Interaction of MLL Amino Terminal Sequences with Menin Is Required for Transformation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 664-664
Author(s):  
Jay L. Hess ◽  
Zhaohai Yang ◽  
Haoren Wang ◽  
Ya-Xiong Chen ◽  
Thomas A. Milne ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Rna Polymerase Ii ◽  
Fusion Proteins ◽  
Target Genes ◽  
Hox Genes ◽  
Dominant Negative ◽  
Similar Distribution ◽  
Genetic Ablation ◽  
Amino Terminal ◽  
Coding Regions

Abstract Rearrangements of the mixed lineage leukemia gene MLL are associated with aggressive lymphoid and myeloid leukemias. The resulting MLL fusion proteins enforce high-level expression of HOX genes including HOX A7 and HOX A9 and the HOX cofactor MEIS1, which is pivotal for leukemogenesis. The mechanism by which this occurs and the relationship to normal MLL function is unknown. MLL and MLL fusion proteins bind with a similar distribution in hematopoietic cells at both promoters and coding sequences of target genes. Our studies suggest that a major mechanism of regulating MLL, which is expressed throughout hematopoiesis, is through modulating it’s binding to target promoters. MLL binds directly to the promoters and coding regions of HOX A7, HOX A9, and MEIS1 only in myeloblasts and not in neutrophils, indicating MLL is physically associated with genes only when they are actively transcribed. Expression of A cluster HOX loci and MEIS1 remains persistently elevated when MLL-ENL or dimerized MLL fusion proteins are expressed. Expression of either fusion protein is associated with increased binding of wild type MLL accompanied by increases in histone acetylation and histone H3 lysine 4, marks that are normally almost completely erased during myeloid differentiation. In addition MLL-ENL induces increased lysine 79 methylation. Both MLL and MLL fusion proteins interact with the tumor suppressor menin via sequences in the extreme amino terminus of MLL. In addition both proteins physically interact with RNA polymerase II, which shows abnormal pausing in the coding regions of HOX genes in Mll null cells. Genetic ablation of menin or expression of a dominant negative inhibitor of the MLL-menin interaction inhibits the growth of MLL fusion protein transformed cells. These findings suggest MLL fusion proteins act in concert with menin, MLL and other coactivators to deregulate HOX gene expression pivotal for transformation.

MLL Fusion Protein Driven AML Is Selectively Inhibited by Targeted Disruption of the MLL-PAFc Interaction

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 56-56 ◽  
Author(s):  
Andrew G. Muntean ◽  
Eric M Granowicz ◽  
Jay L. Hess
Keyword(s):  
Fusion Protein ◽  
Colony Formation ◽  
Fusion Proteins ◽  
Target Genes ◽  
Hox Genes ◽  
Conflicts Of Interest ◽  
Dominant Negative ◽  
Conditional Knockout ◽  
Transformed Cells ◽  
Mouse Bone Marrow

Abstract Abstract 56 Balanced chromosomal translocations of the MLL gene located on chromosome 11q23 result in the expression of a chimeric fusion proteins with enhanced transcriptional activity. The HOX genes and their co-factors, such as MEIS1 and PBX2, are critical downstream targets of MLL fusion proteins and essential for transformation. Previously we showed MLL fusion proteins are critically dependent on a direct interaction with the RNA Pol II Associated Factor complex (PAFc). PAFc is a protein complex important for the initiation, elongation and termination of transcription. It is also necessary for histone H2B K120 mono-ubiquitination through the direct recruitment of the BRE1/RAD6 E3 ubiquitin ligase complex. MLL fusion proteins make two direct contacts with the PAF1 and CTR9 subunits of the PAFc that are crucial for MLL fusion protein mediated transformation. Deletion of regions of MLL that interact with PAFc abrogates AML in mouse bone marrow transplantation assays. Here we tested the general requirement for PAFc in AML using a conditional knockout mouse model of one component of PAFc, Cdc73. These studies show that PAFc is necessary for growth of both E2A-HLF and MLL-AF9 transformed cells. Excision of Cdc73 leads to decreased expression of the MLL target genes Hoxa9 and Meis1, decreased colony formation and decreased proliferation of leukemic blasts and ultimately apoptosis. We then performed chromatin immunoprecipitation assays to assess the binding of PAFc and MLL to target loci with and without Cdc73. Excision of Cdc73 leads to a rapid decrease in association of PAFc as well as MLL fusion proteins and wild type MLL at target loci confirming that proper targeting of MLL fusion proteins requires PAFc. A decrease in H3K4me3 and H2Bub is also observed and consistent with a role of PAFc in the deposition of these epigenetic marks. We then sought to disrupt the MLL-PAFc interaction through expression of a small 40 amino acid fragment of MLL that interacts with the PAF1 subunit of PAFc. As the MLL-PAFc interaction involves interactions between MLL and both CTR9 and PAF1, it was unknown whether targeting one interaction site would be sufficient to disrupt transformation. Indeed, expression of the short fragment encompassing the pre-CxxC region of MLL acts as a dominant negative and disrupts the MLL-PAFc interaction, significantly decreasing Hox gene expression, colony formation and cell proliferation of MLL-AF9 transformed cells. Importantly, expression of the MLL fragment selectively inhibited MLL fusion mediated leukemic transformation and cell growth while the growth and proliferation of E2A-HLF cells is unaffected. Together these data show that targeting the MLL-PAFc interaction with a small MLL fragment can act as a dominant negative and selectively inhibit the growth of AML cells transformed with MLL fusion proteins. These data also suggest the MLL-PAF1 interaction surface is a promising region for therapeutic targeting. Disclosures: No relevant conflicts of interest to declare.

Leukemogenic MLL Fusion Proteins Promote MLL Association with HOX Loci Resulting in Persistent Expression.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 386-386 ◽  
Author(s):  
Thomas A. Milne ◽  
Mary Ellen Martin ◽  
Robert K. Slany ◽  
Jay L. Hess
Keyword(s):  
Rna Polymerase Ii ◽  
Histone Modifications ◽  
Fusion Proteins ◽  
Target Genes ◽  
Hox Genes ◽  
Transcriptional Elongation ◽  
Major Mechanism ◽  
Coding Regions ◽  
High Level ◽  
Target Promoters

Abstract Rearrangements of the mixed lineage leukemia gene MLL are associated with aggressive lymphoid and myeloid leukemias. The resulting MLL fusion proteins enforce high-level expression of HOX genes including HOX A7 and HOX A9 and the HOX cofactor MEIS1, which is pivotal for leukemogenesis. The mechanism by which this occurs and the relationship to normal MLL function is unknown. To address this we performed a detailed study of where MLL and MLL fusion proteins bind at target genes in hematopoietic cells using quantitative chromatin immunoprecipitation (ChIP). In addition we characterized the histone modifications at the HOX A9 locus that occur with normal down modulation of HOX A9 expression during hematopoietic differentiation and how this is perturbed by induction of conditionally transforming MLL fusion proteins. These studies suggest that a major mechanism of regulating MLL, which is expressed throughout hematopoiesis, is through modulating it’s binding to target promoters. MLL binds directly to the promoters and coding regions of HOX A7, HOX A9, and MEIS1 only in myeloblasts and not in neutrophils, indicating MLL is physically associated with genes only when they are actively transcribed. Expression of A cluster HOX loci and MEIS1 is transiently increased by the addition of IL-3 but remains persistently elevated when MLL-ENL or dimerized MLL fusion proteins are expressed. Expression of either fusion protein is associated with increased binding of wild type MLL accompanied by increases in histone acetylation and histone H3 lysine 4 methylation, marks that are normally almost completely erased during myeloid differentiation. In addition MLL-ENL induces increased lysine 79 methylation. MLL extensively colocalizes with RNA polymerase II, which shows abnormal pausing in the coding regions of HOX genes in Mll null cells. These findings suggest MLL fusion proteins maintain expression of HOX genes critical for leukemogenesis through increased MLL binding resulting in both MLL dependent histone modifications and lysine 79 methylation and ultimately promotion of transcriptional elongation.

Targeting Menin-MLL Interaction to Inhibit MLL Fusion Oncoproteins in Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2497-2497
Author(s):  
Jolanta Grembecka ◽  
Shihan He ◽  
Aibin Shi ◽  
Trupta Purohit ◽  
Andrew G. Muntean ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Acute Leukemia ◽  
Protein Interaction ◽  
Fusion Proteins ◽  
Target Genes ◽  
Hox Genes ◽  
Conflicts Of Interest ◽  
Acute Leukemias ◽  
In Vivo Models ◽  
Novel Therapeutic

Abstract Abstract 2497 Chromosomal translocations that affect the MLL (Mixed Lineage Leukemia) proto-oncogene occur in aggressive acute leukemias, both in children and adults. Fusion of MLL to one of more than 50 partner genes results in generation of the MLL fusion oncoprotein, which upregulates expression of HOX genes required for normal hematopoiesis, and ultimately leads to the development of acute leukemia. Patients harboring translocations of MLL gene suffer from very aggressive leukemias and respond poorly to available therapies, emphasizing the urgent need for novel therapeutic treatments. All oncogenic MLL fusion proteins have a preserved N-terminal fragment of MLL that interacts with menin, a tumor suppressor protein encoded by MEN1 (Multiple Endocrine Neoplasia 1) gene. Importantly, the menin-MLL fusion protein interaction is critical to the leukemogenic activity of MLL fusion proteins and misregulation of HOXA9 genes, and therefore it represents a valuable molecular target for therapeutic intervention. Selective targeting of the protein-protein interaction between menin and MLL fusion proteins with small molecules could block the oncogenic activity of MLL fusion proteins and inhibit development of acute leukemia. To identify small molecule inhibitors of the menin-MLL interaction we have performed a High Throughput Screen of 350,000 compounds using a collection of biochemical assays and biophysical methods. This resulted in several classes of compounds that specifically bind to menin and inhibit the menin-MLL interaction both in vitro and in human cells. We then applied medicinal chemistry approaches to develop analogues of selected lead candidates, resulting in very potent compounds that inhibit the menin-MLL interaction with nanomolar affinities. To evaluate potency, specificity and mechanism of action of these compounds we used a broad collection of cellular assays. These compounds selectively inhibit proliferation of the MLL leukemia cells, strongly induce apoptosis and differentiation of these cells. Importantly, these compounds substantially downregulate expression of HOXA9 and MEIS1 genes that are downstream targets of MLL fusion proteins required for their leukemogenicity, and they also deplete the menin-MLL fusion protein complex from the target genes. Furthermore, the compounds that we developed specifically inhibit the MLL fusion protein mediated oncogenic transformation. All these effects closely recapitulate the effects observed upon acute loss of menin or disruption of the menin-MLL fusion protein interaction using genetic manipulations, demonstrating highly specific mode of action for these compounds. Our current efforts are focused to assess the effect of these compounds in in vivo models of MLL leukemia and evaluate their utility as future drug candidates for acute leukemias. This may provide a novel therapeutic approach for the treatment of very aggressive leukemias with MLL translocations. Disclosures: No relevant conflicts of interest to declare.

GST-lamin fusion proteins act as dominant negative mutants in Xenopus egg extract and reveal the function of the lamina in DNA replication

1997 ◽  
Vol 110 (20) ◽  
pp. 2507-2518 ◽  
Author(s):  
D.J. Ellis ◽  
H. Jenkins ◽  
W.G. Whitfield ◽  
C.J. Hutchison
Keyword(s):  
Dna Replication ◽  
Fusion Protein ◽  
Fusion Proteins ◽  
Signal Sequence ◽  
Nuclear Lamina ◽  
Dominant Negative ◽  
Amino Terminal ◽  
Head Domain ◽  
Egg Extract ◽  
Egg Extracts

A cDNA encoding Xlamin B1 was cloned from a whole ovary mRNA by RT-PCR. GST-lamin fusion constructs were generated from this cDNA by first creating convenient restriction sites within the Xlamin B1 coding sequence, using PCR directed mutagenesis, and then sub-cloning relevant sequences into pGEX-4T-3. Two expression constructs were made, the first, termed delta 2+ lacked sequences encoding the amino-terminal ‘head domain’ of lamin B1 but included sequences encoding the nuclear localization signal sequence (NLS). The second expression construct, termed delta 2-, lacked sequences encoding the amino-terminal ‘head domain’ as well as sequences encoding the NLS. Purified fusion proteins expressed from these constructs, when added to egg extracts prior to sperm pronuclear assembly, formed hetero-oligomers with the endogenous lamin B3. The delta 2+ fusion protein prevented nuclear lamina assembly but not nuclear membrane assembly. The resulting nuclei were small (approximately 10 microns in diameter), did not assemble replication centers and failed to initiate DNA replication. When the delta 2- fusion protein was added to egg extracts prior to sperm pronuclear assembly, lamina assembly was delayed but not prevented. The resulting nuclei although small (approximately 12 microns), did form replication centers and initiated DNA replication. When added to egg extracts after sperm pronuclear assembly was completed delta 2+, but not delta 2-, entered the pre-formed nuclei causing lamina disassembly. However, the disassembly of the lamina by delta 2+ did not result in the disruption of replication centers and indeed these centres remained functional. These results are consistent with the hypothesis that lamina assembly precedes and is required for the formation of replication centers but does not support those centers directly.

scholarly journals Presequence and mature part of preproteins strongly influence the dependence of mitochondrial protein import on heat shock protein 70 in the matrix.

1993 ◽  
Vol 123 (1) ◽  
pp. 119-126 ◽  
Author(s):  
W Voos ◽  
B D Gambill ◽  
B Guiard ◽  
N Pfanner ◽  
E A Craig
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Fusion Protein ◽  
Fusion Proteins ◽  
Dual Role ◽  
Intermembrane Space ◽  
Heme Binding ◽  
Membrane Translocation ◽  
Amino Terminal ◽  
The Matrix

To test the hypothesis that 70-kD mitochondrial heat shock protein (mt-hsp70) has a dual role in membrane translocation of preproteins we screened preproteins in an attempt to find examples which required either only the unfoldase or only the translocase function of mt-hsp70. We found that a series of fusion proteins containing amino-terminal portions of the intermembrane space protein cytochrome b2 (cyt. b2) fused to dihydrofolate reductase (DHFR) were differentially imported into mitochondria containing mutant hsp70s. A fusion protein between the amino-terminal 167 residues of the precursor of cyt. b2 and DHFR was efficiently transported into mitochondria independently of both hsp70 functions. When the length of the cyt. b2 portion was increased and included the heme binding domain, the fusion protein became dependent on the unfoldase function of mt-hsp70, presumably caused by a conformational restriction of the heme-bound preprotein. In the absence of heme the noncovalent heme binding domain in the longer fusion proteins no longer conferred a dependence on the unfoldase function. When the cyt. b2 portion of the fusion protein was less than 167 residues, its import was still independent of mt-hsp70 function; however, deletion of the intermembrane space sorting signal resulted in preproteins that ended up in the matrix of wild-type mitochondria and whose translocation was strictly dependent on the translocase function of mt-hsp70. These findings provide strong evidence for a dual role of mt-hsp70 in membrane translocation and indicate that preproteins with an intermembrane space sorting signal can be correctly imported even in mutants with severely impaired hsp70 function.

Analysis of Hoxd-13 and Hoxd-11 misexpression in chick limb buds reveals that Hox genes affect both bone condensation and growth

Development ◽  
1997 ◽  
Vol 124 (3) ◽  
pp. 627-636 ◽  
Author(s):  
D.J. Goff ◽  
C.J. Tabin
Keyword(s):  
Growth Phase ◽  
Target Genes ◽  
Hox Genes ◽  
Bone Development ◽  
Tritiated Thymidine ◽  
Retroviral Vectors ◽  
Dominant Negative ◽  
Thymidine Uptake ◽  
Growth Promoters ◽  
Proliferative Zone

Hox genes are important regulators of limb pattern in vertebrate development. Misexpression of Hox genes in chicks using retroviral vectors provides an opportunity to analyze gain-of-function phenotypes and to assess their modes of action. Here we report the misexpression phenotype for Hoxd-13 and compare it to the misexpression phenotype of Hoxd-11. Hoxd-13 misexpression in the hindlimb results in a shortening of the long bones, including the femur, the tibia, the fibula and the tarsometatarsals. Mutations in an alanine repeat region in the N-terminus of Hoxd-13 have recently been implicated in human synpolydactyly (Muragaki, Y., Mundlos, S., Upton, J. and Olsen, B. R. (1996) Science 272, 548–551). N-terminal truncations of Hoxd-13 which lack this repeat were constructed and were found to produce a similar, although slightly milder, misexpression phenotype than the full-length Hoxd-13. The stage of bone development regulated by Hox genes has not previously been examined. The changes in bone lengths caused by Hoxd-13 misexpression are late phenotypes that first become apparent during the growth phase of the bones. Analysis of tritiated thymidine uptake by the tibia and fibula demonstrates that Hox genes can pattern the limb skeleton by regulating the rates of cell division in the proliferative zone of growing cartilage. Hoxd-11, in contrast to Hoxd-13, acts both at the initial cartilage condensation phase in the foot and during the later growth phase in the lower leg. Ectopic Hoxd-13 appears to act in a dominant negative manner in regions where it is not normally expressed. We propose a model in which all Hox genes are growth promoters, regulating the expression of the same target genes, with some Hox genes being more effective promoters of growth than other Hox genes. According to this model, the overall rate of growth in a given region is the result of the combined action of all of the Hox genes expressed in that region competing for the same target genes.

scholarly journals Expression and purification of functional recombinant epitopes for the platelet antigens, PlA1 and PlA2

Blood ◽  
1994 ◽  
Vol 84 (4) ◽  
pp. 1157-1163 ◽  
Author(s):  
EA Barron-Casella ◽  
TS Kickler ◽  
OC Rogers ◽  
JF Casella
Keyword(s):  
Amino Acids ◽  
Fusion Protein ◽  
Fusion Proteins ◽  
Disulfide Bonds ◽  
Affinity Purification ◽  
Platelet Glycoprotein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Glutathione S Transferase ◽  
Amino Terminal ◽  
Posttransfusion Purpura

Abstract The platelet antigens, PlA1 and PlA2, are responsible for most cases of posttransfusion purpura (PTP) and neonatal alloimmune thrombocytopenia (NAIT) in the caucasian population and are determined by two allelic forms of the platelet glycoprotein GPIIIa gene. To study the interaction between these antigens and their respective antibodies, we inserted the sequence that encodes the signal peptide and the N- terminal 66 amino acids of the PlA1 form of GPIIIa into the expression vector pGEX1. To express the PlA2 antigen, nucleotide 196 of the PlA1 coding sequence was mutated to the PlA2 allelic form. When transformed and induced in Escherichia coli, the two constructs produce glutathione S-transferase (GST)/N-terminal GPIIIa fusion proteins, one containing leucine at position 33 (PlA1), the other proline (PlA2). These proteins are easily purified in milligram quantities using glutathione-Sepharose and react specifically with their respective antibodies by immunoblot and enzyme-linked immunosorbent assay. Antigenicity of the PlA1 fusion protein in reduced glutathione increases with time; moreover, the addition of oxidized glutathione accelerates this process, presumably because of formation of the native disulfide bonds. Neutralization assays indicate that the PlA1 fusion protein competes for all of the anti-PlA1 antibody in the serum of patients with PTP and NAIT that is capable of interacting with the surface of intact platelets. This study shows that the GST/N-terminal GPIIIa fusion proteins contain conformational epitopes that mimic those involved in alloimmunization, and that regions other than the amino terminal 66 amino acids of GPIIIa are not likely to contain or be required for the development of functional PlA1 epitopes. Furthermore, these recombinant proteins can be used for the affinity-purification of clinical anti-PlA1 antibodies and specific antibody identification by western blotting, making them useful in the diagnosis of patients alloimmunized to PlA1 alloantigens.

AML-1/ETO fusion protein is a dominant negative inhibitor of transcriptional repression by the promyelocytic leukemia zinc finger protein

Blood ◽  
2000 ◽  
Vol 96 (12) ◽  
pp. 3939-3947 ◽  
Author(s):  
Ari Melnick ◽  
Graeme W. Carlile ◽  
Melanie J. McConnell ◽  
Adam Polinger ◽  
Scott W. Hiebert ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Zinc Finger ◽  
Transcriptional Repression ◽  
Target Genes ◽  
Zinc Finger Protein ◽  
Promyelocytic Leukemia ◽  
Dominant Negative ◽  
Myelogenous Leukemia ◽  
Promyelocytic Leukemia Zinc Finger ◽  
Dominant Negative Inhibitor

Abstract The AML-1/ETO fusion protein, created by the (8;21) translocation in M2-type acute myelogenous leukemia (AML), is a dominant repressive form of AML-1. This effect is due to the ability of the ETO portion of the protein to recruit co-repressors to promoters of AML-1 target genes. The t(11;17)(q21;q23)-associated acute promyelocytic leukemia creates the promyelocytic leukemia zinc finger PLZFt/RARα fusion protein and, in a similar manner, inhibits RARα target gene expression and myeloid differentiation. PLZF is expressed in hematopoietic progenitors and functions as a growth suppressor by repressing cyclin A2 and other targets. ETO is a corepressor for PLZF and potentiates transcriptional repression by linking PLZF to a histone deacetylase-containing complex. In transiently transfected cells and in a cell line derived from a patient with t(8;21) leukemia, PLZF and AML-1/ETO formed a tight complex. In transient assays, AML-1/ETO blocked transcriptional repression by PLZF, even at substoichiometric levels relative to PLZF. This effect was dependent on the presence of the ETO zinc finger domain, which recruits corepressors, and could not be rescued by overexpression of co-repressors that normally enhance PLZF repression. AML-1/ETO also excluded PLZF from the nuclear matrix and reduced its ability to bind to its cognate DNA-binding site. Finally, ETO interacted with PLZF/RARα and enhanced its ability to repress through the RARE. These data show a link in the transcriptional pathways of M2 and M3 leukemia.

scholarly journals Regulation of the Resident Chromosomal Copy of c-myc by c-Myb Is Involved in Myeloid Leukemogenesis

2000 ◽  
Vol 20 (6) ◽  
pp. 1970-1981 ◽  
Author(s):  
M. Schmidt ◽  
V. Nazarov ◽  
L. Stevens ◽  
R. Watson ◽  
L. Wolff
Keyword(s):  
Estrogen Receptor ◽  
Fusion Protein ◽  
Cell Line ◽  
Insertional Mutagenesis ◽  
Target Genes ◽  
Growth Arrest ◽  
Dominant Negative ◽  
In Vitro Assays ◽  
Protein Synthesis Inhibitor ◽  
Myeloid Lineage

ABSTRACT c-myb is a frequent target of retroviral insertional mutagenesis in murine leukemia virus-induced myeloid leukemia. Induction of the leukemogenic phenotype is generally associated with inappropriate expression of this transcriptional regulator. Despite intensive investigations, the target genes of c-myb that are specifically involved in development of these myeloid lineage neoplasms are still unknown. In vitro assays have indicated that c-myc may be a target gene of c-Myb; however, regulation of the resident chromosomal gene has not yet been demonstrated. To address this question further, we analyzed the expression of c-mycin a myeloblastic cell line, M1, expressing a conditionally active c-Myb–estrogen receptor fusion protein (MybER). Activation of MybER both prevented the growth arrest induced by interleukin-6 (IL-6) and rapidly restored c-myc expression in nearly terminal differentiated cells that had been exposed to IL-6 for 3 days. Restoration occurred in the presence of a protein synthesis inhibitor but not after a transcriptional block, indicating that c-myc is a direct, transcriptionally regulated target of c-Myb. c-myc is a major target that transduces Myb's proliferative signal, as shown by the ability of a c-Myc–estrogen receptor fusion protein alone to also reverse growth arrest in this system. To investigate the possibility that this regulatory connection contributes to Myb's oncogenicity, we expressed a dominant negative Myb in the myeloid leukemic cell line RI-4-11. In this cell line, c-myb is activated by insertional mutagenesis and cannot be effectively down regulated by cytokine. Myb's ability to regulate c-myc's expression was also demonstrated in these cells, showing a mechanism through which the proto-oncogene c-mybcan exert its oncogenic potential in myeloid lineage hematopoietic cells.

The CALM/AF10 Fusion: Molecular Analysis of the Fusion Transcripts in 13 Cases of AML and ALL; Gene Expression Profiling Reveals HOX Gene Deregulation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2895-2895 ◽  
Author(s):  
Alexandre Krause ◽  
Alexander Kohlmann ◽  
Torsten Haferlach ◽  
Claudia Schoch ◽  
Susanne Schnittger ◽  
...  
Keyword(s):  
Gene Expression ◽  
Fusion Protein ◽  
Hox Genes ◽  
Lymphoblastic Leukemia ◽  
Critical Role ◽  
Gene Clusters ◽  
Dominant Negative ◽  
Nucleotide Position ◽  
Gamma Delta ◽  
Hox Gene

Abstract The t(10;11)(p13;q14) is a recurring translocation associated with the CALM/AF10 fusion gene which is found in undifferentiated leukemia, acute myeloid leukemia, acute lymphoblastic leukemia and malignant lymphoma with poor prognosis. The CALM/AF10 fusion protein was reported to be the most common fusion protein in T-ALL with TCR gamma delta rearrangement. We have analyzed samples from 9 patients with different types of leukemia: case 1 (AML M2), case 2 (AML M0), case 3 (Pre T-ALL), case 4 (Acute Undifferentiated Leukemia), case 5 (PreT-ALL), case 6 and 7 (ProT-ALL), case 8 (T-ALL), case 9 (AML), with a t(10;11) translocation suggesting a CALM/AF10-rearrangement. The samples were analyzed for the presence of the CALM/AF10 and AF10/CALM mRNA by RT-PCR and sequence analysis. All these patients were found positive for the CALM/AF10 fusion. In addition, we analyzed a series of twenty-nine patients with T-ALL with gamma delta rearrangement. Among these patients, four were positive for CALM/AF10 transcripts, indicating a high incidence of CALM/AF10 fusions in this group of leukemia. We found three different breakpoints in CALM at nucleotide 1926, 2091 and a new exon, with 106 bases inserted after nt 2064 of CALM in patient 4. In AF10 four breakpoints were identified: at nucleotide position 424, 589, 883 and 979. In seven patients it was also possible to amplify the reciprocal AF10/CALM fusion transcript (case 1, 3, 4, 8, 9, 10 and 11). There was no correlation between disease phenotype and breakpoint location. The patients were 5 to 46 years old (median 25). Ten CALM/AF10 positive patients were further analyzed using oligonucleotide microarrays representing 33,000 different genes (U133 set, Affymetrix). Analysis of microarray gene expression signatures of these patients revealed high expression levels of the homeobox gene MEIS1 and the HOXA cluster genes HOXA1, HOXA4, HOXA5, HOXA7, HOXA9, and HOXA10. The overexpression of HOX genes seen in these CALM/AF10 positive leukemias is reminiscent of the pattern seen in leukemias with rearrangements of the MLL gene, and complex aberrant karyotypes suggesting a common effector pathway (i.e. HOX gene deregulation) for these diverse leukemias. It is known that alhambra, the Drosophila homologue of AF10 can act on polycomb group responsive elements, which play a critical role in the regulation of the HOX gene clusters. It is thus conceivable that the CALM/AF10 fusion proteins acts in a dominant negative fashion on wild type AF10 function relieving the repression that is presumably normally exerted by AF10 on the expression of HOX genes.

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