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1
Wajapeyee, Narendra, Shu-Zong Wang, Ryan W. Serra, Peter D. Solomon, Arvindhan Nagarajan, Xiaochun Zhu, and Michael R. Green. "Senescence induction in human fibroblasts and hematopoietic progenitors by leukemogenic fusion proteins." Blood 115, no. 24 (June 17, 2010): 5057–60. http://dx.doi.org/10.1182/blood-2009-09-245928.
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Abstract Hematologic malignancies are typically associated with leukemogenic fusion proteins, which are required to maintain the oncogenic state. Previous studies have shown that certain oncogenes that promote solid tumors, such as RAS and BRAF, can induce senescence in primary cells, which is thought to provide a barrier to tumorigenesis. In these cases, the activated oncogene elicits a DNA damage response (DDR), which is essential for the senescence program. Here we show that 3 leukemogenic fusion proteins, BCR-ABL, CBFB-MYH11, and RUNX1-ETO, can induce senescence in primary fibroblasts and hematopoietic progenitors. Unexpectedly, we find that senescence induction by BCR-ABL and CBFB-MYH11 occurs through a DDR-independent pathway, whereas RUNX1-ETO induces senescence in a DDR-dependent manner. All 3 fusion proteins activate the p38 MAPK pathway, which is required for senescence induction. Our results reveal diverse pathways for oncogene-induced senescence and further suggest that leukemias harbor genetic or epigenetic alterations that inactivate senescence induction genes.
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Eguchi, Mariko, Minenori Eguchi-Ishimae, and Mel Greaves. "The small oligomerization domain of gephyrin converts MLL to an oncogene." Blood 103, no. 10 (May 15, 2004): 3876–82. http://dx.doi.org/10.1182/blood-2003-11-3817.
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Abstract The MLL (mixed lineage leukemia) gene forms chimeric fusions with a diverse set of partner genes as a consequence of chromosome translocations in leukemia. In several fusion partners, a transcriptional activation domain appears to be essential for conferring leukemogenic capacity on MLL protein. Other fusion partners, however, lack such domains. Here we show that gephyrin (GPHN), a neuronal receptor assembly protein and rare fusion partner of MLL in leukemia, has the capacity as an MLL-GPHN chimera to transform hematopoietic progenitors, despite lack of transcriptional activity. A small 15–amino acid tubulin-binding domain of GPHN is necessary and sufficient for this activity in vitro and in vivo. This domain also confers oligomerization capacity on MLL protein, suggesting that such activity may contribute critically to leukemogenesis. The transduction of MLL-GPHN into hematopoietic progenitor cells caused myeloid and lymphoid lineage leukemias in mice, suggesting that MLL-GPHN can target multipotent progenitor cells. Our results, and other recent data, provide a mechanism for oncogenic conversion of MLL by fusion partners encoding cytoplasmic proteins.
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Yokoyama, Akihiko, and Michael Cleary. "The Menin Tumor Suppressor Functions as a Molecular Adapter That Tethers LEDGF to MLL Proteins in Leukemogenesis." Blood 112, no. 11 (November 16, 2008): 1795. http://dx.doi.org/10.1182/blood.v112.11.1795.1795.
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Abstract MLL gene rearrangements are present in 5 to 10% of acute leukemias, which are generally associated with a poor prognosis. Chromosomal translocations at the MLL locus create MLL fusion genes that constitute 5’ portions of MLL and 3’ portions of partner genes. The resultant MLL fusion proteins transform myeloid progenitors by inappropriately activating a HOX-associated self renewal program. However, the molecular mechanisms underlying MLL oncoprotein function are not fully understood. Previously we identified menin as a component of the MLL macromolecular complex. Menin is a product of the MEN1 tumor suppressor gene, whose loss of function causes multiple endocrine neoplasia type 1 (MEN1). Despite its tumor suppressor role in endocrine tissues, menin functions as an essential oncogenic cofactor for MLL fusion protein-dependent leukemogenesis. This notion was supported by three lines of evidence: MLL fusion proteins lacking the menin binding motif do not transform myeloid progenitors; inactivation of menin causes growth arrest and subsequent differentiation of MLL oncogene transformed cells; and menin is required for MLL oncogene dependent transcriptional activation of HOX genes. These findings raised a fundamental question: how does menin contribute to MLL-dependent transcription? Because menin lacks known functional motifs, its molecular functions could not be deduced from its structure. We hypothesized that menin may function as an adapter that tethers MLL to unknown associated factors. To identify such associated factors, we performed affinity purification of the MLL-ENL/menin complex from nuclear extracts of cells that transiently over-expressed both MLL-ENL and menin. Mass spectrometry identified LEDGF, originally identified as a transcriptional co-activator, in the purified material as a novel associated factor. LEDGF associates conjointly with the MLL/menin complex but not with MLL or menin alone, supporting the hypothesis that menin plays an adapter role to bridge MLL and LEDGF. Further analysis revealed that LEDGF is critical for MLL fusion protein-dependent leukemogenesis. Fine mapping of the domain responsible for LEDGF binding determined MLL residues 109–153 as the LEDGF binding domain (LBD). Mutations in LBD resulted in loss of oncogenic activity of MLL fusion proteins. Moreover, knock down of LEDGF in MLL-transformed cells caused growth arrest and differentiation in the same manner as menin knock down. These results demonstrate that LEDGF is also required for the initiation and maintenance of MLL fusion protein-dependent transformation. In contrast to menin, LEDGF has a distinctive functional motif (the PWWP domain), which reportedly has chromatin binding activity. To further confirm that menin is an adapter that links MLL and LEDGF, we examined the oncogenic functions of an artificial MLL fusion protein whose menin binding motif is replaced by the PWWP domain of LEDGF. This PWWP-MLL-ENL fusion protein does not associate with menin because it lacks the menin binding motif, nevertheless transforms myeloid progenitors. Chromatin immunoprecipitation experiments show that the PWWP-MLL- ENL fusion protein localizes at the HOXA9 locus while menin is absent. Moreover, myeloid progenitors transformed by the PWWP-MLL-ENL fusion protein continue to proliferate after menin is genetically inactivated. Thus covalent tethering of the PWWP domain fully compensates for loss of menin’s cofactor function. Therefore, menin’s only role in MLL-associated leukemogenesis is to tether LEDGF to MLL fusion proteins. In summary, this study identifies a previously unknown essential oncogenic cofactor of MLL fusion proteins and proposes a stepwise association model in which the MLL fusion protein first associates with menin, then recruits LEDGF to its complex to become functionally active.
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Yan, Zhengwei, Karthigayan Shanmugasundaram, Dongwen Ma, Jiayu Luo, Shiwen Luo, and Hai Rao. "The N-terminal domain of the non-receptor tyrosine kinase ABL confers protein instability and suppresses tumorigenesis." Journal of Biological Chemistry 295, no. 27 (May 21, 2020): 9069–75. http://dx.doi.org/10.1074/jbc.ra120.012821.
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Chromosome translocation can lead to chimeric proteins that may become oncogenic drivers. A classic example is the fusion of the BCR activator of RhoGEF and GTPase and the ABL proto-oncogene nonreceptor tyrosine kinase, a result of a chromosome abnormality (Philadelphia chromosome) that causes leukemia. To unravel the mechanism underlying BCR-ABL–mediated tumorigenesis, here we compared the stability of ABL and the BCR-ABL fusion. Using protein degradation, cell proliferation, 5-ethynyl-2-deoxyuridine, and apoptosis assays, along with xenograft tumor analysis, we found that the N-terminal segment of ABL, which is lost in the BCR-ABL fusion, confers degradation capacity that is promoted by SMAD-specific E3 ubiquitin protein ligase 1. We further demonstrate that the N-terminal deletion renders ABL more stable and stimulates cell growth and tumorigenesis. The findings of our study suggest that altered protein stability may contribute to chromosome translocation-induced cancer development.
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Grembecka, Jolanta, Shihan He, Aibin Shi, Trupta Purohit, Andrew G. Muntean, Xiaoqin Li, Thomas Hartley, Duxin Sun, Jay L. Hess, and Tomasz Cierpicki. "Targeting Menin-MLL Interaction to Inhibit MLL Fusion Oncoproteins in Leukemia." Blood 118, no. 21 (November 18, 2011): 2497. http://dx.doi.org/10.1182/blood.v118.21.2497.2497.
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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.
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Su, Kang-Yi, Bing-Ching Ho, Gee-Chen Chang, Hsuan-Yu Chen, Pan-Chyr Yang, and Sung-Liang Yu. "Multiplex ALK, RET, and ROS1 fusion mutation detection in FFPE from lung cancer patients by MALDI-TOF mass spectrometry." Journal of Clinical Oncology 35, no. 15_suppl (May 20, 2017): e13103-e13103. http://dx.doi.org/10.1200/jco.2017.35.15_suppl.e13103.
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e13103 Background: Approximately 3-7% of lung tumors harbor anaplastic lymphoma kinase (ALK) fusions in the subgroup of non-small cell lung cancer (NSCLC). In addition to echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion, TRK-fused gene (TFG)-ALK, kinesin family member 5B (KIF5B)-ALK and kinesin light chain 1 (KLC1)-ALK had been reported in lung cancer. On the other hand, RET proto-oncogene (RET) and ROS proto-oncogene 1 (ROS1) fusion proteins also have prevalence in lung cancer. Food and Drug Administration (FDA)-approved several target drugs are available to treat patients with fusion mutations. Therefore, the diagnosis of ALK, RET or ROS1 fusion genes shows quite important. However, nowadays methods of detecting fusions such as fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) are limited to technique, low sensitivity, sample quality as well as subtype classification. Methods: We established nucleotide MALDI-TOF mass spectrometry based multiplex detection platform to distinguish major types including 9 types of EML4-ALK, 5 types of ALK, 5 types of RET and 8 types ROS1 fusions. Results: The detection limitation was about less 1% mutant cells among wild-type cells. In the pilot testing, we used 2 patients’ cell cDNA and 4 patients’ lung FFPE samples cDNA, which had been diagnosed as ALK fusion before, to be detected by this panel, and then identified their variant types successfully. Furthermore, one patient harbored CCDC6-RET fusion mutation was identified by our platform and confirmed by Sanger Sequencing. Conclusions: Taken together, this new panel has high sensitivity and allows little and poor quality samples for detecting. The correlation between clinical characteristics and fusion subtypes can be further investigated by utilizing this platform in the future. Also, the detection panel can be revised based on clinical needs by removing/adding probes.
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Deneen, Benjamin, Scott M. Welford, Thu Ho, Felicia Hernandez, Irwin Kurland, and Christopher T. Denny. "PIM3 Proto-Oncogene Kinase Is a Common Transcriptional Target of Divergent EWS/ETS Oncoproteins." Molecular and Cellular Biology 23, no. 11 (June 1, 2003): 3897–908. http://dx.doi.org/10.1128/mcb.23.11.3897-3908.2003.
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ABSTRACT Despite significant structural diversity, present evidence suggests that EWS/ETS fusion proteins promote oncogenesis by transcriptionally modulating a common set of target genes. In order to identify these genes, microarray expression analyses were performed on NIH 3T3 polyclonal populations expressing one of three EWS/ETS fusion genes. The majority of these genes can be grouped into seven functional categories, including cellular metabolism and signal transduction. The biologic significance of these target genes was pursued. The effects of modulating genes involved in metabolism were assessed by flux studies and demonstrated shifts in glucose utilization and lactate production as a result of EWS/FLI1 expression. The proto-oncogene coding for serine/threonine kinase PIM3 was found to one of several genes encoding signal transduction proteins that were up-regulated by EWS/ETS fusions. PIM3 was found to be expressed in a panel of human Ewing's family tumor cell lines. Forced expression of PIM3 promoted anchorage-independent growth. Coexpression of a kinase-deficient PIM3 mutant attenuated EWS/FLI1-mediated NIH 3T3 tumorigenesis in immunodeficent mice.
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Szulzewsky, Frank, Pia Hoellerbauer, Hua-Jun Wu, P. J. Cimino, Franziska Michor, Patrick Paddison, Valeri Vasioukhin, and Eric Holland. "GENE-04. THE ONCOGENIC FUNCTIONS OF YAP1-GENE FUSIONS CAN BE INHIBITED BY DISRUPTION OF YAP1-TEAD INTERACTION." Neuro-Oncology 21, Supplement_6 (November 2019): vi98. http://dx.doi.org/10.1093/neuonc/noz175.406.
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Abstract Supratentorial ependymoma can be sub-stratified into clinically relevant subtypes characterized by distinct molecular features. The subtype defined by high YAP1 activity harbored two distinct YAP1 gene fusions, YAP1-MAMLD1 and YAP1-FAM118B. In addition, YAP1 gene fusions have been detected in several other cancer types, including Epithelioid Hemangioendothelioma and Endocervical Adenocarcinoma. YAP1 is a key transcriptional co-activator and proto-oncogene that is negatively regulated by the Hippo pathway. Here, we show that both YAP1-MAMLD1 and YAP1-FAM118B, as well as additional YAP1 fusion genes found in other cancer types, are potent oncogenic drivers that cause tumor formation in the brain and the hindlimb in mice upon overexpression by somatic cell gene transfer. Using different in vitro assays, including Luciferase, RNA-, and ChIP Seq, we show that both the N-terminal YAP1 part and the C-terminal fusion partners exert activity. We can show that the YAP1 activity still relies on the binding to TEAD transcription factors, whereas the C terminal activity does not. Furthermore, the different fusion proteins have become independent from negative Hippo pathway signaling by constitutive nuclear localization and protection from degradation. In addition, by introducing point mutations and truncations to block the YAP1 and the MAMLD1 function we can show that the activity of both halves contributes to the oncogenic function of YAP1-MAMLD1. Using in vitro and in vivo assays we can show that pharmacological and genetic ablation of YAP-TEAD interaction diminishes the oncogenic potential of the fusions, indicating that this might be a potential therapeutic approach for these tumors in the future.
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Park, Steven T., Garry P. Nolan, and Xiao-Hong Sun. "Growth Inhibition and Apoptosis Due to Restoration of E2A Activity in T Cell Acute Lymphoblastic Leukemia Cells." Journal of Experimental Medicine 189, no. 3 (February 1, 1999): 501–8. http://dx.doi.org/10.1084/jem.189.3.501.
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Two models have been proposed for the molecular mechanism by which the Tal1 oncogene causes T cell acute lymphoblastic leukemia (T-ALL). The activation model suggests that Tal1 as heterodimers with the E2A transcription factor activates the expression of oncogenes. The inhibition model postulates that Tal1 interferes with the tumor-suppressing function of E2A. In the Jurkat T cell line, originally derived from a patient with T-ALL, Tal1 is complexed with E2A proteins and the transcriptional activity of E2A is very low. When E2A activity was restored by expressing an E2A–Tal1 fusion protein, E-T/2, the Jurkat cells underwent growth arrest and subsequently apoptosis, thus supporting the inhibition model and suggesting that E2A loss may contribute to leukemic progression.
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Bocchia, M., PA Wentworth, S. Southwood, J. Sidney, K. McGraw, DA Scheinberg, and A. Sette. "Specific binding of leukemia oncogene fusion protein peptides to HLA class I molecules." Blood 85, no. 10 (May 15, 1995): 2680–84. http://dx.doi.org/10.1182/blood.v85.10.2680.bloodjournal85102680.
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Many human leukemias are characterized by chromosomal translocations yielding hybrid RNAs capable of encoding fusion chimeric proteins. The unique amino acid sequences found in these oncogenic fusion proteins represent true tumor-specific antigens that are potentially immunogenic. Although these leukemia-specific fusion proteins have an intracellular location, they might be recognized immunologically by T lymphocytes if peptides derived from the unique sequences are capable of presentation by the major histocompatibility complex (MHC) molecules on leukemic cells. The ability of a series of synthetic peptides corresponding to the junctional sequences of chronic myelogenous leukemia (CML)-derived bcr-abl and acute promyelocytic leukemia (APL)-derived PML-RAR alpha fusion proteins to bind to purified class I molecules was studied. A series of 152 peptides 8, 9, 10, and 11 amino acids in length, spanning the b3a2 and b2a2 breakpoints for CML and PML-RAR alpha A and B breakpoints for APL were analyzed for HLA A1, A2.1, A3.2, A11, A24, B7, B8, and B27 binding motifs. Twenty-one CML peptides and 4 APL peptides were predicted to be potential HLA class I binders. The peptides were tested for binding to appropriate purified HLA molecules in a competition radioimmunoassay. Four peptides derived from b3a2 CML breakpoint bound with high (< 50 nmol/L) or intermediate (< or = 500 nmol/L) affinity to HLA A3, A11, and B8. None of the CML b2a2 or PML-RAR alpha A or B junctional peptides showed affinity of this magnitude for the HLA class I molecules tested. This is the first evidence that tumor-specific breakpoint peptides can bind human MHC class I molecules and provides a rationale for developing a therapeutic vaccine strategy.
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Lin, Yi-Hui, Purvi M. Kakadia, Ying Chen, Ya-Qiang Li, Aniruddha J. Deshpande, Christian Buske, Kang-Ling Zhang, Yi Zhang, Guo-Liang Xu, and Stefan K. Bohlander. "Global reduction of the epigenetic H3K79 methylation mark and increased chromosomal instability in CALM-AF10–positive leukemias." Blood 114, no. 3 (July 16, 2009): 651–58. http://dx.doi.org/10.1182/blood-2009-03-209395.
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Abstract Chromosomal translocations generating fusion proteins are frequently found in human leukemias. The fusion proteins play an important role in leukemogenesis by subverting the function of one or both partner proteins. The leukemogenic CALM-AF10 fusion protein is capable of interacting with the histone H3 lysine 79 (H3K79)–specific methyltransferase hDOT1L through the fused AF10 moiety. This interaction leads to local H3K79 hypermethylation on Hoxa5 loci, which up-regulates the expression of Hoxa5 and contributes to leukemogenesis. However, the long latency of leukemogenesis of CALM-AF10 transgenic mice suggests that the direct effects of fusion oncogene are not sufficient for the induction of leukemia. In this study, we show that the CALM-AF10 fusion protein can also greatly reduce global H3K79 methylation in both human and murine leukemic cells by disrupting the AF10-mediated association of hDOT1L with chromatin. Cells with reduced H3K79 methylation are more sensitive to γ-irradiation and display increased chromosomal instability. Consistently, leukemia patients harboring CALM-AF10 fusion have more secondary chromosomal aberrations. These findings suggest that chromosomal instability associated with global epigenetic alteration contributes to malignant transformation in certain leukemias, and that leukemias with this type of epigenetic alteration might benefit from treatment regimens containing DNA-damaging agents. This study is registered with www.clinicaltrials.gov as NCT00266136.
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Butler, Lisa H., Robert Slany, Xiangmin Cui, Michael L. Cleary, and David Y. Mason. "The HRX Proto-oncogene Product Is Widely Expressed in Human Tissues and Localizes to Nuclear Structures." Blood 89, no. 9 (May 1, 1997): 3361–70. http://dx.doi.org/10.1182/blood.v89.9.3361.
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Abstract Chromosomal rearrangement of the HRX (MLL, ALL-1, Htrx) gene situated at chromosome band 11q23 is one of the most frequent genetic changes in infant leukemias of myeloid and lymphoid lineage and in treatment-induced secondary leukemias. The HRX gene codes for a predicted 431-kD protein that shows significant homology to the Drosophila trithorax protein, an Hox epigenetic regulator. Typically, the region encoding the HRX gene is rearranged, mostly in reciprocal translocations with a number of partners, resulting in a range of fusion genes. However, this is not the only abnormality affecting HRX because partial duplication of the gene, as well as interstitial deletions, can occur. Despite extensive studies of HRX at the genetic level, the protein products of the HRX gene and their patterns of expression in normal and leukemic cells remain uncharacterized. In this study we analyzed the distribution and localization of HRX proteins in cell lines and human tissues, using both polyclonal and monoclonal antibodies. The specificity of these reagents was confirmed using cells transfected with the HRX-ENL fusion gene. Western blot analyses of protein extracts from cells carrying the t(11; 19) and t(4; 11) translocations showed HRX chimeric proteins whose migrations corresponded to the sizes predicted from analyses of translocation-induced fusion mRNAs expressed by the derivative 11 chromosomes. Immunocytochemical analysis showed a punctate distribution of wild-type and chimeric HRX proteins within cell nuclei, suggesting that HRX localizes to nuclear structures in cells with and without 11q23 translocations. Nuclear staining was found in the majority of tissues studied with the strongest reactivity in cerebral cortex, kidney, thyroid, and lymphoid tissues. Thus, HRX is widely expressed in most cell types including hematopoietic cells, a finding that precludes an immunocytochemical approach for diagnosis of leukemias bearing 11q23 structural abnormalities.
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Werner, Michael T., Chen Zhao, Qian Zhang, and Mariusz A. Wasik. "Nucleophosmin-anaplastic lymphoma kinase: the ultimate oncogene and therapeutic target." Blood 129, no. 7 (February 16, 2017): 823–31. http://dx.doi.org/10.1182/blood-2016-05-717793.
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Abstract Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase physiologically expressed by fetal neural cells. However, aberrantly expressed ALK is involved in the pathogenesis of diverse malignancies, including distinct types of lymphoma, lung carcinoma, and neuroblastoma. The aberrant ALK expression in nonneural cells results from chromosomal translocations that create novel fusion proteins. These protein hybrids compose the proximal part of a partner gene, including its promoter region, and the distal part of ALK, including the coding sequence for the entire kinase domain. ALK was first identified in a subset of T-cell lymphomas with anaplastic large cell lymphoma (ALCL) morphology (ALK+ ALCL), the vast majority of which harbor the well-characterized nucleophosmin (NPM)-ALK fusion protein. NPM-ALK co-opts several intracellular signal transduction pathways, foremost being the STAT3 pathway, normally activated by cytokines from the interleukin-2 (IL-2) family to promote cell proliferation and to inhibit apoptosis. Many genes and proteins modulated by NPM-ALK are also involved in evasion of antitumor immune response, protection from hypoxia, angiogenesis, DNA repair, cell migration and invasiveness, and cell metabolism. In addition, NPM-ALK uses epigenetic silencing mechanisms to downregulate tumor suppressor genes to maintain its own expression. Importantly, NPM-ALK is capable of transforming primary human CD4+ T cells into immortalized cell lines indistinguishable from patient-derived ALK+ ALCL. Preliminary clinical studies indicate that inhibition of NPM-ALK induces long-lasting complete remissions in a large subset of heavily pretreated adult patients and the vast majority of children with high-stage ALK+ ALCL. Combining ALK inhibition with other novel therapeutic modalities should prove even more effective.
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Duelli, Dominik M., Stephen Hearn, Michael P. Myers, and Yuri Lazebnik. "A primate virus generates transformed human cells by fusion." Journal of Cell Biology 171, no. 3 (November 7, 2005): 493–503. http://dx.doi.org/10.1083/jcb.200507069.
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Amodel that explains both the origin and sporadic nature of cancer argues that cancer cells are a chance result of events that cause genomic and epigenetic variability. The prevailing view is that these events are mutations that affect chromosome segregation or stability. However, genomic and epigenetic variability is also triggered by cell fusion, which is often caused by viruses. Yet, cells fused by viruses are considered harmless because they die. We provide evidence that a primate virus uses both viral and exosomal proteins involved in cell fusion to produce transformed proliferating human cells. Although normal cells indeed fail to proliferate after fusion, expression of an oncogene or a mutated tumor suppressor p53 in just one of the fusion partners is sufficient to produce heterogeneous progeny. We also show that this virus can produce viable oncogenically transformed cells by fusing cells that are otherwise destined to die. Therefore, we argue that viruses can contribute to carcinogenesis by fusing cells.
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Anobile, Jonathan M., Vaithilingaraja Arumugaswami, Danielle Downs, Kirk Czymmek, Mark Parcells, and Carl J. Schmidt. "Nuclear Localization and Dynamic Properties of the Marek's Disease Virus Oncogene Products Meq and Meq/vIL8." Journal of Virology 80, no. 3 (February 1, 2006): 1160–66. http://dx.doi.org/10.1128/jvi.80.3.1160-1166.2006.
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ABSTRACT Marek's disease virus (MDV) is an avian herpesvirus that causes T-cell lymphomas and immune suppression in susceptible chickens. At least one gene product, MDV Eco Q-encoded protein (Meq), is essential for the oncogenicity of MDV. Alternative splicing permits the meq gene to give rise to two major transcripts encoding proteins designated Meq and Meq/vIL8. Meq is a basic leucine zipper protein capable of modulating transcription. The Meq/vIL8 protein retains a modified leucine zipper, along with the mature receptor-binding portion of vIL8, but lacks the domain of Meq responsible for transcriptional modulation. In this report, we describe studies using fusions between either Meq or Meq/vIL8 and fluorescent proteins to characterize the distribution and properties of these products in chicken embryo fibroblasts (CEFs). Meq and Meq/vIL8 both localized to the nucleoplasm, nucleoli, and Cajal bodies of transfected cells. Similar distributions were found for fluorescent fusion proteins and native Meq or Meq/vIL8. Fluorescence recovery after photobleaching and photoactivatable green fluorescent protein revealed that Meq exhibited mobility properties similar to those of other transcription factors, while Meq/vIL8 was far less mobile. In addition, fluorescence resonance energy transfer studies indicated the formation of Meq/vIL8 homodimers in CEFs. Time lapse studies revealed the coordinated elimination of a portion of Meq and Meq/vIL8 from the nucleus. Our data provide new insight regarding the dynamic cellular properties of two forms of a herpesvirus-encoded oncoprotein and suggest that these forms may have fundamentally different functions in MDV-infected cells.
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Grembecka, Jolanta, Amalia Marie Belcher, and Tomasz Cierpicki. "Molecular Basis of Menin-MLL Interaction: Implication for Targeted Therapies in MLL Leukemias." Blood 114, no. 22 (November 20, 2009): 3775. http://dx.doi.org/10.1182/blood.v114.22.3775.3775.
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Abstract Abstract 3775 Poster Board III-711 Chromosomal translocations that affect the proto-oncogene MLL (Mixed Lineage Leukemia) occur in aggressive human acute leukemias, both AML and ALL, affecting children and adults. The normal MLL protein plays a key role in regulation of HOX genes expression, which are required for proper hematopoiesis. This function is frequently impaired by a fusion of MLL with one of 60 alternative partner genes to form a chimeric oncogene encoding MLL fusion proteins. MLL fusions upregulate HOX genes expression resulting in a blockage of blood cell differentiation that ultimately leads to acute leukemia. Patients with MLL rearrangement poorly respond to available treatments, emphasizing the urgent need to develop novel therapies to treat these leukemias. The leukemogenic activity of MLL fusions is dependent on association with menin, a protein encoded by the MEN1 (Multiple Endocrine Neoplasia I) gene. The menin binding motif is localized at the N-terminus of MLL and therefore it is retained in all MLL fusion proteins. The removal of this motif from MLL oncoproteins abrogates the ability to develop leukemia in mice. Menin functions as an essential oncogenic cofactor in MLL related leukemias and selective targeting of the menin-MLL interaction might represent a novel valuable therapeutic approach for the treatment of the MLL-related leukemias. To understand the molecular basis of how MLL-fusion proteins interact with menin, we carried out detailed in vitro characterization of menin binding to N-terminus of MLL using a collection of biochemical, biophysical and structural biology approaches. We demonstrated that 46 long N-terminal amino acid fragment of MLL very strongly associates with menin with low-nanomolar binding affinity. Employing the NMR spectroscopy, we identified the presence of two separate menin binding motifs within this MLL fragment, MBM1 (menin binding motif 1) and MBM2 (menin binding motif 2), which are separated by a poly-glycine linker. Peptides corresponding to both motifs are capable to independently interact with menin indicating the presence of two separate MLL binding sites on menin. Furthermore, the MBM1 binds to menin with 20-fold higher affinity compared to MBM2. Interestingly, we demonstrated that binding of one of the MBM peptides to menin negatively regulates binding of the second peptide most likely through the mechanism of an allosteric regulation. To aid in rational design of small molecule inhibitors of the menin-MLL interaction we characterized the conformation of the high affinity motif (MBM1) of MLL in a menin bound conformation using NMR spectroscopy. Furthermore, by applying both mutational studies and binding affinity measurements we identified that the most critical amino acids of MBM1 involved in interaction with menin comprise the RFPARP fragment of MLL. Overall, for the first time, we are providing detailed characterization and molecular basis of the MLL interaction with menin, which will be invaluable for development of therapeutically useful inhibitors selectively targeting this interaction. Disclosures: No relevant conflicts of interest to declare.
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Kuravi, Sudhakiranmayi, Preethi Kunchala, Siddhartha Ganguly, Sunil Abhyankar, Yogen Saunthararajah, Kojo S. J. Elenitoba-Johnson, Jensen Roy, Joseph McGuirk, and Ramesh Balusu. "NPM1-TYK2 Fusion Is an Oncogene and a Novel HSP90-Client Protein." Blood 128, no. 22 (December 2, 2016): 4127. http://dx.doi.org/10.1182/blood.v128.22.4127.4127.
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Abstract Cutaneous CD30-positive lymphoproliferative disorders (LPDs), the second most common type of cutaneous T-cell lymphoma include a clinicopathologic spectrum of benign lymphomatoid papulosis (LYP) and primary cutaneous anaplastic large-cell lymphoma (ALCL). NPM1-TYK2 is the first identified tyrosine kinase chromosomal translocation in CD30-positive LPDs. Understanding the deregulated functions resulting from new chromosomal translocations in these neoplasms is essential to discover novel therapeutic approaches. Nucleophosmin 1 (NPM1) is a nucleolar phosphoprotein, which functions as a molecular chaperone for proteins and nucleic acids. Tyrosine kinase 2 (TYK2) is a non-receptor tyrosine kinase that belongs to the family of Janus Kinases (JAKs). NPM1-TYK2 is an 81 kDa fusion protein comprising of NPM1 (1-257 amino acids) and the kinase domain of TYK2 (726-1187 amino acids). Since NPM1-TYK2 is a recently discovered fusion gene, we conducted experiments to determine its oncogenic potential. Transduction of lentiviral particles bearing NPM1-TYK2 gene transformed the interleukin-3 (IL-3) dependent Ba/F3 cell line to IL-3 independent growth through constitutive activation of TYK2 kinase and downstream STAT signaling. We further evaluated the role of NPM1 as part of fusion protein in NPM1-TYK2 mediated oncogenicity. Transfection of NPM1-TYK2 deletion construct lacking NPM1 in HEK293 cells resulted in significant decrease in TYK2 kinase activity in the cells compared to full-length NPM1-TYK2 transfected cells. Currently, there are no effective small molecule inhibitors for NPM1/ TYK2 in the clinical trials. Hence, there is a clinical unmet need to develop novel, targeted therapies for NPM1-TYK2 driven lymphomas. As TYK2 is an established HSP90 client protein, the rationale for this study is to investigate the ability of HSP90 to regulate the stability and kinase activity of NPM1-TYK2 fusion protein. HSP90 is a master chaperone involved in the proper folding and maturation of a variety of oncogenic kinases and perturbing HSP90 function is emerging as a promising approach for cancer therapy. We used Myla cells (expressing endogenous NPM1-TYK2 fusion gene) to investigate the status of NPM1-TYK2 as a HSP90 client protein. Here we report that the treatment of cells with small molecule HSP90 inhibitor, 17-AAG promotes degradation of NPM1-TYK2 fusion protein resulting in downregulation of its oncogenically induced downstream STAT signaling pathways. Mechanistically, dephosphorylation of STAT1, 3, and 5 following 17-AAG treatment resulted in apoptotic cell death of the lymphoma cells. The results obtained using 17-AAG, were further corroborated using other HSP90 small molecule inhibitors AUY922 and PU-H71. Immunoprecipitation studies clearly demonstrate that 17-AAG treatment disrupts interaction between HSP90 and NPM1-TYK2 chimeric protein. Collectively, these findings provide an evidence for NPM1-TYK2 oncogenicity and therapeutic potential of HSP90 inhibitors for the treatment of a subset of cutaneous CD30-positive lymphoproliferative disorder patients expressing NPM1-TYK2 chimeric gene. Disclosures Ganguly: Onyx: Speakers Bureau; Seattle Genetics: Speakers Bureau; Janssen: Research Funding.
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Todorova, Roumiana. "Functional Interactions in Transcription and Splicing of Ewing’s Sarcoma." ISRN Genetics 2013 (July 22, 2013): 1–6. http://dx.doi.org/10.5402/2013/184063.
Full textAbstract:
Ewing’s sarcoma (EWS) protein is a member of the TET (TLS/EWS/TAF15) family of RNA and DNA-binding proteins with unknown cellular role. EWS protein is encoded by the EWS oncogene on chromosome 22q12, a target of chromosomal translocations in Ewing’s sarcoma tumors. The exact mechanism of EWS participation in gene expression and pathogenesis of the resulting cancers is not defined. The binding partners of native EWS and EWS fusion proteins (EFPs) are described schematically in a model, an attempt to link the transcription with the splicing. The experimental data about the partnerships of EWS and EFPs are summarized, which may lead to better understanding of their function.
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Rao, Chaitra, Dianna H. Huisman, Heidi M. Vieira, Danielle E. Frodyma, Beth K. Neilsen, Binita Chakraborty, Suzie K. Hight, Michael A. White, Kurt W. Fisher, and Robert E. Lewis. "A Gene Expression High-Throughput Screen (GE-HTS) for Coordinated Detection of Functionally Similar Effectors in Cancer." Cancers 12, no. 11 (October 27, 2020): 3143. http://dx.doi.org/10.3390/cancers12113143.
Full textAbstract:
Genome-wide, loss-of-function screening can be used to identify novel vulnerabilities upon which specific tumor cells depend for survival. Functional Signature Ontology (FUSION) is a gene expression-based high-throughput screening (GE-HTS) method that allows researchers to identify functionally similar proteins, small molecules, and microRNA mimics, revealing novel therapeutic targets. FUSION uses cell-based high-throughput screening and computational analysis to match gene expression signatures produced by natural products to those produced by small interfering RNA (siRNA) and synthetic microRNA libraries to identify putative protein targets and mechanisms of action (MoA) for several previously undescribed natural products. We have used FUSION to screen for functional analogues to Kinase suppressor of Ras 1 (KSR1), a scaffold protein downstream of Ras in the Raf-MEK-ERK kinase cascade, and biologically validated several proteins with functional similarity to KSR1. FUSION incorporates bioinformatics analysis that may offer higher resolution of the endpoint readout than other screens which utilize Boolean outputs regarding a single pathway activation (i.e., synthetic lethal and cell proliferation). Challenges associated with FUSION and other high-content genome-wide screens include variation, batch effects, and controlling for potential off-target effects. In this review, we discuss the efficacy of FUSION to identify novel inhibitors and oncogene-induced changes that may be cancer cell-specific as well as several potential pitfalls within FUSION and best practices to avoid them.
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Million, Ryan P., and Richard A. Van Etten. "The Grb2 binding site is required for the induction of chronic myeloid leukemia-like disease in mice by the Bcr/Abl tyrosine kinase." Blood 96, no. 2 (July 15, 2000): 664–70. http://dx.doi.org/10.1182/blood.v96.2.664.014k52_664_670.
Full textAbstract:
The BCR/ABL oncogene results from a balanced translocation between chromosomes 9 and 22 and is found in patients with chronic myeloid leukemia (CML) and in some patients with acute B-lymphoid leukemia. The Bcr/Abl fusion protein is a constitutively active tyrosine kinase that stimulates several intracellular signaling pathways, including activation of Ras through direct binding of the SH2-containing adapter protein Grb2 to Bcr tyrosine 177. A tyrosine-to-phenylalanine mutation (Y177F) at this site blocks the co-association of Bcr/Abl and Grb2 in vivo and impairs focus formation by Bcr/Abl in fibroblasts. However, the Bcr/Abl Y177F mutant can transform hematopoietic cell lines and primary bone marrow cells in vitro, so the importance of the Bcr/Abl–Grb2 interaction to myeloid and lymphoid leukemogenesis in vivo is unclear. We have recently demonstrated the efficient induction of CML-like myeloproliferative disease by BCR/ABL in a murine bone marrow transduction/transplantation model system. The Y177F mutation greatly attenuates the myeloproliferative disease induced by BCR/ABL, with mice developing B- and T-lymphoid leukemias of longer latency. In addition, the v-abl oncogene of Abelson murine leukemia virus, whose protein product lacks interaction with Grb2, is completely defective for the induction of CML-like disease. These results suggest that direct binding of Grb2 is required for the efficient induction of CML-like myeloproliferative disease by oncogenic Abl proteins.
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Yokoyama, Akihiko, Tim Somervaille, and Michael L. Cleary. "The Menin Tumor Suppressor Protein Is an Essential Oncogenic Cofactor for MLL-Associated Leukemogenesis." Blood 106, no. 11 (November 16, 2005): 665. http://dx.doi.org/10.1182/blood.v106.11.665.665.
Full textAbstract:
Abstract The Mixed Lineage Leukemia (MLL) protein is a histone methyltransferase that is mutated in clinically and biologically distinctive subsets of acute leukemia. MLL normally associates with a cohort of highly conserved cofactors to form a macromolecular complex that includes menin, a product of the MEN1 tumor suppressor gene, which is mutated in heritable and sporadic endocrine tumors. We demonstrate here that oncogenic MLL fusion proteins retain an ability to stably associate with menin through a high-affinity, amino-terminal, conserved binding motif and that this interaction is required for the initiation of MLL-mediated leukemogenesis. Furthermore, menin is essential for maintenance of MLL-associated but not other oncogene induced myeloid transformation. Acute genetic ablation of menin reverses aberrant Hox gene expression mediated by MLL-menin promoter-associated complexes, and specifically abrogates the differentiation arrest and oncogenic properties of MLL-transformed leukemic blasts. These results demonstrate that a human oncoprotein is critically dependent on direct physical interaction with a tumor suppressor protein for its oncogenic activity, validate a potential target for molecular therapy, and suggest central roles for menin in altered epigenetic functions underlying the pathogenesis of hematopoietic cancers.
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KOVAL, P. Anatolii, A. Vicky BLAKESLEY, T. Charles ROBERTS, Yehiel ZICK, and Derek LeROITH. "Interaction in vitro of the product of the c-Crk-II proto-oncogene with the insulin-like growth factor I receptor." Biochemical Journal 330, no. 2 (March 1, 1998): 923–32. http://dx.doi.org/10.1042/bj3300923.
Full textAbstract:
The Crk proto-oncogene product is an SH2 and SH3 domain-containing adaptor protein. We have previously demonstrated that Crk-II becomes rapidly tyrosine-phosphorylated in response to stimulation with insulin-like growth factor I (IGF-I) and might be involved in the IGF-I receptor signalling pathway. To determine whether this involvement includes the direct interaction of Crk-II with the cytoplasmic region of the receptor, studies were performed in vitro with glutathione S-transferase (GST) fusion proteins containing various domains of Crk-II. The kinase assay in vitro showed that activated IGF-I receptors efficiently phosphorylated the GST-Crk-II fusion protein. This phosphorylation was dependent on the presence of the SH2 domain and Tyr-221 located in the spacer region between the two SH3 domains. Mutation of Tyr-221 not only prevented phosphorylation of GST-Crk in vitro, but also significantly increased the ability of GST-Crk proteins to co-precipitate activated IGF-I receptors from total cell lysates. Additional binding experiments in vitro showed that Crk-II might interact with the phosphorylated IGF-I receptor through its SH2 domain. To elucidate which region of the IGF-I receptor interacts with Crk-II, a peptide association assay was used in vitro. Different domains of the IGF-I receptor were expressed as (His)6-tagged fusion peptides, phosphorylated with activated wheat germ agglutinin-purified IGF-I receptors and tested for association with GST-Crk-II fusion proteins. Using wild-type as well as mutated peptides, we showed that the SH2 domain of Crk-II preferentially binds the peptide encoding the juxtamembrane region of the IGF-I receptor. Phosphorylation of Tyr-950 and Tyr-943 of the receptor is important for this interaction. These findings allow us to propose a model of direct interaction of Crk-II and the IGF-I receptor in vivo. On activation of the IGF-I receptor, Crk-II binds to phosphorylated tyrosine residues, especially in the juxtamembrane region. As a result of this binding, the IGF-I receptor kinase phosphorylates Tyr-221 of Crk-II, resulting in a change in intramolecular folding and binding of the SH2 domain to the phosphorylated Tyr-221, which causes rapid disassociation of the Crk-II-IGF-I receptor complex.
23
Bunker, C. A., and R. E. Kingston. "Transcriptional repression by Drosophila and mammalian Polycomb group proteins in transfected mammalian cells." Molecular and Cellular Biology 14, no. 3 (March 1994): 1721–32. http://dx.doi.org/10.1128/mcb.14.3.1721.
Full textAbstract:
The Polycomb group (Pc-G) genes are essential for maintaining the proper spatially restricted expression pattern of the homeotic loci during Drosophila development. The Pc-G proteins appear to function at target loci to maintain a state of transcriptional repression. The murine oncogene bmi-1 has significant homology to the Pc-G gene Posterior sex combs (Psc) and a highly related gene, Suppressor two of zeste [Su(z)2]. We show here that the proteins encoded by bmi-1 and the Pc-G genes Polycomb (Pc) and Psc as well as Su(z)2 mediate repression in mammalian cells when targeted to a promoter by LexA in a cotransfection system. These fusion proteins repress activator function by as much as 30-fold, and the effect on different activation domains is distinct for each Pc-G protein. Repression is observed when the LexA fusion proteins are bound directly adjacent to activator binding sites and also when bound 1,700 bases from the promoter. These data demonstrate that the products of the Pc-G genes can significantly repress activator function on transiently introduced DNA. We suggest that this function contributes to the stable repression of targeted loci during development.
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Bunker, C. A., and R. E. Kingston. "Transcriptional repression by Drosophila and mammalian Polycomb group proteins in transfected mammalian cells." Molecular and Cellular Biology 14, no. 3 (March 1994): 1721–32. http://dx.doi.org/10.1128/mcb.14.3.1721-1732.1994.
Full textAbstract:
The Polycomb group (Pc-G) genes are essential for maintaining the proper spatially restricted expression pattern of the homeotic loci during Drosophila development. The Pc-G proteins appear to function at target loci to maintain a state of transcriptional repression. The murine oncogene bmi-1 has significant homology to the Pc-G gene Posterior sex combs (Psc) and a highly related gene, Suppressor two of zeste [Su(z)2]. We show here that the proteins encoded by bmi-1 and the Pc-G genes Polycomb (Pc) and Psc as well as Su(z)2 mediate repression in mammalian cells when targeted to a promoter by LexA in a cotransfection system. These fusion proteins repress activator function by as much as 30-fold, and the effect on different activation domains is distinct for each Pc-G protein. Repression is observed when the LexA fusion proteins are bound directly adjacent to activator binding sites and also when bound 1,700 bases from the promoter. These data demonstrate that the products of the Pc-G genes can significantly repress activator function on transiently introduced DNA. We suggest that this function contributes to the stable repression of targeted loci during development.
25
Nair, N., R. J. Davis, and H. L. Robinson. "Protein tyrosine kinase activities of the epidermal growth factor receptor and ErbB proteins: correlation of oncogenic activation with altered kinetics." Molecular and Cellular Biology 12, no. 5 (May 1992): 2010–16. http://dx.doi.org/10.1128/mcb.12.5.2010.
Full textAbstract:
We have compared the protein tyrosine kinase activities of the chicken epidermal growth factor receptor (chEGFR) and three ErbB proteins to learn whether cancer-activating mutations affect the kinetics of kinase activity. In immune complex assays performed in the presence of 15 mM Mn2+, ErbB proteins and the chEGFR exhibited highly reproducible tyrosine kinase activity. Under these conditions, the ErbB and chEGFR proteins had similar apparent Km [Km(app)] values for ATP. The ErbB proteins appeared to be activated, as they had at least 3-fold-higher relative Vmax(app) for autophosphorylation and approximately 2-fold higher relative Vmax(app) for the phosphorylation of the exogenous substrate TK6 (a bacterially expressed fusion protein containing the C-terminal domain of the human EGFR). The ErbB kinases had both higher Km(app) and higher Vmax(app) for the phosphorylation of the exogenous substrate TK6 than did the chEGFR. The ratios of the Vmax(app) to the Km(app) for TK6 phosphorylation suggested that the ErbB proteins had lower catalytic efficiencies for the exogenous substrate than did the chEGFR. The three tested ErbB proteins had cytoplasmic domain mutations that conferred distinctive disease potentials. These mutations did not affect the kinetics for the phosphorylation of the exogenous substrate TK6. Two of the ErbB proteins contained all of the sites used for autophosphorylation. In these, a mutation that broadened oncogenic potential to endothelial cells caused an additional increase in Vmax(app) for autophosphorylation. Thus, mutations that change the EGFR into an ErbB oncogene cause multiple changes in the kinetics of protein tyrosine kinase activity.
26
Nair, N., R. J. Davis, and H. L. Robinson. "Protein tyrosine kinase activities of the epidermal growth factor receptor and ErbB proteins: correlation of oncogenic activation with altered kinetics." Molecular and Cellular Biology 12, no. 5 (May 1992): 2010–16. http://dx.doi.org/10.1128/mcb.12.5.2010-2016.1992.
Full textAbstract:
We have compared the protein tyrosine kinase activities of the chicken epidermal growth factor receptor (chEGFR) and three ErbB proteins to learn whether cancer-activating mutations affect the kinetics of kinase activity. In immune complex assays performed in the presence of 15 mM Mn2+, ErbB proteins and the chEGFR exhibited highly reproducible tyrosine kinase activity. Under these conditions, the ErbB and chEGFR proteins had similar apparent Km [Km(app)] values for ATP. The ErbB proteins appeared to be activated, as they had at least 3-fold-higher relative Vmax(app) for autophosphorylation and approximately 2-fold higher relative Vmax(app) for the phosphorylation of the exogenous substrate TK6 (a bacterially expressed fusion protein containing the C-terminal domain of the human EGFR). The ErbB kinases had both higher Km(app) and higher Vmax(app) for the phosphorylation of the exogenous substrate TK6 than did the chEGFR. The ratios of the Vmax(app) to the Km(app) for TK6 phosphorylation suggested that the ErbB proteins had lower catalytic efficiencies for the exogenous substrate than did the chEGFR. The three tested ErbB proteins had cytoplasmic domain mutations that conferred distinctive disease potentials. These mutations did not affect the kinetics for the phosphorylation of the exogenous substrate TK6. Two of the ErbB proteins contained all of the sites used for autophosphorylation. In these, a mutation that broadened oncogenic potential to endothelial cells caused an additional increase in Vmax(app) for autophosphorylation. Thus, mutations that change the EGFR into an ErbB oncogene cause multiple changes in the kinetics of protein tyrosine kinase activity.
27
Million, Ryan P., and Richard A. Van Etten. "The Grb2 binding site is required for the induction of chronic myeloid leukemia-like disease in mice by the Bcr/Abl tyrosine kinase." Blood 96, no. 2 (July 15, 2000): 664–70. http://dx.doi.org/10.1182/blood.v96.2.664.
Full textAbstract:
Abstract The BCR/ABL oncogene results from a balanced translocation between chromosomes 9 and 22 and is found in patients with chronic myeloid leukemia (CML) and in some patients with acute B-lymphoid leukemia. The Bcr/Abl fusion protein is a constitutively active tyrosine kinase that stimulates several intracellular signaling pathways, including activation of Ras through direct binding of the SH2-containing adapter protein Grb2 to Bcr tyrosine 177. A tyrosine-to-phenylalanine mutation (Y177F) at this site blocks the co-association of Bcr/Abl and Grb2 in vivo and impairs focus formation by Bcr/Abl in fibroblasts. However, the Bcr/Abl Y177F mutant can transform hematopoietic cell lines and primary bone marrow cells in vitro, so the importance of the Bcr/Abl–Grb2 interaction to myeloid and lymphoid leukemogenesis in vivo is unclear. We have recently demonstrated the efficient induction of CML-like myeloproliferative disease by BCR/ABL in a murine bone marrow transduction/transplantation model system. The Y177F mutation greatly attenuates the myeloproliferative disease induced by BCR/ABL, with mice developing B- and T-lymphoid leukemias of longer latency. In addition, the v-abl oncogene of Abelson murine leukemia virus, whose protein product lacks interaction with Grb2, is completely defective for the induction of CML-like disease. These results suggest that direct binding of Grb2 is required for the efficient induction of CML-like myeloproliferative disease by oncogenic Abl proteins.
28
Druker, B., K. Okuda, U. Matulonis, R. Salgia, T. Roberts, and JD Griffin. "Tyrosine phosphorylation of rasGAP and associated proteins in chronic myelogenous leukemia cell lines." Blood 79, no. 9 (May 1, 1992): 2215–20. http://dx.doi.org/10.1182/blood.v79.9.2215.2215.
Full textAbstract:
Abstract The Philadelphia chromosome (Ph1), detected in virtually all cases of chronic myelogenous leukemia, is formed by a reciprocal translocation between chromosomes 9 and 22 that fuses BCR encoded sequences upstream of exon 2 of c-ABL. This oncogene produces a fusion protein (p210BCR/ABL) in which the ABL tyrosine kinase activity is elevated. This elevated kinase activity is essential for transformation, but the mechanisms involved are unknown. We report here that p21ras GTPase activating protein (rasGAP) or rasGAP-associated proteins p190 and p62 are phosphorylated on tyrosine in Ph1 (+) cell lines. Further, rasGAP coimmunoprecipitates with p210BCR/ABL in these cell lines. These results suggest that rasGAP or associated proteins are potential substrates for p210BCR/ABL kinase and thus directly link p210BCR/ABL with a signal transduction pathway known to be activated by hematopoietic growth factors (p21ras).
29
Druker, B., K. Okuda, U. Matulonis, R. Salgia, T. Roberts, and JD Griffin. "Tyrosine phosphorylation of rasGAP and associated proteins in chronic myelogenous leukemia cell lines." Blood 79, no. 9 (May 1, 1992): 2215–20. http://dx.doi.org/10.1182/blood.v79.9.2215.bloodjournal7992215.
Full textAbstract:
The Philadelphia chromosome (Ph1), detected in virtually all cases of chronic myelogenous leukemia, is formed by a reciprocal translocation between chromosomes 9 and 22 that fuses BCR encoded sequences upstream of exon 2 of c-ABL. This oncogene produces a fusion protein (p210BCR/ABL) in which the ABL tyrosine kinase activity is elevated. This elevated kinase activity is essential for transformation, but the mechanisms involved are unknown. We report here that p21ras GTPase activating protein (rasGAP) or rasGAP-associated proteins p190 and p62 are phosphorylated on tyrosine in Ph1 (+) cell lines. Further, rasGAP coimmunoprecipitates with p210BCR/ABL in these cell lines. These results suggest that rasGAP or associated proteins are potential substrates for p210BCR/ABL kinase and thus directly link p210BCR/ABL with a signal transduction pathway known to be activated by hematopoietic growth factors (p21ras).
30
Yassin, Enas R., Anmaar M. Abdul-Nabi, Akiko Takeda, and Nabeel R. Yaseen. "Role of a Conserved Helicase Motif in the Transformation of Primary Human CD34+ Cells by the NUP98-DDX10 Oncogene." Blood 114, no. 22 (November 20, 2009): 2966. http://dx.doi.org/10.1182/blood.v114.22.2966.2966.
Full textAbstract:
Abstract Abstract 2966 Poster Board II-942 NUP98 gene rearrangements occur in acute myeloid leukemia and other hematopoietic malignancies, and result in the expression of fusion proteins. One of the most frequent NUP98 fusions is NUP98-DDX10 that consists of an N-terminal portion of NUP98 and a C-terminal segment of DDX10, a putative DEAD-box RNA helicase. Here we express NUP98-DDX10 in primary human CD34+ hematopoietic cells and show that it localizes within the nucleus in a punctate distribution. It dramatically increases the proliferation and self-renewal of primary human CD34+ cells and disrupts their erythroid and myeloid differentiation. Expression gene profiling shows dysregulation of many genes by NUP98-DDX10 in primary human CD34+ cells starting within 6 h. Comparison of the dysregulome of NUP98-DDX10 to that of another leukemogenic NUP98 fusion, NUP98-HOXA9, reveals a number of genes dysregulated by both oncoproteins, including HOX genes, COX-2, MYCN, angiopoietin-1, renin, HEY1, SOX4, and others, that may account for the induction of AML by these and other NUP98 fusion oncogenes. The HRAGRTAR sequence in the DDX10 portion of NUP98-DDX10 corresponds to a major motif shared by DEAD-box RNA helicases that is required for their ATP binding/hydrolysis, RNA-binding, and helicase functions. Mutating this motif diminished the transforming ability of NUP98-DDX10, indicating that it plays a role in leukemogenesis. These data demonstrate for the first time the transforming ability of NUP98-DDX10 and show that it is partially dependent on one of the consensus helicase motifs of DDX10. They also point to common pathways that may underlie leukemogenesis by different NUP98 fusions. Disclosures: No relevant conflicts of interest to declare.
31
von Lindern, M., S. van Baal, J. Wiegant, A. Raap, A. Hagemeijer, and G. Grosveld. "Can, a putative oncogene associated with myeloid leukemogenesis, may be activated by fusion of its 3' half to different genes: characterization of the set gene." Molecular and Cellular Biology 12, no. 8 (August 1992): 3346–55. http://dx.doi.org/10.1128/mcb.12.8.3346.
Full textAbstract:
The translocation (6;9)(p23;q34) in acute nonlymphocytic leukemia results in the formation of a highly consistent dek-can fusion gene. Translocation breakpoints invariably occur in single introns of dek and can, which were named icb-6 and icb-9, respectively. In a case of acute undifferentiated leukemia, a breakpoint was detected in icb-9 of can, whereas no breakpoint could be detected in dek. Genomic and cDNA cloning showed that instead of dek, a different gene was fused to can, which was named set. set encodes transcripts of 2.0 and 2.7 kb that result from the use of alternative polyadenylation sites. Both transcripts contain the open reading frame for a putative SET protein with a predicted molecular mass of 32 kDa. The set-can fusion gene is transcribed into a 5-kb transcript that contains a single open reading frame predicting a 155-kDa chimeric SET-CAN protein. The SET sequence shows homology with the yeast nucleosome assembly protein NAP-I. The only common sequence motif of SET and DEK proteins is an acidic region. SET has a long acidic tail, of which a large part is present in the predicted SET-CAN fusion protein. The set gene is located on chromosome 9q34, centromeric of c-abl. Since a dek-can fusion gene is present in t(6;9) acute myeloid leukemia and a set-can fusion gene was found in a case of acute undifferentiated leukemia, we assume that can may function as an oncogene activated by fusion of its 3' part to dek, set, or perhaps other genes.
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von Lindern, M., S. van Baal, J. Wiegant, A. Raap, A. Hagemeijer, and G. Grosveld. "Can, a putative oncogene associated with myeloid leukemogenesis, may be activated by fusion of its 3' half to different genes: characterization of the set gene." Molecular and Cellular Biology 12, no. 8 (August 1992): 3346–55. http://dx.doi.org/10.1128/mcb.12.8.3346-3355.1992.
Full textAbstract:
The translocation (6;9)(p23;q34) in acute nonlymphocytic leukemia results in the formation of a highly consistent dek-can fusion gene. Translocation breakpoints invariably occur in single introns of dek and can, which were named icb-6 and icb-9, respectively. In a case of acute undifferentiated leukemia, a breakpoint was detected in icb-9 of can, whereas no breakpoint could be detected in dek. Genomic and cDNA cloning showed that instead of dek, a different gene was fused to can, which was named set. set encodes transcripts of 2.0 and 2.7 kb that result from the use of alternative polyadenylation sites. Both transcripts contain the open reading frame for a putative SET protein with a predicted molecular mass of 32 kDa. The set-can fusion gene is transcribed into a 5-kb transcript that contains a single open reading frame predicting a 155-kDa chimeric SET-CAN protein. The SET sequence shows homology with the yeast nucleosome assembly protein NAP-I. The only common sequence motif of SET and DEK proteins is an acidic region. SET has a long acidic tail, of which a large part is present in the predicted SET-CAN fusion protein. The set gene is located on chromosome 9q34, centromeric of c-abl. Since a dek-can fusion gene is present in t(6;9) acute myeloid leukemia and a set-can fusion gene was found in a case of acute undifferentiated leukemia, we assume that can may function as an oncogene activated by fusion of its 3' part to dek, set, or perhaps other genes.
33
Monica, K., N. Galili, J. Nourse, D. Saltman, and M. L. Cleary. "PBX2 and PBX3, new homeobox genes with extensive homology to the human proto-oncogene PBX1." Molecular and Cellular Biology 11, no. 12 (December 1991): 6149–57. http://dx.doi.org/10.1128/mcb.11.12.6149.
Full textAbstract:
Two new homeobox genes, PBX2 and PBX3, were isolated on the basis of their extensive homology to PBX1, a novel human homeobox gene involved in t(1;19) translocation in acute pre-B-cell leukemias. The predicted Pbx2 and Pbx3 proteins are 92 and 94% identical to Pbx1 over a large region of 266 amino acids within and flanking their homeodomains, but all three proteins diverge significantly near their amino and carboxy termini. Chromosome in situ hybridizations demonstrated that the PBX genes are not clustered but map to separate chromosomal loci: PBX1, 1q23; PBX2, 3q22-23; PBX3, 9q33-34. Expression of PBX2 or PBX3 was not restricted to particular states of differentiation or development, as mRNA transcripts of these genes were detected in most fetal and adult tissues and all cell lines, unlike PBX1, which is not expressed in lymphoid cell lines. Similar to PBX1 RNA, PBX3 RNA is alternatively spliced to yield two translation products with different carboxy termini, a feature not observed for PBX2. Their extensive sequence similarity and widespread expression suggest a generalized, overlapping role for Pbx proteins in most cell types. Differences in their amino and carboxy termini may modulate their activities, mediated in part by differential splicing and, for PBX1, protein fusion following t(1;19) chromosomal translocation.
34
Monica, K., N. Galili, J. Nourse, D. Saltman, and M. L. Cleary. "PBX2 and PBX3, new homeobox genes with extensive homology to the human proto-oncogene PBX1." Molecular and Cellular Biology 11, no. 12 (December 1991): 6149–57. http://dx.doi.org/10.1128/mcb.11.12.6149-6157.1991.
Full textAbstract:
Two new homeobox genes, PBX2 and PBX3, were isolated on the basis of their extensive homology to PBX1, a novel human homeobox gene involved in t(1;19) translocation in acute pre-B-cell leukemias. The predicted Pbx2 and Pbx3 proteins are 92 and 94% identical to Pbx1 over a large region of 266 amino acids within and flanking their homeodomains, but all three proteins diverge significantly near their amino and carboxy termini. Chromosome in situ hybridizations demonstrated that the PBX genes are not clustered but map to separate chromosomal loci: PBX1, 1q23; PBX2, 3q22-23; PBX3, 9q33-34. Expression of PBX2 or PBX3 was not restricted to particular states of differentiation or development, as mRNA transcripts of these genes were detected in most fetal and adult tissues and all cell lines, unlike PBX1, which is not expressed in lymphoid cell lines. Similar to PBX1 RNA, PBX3 RNA is alternatively spliced to yield two translation products with different carboxy termini, a feature not observed for PBX2. Their extensive sequence similarity and widespread expression suggest a generalized, overlapping role for Pbx proteins in most cell types. Differences in their amino and carboxy termini may modulate their activities, mediated in part by differential splicing and, for PBX1, protein fusion following t(1;19) chromosomal translocation.
35
Telegeev, Gennady D., Anna N. Dubrovska, Mykhaylo V. Dybkov, and Stanislav S. Maliuta. "Influence of BCR/ABL fusion proteins on the course of Ph leukemias." Acta Biochimica Polonica 51, no. 3 (September 30, 2004): 845–49. http://dx.doi.org/10.18388/abp.2004_3568.
Full textAbstract:
The hallmark of chronic myeloid leukemia (CML) and a subset of acute lymphoblastic leukemia (ALL) is the presence of the Philadelphia chromosome as a result of the t(9;22) translocation. This gene rearrangement results in the production of a novel oncoprotein, BCR/ABL, a constitutively active tyrosine kinase. There is compelling evidence that the malignant transformation by BCR/ABL is critically dependent on its Abl tyrosine kinase activity. Also the bcr part of the hybrid gene takes part in realization of the malignant phenotype. We supposed that additional mutations accumulate in this region of the BCR/ABL oncogene during the development of the malignant blast crisis in CML patients. In ALL patients having p210 fusion protein the mutations were supposed to be preexisting. Sequencing of PCR product of the BCR/ABL gene (Dbl, PH region) showed that along with single-nucleotide substitutions other mutations, mostly deletions, had occurred. In an ALL patient a deletion of the 5th exon was detected. The size of the deletions varied from 36 to 220 amino acids. For one case of blast crisis of CML changes in the character of actin organization were observed. Taking into account the functional role of these domains in the cell an etiological role of such mutations on the disease phenotype and leukemia progression is plausible.
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Bocchia, M., T. Korontsvit, Q. Xu, S. Mackinnon, SY Yang, A. Sette, and DA Scheinberg. "Specific human cellular immunity to bcr-abl oncogene-derived peptides." Blood 87, no. 9 (May 1, 1996): 3587–92. http://dx.doi.org/10.1182/blood.v87.9.3587.bloodjournal8793587.
Full textAbstract:
Chronic myelogenous leukemia (CML) cells are characterized by a t(9;22) translocation, which can encode one of two chimeric P210 bcr-abl fusion proteins, comprising products of either the b2a2 or the b3a2 exon junction. The junctional sequences represent potentially immunogenic tumor-specific antigens. Despite their intracellular location, the fusion proteins might be recognized immunologically by T lymphocytes if peptides, derived from these unique sequences, are capable of presentation by the major histocompatibility complex molecules. We previously found that four peptides, 9 to 11 amino acids long, spanning the b3a2 CML breakpoint bind with high or intermediate affinity to purified HLA class I molecules A3, A11, B8, or both A3 and A11. We tested the ability of these peptides to elicit specific class I restricted cytotoxic T lymphocytes (CTLs) in vitro in HLA-matched healthy donors. In addition, a longer b3a2 CML-breakpoint-derived peptide, 25 aminoacids in length (b3a2–25), was studied for its ability to induce peptide-specific, class II-mediated, T-cell proliferation. In four of four HLA-A3 donors tested, CML-A3/A11-peptide specific CTLs were induced that killed an allogeneic HLA-A3-matched peptide pulsed leukemia cell line. In two of three HLA-A3 donors, the CML-A3/A11 peptide was able to induce killing of autologous and allogeneic HLA- matched peptide-pulsed peripheral blood mononuclear cells (PBMC). CML- A3 peptide induced peptide specific CTLs in one of the four HLA A3 donors tested. No killing was observed in two HLA-B8 and two HLA-A11 donors. PBMC from seven donors were also tested for anti b3a2–25 peptide proliferation in a thymidine incorporation assay. Specific proliferation was detected in three donors, all of the HLA-DR11 haplotype. These data represent the first evidence of a cytolytic human immune response against CML bcr-abl oncogene-derived peptides and provide a rationale for developing peptide-based vaccines for this disease.
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Sehr, Peter, Michael Pawlita, and Joe Lewis. "Evaluation of Different Glutathione S-Transferase–Tagged Protein Captures for Screening E6/E6AP Interaction Inhibitors Using AlphaScreen®." Journal of Biomolecular Screening 12, no. 4 (March 22, 2007): 560–67. http://dx.doi.org/10.1177/1087057107301246.
Full textAbstract:
Human papillomavirus (HPV) infection is responsible for the development of cervical cancer and its premalignant lesions in women. The virus-encoded oncogene E6 is a promising target for an anti-HPV drug therapy. The authors describe the development of a homogenous screening assay for inhibitors of the E6 interaction with its cellular target, the E6-associated protein (E6AP), based on AlphaScreen® technology. The E6 protein was expressed and purified as glutathione S-transferase (GST) fusion protein, and the binding to a biotinylated E6AP peptide was monitored using GST-detecting Acceptor beads coated either with anti-GST antibody or glutathione. After optimization of the assay conditions, a commercial library of 3000 compounds was screened for inhibitors. Active compounds were retested and counterscreened for E6/E6AP specificity using biotinylated GST as a control protein. The results obtained with both types of GST-detecting reagents correlated very well and demonstrated the great potential of the newly developed glutathione-coated Acceptor beads as a detection reagent for GST fusion proteins. ( Journal of Biomolecular Screening 2007:560-567)
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Bigler, J., and R. N. Eisenman. "c-erbA encodes multiple proteins in chicken erythroid cells." Molecular and Cellular Biology 8, no. 10 (October 1988): 4155–61. http://dx.doi.org/10.1128/mcb.8.10.4155.
Full textAbstract:
To identify and characterize the proteins encoded by the erbA proto-oncogene, we expressed the C-terminal region of v-erbA in a bacterial trpE expression vector system and used the fusion protein to prepare antiserum. The anti-trp-erbA serum recognized the P75gag-erbA protein encoded by avian erythroblastosis virus and specifically precipitated six highly related proteins ranging in size from 27 to 46 kilodaltons from chicken embryonic erythroid cells. In vitro translation of a chicken erbA cDNA produced essentially the same pattern of proteins. Partial proteolytic maps and antigenicity and kinetic analyses of the in vivo and in vitro proteins indicated that they are related and that the multiple bands are likely to arise from internal initiations within c-erbA to generate a nested set of proteins. All of the c-erbA proteins are predominantly associated with chicken erythroblast nuclei. However, Nonidet P-40 treatment resulted in extraction of the three smaller proteins, whereas the larger proteins were retained. During differentiation of erythroid cells in chicken embryos, we found maximal levels of c-erbA protein synthesis at days 7 to 8 of embryogenesis. By contrast, c-erbA mRNA levels remained essentially constant from days 5 to 12. Together, our results indicate that posttranscriptional or translational mechanisms are involved in regulation of c-erbA expression and in the complexity of its protein products.
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Bigler, J., and R. N. Eisenman. "c-erbA encodes multiple proteins in chicken erythroid cells." Molecular and Cellular Biology 8, no. 10 (October 1988): 4155–61. http://dx.doi.org/10.1128/mcb.8.10.4155-4161.1988.
Full textAbstract:
To identify and characterize the proteins encoded by the erbA proto-oncogene, we expressed the C-terminal region of v-erbA in a bacterial trpE expression vector system and used the fusion protein to prepare antiserum. The anti-trp-erbA serum recognized the P75gag-erbA protein encoded by avian erythroblastosis virus and specifically precipitated six highly related proteins ranging in size from 27 to 46 kilodaltons from chicken embryonic erythroid cells. In vitro translation of a chicken erbA cDNA produced essentially the same pattern of proteins. Partial proteolytic maps and antigenicity and kinetic analyses of the in vivo and in vitro proteins indicated that they are related and that the multiple bands are likely to arise from internal initiations within c-erbA to generate a nested set of proteins. All of the c-erbA proteins are predominantly associated with chicken erythroblast nuclei. However, Nonidet P-40 treatment resulted in extraction of the three smaller proteins, whereas the larger proteins were retained. During differentiation of erythroid cells in chicken embryos, we found maximal levels of c-erbA protein synthesis at days 7 to 8 of embryogenesis. By contrast, c-erbA mRNA levels remained essentially constant from days 5 to 12. Together, our results indicate that posttranscriptional or translational mechanisms are involved in regulation of c-erbA expression and in the complexity of its protein products.
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Feldman, Brian J., Tracy Hampton, and Michael L. Cleary. "A carboxy-terminal deletion mutant of Notch1accelerates lymphoid oncogenesis in E2A-PBX1transgenic mice." Blood 96, no. 5 (September 1, 2000): 1906–13. http://dx.doi.org/10.1182/blood.v96.5.1906.
Full textAbstract:
Abstract PBX1 is a proto-oncogene that plays important roles in pattern formation during development. It was discovered as a fusion with the E2A gene after chromosomal translocations in a subset of acute leukemias. The resulting E2a-Pbx1 chimeric proteins display potent oncogenic properties that appear to require dimerization with Hox DNA binding partners. To define molecular pathways that may be impacted by E2a-Pbx1, a genetic screen consisting of neonatal retroviral infection was used to identify genes that accelerate development of T-cell tumors in E2A-PBX1 transgenic mice. Retroviral insertions in the Notch1 gene were observed in 88% of tumors arising with a shortened latency. Among these, approximately half created a NotchIC allele, encoding the intracellular, signaling portion of Notch1, suggesting a synergistic interaction between the Notch and E2a-Pbx1 pathways in oncogenesis. The remaining proviral insertions involvingNotch1 occurred in a more 3′ exon, resulting in truncating mutations that deleted the carboxy-terminal region ofNotch1 containing negative regulatory sequences (Notch1ΔC). In contrast toNotchIC, forced expression ofNotch1ΔC in transgenic mice did not perturb thymocyte growth or differentiation. However, mice transgenic for both the E2A-PBX1 and Notch1ΔC genes displayed a substantially shortened latency for tumor development compared with E2A-PBX1 single transgenic mice. These studies reveal a novel mechanism for oncogenic activation ofNotch1 and demonstrate a collaborative relationship between 2 cellular oncogenes that also contribute to cell fate determination during embryonic development.
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Feldman, Brian J., Tracy Hampton, and Michael L. Cleary. "A carboxy-terminal deletion mutant of Notch1accelerates lymphoid oncogenesis in E2A-PBX1transgenic mice." Blood 96, no. 5 (September 1, 2000): 1906–13. http://dx.doi.org/10.1182/blood.v96.5.1906.h8001906_1906_1913.
Full textAbstract:
PBX1 is a proto-oncogene that plays important roles in pattern formation during development. It was discovered as a fusion with the E2A gene after chromosomal translocations in a subset of acute leukemias. The resulting E2a-Pbx1 chimeric proteins display potent oncogenic properties that appear to require dimerization with Hox DNA binding partners. To define molecular pathways that may be impacted by E2a-Pbx1, a genetic screen consisting of neonatal retroviral infection was used to identify genes that accelerate development of T-cell tumors in E2A-PBX1 transgenic mice. Retroviral insertions in the Notch1 gene were observed in 88% of tumors arising with a shortened latency. Among these, approximately half created a NotchIC allele, encoding the intracellular, signaling portion of Notch1, suggesting a synergistic interaction between the Notch and E2a-Pbx1 pathways in oncogenesis. The remaining proviral insertions involvingNotch1 occurred in a more 3′ exon, resulting in truncating mutations that deleted the carboxy-terminal region ofNotch1 containing negative regulatory sequences (Notch1ΔC). In contrast toNotchIC, forced expression ofNotch1ΔC in transgenic mice did not perturb thymocyte growth or differentiation. However, mice transgenic for both the E2A-PBX1 and Notch1ΔC genes displayed a substantially shortened latency for tumor development compared with E2A-PBX1 single transgenic mice. These studies reveal a novel mechanism for oncogenic activation ofNotch1 and demonstrate a collaborative relationship between 2 cellular oncogenes that also contribute to cell fate determination during embryonic development.
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Hempstead, B. L., R. B. Birge, J. E. Fajardo, R. Glassman, D. Mahadeo, R. Kraemer, and H. Hanafusa. "Expression of the v-crk oncogene product in PC12 cells results in rapid differentiation by both nerve growth factor- and epidermal growth factor-dependent pathways." Molecular and Cellular Biology 14, no. 3 (March 1994): 1964–71. http://dx.doi.org/10.1128/mcb.14.3.1964.
Full textAbstract:
The transforming gene of the avian sarcoma virus CT10 encodes a fusion protein (p47gag-crk or v-Crk) containing viral Gag sequences fused to cellular sequences consisting primarily of Src homology regions 2 and 3 (SH2 and SH3 sequences). Here we report a novel function of v-Crk in the mammalian pheochromocytoma cell line, PC12, whereby stable expression of v-Crk induces accelerated differentiation, as assessed by induction of neurites following nerve growth factor (NGF) or basic fibroblast growth factor (bFGF) treatment compared with the effect in native PC12 cells. Surprisingly, however, these cells also develop extensive neurite processes after epidermal growth factor (EGF) stimulation, an event which is not observed in native PC12 cells. Following EGF or NGF stimulation of the v-CrkPC12 cells, the v-Crk protein itself became tyrosine phosphorylated within 1 min. Moreover, in A431 cells or TrkA-PC12 cells, which overexpress EGF receptors and TrkA, respectively, a GST-CrkSH2 fusion protein was indeed capable of binding these receptors in a phosphotyrosine-dependent manner, suggesting that v-Crk can directly couple to receptor tyrosine kinase pathways in PC12 cells. In transformed fibroblasts, v-Crk binds to specific tyrosine-phosphorylated proteins of p130 and paxillin. Both of these proteins are also complexed to v-Crk in PC12 cells, as evidenced by their coprecipitation with v-Crk in detergent lysates, suggesting that common effector pathways may occur in both cell types. However, whereas PC12 cellular differentiation can occur solely by overexpression of the v-Src or oncogenic Ras proteins, that induced by v-Crk requires a growth factor stimulatory signal, possibility in a two-step process.
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Hempstead, B. L., R. B. Birge, J. E. Fajardo, R. Glassman, D. Mahadeo, R. Kraemer, and H. Hanafusa. "Expression of the v-crk oncogene product in PC12 cells results in rapid differentiation by both nerve growth factor- and epidermal growth factor-dependent pathways." Molecular and Cellular Biology 14, no. 3 (March 1994): 1964–71. http://dx.doi.org/10.1128/mcb.14.3.1964-1971.1994.
Full textAbstract:
The transforming gene of the avian sarcoma virus CT10 encodes a fusion protein (p47gag-crk or v-Crk) containing viral Gag sequences fused to cellular sequences consisting primarily of Src homology regions 2 and 3 (SH2 and SH3 sequences). Here we report a novel function of v-Crk in the mammalian pheochromocytoma cell line, PC12, whereby stable expression of v-Crk induces accelerated differentiation, as assessed by induction of neurites following nerve growth factor (NGF) or basic fibroblast growth factor (bFGF) treatment compared with the effect in native PC12 cells. Surprisingly, however, these cells also develop extensive neurite processes after epidermal growth factor (EGF) stimulation, an event which is not observed in native PC12 cells. Following EGF or NGF stimulation of the v-CrkPC12 cells, the v-Crk protein itself became tyrosine phosphorylated within 1 min. Moreover, in A431 cells or TrkA-PC12 cells, which overexpress EGF receptors and TrkA, respectively, a GST-CrkSH2 fusion protein was indeed capable of binding these receptors in a phosphotyrosine-dependent manner, suggesting that v-Crk can directly couple to receptor tyrosine kinase pathways in PC12 cells. In transformed fibroblasts, v-Crk binds to specific tyrosine-phosphorylated proteins of p130 and paxillin. Both of these proteins are also complexed to v-Crk in PC12 cells, as evidenced by their coprecipitation with v-Crk in detergent lysates, suggesting that common effector pathways may occur in both cell types. However, whereas PC12 cellular differentiation can occur solely by overexpression of the v-Src or oncogenic Ras proteins, that induced by v-Crk requires a growth factor stimulatory signal, possibility in a two-step process.
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RIBON, Vered, and Alan R. SALTIEL. "Insulin stimulates tyrosine phosphorylation of the proto-oncogene product of c-Cbl in 3T3-L1 adipocytes." Biochemical Journal 324, no. 3 (June 15, 1997): 839–46. http://dx.doi.org/10.1042/bj3240839.
Full textAbstract:
We report here that the product of the c-Cbl proto-oncogene is prominently tyrosine phosphorylated in response to insulin in 3T3-L1 adipocytes. The tyrosine phosphorylation of c-Cbl reaches a maximum within 1–2 min after stimulation by insulin and gradually declines thereafter. The tyrosine phosphorylation of c-Cbl was also observed after treatment of 3T3-L1 adipocytes with epidermal growth factor, whereas platelet-derived growth factor had no effect. After insulin-dependent tyrosine phosphorylation, c-Cbl specifically associates with fusion proteins containing the Src homology 2 (SH2) domains of Crk and the Fyn tyrosine kinase, but not with fusion proteins containing the SH2 domains of either the p85 subunit of phosphatidylinositol 3′-kinase or the tyrosine phosphatase SHPTP2/Syp. Furthermore insulin stimulates the association of c-Cbl with endogenous c-Crk and Fyn in intact 3T3-L1 adipocytes. The tyrosine phosphorylation of c-Cbl is regulated during adipocyte differentiation. Neither insulin-like growth factor 1 nor insulin stimulated the tyrosine phosphorylation of c-Cbl in 3T3-L1 fibroblasts. Moreover, c-Cbl is not tyrosine phosphorylated in response to insulin in cells expressing high levels of the human insulin receptor, or in hepatocytes, despite comparable levels of c-Cbl expression. These results suggest that c-Cbl might have a novel function in the regulation of insulin receptor intracellular signalling in 3T3-L1 adipocytes.
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Slany, Robert K., Claudia B. Bittner, Deniz T. Zeisig, Christian Bach, and Jay L. Hess. "c-Myb Is an Essential Downstream Target for Homeobox Mediated Transformation of Hematopoietic Cells." Blood 106, no. 11 (November 16, 2005): 666. http://dx.doi.org/10.1182/blood.v106.11.666.666.
Full textAbstract:
Abstract Overexpression of Hox genes is frequently associated with leukemogenic transformation of hematopoietic cells. In particular the abdominal-type HoxA genes together with their homeodomain containing dimerization partners of the TALE (three amino acid loop extension) family are well documented oncogenes in various hematologic malignancies. Paradigmatic examples are Hoxa9 and Meis1 that are coactivated by retroviral insertions in the BXH2 mouse leukemia model. In addition Hoxa9 is a fusion partner of NUP98 in acute myeloid leukemias with a t(7;11) and Meis1 as well as Hoxa9 are critical targets of MLL (mixed lineage leukemia) fusion proteins. Despite their important role in leukemogenesis little is known how they exert their transforming function. Here we used a novel system of transient transformation by an inducible oncoprotein to generate populations of primary hematopoietic cells that overexpress Hoxa9 in combination with Meis1. The RNA inventory of these cells was compared to control cells by Affymetrix array analysis. Interestingly the RNA of c-Myb (myeloblastosis leukemia virus oncogene) and of a known c-myb downstream target (Gstm1) was amongst the top ten genes upregulated by Hoxa9/Meis1. c-Myb overexpression was verified by Northern blot and quantitative RT-PCR. c-Myb levels also responded to the introduction of MLL-ENL, an oncoprotein that upregulates Hoxa9 and Meis1. Small interfering RNAs directed against c-Myb suppressed transformation by MLL-ENL but had no effect on transformation of hematopoietic precursors by E2A-HLF an oncogene that does not work by increasing Hoxa9/Meis1 levels. The anti c-Myb siRNA effect was abrogated by coexpression of a c-Myb derivative with a mutated siRNA target site. The coexpression of a dominant negative c-Myb mutant had a similar but weaker effect on MLL-ENL mediated transformation. Ectopic expression of c-Myb in hematopoietic precursor cells induced a mild differentiation block. However, c-Myb alone was not able to give a positive readout in a serial replating assay under conditions where the combined overexpression of Hoxa9 together with Meis1 was clearly transforming. In summary the results suggest that c-Myb is essential but not sufficient for the oncogenic activity of Hoxa9/Meis1.
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Smith, Lan-Lan, Jenny Yeung, Bernd B. Zeisig, Ivo Huijbers, Nik Popov, Josephine Barnes, Amanda J. Wilson, et al. "Hoxa9 Suppresses Cellular Senescence and Sustains the Development of Leukemic Stem Cells Induced by Fusion Proteins In the Absence of Bmi1." Blood 116, no. 21 (November 19, 2010): 1578. http://dx.doi.org/10.1182/blood.v116.21.1578.1578.
Full textAbstract:
Abstract Abstract 1578 While self renewal is an essential feature for the maintenance of both normal hematopoietic stem cells (HSCs) and leukemic stem cells (LSCs), very little is known about the underlying molecular pathways. Here we report a critical functional interplay between Bmi1 and Hox in establishment of HSCs and LSCs. Using Bmi1-/- bone marrow cells, we observe that leukemia-associated fusion proteins have distinctive Bmi1 requirements. AML1-ETO (AE) and PLZF-RARα (PR) fail to transform Bmi1-/- primary hematopoietic cells, and induce expression of p16/Arf leading to oncogene-induced senescence (OIS). In contrast, MLL-AF9 driving expression of multiple Hox genes can bypass oncogene-induced senescence and exhibits modest Bmi1-dependence for establishment of LSCs, which can induce leukemia upon serial transplants. Since members of Hox genes with proclaimed self-renewal property are specifically up-regulated by MLL fusions in patient samples and our murine models, we asked the question if these Hox genes may partly compensate the functions associated with the loss of Bmi1. To this end, we generated compound Bmi1-/-Hoxa9-/- mice, which have even more compromised hematopoietic stem cell/progenitor compartments than those of Bmi1-/- or Hoxa9-/- mice. Bmi1-/-Hoxa9-/- mice have a greater than eight-fold reduction in the absolute number of Lin-Sca+kit+ (LSK) in the bone marrow as compared to Bmi1-/- mice and a very significant forty-fold reduction for long term hematopoietic stem cells (LT-HSC). More importantly, while MAF9 is able to transform wild type, Bmi1-/- and Hoxa9-/-, it fails to transform Bmi1-/-Hoxa9-/- cells for establishment of LSCs, which can however be resurrected by re-expression of either Bmi1 or Hoxa9, indicating a critical functional interplay between these protein in development of MLL LSCs. Consistent with the known function of Bmi1 in suppressing cellular senescence and the expression of p16/Arf loci, we showed that Hoxa9 alone can also inhibit replicative senescence and Ras-induced senescence in primary human fibroblast. Forced expression of Hoxa9 can suppress p16/Arf expression, as well as cellular senescence induced by AE and PR in Bmi1-/- cells. Together, these results reveal a previously unrecognized functional interplay between Hox and Bmi1 in regulating cell senescence and development of LSCs induced by fusion proteins, which also suggests that synergistic targeting of both molecules may be required for certain LSCs. Disclosures: No relevant conflicts of interest to declare.
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Brown, Geoffrey, and Kevin Petrie. "The RARγ Oncogene: An Achilles Heel for Some Cancers." International Journal of Molecular Sciences 22, no. 7 (March 31, 2021): 3632. http://dx.doi.org/10.3390/ijms22073632.
Full textAbstract:
Cancer “stem cells” (CSCs) sustain the hierarchies of dividing cells that characterize cancer. The main causes of cancer-related mortality are metastatic disease and relapse, both of which originate primarily from CSCs, so their eradication may provide a bona fide curative strategy, though there maybe also the need to kill the bulk cancer cells. While classic anti-cancer chemotherapy is effective against the dividing progeny of CSCs, non-dividing or quiescent CSCs are often spared. Improved anti-cancer therapies therefore require approaches that target non-dividing CSCs, which must be underpinned by a better understanding of factors that permit these cells to maintain a stem cell-like state. During hematopoiesis, retinoic acid receptor (RAR) γ is selectively expressed by stem cells and their immediate progeny. It is overexpressed in, and is an oncogene for, many cancers including colorectal, renal and hepatocellular carcinoma, cholangiocarcinomas and some cases of acute myeloid leukemia that harbor RARγ fusion proteins. In vitro studies suggest that RARγ-selective and pan-RAR antagonists provoke the death of CSCs by necroptosis and point to antagonism of RARγ as a potential strategy to treat metastatic disease and relapse, and perhaps provide a cure for some cancers.
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Chou, M. M., J. E. Fajardo, and H. Hanafusa. "The SH2- and SH3-containing Nck protein transforms mammalian fibroblasts in the absence of elevated phosphotyrosine levels." Molecular and Cellular Biology 12, no. 12 (December 1992): 5834–42. http://dx.doi.org/10.1128/mcb.12.12.5834.
Full textAbstract:
We have established the human nck sequence as a new oncogene. Nck encodes one SH2 and three SH3 domains, the Src homology motifs found in nonreceptor tyrosine kinases, Ras GTPase-activating protein, phosphatidylinositol 3-kinase, and phospholipase C-gamma. Overexpression of human nck in 3Y1 rat fibroblasts results in transformation as judged by alteration of cell morphology, colony formation in soft agar, and tumor formation in nude BALB/c mice. However, overexpression of nck does not induce detectable elevation of the phosphotyrosine content of specific proteins, as is observed for v-crk, another SH2/SH3-containing oncogene. Despite this fact, we demonstrate that Nck retains the ability to bind tyrosine phosphorylated proteins in vitro, using a fusion protein of Nck with glutathione-S-transferase (GST). Moreover, when incubated with lysates prepared from v-src-transformed 3Y1 cells or the nck-overexpressing cell lines, GST-Nck binds to both p60v-src and serine/threonine kinases, respectively. Although phosphotyrosine levels are not elevated in the nck-expressing fibroblasts, vanadate treatment of these cells results in a phosphotyrosine pattern that is altered from the parental 3Y1 pattern, suggestive of a perturbation of indigenous tyrosine kinase pathways. These results suggest the possibility that human nck induces transformation in 3Y1 fibroblasts by virtue of its altered affinity or specificity for the normal substrates of its rat homolog and that Nck may play a role in linking tyrosine and serine/threonine kinase pathways within the cell.
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Chou, M. M., J. E. Fajardo, and H. Hanafusa. "The SH2- and SH3-containing Nck protein transforms mammalian fibroblasts in the absence of elevated phosphotyrosine levels." Molecular and Cellular Biology 12, no. 12 (December 1992): 5834–42. http://dx.doi.org/10.1128/mcb.12.12.5834-5842.1992.
Full textAbstract:
We have established the human nck sequence as a new oncogene. Nck encodes one SH2 and three SH3 domains, the Src homology motifs found in nonreceptor tyrosine kinases, Ras GTPase-activating protein, phosphatidylinositol 3-kinase, and phospholipase C-gamma. Overexpression of human nck in 3Y1 rat fibroblasts results in transformation as judged by alteration of cell morphology, colony formation in soft agar, and tumor formation in nude BALB/c mice. However, overexpression of nck does not induce detectable elevation of the phosphotyrosine content of specific proteins, as is observed for v-crk, another SH2/SH3-containing oncogene. Despite this fact, we demonstrate that Nck retains the ability to bind tyrosine phosphorylated proteins in vitro, using a fusion protein of Nck with glutathione-S-transferase (GST). Moreover, when incubated with lysates prepared from v-src-transformed 3Y1 cells or the nck-overexpressing cell lines, GST-Nck binds to both p60v-src and serine/threonine kinases, respectively. Although phosphotyrosine levels are not elevated in the nck-expressing fibroblasts, vanadate treatment of these cells results in a phosphotyrosine pattern that is altered from the parental 3Y1 pattern, suggestive of a perturbation of indigenous tyrosine kinase pathways. These results suggest the possibility that human nck induces transformation in 3Y1 fibroblasts by virtue of its altered affinity or specificity for the normal substrates of its rat homolog and that Nck may play a role in linking tyrosine and serine/threonine kinase pathways within the cell.
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Isnard, Patricia, Nathalie Coré, Philippe Naquet, and Malek Djabali. "Altered lymphoid development in mice deficient for the mAF4 proto-oncogene." Blood 96, no. 2 (July 15, 2000): 705–10. http://dx.doi.org/10.1182/blood.v96.2.705.
Full textAbstract:
Abstract Some chromosomal translocations in acute leukemias involve the fusion of the trithorax-related protein Mll (also called HRX, All1, Htrx,) with a variety of heterologous proteins. In acute lymphoblastic leukemia associated with the t(4;11)(q21;q23) translocation, the4q21 gene that fuses with Mll is AF4. To gain insight into the potential role of AF4 in leukemogenesis and development, this gene was inactivated by homologous recombination in mice. As expected from the tissue distribution of the AF4 transcript, development of both B and T cells is affected in AF4 mutant mice. A severe reduction of the thymic double positive CD4/CD8 (CD4+/CD8+) population was observed; in addition most double- and single-positive cells expressed lower levels of CD4 and CD8 coreceptors. Most importantly, the reconstitution of the double-positive compartment by expansion of the double-negative cell compartment was severely impaired in these mutant mice. In the bone marrow pre-B and mature B-cell numbers are reduced. These results demonstrate that the function of the mAF4 gene is critical for normal lymphocyte development. This raises the possibility that the disruption of the normal AF4 gene or its association with Mll function by translocation may orient the oncogenic process toward the lymphoid lineage. This represents the first functional study using a knock-out strategy on one of the Mll partner genes in translocation-associated leukemias.