Disruption of the SCL gene by a t(1;3) translocation in a patient with T cell acute lymphoblastic leukemia.

SCL gene disruptions are the most common chromosomal abnormality associated with the development of T cell acute lymphoblastic leukemia (ALL). Such disruptions can be the result of t(1;14) and t(1;7) translocations, as well as a cytogenetically undetectable interstitial deletion of chromosome 1. We present here a case of T cell ALL with a t(1;3)(p34;p21) translocation that also disrupts the SCL locus and leads to dysregulated SCL gene expression. This translocation, similar to previously reported SCL gene disruptions, appears to have been mediated, at least in part, by the V(D)J recombinase complex, since cryptic heptamer recognition sequences, as well as nontemplated N region nucleotide addition, are present at the breakpoints. The t(1;3) also disrupts a region on chromosome 3 characterized by alternating purine and pyrimidine residues, which can form a Z-DNA structure, reported to be prone to recombination events. A previously undescribed, evolutionarily conserved transcript unit is detected within 8 kb of the breakpoint on chromosome 3. This report extends the spectrum of recognized SCL translocations associated with T cell ALL, and underscores the contention that dysregulated SCL expression may be a causal event in T cell ALL.

Demonstration of delta rec-pseudo J alpha rearrangement with deletion of the delta locus in a human stem-cell leukemia.

It has been hypothesized that a rearrangement between the delta recombining element (delta Rec) and a pseudo J alpha gene serves to delete the TCR-delta locus before rearrangement of the TCR-alpha genes. We have now sequenced a direct, site-specific rearrangement between the delta Rec element and a pseudo J alpha gene in a human leukemic stem-cell line. Putative "N-sequence" addition was noted at the site of recombination, suggesting that this event occurred at a time when the enzyme(s) involved in N-region addition were active in this cell. This provides support for the view that deletion of the TCR-delta locus is required before rearrangement of the TCR-alpha chain genes.

Disruption of the human SCL locus by "illegitimate" V-(D)-J recombinase activity.

A fusion complementary DNA in the T cell line HSB-2 elucidates a provocative mechanism for the disruption of the putative hematopoietic transcription factor SCL. The fusion cDNA results from an interstitial deletion between a previously unknown locus, SIL (SCL interrupting locus), and the 5' untranslated region of SCL. Similar to 1;14 translocations, this deletion disrupts the SCL 5' regulatory region. This event is probably mediated by V-(D)-J recombinase activity, although neither locus is an immunoglobulin or a T cell receptor. Two other T cell lines, CEM and RPMI 8402, have essentially identical deletions. Thus, in lymphocytes, growth-affecting genes other than immune receptors risk rearrangements.

Gene expression profiling of precursor T-cell lymphoblastic leukemia/lymphoma identifies oncogenic pathways that are potential therapeutic targets.

We compared the gene expression pattern of thymic tumors from precursor T-cell lymphoblastic lymphoma/leukemia (pre-T LBL) that arose in transgenic mice that overexpressed SCL, LMO1 or NUP98-HOXD13 (NHD13) with that of thymocytes from normal littermates. Only two genes, Ccl8 and Mrpl38, were consistently more than fourfold overexpressed in pre-T LBL from all three genotypes analyzed, and a single gene, Prss16 was consistently underexpressed. However, we identified a number of genes, such as Cfl1, Tcra, Tcrb, Pbx3, Eif4a, Eif4b and Cox8b that were over or under-expressed in pre-T LBL that arose in specific transgenic lines. Similar to the situation seen with human pre-T LBL, the SCL/LMO1 leukemias displayed an expression profile consistent with mature, late cortical thymocytes, whereas the NHD13 leukemias displayed an expression profile more consistent with immature thymocytes. We evaluated two of the most differentially regulated genes as potential therapeutic targets. Cfl1 was specifically overexpressed in SCL-LMO1 tumors; inactivation of Cfl1 using okadaic acid resulted in suppression of leukemic cell growth. Overexpression of Ccl8 was a consistent finding in all three transgenic lines, and an antagonist for the Ccl8 receptor-induced death of leukemic cell lines, suggesting a novel therapeutic approach.

Mouse embryonic stem cells that express a NUP98-HOXD13 fusion protein are impaired in their ability to differentiate and can be complemented by BCR-ABL.

NUP98-HOXD13 (NHD13) fusions have been identified in patients with myelodysplastic syndrome, acute myelogenous leukemia and chronic myeloid leukemia blast crisis. We generated 'knock-in' mouse embryonic stem (ES) cells that express a NHD13 fusion gene from the endogenous murine NUP98 promoter, and used an in vitro differentiation system to differentiate the ES cells to hematopoietic colonies. Replating assays demonstrated that the partially differentiated NHD13 ES cells were immortal, and two of these cultures were transferred to liquid culture. These cell lines are partially differentiated immature hematopoietic cells, as determined by morphology, immunophenotype and gene expression profile. Despite these characteristics, they were unable to differentiate when exposed to high concentrations of erythropoietin (Epo), granulocyte colony-stimulating factor or macrophage colony-stimulating factor. The cell lines are incompletely transformed, as evidenced by their dependence on interleukin 3 (IL-3), and their failure to initiate tumors when injected into immunodeficient mice. We attempted genetic complementation of the NHD13 gene using IL-3 independence and tumorigenicity in immunodeficient mice as markers of transformation, and found that BCR-ABL successfully transformed the cell lines. These findings support the hypothesis that expression of a NHD13 fusion gene impairs hematopoietic differentiation, and that these cell lines present a model system to study the nature of this impaired differentiation.

Structural characterization of SIL, a gene frequently disrupted in T-cell acute lymphoblastic leukemia.

The SIL (SCL interrupting locus) gene was initially discovered at the site of a genomic rearrangement in a T-cell acute lymphoblastic leukemia cell line. This rearrangement, which occurs in a remarkably site-specific fashion, is present in the leukemic cells of 16 to 26% of patients with T-cell acute lymphoblastic leukemia. We have now cloned a normal SIL cDNA from a cell line which does not carry the rearrangement. The SIL cDNA has a long open reading frame of 1,287 amino acids, with a predicted molecular size of 143 kDa. The predicted protein is not homologous with any previously described protein; however, a potential eukaryotic topoisomerase I active site was identified. Cross-species hybridization using a SIL cDNA probe indicated that the SIL gene was conserved in mammals. A survey of human and murine cell lines and tissues demonstrated SIL mRNA to be ubiquitously expressed, at low levels, in hematopoietic cell lines and tissues. With the exception of 11.5-day-old mouse embryos, SIL mRNA was not detected in nonhematopoietic tissues. The genomic structure of SIL was also analyzed. The gene consists of 18 exons distributed over 70 kb, with the 5' portion of the gene demonstrating alternate exon utilization.

DNA cleavage within the MLL breakpoint cluster region is a specific event which occurs as part of higher-order chromatin fragmentation during the initial stages of apoptosis.

A distinct population of therapy-related acute myeloid leukemia (t-AML) is strongly associated with prior administration of topoisomerase II (topo II) inhibitors. These t-AMLs display distinct cytogenetic alterations, most often disrupting the MLL gene on chromosome 11q23 within a breakpoint cluster region (bcr) of 8.3 kb. We recently identified a unique site within the MLL bcr that is highly susceptible to DNA double-strand cleavage by classic topo II inhibitors (e.g., etoposide and doxorubicin). Here, we report that site-specific cleavage within the MLL bcr can be induced by either catalytic topo II inhibitors, genotoxic chemotherapeutic agents which do not target topo II, or nongenotoxic stimuli of apoptotic cell death, suggesting that this site-specific cleavage is part of a generalized cellular response to an apoptotic stimulus. We also show that site-specific cleavage within the MLL bcr can be linked to the higher-order chromatin fragmentation that occurs during the initial stages of apoptosis, possibly through cleavage of DNA loops at their anchorage sites to the nuclear matrix. In addition, we show that site-specific cleavage is conserved between species, as specific DNA cleavage can also be demonstrated within the murine MLL locus. Lastly, site-specific cleavage during apoptosis can also be identified at the AML1 locus, a locus which is also frequently involved in chromosomal rearrangements present in t-AML patients. In conclusion, these results suggest the potential involvement of higher-order chromatin fragmentation which occurs as a part of a generalized apoptotic response in a mechanism leading to chromosomal translocation of the MLL and AML1 genes and subsequent t-AML.

The SCL gene is formed from a transcriptionally complex locus.

We describe the structural organization of the human SCL gene, a helix-loop-helix family member which we believe plays a fundamental role in hematopoietic differentiation. The SCL locus is composed of eight exons distributed over 16 kb. SCL shows a pattern of expression quite restricted to early hematopoietic tissues, although in malignant states expression of the gene may be somewhat extended into later developmental stages. A detailed analysis of the transcript(s) arising from the SCL locus revealed that (i) the 5' noncoding portion of the SCL transcript, which resides within a CpG island, has a complex pattern of alternative exon utilization as well as two distinct transcription initiation sites; (ii) the 5' portions of the SCL transcript contain features that suggest a possible regulatory role for these segments; (iii) the pattern of utilization of the 5' exons is cell lineage dependent; and (iv) all of the currently studied chromosomal aberrations that affect the SCL locus either structurally or functionally eliminate the normal 5' transcription initiation sites. These data suggest that the SCL gene, and specifically its 5' region, may be a target for regulatory interactions during early hematopoietic development.

Disordered T-cell development and T-cell malignancies in SCL LMO1 double-transgenic mice: parallels with E2A-deficient mice.

The gene most commonly activated by chromosomal rearrangements in patients with T-cell acute lymphoblastic leukemia (T-ALL) is SCL/tal. In collaboration with LMO1 or LMO2, the thymic expression of SCL/tal leads to T-ALL at a young age with a high degree of penetrance in transgenic mice. We now show that SCL LMO1 double-transgenic mice display thymocyte developmental abnormalities in terms of proliferation, apoptosis, clonality, and immunophenotype prior to the onset of a frank malignancy. At 4 weeks of age, thymocytes from SCL LMO1 mice show 70% fewer total thymocytes, with increased rates of both proliferation and apoptosis, than control thymocytes. At this age, a clonal population of thymocytes begins to populate the thymus, as evidenced by oligoclonal T-cell-receptor gene rearrangements. Also, there is a dramatic increase in immature CD44(+) CD25(-) cells, a decrease in the more mature CD4(+) CD8(+) cells, and development of an abnormal CD44(+) CD8(+) population. An identical pattern of premalignant changes is seen with either a full-length SCL protein or an amino-terminal truncated protein which lacks the SCL transactivation domain, demonstrating that the amino-terminal portion of SCL is not important for leukemogenesis. Lastly, we show that the T-ALL which develop in the SCL LMO1 mice are strikingly similar to those which develop in E2A null mice, supporting the hypothesis that SCL exerts its oncogenic action through a functional inactivation of E proteins.

t(9;11)(p22;p15) in acute myeloid leukemia results in a fusion between NUP98 and the gene encoding transcriptional coactivators p52 and p75-lens epithelium-derived growth factor (LEDGF).

A t(9;11)(p22;p15) chromosomal translocation was identified in an adult patient with de novo acute myelogenous leukemia. Fluorescence in situ hybridization and Southern blot analysis mapped the 11p15 break-point to the NUP98 gene. Using 3' rapid amplification of cDNA ends, we have identified a chimeric mRNA that fused the NUP98 FXFG repeats in frame to the COOH-terminal portion of the gene encoding the transcriptional coactivators p52 and p75, also known as lens epithelium-derived growth factor (LEDGF). As expected, both NUP98-p52 and NUP98-p75 (LEDGF) chimeric mRNAs were detected by reverse transcription-PCR; however, the reciprocal p52/p75 (LEDGF)-NUP98 fusion mRNA was not detected. Our results demonstrate that this is the most 5' NUP98 fusion reported and reveal a previously unrecognized genetic target, the gene encoding p52/p75 (LEDGF).

Cloning and characterization of TCTA, a gene located at the site of a t(1;3) translocation.

We have cloned and characterized a novel gene at the site of a t(1;3)(p34;p21) translocation breakpoint in T-cell acute lymphoblastic leukemia. A cDNA for this gene, for which we propose the designation TCTA (T-cell leukemia translocation-associated gene), has been cloned. TCTA mRNA is expressed ubiquitously in normal tissues, with the highest levels of expression seen in the kidney. The TCTA gene is conserved throughout evolution in organisms ranging from Drosophila to humans. A short open reading frame encodes a predicted M(r) 12,000 protein without strong homology to any previously reported proteins. Of note, genomic Southern blots demonstrated a reduced TCTA signal in three of four small cell lung cancer cell lines tested, suggesting loss of one of the two copies of the gene.

scid Thymocytes with TCRbeta gene rearrangements are targets for the oncogenic effect of SCL and LMO1 transgenes.

SCL and LMO1 were both discovered by virtue of their activation by chromosomaltranslocation in patients with T-cell acute lymphoblastic leukemia (T-ALL). Overexpression of SCL and LMO1 in the thymus of transgenic mice leads to T-ALL at a young age. scid (severe combined immunodeficient) mice are unable to efficiently recombine antigen receptor genes and consequently display a developmental block at the CD4-CD8- to CD4+CD8+ transition. To test the hypothesis that this developmental block would protect SCL/LMO1 transgenic mice from developing T-ALL, we crossed the SCL and LMO1 transgenes onto a scid background. The age of onset for T-ALL in the SCL/LMO1/scid mice was significantly delayed (P < 0.001) compared with SCL/LMO1/wild-type mice. Intriguingly, all of the SCL/LMO1/scid malignancies displayed clonal, in-frame TCRbeta gene rearrangements. Taken together, these findings suggest that the "leaky" scid thymocyte that undergoes a productive TCRbeta gene rearrangement is susceptible to the oncogenic action of SCL and LMO1 and additionally suggests that TCRbeta gene rearrangements may be required for the oncogenic action of SCL and LMO1.

NUP98-HOXD13 gene fusion in therapy-related acute myelogenous leukemia.

A novel chromosomal translocation, t(2;11)(q31;p15), was identified in a patient with therapy-related acute myelogenous leukemia (t-AML). Fluorescence in situ hybridization experiments mapped the breakpoint near NUP98; Southern blot analysis demonstrated that the nucleoporin gene NUP98 was disrupted by this translocation. We used rapid amplification of cDNA ends to identify a chimeric mRNA. An in-frame, chimeric mRNA that fused NUP98 sequences to the homeobox gene HOXD13 was cloned; the predicted fusion protein contains both the GLFG repeats from NUP98 as well as the homeodomain from HOXD13. The NUP98-HOXD13 fusion is structurally similar to the NUP98-HOXA9 fusion previously identified in patients with AML, leading to the speculation that NUP98-homeobox gene fusions may be oncogenic. Moreover, this report, along with a recent study that demonstrated NUP98-DDX10 fusions in patients with t-AML, raises the possibility that NUP98 may be a previously unsuspected target for chromosomal translocations in patients with t-AML.

SCL binds the human homologue of DRG in vivo.

A suspected oncoprotein, human development regulated GTP-binding protein (DRG) has never been identified though homologues were found in mouse, Xenopus, Drosophila, yeast and Halobacteria. During a search for SCL binding partners using the yeast 2-hybrid system, we isolated two independent cDNA clones (clone L51 and clone V3) of the human DRG homologue from human fetal liver and human thymus cDNA libraries. Only one amino acid difference was found between human and mouse DRG proteins. Although a human DRG has been previously deposited in the SWISS-PROT Database, we believe that we have cloned the bona fide human DRG based on the highly conserved primary amino acid structure between our cloned human homologue and the mouse DRG.

Disruption of T cell regulatory pathways is necessary for immunotherapeutic cure of T cell acute lymphoblastic leukemia in mice.

Acute lymphoblastic leukemia (ALL) is the most common cancer in children. In recent years, the outcome has been globally improved by current therapies, but it remains poor in patients with high, persistent residual disease following the first course of chemotherapy, prompting evaluation of the possible beneficial effects of immunotherapy protocols. In this study, we hypothesized that the disruption of two immunoregulatory pathways controlling the auto-reactive T cell response might synergize with dendritic cell-based immunotherapy of the disease, which is considered to be poorly immunogenic. In this study, we used TAL1xLMO1 leukemia cells adoptively transferred in mice, to generate murine leukemia with poorly immunogenic cells as a model for human T-ALL. Subsequently, these animals were treated with several different immunotherapeutic protocols. We compared the efficiency of a classical, dendritic cell-based immunotherapy (injection of dendritic cells loaded with tumor-derived antigenic products), to a combined treatment associating injection of antigen-loaded dendritic cells and disruption of the two immunoregulatory pathways: CD25+ suppressive T cells and cytotoxic T lymphocyte-associated antigens (CTLA-4). We show that this combined treatment resulted in cure, concomitantly with in vivo generation of immune memory, and TNF-alpha secretion. This study demonstrates that the disruption of these two immunoregulatory pathways synergized with immunostimulation by dendritic cells loaded with tumor-derived antigens, and paves the way for the testing of this combination in clinical trials.

NUP98 gene fusions in hematologic malignancies.

Acute leukemia is associated with a wide spectrum of recurrent, non-random chromosomal translocations. Molecular analysis of the genes involved in these translocations has led to a better understanding of both the causes of chromosomal rearrangements as well as the mechanisms of leukemic transformation. Recently, a number of laboratories have cloned translocations involving the NUP98 gene on chromosome 11p15.5, from patients with acute myelogenous leukemia (AML), myelodysplastic syndrome (MDS), chronic myelogenous leukemia (CML), and T cell acute lymphoblastic leukemia (T-ALL). To date, at least eight different chromosomal rearrangements involving NUP98 have been identified. The resultant chimeric transcripts encode fusion proteins that juxtapose the N-terminal GLFG repeats of NUP98 to the C-terminus of the partner gene. Of note, several of these translocations have been found in patients with therapy-related acute myelogenous leukemia (t-AML) or myelodysplastic syndrome (t-MDS), suggesting that genotoxic chemotherapeutic agents may play an important role in generating chromosomal rearrangements involving NUP98.

Topoisomerase II inhibitors induce DNA double-strand breaks at a specific site within the AML1 locus.

Treatment-related acute myeloid leukemia (t-AML) following successful therapy of a primary malignancy has been recognized with increasing frequency among cancer survivors over the past several years. Many of these t-AML cases are associated with the use of intensive chemotherapy regimens that employ one or more agents which target eukaryotic topoisomerase II (topo II), and demonstrate non-random chromosomal translocations involving either the MLL (ALL-1, HRX) gene at 11q23 or the AML1 gene at 21q22. Although many investigators have speculated that these translocations are induced by the therapeutic use of topo II inhibitors, the molecular sequence of events by which topo II inhibitors might induce a chromosomal translocation are not well understood. We describe here the reproducible induction of highly specific, double-strand DNA cleavage at a specific site within the AML1 locus by topo II inhibitors. This DNA cleavage, which maps to a region of the AML1 locus frequently disrupted by chromosomal translocations, can be induced in several cell lines, with multiple different topo II inhibitors, indicating that this phenomenon is not restricted to a specific cell type or specific topo II inhibitor. It is conceivable that site-specific double-strand DNA cleavage within the AML1 locus induced by topo II inhibitors represents the initial molecular event leading to a chromosomal translocation and t-AML.

Development and characterization of T cell leukemia cell lines established from SCL/LMO1 double transgenic mice.

We have established a panel of nine immortal cell lines from T cell malignancies which arose in mice transgenic for the SCL and LMO1 genes. Cells from the primary malignancies initially grew very slowly in vitro, loosely attached to a stromal layer, before gaining the ability to proliferate independently. Upon gaining the ability to proliferate in the absence of a stromal layer, these cell lines grew rapidly, doubling every 14-23 h, to a very high density, approaching 10(7) cells/ml. Whereas the tumors which arise in SCL/LMO1 double transgenic mice are typically diploid or pseudodiploid, the cell lines were all grossly aneuploid, suggesting the possibility that additional genetic events were selected for in vitro. Given that SCL and LMO1 gene activation are both commonly seen in human patients with T cell acute lymphoblastic leukemia, these cell lines may be a useful in vitro model for the human disease.

Effect of ischemia and reperfusion on protein oxidation in isolated rabbit hearts.

Reactive oxygen species and other oxidants are involved in the mechanism of postischemic contractile dysfunction, known as myocardial stunning. The present study investigated the oxidative modification of cardiac proteins in isolated Langendorff-perfused rabbit hearts subjected to 15 min normothermic ischemia followed by 10 min reperfusion. Reperfusion under these conditions resulted in only 61.8+/-2.7 % recovery of developed pressure relative to preischemic values and this mechanical dysfunction was accompanied by oxidative damage to cardiac proteins. The total sulfhydryl group content was significantly reduced in both ventricle homogenates and mitochondria isolated from stunned hearts. Fluorescence measurements revealed enhanced formation of bityrosines and conjugates of lipid peroxidation-end products with proteins in cardiac homogenates, whereas these parameters were unchanged in the mitochondrial fraction. Reperfusion did not alter protein surface hydrophobicity, as detected by a fluorescent probe 1-anilino-8-naphthalenesulfonate. Our results indicate that oxidation of proteins in mitochondria and possibly in other intracellular structures occurs during cardiac reperfusion and might contribute to ischemia-reperfusion injury.

Molecular biology of acute lymphocytic leukemia.

Acute lymphoblastic leukemia (ALL) has been extensively studied and characterized at a molecular level. The identification and characterization of molecular lesions associated with ALL has led to advances in classification and prognosis of ALL, which in turn has been useful in stratifying ALL patients for treatment purposes. However, perhaps of greater long-term significance are insights into the mechanisms of leukemogenesis that have been provided by characterization of ALL at a molecular level. This review focuses on general themes and mechanisms of disease, concentrating on recent advances in the field, including the relationship of deregulated transcription factors to ALL, probable mechanisms of lymphoid translocations, and newly characterized genetic lesions, such as those involving tumor suppressor genes, 11q23 abnormalities, and 12p12 abnormalities.