Littérature scientifique sur le sujet « ETV6-RT »

Abstract The dic(9;12)(p12;p11.2) has been described as a rare cytogenetic abnormality in pediatric precursor B-cell ALL. Initial studies suggested that the rearrangement is associated with a favorable outcome, and recent studies demonstrated the presence of a PAX5-ETV6 fusion gene was associated with this cytogenetic abnormality. Twenty cases with a cytogenetic dic(9;12) were identified in the Children’s Oncology Group (COG) cytogenetics databases. FISH analysis with the ETV6-RUNX1 (TEL-AML1) probes was done on 12 of these samples. Five cases were positive for fusion, indicating a cryptic t(12;21)(p13;q22), and also had loss of the ETV6 probe from the chromosome 12 not involved in the t(12;21). Seven cases were negative for fusion and had loss of an ETV6 signal, although one of the latter had a diminished ETV6 signal identified. To determine whether both PAX5-ETV6 and ETV6-RUNX1 rearrangements occurred in some patients, a diagnostic sample from each patient was analyzed by RT-PCR for the PAX5-ETV6 and ETV6-RUNX1 fusion genes. Primers from exon 3 of PAX5 and exon 3 of ETV6 were used for the PAX5-ETV6 analysis and from exon 5 of ETV6 and exon 4 of RUNX1 for the ETV6-RUNX1 analysis. Of the 20 cases, only 8 were RT-PCR positive for the PAX5-ETV6 fusion with the above primers; however, an additional 2 were RT-PCR positive with alternate primers, and all 10 of these were negative for the ETV6-RUNX1 fusion by RT-PCR. Of the remaining 10 patients, 9 were RT-PCR positive for the ETV6-RUNX1 fusion, including all of the ETV6-RUNX1 cases positive by FISH. The gene rearrangement associated with the dic(9;12) in these cases is not known. One patient was negative for both fusions by RT-PCR, negative by FISH for ETV6-RUNX1 rearrangement, yet had loss of an ETV6 signal. No cytogenetic differences could be seen between the 2 groups, either in the appearance of the dic(9;12) or in the other abnormalities identified. These results demonstrate the presence of two mutually exclusive dic(9;12) rearrangements in pediatric ALL; one associated with ETV6-RUNX1 rearrangement and one resulting in PAX5-ETV6 fusion. Both PAX5-ETV6 and ETV6-RUNX1 rearrangements are associated with a favorable prognosis. However, molecular analysis of the dic(9;12) patients must be performed to determine whether the dicentric chromosome results in PAX5-ETV6 fusion or whether the case has ETV6-RUNX1 fusion.

Abstract The t(12;13)(p11-13:q11-14) is a recurring chromosomal abnormality found in different types of hematological malignancies. Three genes on 13q12-14, CDX2, FLT3, and TTL have been identified as fusion partners of ETV6(TEL) on 12p13. These genes create different forms of fusion transcripts with ETV6. In the present study, we identified novel types of ETV6-TTL fusion transcripts in a case of a 9 year-old female who developed myeloid/NK cell precursor acute leukemia with t(12;13)(p13;q14). Cytogenetic analyses of the leukemic cells of the patient using a regular G-banding revealed 47, XX, del(2)(q?), -9, der(12)t(12;13)(p13;q14), add(13)(q1?2), +mar1, +mar2 [13/20]. Fluorescence in situ hybridization using YAC clone revealed that ETV6 gene was split in metaphase chromosomes of patient’s leukemic cells. We performed reverse-transcriptase-polymerase chain reaction (RT-PCR) analysis to detect the fusion transcripts of ETV6-CDX2, ETV6-FLT3 or TTL-ETV6, however, no fusion transcripts of previously described types were detected. We next performed RT-PCR analysis using various sets of primers to detect unknown types of fusion transcripts involving these genes, and detected novel types of fusion transcripts of ETV6-TTL. These fusion transcripts consisted of exons 1 to 5 of ETV6 and exons 5 to 8 of TTL, and exons 1 to 6 of ETV6 and exon 9 or exons 8 to 9 of TTL. No reciprocal fusion transcripts were detected. Predicted fusion proteins consisted of N-terminal ETV6 lacking whole or part of ETS-binding domain and C-terminal TTL. Previous report showed major type of TTL-ETV6 fusion transcript consisted of exons 1 to 5 of TTL and exons 2 to 8 of ETV6 which contained both helix-loop-helix and ETS-binding domains. These results suggested that novel types of ETV6-TTL act as different fusion proteins from previously reported TTL-ETV6 in leukemogenesis. At the cytogenetic level, it may be difficult to distinguish ETV6-CDX2, ETV6-FLT3, and ETV6-TTL. Further accumulation of the patients with t(12;13) and further analysis of these novel types of fusion transcripts may clarify the pathogenesis of t(12;13)-leukemia.

Abstract The ETV6 gene (also known as TEL) is the main target of chromosomal translocations affecting chromosome band 12p13. The rearrangements fuse ETV6 to a wide variety of partner genes in both myeloid and lymphoid malignancies. We report here 4 new cases of acute myeloid leukemia (AML) with very immature myeloblasts (French-American-British [FAB]-M0) and with a t(4;12)(q11-q12;p13). In all cases, ETV6 was found recombined to a new gene, homologous to the mouse Brx gene. The gene was named BTL (Brx-likeTranslocated in Leukemia). Reverse transcriptase-polymerase chain reaction (RT-PCR) experiments indicate that the expression of the BTL-ETV6 transcript, but not of the reciprocal ETV6-BTL transcript, is a common finding in these leukemias. In contrast to the majority of other ETV6 fusions, both the complete helix-loop-helix (HLH) and ETS DNA binding domains of ETV6 are present in the predicted BTL-ETV6 fusion protein, and the chimeric gene is transcribed from theBTL promoter.

The ETV6 gene (also known as TEL) is the main target of chromosomal translocations affecting chromosome band 12p13. The rearrangements fuse ETV6 to a wide variety of partner genes in both myeloid and lymphoid malignancies. We report here 4 new cases of acute myeloid leukemia (AML) with very immature myeloblasts (French-American-British [FAB]-M0) and with a t(4;12)(q11-q12;p13). In all cases, ETV6 was found recombined to a new gene, homologous to the mouse Brx gene. The gene was named BTL (Brx-likeTranslocated in Leukemia). Reverse transcriptase-polymerase chain reaction (RT-PCR) experiments indicate that the expression of the BTL-ETV6 transcript, but not of the reciprocal ETV6-BTL transcript, is a common finding in these leukemias. In contrast to the majority of other ETV6 fusions, both the complete helix-loop-helix (HLH) and ETS DNA binding domains of ETV6 are present in the predicted BTL-ETV6 fusion protein, and the chimeric gene is transcribed from theBTL promoter.

Abstract The chromosomal translocation t(7;12) was described recently in MLL negative acute myeloid leukemias of infancy associated with a very poor clinical outcome. The rearrangement involves the genes HLXB9 (7q36) and ETV6 (12p13) with a fusion transcript of exon1/HLXB9 and exon3/ETV6. An alternative in frame splicing variant of exon1/HLXB9 to exon2/ETV6 is also detectable. Leukemic bone marrow samples of 42 infants, diagnosed in Germany with AML, were screened for the fusion transcript HLXB9/ETV6 with RT-PCR. Inclusion criteria were diagnosis of AML, age<2 years, no MLL/AF9, MLL/AF4 or MLL/ENL rearrangement in RT-PCR, no chromosomal 11q23 rearrangement and no trisomy 21. Positive samples were cloned and sequenced. A fusion transcript was found in approx. ~17% of patients. Common features of HLXB9/ETV6 positive leukemias in this cohort are poorly differentiated FAB subtypes, trisomy 19 karyotype of the leukemic cells, coexpression of the T-cell markers CD4 and CD7 as well as high expression of the un-rearranged HLXB9 gene. Quantitative PCR showed no upregulation of HOXA9 and MEIS1 in HLXB9/ETV6 positive patients as reported for MLL+ leukemias. The mechanism of HLXB9/ETV6 induced leukemogenesis remains unknown. We show that in vitro retroviral transduction of murine hematopoietic precursor cells with either HLXB9/ETV6 or HLXB9 or ETV6/RUNX1 was not transforming. Furthermore altered activity of the transcription factor family NF-kappaB has been demonstrated for various types of cancer including acute myeloid leukemias. Therefore we analysed the impact of the HLXB9/ETV6 fusion on the NF-kappaB signaling pathway in a 293T cell-system. HLXB9/ETV6 and HLXB9 showed inhibition of the NF-kappaB dependent transcription signal in a reporter assay, whereas the typical ALL-fusion ETV6/RUNX1 showed no influence. Analysis of nuclear extracts by western blot confirmed, that the inhibition of the NF-kappaB signal is due to a reduced nuclear translocation of the non-canonical NF-kappaB components p52 and RelB, whereas the canonical pathway remains not influenced. Conclusion: The HLXB9/ETV6 fusion transcript can be found in 17% of infants with MLL-negative AML in a German cohort. HLXB9/ETV6 positive leukemias show no increased expression on HOXA9 and MEIS1, but coexpression of T-cell markers and inhibition of the non-canonical NF-kappaB pathway on protein level. HLXB9/ETV6 did not induce malignant transformation in murine hematopoietic precursor cells.

Abstract [Background] ETV6-FRK is a rare kinase-related fusion gene which was identified only in acute myeloid leukemia (Hosoya N, et al. Genes Chromosomes Cancer. 2005). Herein, we firstly identified ETV6-FRK fusion gene in a patient with high-risk pediatric B cell precursor ALL (B-ALL). Because FRK is Src family tyrosine kinase, we performed functional analysis of ETV6-FRK to establish molecular targeting therapy. [Patient] A 11-year-old boy with B-ALL was refractory to conventional chemotherapy and received allogeneic bone marrow transplantation (allo-BMT) following two courses of blinatumomab. This patient maintains complete remission for three months after allo-BMT. Cytogenetic analysis demonstrated t(6;12)(q21;p13) as a part of complex karyotype. Targeted capture mRNA sequencing identified ETV6-FRK fusion transcript in this patient. [Materials and methods] ETV6-FRK fusion was validated by RT-PCR of the diagnostic leukemic sample from this patient. Full length of ETV6-FRK cDNA was cloned into retroviral vector with Tet-On system. Then, Ba/F3 cells, which are IL-3 dependent murine pro B-ALL cells, were transduced with retroviral vector to establish Ba/F3 cells expressing ETV6-FRK (Ba/F3-ETV6-FRK) under doxycycline (DOX) dependent manner. Ba/F3-ETV6-FRK was analyzed whether IL-3 independent growth was achieved. To determine whether aberrant activation of FRK-STAT pathway as the downstream effects of ETV6-FRK, activation of FRK-STAT pathway was evaluated by western blot. Finally, in cytotoxic assay, proliferation of Ba/F3-ETV6-FRK was assessed under the media with various concentrations of dasatinib, a tyrosine kinase inhibitor. [Results and discussions] Sequencing of RT-PCR product revealed that ETV6 exon 4 was fused in-frame to FRK exon 3, creating an ETV6-FRK fusion gene. The ETV6-FRK fusion gene produced a chimeric protein consisting of the entire pointed (PNT) oligomerization domain of ETV6 and the kinase domain of FRK (Fig 1). The expression of ETV6-FRK in Ba/F3 cells under DOX dependent manner was confirmed by western blot. Ba/F3-ETV6-FRK proliferated without IL-3 in contrast to Ba/F3 cells not expressing ETV6-FRK (p<0.01), suggesting ETV6-FRK had proliferation activity. Western blot analysis revealed constitutive phosphorylation of tyrosine residues of ETV6-FRK and STAT5/STAT3/STAT1, suggesting constitutive activation of FRK-STATs pathway was associated with IL-3 independent proliferation activity of ETV6-FRK. Considering that dasatinib, which is Src-kinase inhibitor, could block constitutive phosphorylation of ETV6-FRK, we hypothesized that dasatinib might block the IL-3 independent proliferation of Ba/F3-ETV6-FRK. In vitro killing assay showed that dasatinib suppressed efficiently the proliferation of Ba/F3-ETV6-FRK with 50% inhibitory concentration (IC50) 1.64 ± 0.02 nM, although dasatinib didn't show any effect on Ba/F3 cells not expressing ETV6-FRK (Fig. 2). Annexin V assay determined that 35.8 ± 6.9 % of Ba/F3-ETV6-FRK with dasatinib (10nM, 48hrs) were apoptotic than Ba/F3 cells not expressing ETV6-FRK (Fig. 3, p<0.01). These findings suggested that dasatinib abolished the proliferation activity of ETV6-FRK selectively. [Conclusion] We identified the first patient of pediatric high-risk B-ALL harboring ETV6-FRK fusion by targeted capture mRNA sequencing, who was refractory to the conventional chemotherapy. We also provide the first evidence that dasatinib could abrogate proliferation activity of ETV6-FRK in vitro, suggesting that dasatinib might be effective for the patient with B-ALL carrying a ETV6-FRK fusion. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Objectives: Macrophages play a critical role in atherosclerosis by contributing to plaque development, local inflammation, and thrombosis. Elucidation of the molecular cascades in atherosclerotic macrophages is important for preventing and treating atherosclerosis. This study aims to deepen the understanding of the mechanisms that regulate the function of aorta macrophage in atherosclerosis. Methods: In the current study, the expression and function of ETS variant transcription factor 6 (ETV6) in aorta macrophages in a mouse atherosclerosis model. Aorta macrophages were enriched by flow cytometry. ETV6 expression was analyzed by quantitative RT-PCR. The role of ETV6 in macrophage-mediated pro-inflammatory response was evaluated both in vitro and in vivo after ETV6 silencing. Results: A remarkable elevation of ETV6 in aorta macrophages of atherosclerotic mice was observed. In addition, in vitro analysis indicated that oxidized low-density lipoprotein (oxLDL) up-regulated ETV6 in macrophages via the NF-κB pathway. ETV6 silencing suppressed oxLDL-induced expression of IL-1β, IL-6, and TNF-α in macrophages in vitro. However, ETV6 silencing did not impact the uptake of either oxLDL or cholesterol by macrophages. Furthermore, ETV6 silencing suppressed oxLDL-induced activation of the NF-κB pathway in macrophages, as evidenced by less phosphorylation of IKKβ and NF-κB p65, more cytoplasmic IκBα, and lower nuclear NF-κB p65. Moreover, ETV6 silencing inhibited the production of IL-1β and TNF-α in aorta macrophages in vivo. Conclusion: ETV6 supports macrophage-mediated inflammation in atherosclerotic aortas. This is a novel mechanism regulating the pro-inflammatory activity of atherosclerotic macrophages.

Abstract Disruption of the transcription factor ETV6 (TEL) is believed to play a pivotal role in the development of many types of leukaemia. As well as there being more than 15 ETV6 fusion gene partners described to date, there are other genetic alterations of ETV6 including deletions, point mutations and disruption in the ETV6 promoter region that may contribute to the malignant phenotype. We generated two Etv6 knock-out mouse models (Met1 and Met43) to investigate the role of Etv6 in mouse embryonic development and two knock-in models carrying the t(12;21) and t(9;21) fusion genes to investigate the role of the Etv6-RUNX1 and Etv6-ABL fusion oncoproteins in leukaemogenesis. Etv6-Met1−/− was compatible with full embryonic development. However, from a total of 382 animals generated from the Etv6-Met1+/− cross only 39 were identified as homozygous suggesting some embryonic lethality. A second knock-out construct (Etv6-Met43) was generated in which the second transcription start site at AA position 43 was removed. Of the 235 offspring genotyped from the Etv6-Met43+/− cross, no homozygous animals were identified. Further investigation revealed that Etv6-Met43−/− foetuses lacked definitive haematopoiesis and died between dE10.5–11.5. These data indicate that an Etv6 transcript generated from the methionine at AA position 43 can partially rescue the Etv6-Met1−/− phenotype. No mice homozygous for the Etv6-RUNX1 were identified from the 628 offspring genotyped from the Etv6-RUNX1+/− cross. Further investigation revealed that Etv6-RUNX1−/− foetuses displayed a similar phenotype to the Etv6-Met43−/− animals and died in-utero at a similar time. Etv6-RUNX1+/− animals were fertile and remained well for the first year of life. However, after this time in a group of 91 animals it was noticed that among 41 Etv6-RUNX1+/− animals, the majority were dying from a blastic haematological tumour formation best identified in spleens, at a higher incidence than in the 51 wild-type littermates. Chimeric mice targeted with the Etv6-ABL construct remained well for a short period after birth but all subsequently sickened and died within the first year of life. Upon examination peripheral blood as well as post-mortem organs showed a homogeneous infiltration of granulocytic cells resembling a chronic myeloid proliferation. RT-PCR on RNA isolated from the peripheral blood, bone marrow and spleen of three chimeric animals showed the presence of the fusion gene. Our study demonstrates that Etv6 is essential for mouse embryonic development. The role of Etv6 in malignant transformation is complex and would appear to be dependent on the function of the partner fusion gene rather than on the common Etv6 part of the protein.

Abstract The Ets variant gene 6 (ETV6/TEL) gene is rearranged in the majority of patients with 12p13 translocations fused to a number of different partners. We present here a case of acute myeloid leukemia M4 with eosinophilia (AML-M4Eo) positive for the CBFb/MYH11 rearrangement and carrying a t(1;12)(q25;p13) that involves the ETV6 gene at 12p13. By 3′rapid amplification of cDNA ends-polymerase chain reaction (3′RACE-PCR), a novel fusion transcript was identified between the ETV6 and the Abelson-related gene (ARG) at 1q25, resulting in a chimeric protein consisting of the HLH oligomerization domain of ETV6 and the SH2, SH3, and protein tyrosine kinase (PTK) domains of ARG. The reciprocal transcript ARG-ETV6 was also detected in the patient RNA by reverse transcriptase-polymerase chain reaction (RT-PCR), although at a lower expression level. The ARG gene encodes for a nonreceptor tyrosine kinase characterized by high homology with c-Abl in the TK, SH2, and SH3 domains. This is the first report on ARGinvolvement in a human malignancy.

The Ets variant gene 6 (ETV6/TEL) gene is rearranged in the majority of patients with 12p13 translocations fused to a number of different partners. We present here a case of acute myeloid leukemia M4 with eosinophilia (AML-M4Eo) positive for the CBFb/MYH11 rearrangement and carrying a t(1;12)(q25;p13) that involves the ETV6 gene at 12p13. By 3′rapid amplification of cDNA ends-polymerase chain reaction (3′RACE-PCR), a novel fusion transcript was identified between the ETV6 and the Abelson-related gene (ARG) at 1q25, resulting in a chimeric protein consisting of the HLH oligomerization domain of ETV6 and the SH2, SH3, and protein tyrosine kinase (PTK) domains of ARG. The reciprocal transcript ARG-ETV6 was also detected in the patient RNA by reverse transcriptase-polymerase chain reaction (RT-PCR), although at a lower expression level. The ARG gene encodes for a nonreceptor tyrosine kinase characterized by high homology with c-Abl in the TK, SH2, and SH3 domains. This is the first report on ARGinvolvement in a human malignancy.