Lymphoid Myeloid Leukemia Gene Research Articles

MLL and CALM Are Fused to AF10 in Morphologically Distinct Subsets of Acute Leukemia With Translocation t(10;11): Both Rearrangements Are Associated With a Poor Prognosis

AbstractThe translocation t(10;11)(p13;q14) has been observed in acute lymphoblastic leukemia (ALL) as well as acute myeloid leukemia (AML). A recent study showed a MLL/AF10fusion in all cases of AML with t(10;11) and various breakpoints on chromosome 11 ranging from q13 to q23. We recently cloned CALM (Clathrin Assembly Lymphoid Myeloid leukemia gene), the fusion partner of AF10 at 11q14 in the monocytic cell line U937. To further define the role of these genes in acute leukemias, 10 cases (9 AML and 1 ALL) with cytogenetically proven t(10;11)(p12-14;q13-21) and well-characterized morphology, immunophenotype, and clinical course were analyzed. Interphase fluorescence in situ hybridization (FISH) was performed with 2 YACs flanking the CALM region, a YAC contig of the MLLregion, and a YAC spanning the AF10 breakpoint. Rearrangement of at least one of these genes was detected in all cases with balanced t(10;11). In 4 cases, including 3 AML with immature morphology (1 AML-M0 and 2 AML-M1) and 1 ALL, the signals of the CALM YACS were separated in interphase cells, indicating a translocation breakpoint within the CALM region. MLL was rearranged in 3 AML with myelomonocytic differentiation (2 AML-M2 and 1 AML-M5), including 1 secondary AML. In all 3 cases, a characteristic immunophenotype was identified (CD4+, CD13−, CD33+, CD65s+).AF-10 was involved in 5 of 6 evaluable cases, including 1 case without detectable CALM or MLL rearrangement. In 2 complex translocations, none of the three genes was rearranged. All cases had a remarkably poor prognosis, with a mean survival of 9.6 ± 6.6 months. For the 7 AML cases that were uniformly treated according to the AMLCG86/92 protocols, disease-free and overall survival was significantly worse than for the overall study group (P = .03 and P = .01, respectively). We conclude that the t(10;11)(p13;q14) indicates CALM and MLL rearrangements in morphologically distinct subsets of acute leukemia and may be associated with a poor prognosis.

Abstract 3064: Epigenetic manipulation can sensitize AML cells to differentiate with ATRA

Abstract Human acute myeloid leukemia (AML), the most common type of acute leukemia, has approximately eight subtypes, many of which have poor prognosis. Most of these subtypes are associated with specific, recurrent chromosome translocations. These translocations result in fusion genes, which encode oncoproteins that block differentiation and promote proliferation of immature cells. The Myeloid Lymphoid Leukemia gene (MLL) is frequently involved in these translocations, and is considered a driver of the AML. Differentiation promoting drugs, such as all-trans-retinoic acid (ATRA) are an attractive alternative to cytotoxic chemotherapy, but few types of AML, other than acute promyelocytic leukemia (APL), respond to ATRA. We hypothesize that specific genes must be activated or inhibited in AML for drugs like ATRA to induce differentiation, and that gene activation or inhibition may be the result of specific epigenetic modification. We also hypothesize that AML with different genetic alterations may respond differently to specific epigenetic inhibitors. Our initial studies have focused on two MLL-driven AML cell lines, MV4;11 and THP-1, showing translocations t(4;11) and t(9;11), respectively, and one non-MLL related AML cell line, U937. MV4;11, THP-1, and U937 were treated with specific epigenetic modifiers, including tranylcypromine (TCP), an inhibitor of histone demethylase KMD1A/LSD1, N-acetyl-dinaline (CI-994), a general histone deacetylase inhibitor, and 3-deazaneplanocin A (DZNep), a S-adenosylhomocystein hydrolase inhibitor that depletes EZH2 and thus its associated H3K27me3 activity. Evidence for differentiation was noted in a variety of assays, including reduced cell proliferation as measured directly and by MTT assay, upregulation of myeloid-specific cell surface markers such as CD11b as measured by fluorescence-activated cell sorting (FACS), myeloid-related nuclear morphological changes noted with cytospin analysis of cells, and decreased AML-associated gene expression with qPCR. All three drugs seemed to sensitize U937 and THP-1 cells to differentiate when treated with ATRA. MV4;11 cells were sensitized to differentiate by CI-994 and DZNep with or without ATRA, although there was no indication of CD11b upregulation. However, MV4;11 was not sensitized by TCP to differentiate with or without ATRA. These experiments suggest epigenetic inhibitors may increase sensitivity to ATRA differentiation therapy, but since the two MLL cell lines responded differently the response may still be dependent on the specific MLL partner gene driving the AML. Citation Format: Kalsi Heimdal, Bikalpa Ghimire, Edjay Ralph Hernandez, Heidi J. Gill Super. Epigenetic manipulation can sensitize AML cells to differentiate with ATRA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3064.

Abstract 870: Effects of epigenetic modifier inhibitors on AML cell sensitivity to differentiation therapy

Abstract Human acute myeloid leukemia (AML), the most common type of acute leukemia, has approximately eight subtypes, many of which have poor prognosis. Most of these subtypes are associated with specific, recurrent chromosome translocations. These translocations result in fusion genes, which encode oncoproteins that block differentiation and promote proliferation of immature cells. The Myeloid Lymphoid Leukemia gene (MLL) is frequently involved in these translocations, and is considered a driver of the AML. Differentiation promoting drugs, such as all-trans-retinoic acid (ATRA) are an attractive alternative to cytotoxic chemotherapy, but few types of AML, other than acute promyelocytic leukemia (APL), respond to ATRA. We hypothesize that specific genes must be activated or inhibited in AML for drugs like ATRA to induce differentiation, and that gene activation or inhibition may be the result of specific epigenetic modification. We also hypothesize that AML with different genetic alterations may respond differently to specific epigenetic inhibitors. Our initial studies have focused on one AML cell line, MV4;11, with alterations in the MLL gene, and one non-MLL related AML cell line, U937. MV4;11 and U937 were treated with specific epigenetic modifiers, including tranylcypromine (TCP), an inhibitor of histone demethylase KMD1A/LSD1, and CI-994, a general histone deacetylase inhibitor. Evidence for differentiation was noted in a variety of assays, including reduced cell proliferation as measured directly and by MTT assay, upregulation of myeloid-specific cell surface markers such as CD11b as measured by fluorescence-activated cell sorting (FACS), and myeloid-related nuclear morphological changes noted with cytospin analysis of cells. Both CI-994 and TCP seemed to sensitize U937 cells to differentiate when treated with ATRA as measured by all indicators of differentiation. MV4;11 cells were sensitized to differentiation by CI-994 with or without ATRA, although there was no indication of CD11b upregulation. However, MV4;11 was not sensitized by TCP to differentiate with ATRA. These experiments suggest epigenetic inhibitors may increase sensitivity to differentiation therapy, but that the response may still be dependent on the specific genetic alteration driving the AML. Our studies suggest that in MLL-driven leukemia, the MLL fusion oncoprotein may override genetic manipulation resulting from treatment with epigenetic inhibitors, preventing differentiation by ATRA. Citation Format: Kalsi K. Heimdal, Edjay Ralph A. Hernandez, Heidi J. Gill Super. Effects of epigenetic modifier inhibitors on AML cell sensitivity to differentiation therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 870.

Rearrangement of the Myeloid/Lymphoid Leukemia Gene in Therapy-Related Myelodysplastic Syndrome in Patients Previously Treated with Agents Targeting DNA Topoisomerase II

Background: Therapy-related acute myeloid leukemias (t-AML), with balanced translocations affecting the 11q23 point in the myeloid/lymphoid leukemia (MLL) gene, are one of the most serious complications of treatments with topoisomerase II inhibitors. However, only a few reports of t-AML exist. We aimed to study if these translocations are cumulative-dose-dependent, their frequency in therapy-related myelodysplastic syndrome and the relationship between their presence, the type of therapy and the response criteria. Methods: This retrospective study included 120 patients with various malignancies (108 non-Hodgkin’s lymphoma, 8 Hodgkin’s disease and 4 neuroblastoma) in remission, being treated with topoisomerase 2 inhibitors; 74 had been diagnosed with therapy-related myelodysplasia and 46 did not have dysplasia. All bone marrow biopsy samples were evaluated by fluorescence in situ hybridization for 11q23 point breakage in the MLL gene. Results: MLL gene rearrangement frequency was 6% in dysplastic versus 2% in nondysplastic groups; p < 0.001. It was associated with a worse overall survival (mean 13 ± 2 vs. 39 ± 3 months, log-rank p value <0.0001). It was dose-dependent with a cut-off value of 290 mg/kg of topoisomerase II inhibitors as assessed by ROC curve (area under the curve 0.84 ± 0.05, p < 0.0001). Conclusions: It is proposed that the MLL gene is etiopathogenetically relevant for hematological neoplasias transformation and survival.

Diagnostic Usefulness of Genomic Breakpoint Analysis of Various Gene Rearrangements in Acute Leukemias: A Perspective of Long Distance– or Long Distance Inverse-PCR–based Approaches

Diagnostic Usefulness of Genomic Breakpoint Analysis of Various Gene Rearrangements in Acute Leukemias: A Perspective of Long Distance– or Long Distance Inverse-PCR–based Approaches

Cytogenetic manifestation of chromosome 11 duplication/amplification in acute myeloid leukemia

Gene amplification is a frequent genetic abnormality in solid tumors, and many oncogenes are activated in this way. In acute myeloid leukemia (AML), a frequent target of gene amplification is chromosome 11, particularly chromosome region 11q23, including the MLL (myeloid/lymphoid leukemia) gene. However, the number of other amplicons from the long arm of chromosome 11 has also been described. Duplication/amplification of chromosome 11 was found by cytogenetic methods in 10 of 119 newly diagnosed patients with AML. The amplification was presented as: amplification including only the 5′ segment of the MLL gene (1 patient), trisomy 11 (3 patients), partial trisomy 11q (2 patients), isochromosome 11q (1 patient), and multiple amplification of specific regions (3 patients). In two cases, amplification involved parts of not only long arm but also of short arm of the chromosome 11: 11p15 and 11p11.1 to 11p13.

PICALM (phosphatidylinositol binding clathrin assembly protein)

Review on CALM (clathrin assembly lymphoid myeloid leukemia gene), with data on DNA, on the protein encoded, and where the gene is implicated.

5′ MLL Gene Deletion in a Case with Childhood Acute Lymphoblastic Leukemia

Myeloid/lymphoid leukemia (MLL) gene rearrangements are high risk cytogenetic characteristics of acute lymphoblastic leukemia (ALL). Translocations of this gene are well defined, and their impact on the patient’s prognosis is well known, but deletions of the same region are rare, and little is known about their prognostic significance and the significance of their accompanying translocations. Here we present a case of childhood ALL with a deletion of the 5’region of the MLL gene detected by fluorescence in situ hybridization (FISH) analysis. This result also confirmed the sensitivity and efficiency of FISH analysis.

Early Target Genes of CALM/AF10 as Revealed by Gene Expression Profiling.

The t(10;11)(p12;q14) is a recurring chromosomal translocation that is found in acute myeloid and acute lymphoblastic leukemia as well as in malignant lymphoma. This translocation results in the fusion of AF10, a putative zinc finger transcription factor containing an N-terminal LAP/PHD zinc finger motif, a nuclear localization signal, an AT-hook domain, and a leucine zipper and with the CALM gene (Clathrin assembly protein lymphoid myeloid leukemia gene) that encodes a clathrin assembly protein. In the monocytic cell line U937 both the CALM/AF10 and the AF10/CALM fusion mRNAs can be identified. The CALM/AF10 fusion mRNA codes for the CALM/AF10 fusion protein which contains almost the complete CALM protein fused in frame to about 90% of the AF10 protein without the N terminal two PHD zinc fingers. CALM/AF10 is highly leukemogenic with its expression in primary bone marrow cells leading to the development of an aggressive acute leukemia with a short latency of 10 weeks in a murine bone marrow transplant model. We set out to identify immediate target genes of CALM/AF10. The fusion gene was cloned into pRTS-1, an episomally replicating tetracycline/doxycycline inducible (tet-on) expression vector. The construct was stably transfected into the Burkitt's lymphoma B cell line DG75. The expression of CALM/AF10 after induction was confirmed by RT-PCR. Gene expression profiling experiments were conducted using the Affymetrix® Human Genome U133 Plus 2.0 Array analyzing non-induced and induced (24 and 72 hours after induction) samples. As controls, DG75 cells transfected with the pRTS-1 vector without the CALM/AF10 fusion gene were used. The results were analyzed using the R statistical package and dChip (DNA Chip Analyzer) software. The expression of 1237 genes was found to be changed at least 2 fold 24 hours after the induction of CALM/AF10. 594 (48%) genes were downregulated, while 643 (52%) were upregulated. Downregulated genes included genes involved in DNA repair (DDB2, TOP2A, BRCA1), cell cycle check point control (CCNE2, CHEK1, CDC2), and chromosome maintenance (MCM3, MCM7, MCM10). Upregulated genes included signal transduction molecules like RAB8B (a member of the RAS oncogene family) and STAT family members (STAT1 and STAT2), chromatin remodeling factors like BAZ2A (bromodomain adjacent to zinc finger domain, 2A), and MAML3 (master mind like 3), a positive regulator of Notch signaling. Pathway analysis using KegArray showed an enrichment of differentially regulated genes in processes like cell cycle regulation and DNA replication and repair. Interestingly, no significant upregulation of Hox genes was observed, as was previously reported in a study of CALM/AF10 positive patient samples (Dik et al., 2005). The changes in the expression levels of some selected genes were confirmed using a Taqman® Low Density Array (LDA). This analysis showed a statistically significant correlation (r = 0.78, p = 0.001) between the real time PCR results and the expression levels obtained from the Affymetrix arrays. Our results demonstrate that the expression of the leukemogenic CALM/AF10 fusion protein leads to a severe deregulation of critical cellular processes giving a first hint at the direct target genes of this fusion protein.

Expression of Alternative CALM-AF10 Fusions Causes Different Hematological Malignancies in Mice.

The clathrin assembly lymphoid myeloid leukemia gene, CALM, was first described as a translocation partner for AF10 in morphologically distinct subsets of acute leukemia with a recurring 10;11 translocation. As the name implies, CALM is involved in the regulation of clathrin-mediated endocytosis. We hypothesize that expression of the CALM-AF10 protein disrupts endocytosis of growth receptors, leading to constitutive activation and, thereby, contributes to the development of leukemia. The CALM-AF10 fusion transcripts are characterized by two breakpoints in CALM at positions 1926 and 2091. Using the 293T human renal epithelial cell line as a model, we demonstrated that CALM1926-AF10 localized diffusely to the cytoplasm, whereas CALM2091-AF10 had a more prominent punctate appearance. This difference in distribution was likely attributable to protein domains in CALM rather than AF10, which is a transcription factor that localizes to the nucleus. The difference in localization extended to their effects on endocytosis. Whereas ∼4–8% of cells expressing truncated CALM2091, CALM1926, or AF10 displayed impaired transferrin uptake, this value was ∼60% in cells expressing CALM2091-AF10 and only ∼13% in cells expressing CALM1926-AF10. Next, we used a retroviral transduction-transplantation murine model to test the impact of CALM-AF10 expression on the development of leukemia. Mice transplanted with either CALM1926-AF10 or CALM2091-AF10 transduced fetal liver cells developed disease within a short period of time (∼8 to 21 weeks post-transplantation). Interestingly, expression of CALM1926-AF10 led to the development of a myeloproliferative disease (MPD)-like leukemia, with a predominance of mature neutrophils, whereas expression of CALM2091-AF10 led to the development of an acute myeloid leukemia (AML) with >30% immature myeloid blasts, with one exception. One mouse transplanted with progenitor cells expressing CALM2091-AF10 developed a MPD accompanied by a granulocytic sarcoma; however, examination of RNA from the spleen of this diseased mouse revealed that it had undergone a splicing event consistent with the size expected for the 1926 breakpoint. Both diseases were transplantable to secondary recipients confirming the leukemic nature of the CALM-AF10 expressing cells. Previous studies show that patients with the t(10;11) (p13;q14-21) develop either AML or T cell-acute lymphoblastic leukemia (T-ALL). Most of the patients with T-ALL express the CALM1926-AF10 fusion. Although there are differences in presentation between humans and mice, the hematological disease may be a reflection of the conditions of the mouse model. To identify possible secondary cooperating mutations, spectral karyotyping analysis of the mouse AMLs and MPDs was performed. However, to date we have not identified any significant abnormalities. We hypothesize that defective endocytosis synergizes with transcriptional deregulation, leading to an aggressive form of leukemia.

The Four and a Half LIM Domain Protein 2 (FHL2) Interacts with CALM and Is Highly Expressed in AML with Complex Aberrant Karyotpes.

The balanced chromosomal translocation t(10;11)(p13;q14) results in the CALM/AF10 fusion gene. This translocation is found in acute myeloid leukemia (AML), T-cell acute lymphoblastic leukaemia (T-ALL) and malignant lymphoma. The CALM/AF10 fusion gene has recently been shown to cause an aggressive biphenotypic leukemia in a murine bone marrow transplant model. The CALM (Clathrin Assembly Lymphoid Myeloid leukemia gene) gene product is a clathrin assembly protein which plays a role in clathrin mediated endocytosis and trans Golgi network trafficking. AF10 is a putative transcription factor most likely involved in processes related to chromatin organization and has polycomb group gene like properties.To learn more about the function of the CALM/AF10 fusion protein, we searched for protein interaction partners of CALM. In a yeast two hybrid screen the four and a half LIM domain protein FHL2 was identified as putative CALM interacting partner. The CALM FHL2 interaction was confirmed by co-transformation assay in yeast and by GST-pulldown experiments. The FHL2 interaction domain of CALM was mapped to amino acids 294 to 335 of CALM using the yeast two hybrid assay. In co-localization studies with transiently expressed fluorescent protein tagged CALM and FHL2, both proteins showed cytoplasmatic localization. Expression analysis (Affymetrix based) in different AML subtypes showed a significantly higher expression of FHL2 in AML with complex aberrant karyotypes compared to AML with normal karyotypes or balanced chromosomal translocations like the t(8;21), inv(16) or t(15;17).FHL2, which is also known as DRAL (downregulated in rhabdomyosarcoma LIM protein), is a TP53 responsive gene known to interact with numerous proteins in both the nucleus and the cytoplasm and can function as a transcriptional cofactor. Known FHL2 interactors include TP53, BRCA1, PLZF (promyelocytic leukemia zinc finger protein), the proto-oncogene SKI1 and beta-catenin. High expression of FHL2 in breast cancer has recently been shown to be associated with an adverse prognosis. CALM has been shown to shuttle between the nucleus and the cytoplasm because inhibition of CREM-mediated nuclear export by leptomycin B leads to the accumulation of CALM in the nucleus. Reporter gene assays using a GAL4-DNA binding domain CALM fusion protein and a GAL4 responsive luciferase reporter were able to demonstrate a transcriptional activation function of CALM. We are currently investigation the effect of FHL2 co-expression on this aspect of the CALM function.It is thus conceivable that FHL2 is playing an important role in CALM/AF10-mediated leukemogenesis by tethering the CALM/AF10 fusion protein to various nuclear transcription factor complexes.

Interaction of the Leukemogenic CALM/AF10 Fusion Protein with the Hematopoietic Key Regulator Ikaros.

The focus of our research group is the study of the t(10;11)(p13;q14) translocation that leads to the fusion of the proteins CALM and AF10. This translocation can be found in acute lymphoblastic leukemia (ALL), acute myeloid leukemia (ALL) and also in malignant lymphomas. In some patients the t(10;11) is the only cytogenetic abnormality which indicates that the CALM/AF10 fusion is a causal event during leukemogenesis. Previous studies of our group have shown that the expression of CALM/AF10 in hematopoietic stem cells triggers the development of an aggressive leukemia in a murine bone marrow transplantation model. CALM (Clathrin Assembly Lymphoid Myeloid leukemia gene) has a function in Clathrin mediated endocytosis. AF10, a putative transcription factor with a PHD-Motive (Plant Homeo Domain) and a Leucine Zipper domain, was initially identified as fusion partner of MLL. The underlying mechanism of CALM/AF10 dependent leukemogenesis, however, remains mostly unknown. Recently we could show that AF10 interacts with the transcription factor Ikaros (ZNFN1A1) in yeast-two-hybrid assays. Interestingly, Ikaros is a key regulator of hematopoesis, required for normal differentiation and proliferation of B- and T-lymphocytes. The structure of the protein is characterized by a DNA-binding and an oligomerisation domain. Through interaction with many factors in the cell nucleus, Ikaros can act both as activator and repressor of transcription. In various forms of ALL as well as chronic myeloid leukemia (CML) an aberrant expression pattern of Ikaros has been found. In a murine model the expression of a dominant negative isoform of Ikaros causes leukemias and lymphomas. Using various AF10 deletion mutants in the yeast, the Ikaros interaction domain of AF10 was mapped to the Leucine Zipper domain of AF10 which has also been shown to be required for malignant transformation by the MLL/AF10 fusion protein. Overexpression of fluorescently labelled proteins reveals a similar distribution pattern of AF10 and Ikaros in the nucleus, whereas in the presence of CALM/AF10 Ikaros appears to be localized predominately in the cytoplasm. The interaction between AF10 and Ikaros has been confirmed by GST-pull-down assays. In order to further study this interaction and its role in leukemogenesis we have raised monoclonal antibodies against the C-terminus of AF10. These antibodies are currently established for Western Blot analysis and Immunoprecipitation experiments. Reporter gene assays are carried out to measure the impact of CALM/AF10 on Ikaros' function as repressor or activator of transcription. These studies may provide new insights into the mechanism of CALM/AF10 induced leukemia and thereby facilitate the development of new therapies.

The novel CALM interactor CATS influences the subcellular localization of the leukemogenic fusion protein CALM/AF10

The Clathrin Assembly Lymphoid Myeloid leukemia gene (CALM or PICALM) was first identified as the fusion partner of AF10 in the t(10;11)(p13;q14) translocation, which is observed in acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL) and malignant lymphoma. The CALM/AF10 fusion protein plays a crucial role in t(10;11)(p13;q14) associated leukemogenesis. Using the N-terminal half of CALM as a bait in a yeast two-hybrid screen, a novel protein named CATS (CALM interacting protein expressed in thymus and spleen) was identified. Multiple tissue Northern blot analysis showed predominant expression of CATS in thymus, spleen and colon. CATS codes for two protein isoforms of 238 and 248 amino acids (aa). The interaction between CALM and CATS could be confirmed using pull down assays, co-immunoprecipitation and colocalization experiments. The CATS interaction domain of CALM was mapped to aa 221-335 of CALM. This domain is contained in the CALM/AF10 fusion protein. CATS localizes to the nucleus and shows a preference for nucleoli. Expression of CATS was able to markedly increase the nuclear localization of CALM and of the leukemogenic fusion protein CALM/AF10. The possible implications of these findings for CALM/AF10-mediated leukemogenesis are discussed.

Gene Rearrangements in Bone Marrow Cells of Patients with Acute Myelogenous Leukemia

At diagnosis, clonal gene rearrangement probes [retinoic acid receptor (RAR)-α, major breakpoint cluster region (M-bcr), immunoglobulin (Ig)-JH, T cell receptor (TcR)-β, myeloid lymphoid leukemia (MLL) or cytokine genes (GM-CSF, G-CSF, IL-3)] were detected in bone marrow samples from 71 of 153 patients with acute myelogenous leukemia (AML) (46%): in 41 patients with primary AML (pAML) (58%) and in 30 patients with secondary AML (42%). In all cases with promyelocytic leukemia (AML-M3) RAR-α gene rearrangements were detected (n = 9). Gene rearrangements in the Ig-JH or the TcR-β or GM-CSF or IL-3 or MLL gene were detected in 12, 10, 16 and 12% of the cases, respectively, whereas only few cases showed gene rearrangements in the M-bcr (6%) or G-CSF gene (3%). Survival of pAML patients with TcR-β gene rearrangements was longer and survival of pAML patients with IL-3 or GM-CSF gene rearrangement was shorter than in patients without those rearrangements. No worse survival outcome was seen in patients with rearrangements in the MLL, Ig-JH or M-bcr gene. In remission of AML (CR), clonal gene rearrangements were detected in 23 of 48 cases (48%) if samples were taken once in CR, in 23 of 26 cases (88%) if samples were taken twice in CR and in 23 of 23 cases (100%) if samples were studied three times in CR. All cases with gene rearrangements at diagnosis showed the same kind of rearrangement at relapse of the disease (n = 12). Our data show that (1) populations with clonal gene rearrangements can be regularly detected at diagnosis, in CR and at relapse of AML. (2) Certain gene rearrangements that are detectable at diagnosis have a prognostic significance for the patients’ outcome. Our results point out the significance of gene rearrangement analyses at diagnosis of AML in order to identify ‘poor risk’ patients – independently of the karyotype. Moreover, the persistence of clonal cells in the further course of AML can be studied by gene rearrangement analysis.

An In Vivo Topoisomerase II Cleavage Site and a DNase I Hypersensitive Site Colocalize Near Exon 9 in the MLLBreakpoint Cluster Region

The human myeloid-lymphoid leukemia gene, MLL (also calledALL-1, Htrx, or HRX ), maps to chromosomal band 11q23. MLL is involved in translocations that result in de novo acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), mixed lineage leukemia, and also in therapy AML (t-AML) and therapy ALL (t-ALL) resulting from treatment with DNA topoisomerase II (topo II) targeting drugs. MLL can recombine with more than 30 other chromosomal bands, of which 16 of the partner genes have been cloned. Breaks in MLL occur in an 8.3-kb breakpoint cluster region (BCR) encompassing exons 5 through 11. We recently demonstrated that 75% of de novo patient breakpoints in MLL mapped in the centromeric half of the BCR between two scaffold-associated regions (SAR), whereas 75% of the t-AML patient breakpoints mapped to the telomeric half of the BCR within a strong SAR. We have mapped additional structural elements in the BCR. An in vivo DNA topo II cleavage site (induced with several different drugs that target topo II) mapped near exon 9 in three leukemia cell lines. A strong DNase I hypersensitive site (HS) also mapped near exon 9 in four leukemia cell lines, including two in which MLL was rearranged [a t(6;11) and a t(9;11)], and in two lymphoblastoid cell lines with normalMLL. Two of the leukemia cell lines also showed an in vivo topo II cleavage site. Our results suggest that the chromatin structure of the MLL BCR may influence the location of DNA breaks in both de novo and therapy-related leukemias. We propose that topo II is enriched in the MLL telomeric SAR and that it cleaves the DNase I HS site after treatment with topo II inhibitors. These events may be involved in recombination associated with t-AML/t-ALL breakpoints mapping in the MLL SAR.

Hematologic malignancies with the t(10;11) (p13;q21) have the same molecular event and a variety of morphologic or immunologic phenotypes.

Previous studies described the t(10;11)(p13-14;q14-21) as a recurring translocation associated with T-cell acute lymphoblastic leukemia (ALL). This translocation has also been reported in monocytic leukemia or ALL with a very early pre-B phenotype. However, whether these cytogenetically similar translocations involve the same molecular breakpoint is unknown. Using fluorescence in situ hybridization (FISH) with a series of probes on 11q, we mapped the 11q breakpoint of the U937 cell line, which was derived from a patient with diffuse histiocytic lymphoma and was shown by FISH to have the t(10;11)(p13-14;q14-21). Subsequently, we identified a yeast artificial chromosome (YAC) clone, y960g8, that included the breakpoint on 11q. From this YAC, we isolated a PI clone, P91B1, that was split by the 10;11 translocation. We studied four patients with a t(10;11), one of whom had acute monocytic leukemia (AMoL), one had acute lymphoblastic leukemia (ALL), one had lymphoblastic lymphoma (LBL), and one had granulocytic sarcoma, by using FISH with y960g8 and P91B1. Y960g8 and P91B1 were split by the translocation in each patient. We showed that P91B1 included a recently identified gene, CALM (Clathrin Assembly Lymphoid Myeloid leukemia gene), and that AF10 was also rearranged in each patient by FISH when we used y807b3, which contains the AF10 gene. These findings indicate that hematologic malignant diseases with fusion of AF10 and CALM show various morphologic and immunologic phenotypes, suggesting that this fusion occurs in multipotential or very early precursor cells.

The t(10;11)(p13;q14) in the U937 cell line results in the fusion of the AF10 gene and CALM, encoding a new member of the AP-3 clathrin assembly protein family.

The translocation t(10;11)(p13;q14) is a recurring chromosomal abnormality that has been observed in patients with acute lymphoblastic leukemia as well as acute myeloid leukemia. We have recently reported that the monocytic cell line U937 has a t(10;11)(p13;q14) translocation. Using a combination of positional cloning and candidate gene approach, we cloned the breakpoint and were able to show that AF10 is fused to a novel gene that we named CALM (Clathrin Assembly Lymphoid Myeloid leukemia gene) located at 11q14. AF10, a putative transcription factor, had recently been cloned as one of the fusion partners of MLL. CALM has a very high homology in its N-terminal third to the murine ap-3 gene which is one of the clathrin assembly proteins. The N-terminal region of ap-3 has been shown to bind to clathrin and to have a high-affinity binding site for phosphoinositols. The identification of the CALM/AF10 fusion gene in the widely used U937 cell line will contribute to our understanding of the malignant phenotype of this line.