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Epigenetic alterations of chromatin structure affect chromatin accessibility and collaborate with genetic alterations in the development of cancer. Lysine demethylase 4B (KDM4B) has been identified as a JmjC domain‐containing epigenetic modifier that possesses histone demethylase activity. Although recent studies have demonstrated that KDM4B positively regulates the pathogenesis of multiple types of solid tumors, the tissue specificity and context dependency have not been fully elucidated. In this study, we investigated gene expression profiles established from clinical samples and found that KDM4B is elevated specifically in acute myeloid leukemia (AML) associated with chromosomal translocation 8;21 [t(8;21)], which results in a fusion of the AML1 and the eight‐twenty‐one (ETO) genes to generate a leukemia oncogene, AML1‐ETO fusion transcription factor. Short hairpin RNA‐mediated KDM4B silencing significantly reduced cell proliferation in t(8;21)‐positive AML cell lines. Meanwhile, KDM4B silencing suppressed the expression of AML1‐ETO‐inducible genes, and consistently perturbed chromatin accessibility of AML1‐ETO‐binding sites involving altered active enhancer marks and functional cis‐regulatory elements. Notably, transduction of murine KDM4B orthologue mutants followed by KDM4B silencing demonstrated a requirement of methylated‐histone binding modules for a proliferative surge. To address the role of KDM4B in leukemia development, we further generated and analyzed Kdm4b conditional knockout mice. As a result, Kdm4b deficiency attenuated clonogenic potential mediated by AML1‐ETO and delayed leukemia progression in vivo. Thus, our results highlight a tumor‐promoting role of KDM4B in AML associated with t(8;21).

Key Points c-KIT activating mutations cause resistance to PARP inhibitor in AML1-ETO–positive leukemias. c-KIT inhibitor avapritinib downregulates BRCA1/2 and DNA-PK catalytic subunit to restore the sensitivity to PARP inhibitor.

Highlights l Performed DNA-based ddPCR to accurately monitor the MRD of AE (+) AML. l Identified quantification of the fusion gene correlates strongly with clinical prognosis., Relapse of childhood AML1-ETO (AE) acute myeloid leukemia is the most common cause of treatment failure. Optimized minimal residual disease monitoring methods is required to prevent relapse. In this study, we used next-generation sequencing to identify the breakpoints in the fusion gene and the DNA-based droplet digital PCR (ddPCR) method was used for dynamic monitoring of AE-DNA. The ddPCR technique provides more sensitive and precise quantitation of the AE gene during disease progression and relapse. Quantification of the AE fusion gene by ddPCR further contributes to improved prognosis. Our study provides valuable methods for dynamic surveillance of AE fusion DNA and assistance in determining the prognosis.

Blast crisis develops in a minority of patients with chronic myeloid leukemia even in the era of tyrosine kinase inhibitor (TKI) therapy. Reports suggest that we know little about the mechanism of BCR-ABL and AML1-ETO co-expression in blast crisis of chronic myeloid leukemia, and that other chromosomal abnormalities also coexist. Here, we document an unusual and interesting case of a 51-year-old female diagnosed in the chronic phase of chronic myeloid leukemia. After undergoing TKI treatment for 3 months, her bone marrow aspirates in the chronic phase had transformed to blast crisis. Molecular genetic testing indicated she was positive for p210 form of BCR-ABL (copy number decreased from 108.91% to 56.96%) and AML1-ETO fusion (copy number, 5.65%) genes and had additional chromosomal abnormalities of t(8; 21)(q22; q22)/t(9; 22)(q34; q11), t(2; 5)(p24; q13) and an additional +8 chromosome.

Coexistence of AML1-ETO and PML-RARa has rarely been reported, and there is limited information on a special subtype of AML characterized by co-expression of AML1-ETO and PML-RARa. Herein, we prese...

Background: Deregulation of self-renewal and differentiation programs are central to the pathogenesis of hematologic malignancies. MicroRNAs (miRNAs) represent a large class of post-transcriptional regulators that mediate repression of multiple target mRNAs and are frequently deregulated in acute myeloid leukemia (AML). From our previous in vivo miRNA enforced expression screen in human hematopoietic stem and progenitor cells (HSPC), we identified miR-130a as a regulator of self-renewal and lineage specification. Enforced expression of miR-130a in human cord blood (CB) derived HSPC caused an expansion of HSC, block in erythroid differentiation and abnormal myelopoiesis in xenografts. Thus, we examined miR-130a expression in AML and found miR-130a to be specifically upregulated in t(8,21) AML. The translocation t(8,21) is one of the most common karyotypic abnormalities in AML, accounting up to 10% of all AML cases. The consequence of this translocation is a fusion of AML1 and ETO genes, resulting in a formation of the AML1-ETO (AE) oncofusion protein, which acts as a dominant repressor of the wild type AML1/RUNX1. The ETO moiety mediates the recruitment of the nuclear corepressor (NCoR) and histone deacetylases (HDAC1-3) to block RUNX1 target gene expression. This prevents myeloid maturation, apoptosis and promotes leukemogenesis. Here we investigated the molecular mechanism of miR-130a in t(8,21) AML and how it contributes to the leukemogenesis of this AML subtype. Results: Using the TCGA dataset and our PMCC patient cohort, we identified miR-130a to be upregulated in t(8,21) AML and high miR-130a expression was associated with worse patient overall survival. To interrogate the functional significance of elevated miR-130a in t(8,21) AML, we performed knock-down (KD) experiments in the Kasumi-1 cell line, which represents a well characterized model system for t(8,21) AML. Notably, KD of miR-130a induced a significant reduction in the CD34+ cell population and an increase in differentiated CD11b+CD15+ and pro-apoptotic annexin V+ cells. We next examined the impact of miR-130a KD in CD34+ blasts from primary t(8,21) AML patient samples. In line with our findings in the Kasumi-1 cells, miR-130a KD decreased the proportion of CD34+ cells and increased the proportion of differentiated CD11b+CD15+ blasts. To investigate the effect of miR-130a KD on leukemic engraftment in vivo, we transduced CD34+ blasts from 2 patient samples and transplanted them into NSG-GF mice. miR-130a KD decreased leukemic engraftment and the proportion of transduced cells, corroborating the functional significance of high miR-130a expression in t(8,21) AML. To investigate the mechanistic action of miR-130a, we performed label-free semi-quantitative proteomics in human CB derived HSPC to uncover miR-130a targets. Surprisingly, we found the beta subunit of RUNX1, CBFb, and Transducin Beta Like 1 X-Linked Receptor 1, TBL1XR1, to be among the most repressed targets. TBL1XR1 is a component of the nuclear receptor corepressor (NCoR) complex and is involved in NCoR degradation. Thus, we performed western and immunoprecipitations (IP) assays in Flag-AE Kasumi-1 cells following miR-130a KD to examine changes in the expression of proteins associated with the AE complex. We observed increased expression of CBFβ, TBL1XR1 and CEBPA with miR-130a KD. Notably, miR-130a KD resulted in a dramatic decrease of NCoR protein levels. IP of Flag-AE showed decreased association of CBFβ and NCoR with AE, despite unaltered protein levels of AE. To investigate changes in binding occupancy of Flag-AE after miR-130a KD, we performed Cleavage Under the Targets and Release Using Nuclease (CUT&RUN) assay. Surprisingly, we observed 2-fold gain of AE sites in miR-130a KD sample compared to control. De novo motif enrichment analysis showed loss of motives for ETS and HOX transcription factors known to associate with AE following miR-130a KD. Genomic distribution of the peaks revealed a dramatic shift of AE peaks away from the promoter region to introns in miR-130a KD. Pathway enrichment analysis of the unique peaks gained in miR-130a KD showed stress responses and organelle disassembly, in line with the differentiation phenotype observed with miR-130a KD. Collectively, we uncovered a novel mechanism by which miR-130a reinforces the aberrant AE molecular program by controlling the composition and binding of the AE complex. Disclosures Dick: Bristol-Myers Squibb/Celgene: Research Funding.

Author(s): Marneth, Anna E; Botezatu, Lacramioara; Hones, Judith M; Israel, Jimmy CL; Schutte, Judith; Vassen, Lothar; Lams, Robert F; Bergevoet, Saskia M; Groothuis, Laura; Mandoli, Amit; Martens, Joost HA; Huls, Gerwin; Jansen, Joop H; Duhrsen, Ulrich; Berg, Tobias; Moroy, Tarik; Wichmann, Christian; Lo, Mia-Chia; Zhang, Dong-Er; van der Reijden, Bert A; Khandanpour, Cyrus

The AML1/ETO onco-fusion protein is crucial for the genesis of t(8;21) acute myeloid leukemia (AML) and is well documented as a transcriptional repressor through dominant-negative effect. However, little is known about the transactivation mechanism of AML1/ETO. Through large cohort of patient's expression level data analysis and a series of experimental validation, we report here that AML1/ETO transactivates c-KIT expression through directly binding to and mediating the long-range interaction between the promoter and intronic enhancer regions of c-KIT. Gene expression analyses verify that c-KIT expression is significantly high in t(8;21) AML. Further ChIP-seq analysis and motif scanning identify two regulatory regions located in the promoter and intronic enhancer region of c-KIT, respectively. Both regions are enriched by co-factors of AML1/ETO, such as AML1, CEBPe, c-Jun, and c-Fos. Further luciferase reporter assays show that AML1/ETO trans-activates c-KIT promoter activity through directly recognizing the AML1 motif and the co-existence of co-factors. The induction of c-KIT promoter activity is reinforced with the existence of intronic enhancer region. Furthermore, ChIP-3C-qPCR assays verify that AML1/ETO mediates the formation of DNA-looping between the c-KIT promoter and intronic enhancer region through the long-range interaction. Collectively, our data uncover a novel transcriptional activity mechanism of AML1/ETO and enrich our knowledge of the onco-fusion protein mediated transcription regulation.

Rationale: Acute myeloid leukemia (AML) is a malignantly clonal and highly heterogeneous disease. Although the treatment of AML has brought promising outcomes for younger patients, prognosis of the elderly remains dismal. Innovative regimens are increasingly necessary to be investigated. Patient concerns: We present an 86-year-old AML patient with fever, cough, and sputum production. Diagnoses: A diagnosis of AML with maturation (AML-M2) and AML1/ETO was made. Interventions: The patient was treated with a regimen of Homoharringtonine coupled with arsenic trioxide. Outcomes: The AML-M2 patient with AML1/ETO achieved incomplete remission, but showed few toxic side effects and improved survival. Besides, we analyzed the dynamic counts of complete blood cells during the treatment. The count of white blood cell had a positive correlation with the percentage of blast cells (r = 0.65), both of which had a negative correlation with the percentage of segmented neutrophils (r = –0.63, –0.89). Lessons: Homoharringtonine and arsenic trioxide may induce both the apoptosis and differentiation of leukemic cells in AML-M2 with AML1/ETO.

Onset of acute myeloid leukemia (AML) has been accounted for by cooperation between multiple genetic alterations which induce abnormal control of various cellular pathways. Among the previously listed leukemogenic lesions, AML1-ETO fusion (AE) generated by translocation (8;21) (q22;q22) is one of the common mutations observed in 20-40% of patients. AE affects transcriptional regulation associated with hematopoietic differentiation, while 60% of AE-positive AML cases are shown to have together other types of mutation of genes involved in cell proliferation, such as receptor tyrosine kinase (RTK) c-kit and FLT3. However, the detailed mechanisms of how they work in the very early stages of leukemogenesis and what unknown "cooperative " cues function in those periods. From this viewpoint, in order to identify novel cellular molecules involved in the acquisition of leukemic phenotypes, we have conducted the gene-trap strategy-based phenomic screen in the use of pluripotent stem cell (PSC)-derived hematopoietic culture. Through our screening, we found that the functional knockdown of a gene NSFL1c, also known as p47, enhanced the activities of hematopoietic progenitor cells harboring AE to show leukemic phenotype both in vivo and in vitro: Cells differentiated from AE-PSCs which have additionally the poly A trapping sequence inserted in NSFL1c locus showed doubled efficiency in engraftment into immunodeficient NOG mice than cells without trapping (3.1% v.s. 1.3%, 16 weeks after intra bone marrow transplantation), and also showed the significantly higher colony replating efficacy in methylcellulose colony forming assay. In order to clear what lineages of cells were most responsible for those phenomena, we next performed those colony forming experiments after sorting of CD34+CD43+CD13- immature cells, CD34+/-CD43+CD13+ myeloid cells and CD71+CD41+ erythro-megakaryocytic subpopulations of cells. As a result, CD34+/-CD43+CD13+ myeloid precursors showed the strongest tendencies to emerge highly proliferative clones followed by CD34+CD43+CD13- immature progenitors in the presence of NSF1c trapping. Interestingly, those activities were cancelled in the absence of AE expression. NSFL1c is one of the component of NEMO complex which binds to ubiquitinated NEMO and induces its degradation, resulting in reduced IKK and elevated NFkB activities. In AML, elevated NF-κB pathways have been detected in more than 30% of patients. Although NF-κB signaling networks have proved induced by inflammatory and immune signals, and previous studies in vivo and in vitro showed their abnormal activities make leukemia cells escape from cell death and go into abnormal proliferation, the detailed mechanisms of how they are involved in the pathogenesis, in particular at the very early stages of the leukemogenesis, are well not defined. Our data therefore reflect the novel mechanisms behind the deviation of progenitor cell fate from normal to abnormal pathway leading to the emergence of leukemic initiating cells, and suggested the myeloid-biased leukemogenic potentials. Disclosures No relevant conflicts of interest to declare.

A novel exon in AML1-ETO negatively influences the clonogenic potential of the t(8;21) in acute myeloid leukemia

AML1-ETO fusion protein is observed in approximately 12% of acute myeloid leukemia. In the present research, we found that AML1-ETO is able to inhibit Sp1 transactivity. We also found that this inhibition of Sp1 transactivity by AML1-ETO is achieved by interaction between Sp1 and RUNT domain of AML1. AML1b is able to abrogate the inhibition of AML1-ETO. Since Sp1 is involved in hematopoietic cell differentiation, we proposed that AML1-ETO promotes leukemogenesis by blocking cell differentiation through inhibition of Sp1 transactivity.Structured summaryMINT-6549474: AML1-ETO (genbank_protein_gi:AAB34820) physically interacts (MI:0218) with Sp1 (uniprotkb:P08047) by anti bait coimmunoprecipitation (MI:0006)MINT-6549439: Sp1 (uniprotkb:P08047) physically interacts (MI:0218) with AML1-ETO (uniprotkb:AAB34820) by anti tag coimmunoprecipitation (MI:0007)MINT-6549458: Sp1 (uniprotkb:P08047) physically interacts (MI:0218) with AML1a (uniprotkb:Q01196-2) by anti tag coimmunoprecipitation (MI:0007)

t(8;21)(q22;q22) results in the AML1-ETO (A1E) fusion gene and is a common cytogenetic abnormality in acute myeloid leukemia (AML). Although insertions at the breakpoint region of the A1E fusion transcripts have been reported, additional structural alterations are largely uncharacterized. By RT-PCR amplifications and DNA sequencing, numerous in-frame and out-of-frame AML1b-ETO and AML1c-ETO transcripts were identified in 13 pediatric t(8;21) AMLs, likely resulting from alternate splicing, internal deletions and/or breakpoint region insertions involving both the AML1 (RUNX1) and ETO regions. The in-frame A1E fusion transcript forms represented minor forms. These structure alterations were found in AML1c-ETO but not AML1b-ETO transcripts in two adult t(8;21) AMLs. Although no analogous alterations were detected in native AML1b transcripts, identical alterations in native ETO transcripts were identified. When transfected into HeLa cells, only AML1b, and not the in-frame A1E forms, transactivated the GM-CSF promoter. In co-transfection experiments, the effects of A1E proteins on GM-CSF transactivation by AML1b ranged from repressive to activating. Our results demonstrate a remarkable and unprecedented heterogeneity in A1E fusion transcripts in t(8;21) myeloblasts and suggest that synthesis of alternate A1E transcript and protein forms can significantly impact the regulation of AML1 responsive genes.

Conventional/molecular cytogenetics is important in identification of genomic abnormalities, for prognostication and in risk stratification of de novo patients with acute myeloid leukemias (AML). Here we present an AML M2 case showing the sole karyotypic abnormality, the rare interstitial deletion in the long arm of chromosome 9 with the loss of segment q12–q13.

Acute myeloid leukaemia (AML) with the t(8;21)(q22;q22) is deemed to be a 'good-risk' disease. 396 patients with AML at diagnosis were screened for the presence of t(8;21) and AML1/ETO fusion transcripts by cytogenetic and RT-PCR techniques respectively. 32 cases of t(8;21) were detected, all of which were also PCR positive. A further 19 cases were detected at the molecular level, predominantly but not exclusively in M1 and M2 FAB types. Approximately 12% of all new cases of AML are estimated to have AML1/ETO fusion transcripts and it is suggested that molecular screening should be performed in all cases with the possible exception of the M3 FAB type.

AML1/ETO and POU4F1 synergy drives B-lymphoid gene expression typical of t(8;21) acute myeloid leukemia

To the editor: Identification of interacting proteins essential for oncogenic functions of leukemia-associated transcription factors is important for understanding the underlying transformation mechanisms and designing effective cancer therapeutics.[1][1] We have recently found that homo

NHR4 domain mutations of ETO are probably very infrequent in AML1–ETO positive myeloid leukemia cells

AML1-ETO and TEL-AML1 are chimeric proteins resulting from the t(8;21)(q22;q22) in acute myeloid leukemia, and the t(12;21)(p13;q22) in pre-B-cell leukemia, respectively. The Runt domain of AML1 in both proteins mediates DNA binding and heterodimerization with the core binding factor β (CBFβ) subunit. To determine whether CBFβ is required for AML1-ETO and TEL-AML1 activity, we introduced amino acid substitutions into the Runt domain that disrupt heterodimerization with CBFβ but not DNA binding. We show that CBFβ contributes to AML1-ETO's inhibition of granulocyte differentiation, is essential for its ability to enhance the clonogenic potential of primary mouse bone marrow cells, and is indispensable for its cooperativity with the activated receptor tyrosine kinase TEL-PDGFβR in generating acute myeloid leukemia in mice. Similarly, CBFβ is essential for TEL-AML1's ability to promote self-renewal of B cell precursors in vitro. These studies validate the Runt domain/CBFβ interaction as a therapeutic target in core binding factor leukemias.

We analyze the prevalence and clinical features of a group of patients with t(8;21) (q22;q22) acute myeloblastic leukemia, identified in a single institution in México over a 10-year period. Fifteen patients presented at the Centro de Hematología y Medicina Interna de Puebla from February 1995 to August 2005; only nine were treated and followed in the institution. Median age was 24 years, (range 7-49); there was only one male. According to the French-American-British (FAB) morphological classification of leukemia, the morphology was M2 in four cases, M4 in three cases, M3 in one case and M0 in one. In addition to the myeloid markers, lymphoid markers were identified in 6 patients. Patients were induced to remission with combined chemotherapy and three subsequently underwent bone marrow transplantation (BMT). The median overall and disease-free survival has not been reached, being above 3390 days, the probability of survival at this time was 73%. In this single-center experience in México, we found that the t(8;21) (q22;q22) variant of leukemia was more frequent than in Caucasian populations, that the co-expression of lymphoid markers in the blast cells is very frequent and that this malignancy is associated with a relatively good prognosis.

In this issue of Blood, Chou et al1 and Pulikkan et al2 reveal that the (8;21)(q22;q22) translocation product AML1-ETO (RUNX1-RUNX1T1) leads to enhanced JAK/STAT signaling downstream of myeloproliferative leukemia (MPL) virus, which in turn contributes to increased survival of tumor cells. Hijacking of the JAK/STAT pathway through up-regulation of wild-type MPL is a new paradigm for hematologic malignancies.

Acute myeloid leukemia (AML) is a heterogeneous disease, with individual cases showing variability in clinical presentation, blast-cell morphology, therapeutic response, and long-term prognosis. One of the most common cytogenetic abnormalities described in AML is t(8;21)(q22;q22) found in 10% to 15

[Comment on Fazi et al, page 4432][1]In this issue of Blood , Fazi and colleagues demonstrate that ligand-activated transcription via retinoic acid receptor α, which is important for proper myelomonocytic differentiation, is blocked by the AML1/ETO fusion oncoprotein. Approximately 15% of all

JAK2 seems to be a typical cooperating mutation in therapy-related t(8;21)/ AML 1- ETO -positive AML

To the Editor : We have evaluated the occurrence of the AML1/ETO fusion transcripts by reverse transcription-polymerase chain reaction (RT-PCR) analysis[1][1] in 7 patients with t(8; 21)(q22; q22) acute myeloid leukemia (AML) who underwent allogeneic bone marrow transplantation (BMT; n = 6) or