Suppression of MLL-AF4 by Small Interfering RNAs Inhibits Proliferation and Induces Apoptosis in T(4;11)-Positive SEM Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4433-4433
Author(s):  
Johann Greil ◽  
Maria Thomas ◽  
Christian Huenefeld ◽  
Hans-Peter Vornlocher ◽  
Philipp Hadwiger ◽  
...  
Keyword(s):  
Fusion Gene ◽  
Chromosomal Translocation ◽  
Fusion Transcript ◽  
Derivative Chromosome ◽  
Mrna Levels ◽  
Small Interfering Rnas ◽  
Chromosome 11 ◽  
Inhibition Of Proliferation ◽  
Treatment Concepts ◽  
Cell Cycle Transition

Abstract The reciprocal chromosomal translocation t(4;11)(q21;q23) creates the fusion genes MLL-AF4 and AF4-MLL located on derivative chromosome 11 or derivative chromosome 4, respectively. We used small interfering RNAs to suppress the MLL-AF4 fusion gene in the t(4;11)-positive leukaemic cell line SEM. Electroporation of SEM cells with MLL-AF4 siRNAs caused a more than two fold transient decrease in MLL-AF4 mRNA levels, which lasted for three days. The reduction in MLL-AF4 fusion transcript levels were associated with a severely diminished clonogenicity, inhibition of proliferation and of G1-S cell cycle transition and induction of apoptosis. Therefore, MLL-AF4 siRNAs are not only useful to study the functions of MLL-AF4 in leukaemogenesis, but may be also promising agents for novel treatment concepts for t(4;11)-associated leukaemias.

scholarly journals Identification of a Novel Oncogenic Fusion Gene SPON1-TRIM29 in Clinical Ovarian Cancer That Promotes Cell and Tumor Growth and Enhances Chemoresistance in A2780 Cells

2022 ◽  
Vol 23 (2) ◽  
pp. 689
Author(s):  
Saya Nagasawa ◽  
Kazuhiro Ikeda ◽  
Daisuke Shintani ◽  
Chiujung Yang ◽  
Satoru Takeda ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Rna Sequencing ◽  
Anticancer Drugs ◽  
Fusion Gene ◽  
Xenograft Model ◽  
Fusion Transcript ◽  
Serous Carcinoma ◽  
Fusion Genes ◽  
Sequencing Data ◽  
Chromosome 11

Gene structure alterations, such as chromosomal rearrangements that develop fusion genes, often contribute to tumorigenesis. It has been shown that the fusion genes identified in public RNA-sequencing datasets are mainly derived from intrachromosomal rearrangements. In this study, we explored fusion transcripts in clinical ovarian cancer specimens based on our RNA-sequencing data. We successfully identified an in-frame fusion transcript SPON1-TRIM29 in chromosome 11 from a recurrent tumor specimen of high-grade serous carcinoma (HGSC), which was not detected in the corresponding primary carcinoma, and validated the expression of the identical fusion transcript in another tumor from a distinct HGSC patient. Ovarian cancer A2780 cells stably expressing SPON1-TRIM29 exhibited an increase in cell growth, whereas a decrease in apoptosis was observed, even in the presence of anticancer drugs. The siRNA-mediated silencing of SPON1-TRIM29 fusion transcript substantially impaired the enhanced growth of A2780 cells expressing the chimeric gene treated with anticancer drugs. Moreover, a subcutaneous xenograft model using athymic mice indicated that SPON1-TRIM29-expressing A2780 cells rapidly generated tumors in vivo compared to control cells, whose growth was significantly repressed by the fusion-specific siRNA administration. Overall, the SPON1-TRIM29 fusion gene could be involved in carcinogenesis and chemotherapy resistance in ovarian cancer, and offers potential use as a diagnostic and therapeutic target for the disease with the fusion transcript.

Single-translocation and double-chimeric transcripts: detection of NUP98-HOXA9 in myeloid leukemias withHOXA11 or HOXA13 breaks of the chromosomal translocation t(7;11)(p15;p15)

Blood ◽  
2002 ◽  
Vol 99 (4) ◽  
pp. 1428-1433 ◽  
Author(s):  
Takashi Fujino ◽  
Akitaka Suzuki ◽  
Yoshikazu Ito ◽  
Kazuma Ohyashiki ◽  
Yoshiaki Hatano ◽  
...  
Keyword(s):  
Fusion Gene ◽  
Leukemia Cell ◽  
Chromosomal Translocation ◽  
Myelogenous Leukemia ◽  
Pcr Analysis ◽  
Chromosome 11 ◽  
Reverse Transcription Pcr ◽  
Class 1 ◽  
Chromosomal Breakpoints ◽  
Acute Myelogenous

It has been demonstrated that the chromosomal translocation t(7;11)(p15;p15) in patients with human acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) invariably involves fusion of the nucleoporin gene, NUP98, on chromosome 11 and the class 1 HOX gene, HOXA9, on chromosome 7, and that the fusion gene NUP98-HOXA9 is an important gene in myeloid leukemogenesis. Here are reported 2 novel chromosome 7p15 targets of the t(7;11)(p15;p15) chromosomal translocation in 2 patients with CML and myelodysplastic syndrome (MDS). Southern blot and polymerase chain reaction (PCR) analyses of leukemia cell DNA failed to show rearrangement of HOXA9,whereas NUP98 was found to be rearranged in both cases. Reverse transcription-PCR analysis using a NUP98 primer and a degenerate primer corresponding to the third helix of the homeodomain of HOXA demonstrated that NUP98 was fused in-frame to HOXA11 in the patient with CML and toHOXA13 in the patient with MDS. The chromosomal breakpoints on 7p15 were located within introns of HOXA11 orHOXA13 genes. In both patients chimericNUP98-HOXA9 transcripts were also observed. These findings suggest that AbdB-type HOXA genes are common targets of t(7;11)(p15;p15) chromosomal translocations and that a single translocation can produce more than oneNUP98-HOXA fusion gene, presumably because of altered splicing.

Depletion of the Leukemic Fusion Protein MLL-AF4 with Short Interfering RNAs (siRNAs) Affects Post-Translational Modification of Aldolase A.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4341-4341
Author(s):  
Johann Greil ◽  
Andreas Gessner ◽  
Maria Thomas ◽  
Olaf Heidenreich
Keyword(s):  
Protein Pattern ◽  
Chromosomal Translocation ◽  
Global Changes ◽  
Mrna Levels ◽  
Post Translational Modification ◽  
Inhibition Of Proliferation ◽  
Protein Levels ◽  
Mass Spectrometry Identification ◽  
Aldolase A

Abstract The chromosomal translocation t(4;11) marks a therapy-resistant infant leukemia with very poor prognosis. It results in the expression of two fusion-proteins, MLL-AF4 and AF4-MLL. We addressed the role of MLL-AF4 in t(4;11) positive SEM cells by siRNA-mediated suppression. Depletion of MLL-AF4 results in induction of apoptosis, inhibition of proliferation, decrease in colony formation and diminished leukemic engraftment in vivo. Currently, we are analyzing global changes in protein expression. For that, we compare the proteome of MLL-AF4 depleted SEM cells with those of control cells. The analysis is performed by 2D-gelelectrophoresis followed by mass spectrometry identification and immunoblot validation of differentially expressed spots. One of these spots was identified as Aldolase A. Comparison of MLL-AF4 depleted SEM cells with control cells showed neither change in mRNA levels nor in absolute protein levels of Aldolase A. Two-dimensional western blotting, however, revealed differences in the protein pattern, suggesting changes in Aldolase A modifications upon MLL-AF4 depletion. These analyses will provide us with a better insight into the effects of siRNA-mediated MLL-AF4 knockdown on the proteome, and may enable us to identify new targets for molecular therapeutic approaches.

Isolation of mutations that act in trans to alter expression from a yeast hsp70 promoter

1988 ◽  
Vol 8 (8) ◽  
pp. 3423-3431
Author(s):  
R C Findly ◽  
H Alavi ◽  
T Platt
Keyword(s):  
Heat Shock ◽  
Fusion Gene ◽  
Fusion Transcript ◽  
Mrna Levels ◽  
Hsp70 Promoter ◽  
Transcript Stability ◽  
In Trans ◽  
Selection For ◽  
Rna Blots ◽  
Beta Galactosidase

Transcription of SSA1 (formerly YG100), a member of the hsp70 gene family in Saccharomyces cerevisiae, increases dramatically upon heat shock. An expression vector in which the promoter of SSA1 is fused to the Escherichia coli galactokinase gene (galK) was constructed and transformed into a galactokinase-deficient yeast strain. The transformants grew on galactose at 23 degrees C, but increased expression of the SSA1-galK fusion gene inhibited growth of cells on galactose at 37 degrees C. Selection for survivors under nonpermissive conditions yielded a class of mutants, termed HSR (for heat shock regulation), which showed reduced levels of expression of the hsp70-galK gene fusion as determined by measurement of galactokinase activity. Similar effects on beta-galactosidase activity were obtained when an SSA1-lacZ fusion vector was introduced into the mutants, suggesting action in trans through the SSA1 promoter. Analysis of Northern (RNA) blots demonstrated that the reduction in expression was a result of decreased mRNA levels for the fusion gene. In addition, mRNA levels of the endogenous SSA1 gene are reduced in an HSR mutant. Genetic analysis has shown that these mutations act in trans and affect both transcription from the SSA1 promoter and turnover of the fusion transcript. These are the first trans-acting mutations known to affect directly the transcriptional regulation and transcript stability of heat shock genes in eucaryotes.

scholarly journals Isolation of mutations that act in trans to alter expression from a yeast hsp70 promoter.

1988 ◽  
Vol 8 (8) ◽  
pp. 3423-3431 ◽  
Author(s):  
R C Findly ◽  
H Alavi ◽  
T Platt
Keyword(s):  
Heat Shock ◽  
Fusion Gene ◽  
Fusion Transcript ◽  
Mrna Levels ◽  
Hsp70 Promoter ◽  
Transcript Stability ◽  
In Trans ◽  
Selection For ◽  
Rna Blots ◽  
Beta Galactosidase

Transcription of SSA1 (formerly YG100), a member of the hsp70 gene family in Saccharomyces cerevisiae, increases dramatically upon heat shock. An expression vector in which the promoter of SSA1 is fused to the Escherichia coli galactokinase gene (galK) was constructed and transformed into a galactokinase-deficient yeast strain. The transformants grew on galactose at 23 degrees C, but increased expression of the SSA1-galK fusion gene inhibited growth of cells on galactose at 37 degrees C. Selection for survivors under nonpermissive conditions yielded a class of mutants, termed HSR (for heat shock regulation), which showed reduced levels of expression of the hsp70-galK gene fusion as determined by measurement of galactokinase activity. Similar effects on beta-galactosidase activity were obtained when an SSA1-lacZ fusion vector was introduced into the mutants, suggesting action in trans through the SSA1 promoter. Analysis of Northern (RNA) blots demonstrated that the reduction in expression was a result of decreased mRNA levels for the fusion gene. In addition, mRNA levels of the endogenous SSA1 gene are reduced in an HSR mutant. Genetic analysis has shown that these mutations act in trans and affect both transcription from the SSA1 promoter and turnover of the fusion transcript. These are the first trans-acting mutations known to affect directly the transcriptional regulation and transcript stability of heat shock genes in eucaryotes.

The T(4;11) Fusion Protein MLL/AF4 Regulates TERT Expression

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3111-3111
Author(s):  
Andreas Gessner ◽  
Maria Thomas ◽  
Patricia Garrido Castro ◽  
Olaf Heidenreich ◽  
Johann Greil
Keyword(s):  
Cell Lines ◽  
Molecular Mechanisms ◽  
Fusion Transcript ◽  
Telomerase Activity ◽  
Small Interfering Rnas ◽  
Inhibition Of Proliferation ◽  
Knock Down ◽  
Protein Levels ◽  
Induced Apoptosis ◽  
Tert Expression

Abstract About 50% of all infants suffering from acute lymphoblastic leukaemia (ALL) show the translocation t(4;11)(q21;q23) which creates the fusion genes MLL/AF4 and AF4/MLL. This reciprocal translocation identifies a therapy resistant form of leukaemia with a poor prognosis. In order to gain a better insight into the molecular mechanisms of t(4;11) leukaemias we used the two ALL cell lines SEM and RS4;11, both harbouring the t(4;11) translocation, however with different fusion sites. Specific small interfering RNAs (siRNAs) against the two fusion site variants of the MLL/AF4 fusion transcript were designed and transfected into the respective cells via electroporation, using very mild conditions. Serial electroporations at two-day intervals resulted in sustained depletion of the MLL/AF4 transcript up to 70–80%, and depending on the experimental setup, cells were analysed after two or three electroporations. Knock-down of MLL/AF4 resulted in strong inhibition of proliferation and clonogenicity in the two t(4;11)-positive cell lines SEM and RS4;11, along with induction of apoptosis. MLL/AF4 depletion resulted in a 65% decrease in telomerase activity in both SEM and RS4;11 cells, with telomerase reverse transcriptase (TERT) expression being reduced twofold on both transcript and protein levels. Notably, TERT reduction was even stronger (90% depletion) when apoptosis caused by MLL/AF4 knock-down was suppressed with the caspase inhibitor zVAD. In contrast, levels of the RNA component of the telomerase, TERC, were not affected by MLL/AF4 knock-down. Additionally, MLL/AF4 knock-down was associated with reduced expression of several members of the HOXA gene cluster, HOXA6 (65% reduction), HOXA7 (85% reduction), HOXA9 (60% reduction) and HOXA10 (75% reduction). Interestingly, siRNA-mediated knock-down of the MLL/AF4 target gene HOXA7 also induced apoptosis and resulted in a 70% decrease of TERT levels in two t(4;11) positive cell lines without affecting MLL/AF4. Chromatin immunoprecipitation assays revealed HOXA7 binding to the promoter of TERT. Therefore, MLL/AF4 regulates TERT expression, at least in part, via HOXA7. These data suggest that t(4;11) positive cells with substantially lower TERT expression undergo apoptosis, and that TERT may play an antiapoptotic role in t(4;11) positive ALL. Furthermore, these studies identify TERT as a putative new therapeutic target in this therapy-resistant infant leukaemia.

Dermatofibrosarcoma protuberans (DFSP) in six patients with ADA-SCID

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. 10570-10570
Author(s):  
C. Kesserwan ◽  
R. Sokolic ◽  
E. Cowen ◽  
E. Garabedian ◽  
S. Pittaluga ◽  
...  
Keyword(s):  
Fusion Gene ◽  
Severe Combined Immunodeficiency ◽  
Subcutaneous Fat ◽  
Chromosomal Translocation ◽  
Dermatofibrosarcoma Protuberans ◽  
Fusion Transcript ◽  
Skin Tumor ◽  
Rt Pcr ◽  
Spindle Cell Proliferation ◽  
Repair Defect

10570 Background: DFSP is a rare malignant skin tumor associated with a characteristic chromosomal translocation (t(17;22)(q22;q13)), resulting in the COL1A1-PDGFB fusion gene. We originally diagnosed DFSP in two patients affected with a rare form of severe combined immunodeficiency due to adenosine deaminase deficiency (ADA-SCID). The association of these two rare conditions has been described in two other cases, which prompted us to screen for DFSP systematically in patients with ADA-SCID. Methods: Eight ADA-SCID patients were evaluated with complete dermatological exam and skin biopsy. Molecular analysis (FISH and/or RT-PCR) and karyotype were performed when possible. Results: Six patients (age 2, 2, 5, 9, 12 and 22 years) were found to have DFSP. Five patients had between 4 and 12 multicentric lesions over the trunk and extremities. One patient had a single lesion. Most lesions appeared as 2–15 mm tan atrophic plaques. Nodular lesions were present in 3 patients. All lesions showed a spindle cell proliferation of the dermis, extending into the subcutaneous fat. A storiform pattern was only noticed in one adult patient. In all cases, CD34 expression was diffusely positive and FXIIIa was negative. Karyotype showed t(17;22)(q22;q13) in the 2 patients in whom it was performed. FISH was positive for COL1A1-PDGFB in 2 of 4 patients studied. RT-PCR showed the COL1A1-PDGFB fusion transcript in one case in which FISH was inconclusive.FISH and RT-PCR analyses are being conducted in 2 and 5 remaining cases, respectively. Conclusions: We describe a previously unrecognized association between multicentric DFSP and ADA-SCID. Multicentricity of DFSP to this extent has not previously been reported . We hypothesize that t(17;22)(q22;q13) may arise due to the known DNA repair defect in ADA-SCID and that the known dermal overexpression of PDGFB in this condition may favor the development of fibrotic tumors, as opposed to other skin cancers. Our observations can provide further insight into the pathogenesis of DFSP and should facilitate early diagnosis of DFSP in ADA-SCID. No significant financial relationships to disclose.

Decrease of Malignant Potential of t(8;21) Positive Cells after Stable Expression of RUNX-CBFA2T1-Specific Small Interfering RNA.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4274-4274
Author(s):  
Bettina Drescher ◽  
Kerstin Goerlich ◽  
Dagmar Reile ◽  
Gerhard Heil ◽  
Olaf Heidenreich ◽  
...  
Keyword(s):  
Histone Deacetylases ◽  
Fusion Gene ◽  
Chimeric Protein ◽  
Chromosomal Translocation ◽  
Surface Expression ◽  
Stable Expression ◽  
Target Cells ◽  
Small Interfering Rnas ◽  
Sirna Sequence ◽  
Semisolid Medium

Abstract The reciprocal chromosomal translocation t(8;21) results in the fusion of the DNA-binding domain of the transcription factor RUNX1 (also called AML1 or CBFα) to CBFA2T1 (also called MTG8 or ETO). The chimeric protein RUNX1-CBFA2T1 interferes with hematopoietic gene expression by recruiting histone deacetylases via N-CoR and mSin3. In addition, impairment of the function of transcription factors such as C/EBPα has been described. To further investigate the function of RUNX1-CBFA2T1 in the development of leukemia, the expression of the fusion protein was inhibited by small interfering RNAs (siRNAs). For stable expression of the siRNA in the target cells, a RUNX1-CBFA2T1-specific siRNA-sequence was cloned into a lentivial vector as short-hairpin-RNA (shRNA). Subsequently, the t(8;21) positive Kasumi-1 cell line was infected with an effeciency of >95% as detected by expression of the GFP reporter gene. As a control, an shRNA targeting luciferase mRNA was used. Expression of the anti-RUNX1-CBFA2T1-shRNA in the Kasumi-1 cells led to a marked reduction of mRNA and protein expression of the fusion gene, whereas the expression of the wildtype RUNX1 gene was not affected. The surface expression of the M-CSF-Receptor, which is a known target gene of C/EBPα, was increased in the RUNX1-CBF2T1 depleted cells. Also, Kasumi-1 cells treated with the shRNA displayed a decrease in CD34 surface expression. In parallel, CD34 mRNA expression was reduced to 10%. To analyze, if CD34 downregulation of the Kasumi-1 cells after RUNX1-CBF2T1 depletion correlates with a loss of progenitor status, the clonogenicity of the cells in semisolid medium was investigated. In Kasumi-1 cells treated with the shRNA against RUNX1-CBFA2T1 the number of spontaneous colonies after 14 days of culture was reduced to about 30% in comparison to cells expressing the control shRNA. In conclusion, these experiments show that RUNX1-CBF2T1 expression can be stably suppressed in t(8;21) positive cells by endogenously expressed shRNAs thereby reducing their clonogenic potential.

Specific inhibition of bcr-abl gene expression by small interfering RNA

Blood ◽  
2003 ◽  
Vol 101 (4) ◽  
pp. 1566-1569 ◽  
Author(s):  
Michaela Scherr ◽  
Karin Battmer ◽  
Thomas Winkler ◽  
Olaf Heidenreich ◽  
Arnold Ganser ◽  
...  
Keyword(s):  
Myeloid Leukemia ◽  
Small Interfering Rna ◽  
Fusion Gene ◽  
Lymphoblastic Leukemia ◽  
Mrna Levels ◽  
Specific Inhibition ◽  
Primary Cells ◽  
Small Interfering Rnas ◽  
Cd34 Cells ◽  
Interfering Rna

Small interfering RNAs (siRNAs) were designed to target thebcr-abl oncogene, which causes chronic myeloid leukemia (CML) and bcr-abl–positive acute lymphoblastic leukemia (ALL). Chemically synthesized anti–bcr-abl siRNAs were selected using reporter gene constructs and were found to reduce bcr-abl mRNA up to 87% in bcr-abl–positive cell lines and in primary cells from CML patients. This mRNA reduction was specific for bcr-abl because c-abl and c-bcr mRNA levels remained unaffected. Furthermore, protein expression of BCR-ABL and of laminA/C was reduced by specific siRNAs up to 80% in bcr-abl–positive and normal CD34+ cells, respectively. Finally, anti–bcr-abl siRNA inhibited BCR-ABL–dependent, but not cytokine-dependent, proliferation in a bcr-abl–positive cell line. These data demonstrate that siRNA can specifically and efficiently interfere with the expression of an oncogenic fusion gene in hematopoietic cells.

scholarly journals Transformation of an Unclassified Myeloproliferative Neoplasm with a RareBCR-JAK2Fusion Transcript Resulting from the Translocation (9;22)(p24;q11)

2015 ◽  
Vol 2015 ◽  
pp. 1-5
Author(s):  
A. N. Chamseddine ◽  
P. Etancelin ◽  
D. Penther ◽  
F. Parmentier ◽  
C. Kuadjovi ◽  
...  
Keyword(s):  
Fusion Gene ◽  
Myeloproliferative Neoplasms ◽  
Present Report ◽  
Myeloproliferative Neoplasm ◽  
Strong Support ◽  
Fusion Transcript ◽  
Derivative Chromosome ◽  
Aggressive Clinical Course

BCR-ABL1negative myeloproliferative neoplasms (MPNs) are known to contain alterations of the tyrosine kinase JAK2 (located on 9p24) that result in constitutive activation of the encoded protein. JAK2 fusions are reported in acute and chronic leukemias of myeloid and lymphoid phenotypes. Here, we report an unclassified case of MPN (MPN-U) showing a t(9;22)(p24;q11), which generates aBCR-JAK2fusion gene by fusing theBCRat intron 13 toJAK2at intron 17 on the derivative chromosome 22. Most reported JAK2 fusions cases reveal an aggressive clinical course and long-term remissions have only been achieved after allogeneic stem cell transplantation (ASCT). To the best of our knowledge, this is the thirteenth case reported worldwide to describe aBCR-JAK2fusion transcript in MPN-U. The present report revealed a sustained complete clinical, hematologic, and cytogenetic remission 35 months after diagnosis and ~24 months after ASCT. RegardingBCR-ABL1  negativeMPN patients this case report provides strong support for a role ofJAK2activation in the oncogenesis and suggests a possible diagnostic and therapeutic target that should be investigated.

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