RUNX1/AML1 DNA Binding Domain and ETO/MTG8 NHR2 Dimerization Domain Are Critical to AML1−ETO9a Leukemogenesis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 772-772
Author(s):  
Ming Yan ◽  
Scott Hiebert ◽  
Dong-Er Zhang
Keyword(s):  
Acute Myeloid Leukemia ◽  
Dna Binding ◽  
Median Survival ◽  
Myeloid Leukemia ◽  
Binding Domain ◽  
Chromosome 8 ◽  
Chromosome 21 ◽  
Dna Binding Domain ◽  
Dimerization Domain ◽  
Acute Myeloid

Abstract The 8;21 translocation, which involves the gene encoding the RUNX family DNA binding transcription factor AML1 (RUNX1) on chromosome 21 and the ETO (MTG8) gene on chromosome 8, generates AML1−ETO fusion proteins. Previous analyses have demonstrated that full length AML1−ETO blocks AML1 function and requires additional mutagenic events to promote leukemia in mice. More recently, we have identified an alternatively spliced form of AML1−ETO, AML1−ETO9a, from t(8;21) AML patient samples (Yan et al. Nat. Med.12:945–949, 2006). AML1−ETO9a lacks the C−terminal NHR3 and NHR4 domains of AML1−ETO and is highly leukemogenic in mice. Here, we report that the AML1 DNA binding domain and the ETO NHR2 dimerization domain, but not the ETO NHR1 domain are critical for the induction of acute myeloid leukemia by AML1−ETO9a. Using retroviral mediated gene expression and hematopoietic cell transplantation in recipient mice, we examined AML1−ETO9a, AML1−ETO9a without the NHR1 domain [AML1−ETO9a (dNHR1)] or the NHR2 domain [AML1−ETO9a(dNHR2)], without a histone deacetylase/Sin3A interacting domain between NHR1 and NHR2 [AML1−ETO9a(d350–428)], and mutant AML1−ETO9a proteins that have lost the capacity to bind DNA [AML1−ETO9a(L148D)] and [AML1−ETO9a(R173Q)] in leukemogenesis. All of the mice transplanted with AML1−ETO9a (n =11) and AML1−ETO9a(dNHR1) (n = 12) expressing cells developed acute myeloid leukemia with a similar phenotype (Lin−/c−kit+) within 21 weeks. The median survival times of mice with AML1−ETO9a and AML1−ETO9a(dNHR1) are 9.4 weeks and 10.5 weeks, respectively. Furthermore, all of the mice expressing AML1−ETO9a(d350–428) (n = 11) also developed leukemia with a median survival time of 17.2 weeks. Significant numbers of AML1−ETO9a(d350–428) expressing cells are positive for myeloid markers CD11b and Gr1 in these leukemic mice. In contrast, none of the mice with AML1−ETO9a(dNHR2) (n = 14), AML1−ETO9a(L148D) (n = 8), and AML1−ETO9a(R173Q) (n = 8) expressing hematopoietic cells developed leukemia. Taken together, these data suggest that the AML1 DNA binding domain and the ETO NHR2 domain are required for AML1−ETO9a induced leukemia development and the region between amino acids 350 and 428 of AML1−ETO9a also affects the differentiation stage and latency of leukemogenesis.

t(8;21) Breakpoints are Clustered between Alternatively Spliced Exons of MTG8

1995 ◽  
Vol 89 (3) ◽  
pp. 215-218 ◽  
Author(s):  
Jane E. Tighe ◽  
Franco Calabi
Keyword(s):  
Dna Binding ◽  
Acute Myeloid Leukaemia ◽  
Myeloid Leukaemia ◽  
Binding Domain ◽  
Chromosome 8 ◽  
Chromosome 21 ◽  
Dna Binding Domain ◽  
Alternatively Spliced ◽  
Alternatively Spliced Exons ◽  
Acute Myeloid

1. The (8;21) translocation, which is consistently associated with a subgroup of acute myeloid leukaemia, involves two loci: runt on chromosome 21 and MTG8 on chromosome 8. 2. Breakpoints in runt fall within a single intron that is located immediately downstream of a phylogenetically conserved DNA-binding domain (the ‘runt box’). 3. We now show that most breakpoints on chromosome 8 fall within a region between two alternative 5′ MTG8 exons. Thus, we predict that chimaeric genes on both the derivative(8) and the derivative(21) chromosomes have the potential to be transcriptionally active.

Heterozygous PU.1 mutations are associated with acute myeloid leukemia

Blood ◽  
2002 ◽  
Vol 100 (3) ◽  
pp. 998-1007 ◽  
Author(s):  
Beatrice U. Mueller ◽  
Thomas Pabst ◽  
Motomi Osato ◽  
Norio Asou ◽  
Lisa M. Johansen ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Dna Binding ◽  
Myeloid Leukemia ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Colony Stimulating Factor ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor ◽  
Acute Myeloid

Abstract The transcription factor PU.1 is required for normal blood cell development. PU.1 regulates the expression of a number of crucial myeloid genes, such as the macrophage colony-stimulating factor (M-CSF) receptor, the granulocyte colony-stimulating factor (G-CSF) receptor, and the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor. Myeloid cells derived from PU.1−/− mice are blocked at the earliest stage of myeloid differentiation, similar to the blast cells that are the hallmark of human acute myeloid leukemia (AML). These facts led us to hypothesize that molecular abnormalities involving the PU.1 gene could contribute to the development of AML. We identified 10 mutant alleles of the PU.1 gene in 9 of 126 AML patients. The PU.1 mutations comprised 5 deletions affecting the DNA-binding domain, and 5 point mutations in 1) the DNA-binding domain (2 patients), 2) the PEST domain (2 patients), and 3) the transactivation domain (one patient). DNA binding to and transactivation of the M-CSF receptor promoter, a direct PU.1 target gene, were deficient in the 7 PU.1 mutants that affected the DNA-binding domain. In addition, these mutations decreased the ability of PU.1 to synergize with PU.1-interacting proteins such as AML1 or c-Jun in the activation of PU.1 target genes. This is the first report of mutations in the PU.1 gene in human neoplasia and suggests that disruption of PU.1 function contributes to the block in differentiation found in AML patients.

Isoform Specific Regulation of Acute Myeloid Leukemia Maintenance By Wilms Tumor 1

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1232-1232
Author(s):  
Sandeep Potluri ◽  
Salam A. Assi ◽  
Suyin Paulynn Chin ◽  
Anetta Ptasinska ◽  
Daniel Coleman ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Amino Acids ◽  
Acute Myeloid Leukemia ◽  
Dna Binding ◽  
Myeloid Leukemia ◽  
Wilms Tumor ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Wilms Tumor 1 ◽  
Acute Myeloid

The gene encoding Wilms Tumor 1 (WT1) is recurrently upregulated in Acute Myeloid Leukemia (AML). WT1 transcript burden is associated with primary refractory disease and with relapse when there is detectable WT1 minimal residual disease (Lambert et al., Oncotarget, 2014). We have recently described the gene regulatory networks in purified blasts from AML patients with various driver mutations (Assi et al., Nature Genetics, 2019) and found that WT1 was a key transcription factor node in all networks examined. Furthermore, in knockdown experiments as part of a shRNA depletion screen against transcription factors in vitro and in murine xenotransplantation experiments, we showed that WT1 was essential for leukemic maintenance (Martinez-Soria et al., Cancer Cell, 2018). WT1 produces 8 primary isoforms, all of which are expressed in hematopoietic cells by employing one of two transcription start sites and alternative splicing in exon 5 and exon 9. To further understand how WT1 maintains leukemia, we cloned each of the 8 isoforms into a doxycycline-inducible vector and transduced them into a t(8;21) AML cell line, Kasumi-1, which well recapitulates primary t(8;21) AML (Ptasinska et al., Leukemia, 2014). We carefully overexpressed each of the 8 WT1 isoforms to levels seen in AML patient samples and functionally and molecularly characterised them. Overexpression of isoforms lacking 3 amino acids in the zinc finger DNA binding domain (WT1 -KTS) significantly reduced cell growth and colony formation, increased apoptosis, caused a G1 cell cycle arrest and caused myeloid differentiation of leukemic blasts. However, overexpression of isoforms with the 3 amino acids (WT1 +KTS) in the DNA-binding domain had the opposite effects, thus having a major role in the maintenance of a leukemic phenotype. Also, we found that the expression of WT1 +KTS isoforms in purified blasts from patients was higher than WT1 -KTS isoforms and may therefore permit leukemic maintenance. We did not find any functional differences between isoforms employing alternate transcriptional start sites or between exon 5 splice variants. We then performed the first ever WT1 chromatin immunoprecipitation (ChIP-seq) in AML and show that WT1 -KTS shows increased binding as compared to WT1 +KTS with 9451 unique WT1 binding sites in WT1 -KTS expressing cells and only 1355 sites in WT1 +KTS expressing cells with 2391 sites overlapping between the isoforms. This finding suggests that WT1 isoforms compete for binding at multiple sites and may explain why the ratio of expression between WT1 isoforms is critical (Calabrese et al., Blood, 2012). We also correlated WT1 ChIP-seq peaks to transcription as assayed by RNA-seq. We found that differentially bound cis-regulatory elements are associated with altered expression of genes involved in proliferation, cell cycle, apoptosis and differentiation pathways, explaining many of the functional alterations in cellular behaviour that we see. Several vaccine studies and more recently a T cell receptor study targeted against WT1 have been undertaken but they have shown mixed efficacy in AML (Chapuis et al, Nature Medicine, 2019). Since we find that different isoforms of WT1 have antagonistic effects, we hypothesise that a more effective therapeutic strategy would be to selectively target only WT1 +KTS isoforms. Disclosures No relevant conflicts of interest to declare.

scholarly journals RUNX1/AML1 DNA-binding domain and ETO/MTG8 NHR2-dimerization domain are critical to AML1-ETO9a leukemogenesis

Blood ◽  
2009 ◽  
Vol 113 (4) ◽  
pp. 883-886 ◽  
Author(s):  
Ming Yan ◽  
Eun-Young Ahn ◽  
Scott W. Hiebert ◽  
Dong-Er Zhang
Keyword(s):  
Dna Binding ◽  
Binding Domain ◽  
Chromosome 8 ◽  
Chromosome 21 ◽  
Valuable Insight ◽  
Dna Binding Domain ◽  
Dimerization Domain ◽  
Gene Encoding ◽  
Alternatively Spliced ◽  
Insight Into

Abstract The 8;21 translocation, which involves the gene encoding the RUNX family DNA-binding transcription factor AML1 (RUNX1) on chromosome 21 and the ETO (MTG8) gene on chromosome 8, generates AML1-ETO fusion proteins. Previous analyses have demonstrated that full-length AML1-ETO blocks AML1 function and requires additional mutagenic events to promote leukemia. More recently, we have identified an alternatively spliced form of AML1-ETO, AML1-ETO9a, from t(8;21) acute myeloid leukemia (AML) patient samples. AML1-ETO9a lacks the C-terminal NHR3 and NHR4 domains of AML1-ETO and is highly leukemogenic in the mouse model. Here, we report that the AML1 DNA-binding domain and the ETO NHR2-dimerization domain, but not the ETO NHR1 domain, are critical for the induction of AML by AML1-ETO9a. A region between NHR1 and NHR2 affects latency of leukemogenesis. These results provide valuable insight into further analysis of the molecular mechanism of t(8;21) in leukemogenesis.

scholarly journals Translocation breakpoints are clustered on both chromosome 8 and chromosome 21 in the t(8;21) of acute myeloid leukemia

Blood ◽  
1993 ◽  
Vol 81 (3) ◽  
pp. 592-596 ◽  
Author(s):  
JE Tighe ◽  
A Daga ◽  
F Calabi
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Chromosome 8 ◽  
Chromosome 21 ◽  
Novel Gene ◽  
Translocation Breakpoints ◽  
Acute Myeloid

Abstract The t(8;21)(q22;q22) is consistently associated with acute myeloid leukemia (AML) M2. Recent data have suggested that breakpoints on chromosome 21 are clustered within a single intron of a novel gene, AML1, just downstream of a region of homology to the runt gene of D melanogaster. In this report, we confirm rearrangement at the same location in at least 12 of 18 patients with t(8;21). Furthermore, we have isolated recombinant clones spanning the breakpoint regions on both the der(8) and the der(21) from one patient. By using a chromosome 8 probe derived from these clones, we show that t(8;21) breakpoints are also clustered on chromosome 8.

scholarly journals Translocation breakpoints are clustered on both chromosome 8 and chromosome 21 in the t(8;21) of acute myeloid leukemia

Blood ◽  
1993 ◽  
Vol 81 (3) ◽  
pp. 592-596 ◽  
Author(s):  
JE Tighe ◽  
A Daga ◽  
F Calabi
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Chromosome 8 ◽  
Chromosome 21 ◽  
Novel Gene ◽  
Translocation Breakpoints ◽  
Acute Myeloid

The t(8;21)(q22;q22) is consistently associated with acute myeloid leukemia (AML) M2. Recent data have suggested that breakpoints on chromosome 21 are clustered within a single intron of a novel gene, AML1, just downstream of a region of homology to the runt gene of D melanogaster. In this report, we confirm rearrangement at the same location in at least 12 of 18 patients with t(8;21). Furthermore, we have isolated recombinant clones spanning the breakpoint regions on both the der(8) and the der(21) from one patient. By using a chromosome 8 probe derived from these clones, we show that t(8;21) breakpoints are also clustered on chromosome 8.

scholarly journals Persistence of the 8;21 translocation in patients with acute myeloid leukemia type M2 in long-term remission

Blood ◽  
1993 ◽  
Vol 82 (3) ◽  
pp. 712-715 ◽  
Author(s):  
G Nucifora ◽  
RA Larson ◽  
JD Rowley
Keyword(s):  
Acute Myeloid Leukemia ◽  
Complete Remission ◽  
Myeloid Leukemia ◽  
Fusion Gene ◽  
Fusion Transcript ◽  
Chromosome 8 ◽  
Chromosome 21 ◽  
First Relapse ◽  
Acute Myeloid

The translocation between chromosomes 8 and 21, t(8;21) (q22;q22), is the most frequent abnormality in acute myeloid leukemia (AML) with French-American-British type M2 (FAB-M2) morphology. The breakpoints in this translocation have been characterized at the molecular level, and the genes involved are AML1 on chromosome 21 and ETO on chromosome 8. The rearrangement of the two chromosomes results in a fusion gene and in the production of a consistent fusion transcript on the der(8) chromosome. We have used oligonucleotide primers derived from both sides of the fusion cDNA junction and reverse transcription-polymerase chain reaction (RT-PCR) to analyze six AML-M2 patients with a t(8;21) during various stages of their disease. Two patients studied at diagnosis and one studied at first relapse are alive off therapy and in continuous complete remission for 83 to 94 months. We have detected the AML/ETO fusion transcript in recent peripheral blood samples from each of them. Three other patients also had a fusion transcript detected after 1 to 4 months in remission. Two of these patients subsequently relapsed and died whereas the third patient is alive and in continuous complete remission 70 months later. Thus, our preliminary data suggest that cells with the translocation are still circulating in t(8;21) patients in long-term remission. This finding raises serious questions regarding the interpretation of positive results obtained only with this technique that may not be suitable to decide appropriate further treatment for patients in clinical remission.

Heterogeneous Chromosomal Mechanisms Generating the 5′RUNX1/3′CBFA2T1 Gene in Acute Myeloid Leukemia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4272-4272
Author(s):  
Giorgina Specchia ◽  
Francesco Albano ◽  
Luisa Anelli ◽  
Antonella Zagaria ◽  
Arcangelo Liso ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Fusion Gene ◽  
Chromosome 8 ◽  
Chromosome 21 ◽  
Derivative Chromosome ◽  
Molecular Cytogenetic ◽  
Karyotypic Abnormality ◽  
Fusion Signal ◽  
Acute Myeloid

Abstract Translocation t(8;21)(q22;q22) is a common karyotypic abnormality detected in about 15% of Acute Myeloid Leukemia (AML) cases. The rearrangement results in fusion of the RUNX1 (also known as AML1) and CBFA2T1 (also known as ETO) genes generating a 5′RUNX1/3′CBFA2T1 transcriptionally active fusion gene on derivative chromosome 8. In 1 to 8.5% of AML cases insertions events generating a 5′RUNX1/3′CBFA2T1 fusion gene have been reported, whereas the occurrence of inversions accompanying the t(8;21) has never been observed. We report a screening of 82 AML cases bearing the RUNX1/CBFA2T1 rearrangement detected by RT-PCR; all cases were tested by Fluorescence In Situ Hybridization (FISH) with BAC and PAC clones specific for CBFA2T1 and RUNX1 genes. This analysis allowed us to reveal five cases with ins(21;8), one with ins(8;21), and two with a pericentric chromosome 8 inversion followed by a t(8;21) translocation. A detailed molecular cytogenetic characterization of breakpoints has been performed in all cases. FISH co-hybridization experiments with CBFA2T1 and RUNX1 probes revealed the presence of a functional fusion gene on the der(21) instead of the der(8) chromosome in five cases with ins(21;8); a single fusion signal on the der(8) chromosome was detected in the case with ins(8;21). The use of the same clones in FISH studies showed the presence of a single unexpected fusion signal on the 8p derivative chromosome in addition to faint CBFA2T1 and RUNX1 signals on the long arm of der(8) and der(21) chromosomes, respectively. These results suggested that a pericentric chromosome 8 inversion involving CBFA2T1 gene occurred and that the chromosome 21 was rearranged with the 8p derivative chromosome. Appropriate chromosome 21 and 8 BAC clones were employed to precisely define the size of inserted regions in cases with insertions and the breakpoint on the 8p derivative chromosome in cases showing pericentric chromosome 8 inversion. The insertion size turned out to be very heterogeneous, ranging from a minimum of 2.4 Mb to a maximum of 44 Mb. In both cases with chromosome 8 inversion, the CBFA2T1 gene represents the breakpoint at the chromosome 8 long arm whereas the 8p breakpoint showed different mapping positions in 8p21.3 and 8p21.1, respectively. Our results illustrate that (1) heterogeneous mechanisms can lead to the generation of the 5′RUNX1/3′CBFA2T1 chimeric gene; (2) molecular cytogenetic techniques may identify cryptic chromosomal changes, not detected by conventional cytogenetic analysis; (3) the crucial role of the 5′RUNX1/3′CBFA2T1 fusion gene in leukemogenesis does not depend on its location.

scholarly journals Persistence of the 8;21 translocation in patients with acute myeloid leukemia type M2 in long-term remission

Blood ◽  
1993 ◽  
Vol 82 (3) ◽  
pp. 712-715 ◽  
Author(s):  
G Nucifora ◽  
RA Larson ◽  
JD Rowley
Keyword(s):  
Acute Myeloid Leukemia ◽  
Complete Remission ◽  
Myeloid Leukemia ◽  
Fusion Gene ◽  
Fusion Transcript ◽  
Chromosome 8 ◽  
Chromosome 21 ◽  
First Relapse ◽  
Acute Myeloid

Abstract The translocation between chromosomes 8 and 21, t(8;21) (q22;q22), is the most frequent abnormality in acute myeloid leukemia (AML) with French-American-British type M2 (FAB-M2) morphology. The breakpoints in this translocation have been characterized at the molecular level, and the genes involved are AML1 on chromosome 21 and ETO on chromosome 8. The rearrangement of the two chromosomes results in a fusion gene and in the production of a consistent fusion transcript on the der(8) chromosome. We have used oligonucleotide primers derived from both sides of the fusion cDNA junction and reverse transcription-polymerase chain reaction (RT-PCR) to analyze six AML-M2 patients with a t(8;21) during various stages of their disease. Two patients studied at diagnosis and one studied at first relapse are alive off therapy and in continuous complete remission for 83 to 94 months. We have detected the AML/ETO fusion transcript in recent peripheral blood samples from each of them. Three other patients also had a fusion transcript detected after 1 to 4 months in remission. Two of these patients subsequently relapsed and died whereas the third patient is alive and in continuous complete remission 70 months later. Thus, our preliminary data suggest that cells with the translocation are still circulating in t(8;21) patients in long-term remission. This finding raises serious questions regarding the interpretation of positive results obtained only with this technique that may not be suitable to decide appropriate further treatment for patients in clinical remission.

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