Humans ; Leukemia, Myeloid, Acute/genetics* ; Myeloid-Lymphoid Leukemia Protein/genetics* ; Oncogene Proteins, Fusion/genetics* ; Translocation, Genetic

Humans ; Child ; Myeloid-Lymphoid Leukemia Protein/genetics* ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; Ubiquitin Thiolesterase

OBJECTIVETo establish the ADAR1 (adenosine deaminase that act on RNA 1) knockout MLL-AF9 acute myeloid leukemia (AML) mouse model, and to preliminarily investigate the effects of ADAR1 deletion on the development of AML.

METHODSThe lineage⁻ (Lin⁻) cells of ER-CreADAR1(lox/lox) mice and their ADAR1(lox/lox) counterparts were enriched by magnetic activated cell sorting (MACS) and then transduced with retrovirus carrying MSCV- MLL/AF9-IRES-GFP fusion gene. The efficiency of transduction was detected by flow cytometry, and equal number of GFP⁺ cells were transplanted into lethally irradiated recipient mice. The recipient mice were treated with tamoxifen at 48 hours after transplantation to induce ADAR1 knockout and divided into following groups: experimental group (ER-Cre;ADAR1(lox/lox)+tamoxifen), control groups ((1)ER-Cre;ADAR1(lox/lox)+vechile, (2)ADAR1(lox/lox)+tamoxifen, (3)ADAR1(lox/lox)+vechile). The percentage of GFP⁺ cells in peripheral blood was examined at 10, 15 and 20 days respectively after transplantation and the survival of the recipient mice was observed. In vitro study, ER-Cre;ADAR1(lox/lox) and ADAR1(lox/lox) AML cells were cultured and the apoptosis rates of these cells 48 hours after 4-hydroxytamoxifen treatment were examined.

RESULTSThe ADAR1 deletion MLL-AF9 AML mouse model was successfully established. Deletion of ADAR1 could decrease the percentage of GFP⁺ cells in the peripheral blood and significantly prolong the survival rate of recipient mice（P<0.05）. In vitro study showed that the cultured total cell number, percentage of GFP⁺ cells decreased and the apoptosis rate of AML cells increased.

CONCLUSIONAblation of ADAR1 could delay the progression of AML in recipient mice. ADAR1 plays a critical role in the development and maintenance of murine MLL-AF9 AML.

Adenosine Deaminase ; Animals ; Apoptosis ; Disease Models, Animal ; Leukemia, Myeloid, Acute ; Mice ; Myeloid-Lymphoid Leukemia Protein ; Tamoxifen ; analogs & derivatives

OBJECTIVE: To investigate the role of bone marrow vascular niche in the development of MLL-AF9 acute myeloid leukemia (AML). METHODS: Transplantation experiments were performed to establish non-radiated MLL-AF9 AML model; the half-bone immunofluorescence staining and tile-scan imaging of two-photon confocal microscopy were used to obtain the data of 3 main bone marrow niche cells; flow cytometry analysis was performed to characterize leukemia cells in different anatomical sites. RESULTS: In the early stage of MLL-AF9 AML, the proportion of leukemia cells in the metaphysis of the femur was significantly higher than that in the diaphysis. The detection of apoptosis and proliferation rate of leukemia cells showed that the percentage of leukemia cells in metaphysis significantly decreased, and the proliferation (S/G/M phase) was also significantly more active. These different features of leukemia cells may relate with different bone marrow microenvironment. The image data of 3 major components of bone marrow niche (endothelial cells, endosteum, megakaryocytes) showed different distribution of blood vessels in metaphysis and diaphysis. Furtherly comparing the spatial distance between leukemia cells and endothelial cells, endosteum, megakaryocytes indicated that leukemia cells are closer to the blood vessels, suggesting the important role of blood vessels in the development of leukemia. Glucose uptake assays and intracellular ROS detection showed that the supportive role of blood vessels for leukemia cells did not related with nutrient metabolism pathway. CONCLUSION The vascular niche plays an important role in the development of leukemia, and does not relate with the transport of nutrients and the elimination of metabolic waste, instead, which may relate with perivascular cytokines or other vascular functions.
Acute Disease ; Apoptosis ; Bone Marrow ; Bone Marrow Cells ; Humans ; Leukemia, Myeloid, Acute ; Myeloid-Lymphoid Leukemia Protein ; Oncogene Proteins, Fusion

OBJECTIVETo study the clinical and laboratory features of childhood acute leukemia (AL) with MLL gene rearrangements.

METHODSSixteen of 298 cases of childhood AL with MLL rearrangements were studied by using MLL dual-color FISH, multiplex RT-PCR with 13 pairs of primers in combination with R banding karyotype analysis and cell immunophenotyping by flow cytometry.

RESULTSSixteen cases of childhood AL with MLL rearrangements accounted for 5.4% of 298 AL patients, and 56.3% of infant ALs. Among 106 cases analyzed by multiplex RT-PCR, MLL gene rearrangements were found in 11 cases, including MLL/AF4 fusion gene in 2, MLL/AF6 fusion gene in 1, MLL/AF6 and MLL/ELL combined with MLL/ AFX or HOX11 in one case each, MLL/AF9 in 2, MLL/AF10 in 1, MLL/ELL in 2. MLL partial tandem duplication in 1 and activated HOX11 in 1. In 27 cases assayed by FISH, 9 cases (36.0%) were demonstrated MLL gene rearrangements. In 16 patients with MLL gene rearrangements, 14 (87.5%) exhibited clonal chromosome abnormalities involved chromosome 11 in 11 cases: being t(4;11) in 2, t(6;11), t(8;11), t(7;8;11), t(9;11) in each trisomy 11 in 2 and 11q--in 3 cases. Among these 16 patients, 11 were B-ALL, and 5 AML-M5, 3 of the latter were CD7+ and CD2+. Of these 16 patients, 8 received chemotherapy and 7 of them achieved complete remission, while the other 8 patients gave up treatment.

CONCLUSIONMultiplex RT-PCR combined with FISH provided a more accurate and sensitive method for detection of MLL gene rearrangements. Finding out MLL gene rearrangement is of most importance in guiding therapy and predicting prognosis in childhood AL.

Adolescent ; Child ; Child, Preschool ; Female ; Gene Rearrangement ; Humans ; In Situ Hybridization, Fluorescence ; Infant ; Leukemia ; genetics ; Male ; Myeloid-Lymphoid Leukemia Protein ; genetics

Mixed lineage leukemia proteins (MLL) are the key histone lysine methyltransferases that regulate expression of diverse genes. Aberrant activation of MLL promotes leukemia as well as solid tumors in humans, highlighting the urgent need for the development of an MLL inhibitor. We screened and isolated MLL1-binding ssRNAs using SELEX (Systemic Evolution of Ligands by Exponential enrichment) technology. When sequences in sub-libraries were obtained using next-generation sequencing (NGS), the most enriched aptamers—APT1 and APT2—represented about 30% and 26% of sub-library populations, respectively. Motif analysis of the top 50 sequences provided a highly conserved sequence: 5′-A[A/C][C/G][G/U][U/A]ACAGAGGG[U/A]GG[A/C] GAGUGGGU-3′. APT1, APT2, and APT5 embracing this motif generated secondary structures with similar topological characteristics. We found that APT1 and APT2 have a good binding activity and the analysis using mutated aptamer variants showed that the site information in the central region was critical for binding. In vitro enzyme activity assay showed that APT1 and APT2 had MLL1 inhibitory activity. Three-dimensional structure prediction of APT1-MLL1 complex indicates multiple weak interactions formed between MLL1 SET domain and APT1. Our study confirmed that NGS-assisted SELEX is an efficient tool for aptamer screening and that aptamers could be useful in diagnosis and treatment of MLL1-mediated diseases.
Aptamers, Nucleotide ; Conserved Sequence ; Diagnosis ; Histones ; Humans ; In Vitro Techniques ; Leukemia ; Ligands ; Lysine ; Mass Screening ; Methyltransferases ; Myeloid-Lymphoid Leukemia Protein ; RNA

BACKGROUNDPartial tandem duplication of mixed lineage leukaemia (MLL-PTD) is detected both in patients with acute leukemia and in healthy people. However, MLL-PTD in relatives of patients with MLL-PTD has not been reported. The objective of this study was to investigate the expression of MLL-PTD in patients with acute leukemia and in their relatives.

METHODSThe bone marrow or peripheral blood was collected from patients with acute leukaemia and their relatives. Nested reverse transcription-polymerase chain reaction (RT-PCR) was applied to detect the mRNA expression of the MLLPTD fused gene, and further confirm in genomic DNA level.

RESULTSAnalysing MLL-PTD in case 1, the patient's older brother and his younger brother were positive, while his mother and his son were negative. The exon type in case 1 was e9/3 fusion, but in his older brother, it was e9/3 and e11/3 fusion, and in his younger brother, it was e9/3, e10/3, and e11/3 fusion. MLL-PTD in case 2 was negative, but in the patient's older sister was positive, and the exon type was e9/3, e10/3, and e11/3.

CONCLUSIONSThe expression of MLL-PTD was present in cases with acute leukaemia with a single expression type. However, various expression types were detected in their healthy relatives. MLL-PTD can couple with other chromosome aberrations, and its impact on disease prognosis remains to be studied further.

Acute Disease ; Chromosome Aberrations ; Female ; Humans ; Leukemia, Myeloid, Acute ; genetics ; Male ; Myeloid-Lymphoid Leukemia Protein ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

UNLABELLEDThis study was aimed to explore the value of detecting the expression levels of MLL-AF9 (mixed lineage leukemia, MLL) fusion gene during the treatment of acute myeloid leukemia (AML) by real-time fluorescence quantitative PCR (RQ-PCR), and to evaluate its prognostic significance in monitoring minimal residual disease (MRD). The expression levels of 11 patients with MLL-AF9 fusion gene positive were detected precisely by RQ-PCR during the treatment in order to analyze the correlation of detection results with clinical manifestations. The results showed that the expression levels of MLL-AF9 fusion gene in patients at initial diagnosis were 1.3%-55.28%.

RESULTSobtained from 5 patients who received chemotherapy alone during the interval between first and second courses of chemotherapy indicated that 2 patients with <0.1% of MLL-AF9 fusion gene expression levels all achieved hematologic complete remission and survived, while the remaining 3 patients with ≥ 0.1% of MLL-AF9 fusion gene did not achieve hematologic complete remission and only 1 case survived. Moreover, results obtained from 6 transplant patients within a month before the transplantation suggested that 4 of them with < 0.1% of MLL-AF9 fusion gene expression levels survived without relapses, while the remaining 2 patients with ≥ 0.1% of MLL-AF9 fusion gene expression levels relapsed and died. Besides, MLL-AF9 fusion gene expression levels were ≥ 0.1% within one month before the morphological relapse of bone marrow in 2 recurrent patients. It is concluded that the detecting the expression level of MLL-AF9 fusion gene by RQ-PCR is an effective and accurate method to quantify and monitor the MRD level of MLL-AF9 gene positive AML patients and may be used for early detecting molecular relapse of AML.

Humans ; Leukemia, Myeloid, Acute ; diagnosis ; genetics ; Myeloid-Lymphoid Leukemia Protein ; genetics ; Neoplasm, Residual ; genetics ; Oncogene Proteins, Fusion ; genetics ; Prognosis ; Real-Time Polymerase Chain Reaction ; methods

This study was purposed to investigate the incidence of mixed lineage leukemia (MLL) gene rearrangement and partner gene types as well as the clinical features and prognosis of acute leukemia (AL) with this rearrangement through detection in adult AL using combination of 3 techniques, and to evaluate the clinical value of this combination detection. The MLL gene rearrangement in 183 cases of adult AL was detected by combination of conventional cytogenetics, split signal FISH and multiplex nested PCR. The results showed that the incidence of MLL rearrangements in adult patients with AL was low (8.2%), and MLL-AF4 fusion gene was most common and predominant in acute lymphoblastic leukemia (ALL), while the MLL-AF6 and MLL-AF9 were most frequent in acute myeloid leukemia (AML). Extramedullary involvements were found in 40% of MLL-rearranged AL patients, and 33.3% of patients with MLL-rearranged AL reached to complete remission within 30 days during induction chemotherapy. In addition, in this cohort of MLL-rearranged adult AL patients, the 3-month relapse rate and 6-month overall survival rate were 50.0% and 50.0% respectively. It is concluded that the rate of missed diagnosis of CC technique for patients with MLL-rearranged AL reached to 60% in this study, while the combination of CC, FISH and multiplex nested PCR has been confirmed to have important significance for evaluating prognosis and conducting clinical therapy of patients with MLL-rearranged AL.
Adolescent ; Adult ; Aged ; Female ; Gene Rearrangement ; Humans ; Leukemia, Myeloid, Acute ; genetics ; Male ; Middle Aged ; Myeloid-Lymphoid Leukemia Protein ; genetics ; Oncogene Proteins, Fusion ; genetics ; Young Adult

OBJECTIVE: To investigate the clinical features and prognosis of acute myeloid leukemia (AML) patients with the mixed lineage leukemia (MLL) gene rearrangements AF6 (MLL-AF6) positive. METHODS: In the study, 11 patients who were newly diagnosed with MLL-AF6 positive AML were analyzed retrospectively, related literature was reviewed to clarify the clinical features and prognosis of MLL-AF6 positive patients. RESULTS: Among the 11 patients, there were 6 males and 5 females, with a median age of 36 years. Six patients were diagnosed with AML M5 and five with M4 according to FAB classification (French-American-British classification systems). Gingival swelling and pain occurred in 6 cases and fever occurred in 5 cases. At first diagnosis, the median white blood cells were 55.5×10⁹/L. Immunotype showed the expression of myeloid/monocyte and early stem cell series antigens. The expression level of MLL-AF6 fusion gene (real-time quantitative PCR) was 14.2%-214.5%, and 6/11 cases (54.5%) were associated with high EVI1 gene expression. Mutations of KRAS, TET2, ASXL1, TP53, DNMT3A, and FLT3-ITD were detected by next generation sequencing (NGS) in 4 patients. Chromosome G banding examination showed that 2 cases were t(6;11)(q27, q23) with complex karyotype abnormality, 4 cases with +8 abnormality and 2 cases with normal karyotype. Hematological complete remission (CR) was achieved in 8/11 patients (72.7%) after conventional induction chemotherapy, and primary drug resistance was observed in 3 patients. Two of the eight patients with CR were negative for minimal residual disease (MRD), with a median CR duration of 4.5 months. Two patients with positive MRD and three patients with refractory recurrence underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT), but all died due to leukemia progression. At the end of follow-up on December 1, 2019, 2 patients were alive and 9 died, with median survival time of 9 months. CONCLUSION The AML patients with MLL-AF6 positive were mostly young, the majority of FAB types were M4 and M5, and most of the patients often had fever as the first symptom, with increased white blood cells, accompanied by organ infiltration, and high EVI1 gene expression. The hematological remission rate of routine chemotherapy is not low, but it is difficult to achieve molecular remission, most of which have early recurrence. Early allo-HSCT in a molecular negative state may prolong the CR duration.
Adult ; Female ; Hematopoietic Stem Cell Transplantation ; Humans ; Leukemia, Myeloid, Acute/genetics* ; Male ; Myeloid-Lymphoid Leukemia Protein/genetics* ; Oncogene Proteins, Fusion/genetics* ; Prognosis ; Remission Induction ; Retrospective Studies