Fusion Partner Genes Research Articles

NUP98 Is Fused to Topoisomerase (DNA) IIβ 180 kDa (TOP2B) in a Patient with Acute Myeloid Leukemia with a New t(3;11)(p24;p15)

The nucleoporin 98 kDa (NUP98) gene has been reported to be fused to 17 different partner genes in various hematologic malignancies with 11p15 aberrations. Cytogenetic analysis of an adult de novo acute myelogenous leukemia (M5a) revealed a t(3;11)(p24;p15), suggesting rearrangement of NUP98 with a novel partner gene. Fluorescence in situ hybridization (FISH) was used to confirm the involvement of NUP98 in the t(3;11)(p24;p15). Selection of possible NUP98 partner genes was done by computer-aided analysis of the 3p24 region using the University of California Santa Cruz genome browser. Fusion gene-specific FISH and reverse transcription-PCR analyses were done to verify the presence of the new NUP98 fusion. FISH analysis using a NUP98-specific clone showed a split signal, indicating that the NUP98 gene was affected by the translocation. Of the genes localized at 3p24, TOP2B was selected as a possible fusion partner candidate gene. Dual-color fusion gene-specific FISH and reverse transcription-PCR analysis verified that NUP98 was indeed fused to TOP2B. In addition to reciprocal NUP98-TOP2B and TOP2B-NUP98 in-frame fusion transcripts, an alternatively spliced out-of-frame TOP2B-NUP98 transcript that resulted in a premature stop codon was detected. Analysis of the genomic breakpoints revealed typical signs of nonhomologous end joining resulting from error-prone DNA repair. TOP2B encodes a type II topoisomerase, which is involved in DNA transcription, replication, recombination, and mitosis, and besides TOP1, represents the second NUP98 fusion partner gene that belongs to the topoisomerase gene family. This finding emphasizes the important role of topoisomerases in malignant transformation processes.

HOXA gene cluster rearrangement in a t(7;9)(p15;q34) in a child with MDS

We describe the molecular characterization of a t(7;9)(p15;q34) found in a 15-month-old female patient, diagnosed with refractory anemia with excess blasts in transformation (RAEBt), in progression to acute myeloid leukemia (AML) M7. Molecular characterization of the 7p15 breakpoint showed that this was localized within a fully sequenced PAC clone RP1-170O19 containing the HOXA4 to HOXA13 genes and the EVX1 gene. The 9q34 breakpoint was mapped distal to ABL1 and proximal to NOTCH1 excluding their involvement as fusion gene partners. Our findings suggest a causal role for HOXA genes in childhood myelodysplasia and warrant investigation of this locus in a larger series of patients.

Partial tandem duplication of MLL gene in acute myeloid leukemia with translocation (11;17)(q23;q12–21)

Translocation 11q23 and MLL gene rearrangements are commonly observed in acute myeloid leukemia (AML) in association with the myelomonocytic or monocytic feature. We describe a case involving a 15-year-old patient with AML characterized by leukemic cells exhibiting translocation (11;17)(q23;q12-21) and MLL gene rearrangement. No fusion partner gene of the MLL gene was identified, including RARalpha(17q12) or AF17 (17q21); however, a partial tandem duplication of the MLL exon 11/exon 10 was detected in leukemic cells via a 3'RACE method for detection of unknown partner genes. The patient has been in remission for more than 2 years without hematopoietic stem cell transplantation.

ELL binding regulates U19/Eaf2 intracellular localization, stability, and transactivation

U19/Eaf2, an androgen-response gene, is downregulated in advanced human prostate cancer specimens and its overexpression can markedly induce apoptosis in prostate cancer cells. Eleven-nineteen Lysine-rich Leukemia (ELL) is an RNA polymerase II transcription elongation factor, initially identified as a fusion partner gene of MLL in the t(11; 19) (q23; p13.1) chromosomal translocation in acute myeloid leukemia. U19/Eaf2 was previously reported as an ELL-associated factor, a potential transcription factor binds to ELL, forming nuclear speckles in vivo. These findings suggest that ELL-U19/Eaf2 interaction is potentially important in prostate cancer progression and/or acute myeloid leukemia. However, the functional significance of U19/Eaf2 interaction with ELL remains unclear. Using co-transfection, co-immunoprecipitation, protein stability assay and transactivation assay, we characterized the consequence of ELL binding to U19/Eaf2. We provide further evidence for U19/Eaf2 as a transcription factor and show that ELL binding is required for nuclear speckle formation of human U19/Eaf2, stabilizes U19/Eaf2 and enhances its transactivation activity. The above observations indicate ELL may be an important factor required for U19/Eaf2 function because U19/Eaf2 nuclear localization and transactivation activity are essential for its function as a transcription factor.

KIAA1509 is a novel PDGFRB fusion partner in imatinib-responsive myeloproliferative disease associated with a t(5;14)(q33;q32)

We report the cloning of a novel PDGFRB fusion gene partner in a patient with a chronic myeloproliferative disorder characterized by t(5;14)(q33;q32), who responded to treatment with imatinib mesylate. Fluorescence in situ hybridization demonstrated that PDGFRB was involved in the translocation. Long distance inversion PCR identified KIAA1509 as the PDGFRB fusion partner. KIAA1509 is an uncharacterized gene with a predicted coiled-coil oligomerization domain with homology to the HOOK family of proteins. The predicted KIAA1509-PDGFRbeta fusion protein contains the KIAA1509 coiled-coil domain fused to the cytoplasmic domain of PDGFRbeta that includes the tyrosine kinase domain. Imatinib therapy resulted in rapid normalization of the patient's blood counts, and subsequent bone marrow biopsies and karyotypic analysis were consistent with sustained complete remission.

Subcellular localization of EEN/endophilin A2, a fusion partner gene in leukaemia.

EEN (extra eleven nineteen), also known as EA2 (endophilin A2), a fusion partner of the MLL (mixed-lineage leukaemia) gene in human acute leukaemia, is a member of the endophilin A family, involved in the formation of endocytic vesicles. We present evidence to show that EEN/EA2 is localized predominantly in nuclei of various cell lines of haemopoietic, fibroblast and epithelial origin, in contrast with its reported cytoplasmic localization in neurons and osteoclasts, and that EEN/EA2 exhibits nucleocytoplasmic shuttling. During the cell cycle, EEN/EA2 shows dynamic localization: it is perichromosomal in prometaphase, co-localizes with the bipolar spindle in metaphase and anaphase and redistributes to the midzone and midbody in telophase. This pattern of distribution coincides with changes in protein levels of EEN/EA2, with the highest levels being observed in G2/M-phase. Our results suggest that distinct subcellular localization of the endophilin A family members probably underpins their diverse cellular functions and indicates a role for EEN/EA2 in the cell cycle.

A diagnostic biochip for the comprehensive analysis of MLL translocations in acute leukemia.

Reciprocal rearrangements of the MLL gene are among the most common chromosomal abnormalities in both Acute Lymphoblastic and Myeloid Leukemia. The MLL gene, located on the 11q23 chromosomal band, is involved in more than 40 recurrent translocations. In the present study, we describe the development and validation of a biochip-based assay designed to provide a comprehensive molecular analysis of MLL rearrangements when used in a standard clinical pathology laboratory. A retrospective blind study was run with cell lines (n=5), and MLL positive and negative patient samples (n=31), to evaluate assay performance. The limits of detection determined on cell line data were 10(-1), and the precision studies yielded 100% repeatability and 98% reproducibility. The study shows that the device can detect frequent (AF4, AF6, AF10, ELL or ENL) as well as rare partner genes (AF17, MSF). The identified fusion transcripts can then be used as molecular phenotypic markers of disease for the precise evaluation of minimal residual disease by RQ-PCR. This biochip-based molecular diagnostic tool allows, in a single experiment, rapid and accurate identification of MLL gene rearrangements among 32 different fusion gene (FG) partners, precise breakpoint positioning and comprehensive screening of all currently characterized MLL FGs.

NUP98-Topoisomerase I acute myeloid leukemia-associated fusion gene has potent leukemogenic activities independent of an engineered catalytic site mutation

AbstractChromosomal rearrangements of the 11p15 locus have been identified in hematopoietic malignancies, resulting in translocations involving the N-terminal portion of the nucleoporin gene NUP98. Fifteen different fusion partner genes have been identified for NUP98, and more than one half of these are homeobox transcription factors. By contrast, the NUP98 fusion partner in t(11;20) is Topoisomerase I (TOP1), a catalytic enzyme recognized for its key role in relaxing supercoiled DNA. We now show that retrovirally engineered expression of NUP98-TOP1 in murine bone marrow confers a potent in vitro growth advantage and a block in differentiation in hematopoietic precursors, evidenced by a competitive growth advantage in liquid culture, increased replating efficient of colony-forming cells (CFCs), and a marked increase in spleen colony-forming cell output. Moreover, in a murine bone marrow transplantation model, NUP98-TOP1 expression led to a lethal, transplantable leukemia characterized by extremely high white cell counts, splenomegaly, and mild anemia. Strikingly, a mutation to a TOP1 site to inactivate the isomerase activity essentially left unaltered the growth-promoting and leukemogenic effects of NUP98-TOP1. These findings, together with similar biologic effects reported for NUP98-HOX fusions, suggest unexpected, overlapping functions of NUP98 fusion genes, perhaps related to common DNA binding properties. (Blood. 2004;104:1127-1136)

Multi-center performance evaluation of a biochip designed to analyze the expression of 11q23/MLL abnormal fusion transcripts in acute leukemia

9577 Background: 11q23/MLL abnormalities are generally associated with unfavorable prognosis in acute myeloid leukemia (AML). More than 50 partners have been described for MLL, and 35 of these partners have been cloned and analyzed at the molecular level to date. The functional role of each specific fusion transcript on the progression and outcome of disease remains to be elucidated. We present here the results of an international multi-center study aimed at assessing the clinical performance of a new biochip based molecular device (MLL FusionChip™ kit) designed to confirm the 11q23 abnormality and identify the MLL fusion gene partner. Methods: Nine laboratories in 7 countries ran 4 sets of 5 assays. Each run included a control sample (positive cell line), 2 negative samples, and 2 positive samples. Sample inclusion criteria were made by current standard diagnostic procedures: MLL status of all samples were characterized by cytogenetics, FISH, and/or RQ-PCR, and RNA sample quality assessed prior to analysis on the MLL FusionChip. Results: Nine different partners were accurately identified, including rare cases such as AF17, AF10, MSF, and P300. Overall agreement between routine diagnostic analysis and the MLL FusionChip was >90%. Conclusions: A previous study showed the analytical performance of this oncodiagnostic device is consistent with its claimed use. Taken together, these results collectively demonstrate the MLL FusionChip provides reliable data that may enrich minimal residual disease (MRD) follow-up in the clinical setting. Further studies are necessary to evaluate the clinical utility of the molecular classification of acute leukemia, and whether additional molecular information acquired with this device will contribute to refinement of the 2001 WHO classification. Expected consequences may be enhanced patient stratification, expansion of personalized therapeutic approaches, and optimized use of molecular targeted-based therapeutics. No significant financial relationships to disclose.

Multi-center performance evaluation of a biochip designed to analyze the expression of 11q23/MLL abnormal fusion transcripts in acute leukemia

9577 Background: 11q23/MLL abnormalities are generally associated with unfavorable prognosis in acute myeloid leukemia (AML). More than 50 partners have been described for MLL, and 35 of these partners have been cloned and analyzed at the molecular level to date. The functional role of each specific fusion transcript on the progression and outcome of disease remains to be elucidated. We present here the results of an international multi-center study aimed at assessing the clinical performance of a new biochip based molecular device (MLL FusionChip™ kit) designed to confirm the 11q23 abnormality and identify the MLL fusion gene partner. Methods: Nine laboratories in 7 countries ran 4 sets of 5 assays. Each run included a control sample (positive cell line), 2 negative samples, and 2 positive samples. Sample inclusion criteria were made by current standard diagnostic procedures: MLL status of all samples were characterized by cytogenetics, FISH, and/or RQ-PCR, and RNA sample quality assessed prior to analysis on the MLL FusionChip. Results: Nine different partners were accurately identified, including rare cases such as AF17, AF10, MSF, and P300. Overall agreement between routine diagnostic analysis and the MLL FusionChip was >90%. Conclusions: A previous study showed the analytical performance of this oncodiagnostic device is consistent with its claimed use. Taken together, these results collectively demonstrate the MLL FusionChip provides reliable data that may enrich minimal residual disease (MRD) follow-up in the clinical setting. Further studies are necessary to evaluate the clinical utility of the molecular classification of acute leukemia, and whether additional molecular information acquired with this device will contribute to refinement of the 2001 WHO classification. Expected consequences may be enhanced patient stratification, expansion of personalized therapeutic approaches, and optimized use of molecular targeted-based therapeutics. No significant financial relationships to disclose.

The Role of NUP98 Gene Fusions in Hematologic Malignancy

Chromosomal aberrations occur with great frequency and some specificity in leukemia and other hematologic malignancies. The most common outcome of these rearrangements is the formation of a fusion gene, comprising portions of 2 genes normally present in the cell. These fusion proteins are presumed to be oncogenic; in many cases, animal models have proven them to be oncogenic. One of the most promiscuous fusion partner genes is the newly identified NUP98 gene, located on chromosome 11p15.5, which to date has been observed fused to 15 different fusion partners. NUP98 encodes a 98 kD protein that is an important component of the nuclear pore complex, which mediates nucleo-cytoplasmic transport of protein and RNA. The fusion partners of NUP98 form 2 distinct groups: homeobox genes and non-homeobox genes. All NUP98 fusions join the N-terminal GLFG repeats of NUP98 to the C-terminal portion of the partner gene, which, in the case of the homeobox gene partners, includes the homeodomain. Clinical findings are reviewed here, along with the findings of several in vivo and in vitro models have been employed to investigate the mechanisms by which NUP98 fusion genes contribute to the pathogenesis of leukemia.

Production and enhanced biological activity of a novel GHRH analog, hGHRH with an N-terminal Pro–Pro extension

Growth Hormone Releasing Hormone (GHRH) is one of the most important hormones in life. Because of its potential clinical importance, its short half-life, and its expensive chemical synthesis, an analog of hGHRH with a prolonged half-life and better activity has been studied for clinical application, especially for the treatment of muscle wasting, type II diabetes, or sleep disorders. The Pro–Pro–hGHRH(1–44) peptide has better activity. The fusion partner gene with 127 amino acid residues of the C-terminus from l-asparaginase was recombined with asp-pro–pro–hGHRH(1–44) gene synthesized by PCR method to form a fusion protein with the unique acid labile linker Asp–Pro. The recombinant protein was expressed to high levels in Escherichia coli BL21 (DE3). The Pro–Pro–hGHRH(1–44) peptide was purified to homogeneity by means of cell disruption, washing, ethanol precipitation, acid hydrolysis, and SP-Sephadex C-25, and Sephadex G-25 column chromatography. The fold of the purification was about 88 times and the yield was 1.1% of the total protein weight of the inclusion body. The peptide molecular mass of 5235.25 Da was determined by ESI mass spectroscopy. Its purity was determined by SDS–PAGE. In the study of the activity, we measured GH release of rat pituitary by using the antiserum kit against human GH. The peptide doses of 0.01, 0.1, 1.0, 7.72, and 20.9 μg/ml used, respectively, released the GH values of 0.1 ± 0.1, 12.5 ± 7.3, 16.6 ± 5.8, 49.8 ± 7.6, and 79.5 ± 5.7 ng/ml whereas their blank controls, respectively, were 0.5 ± 0.8, 4.1 ± 2.6, 3.1 ± 3.1, 4.7 ± 1.8, and 1.2 ± 0.3 ng/ml. The activity results of all dose groups except 0.01 μg/ml Pro–Pro–hGHRH(1–44) group and hGHRH(1–40) group showed that there were significant differences between GH released by the peptide and that by its blank control. With the increase of dosage, the differences were more significant. hGHRH(1–40) showed no measured GH release when the dose was up to 2 μg/ml. The activity results show that the Pro–Pro–hGHRH(1–44) peptide is a potential GH releasing analog.

Rearrangement of the MLL gene and a region proximal to the RARα gene in a case of acute myelocytic leukemia M5 with a t(11;17)(q23;q21)

A case of acute myelocytic leukemia (AML) M5 subtype (French–American–British classification), in a 13-year-old girl showed the abnormal karyotype 46,XX,t(11;17)(q23;q21) in all bone marrow cells analyzed. Rearrangements involving 11q23 are frequent in cases of AML M5 and often involve the MLL gene. Nevertheless, t(11;17)(q23;q21) is very rare in this type of leukemia. In acute promyelocytic leukemia, the RARα gene, located at 17q21, is involved in almost all cases. Fluorescence in situ hybridization studies revealed a deletion of the C-terminal part of the MLL gene and a translocation of the RARα gene on the derivative chromosome 11, proximal to the remaining part of the MLL gene. However, hybridization with the LSI RARA dual color break-apart rearrangement probe showed that the RARα gene was not rearranged in this translocation. This is the first study reporting a t(11;17)(q23;q21) with a deletion distal to MLL gene exon 6 in a case of AML M5. Furthermore, this is the second study that strongly suggests the implication of a gene proximal and close to the RARα locus in a case of AML M5. According to these results, the discovery of new fusion partner genes of MLL and the precise characterization of t(11;17) will be important for the understanding of neoplastic cell differentiation in AML M5.

Molecular and fluorescence in situ hybridization analysis of a 10;11 rearrangement in a case of infant acute monocytic leukemia

Fluorescence in situ hybridization (FISH) analysis in a case of infant acute monocytic leukemia M5 revealed a complex rearrangement between chromosomes 10 and 11, leading to the disruption of the MLL gene. Using two painting probes for chromosomes 10 and 11 and a specific probe for the MLL gene localized on 11q23, we observed a paracentric inversion of the 11q13∼q23 fragment translocated to 10p12. Molecular analysis showed that AF10 localized on 10p12 was the fusion partner gene of MLL in this rearrangement (10;11). This report underlined the usefulness of FISH and molecular techniques in identifying complex rearrangements.

The Leukemia-Associated Gene Mllt1/ENL: Characterization of a Murine Homolog and Demonstration of an Essential Role in Embryonic Development

ABSTRACT MLLT1 (ENL/LTG19) is one of a number of fusion gene partners with the MLL oncogene involved in 11q23 translocations in human leukemia and encodes a transcriptional regulator of unknown function. Leukemias bearing MLL translocations may be myeloid or lymphoid or bear mixed lineage properties; however, those bearing MLL/MLLT1 translocations are predominantly lymphoid, suggesting that MLLT1 may influence the leukemic phenotype. The murine homolog Mllt1 exhibits 86% amino acid sequence identity with the human gene and is broadly expressed in murine tissues and cell lines, with the exception of liver and myeloid cell lines. We have mapped Mllt1 to mouse chromosome 17 band E2 using FISH analysis. The genomic structure and 5′ regulatory sequence of Mllt1 are highly conserved between mouse and human. There is also conservation of the genomic structure, but not the promoter, between MLLT1 and MLLT3/AF9, a homologous gene that is also an MLL translocation partner in human leukemias with a predominant myeloid phenotype. Targeted disruption of Mllt1 in mice leads to embryonic lethality prior to 8.5 dpc. These studies indicate that MLLT1 is involved in essential developmental processes and suggest that expression patterns of MLL fusion partners may influence the lineage of MLL-associated leukemias.

Transcription Factors: Normal and Malignant Development of Blood Cells.

Transcription Factors: Normal and Malignant Development of Blood Cells, edited by Katya Ravid and Jonathan Licht. New York, Wiley-Liss, Inc, 2001; 622 pages. Nobody doubts that hematopoietic differentiation stems from the transcription of genes involved in lineage specificity. Protein-DNA interaction played out by transcription factors and their cognate DNA sequences must be coordinately regulated throughout the process of blood cell development. Vigorous efforts have been made over the last 15 years to elucidate the precise role of individual transcription factors. So much has been learned that it has become overwhelming for clinicians, and even for researchers, to catch up on new discoveries about transcription in hematopoiesis. Transcription Factors: Normal and Malignant Development of Blood Cells, edited by Katya Ravid and Jonathan Licht, is a comprehensive manual that provides information about the major transcription factors for blood cell development and aberrant pathways by translocation. This volume (622 pages and 111 figures), which weighs about one kilogram, has 32 chapters and is divided into six parts. The first part deals with transcription factors relating to the normal differentiation of megakaryocytic and erythroid cells. The topics addressed in Part One are GATA-1 and friend of GATA (FOG), nuclear factor erythroid 2 (NF-E2), TAL1/SCL, erythroid Kruppel-like factor (EKLF), and an overview of transcription factors implicated in megakaryocytic differentiation. Part Two, which focuses on myeloid differentiation, consists of the topics RUNX1(AML1)/CBFB ( -subunit–encoding core binding factor), PU.1, CCAAT/ enhancer-binding proteins, homeobox genes, retinoic acid receptors, and vitamin D3 receptors. Part Three, which addresses lymphoid differentiation, contains eight chapters, and includes information about Ikaros, PU.1, Pax5(BSAP), Janus kinases and STAT families, E2A, Bcl-6, octamer factors, and early B-cell factor (EBF). Each chapter (15-20 pages) is written by researchers in the field, most in the United States and a few in Europe, and can be read easily. Authors have intended to describe their works using culture systems and, to a greater extent, gene-targeted animal models. Knowledge obtained by knockout and knockin methods is reviewed in the chapters and makes this book an up-todate guide for hematologists. Part Four describes transcription factors involved in leukemia due to chromosomal translocation and targets nine factors: retinoic acid receptor (RAR) , inv(16), EVI1, t(8;21), TEL/ETV6, mixed lineage leukemia (MLL), coactivators, LMO2, and acetyltransferases CREB-binding protein (CBP)/p300. Each chapter includes information based on experiments by the authors and other researchers and discusses the structure and function of the gene, its role in differentiation, fusion partner genes, and the function of chimera genes in leukemogenesis. Part Five, “Oncogenesis and Hematopoiesis,” is relatively short, with two chapters, one for myc and myb and another for NFB. Readers will encounter broad descriptions, derived from numerous references, about the structure of these oncogenes and their roles in hematopoiesis and transformation. The last section contains a list of transcription factors implicated in hematopoiesis and a table of chromosomal translocations associated with disruption of transcriptional regulators in leukemia and lymphoma. This section is useful for locating information about the genes in chromosomal translocations that are relevant in the clinical settings of hematological malignancies. The editors summarize their conclusions in the introduction, presenting the general characteristics of transcription factors in hematopoiesis. Their main points are extracted as follows:

TIMELY TOPIC: ANAPLASTIC LYMPHOMA KINASE (ALK) SPREADS ITS INFLUENCE

Anaplastic lymphoma kinase (ALK) is normally not expressed in human tissues except selected sites in the nervous system. Its expression and constitutive activation as a result of a chromosomal translocation involving 2p23 plays a pivotal role in the genesis of anaplastic large cell lymphoma. ALK expression has been instrumental in defining a homogeneous subset from the category of anaplastic large cell lymphoma, characterised by occurrence in young patients, primary systemic presentation, favorable prognosis, a broad morphological spectrum, nuclear and/or cytoplasmic immunostaining for ALK protein, and a number of possible fusion partner genes such as NPM, ATIC, TFG, TPM3 and CLTCL. Recently ALK has been implicated in the genesis of another tumour type, the inflammatory myofibroblastic tumours. The ALK-positive examples occur in children and young adults, involving a variety of sites, such as the abdomen, mesentery, liver, bladder, mediastinum, lung and bone. The partner genes identified in some cases are TPM3 (tropomyosin 3) and TPM4 (tropomyosin 4). These molecular findings also further support the neoplastic nature of at least a subset of inflammatory myofibroblastic tumours. Anaplastic lymphoma kinase (ALK) is normally not expressed in human tissues except selected sites in the nervous system. Its expression and constitutive activation as a result of a chromosomal translocation involving 2p23 plays a pivotal role in the genesis of anaplastic large cell lymphoma. ALK expression has been instrumental in defining a homogeneous subset from the category of anaplastic large cell lymphoma, characterised by occurrence in young patients, primary systemic presentation, favorable prognosis, a broad morphological spectrum, nuclear and/or cytoplasmic immunostaining for ALK protein, and a number of possible fusion partner genes such as NPM, ATIC, TFG, TPM3 and CLTCL. Recently ALK has been implicated in the genesis of another tumour type, the inflammatory myofibroblastic tumours. The ALK-positive examples occur in children and young adults, involving a variety of sites, such as the abdomen, mesentery, liver, bladder, mediastinum, lung and bone. The partner genes identified in some cases are TPM3 (tropomyosin 3) and TPM4 (tropomyosin 4). These molecular findings also further support the neoplastic nature of at least a subset of inflammatory myofibroblastic tumours.

Timely topic: Anaplastic lymphoma kinase (ALK) spreads its influence

Anaplastic lymphoma kinase (ALK) is normally not expressed in human tissues except selected sites in the nervous system. Its expression and constitutive activation as a result of a chromosomal translocation involving 2p23 plays a pivotal role in the genesis of anaplastic large cell lymphoma. ALK expression has been instrumental in defining a homogeneous subset from the category of anaplastic large cell lymphoma, characterised by occurrence in young patients, primary systemic presentation, favorable prognosis, a broad morphological spectrum, nuclear and/or cytoplasmic immunostaining for ALK protein, and a number of possible fusion partner genes such as NPM, ATIC, TFG, TPM3 and CLTCL. Recently ALK has been implicated in the genesis of another tumour type, the inflammatory myofibroblastic tumours. The ALK-positive examples occur in children and young adults, involving a variety of sites, such as the abdomen, mesentery, liver, bladder, mediastinum, lung and bone. The partner genes identified in some cases are TPM3 (tropomyosin 3) and TPM4 (tropomyosin 4). These molecular findings also further support the neoplastic nature of at least a subset of inflammatory myofibroblastic tumours.

Structure of AF3p21, a New Member of Mixed Lineage Leukemia (MLL) Fusion Partner Proteins-Implication for MLL-Induced Leukemogenesis

The Mixed Lineage Leukemia (MLL) gene is frequently rearranged in leukemia, especially in infantile leukemia and therapy-related leukemia. The MLL gene is localized at chromosome 11q23, and is involved in almost all of the chromosomal translocations involving 11q23. Twenty-four fusion partner genes have been identified to date, and the N-terminus of MLL fuses in-frame to the partner genes in all cases. Some of the MLL fusion partner genes encode transcription factors; others encode small GTP binding protein interacting molecules or cytoplasmic proteins, the functions of which are presently unknown. As a result of the diverse features of the MLL fusion partners, the underlying mechanism for leukemogenesis remains obscure. We cloned the MLL fusion partner gene from leukemic cells from a therapy-related leukemia patient with t(3;11)(p21;q23) and designated the gene AF3p21 This patient had a long latency period (9 years) before developing secondary leukemia. The AF3p21 gene encodes a nuclear protein with a molecular mass of 80 kDa, and this protein has SH3 and proline-rich domains. Among MLL fusion partners identified to date, only AF10 and AF17 have a homo-oligomerization domain. AF3p21 also has a homo-oligomerization domain, which was revealed by using a mammalian two-hybrid system. These results suggest that one possible role of the MLL fusion partners is to form an oligomer of truncated MLL. In this review, current knowledge about MLL-involved leukemogenesis is outlined.

DNA structural properties of AF9 are similar to MLL and could act as recombination hot spots resulting in MLL/AF9 translocations and leukemogenesis

The human AF9 gene at 9p22 is one of the most common fusion partner genes with the MLL gene at 11q23, resulting in the t(9;11)(p22;q23). The MLL-AF9 fusion gene is associated with de novo acute myelo-genous leukemia (AML), rarely with acute lymphocytic leukemia (ALL) and with therapy related leukemia (t-AML). The AF9 gene is >100 kb and two patient breakpoint cluster regions (BCRs) have been identified; BCR1 is within intron 4, previously called site A, whereas BCR2 or site B spans introns 7 and 8. Patient breakpoint locations were determined previously by RT-PCR and by genomic DNA cloning. In this study, we defined the exon-intron boundaries and identified several different structural elements in AF9 including a co-localizing in vivo DNA topo II cleavage site and an in vitro DNase I hypersensitive (DNase 1 HS) site in intron 7 in BCR2. Reversibility experiments demonstrated a religation of the topo II cleavage sites. The location of the in vivo topo II cleavage site was confirmed in vitro using a topo II cleavage assay. In addition, two scaffold associated regions (SARs) are located centromeric to the topo II and DNase I HS cleavage sites and border both patient breakpoint regions: SAR1 is located in intron 4, whereas SAR2 encompasses parts of exons 5-7. This study demonstrates that the patient breakpoint regions of AF9 share the same structural elements as the MLL BCR. We describe a DNA breakage and repair model for non-homologous recombination between MLL and its partner genes, particularly AF9.