FLT3 Is Fused to ETV6 in a Myeloproliferative Disorder with a t(12;13)(p13;q12) Translocation.

Abstract The FLT3, located at band 13q12 and encoding a receptor tyrosine kinase (RTK), is one of the most frequently mutated genes in hematologic malignancies including ALL, MDS, and AML. The most common mutation of the FLT3 is an internal tandem duplication in exons 14 and 15, whereas other mutations have also been found at and around codon 835 of exon 20. These activating mutations promote constitutive RTK activity in the absence of ligand, proposing FLT3 as an attractive therapeutic target for directed inhibition. However, many questions with regards to the biology of FLT3 and its role in leukemogenesis remain to be clarified. Despite its highly frequent mutations, FLT3 has never been reported to fuse to any other genes, a phenomenon usually observed in other RTKs. Here, we report a case of a novel fusion gene between FLT3 and ETV6 at 12p13, a well-known target for a number of translocations. The patient, a 68-year-old female, was diagnosed as myeloproliferative disorder with hypereosinophilia in May 2002. Peripheral blood showed WBC 33.6x106/L (3% myelocytes, 33.5% neutrophils, 54% eosinophils, 1.5% basophils, 1.0% monocytes and 7% lymphocytes), Hb 119g/L and platelet counts 5,450x106/L. The bone marrow (BM) was marked hypercellular with 0.9% blasts, 6.0% promyelocytes, 15.6% myelocytes, 8.1% immature eosinophils and 19.2% mature eosinophils. Karyotype of BM cells was 46, XX, t(12;13)(p13.1;q12.3–13)[28]/46, XX[2]. Under the suspicion of Ph-negative CML, she was treated with IFNα with no response. Then, HU was started and her WBC decreased to 30x106/L. FISH analysis showed that the breakpoint at 12p13 occurred within ETV6, while the breakpoints at 13q12 occurred at two locations, within FLT3 or CDX2. To identify the fusion partner of ETV6, 3′-RACE PCR was performed. Sequence analysis of PCR-products revealed 4 types of ETV6/FLT3 transcripts. These fusion transcripts were confirmed by Northern blot analysis. Each ETV6/FLT3 transcript contained the entire helix-loop-helix domain of ETV6 (exons 1 to 4 or 5) and almost all of the functional domains of FLT3 including the tyrosine kinase domain (from exons 14, 16 or 17), suggesting that the resultant chimeric protein would be constitutively activated FLT3 kinase. Of them, three are in-frame fusion, presumably encoding for the approximately 58, 62, and 83 kD fusion proteins. However, Western blot analysis showed only expression of the 58 and 83 kD proteins. RT-PCR detected the reciprocal FLT3/ETV6 transcript, comprising the FLT3 exons 1 to 13 frameshifly fused to the ETV6 exons 6 to 8, within which a stop codon appeared at codon 33- downstream from the fusion point. Functional studies to assess the oncogenic properties of these fusion proteins are now in progress. Our findings provide an evidence that FLT3 is also involved in hematologic malignancies as a fusion gene.

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Fusion of SFQP to ABL in a Patient with a T(1;9)(p34;q34) and Acute Lymphocytic Leukemia.

Blood ◽

10.1182/blood.v104.11.4444.4444 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4444-4444

Author(s):

Nicholas C.P. Cross ◽

Andrew J. Chase ◽

Milton Drachenberg ◽

W. Mark Roberts ◽

Jerry Z. Finklestein ◽

...

Keyword(s):

Complete Remission ◽

Fusion Gene ◽

Chimeric Protein ◽

Sh2 Domain ◽

Lymphocytic Leukemia ◽

Tyrosine Kinase Domain ◽

Fish Analysis ◽

Fusion Partner ◽

Rt Pcr ◽

Polypyrimidine Tract Binding Protein

Abstract We have investigated a child who presented with pre-B ALL and an acquired t(1;9)(p34;q34). BCR-ABL was not detected by RT-PCR or FISH analysis, however FISH did indicate that the ABL gene at 9q34 was disrupted. To identify the putative partner locus in this case, a modified 5′RACE strategy was employed that selected against normal ABL transcripts. Several clones were recovered in which ABL was fused to SFPQ (also known as PSF), a gene mapping to 1p34 that encodes a polypyrimidine tract-binding protein-associated splicing previously identified as a fusion partner of the helix-loop-helix transcription factor TFE3 in papillary renal cell carcinomas. Both SFPQ-ABL and reciprocal ABL-SFPQ transcripts were detectable by RT-PCR, and disruption of these two genes was further confirmed by amplification and sequencing of the forward genomic breakpoint. SFPQ-ABL, the likely oncogenic product, is predicted to encode a protein that retains the coiled coil domain of SFPQ and the entire tyrosine kinase domain and C-terminal sequences of ABL. The breakpoint in ABL was downstream of that seen for other ABL fusion genes and the chimeric protein is predicted to lack the ABL-encoded SH3 domain and part of the SH2 domain. The patient was treated according to the Children’s Cancer Group Protocol 1961 and subsequently received augmented BFM therapy with doxorubicin and double delayed intensification. He achieved complete remission but suffered extramedullary testicular relapse at 4.5 years. Following orchiectomy and intensive chemotherapy he remains in complete remission more than 6 years after diagnosis. We conclude that SFPQ-ABL is a novel fusion gene associated with ALL. Although the patient here responded to conventional chemotherapy, SFPQ-ABL is likely to be sensitive to imatinib and use of this agent might be considered in further cases.

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NUP98 is fused to the NSD3 gene in acute myeloid leukemia associated with t(8;11)(p11.2;p15)

Blood ◽

10.1182/blood.v99.10.3857 ◽

2002 ◽

Vol 99 (10) ◽

pp. 3857-3860 ◽

Cited By ~ 134

Author(s):

Roberto Rosati ◽

Roberta La Starza ◽

Angelo Veronese ◽

Ana Aventin ◽

Christine Schwienbacher ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Hematologic Malignancies ◽

Fish Analysis ◽

Fusion Partner ◽

First Time ◽

Classical Cytogenetics ◽

Split Signal ◽

Acute Myeloid

Fusion between the NUP98 and NSD3genes in a patient with acute myeloid leukemia associated with t(8;11)(p11.2;p15), is reported for the first time. The t(8;11)(p11.2;p15) was identified by classical cytogenetics. Fluorescence in situ hybridization (FISH) analysis revealed a split signal with a mix of BAC 118H17 and 290A12, indicating the translocation disrupted NUP98. FISH restriction at 8p11-12 showed a split of BAC 350N15. Molecular investigations into candidate genes in this BAC showed the NUP98 fusion partner at 8p11.2 was the NSD3 gene. To date the NSD3 gene has never been implicated in hematologic malignancies.

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Transforming and Tumorogenic Activity of JAK2 by Fusion to BCR: Molecular Mechanisms of Action of a Novel BCR-JAK2 Tyrosin-Kinase.

Blood ◽

10.1182/blood.v114.22.4683.4683 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4683-4683

Author(s):

Álvaro Cuesta-Domínguez ◽

Mara Ortega ◽

Cristina Ormazabal ◽

Matilde Santos-Roncero ◽

Marta Galán-Díez ◽

...

Keyword(s):

Tyrosine Kinase ◽

Nude Mice ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Fusion Gene ◽

Lymphoblastic Leukemia ◽

Chimeric Protein ◽

Tyrosine Kinase Domain ◽

Molecular Response

Abstract Abstract 4683 Chromosomal translocations in human tumors frequently produce fusion genes whose chimeric protein products play an essential role in oncogenesis. Recent reports have found a BCR-JAK2 fusion gene in cases of chronic or acute myeloid leukemia, but the protein had not been characterized. We describe a BCR-JAK2 fusion gene by fluorescence in situ hybridization and RT-PCR amplification from bone marrow at diagnosis of a patient with acute lymphoblastic leukemia. After induction therapy, real time PCR showed persistent molecular response correlating with hematological remission maintained up to present. BCR-JAK2 is a 110 KDa chimeric protein containing the BCR oligomerization domain fused to the JAK2 tyrosine-kinase domain. In vitro analysis showed that BCR-JAK2 was constitutively phosphorylated and was located to the cytoplasm. BCR-JAK2 transformed the IL-3-dependent murine hematopoietic cell line Ba/F3 into IL-3 independent growth and induced STAT5b phosphorylation and translocation into the cell nuclei. The treatment with a JAK2 inhibitor abrogated BCR-JAK2 and STAT5b phosphorylation, leading to apoptosis of transformed Ba/F3 cells. To test whether BCR-JAK2 has tumorogenic ability in vivo, we performed experiments with nude mice, in which we injected subcutaneously cells transduced with the control vector and cells expressing BCR-JAK2. Notably, we only obtained tumors in the flank injected with BCR-JAK2 expressing cells, thus confirming the tumorogenic activity of the BCR-JAK2 fusion protein. We conclude that BCR-JAK2 is a new tyrosine-kinase that induces proliferation and cell survival, which can be abrogated by JAK2 inhibitors. In vitro studies demonstrate that BCR-JAK2 displays transforming activity. Moreover, the nude mice model reveals its ability to cause tumors. Disclosures: No relevant conflicts of interest to declare.

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A Novel Fusion Gene NDEL1-Pdgfrb in a Patient with JMML with a New Variant of TKI-Resistant Mutation in the Kinase Domain of PDGFRβ

Blood ◽

10.1182/blood.v124.21.613.613 ◽

2014 ◽

Vol 124 (21) ◽

pp. 613-613 ◽

Cited By ~ 2

Author(s):

Konstantin Byrgazov ◽

Renate Kastner ◽

Michael Dworzak ◽

Gregor Hoermann ◽

Oskar A. Haas ◽

...

Keyword(s):

Amino Acid ◽

Tyrosine Kinase ◽

Fusion Gene ◽

Kinase Domain ◽

Juvenile Myelomonocytic Leukemia ◽

Fusion Partner ◽

Amino Acid Exchange ◽

Inactive Conformation ◽

Clinical Resistance ◽

Acid Exchange

Abstract We have identified a novel fusion gene in an 18-month old child with juvenile myelomonocytic leukemia (JMML) displaying a reciprocal chromosomal translocation t(5;7)(q33;p11.2). Molecular investigation at diagnosis revealed absence of mutations in KRAS, NRAS, PTPN11, or cCBL, but FISH analysis identified a rearrangement involving the PDGFRB gene located on chromosome 5q33. After temporary responses to imatinib (IM) and subsequently nilotinib (NIL) treatment, resistance associated with disease relapses was observed. Employment of the 5’-RACE technique facilitated identification of the PDGFRB fusion partner on chromosome 7p11.2, the NDEL1 gene encoding the nudE neurodevelopmental protein 1-like 1. The NDEL1 gene has not been implicated in any other reciprocal translocation to date, and it is conceivable that its ability to form dimers could drive permanent kinase activation of PDGFRβ. The chimeric mRNA contains the 5´exons 1-5 of NDEL1 fused in frame to the PDGFRB exons 10-22 containing the transmembrane and tyrosine kinase domains. To assess the oncogenicity of the fusion protein, Ba/F3 cells were transduced with the NDEL1-PDGFRB gene construct. The observation of IL3-independent growth confirmed the oncogenic potential of the novel fusion gene. The observed clinical resistance to IM and NIL prompted us to analyze the entire PDGFRB kinase domain for the presence of mutations by Sanger sequencing of overlapping amplicons. A point mutation in the activation (A) loop converting aspartate at the position 850 into glutamate (D850E) was detected in peripheral blood specimens from the time of first and second relapses, but not in the diagnostic sample. The crystal structure of the PDGFRβ TKD is not available, but protein modelling suggested that the mutation D850E destabilizes the inactive confirmation of the A-loop. This notion was in line with the observed clinical resistance to IM and NIL, but suggested sensitivity of the mutant to dasatinib (DAS). To test the predicted TKI responses, Ba/F3 cells transduced with wild type or mutant NDEL1-PDGFRB were tested in MTT assays against a panel of TKIs: Ba/F3-NDEL1-PDGFRBWT cells were sensitive to IM (IC50 = 60 nM), NIL (100 nM), DAS (5 nM), sorafenib (SOR; 20 nM), and ponatinib (PON; 10 nM), but insensitive to bosutinib (BOS; >2500 nM). Conversely, Ba/F3-NDEL1-PDGFRBD850E cells exhibited high resistance to IM (>2500), a 10-fold higher IC50 for NIL (1000 nM) and a 100-fold higher IC50 for SOR (2500 nM), but retained sensitivity to PON (15 nM) and DAS (15 nM). Mutations in the A-loop of different tyrosine kinases such as PDGFRα (D842V) or c-Kit (D816V) associated with resistance to IM have already been described in different tumor entities. However, the mutation D850E in the PDGFRβ TKD with apparent insensitivity to IM, NIL, and SOR revealed a completely different pattern of resistance than the same amino acid exchange at the corresponding site of PDGFRα (D842E). The latter mutation was previously shown to be sensitive to IM, NIL, and SOR with IC50 values of 4, 12.5, and 0.25 nM, respectively. This difference is intriguing because the exchange of a negatively charged amino acid, aspartate, to an amino acid with the same physical properties, glutamate, is not known to exert a major structural effect on the protein conformation, as observed for the D842E mutation in PDGFRα. We speculate that the great difference between the presence of the same amino acid exchange at corresponding positions in PDGFRα and PDGFRβ is the main interaction amino acid partner residue of aspartate at the position +3 which may influence the stability of the A-loop in its inactive conformation. In PDGFRα, it is histidine whose physical interaction with aspartate might not be affected by the change to glutamate. By contrast, the electrostatic bonds between arginine as the +3 residue in PDGFRβ might be greatly weakened by the elongation of the side chain in glutamate in comparison with aspartate, thus destabilizing the inactive conformation of the A-loop resulting in resistance to type II TKIs. To our knowledge, this is the first observation of an exchange between two negatively charged amino acids in a tyrosine kinase associated with a major change in responsiveness to TKI treatment. This finding is currently under further investigation, and may extend our understanding of structural interactions leading to TKI resistance. (Supported by the FWF SFB grant F4705-B20). Disclosures Valent: Novartis: Consultancy, Honoraria, Research Funding.

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Rabaptin-5 is a novel fusion partner to platelet-derived growth factor β receptor in chronic myelomonocytic leukemia

Blood ◽

10.1182/blood.v98.8.2518 ◽

2001 ◽

Vol 98 (8) ◽

pp. 2518-2525 ◽

Cited By ~ 97

Author(s):

Magnus K. Magnusson ◽

Kristin E. Meade ◽

Kevin E. Brown ◽

Diane C. Arthur ◽

Lisa A. Krueger ◽

...

Keyword(s):

Growth Factor ◽

Fusion Protein ◽

Tyrosine Kinase ◽

Fusion Proteins ◽

Growth Regulation ◽

Chronic Myelomonocytic Leukemia ◽

Platelet Derived Growth Factor ◽

Fusion Partner ◽

Β Receptor ◽

Myelomonocytic Leukemia

Abstract Chromosomal translocations involving the platelet-derived growth factor β receptor (PDGFβR) gene have been reported in some patients with chronic myelomonocytic leukemia (CMML). The resultant fusion proteins have constitutive PDGFβR tyrosine kinase activity, but the partner genes previously reported(tel, Huntingtin interacting protein 1[HIP-1], H4/D10S170) have poorly understood roles in the oncogenic activity of the fusion proteins. A novel PDGFβR fusion protein has been characterized in a patient with CMML and an acquired t(5;17)(q33;p13). Southern blot analysis on patient leukemia cells demonstrated involvement of the PDGFβR gene. Using 5′ rapid amplification of complementary DNA ends–polymerase chain reaction (RACE-PCR) on patient RNA, rabaptin-5 was identified as a novel partner fused in-frame to thePDGFβR gene. The new fusion protein includes more than 85% of the native Rabaptin-5 fused to the transmembrane and intracellular tyrosine kinase domains of the PDGFβR. Transduction with a retroviral vector expressing rabaptin-5/PDGFβRtransformed the hematopoietic cell line Ba/F3 to growth factor independence and caused a fatal myeloproliferative disease in mice. Rabaptin-5 is a well-studied protein shown to be an essential and rate-limiting component of early endosomal fusion through interaction with the Ras family GTPases Rab5 and Rab4. The fusion protein includes 3 of 4 coiled-coil domains (involved in homodimerization of native rabaptin-5), 2 caspase-3 cleavage sites, and a binding site for the tumor suppressor gene tuberin (tuberous sclerosis complex-2). Early endosomal transport is critical in regulation of various growth factor receptors, through ligand-induced clathrin-mediated endocytosis, and thus this new fusion protein links together 2 important pathways of growth regulation.

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Identification of a Novel HOOK3-FGFR1 Fusion Gene Involved in Activation of The NF-kappaB Pathway

10.21203/rs.3.rs-1052427/v1 ◽

2021 ◽

Author(s):

Xuehong Zhang ◽

Furong Wang ◽

Fanzhi Yan ◽

Dan Huang ◽

Haina Wang ◽

...

Keyword(s):

Tyrosine Kinase ◽

Sanger Sequencing ◽

Fusion Gene ◽

Chromosome 8 ◽

Antibody Array ◽

Fish Analysis ◽

Qrt Pcr ◽

Healthy Donors ◽

Recurrent Mutations ◽

Nf Kappab

Abstract BackgroundRearrangements involving the fibroblast growth factor receptor 1 (FGFR1) gene result in 8p11 myeloproliferative syndrome (EMS), which is a rare and aggressive hematological malignancy that is often initially diagnosed as myelodysplastic syndrome (MDS). Clinical outcomes are typically poor due to relative resistance to tyrosine kinase inhibitors (TKIs) and rapid transformation to acute leukemia. Deciphering the transcriptomic signature of FGFR1 fusions may open new treatment strategies for FGFR1 rearrangement patients.MethodsDNA sequencing (DNA-seq) was performed for 20 MDS patients and whole exome sequencing (WES) was performed for one HOOK3-FGFR1 fusion positive patient. RNA sequencing (RNA-seq) was performed for 20 MDS patients and 8 healthy donors. Fusion genes were detected using the STAR-Fusion tool. Fluorescence in situ hybridization (FISH), quantitative real-time PCR (qRT-PCR), and Sanger sequencing were used to confirm the HOOK3-FGFR1 fusion gene. The phosphorylation antibody array was performed to validate the activation of nuclear factor-kappaB (NF-kappaB) signaling. ResultsWe identified frequently recurrent mutations of ASXL1 and U2AF1 in the MDS cohort, which is consistent with previous reports. We also identified a novel in-frame HOOK3-FGFR1 fusion gene in one MDS case with abnormal monoclonal B-cell lymphocytosis and ring chromosome 8. FISH analysis detected the FGFR1 break-apart signal in myeloid blasts only. qRT-PCR and Sanger sequencing confirmed the HOOK3-FGFR1 fusion transcript with breakpoints located at the 11th exon of HOOK3 and 10th exon of FGFR1, and Western blot detected the chimeric HOOK3-FGFR1 fusion protein that is presumed to retain the entire tyrosine kinase domain of FGFR1. The transcriptional feature of HOOK3-FGFR1 fusion was characterized by the significant enrichment of the NF-kappaB pathway by comparing the expression profiling of FGFR1 fusion positive MDS with 8 healthy donors and FGFR1 fusion negative MDS patients. Further validation by phosphorylation antibody array also showed NF-kappaB activation, as evidenced by increased phosphorylation of p65 (Ser 536) and of IKBalpha (Ser 32). ConclusionThe HOOK3-FGFR1 fusion gene may contribute to the pathogenesis of MDS and activate the NF-kappaB pathway. These findings highlight a potential novel approach for combination therapy for FGFR1 rearrangement patients.

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Characterization of 14 NUP214-ABL1 Fusions in T-Cell Acute Lymphoblastic Leukaemia (T-ALL) Exhibits a Genomic Heterogeneity.

Blood ◽

10.1182/blood.v108.11.2075.2075 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2075-2075

Author(s):

Carlos Graux ◽

Marina Lafage ◽

Nicole Dastugue ◽

Francine Mugneret ◽

Roland Berger ◽

...

Keyword(s):

T Cell ◽

Tyrosine Kinase ◽

Cultured Cells ◽

Outcome Data ◽

Tyrosine Kinase Domain ◽

Fish Analysis ◽

Rt Pcr ◽

Genomic Amplification ◽

Chromosomal Alterations ◽

Male Predominance

Abstract The recently described episomal amplification of the NUP214-ABL1 fusion in 6% of T-ALL leads to the expression of a constitutively activated chimeric tyrosine kinase sensitive to imatinib. We collected additional cases in order to better characterize this new entity with respect to genetic presentations and clinical course. We collected 14 new NUP214-ABL1 positive cases by FISH (LSI-BCR/ABL1 ES, Vysis and ABL1 break-apart home-made probes) or RT-PCR (fusion transcript) screening. FISH analysis detected episomal amplification of NUP214-ABL1 in 11 patients with a highly variable number of nuclei with amplification (<1% to 90%). Interestingly, one case showed a higher percentage of nuclei with amplification when using frozen non cultured cells rather than cultured fixed cells (10% vs 1%) suggesting loss of episomes during culture. FISH showed also intrachromosomal amplification of NUP214-ABL1 in two cases: one presented a HSR at the original 9q34 site without detectable episomes, the other associated HSR (probably on a chromosome 10), episomes (<1% of nuclei) and 9q34 chromosomal insertions including NUP214 and ABL3′ that encode the tyrosine kinase domain but not ABL5′, on variable chromosomes including 14p (33%). One NUP214-ABL1 RT-PCR positive case did not show any FISH aberration. Median age: 16 y (3–45) with a male predominance (10:4). There were no T-cell lymphoblastic lymphoma. Immunophenotype (EGIL): mature (n=2), cortical (n=6) or pre-T (n=4). Karyotype: structural chromosomal alterations in 8 patients (including 4 with 10q24/HOX11 rearrangements), only numerical chromosomal alterations in 4 (including 2 with + 8), normal in 1, failed in 1. All samples with available information (10/14) showed a HOX11 or HOX11L2 abnormality. Among the 13 cases with available outcome data, we observed 5 early relapses, including both patients with NUP214-ABL1 HSR, and 1 refractory ALL. These observations emphasize the interest of combining both (quantitative) RT-PCR and FISH for the screening and characterisation of NUP214-ABL1 fusion and amplification, demonstrate the coexistence of different NUP214-ABL1 genomic presentations in one patient (episomal amplification, 9q34 insertions, HSR) compatible with the model in which genomic amplification start with episome formation in order to create the NUP214-ABL1 fusion followed by their amplification and optional secondary reintegration, confirm occurrence of NUP214-ABL1 in T-ALL with HOX11 and HOX11L2 involvement, raise the question of the rather worse prognosis for cases with intrachromosomal amplification as previously suggested, raise the signification of minor NUP214-ABL1 clones and variable genomic presentations in the leukemogenesis of this subgroup of T-ALL that could potentially benefit from imatimib. ° on behalf of the GFCH (Groupe Francophone de Cytogénétique Hématologique) and the BCGHO (Belgian Cytogenetic Group for Hematology and Oncology).

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Activation of Nuclear Factor KappaB in ETV6/FLT3-Transformed Ba/F3 Cells Depends on the FLT3 Tyrosine 591.

Blood ◽

10.1182/blood.v110.11.4666.4666 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4666-4666

Author(s):

Hoang Anh Vu ◽

Phan Thi Xinh ◽

Yasuhiko Kano ◽

Takaomi Ishida ◽

Toshiki Watanabe ◽

...

Keyword(s):

Tyrosine Kinase ◽

Fusion Gene ◽

Hematologic Malignancies ◽

Signaling Proteins ◽

Interleukin 3 ◽

Downstream Signaling ◽

Support Cell ◽

Helix Loop Helix ◽

Oligomerization Domain

Abstract The FLT3 gene is one of the most frequently mutated genes in hematologic malignancies. Mutated FLT3 has been found to lead to strong constitutive activation of several signaling proteins such as STAT5, CBL, VAV, SHP2, NF-kB, and AKT. Recently, we have reported that FLT3 contributed to leukemogenesis through generating a ETV6/FLT3 fusion gene in a patient with myeloproliferative disorder and a t(12;13)(p13;q12) translocation, which is the first report to show that FLT3 fusion gene can be involved in leukemogenesis. The ETV6/FLT3 which contained the helix-loop-helix oligomerization domain of ETV6 fused to the juxtamembrane (JM) domain plus tyrosine kinase (TK) domains of FLT3, conferred interleukin-3-independent growth on Ba/F3 cells (Vu HA, et al. LEUKEMIA20:1414–21, 2006). To assess a role of the JM domain of FLT3 in the ETV6/FLT3 protein, we generated a series of ETV6/FLT3 mutants (tyrosine to phenylalanine substitution and deletion of the JM domain or N-terminal region of the TK1 domain). Each mutant was introduced into Ba/F3 cells and downstream signaling as well as cell proliferation was investigated. We found that the wild-type ETV6/FLT3 in Ba/F3 cells was a constitutively activated tyrosine kinase that led to activation of STAT5, AKT, MAPK, and NF-kB pathways as well as up-regulation of PIM-1. Deletion of the JM domain retained compatible level of autophosphorylation of the fusion protein as well as activation of STAT5, AKT and MAPK, suggesting that the JM domain is dispensable for STAT5, MAPK and AKT activations. In contrast, deletion of the JM domain did abrogate interleukin-3-independent growth of Ba/F3 cells, PIM-1 up-regulation, and activation of NF-kB. Importantly, while substitutions of other 3 tyrosines (589, 597, and 599) had no affect on NF-kB activation, the substitution of tyrosine 591 to phenylalanine abrogated this activation. Our results suggest that the tyrosine 591 in JM domain of FLT3 in ETV6/FLT3 is critical for NF-kB activation to support cell survival and PIM-1 up-regulation.

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A novel mutation in FGFR-3 disrupts a putative N-glycosylation site and results in hypochondroplasia

Physiological Genomics ◽

10.1152/physiolgenomics.2000.2.1.9 ◽

2000 ◽

Vol 2 (1) ◽

pp. 9-12 ◽

Cited By ~ 44

Author(s):

ANDREAS WINTERPACHT ◽

KATJA HILBERT ◽

CHRISTIANE STELZER ◽

THORSTEN SCHWEIKARDT ◽

HEINZ DECKER ◽

...

Keyword(s):

Tyrosine Kinase ◽

Novel Mutation ◽

Hot Spot ◽

Three Dimensional ◽

Growth Factor Receptor ◽

Kinase Domain ◽

Glycosylation Site ◽

Dimensional Structure ◽

Tyrosine Kinase Domain ◽

Common Mutation

Winterpacht, Andreas, Katja Hilbert, Christiane Stelzer, Thorsten Schweikardt, Heinz Decker, Hugo Segerer, Jürgen Spranger, and Bernhard Zabel. A novel mutation in FGFR-3 disrupts a putative N-glycosylation site and results in hypochondroplasia. Physiol. Genomics 2: 9–12, 2000.—Fibroblast growth factor receptor 3 (FGFR3) is a glycoprotein that belongs to the family of tyrosine kinase receptors. Specific mutations in the FGFR3 gene are associated with autosomal dominant human skeletal disorders such as hypochondroplasia, achondroplasia, and thanatophoric dysplasia. Hypochondroplasia (HCH), the mildest form of this group of short-limbed dwarfism disorders, results in ∼60% of cases from a mutation in the intracellular FGFR3-tyrosine kinase domain. The remaining cases may either be caused by defects in other FGFR gene regions or other yet unidentified genes. We describe a novel HCH mutation, the first found outside the common mutation hot spot of this condition. This point mutation, an N328I exchange in the extracellular Ig domain III of the receptor, seems to be unique as it affects a putative N-glycosylation site that is conserved between different FGFRs and species. The amino acid exchange itself most probably has no impact on the three-dimensional structure of the receptor domain, suggesting that the phenotype is the result of altered receptor glycosylation and its pathophysiological consequences.

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A Novel FOXP1-PDGFRA Fusion Gene in A Case of Chronic Eosinophilic Leukemia.

Blood ◽

10.1182/blood.v120.21.2830.2830 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2830-2830

Author(s):

Yuka Sugimoto ◽

Akiko Sada ◽

Fumihiko Monma ◽

Kohshi Ohishi ◽

Masahiro Masuya ◽

...

Keyword(s):

Bone Marrow ◽

Peripheral Blood ◽

Blood Cells ◽

Genomic Dna ◽

Low Dose ◽

Fusion Gene ◽

Fish Analysis ◽

Fusion Partner ◽

Rt Pcr ◽

Total Rna

Abstract Abstract 2830 Introduction Myeloid and lymphoid neoplasms with eosinophilia and abnormalities of PDGFRA, PDGFRB, or FGFR1 is a new major category in the 2008 WHO classification of myeloid malignancies. FIP1L1-PDGFRA fusion gene is currently the most common abnormality in this category, but there are some other fusion genes incorporating part of PDGFRA. In a case of myeloproliferative neoplasms (MPN) with eosinophilia and hepatosplenomegaly, karyotype by G-banding and fluorescence in situ hybridization (FISH) for 4q12 rearrangements indicated a PDGFRA rearrangement other than FIP1L1-PDGFRA, and a novel FOXP1-PDGFRA fusion gene was identified. Case presentation A 44-year-old male visited a clinic because of wet cough for one year. His peripheral blood showed leukocytosis of 43.15 × 109 /L with eosinophilia up to 57.5%, mild erythrocytosis (Hb 17.3 g/dL), and thrombocytopenia of 86 × 109 /L. CT scan of the abdomen revealed hepatosplenomegaly. He was referred to our hospital and received oral PSL (1 mg/kg) first, because pulmonary eosinophilic infiltration was suspected by follow-up CT findings. Pulmonary infiltration and his cough disappeared rapidly in a week, but his leukocytosis with eosinophilia was exacerbated again with PSL tapering. His bone marrow at the time of admission disclosed hypercellular marrow with myeloid hyperplasia and eosinophilia, of which karyotype was 46, XY, t(3:4)(p13;q12), inv(9)(p12q13) in all of 20 metaphases. FISH analysis with tricolor 4q12 rearrangement probe set indicated that PDGFRA was disrupted in 97.3% of his peripheral blood cells. These cytogenetic abnormalities of his bone marrow cells suggested involvement of PDGFRA fusion gene except for FIP1L1-PDGFRA and did not disappear after steroid administration for 2 weeks. After low-dose of imatinib (100 mg/day) was started, he achieved a hematological response within 5 days, and PSL could be gradually tapered off. 3 months after therapy, he obtained complete cytogenetic response (CCyR). He has been in CCyR and free of symptoms for more than 6 months with only low-dose imatinib. Methods and Results Genomic DNA and total RNA were isolated from white blood cells in his peripheral blood at diagnosis. Complementary DNA was synthesized from total RNA. FIP1L1-PDGFRA fusion transcript was proved to be negative by RT-PCR. Molecular cloning with 5′-RACE-PCR revealed a novel mRNA in-frame fusion between exon 23 of FOXP1 and a truncated PDGFRA exon12. Reciprocal PDGFRA-FOXP1 transcripts were confirmed by RT-PCR analysis and FOXP1-PDGFRA genomic DNA sequence was determined with genomic PCR. As in the case with FIP1L1-PDGFRA, the breakpoint of PDGFRA in FOXP1-PDGFRA was located between the two tryptophan (W) residues of the putative WW-domain. Meanwhile, the other breakpoint was near inverted repeat in intron 23 of FOXP1, which is presumed to be very fragile site. By FISH analysis after magnetic cell sorting with MicroBeads, the 4q12 abnormality attributed to FOXP1-PDGFRA fusion gene was detected in granulocytes, but not in CD19-positive B or CD3-positive T cells. Discussion In a case with chronic eosinophilia harboring 46, XY, t(3:4)(p13;q12), inv(9)(p12q13), novel FOXP1-PDGFRA fusion gene was identified. Similar karyotypic abnormality harboring t(3:4)(p13;q12) was reported in a case of MPN with chronic eosinophilia, but responsible fusion gene was not identified (Myint H, et al. Br J Haematol. 1995). FOXP1 is a transcription factor which is implicated in a variety of cellular processes and has a role in immune regulation and carcinogenesis (Wlodarska I, et al. Leukemia. 2005). As a fusion partner of FOXP1, PAX5 and ABL1 are reported in cases with acute lymphoblastic leukemia. Thus, this is a first report showing that FOXP1-PDGFRA fusion gene is involved in hematologic malignancy. It is likely that FOXP1-PDGFRA is constitutively activated tyrosine kinase, which does not depend on dimerization but on the disruption of an autoinhibitory juxtamembrane domain encoded by exon 12 of PDGFRA from its structure. Eosinophilia responded well to low dose of imatinib as observed in CEL with FIP1L1-PDGFRA. Conclusion FOXP1-PDGFRA was identified in CEL for the first time. This is the eighth reported fusion gene associated with PDGFRA in CEL so far. Our patient with FOXP1-PDGFRA promptly responded to low-dose of imatinib as same as other cases with PDGFRA abnormalities. Further investigation is still in progress. Disclosures: No relevant conflicts of interest to declare.

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