive literature review. We then analysed data from gene expression studies. These included 312 AML samples in the Microarray Innovations In Leukemia (MILE) dataset. We found a trend between above median levels of BIRC5 being associated with improved overall survival (OS) but this did not reach statistical significance ( p = 0.077, Log-Rank). There was some evidence of a beneficial effect in adjusted analyses where above median levels of BIRC5 were shown to be associated with improved OS ( p = 0.001) including in Core Binding Factor (CBF) patients ( p = 0.03). Above median levels of BIRC5 transcript were associated with improved relapse free survival ( p < 0.0001). Utilisation of a second large cDNA microarray dataset including 306 AML cases, again showed no correlation between BIRC5 levels and OS, but high expression levels of BIRC5 correlated with worse survival in inv(16) patients ( p = 0.077) which was highly significant when datasets A and B were combined ( p = 0.001). In addition, decreased BIRC5 expression was associated with better clinical outcome ( p = 0.004) in AML patients exhibiting CBF mainly due to patients with inv(16) ( p = 0.007). This study has shown that BIRC5 expression plays a role in the survival of AML patients, this association is not apparent when we examine CBF patients as a cohort, but when those with inv(16) independently indicating that those patients with inv(16) would provide interesting candidates for immunotherapies that target BIRC5.

Genomic imprinting is a form of epigenetic regulation and imprinted genes are silenced in a parental-specific manner. Imprinting is associated with various human diseases and cancers, but its roles in leukemogenesis remains elusive. In this study, the expression of a panel of 16 human imprinted genes was investigated using real-time quantitative polymerase chain reaction and 8 of them were further validated in 114 patients newly diagnosed with cytogenetically abnormal-acute myeloid leukemia (CA-AML) and 85 healthy subjects. Our results demonstrated upregulated expression of 8 imprinted genes (C15orf2, COPG2, H19, IGF2, PEG3-AS1, PRIM2, SLC22A3 and ZNF215) was observed in patients with CA-AML (p < 0.001). Patients' survival days were negatively correlated with the expression levels of H19 (p = 0.024), PGE3-AS1 (p = 0.038), and ZNF215 (p = 0.012). Multivariate logistic regression analysis further revealed the expression level ZNF215 can be used as a predictor for five-year survival for patients with CA-AML (p = 0.009) with a hazard ratio of 0.870 (95.0% confident interval: 0.784-0.965). Our results demonstrated that loss of imprinting of imprinted genes is critical for the leukemogenesis of AML under CA condition, and loss of ZNF215 imprinting is associated with poor five-year survival of patients with CA-AML., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The long-noncoding RNA colorectal neoplasia differentially expressed (CRNDE) gene has been considered to be crucial in tumor malignancy. Although CRNDE is highly expressed in acute myeloid leukemia (AML), its mechanism of action remains unknown. In this study, GEPIA and qRT-PCR were performed to confirm the expression of CRNDE in AML samples and cell lines, respectively. CRNDE shRNA vectors were transfected to explore the biological functions of CRNDE. The cell proliferation was assessed by the CCK8 assay, while apoptosis and cell cycle distribution were measured by flow cytometry and Western blotting. The results showed that CRNDE was overexpressed in both AML samples and cell lines. CRNDE silencing inhibited proliferation and increased apoptotic rate and cell cycle arrest of KG-1a cells. The luciferase reporter assay coupled with RIP assay revealed that CRNDE act as a ceRNA. Rescue assays demonstrated that the effects of CRNDE silencing could be reversed by miR-136-5p inhibitors. In conclusion, our results expound that the CRNDE/miR-136-5p/MCM5 axis modulates cell progression and provide a new regulatory network of CRNDE in KG-1a cells., (© 2021. The Author(s).)

The exosomes are involved in intercellular communication via RNA trafficking in human diseases. Hsa&#95;circ&#95;0009910 (circ&#95;0009910) is a novel leukemia-related circular RNA. However, the mechanism of circ&#95;0009910 in acute myeloid leukemia (AML) cell-to-cell communication remained obscure. Expression of circ&#95;0009910, miRNA (miR)-5195-3p and growth factor receptor-bound protein 10 (GRB10) was detected by quantitative real-time polymerase chain reaction and Western blotting. A stable cell coculture model was established and functional experiment was performed using Cell Counting Kit-8 assay, flow cytometry, and Western blotting. The interaction among circ&#95;0009910, miR-5195-3p and GRB10 was confirmed by dual-luciferase reporter assay and RNA immunoprecipitation. As a result, circ&#95;0009910 was upregulated in AML bone marrows and cells (HL-60 and MOLM-13), even higher in AML cells-derived exosomes. Functionally, blocking circ&#95;0009910 via small interfering RNA (siRNA) suppressed cell proliferation and cell cycle progression, but facilitated apoptosis rate of HL-60 and MOLM-13 cells, accompanied with lower B-cell lymphoma 2 (Bcl-2) level and higher Bcl-2-associated X protein (Bax) level. circ&#95;0009910 shuttled via exosomes negatively regulated miR-5195-3p expression by target binding. Furthermore, circ&#95;0009910 knockdown via exosomes and miR-5195-3p overexpression via mimic resulted in similar results of circ&#95;0009910 siRNA in proliferation, apoptosis and cell cycle progression of AML cells. Meanwhile, the role of circ&#95;0009910 knockdown in AML cells was partially reversed by miR-5195-3p deletion, and restoring GRB10 could abrogate miR-5195-3p effect as well. Notably, GRB10 was a downstream target of miR-5195-3p. circ&#95;0009910-containing exosomes mediated proliferation, apoptosis and cell cycle progression of AML cells partially through miR-5195-3p/GRB10 axis., (© 2021 John Wiley & Sons Ltd.)

It was previously reported that PRR34-AS1 was overexpressed in some solid tumors. PRR34-AS1 promoter was shown to have a differential methylation region (DMR), and was hypomethylated in acute myeloid leukemia (AML). Therefore, the present study used real-time quantitative PCR (RQ-PCR) to explore the expression characteristics of PRR34-AS1 in AML. In addition, the correlation between the expression of PRR34-AS1 and clinical prognosis of AML was determined. The findings of this study indicated that high PRR34-AS1 expression was bound up with shorter overall survival (OS) in AML patients (p = 0.002). Moreover, patients with high expression of PRR34-AS1 had significantly lower complete remission (CR) rate compared with those with low expression of PRR34-AS1 after induction chemotherapy. Furthermore, multivariate analysis confirmed that PRR34-AS1 expression was an independent factor affecting CR in whole-AML, non-APL-AML, and CN-AML patients (p = 0.032, 0.039, and 0.036, respectively). Methylation-specific PCR (MSP) and bisulfite sequencing PCR (BSP) were used to explore the methylation status of PRR34-AS1. PRR34-AS1 promoter showed a pattern of hypomethylation in AML patients compared with normal controls (p = 0.122). Notably, of whole-AML and non-APL-AML patients, PRR34-AS1 hypomethylated patients presented a significantly shorter OS than those with a hypermethylated PRR34-AS1 (p = 0.010 and 0.037, respectively). Multivariate analysis confirmed that the hypomethylation of PRR34-AS1 served as an independent prognostic indicator in both whole-cohort AML and non-APL-AML categories (p = 0.057 and 0.018, respectively). In summary, the findings of this study showed that abnormalities in PRR34-AS1 are associated with poor prognosis in AML. Therefore, monitoring this index may be important in the prognosis of AML and can provide information on effective chemotherapy against the disease., (© 2021 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Relapse is associated with therapy resistance and is a major cause of death in acute myeloid leukemia (AML). It is thought to result from the accretion of therapy-refractory leukemic stem cells. Genetic and transcriptional changes that are recurrently gained at relapse are likely to contribute to the increased stemness and decreased therapy responsiveness at this disease stage. Despite the recent approval of several targeted drugs, chemotherapy with cytosine arabinoside and anthracyclines is still the mainstay of AML therapy. Accordingly, a number of studies have investigated genetic and gene expression changes between diagnosis and relapse of patients subjected to such treatment. Genetic alterations recurrently acquired at relapse were identified, but were restricted to small proportions of patients, and their functional characterization is still largely pending. In contrast, the expression of a substantial number of genes was altered consistently between diagnosis and recurrence of AML. Recent studies corroborated the roles of the upregulation of SOCS2 and CALCRL and of the downregulation of MTSS1 and KDM6A in therapy resistance and/or stemness of AML. These findings spur the assumption that functional investigations of genes consistently altered at recurrence of AML have the potential to promote the development of novel targeted drugs that may help to improve the outcome of this currently often fatal disease., Competing Interests: Conflict of interest disclosure The authors declare no competing interests., (Copyright © 2021 ISEH -- Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Metabolic reprogramming is a common feature of many human cancers, including acute myeloid leukemia (AML). However, the upstream regulators that promote AML metabolic reprogramming and the benefits conferred to leukemia cells by these metabolic changes remain largely unknown. We report that the transcription factor ATF3 coordinates serine and nucleotide metabolism to maintain cell cycling, survival, and the differentiation blockade in AML. Analysis of mouse and human AML models demonstrate that ATF3 directly activates the transcription of genes encoding key enzymatic regulators of serine synthesis, one-carbon metabolism, and de novo purine and pyrimidine synthesis. Total steady-state polar metabolite and heavy isotope tracing analyses show that ATF3 inhibition reduces de novo serine synthesis, impedes the incorporation of serine-derived carbons into newly synthesized purines, and disrupts pyrimidine metabolism. Importantly, exogenous nucleotide supplementation mitigates the anti-leukemia effects of ATF3 inhibition. Together, these findings reveal the dependence of AML on ATF3-regulated serine and nucleotide metabolism., Competing Interests: Declaration of interests S.P. is currently an employee of Merck Research Laboratories and R. Gabbasov is currently an employee of Carisma Therapeutics. All other authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Aims: Acute myeloblastic leukemia (AML) is the most common type of acute leukemia in adults. Despite numerous treatment strategies including chemotherapy and radiotherapy, a large number of patients do not respond to treatment and experience relapse. The main problem of these patients is the development of resistance to anti-cancer drugs. Therefore, any endeavor to reduce drug resistance in these patients is of high priority. In general, several mechanisms such as changes in drug metabolic pathways, drug inactivation, drug target alterations and reduced drug accumulation in the cells contribute to drug resistance of cancer cells. In this context, evidence suggests that exosomes could reduce drug resistance by removing drugs from their parent cells. In the present study, we aimed to investigate the effects of exosome release inhibition on the resistance of U937 cells to PEGylated liposomal doxorubicin (PLD)., Main Methods: In order to find a suitable ABCG2 (ATP-binding cassette sub-family G member 2) transporter substrate, virtual screening was performed among a list of drugs used in leukemia and PLD was selected. U937 cells were treated with PLD with/without co-treatment with the exosome release inhibitor, GW4869. Released exosomes within different study groups were isolated and characterized to determine the differences between groups. Doxorubicin presence in the isolated exosomes was also measured by high performance liquid chromatography (HPLC) to confirm drug export through the exosomes. Finally, the effect of exosome inhibition on the cytotoxicity of PLD on U937 cells was determined using different cytotoxicity assays including the standard lactate dehydrogenase (LDH) release assay and the flow cytometric analysis of apoptotic and non-apoptotic cell death., Key Findings: GW4869 treatment caused a significant decrease in the exosome release of U937 cells compared to the untreated cells, as evidenced by the reduction of the protein content of the isolated exosomes (P<0.05). Co-treatment with GW4869 significantly increased cytotoxic cell death in the groups treated with 0.5 and 1 µM PLD, compared to the same groups without GW4869 co-treatment (P<0.05). Interestingly, co-treatment with GW4896 and 0.5 µM PLD was enough to induce the same cytotoxic effect as that of the sole 1 µM PLD group., Significance: Our findings showed that U937 cells increase their resistance against the cytotoxic effects of PLD through the exosome-mediated expelling of the drug. Inhibition of exosome release could prevent PLD efflux and consequently increase the vulnerability of the U937 cells to the cytotoxic effects of PLD. Our results along with prior studies indicate that the integration of exosome release inhibitors into the common PLD-containing chemotherapy regimens could significantly lower the required concentrations of the drug and consequently reduce its associated side effects. Further studies are warranted to identify clinically safe inhibitors and investigate their clinical efficacy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Hekmatirad, Moloudizargari, Moghadamnia, Kazemi, Mohammadnia-Afrouzi, Baeeri, Moradkhani and Asghari.)

Sialic acid-binding immunoglobulin-like lectin (Siglec)-15 has recently been identified as a critical tumour checkpoint, augmenting the expression and function of programmed death-ligand 1. We raised a monoclonal antibody, A9E8, specific for Siglec-15 using phage display. A9E8 stained myeloid leukaemia cell lines and peripheral cluster of differentiation (CD)33 + blasts and CD34 + leukaemia stem cells from patients with acute myeloid leukaemia (AML). By contrast, there was minimal expression on healthy donor leucocytes or CD34 + stem cells from non-AML donors, suggesting targeting Siglec-15 may have significant therapeutic advantages over its fellow Siglec CD33. After binding, A9E8 was rapidly internalised (half-life of 180 s) into K562 cells. Antibodies to Siglec-15 therefore hold therapeutic potential for AML treatment., (© 2021 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

Core-binding factor (CBF)-acute myeloid leukemia (AML) generally have a favorable prognosis. However, approximately 50% of patients experience disease relapse during or after post-remission therapy. Retrospective studies on autologous hematopoietic cell transplantation (AHCT) have shown improved survival with decreased relapse rate in CBF-AML. In this prospective study, we evaluate the outcomes of AHCT following high-dose cytarabine (HiDAC) consolidation in patients with CBF-AML in first complete remission (CR). Adult patients with CBF-AML achieving first CR after induction chemotherapy were eligible for the study. High-dose chemotherapy before AHCT included intravenous busulfan (3.2 mg/kg/day, days - 7 to - 5) and etoposide (400 mg/m 2 /day, days - 3 to - 2). Twenty-nine patients, 17 with t(8;21) and 12 with inv(16), underwent AHCT following 2 or 3 courses of HiDAC consolidation. The estimated 5-year overall and disease-free survival rates were between 89.0% and 82.5%, respectively. The cumulative incidences of relapse and non-relapse mortality were between 17.5% and 0%, respectively. Presence of measurable residual disease (MRD) before AHCT and KIT mutation were significantly associated with relapse after transplantation. In conclusion, the post-remission strategy of AHCT following HiDAC consolidation in CBF-AML was feasible and efficacious. Assays for MRD and KIT mutation may guide selection of patients who will benefit from AHCT in CBF-AML in first CR.

Tyrosine kinase inhibitors (TKIs) are standard therapies for chronic myeloid leukemia (CML) that can eradicate Ph-positive leukemic cells. However, disease control is not achievable in a minority of cases, most commonly due to evolution of TKI-resistant clones. There have also been rare cases of emergence of Ph-negative clones with other cytogenetic abnormalities, and, less commonly, development of Ph-negative acute myeloid leukemia (AML), whose molecular pathogenesis is largely unknown. Here we report molecular features of a patient with Ph + CML who developed Ph-negative AML after showing a major molecular response to dasatinib. A 55-year-old man was diagnosed with CML. He achieved a complete cytogenetic response three months after dasatinib therapy but developed AML with normal karyotype 1 year later. After receiving induction and consolidation chemotherapy for AML, the patient achieved complete remission with no evidence of CML under maintenance with bosutinib. Targeted sequencing of serial bone marrow samples identified mutations in IDH2 and NPM1 in the Ph-negative AML cells, which had not been detected in CML cells. These results suggest that Ph-negative AML in this patient originated from a preleukemic population, which might have expanded during or after the successful elimination of CML clones with TKI therapy.

Mitochondria not only are essential for cell metabolism and energy supply but are also engaged in calcium homeostasis and reactive oxygen species generation and play a key role in apoptosis. As a consequence, functional mitochondrial disorders are involved in many human cancers including acute myeloid leukemia (AML). However, very few data are available on the deregulation of their number and/or shape in leukemic cells, despite the evident link between ultrastructure and function. In this context, we analyzed the ultrastructural mitochondrial parameters (number per cell, mitochondria area, number of cristae/mitochondria, cristal thickness) in five leukemia cell lines (HEL, HL60, K562, KG1, and OCI-AML3) together with the functional assay of their respiratory profile. First, we describe significant differences in basal respiration, maximal respiration, ATP production, and spare respiratory capacity between our cell lines, confirming the various respiratory profiles among leukemia subtypes. Second, we highlight that these variations are obviously associated with significant interleukemia heterogeneity of the number and/or shape of mitochondria. For instance, KG1, characterized by the smallest number of mitochondria together with reduced cristal diameter, had a particularly deficient respiratory profile. In comparison, the HEL and K562 cell lines, both with high respiratory profiles, harbored the largest number of mitochondria/cells with high cristal diameters. Moreover, we report that K562, carrying the ASXL1 mutation, presents significant mitochondria-endoplasmic reticulum deficiency reflected by decreases in the numbers of matrix granules and mitochondria-associated endoplasmic reticulum membrane (MAM) and mitochondrial-derived vesicle (MDV) precursors, which are implicated in the regulatory pathways of cell mortality via the processes of mitophagy and calcium homeostasis. Contrarily, HL60 carried high levels of matrix granules and MAMs and had a higher sensitivity to drugs targeting mitochondria (rotenone/antimycin). We confirm the implication of ASXL1 mutation in this mitochondria dysregulation through the study of transcript expression (from 415 patients with public data) involved in three mitochondrial pathways: (1) endoplasmic reticulum-mitochondria contacts (MAMs), (2) matrix granule homeostasis, and (3) MDV precursor production. Our study offers new and original data on mitochondria structural alterations linked to deregulation of respiration profiles in AMLs and some genetic characteristics, suggesting that modifications of mitochondrial shape and/or number in leukemic cells participate in chemoresistance and could be a targeted mechanism to regulate their proliferative potential., Competing Interests: Conflict of interest disclosure The authors declare that they have no competing interests., (Copyright © 2021 ISEH -- Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

DNMT3A mutations are frequently identified in acute myeloid leukemia (AML) and indicate poor prognosis. Previously, we found that the hotspot mutation DNMT3A R882H could upregulate CDK1 and induce AML in conditional knock-in mice. However, the mechanism by which CDK1 is involved in leukemogenesis of DNMT3A mutation-related AML, and whether CDK1 could be a therapeutic target, remains unclear. In this study, using fluorescence resonance energy transfer and immunoprecipitation analysis, we discovered that increased CDK1 could compete with EZH2 to bind to the PHD-like motif of DNMT3A, which may disturb the protein interaction between EZH2 and DNMT3A. Knockdown of CDK1 in OCI-AML3 cells with DNMT3A mutation markedly inhibited proliferation and induced apoptosis. CDK1 selective inhibitor CGP74514A (CGP) and the pan-CDK inhibitor flavopiridol (FLA) arrested OCI-AML3 cells in the G2/M phase, and induced cell apoptosis. CGP significantly increased CD163-positive cells. Moreover, the combined application of CDK1 inhibitor and traditional chemotherapy drugs synergistically inhibited proliferation and induced apoptosis of OCI-AML3 cells. In conclusion, this study highlights CDK1 overexpression as a pathogenic factor and a potential therapeutic target for DNMT3A mutation-related AML.

The World Health Organization classification and definition of "myeloid sarcoma" is imprecise and misleading. A more accurate term is "extramedullary acute myeloid leukemia tumor (eAML)." The pathogenesis of eAML has been associated with aberrancy of cellular adhesion molecules, chemokine receptors/ligands and RAS-MAPK/ERK signaling. eAML can present with or without synchronous or metachronous intramedullary acute myeloid leukemia (AML) so a bone marrow evaluation is always recommended. Accurate diagnosis of eAML requires tissue biopsy. eAML confined to one or a few sites is frequently treated with local therapy such as radiotherapy. About 75-90% of patients with isolated eAML will develop metachronous intramedullary AML with a median latency period ranging from 4 to 12 months; thus, patients with isolated eAML may also be treated with systemic anti-leukemia therapy. eAML does not appear to have an independent prognostic impact; selection of post-remission therapy including allogeneic hematopoietic cell transplant (alloHCT) is typically guided by intramedullary disease risk. Management of isolated eAML should be individualized based on patient characteristics as well as eAML location and cytogenetic/molecular features. The role of PET/CT in eAML is also currently being elucidated. Improving outcomes of patients with eAML requires further knowledge of its etiology and mechanism(s) as well as therapeutic approaches beyond conventional chemotherapy, ideally in the context of controlled trials., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Identification of genomic mutations by molecular testing plays an important role in diagnosis, prognosis, and treatment of myeloid neoplasms. Next-generation sequencing (NGS) is an efficient method for simultaneous detection of clinically significant genomic mutations with high sensitivity. Various NGS based in-house developed and commercial myeloid neoplasm panels have been integrated into routine clinical practice. However, some genes frequently mutated in myeloid malignancies are particularly difficult to sequence with NGS panels (e.g., CEBPA, CARL, and FLT3). We report development and validation of a 48-gene NGS panel that includes genes that are technically challenging for molecular profiling of myeloid neoplasms including acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and myeloproliferative neoplasms (MPN). Target regions were captured by hybridization with complementary biotinylated DNA baits, and NGS was performed on an Illumina NextSeq500 instrument. A bioinformatics pipeline that was developed in-house was used to detect single nucleotide variations (SNVs), insertions/deletions (indels), and FLT3 internal tandem duplications (FLT3-ITD). An analytical validation study was performed on 184 unique specimens for variants with allele frequencies ≥5%. Variants identified by the 48-gene panel were compared to those identified by a 35-gene hematologic neoplasms panel using an additional 137 unique specimens. The developed assay was applied to a large cohort (n = 2,053) of patients with suspected myeloid neoplasms. Analytical validation yielded 99.6% sensitivity (95% CI: 98.9-99.9%) and 100% specificity (95% CI: 100%). Concordance of variants detected by the 2 tested panels was 100%. Among patients with suspected myeloid neoplasms (n = 2,053), 54.5% patients harbored at least one clinically significant mutation: 77% in AML patients, 48% in MDS, and 45% in MPN. Together, these findings demonstrate that the assay can identify mutations associated with diagnosis, prognosis, and treatment options of myeloid neoplasms even in technically challenging genes., Competing Interests: Sun Hee Rosenthal, Anna Gerasimova, Charles Ma, Hai-Rong Li, Andrew Grupe, Hansook Chong, Allan Acab, Alla Smolgovsky, Renius Owen, Christopher Elzinga, Rebecca Chen, Daniel Sugganth, Anindya Bhattacharya, Frederick Racke, and Felicitas Lacbawan are employees of Quest Diagnostics Nichols Institute. Tracey Freitas, Jennifer Graham, and Kristen Champion are employees of med fusion/Quest Diagnostics. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Acute myeloid leukaemia (AML) is a blood cancer where undifferentiated myeloid cells are increased in the bone marrow and peripheral blood. As AML is dangerous and shows poor prognosis, many researchers categorised the relevant cytogenetic factors according to risk and prognosis. However, the specific reasons for poor cytogenetic factors remain unknown. We analysed a large data set from AML patients and found that TPD52 expression is elevated in patient groups with poor cytogenetic factors. As the amino acid sequence of TPD52 is evolutionally conserved in vertebrates, zebrafish embryos were used to investigate the function of TPD52. Since myeloid-biased haematopoietic stem cells (HSCs) are relevant to AML, the function of TPD52 in the development of HSCs was investigated. We determined that the zebrafish paralog, tpd52, is important for the maintenance of HSCs through regulation of cell proliferation. As tpd52 is linked to cell proliferation in zebrafish embryos, the proliferation-related gene, CD59, was correlated to TPD52 in every AML cohort with a high correlation coefficient. We suggest that TPD52 can be a novel therapeutic target for AML patients with poor cytogenetic factors. Additionally, more studies between TPD52 and CD59 will further increase the value of TPD52 as a novel target., (© 2020 Wiley Periodicals LLC.)

Emerging evidence suggests that aryl hydrocarbon receptor (AHR) promotes the initiation, invasion, progression, and metastasis of cancer cells. However, its effects in patients with myelodysplastic syndrome/acute myeloid leukemia (MDS/AML) remain undefined. In this study, we aimed to investigate the effects of AHR activation on malignant cells in patients with MDS/AML. We found that AHR was expressed aberrantly in patients with MDS/AML. Further studies demonstrated that inhibiting AHR decreased the mitochondrial dehydrogenase content and the mitochondrial membrane potential (MMP) in MDS/AML cells. Activating AHR with L-kynurenine (Kyn) increased AHR expression, which was accompanied by an increase in mitochondrial dehydrogenase content and MMP in MDS/AML cells. Moreover, the expression level of mitochondria-associated mitochondrial transcription factor A was increased after activating AHR with L-Kyn when compared with that in the control group but decreased after inhibiting the AHR signal. Activating AHR in MDS/AML cells enhanced the resistance to cytarabine. These findings indicated that activating the AHR signaling pathway reshaped the metabolism in MDS/AML cells, thus contributing to the resistance to cytarabine., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Clonal haematopoiesis (CH) in patients with acute myeloid leukaemia (AML) may persist beyond attaining complete remission. From a consecutive cohort of 67 patients with nucleophosmin 1-mutated (NPM1 mut ) AML, we identified 50 who achieved NPM1 mut clearance and had parallel multicolour flow cytometry (MFC) and next generation sequencing (NGS). In total, 13 (26%) cleared all mutations, 37 (74%) had persistent CH frequently involving DNA methyltransferase 3α (DNMT3A,70%), tet methylcytosine dioxygenase 2 (TET2, 27%), isocitrate dehydrogenase 2 (IDH2, 19%) and IDH1 (11%). A small number (<1%) of aberrant CD34 + myeloblasts, but immunophenotypically different from original AML blasts [herein referred to as a pre-leukaemic (PL) phenotype], was detected in 17 (49%) patients with CH, but not in any patients with complete clearance of all mutations (P = 0·0037). A PL phenotype was associated with higher mutation burden (P = 0·005). Persistent IDH2 and serine and arginine-rich splicing factor 2 (SRSF2) mutations were exclusively observed in PL + CH + cases (P = 0·016). Persistent dysplasia was seen exclusively in cases with a PL + phenotype (29% vs. none; P = 0·04). The PL + phenotype did not correlate with age, intensity of induction therapy or relapse-free survival. Post-remission CH in the setting of NPM1 mut clearance is common and may result in immunophenotypic changes in myeloid progenitors. It is important to not misinterpret these cells as AML measurable residual disease (MRD)., (© 2021 British Society for Haematology and John Wiley & Sons Ltd.)

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with poor prognosis and overall survival. Clinical investigations show that chronic stress is commonly present in the course of AML and associated with adverse outcome. However, the underlying molecular mechanisms are elusive. In the present study, a chronic restraint stress mouse model was established to evaluate the effect of stress on AML. We found that mice under chronic stress exhibited significantly increased liver and spleen infiltration of leukemic cells and poorer overall survival. This was accompanied by elevated cellular NLR family pyrin domain containing 3 (NLRP3) and interleukin-1β (IL-1β) in the liver or bone marrow, and secreted IL-1β in the plasma, indicating the activation of inflammasomes under chronic restraint stress. High mobility group box 1 (HMGB1) expression was markedly increased in newly diagnosed AML patients, but reduced in complete remission AML patients. The expression level of HMGB1 was positively correlated with NLRP3 mRNA in AML patients. Knockdown of HMGB1 significantly decreased NLRP3 and IL-1β expression in AML cell lines, and secreted IL-1β in supernatant of AML cell culture, while HMGB1 stimulation caused contrary effects. These results implied that HMGB1 could be involved in the regulation of inflammasome activation in AML development. Mice model showed that chronic restraint stress-facilitated proliferation and infiltration of AML cells were largely abrogated by knocking down HMGB1. Knockdown of HMGB1 also ameliorated overall survival and remarkably neutralized NLRP3 and IL-1β expression under chronic restraint stress. These findings provide evidences that chronic stress promotes AML progression via HMGB1/NLRP3/IL-1β dependent mechanism, suggesting that HMGB1 is a potential therapeutic target for AML. KEY MESSAGES: • Chronic restraint stress promoted acute myeloid leukemia (AML) progression and mediated NLRP3 inflammasome activation in xenograft mice. • HMGB1 mediated NLRP3 inflammasome activation in AML cells. • Knockdown of HMGB1 inhibited AML progression under chronic stress in vivo.

How particular bone marrow niche factors contribute to the leukemogenic activities of leukemia-initiating cells (LICs) remains largely unknown. Here, we showed that ATP levels were markedly increased in the bone marrow niches of mice with acute myeloid leukemia (AML), and LICs preferentially localized to the endosteal niche with relatively high ATP levels, as indicated by a sensitive ATP indicator. ATP could efficiently induce the influx of ions into LICs in an MLL-AF9-induced murine AML model via the ligand-gated ion channel P2X7. P2x7 deletion led to notably impaired homing and self-renewal capacities of LICs and contributed to an approximately 5-fold decrease in the number of functional LICs but had no effect on normal hematopoiesis. ATP/P2X7 signaling enhanced the calcium flux-mediated phosphorylation of CREB, which further transactivated phosphoglycerate dehydrogenase (Phgdh) expression to maintain serine metabolism and LIC fates. P2X7 knockdown resulted in a markedly extended survival of recipients transplanted with either human AML cell lines or primary leukemia cells. Blockade of ATP/P2X7 signaling could efficiently inhibit leukemogenesis. Here, we provide a perspective for understanding how ATP/P2X7 signaling sustains LIC activities, which may benefit the development of specific strategies for targeting LICs or other types of cancer stem cells.

Small extracellular vesicles (SEVs) are functional messengers of certain cellular niches that permit noncontact cell communications. Whether niche-specific SEVs fulfill this role in cancer is unclear. Here, we used 7 cell type-specific mouse Cre lines to conditionally knock out Vps33b in Cdh5+ or Tie2+ endothelial cells (ECs), Lepr+ BM perivascular cells, Osx+ osteoprogenitor cells, Pf4+ megakaryocytes, and Tcf21+ spleen stromal cells. We then examined the effects of reduced SEV secretion on progression of MLL-AF9-induced acute myeloid leukemia (AML), as well as normal hematopoiesis. Blocking SEV secretion from ECs, but not perivascular cells, megakaryocytes, or spleen stromal cells, markedly delayed the leukemia progression. Notably, reducing SEV production from ECs had no effect on normal hematopoiesis. Protein analysis showed that EC-derived SEVs contained a high level of ANGPTL2, which accelerated leukemia progression via binding to the LILRB2 receptor. Moreover, ANGPTL2-SEVs released from ECs were governed by VPS33B. Importantly, ANGPTL2-SEVs were also required for primary human AML cell maintenance. These findings demonstrate a role of niche-specific SEVs in cancer development and suggest targeting of ANGPTL2-SEVs from ECs as a potential strategy to interfere with certain types of AML.

Background: Myeloproliferative neoplasms (MPNs) can transform into blast phase MPN (leukemic transformation; MPN-BP), typically via accelerated phase MPN (MPN-AP), in ∼20-25% of the cases. MPN-AP and MPN-BP are characterized by 10-19% and ≥20% blasts, respectively. MPN-AP/BP portend a dismal prognosis with no established conventional treatment. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the sole modality associated with long-term survival., Summary: MPN-AP/BP has a markedly different mutational profile from de novo acute myeloid leukemia (AML). In MPN-AP/BP, TP53 and IDH1/2 are more frequent, whereas FLT3 and DNMT3A are rare. Higher incidence of leukemic transformation has been associated with the most aggressive MPN subtype, myelofibrosis (MF); other risk factors for leukemic transformation include rising blast counts above 3-5%, advanced age, severe anemia, thrombocytopenia, leukocytosis, increasing bone marrow fibrosis, type 1 CALR-unmutated status, lack of driver mutations (negative for JAK2, CALR, or MPL genes), adverse cytogenetics, and acquisition of ≥2 high-molecular risk mutations (ASXL1, EZH2, IDH1/2, SRSF2, and U2AF1Q157). The aforementioned factors have been incorporated in several novel prognostic scoring systems for MF. Currently, elderly/unfit patients with MPN-AP/BP are treated with hypomethylating agents with/without ruxolitinib; these regimens appear to confer comparable benefit to intensive chemotherapy but with lower toxicity. Retrospective studies in patients who acquired actionable mutations during MPN-AP/BP showed positive outcomes with targeted AML treatments, such as IDH1/2 inhibitors, and require further evaluation in clinical trials. Key Messages: Therapy for MPN-AP patients represents an unmet medical need. MF patients, in particular, should be appropriately stratified regarding their prognosis and the risk for transformation. Higher-risk patients should be monitored regularly and treated prior to progression to MPN-BP. MPN-AP patients may be treated with hypomethylating agents alone or in combination with ruxolitinib; also, patients can be provided with the option to enroll in rationally designed clinical trials exploring combination regimens, including novel targeted drugs, with an ultimate goal to transition to transplant., (© 2021 S. Karger AG, Basel.)

Acute myeloid leukemia (AML) is an aggressive, disseminated hematological malignancy associated with clonal selection of aberrant self-renewing hematopoietic stem cells and progenitors and poorly differentiated myeloid blasts. The most prevalent form of leukemia in adults, AML is predominantly an age-related disorder and accounts for more than 10,000 deaths per year in the United States alone. In comparison to solid tumors, AML has an overall low mutational burden, albeit more than 70% of AML patients harbor somatic mutations in genes encoding epigenetic modifiers and chromatin regulators. In the past decade, discoveries highlighting the role of DNA and histone modifications in determining cellular plasticity and lineage commitment have attested to the importance of epigenetic contributions to tumor cell de-differentiation and heterogeneity, tumor initiation, maintenance, and relapse. Orchestration in histone methylation levels regulates pluripotency and multicellular development. The increasing number of reversible methylation regulators being identified, including histone methylation writer, reader, and eraser enzymes, and their implications in AML pathogenesis have widened the scope of epigenetic reprogramming, with multiple drugs currently in various stages of preclinical and clinical trials. AML methylome also determines response to conventional chemotherapy, as well as AML cell interaction within a tumor-immune microenvironment ecosystem. Here we summarize the latest developments focusing on molecular derangements in histone methyltransferases (HMTs) and histone demethylases (HDMs) in AML pathogenesis. AML-associated HMTs and HDMs, through intricate crosstalk mechanisms, maintain an altered histone methylation code conducive to disease progression. We further discuss their importance in governing response to therapy, which can be used as a biomarker for treatment efficacy. Finally we deliberate on the therapeutic potential of targeting aberrant histone methylome in AML, examine available small molecule inhibitors in combination with immunomodulating therapeutic approaches and caveats, and discuss how future studies can enable posited epigenome-based targeted therapy to become a mainstay for AML treatment., Competing Interests: Conflict of interest disclosure The authors declare no conflicts of interest., (Copyright © 2020 ISEH -- Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Introduction: A variety of molecular-targeted drugs have been widely used in hematological malignancies and have shown great advances. Nevertheless, as the use of drugs in clinical practice increases, the problem of relapse or of the disease being refractory to treatment is becoming apparent. This problem is closely related to the C-X-C chemokine receptor 4 (CXCR4)., Areas Covered: This review focuses mainly on the effect of CXCR4 on molecular-targeted drug resistance in hematological malignancies as well as the clinical efficacy of CXCR4 antagonists combined with molecular-targeted drugs. Relevant literatures published between 2006 and 2020 were searched using PubMed/Medline for this review., Expert Opinion: Monoclonal antibodies and non-antibody molecular-targeted drugs provide new therapeutic approaches for B-lineage malignancies and leukemia, but the clinical activity of these drugs is affected by CXCR4. In general, high CXCR4 expression or mutation inhibits the effects of molecular-targeted drugs, but there are exceptions, and in studies of proteasome inhibitors bortezomib (Bz) in multiple myeloma (MM), low CXCR4 expression or loss of CXCR4 was associated with Bz resistance (BzR) and poor treatment outcomes. Given that CXCR4 is a critical mediator of molecular-targeted drug resistance, numerous studies have combined molecular-targeted drugs with CXCR4 antagonists, which synergistically enhance the anti-proliferative/pro-apoptotic effect of molecular-targeted drugs.

Accurate prediction of malignancies is important in choosing therapeutic strategies. Although there are many genetic and cytogenetic prognostic factors for acute myeloid leukemia (AML), prognosis is difficult to predict because of the heterogeneity of AML. Prognostic factors, including messenger RNA (mRNA) expression, have been determined for other malignancies, but not for AML. A total of 402 patients from The Cancer Genome Atlas, GSE12417 (GPL96, 97), and GSE12417 (GPL570) were included in this study. In Kaplan-Meier curve analyses, high expression of family with sequence similarity 213 member A (FAM213A), which activates antioxidant proteins, was associated with worse prognosis of AML. Similar to the results of the survival curve, C-indices and area under the curve values were high. Current prognostic factors of AML, unlike those of other cancers, do not take mRNA expression into consideration. Thus, the development of mRNA-based prognostic factors would be beneficial for accurate prediction of the survival of AML patients. Additionally, in vivo validation using zebrafish revealed that fam213a is important for myelopoiesis at the developmental stage and is a negative regulator of the p53 tumor suppressor gene. The findings implicate fam213a as a novel prognostic factor for AML patients., (© 2020 John Wiley & Sons Ltd.)

Introduction: The development of drug resistance is the main obstacle for successful treatment in acute myeloid leukemia (AML). Noncoding RNAs have been implicated in biological function in AML drug resistance. Aberrant protein glycosylation is associated with AML progression. The aim of the study was to explore the potential regulatory mechanism of lncRNA MEG3/miR-155/ALG9 axis in drug resistance of AML., Methods: QRT-PCR and Western blot were used for comparison analyses of ALG9, MEG3, and miR-155 levels. CCK-8 and colony formation assays were determined for drug sensitivity and proliferative capability of AML cells. Luciferase reporter assay was used to confirm the targets of miR-155., Results: The mannosyltransferase ALG9 and MEG3 was downregulated in peripheral blood mononuclear cells (PBMCs) of M5/multidrug resistance (MDR) AML patients and adriamycin (ADR)-resistant AML cell lines, which determined a positive correlation in AML patients. Low expression of ALG9 and MEG3 predicted poor prognosis of AML patients. The altered level of ALG9 was found corresponding to the drug-resistant phenotype and sphere formation of AML cells. MiR-155 was overexpressed in M5/MDR patients and ADR-resistant AML cells, as well as inversely correlated to ALG9 expression. MEG3 was a direct target of miR-155 and could sponge miR-155 in AML cells. MEG3 interacted with miR-155 to regulate ALG9 expression, which reversed the effects of ALG9 regulation on proliferation and drug resistance in AML cells., Conclusion: MEG3 sponged miR-155 by competing endogenous RNA (ceRNA) mechanism, which further modulated ALG9 expression and AML procession, providing a novel therapeutic target for AML chemoresistance., (© 2020 John Wiley & Sons Ltd.)

CCTC-binding factor (CTCF) is a key regulator of gene expression through organization of the chromatin structure. Still, it is unclear how CTCF binding is perturbed in leukemia or in cancer in general. We studied CTCF binding by chromatin immunoprecipitation sequencing in cells from patients with acute myeloid leukemia (AML) and in normal bone marrow (NBM) in the context of gene expression, DNA methylation, and azacitidine exposure. CTCF binding was increased in AML compared with NBM. Aberrant CTCF binding was enriched for motifs for key myeloid transcription factors such as CEBPA, PU.1, and RUNX1. AML with TET2 mutations was characterized by a particularly strong gain of CTCF binding, highly enriched for gain in promoter regions, while AML in general was enriched for changes at enhancers. There was a strong anticorrelation between CTCF binding and DNA methylation. Gain of CTCF occupancy was associated with increased gene expression; however, the genomic location (promoter vs distal regions) and enrichment of motifs (for repressing vs activating cofactors) were decisive for the gene expression pattern. Knockdown of CTCF in K562 cells caused loss of CTCF binding and transcriptional repression of genes with changed CTCF binding in AML, as well as loss of RUNX1 binding at RUNX1/CTCF-binding sites. In addition, CTCF knockdown caused increased differentiation. Azacitidine exposure caused major changes in CTCF occupancy in AML patient cells, partly by restoring a CTCF-binding pattern similar to NBM. We conclude that AML displays an aberrant increase in CTCF occupancy that targets key genes for AML development and impacts gene expression., (© 2020 by The American Society of Hematology.)

Through a clustered regularly insterspaced short palindromic repeats (CRISPR) screen to identify mitochondrial genes necessary for the growth of acute myeloid leukemia (AML) cells, we identified the mitochondrial outer membrane protein mitochondrial carrier homolog 2 (MTCH2). In AML, knockdown of MTCH2 decreased growth, reduced engraftment potential of stem cells, and induced differentiation. Inhibiting MTCH2 in AML cells increased nuclear pyruvate and pyruvate dehydrogenase (PDH), which induced histone acetylation and subsequently promoted the differentiation of AML cells. Thus, we have defined a new mechanism by which mitochondria and metabolism regulate AML stem cells and gene expression., (© 2020 by The American Society of Hematology.)

This multi-institutional study retrospectively evaluated clinicopathologic and genetic characteristics in 351 patients with core-binding-factor acute myeloid leukemia (CBF-AML), comprising 69 therapy-related (t-CBF-AML) and 282 de novo cases. The T-CBF-AML patients were older, had lower WBC counts, and slightly higher hemoglobin than patients with de novo disease. Secondary cytogenetic abnormalities were more frequent in patients with de novo disease than t-CBF-AML (57.1% vs 41.1%, P = .026). Patients with secondary cytogenetic abnormalities had longer overall survival (OS) than those without abnormalities (median 190 vs 87 months, P = .021); trisomy 8, trisomy 22, and loss of the X or Y chromosome were associated with longer OS. In the 165 cases performed of targeted gene sequencing, pathogenic mutations were detected in 75.7% of cases, and were more frequent in de novo than in therapy-related disease (P = .013). Mutations were found in N/KRAS (37.0%), FLT3 (27.8%), KIT (17.2%), TET2 (4.9%), and ASXL1 (3.9%). The TET2 mutations were associated with shorter OS (P = .012) while N/KRAS mutation was associated with longer OS in t(8;21) AML patients (P = .001). The KIT mutation did not show prognostic significance in this cohort. Although they received similar therapy, t-CBF-AML patients had shorter OS than de novo patients (median 69 vs 190 months, P = .038). In multivariate analysis of all patients, older age and absence of any secondary cytogenetic abnormalities were significant predictors of shorter OS. Among the t-CBF-AML subset, age and hemoglobin were significant on multivariate analysis. This study demonstrated that although de novo and t-CBF-AML patients share many features, t-CBF-AML patients have worse clinical outcome than de novo patients., (© 2020 Wiley Periodicals, Inc.)

Acute myeloid leukemia (AML) disrupts the generation of normal blood cells, predisposing patients to hemorrhage, anemia, and infections. Differentiation and proliferation of residual normal hematopoietic stem and progenitor cells (HSPCs) are impeded in AML-infiltrated bone marrow (BM). The underlying mechanisms and interactions of residual hematopoietic stem cells (HSCs) within the leukemic niche are poorly understood, especially in the human context. To mimic AML infiltration and dissect the cellular crosstalk in human BM, we established humanized ex vivo and in vivo niche models comprising AML cells, normal HSPCs, and mesenchymal stromal cells (MSCs). Both models replicated the suppression of phenotypically defined HSPC differentiation without affecting their viability. As occurs in AML patients, the majority of HSPCs were quiescent and showed enrichment of functional HSCs. HSPC suppression was largely dependent on secreted factors produced by transcriptionally remodeled MSCs. Secretome analysis and functional validation revealed MSC-derived stanniocalcin 1 (STC1) and its transcriptional regulator HIF-1α as limiting factors for HSPC proliferation. Abrogation of either STC1 or HIF-1α alleviated HSPC suppression by AML. This study provides a humanized model to study the crosstalk among HSPCs, leukemia, and their MSC niche, and a molecular mechanism whereby AML impairs normal hematopoiesis by remodeling the mesenchymal niche.

Introduction: The expression of forkhead box N3 (FOXN3), also known as checkpoint suppressor 1 (CHES1), is reduced in many types of tumours. However, the clinical significance of FOXN3 and its potential role in acute myeloid leukaemia (AML) remain largely unknown., Methods: A total of 117 de novo AML patients newly diagnosed between December 2015 and January 2018 were included in this study. The expression of FOXN3 and its clinical significance were analysed in these AML patients., Results: The expression of FOXN3 was significantly downregulated in AML. In addition, lower FOXN3 expression was associated with older age and higher white blood cell counts. Moreover, a close correlation was observed between lower FOXN3 expression and a lower complete remission (CR) rate and shorter overall survival (OS), which was further analysed by multivariate analysis., Conclusion: These data suggest that FOXN3 is a novel biomarker in AML and that lower FOXN3 expression predicts poor chemotherapy response and prognosis in AML., (© 2020 The Authors. International Journal of Laboratory Hematology published by John Wiley & Sons Ltd.)

Acute myeloid leukaemia (AML) is the most common adult acute leukaemia with the lowest survival rate. It is characterised by a build-up of immature myeloid cells anchored in the protective niche of the bone marrow (BM) microenvironment. The CXCL12/CXCR4 axis is central to the pathogenesis of AML as it has fundamental control over AML cell adhesion into the protective BM niche, adaptation to the hypoxic environment, cellular migration and survival. High levels of CXCR4 expression are associated with poor relapse-free and overall survival. The CXCR4 ligand, CXCL12 (SDF-1), is expressed by multiple cells types in the BM, facilitating the adhesion and survival of the malignant clone. Blocking the CXCL12/CXCR4 axis is an attractive therapeutic strategy providing a 'multi-hit' therapy that both prevents essential survival signals and releases the AML cells from the BM into the circulation. Once out of the protective niche of the BM they would be more susceptible to destruction by conventional chemotherapeutic drugs. In this review, we disentangle the diverse roles of the CXCL12/CXCR4 axis in AML. We then describe multiple CXCR4 inhibitors, including small molecules, peptides, or monoclonal antibodies, which have been developed to date and their progress in pre-clinical and clinical trials. Finally, the review leads us to the conclusion that there is a need for further investigation into the development of a 'multi-hit' therapy that targets several signalling pathways related to AML cell adhesion and maintenance in the BM., (© 2020 British Society for Haematology and John Wiley & Sons Ltd.)

Introduction: Acute myeloid leukemia (AML) is the most common acute leukemia in adults. There is growing evidence that microRNAs (miRNAs) provide prognostic information in AML. MiR-204 has a tumor suppressor function, and several studies have proven its role in solid cancers. The aim of this work is to evaluate the level of expression of miR-204 in adults newly diagnosed with AML with normal karyotype and to correlate its level of expression with disease outcome and different prognostic factors., Patients and Methods: The study included 87 adult patients newly diagnosed with AML. Detection of miR-204 was done using RT-PCR in patients and seven age-matched controls., Results: Acute myeloid leukemia patients showed significantly lower miR-204 expression, compared to control group (P = .029). Low miR-204 expression was significantly associated with positive CD34 (P = .017), with poor performance status (PS) (P = .009), and with the presence of diabetes mellitus (DM) (P = .014). Low expression of miR-204 was also significantly associated with shorter overall survival (OS) (P = .020) and disease-free survival (DFS) (P = .013). Low miR-204 expression was identified as an independent prognostic factor for prediction of shorter OS (P = .034) and DFS (P = .027) in AML., Conclusion: To the best of our knowledge; this is the first time to prove the correlation between miR-204 expression and CD34 expression. Further study of this correlation is needed to confirm the role of miR-204 in CD34-positive cells, including leukemic stem cells. This correlation may have therapeutic implications. MiR-204 can be used as a biomarker for PS in AML patients., (© 2020 John Wiley & Sons Ltd.)

Acute myeloid leukemia (AML) is among the top four malignancies in Saudi nationals, and it is the top leukemia subtype worldwide. Resistance to available AML drugs requires the identification of new targets and agents. Hsp90 is one of the emerging important targets in AML, which has a central role in the regulation of apoptosis and cell proliferation through client proteins including the growth factor receptors and cyclin dependent kinases. The objective of the first part of this study is to investigate the putative Hsp90 inhibition activity of three novel previously synthesized quinazolines, which showed HL60 cytotoxicity and VEGFR2 and EGFR kinases inhibition activities. Using surface plasmon resonance, compound 1 (HAA 2020 ) showed better Hsp90 inhibition compared to 17-AAG, and a docking study revealed that it fits nicely into the ATPase site. The objective of the second part is to maximize the anti-leukemic activity of HAA 2020 , which was combined with each of the eleven standard inhibitors. The best resulting synergistic effect in HL60 cells was with the pan cyclin-dependent kinases (CDK) inhibitor dinaciclib, using an MTT assay. Furthermore, the inhibiting effect of the Hsp90α gene by the combination of HAA 2020 and dinaciclib was associated with increased caspase-7 and TNF-α, leading to apoptosis in HL60 cells. In addition, the combination upregulated p27 simultaneously with the inhibition of cyclinD3 and CDK2, leading to abolished HL60 proliferation and survival. The actions of HAA 2020 propagated the apoptotic and cell cycle control properties of dinaciclib, showing the importance of co-targeting Hsp90 and CDK, which could lead to the better management of leukemia.

Objective: miR-194-5p and NEAT1 have been reported to be associated with multiple malignancies, but their roles in acute myeloid leukemia (AML) remains not fully understood., Methods: Bone marrow samples were collected for monocyte separation. qRT-PCR assay was performed to investigate the expression patterns of NEAT1 and miR-194-5p in AML. CCK-8, soft agar colony formation, flow cytometry and transwell assays were employed to explore the biological functions of NEAT1 or miR-194-5p. Methylation PCR was performed to monitor the methylation of NEAT1. Luciferase reporter assay was subjected to verify the relationship between miR-194-5p and DNMT3A. Immunofluorescence and western blotting were performed to detect the alterations of protein expression., Results: NEAT1 and miR-194-5p were both down-regulated in AML. Overexpression of either NEAT1 or miR-194-5p repressed proliferation, induced apoptosis and restrained migration and invasion of AML cells. There was a negative correlation between NEAT1 and DNMT3A in AML. Knockdown of DNMT3A dramatically decreased the methylation of NEAT1. Moreover, DNMT3A was identified as a downstream target of miR-194-5p. Furthermore, down-regulation of DNMT3A rescued the impacts on the malignant phenotypes of NEAT1 inhibition by miR-194-5p inhibitor., Conclusion: Altogether, down-regulation of NEAT1 mediated by miR-194-5p/DNMT3A axis promotes AML progression, which might provide therapeutic targets in AML treatment., Competing Interests: Declaration of competing interest All authors declared no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Objective : Therapeutic advances based on risk stratification and implementation of excellent supportive care measures have significantly improved outcomes for childhood acute myeloid leukemia (AML) over the past 30 years. However, approximately half of all childhood AML cases relapse. Therefore, precise risk stratification is needed for predicting relapse potential. Methods : RNA-seq data of TARGET-AML and corresponding clinical information of pediatric and adolescent AML cases were downloaded from The Cancer Genome Atlas. Clinical information of 156 patients with gene expression data was extracted. The effects of PAX7 expression profiles on overall survival (OS) and event-free survival (EFS) were analyzed. Results : Positive expression of PAX7 indicated shorter OS and EFS, especially in patients older than 14 years. Furthermore, positive PAX7 expression also predicted shorter OS and EFS in intermediate- and low-risk group patients, compared to patients with negative PAX7 expression. In addition, patients who have received allogenic hematopoietic stem cell transplantation (allo-HSCT) in the first complete remission had better outcome than those who did not receive HSCT. Conclusions : Positive PAX7 expression in pediatric and adolescent AML patients indicates a poor outcome. Hence, the detection of PAX7 expression profiles is helpful for further stratification of intermediate- and low-risk groups.

The long noncoding RNA urothelial carcinoma-associated 1 (UCA1) has been reported to sustain the proliferation of acute myeloid leukemia (AML) cells through downregulating cell cycle regulators p27 kip1 . Yet, the foundational mechanism of UCA1 in AML pathologies remains unclear. Herein, we found an escalation of UCA1 expression and suppression of miR-204 expression in pediatric AML patients and cells. UCA1 silencing suppressed cell proliferative abilities, promoted apoptotic rates, decreased Ki67, and increased cleaved caspase-3 in AML cells. Moreover, UCA1 sponged miR-204 and suppressed its expression. UCA1 overexpression inversed the miR-204 suppressed proliferation and promoted apoptosis. UCA1 also boosted the expression of SIRT1, a miR-204 target, via the sponging interaction. Furthermore, miR-204 inhibited inducible nitric oxide synthase and cyclooxygenase-2 expression, while UCA1 overexpression inversed the inhibitory effects in AML cells. Our findings concluded that UCA1 downregulation repressed cell proliferation and promoted apoptosis through inactivating SIRT1 signals by upregulating miR-204 in pediatric AML., (© 2020 Wiley Periodicals, Inc.)

The myelodysplastic syndromes (MDS) are common myeloid malignancies showing frequent progression to acute myeloid leukemia (AML). Pre-mRNA splicing is an essential cellular process carried out by the spliceosome. Mutations in splicing factor genes (including SF3B1, SRSF2, U2AF1 and ZRSR2) occur in over half of MDS patients and result in aberrant pre-mRNA splicing of many target genes, implicating aberrant spliceosome function in MDS disease pathogenesis. Recent functional studies have illuminated the impact on hematopoiesis of some aberrantly spliced target genes associated with splicing factor mutations. Emerging data show that the commonly mutated splicing factors have convergent effects on aberrant splicing of mRNAs that promote NF-κB signaling and on R-loop elevation leading to DNA damage, providing novel insights into MDS disease pathophysiology. It is recognized that the survival of splicing factor mutant cells is dependent on the presence of the wildtype allele, providing a rationale for the use of spliceosome inhibitors in splicing factor mutant MDS. Pre-clinical studies involving E7107 and H3B-8800 have shown the potential of these spliceosome inhibitors for the treatment of splicing factor mutant MDS and AML., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Nucleophosmin (NPM1) is the most commonly mutated gene in acute myeloid leukemia (AML). AML with mutated NPM1 is recognized as a separate entity in the World Health Organization 2016 classification and carries a relatively favorable prognosis. NPM1 mutations are predominantly 4-bp duplications or insertions in the terminal exon that arise through an unknown mechanism. Here we analyze 2430 NPM1 mutations from 2329 adult and 101 pediatric patients to address their origin. We show that NPM1 mutations display the hallmarks of replication slippage, but lack suitable germline microhomology available for priming. Insertion mutations display G/C-rich N-nucleotide tracts, with a significant bias toward polypurine and polypyrimidine stacking (P < .001). These features suggest terminal deoxynucleotidyl transferase (TdT) primes replication slippage through N-nucleotide addition, with longer syntheses manifesting as N-regions. The recurrent type A, type D, and type B mutations require 1, 2, and 3 N-nucleotide extensions of T, CC, and CAT, respectively, with the last nucleotide used as occult microhomology. This TdT-mutator model successfully predicts the relative incidence of the 256 potential 4-bp insertion/duplication mutations at position c.863&#95;864 over 4 orders of magnitude (ρ = 0.484, P < .0001). Children have a different NPM1 mutation spectrum to adults, including a shift away from type A mutations and toward longer N-regions, consistent with higher TdT activity in pediatric myeloid stem cells. These findings complement our FLT3-ITD data, suggesting illegitimate TdT activity contributes to around one-half of AMLs. AML may therefore reflect the price for adaptive immunity., (© 2019 by The American Society of Hematology.)

Pediatric myelodysplastic syndrome (MDS) is an uncommon disease and little is known about the molecular alterations of its development and evolution to acute myeloid leukemia (AML). The Enhancer of Zeste Homolog 2 ( EZH2 ) is the catalytic subunit of Polycomb repressive complex 2 (PCR2). It is a histone methyltransferase, that targets lysine 27 of histone 3. This methylated H3-K27 is usually associated with the silencing of genes that are involved in fundamental cellular processes, such as cell proliferation and differentiation. There are only few studies showing the status of EZH2 expression in patients with MDS and they were performed in adult MDS patients. The aim of this study was to analyze the EZH2 expression in pediatric patients with MDS and its association with karyotypes and evolution to acute myeloid leukemia (AML). We conducted the first study of EZH2 expression in pediatric patients with MDS. Considering the EZH2 expression levels in 42 patients and 17 healthy pediatric donors, it was possible to define three groups of expression in patients: low, intermediate, and high. The intermediate level encompassed patients with normal karyotypes, low level included patients with monosomy 7 and del(7q) and high level included patients with trisomy 8 and del(11q) ( p < 0.0001). Comparing the leukemic evolution, the low expression group presented disease evolution in 100% (8/8) of the cases, the intermediate expression group showed disease evolution in 4.34% (1/23) and in the high expression group, 63.63% (7/11) patients showed evolution from MDS to AML ( p < 0.0001). It is important to note that low and high EZH2 expression are associated with leukemic evolution, however low expression showed a stronger association with evolution from MDS to AML than the high expression. Our results suggest a scale of measure for EZH2 expression in pediatric MDS, where aberrant EZH2 expression may be a potential biomarker of disease evolution., Competing Interests: The authors declare that there are no conflicts of interests regarding the publication of this paper., (Copyright © 2019 Teresa de Souza Fernandez et al.)

MRD technologies increase our ability to measure response in acute myeloid leukemia (AML) beyond the limitations of morphology. When applied in clinical trials, molecular and immunophenotypic MRD assays have improved prognostic precision, providing a strong rationale for their use to guide treatment, as well as to measure its effectiveness. Initiatives such as those from the European Leukemia Network now provide a collaborative knowledge-based framework for selection and implementation of MRD assays most appropriate for defined genetic subgroups. For patients with mutated-NPM1 AML, quantitative polymerase chain reaction (qPCR) monitoring of mutated-NPM1 transcripts postinduction and sequentially after treatment has emerged as a highly sensitive and specific tool to predict relapse and potential benefit from allogeneic transplant. Flow cytometric MRD after induction is prognostic across genetic risk groups and can identify those patients in the wild-type NPM1 intermediate AML subgroup with a very high risk for relapse. In parallel with these data, advances in genetic profiling have extended understanding of the etiology and the complex dynamic clonal nature of AML, as well as created the opportunity for MRD monitoring using next-generation sequencing (NGS). NGS AML MRD detection can stratify outcomes and has potential utility in the peri-allogeneic transplant setting. However, there remain challenges inherent in the NGS approach of multiplex quantification of mutations to track AML MRD. Although further development of this methodology, together with orthogonal testing, will clarify its relevance for routine clinical use, particularly for patients lacking a qPCR genetic target, established validated MRD assays can already provide information to direct clinical practice., Competing Interests: Conflict-of-interest disclosure: C.S.H. has received laboratory research funding from Merck and Sellas. S.D.F. declares no competing financial interests., (© 2019 by The American Society of Hematology. All rights reserved.)

U2 small nuclear RNA auxiliary factor 1 (U2AF1) mutant is the most common molecular biological abnormality in patients with myelodysplastic syndromes. Some studies have reported the prognostic impact of U2AF1 mutant in patients with de novo MDS, with discrepant results, so we do a meta-analysis about the relevant literatures to further investigate their prognostic impact on patients with de novo MDS. We conducted a literature search on databases such as PubMed, Embase, and the Cochrane Library to obtain studies on the prognosis of U2AF1 mutant in patients with de novo MDS published up to August 9, 2018. The primary endpoint was overall survival (OS), and the secondary endpoint was acute myeloid leukemia (AML) transformation. We extracted the hazard ratios (HRs) of OS and AML transformation and their 95% confidence intervals (CIs). Meta-analysis was performed by selecting a fixed-effect model or a random-effects model based on the heterogeneity between studies. A total of 14 cohort studies were included in the final meta-analysis, including 3322 patients with de no MDS, in which 390 patients were associated with U2AF1 mutant. The results showed that U2AF1 mutant had an adverse prognostic impact on OS (HR = 1.84, 95% CI: 1.45-2.33, P < 0.00001) and AML transformation (HR = 2.47, 95% CI: 1.50-4.06, P = 0.0004). U2AF1 mutant was associated with shorter OS in subgroup analyses of low- or intermediate-1-IPSS, U2AF1 S34 and U2AF1 Q157/R156 . Out meta-analysis indicates that U2AF1 mutants are independent, detrimental prognostic factors for OS and AML transformation in patients with de novo MDS, as well as associating with shorter OS in subgroups of low- or intermediate-1-IPSS, U2AF1 S34 and U2AF1 Q157/R156 . Further prospective studies are needed in the future, and subgroup analysis of U2AF1 subgroups is needed to obtain a more reliable basis for the impact of U2AF1 mutant on the prognosis of de novo MDS.

Constitutive activation of FGFR1 as a result of chromosome translocations is responsible for the development of a hematopoietic stem cell disorder that progresses to AML. We have developed a syngeneic mouse model of BCR-FGFR1 driven AML and used RNASeq to define gene expression signatures associated with disease progression. The development of the leukemic stem cells (LSC) is associated with a profound downregulation of specific transcription factors that normally maintain stem cell quiescence as well as cell adhesion and motility gene sets related to confinement to the stem cell niche. A prominent feature of the LSCs is the upregulation of genes involved in T-cell function, activation, migration and development. Despite this apparent T-cell priming in the LSCs, however, the majority of these genes are subsequently inactivated in the leukemic blast cells that derive from them. These studies provide insights into the molecular etiology of development and progression of FGFR1 driven AML., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The interleukin-3 receptor α subunit, CD123, is expressed in many hematologic malignancies including acute myeloid leukemia (AML) and blastic plasmacytoid dendritic cell neoplasm (BPDCN). Tagraxofusp (SL-401) is a CD123-targeted therapy consisting of interleukin-3 fused to a truncated diphtheria toxin payload. Factors influencing response to tagraxofusp other than CD123 expression are largely unknown. We interrogated tagraxofusp resistance in patients and experimental models and found that it was not associated with CD123 loss. Rather, resistant AML and BPDCN cells frequently acquired deficiencies in the diphthamide synthesis pathway, impairing tagraxofusp's ability to ADP-ribosylate cellular targets. Expression of DPH1, encoding a diphthamide pathway enzyme, was reduced by DNA CpG methylation in resistant cells. Treatment with the DNA methyltransferase inhibitor azacitidine restored DPH1 expression and tagraxofusp sensitivity. We also developed a drug-dependent ADP-ribosylation assay in primary cells that correlated with tagraxofusp activity and may represent an additional novel biomarker. As predicted by these results and our observation that resistance also increased mitochondrial apoptotic priming, we found that the combination of tagraxofusp and azacitidine was effective in patient-derived xenografts treated in vivo. These data have important implications for clinical use of tagraxofusp and led to a phase 1 study combining tagraxofusp and azacitidine in myeloid malignancies.