Transcriptional Regulation of RUNX1: An Informatics Analysis

The RUNX1/AML1 gene encodes a developmental transcription factor that is an important regulator of haematopoiesis in vertebrates. Genetic disruptions to the RUNX1 gene are frequently associated with acute myeloid leukaemia. Gene regulatory elements (REs), such as enhancers located in non-coding DNA, are likely to be important for Runx1 transcription. Non-coding elements that modulate Runx1 expression have been investigated over several decades, but how and when these REs function remains poorly understood. Here we used bioinformatic methods and functional data to characterise the regulatory landscape of vertebrate Runx1. We identified REs that are conserved between human and mouse, many of which produce enhancer RNAs in diverse tissues. Genome-wide association studies detected single nucleotide polymorphisms in REs, some of which correlate with gene expression quantitative trait loci in tissues in which the RE is active. Our analyses also suggest that REs can be variant in haematological malignancies. In summary, our analysis identifies features of the RUNX1 regulatory landscape that are likely to be important for the regulation of this gene in normal and malignant haematopoiesis.

Aberrant AML1 gene expression in the diagnosis of childhood leukemias not characterized by AML1-involved cytogenetic abnormalities

The AML1 ( acute myeloid leukemia 1) gene, a necessary prerequisite of embryonic hematopoiesis and a critical regulator of normal hematopoietic development, is one of the most frequently mutated genes in human leukemia, involving over 50 chromosome translocations and over 20 partner genes. In the few existing studies investigating AML1 gene expression in childhood leukemias, aberrant upregulation seems to specifically associate with AML1 translocations and amplifications. The aim of this study was to determine whether overexpression also extends to other leukemic subtypes than the ones karyotypically involving AML1. We use quantitative real-time polymerase chain reaction methodology to investigate gene expression in 100 children with acute leukemias and compare them to those of healthy controls. We show that in childhood acute lymphoblastic leukemia, AML1 gene overexpression is associated with a variety of leukemic subtypes, both immunophenotypically and cytogenetically. Statistically significantly higher transcripts of the gene were detected in the acute lymphoblastic leukemia group as compared to the acute myeloid leukemia group, where AML1 overexpression appeared to associate with cytogenetic abnormalities additional to those that engage the AML1 gene, or that are reported as showing a “normal” karyotype. Collectively, our study shows that AML1 gene overexpression characterizes a broader range of leukemic subtypes than previously thought, including various maturation stages of B-cell acute lymphoblastic leukemia and cytogenetic types additional to those involving the AML1 gene.

Association of the RUNX1/AML1 Gene Mutation and Familial MDS/AML: A Case Report and Review of Literature

Myelodysplastic syndrome (MDS) and Acute Myeloid Leukemia (AML) are mostly sporadic hematopoetic malignancies, which generally thought inheritable, though specific gene abnormalities may play a role in the progression of myelodysplasia. Recently, many reports have revealed that mutations in the RUNX1/AML1 gene are involved in defective hematopoiesis. Moreover, patients with such mutations have a significant poorer prognosis than cases without RUNX1 mutations. Since familial occurrence of MDS/AML is rare, there are few reports about the relationship between RUNX1 mutations and familial MDS/AML. In the study of a familial MDS/AML, we found a61-year-old male with MDS who developed into AML later. He had a history of leukocytopenia and thrombocytopenia for decades before developing into pancytopenia. We identified a RUNX1mutationin this patient through sequencing. Furthermore, the same mutation was present in his son (II-1) who suffered from MDS as well as his granddaughter. Intriguingly, II-1 was 10years old when diagnosed with MDS, nearly 50 years earlier than his father. Myelodysplastic features in bone marrow sections of these two patients were not obvious in the early stage. They mainly showed active erythrocyte proliferation, together with an increased mean corpuscular volume, which can indicate inflection points in MDS progression. To date, patients with familial MDS had no antecedent hematological abnormalities in the early stage. In this study, we found a new inheritable mutation of the RUNX1 gene in the familial MDS which is frequently deregulated in hematological malignancies. As RUNX1 was present in the early stage of familial MDS, this genetic mutation may serve as an early diagnostic marker and contribute to the treatment of MDS/AML. Disclosures No relevant conflicts of interest to declare.

Implication of HOXA9, MEIS1, AML1 and IRF4 In Childhood Acute Leukemia (AL) In Greek Patients

Abstract 4643 Introduction: Childhood AL is characterized by abnormal chromosomal translocations and gene fusions. The most common cytogenetic aberrations are: TEL/AML1 (25%), E2A/PBX1 (4.8%), BCR/ABL (1.6%), MLL rearrangements (1.6%) and AML1/ETO (exclusively found in acute myeloid leukemia (AML)). Their high prognostic significance determines the intensification (eg. for MLL rearrangements) or de-intensification (eg. for TEL/AML1) of therapy, even though the mechanisms through which these fusions affect the prognostic outcome are largely unknown. However, the majority (~70%) of childhood patients is negative for these translocations and, even though most present with numeric cytogenetic abnormalities (eg. aneuploidies) at diagnosis, they are treated with the same therapeutic protocols as those with known translocations. Only BCR/ABL+ AL patients receive individualized treatment (addition of imatinib mesylate) that offers a better prognostic outcome, thus representing an example of how childhood leukemia can benefit from individualized therapy. The study of the genes implicated in leukemic lesions can give insight into the pathogenesis of leukemia and hence provide new diagnostic markers and therapeutic targets. It is therefore fundamental to make scientific hypotheses based on the behaviour of such genes and then perform experiments to validate or reject them. In the present study we investigate the expression of 4 such genes: HOXA9 and MEIS1, implicated in both normal hematopoiesis and MLL+ leukemias; AML1, an important regulator of B-cell differentiation and the most frequent target of chromosomal translocations; and IRF4, a transcription factor of lymphocyte differentiation, also implicated in leukemias and lymphomas. The aim of this study is to examine whether there is a correlation between the expression of the above genes and the leukemic cytogenetic profile. Materials and Methods: RNA was extracted with Trizol (Invitrogen Inc.) from bone marrow samples of 43 newly diagnosed children with leukemia (39 acute lymphoblastic leukemia (ALL) and 4 AML), 20 healthy children (controls) and from 4 cell lines, each representing a different type of childhood leukemia: CCRF-CEM (T-cell ALL), CCRF-SB (B-cell ALL), Reh (non-T, non-B ALL) and THP-1 (acute monocytic leukemia). Gene expression was investigated with Real-Time Reverse Transcription PCR (qRT-PCR), using the Plexor one-step qRT-PCR kit (Promega Inc.). Fluorescence in situ hybridization (FISH) was performed with dual-color probes (Abbott Molecular, Inc.). Results: Cytogenetic data were available for 41 patients (pts): TEL/AML1+: 5 pts (ALL) (12.2%), MLL+: 4 pts (3 ALL and 1 AML) (9.7%), BCR/ABL+: 3 pts (ALL) (7.3%), E2A/PBX+: 2 pts (ALL) (4.8%), AML1/ETO+: 1 pt (AML); the remaining 26 patients (63%) were negative for these fusions but 6 (14.6%) had extra copies of the AML1 gene. Gene expression was as follows: HOXA9: up-regulated in 20 patients (46.51%) and the CCRF-SB, Reh and THP-1 cell lines; MEIS1: up-regulated in 19 patients (44.19%); AML1: up-regulated in 21 patients (48.84%) and all 4 cell lines; IRF4: up-regulated in 25 patients (58.14%) and down-regulated in the CCRF-SB cell line. Simultaneous co-expression of HOXA9 and MEIS1 was present in 15 patients (34.88%). Of the 6 patients having extra copies of AML1, only one showed over-expression of the gene. Conclusions: In our group of patients the frequency of the TEL/AML1 fusion appears to be less, whereas the frequency of the MLL rearrangement appears to be higher, than that reported for western European countries. We find all of the 4 genes studied significantly up-regulated in certain groups of patients, as compared to controls, regardless of leukemic subtype. Up-regulation of HOXA9 and MEIS1 did not appear to be an exclusive characteristic of the MLL+ group. In the case of the AML1 gene, we find that gene amplification does not correlate with over-expression of the gene. In addition, IRF4, a known tumor suppressor gene which would be expected to be down-regulated in newly diagnosed leukemias, seems to be the one most frequently up-regulated (58.14% of patients). Our findings lead us to suggest that there might be common patterns of aberrant gene expression among childhood leukemia patients regardless of cytogenetic subtype. Such patterns could be further investigated in an attempt to gain insight into the etiology of leukemogenesis. Disclosures: No relevant conflicts of interest to declare.

Translocation ETS leukemia-acute myeloid leukemia 1 (TEL-AML1) gene fusion in childhood acute lymphoblastic leukemia

Background Acute lymphoblastic leukemia (ALL) in childrenis a heterogeneous disease with different subtypes based on their cellular and molecular characteristics. This condition wouldinfluence the treatment outcome and subsequent risk for relapse. Accurate assignment of individual patients to risk groups is a critical issue for better outcome. TEL-AML1 gene fusion is themost frequent in childhood ALL.Objective The aim of this study was to investigate the incidenceofTEL-AML1 children with ALL in Sardjito Hospital.Methods This was a cross sectional study. In this preliminarystudy, we used nested reverse-transcriptase polymerase chainreaction (RT-PCR) to analyze the present of TEL-AML1 genefusion in bone marrow sample of childhood ALL patients.Results We analyzed 41 samples. Out of these, 30 (73%) wereamplified. Twenry three out of 30 ALL patients with good medicalrecord were analyzed for this gene fusion. Out of 30 patients, there were five patients (17%) with TEL-AML1-positive gene fusion and 25 (83%) were TEL-AML1-negative. Among five patients with TEL-AML1-positive gene fusion, four patients (80%) were one year to less than 10 year old. All of the patients (100%) were with leukocyte < 50x109/L.Conclusions TEL-AML1 gene fusion was found in 17 % ofsamples. This gene fusion was more frequent in standard risk group (based on age and leukocyte). These data must be clarified with more samples. RT-PCR must be apply in all center as one part of improving diagnostic quality, especially in managing leukemia patients.