8p12 stem cell myeloproliferative disorder: the FOP-fibroblast growth factor receptor 1 fusion protein of the t(6;8) translocation induces cell survival mediated by mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt/mTOR pathways.

The FOP-fibroblast growth factor receptor 1 (FGFR1) fusion protein is expressed as a consequence of a t(6;8) (q27;p12) translocation associated with a stem cell myeloproliferative disorder with lymphoma, myeloid hyperplasia and eosinophilia. In the present report, we show that the fusion of the leucine-rich N-terminal region of FOP to the catalytic domain of FGFR1 results in conversion of murine hematopoietic cell line Ba/F3 to factor-independent cell survival via an antiapoptotic effect. This survival effect is dependent upon the constitutive tyrosine phosphorylation of FOP-FGFR1. Phosphorylation of STAT1 and of STAT3, but not STAT5, is observed in cells expressing FOP-FGFR1. The survival function of FOP-FGFR1 is abrogated by mutation of the phospholipase C gamma binding site. Mitogen-activated protein kinase (MAPK) is also activated in FOP-FGFR1-expressing cells and confers cytokine-independent survival to hematopoietic cells. These results demonstrate that FOP-FGFR1 is capable of protecting cells from apoptosis by using the same effectors as the wild-type FGFR1. Furthermore, we show that FOP-FGFR1 phosphorylates phosphatidylinositol 3 (PI3)-kinase and AKT and that specific inhibitors of PI3-kinase impair its ability to promote cell survival. In addition, FOP-FGFR1-expressing cells show constitutive phosphorylation of the positive regulator of translation p70S6 kinase; this phosphorylation is inhibited by PI3-kinase and mTOR (mammalian target of rapamycin) inhibitors. These results indicate that translation control is important to mediate the cell survival effect induced by FOP-FGFR1. Finally, FOP-FGFR1 protects cells from apoptosis by survival signals including BCL2 overexpression and inactivation of caspase-9 activity. Elucidation of signaling events downstream of FOP-FGFR1 constitutive activation provides insight into the mechanism of leukemogenesis mediated by this oncogenic fusion protein.

WNT pathway and mammary carcinogenesis: loss of expression of candidate tumor suppressor gene SFRP1 in most invasive carcinomas except of the medullary type.

Secreted Frizzled-related protein 1 (SFRP1) encodes a member of a protein family that contains a cysteine-rich domain similar to the WNT-binding site of Frizzled receptors and regulates the WNT pathway. The WNT pathway is frequently altered in human cancers. We have defined the pattern of SFRP1 mRNA expression in the progression of breast cancer. We show that SFRP1 is expressed in the epithelial component of normal breast, in the in situ component of ductal carcinomas and is lost in more than 80% of invasive breast carcinomas except the medullary type. Loss of SFRP1 expression is correlated with the presence of hormonal receptors. Conversely, the maintenance of SFRP1 in carcinomas is correlated with the presence of lymphoplasmocytic stroma. No significant association was observed between SFRP1 status and the level of apoptosis in tumoral cells.

Molecular and functional characterization of the stress-induced protein (SIP) gene and its two transcripts generated by alternative splicing. SIP induced by stress and promotes cell death.

We have used a quantitative fluorescent cDNA microarray hybridization approach to identify pancreatic genes induced by the cellular stress promoted by acute pancreatitis in the mouse. We report the cloning and characterization of one of them that encodes the stress-induced proteins (SIP). The mouse SIP gene is organized into five exons and expands over approximately 20 kilobase pairs. Exon 4 (38 base pairs) is alternatively spliced to generate two transcripts. Northern blot and in situ hybridization showed that both SIP mRNAs are rapidly and strongly induced in acinar cells of the pancreas with acute pancreatitis. They are also constitutively expressed in several other tissues, although with different ratios. They encode proteins of 18 and 27 kDa (SIP(18) and SIP(27)). SIP(27) is identical to the thymus-expressed acidic protein (TEAP) protein, formerly described as a thymus-specific protein. Expression of the SIP(18) and SIP(27)/EGFP or V5 fusion proteins showed that both are nuclear factors. We monitored SIP expression in NIH3T3 cells submitted to various stress agents. UV stress, base damaging, mutagenic stress, ethanol, heat shock, and oxidative stress induced the concomitant expression of SIP(18) and SIP(27) mRNAs. Finally, transient transfection of SIP(18) and SIP(27) expression plasmids induced death by apoptosis in COS7 cells as measured by terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling staining. In conclusion, the SIP gene is an important element of cellular stress response. It is expressed in many tissues and induced by a variety of stress agents affecting many cellular pathways. SIP generates, by alternative splicing, two nuclear proteins that can promote cell death by apoptosis.

The 8p12 myeloproliferative disorder. t(8;19)(p12;q13.3): a novel translocation involving the FGFR1 gene.

Translocations affecting the chromosomal locus 8p12 are hallmarks of an atypical stem cell myeloproliferative disorder. These events disrupt the fibroblast growth factor receptor 1 (FGFR1) gene and fuse the FGFR1 C-terminus catalytic domain with unrelated proteins. Here, we report on the characterization of the 19q13.3 locus as the fifth FGFR1 chromosomal partner.

MOZ is fused to p300 in an acute monocytic leukemia with t(8;22).

We report on the fusion of the monocytic leukemia zinc finger protein (MOZ) gene to the adenoviral E1A-associated protein p300 (p300) gene in acute monocytic leukemia M5 associated with a t(8;22)(p11;q13) translocation. We studied two patients with double-color fluorescence in situ hybridization (FISH) using the yeast artificial chromosome 176C9 and the bacterial artificial chromosome clone H59D10 specific to the MOZ and p300 genes, respectively. Both probes were split in the patients' chromosome metaphase cells, and the two derivative chromosomes were each labeled with both probes. We showed by Southern blot the rearrangement of the MOZ gene, and cloned the fusion transcripts in one patient carrying the t(8;22) by reverse transcription-polymerase chain reaction using MOZ- and p300-specific primers. Both fusion transcripts were expressed. This result defines a novel reciprocal translocation involving two acetyltransferases, MOZ and p300, resulting in an abnormal transcriptional co-activator that could play a critical role in leukemogenesis.

"Paleogenomics": looking in the past to the future.

The complete sequence of the human and other vertebrate and nonvertebrate genomes provide a wealth of information on the organization, relationships and evolution of the metazoans. Soon the fine structure of our innermost biological identity will be unveiled and what has so far remained deep and secret will shine like an unearthed treasure and shape and fuel our future quests. A key treasure, for many molecular scientists interested in molecular evolution and development would be the knowledge of the genome of the ancestral precursor of all metazoans. In the absence of fossil DNA, this knowledge will forever remain a yearning for dreamy molecular biologists. And yet, will not the power of deduction and reconstitution of information gained through man's sophisticated technologies one day recreate a "virtual" metazoan ancestor?

FGFR1 is fused to the centrosome-associated protein CEP110 in the 8p12 stem cell myeloproliferative disorder with t(8;9)(p12;q33).

The hallmark of the 8p12 stem cell myeloproliferative disorder (MPD) is the disruption of the FGFR1 gene, which encodes a tyrosine kinase receptor for members of the fibroblast growth factor family. FGFR1 can be fused to at least 3 partner genes at chromosomal regions 6q27, 9q33, or 13q12. We report here the cloning of the t(8;9)(p12;q33) and the detection of a novel fusion betweenFGFR1 and the CEP110 gene, which codes for a novel centrosome-associated protein with a unique cell-cycle distribution. CEP110 is widely expressed at various levels in different tissues and is predicted to encode a 994-amino acid coiled-coil protein with 4 consensus leucine zippers [L-X(6)-L-X(6)-L-X(6)-L]. Both reciprocal fusion transcripts are expressed in the patient's cells. The CEP110-FGFR1 fusion protein encodes an aberrant tyrosine kinase of circa 150-kd, which retains most of CEP110 with the leucine zipper motifs and the catalytic domain of FGFR1. Transient expression studies show that the CEP110-FGFR1 protein has a constitutive kinase activity and is located within the cell cytoplasm. (Blood. 2000;95:1788-1796)

[FGFR1 and MOZ, two key genes involved in malignant hemopathies linked to rearrangements within the chromosomal region 8p11-12]. / FGFR1 et MOZ, deux gènes clés dans les hémopathies malignes associées à des réarrangements de la région chromosomique 8p11-12.

Two distinct clinical syndromes have been associated with the p11.12 region of the short arm of chromosome 8: stem-cell myeloproliferative disorder (B-or T-cell lymphoblastic leukemia/lymphoma with myeloid hyperplasia and peripheral blood eosinophilia) and acute myeloid leukemia (myelomonocytic or monocytic with erythrophagocytosis). The FGFR1 and MOZ genes are rearranged in these diseases and encode one of the four fibroblast growth factor receptors and a member of a novel histone acetyltransferase family, respectively. The predicted fusion proteins that are putatively oncogenic - FOP-FGFR1, CEP110-FGFR1, and FIM-FGFR1 - and - MOZ-CBP, MOZ-p300, and MOZ-TIF2 - lead to tumorigenesis through distinct pathways. The constitutive kinase activity triggered by dimerization mediated by the protein-protein interaction motifs of the FGFR1 protein partner regardless of external stimuli and the delocalization of the fusion proteins compared to their normal counterparts may lead to tumorigenesis presumably by inducing inappropriate recruitment in the cytoplasm of signaling substrates. Currently, little is known about the precise role of MOZ in the regulation of gene transcription. However, all the aberrant proteins described to date retain the MOZ histone acetyltransferase domain fused to that of the transcription coactivators CBP, p300, and TIF2. The fusion of two acetyltransferases whose activity may be mistargetted or misregulated could be a critical event in leukemogenesis. The increasing number of translocations affecting FGFR1 and MOZ strongly suggest their involvement in oncogenic processes and point to these proteins as potential therapeutical targets.

gamma-heregulin is the product of a chromosomal translocation fusing the DOC4 and HGL/NRG1 genes in the MDA-MB-175 breast cancer cell line.

gamma-heregulin is a recently described novel isoform of the heregulin/neuregulin class of EGF-like ligands that bind to and activate receptors of the ErbB family. Deregulated signaling through the heregulin-ErbB pathway is thought to be implicated in the development of a subset of human breast cancers. gamma-heregulin has been found to be expressed in the culture supernatant of MDA-MB-175, a breast carcinoma cell line. gamma-heregulin is characterized by the presence of a large N-terminal peptide extension that is not found in other heregulin isoforms. Here we report that this unique N-terminal extension of gamma-heregulin is identical to the N-terminus of DOC4, a product of a recently identified CHOP-dependent stress-induced gene. Human DOC4 and the heregulin-encoding genes map to different chromosomes and the MDA-MB-175 cell line contains a chromosomal translocation that leads to the fusion of DOC4 and HGL, on chromosomes 11 and 8, respectively. Thus, gamma-heregulin is a product of a mutant fusion gene and not a bona fide normal isoform. We speculate that the mutation may be selected for by virtue of its ability to activate ErbB signaling through the production of an autocrine ligand.

A case of inv(8)(p11q24) associated with acute myeloid leukemia involves the MOZ and CBP genes in a masked t(8;16).

We report on a novel chromosomal aberration, inv(8)(p11q24), in an M5 acute myeloid leukemia. We show by fluorescence in situ hybridization and Southern blot analyses that a t(8;16)(p11;p13) is masked by this inversion. The translocation targets the MOZ gene from the 8p11 and the CBP gene from the 16p13 chromosomal regions. The breakpoints occur in the MOZ region encoding the acidic domain and in the 5' end of the CBP gene. These results provide further evidence for the multiple contribution of both MOZ and CBP genes in acute leukemias. Genes Chromosomes Cancer 26:161-165, 1999.

Characterization of FIM-FGFR1, the fusion product of the myeloproliferative disorder-associated t(8;13) translocation.

The t(8;13) translocation found in a rare type of stem cell myeloproliferative disorder generates a constitutively activated tyrosine kinase containing N-terminal sequence encoded by the FIM gene linked to the FGFR1 kinase domain. Here we have further characterized FIM and FIM-FGFR1 proteins. Firstly, we have studied their respective subcellular localization. We show that FIM has nuclear and nucleolar localization, whereas FIM-FGFR1 is mainly cytoplasmic. Within the nucleolus, FIM colocalizes with the upstream binding factor in interphasic cells, indicating that FIM may be involved in the regulation of rRNA transcription. We demonstrate that the targetting of FIM to the nucleus depends upon its C-terminal region, which is absent in the cytoplasmic FIM-FGFR1 protein. Secondly, we demonstrate that FIM-FGFR1 has constitutive dimerization capability mediated by the FIM N-terminal sequences. Finally, we show that FIM-FGFR1 promotes survival of pro-B Ba/F3 cells after interleukin-3 withdrawal, whereas ligand-activated FGFR1 induced not only cell survival but also interleukin-3 independence. Taken together, these results indicate that FIM-FGFR1 is activated by dimerization as a cytoplasmic kinase and suggest that FIM-FGFR1 partially signals through the FGFR1 pathways.

Differential expression assay of chromosome arm 8p genes identifies Frizzled-related (FRP1/FRZB) and Fibroblast Growth Factor Receptor 1 (FGFR1) as candidate breast cancer genes.

Deletions and amplifications are frequent alterations of the short arm of chromosome 8 associated with various types of cancers, including breast cancers. This indicates the likely presence of tumor suppressor genes and oncogenes. In the present study, we have used the expressed sequence tag (EST) map of 8p11-21 to assemble a set of available cDNAs representing genes from this region. DNA arrays were prepared for expression analysis and search for genes potentially involved in breast cancer. Underexpresion in tumoral breast cells (versus normal breast) was observed for 15 transcripts. Among these, the Frizzled-related gene FRP1/FRZB, was turned off in 78% of breast carcinomas, suggesting that the lack of its product may be associated with malignant transformation. Overexpression in tumoral breast cells was observed for 13 genes. The FGFR1 gene, that encodes a tyrosine kinase receptor for members of the fibroblast growth factor family, was identified as a good candidate for one amplification unit. Taken together, our results demonstrate that such a strategy can rapidly identify genes with an altered pattern of expression and provide candidate genes for malignancies.

The t(6;8)(q27;p11) translocation in a stem cell myeloproliferative disorder fuses a novel gene, FOP, to fibroblast growth factor receptor 1.

In patients with an atypical stem-cell myeloproliferative disorder with lymphoma (B or T cell), myeloid hyperplasia, and eosinophilia, the chromosome 8p11-12 region is the site of a recurrent breakpoint that can be associated with three different partners, 6q27, 9q32-34, and 13q12. Rearrangements are supposed to affect a pluripotent stem cell capable of myeloid and lymphoid differentiation and to involve the same 8p11-12 gene. The t(8;13) translocation has recently been shown to result in a fusion between the FGFR1 gene that encodes a tyrosine kinase receptor for fibroblast growth factors and a novel gene, FIM (also called RAMP or ZNF198), belonging to a novel family of zinc finger genes. In the present study, we have cloned the t(6;8)(q27;p11) translocation in two patients and found a fusion between FGFR1 and a novel gene, FOP (FGFR1 Oncogene Partner), located on chromosome band 6q27. This gene is alternatively spliced and ubiquitously expressed. It encodes a protein containing two regions of putative leucine-rich repeats putatively folding in alpha-helices and separated by a hydrophobic spacer. The two reciprocal fusion transcripts were evidenced by reverse transcription-polymerase chain reaction in the tumoral cells of the patients. The predicted chimeric FOP-FGFR1 protein contains the FOP N-terminus leucine-rich region fused to the catalytic domain of FGFR1. It may promote hematopoietic stem cell proliferation and leukemogenesis through a constitutive phosphorylation and activation of the downstream pathway of FGFR1.

Ancient large-scale genome duplications: phylogenetic and linkage analyses shed light on chordate genome evolution.

Paralogous genes from several families were found in four human chromosome regions (4p16, 5q33-35, 8p12-21, and 10q24-26), suggesting that their common ancestral region underwent several rounds of large-scale duplication. Searches in the EMBL databases, followed by phylogenetic analyses, showed that cognates (orthologs) of human duplicated genes can be found in other vertebrates, including bony fishes. In contrast, within each family, only one gene showing the same high degree of similarity with all the duplicated mammalian genes was found in nonvertebrates (echinoderms, insects, nematodes). This indicates that large-scale duplications occurred after the echinoderms/chordates split and before the bony vertebrate radiation. It has been suggested that two rounds of gene duplication occurred in the vertebrate lineage after the separation of Amphioxus and craniate (vertebrates + Myxini) ancestors. Before these duplications, the genes that have led to the families of paralogous genes in vertebrates must have been physically linked in the craniate ancestor. Linkage of some of these genes can be found in the Drosophila melanogaster and Caenorhabditis elegans genomes, suggesting that they were linked in the triploblast Metazoa ancestor.

Chromosome region 8p11-p21: refined mapping and molecular alterations in breast cancer.

Several genes, most of them unknown, of the short arm of chromosome 8 are involved in malignant diseases. Numerous studies have implicated a portion of the 8p11-p21 region as the location of one or more tumor suppressor genes involved in a variety of human cancers, including breast cancer. We and others have reported linkage analyses suggesting the presence of a putative breast cancer susceptibility gene. Furthermore, several oncogenes of the 8p11-p12 region are involved in reciprocal translocations in myeloproliferative and myelodysplastic disorders and in amplification in breast cancer. To facilitate the analysis of the 8p11-p21 region and the cloning of candidate oncogenes and tumor suppressor genes, a high-resolution physical and transcriptional map was established with 39 yeast artificial chromosomes and 94 markers, including so-called sequence-tagged sites and expressed sequence-tagged sites derived from either known genes or expressed sequence tags corresponding to unidentified transcripts. In addition, four novel transcripts were identified and localized precisely within the map. This transcription map provides a detailed description of gene order for the 8p11-p21 region and will be helpful in the identification of candidate genes for diseases. From this basis, we refined the mapping of two types of molecular alterations that occur at 8p11-p21 in sporadic breast cancers, i.e., amplification and deletion.

Fibroblast growth factor receptor 1 is fused to FIM in stem-cell myeloproliferative disorder with t(8;13).

Chromosome 8p11-12 is the site of a recurrent breakpoint in a myeloproliferative disorder that involves lymphoid (T- or B-cell), myeloid hyperplasia and eosinophilia, and evolves toward acute leukemia. This multilineage involvement suggests the malignant transformation of a primitive hematopoietic stem cell. In this disorder, the 8p11-12 region is associated with three different partners 6q27, 9q33, and 13q12. We describe here the molecular characterization of the t(8;13) translocation that involves the FGFR1 gene from 8p12, encoding a tyrosine kinase receptor for members of the fibroblast growth factor family, and a gene from 13q12, tentatively named FIM (Fused In Myeloproliferative disorders). FIM is related to DXS6673E, a candidate gene for X-linked mental retardation in Xq13.1; this defines a gene family involved in different human pathologies. The two reciprocal fusion transcripts, FIM/FGFR1 and FGFR1/FIM are expressed in the malignant cells. The FIM/FGFR1 fusion protein contains the FIM putative zinc finger motifs and the catalytic domain of FGFR1. We show that it has a constitutive tyrosine kinase activity.

t(6;8), t(8;9) and t(8;13) translocations associated with stem cell myeloproliferative disorders have close or identical breakpoints in chromosome region 8p11-12.

A stem-cell myeloproliferative disorder involving T- and B-cell, and myeloid lineages, is associated with three different translocations with a breakpoint in region p11-12 of chromosome 8: t(6;8)(q27;p11), t(8;9)(p11;q33), and t(8;13)(p12;q12), respectively. Using fluorescence in situ hybridization (FISH), we have analysed blood cells from a series of five patients carrying these different translocations. We have identified cosmids from chromosome region 8p11-12 that span the breakpoint in all the cases. They are specific for the FCFR1 gene that encodes a receptor for members of the FGF family. The breakpoint was further detected by Southern and pulsed-field gel electrophoresis analyses with probes from the FGFR1 locus.