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Gastrointestinal stromal tumors (GISTs) are typically characterized by activating mutations of the KIT proto-oncogene receptor tyrosine kinase (KIT) or platelet-derived growth factor receptor alpha (PDGFRA). Recently, the neurotrophic tyrosine receptor kinase (NTRK) fusion was reported in a small subset of wild-type GIST. We examined trk IHC and NTRK gene expressions in GIST. Pan-trk immunohistochemistry (IHC) was positive in 25 (all 16 duodenal and 9 out of 16 small intestinal GISTs) of 139 cases, and all pan-trk positive cases showed diffuse and strong expression of c-kit. Interestingly, all of these cases showed only trkB but not trkA/trkC expression. Cap analysis of gene expression (CAGE) analysis identified increased number of genes whose promoters were activated in pan-trk/trkB positive GISTs. Imbalanced expression of NTRK2, which suggests the presence of NTRK2 fusion, was not observed in any of trkB positive GISTs, despite higher mRNA expression. TrkB expression was found in duodenal GISTs and more than half of small intestinal GISTs, and this subset of cases showed poor prognosis. However, there was not clear difference in clinical outcomes according to the trkB expression status in small intestinal GISTs. These findings may provide a possible hypothesis for trkB overexpression contributing to the tumorigenesis and aggressive clinical outcome in GISTs of duodenal origin., (© 2024. The Author(s).)

A number of studies have reported frequent incidence of c-kit gene mutations in association with core binding factor acute myeloid leukemia (CBF-AML). These genetic changes have become important prognostic predictors in patients with abnormal karyotype. Aim of this study was the detection of nucleotide alterations in newly diagnosed acute myeloid leukemia patients for three exons of c-kit gene, including cytogenetically normal patients. Thirty-one de novo AML patients were screened for any possible variations in exon 8, 11 and 17 sequences of c-kit proto-oncogene leading to amino acid substitutions or frame shift. Sanger sequencing method was employed followed by sequence analysis. Mutation data was then correlated with clinical and hematological parameters of patients and prognostic significance of genetic changes was assessed as well. The computational tools were then used to further understand the extent of damage caused by these mutations to c-kit protein. Fifteen (48.4%) mutant patients were observed with single, double or multiple mutations in one, two or all three exons studied. The analysis revealed eight new alterations which were not reported previously. Significant variation among mutant and non-mutant group of patients was observed with respect to FAB subtypes (x 2  = 12.524, p = 0.029), Spleen size (x 2  = 4.288, p = 0.038) and Red blood cell count (x 2  = 8.447, p = 0.007). The survival analysis indicates poor overall and event free survival outcomes in mutant individuals. Furthermore, the in silico analysis suggests that changes in nucleotide sequences can possibly damage the protein structure and effect it's function. This study emphasizes the need to consider screening of c-kit gene alterations not only in CBF-AML but in cytogenetically normal AML patients as well. In current investigation the effect of mutation Arg420Gly on structure and function of c-kit protein was investigated, as this was the most observed substitution in present cohort. Various bioinformatics tools and techniques were employed, which determined that Arg420Gly is possibly non-pathogenic mutation., (© 2022. The Author(s).)

Combination therapies or multi-targeted drugs have been pointed out as an option to prevent the emergence of resistant clones, which could make long-term treatment more effective and translate into better clinical outcomes for cancer patients. The NT157 compound is a synthetic tyrphostin that leads to long-term inhibition of IGF1R/IRS1-2-, STAT3- and AXL-mediated signaling pathways. Given the importance of these signaling pathways for the development and progression of lung cancer, this disease becomes an interesting model for generating preclinical evidence on the cellular and molecular mechanisms underlying the antineoplastic activity of NT157. In lung cancer cells, exposure to NT157 decreased, in a dose-dependent manner, cell viability, clonogenicity, cell cycle progression and migration, and induced apoptosis (p < 0.05). In the molecular scenario, NT157 reduced expression of IRS1 and AXL and phosphorylation of p38 MAPK, AKT, and 4EBP1. Besides, NT157 decreased expression of oncogenes BCL2, CCND1, MYB, and MYC and increased genes related to cellular stress and apoptosis, JUN, BBC3, CDKN1A, CDKN1B, FOS, and EGR1 (p < 0.05), favoring a tumor-suppressive cell signaling network in the context of lung cancer. Of note, JNK was identified as a key kinase for NT157-induced IRS1 and IRS2 phosphorylation, revealing a novel axis involved in the mechanism of action of the drug. NT157 also presented potentiating effects on EGFR inhibitors in lung cancer cells. In conclusion, our preclinical findings highlight NT157 as a putative prototype of a multitarget drug that may contribute to the antineoplastic arsenal against lung cancer., (© 2022. The Author(s).)

Myeloid epithelial reproductive proto-oncogene tyrosine kinase (MERTK) plays an essential role in modulating cancer immune tolerance by regulating macrophage efferocytosis. Studies are underway to develop small-molecule chemicals that inhibit MERTK as cancer immunotherapeutic agents, but these efforts are in their early stages. This study identified BMS794833, whose primary targets are MET and VEGFR2, as a potent MERTK inhibitor and developed a real-time efferocytosis monitoring system. The X-ray cocrystal structure revealed that BMS794833 was in contact with the ATP-binding pocket and the allosteric back pocket, rendering MERTK inactive. Homogeneous time-resolved fluorescence kinetic and Western blotting analyses showed that BMS794833 competitively inhibited MERTK activity in vitro and inhibited the autophosphorylation of MERTK in macrophages. We developed a system to monitor MERTK-dependent efferocytosis in real time, and using this system, we confirmed that BMS794833 significantly inhibited the efferocytosis of differentiated macrophages. Finally, BMS794833 significantly inhibited efferocytosis in vivo in a mouse model. These data show that BMS794833 is a type II MERTK inhibitor that regulates macrophage efferocytosis. In addition, the real-time efferocytosis monitoring technology developed in this study has great potential for future applications., (© 2022. The Author(s).)

In spite of extensive research and advances on the molecular biology of melanoma, the process of melanocytic differentiation or its relationship with proliferation is poorly understood. The role of proto-oncogenes in normal melanocyte biology is also intriguing. Proto-oncogene MYC is overexpressed in 40% of melanomas. It has been suggested that MYC can mediate senescence bypass in malignant melanocytes, an important event in melanoma development, likely in cooperation with other oncogenic pathways. However, despite the apparent importance of MYC in melanoma, its functions in normal melanocytes are unknown. We have overexpressed MYC in freshly isolated human primary melanocytes and studied the effects on melanocytic proliferation and differentiation. MYC promoted a transient activation of melanocytes including cell cycle entry, DNA damage and cell migration. Subsequently, MYC induced melanogenesis, increased cellular size and complexity and senescence. Interestingly, we also found strong expression of MYC in regions of human nevi displaying high pigmentation and high expression of senescence marker p16. The results altogether show that MYC drives melanocytic differentiation and suggest that senescence is associated with differentiation. We discuss the implications into the mechanisms governing melanocytic differentiation and the development of melanoma., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The copy number (CN) gain of protooncogenes is a frequent finding in gastric carcinoma (GC), but its prognostic implication remains elusive. The study aimed to characterize the clinicopathological features, including prognosis, of GCs with copy number gains in multiple protooncogenes. Three hundred thirty-three patients with advanced GC were analyzed for their gene ratios in EGFR, GATA6, IGF2, and SETDB1 using droplet dPCR (ddPCR) for an accurate assessment of CN changes in target genes. The number of GC patients with 3 or more genes with CN gain was 16 (4.8%). Compared with the GCs with 2 or less genes with CN gain, the GCs with 3 or more CN gains displayed more frequent venous invasion, a lower density of tumor-infiltrating lymphocytes, and lower methylation levels of L1 or SAT-alpha. Microsatellite instability-high tumors or Epstein-Barr virus-positive tumors were not found in the GCs with 3 or more genes with CN gain. Patients of this groups also showed the worst clinical outcomes for both overall survival and recurrence-free survival, which was persistent in the multivariate survival analyses. Our findings suggest that the ddPCR-based detection of multiple CN gain of protooncogenes might help to identify a subset of patients with poor prognosis.

Ribosome profiling revealed widespread translational activity at upstream open reading frames (uORFs) and validated uORF-mediated translational control as a commonly repressive mechanism of gene expression. Translational activation of proto-oncogenes through loss-of-uORF mutations has been demonstrated, yet a systematic search for cancer-associated genetic alterations in uORFs is lacking. Here, we applied a PCR-based, multiplex identifier-tagged deep sequencing approach to screen 404 uORF translation initiation sites of 83 human tyrosine kinases and 49 other proto-oncogenes in 308 human malignancies. We identified loss-of-function uORF mutations in EPHB1 in two samples derived from breast and colon cancer, and in MAP2K6 in a sample of colon adenocarcinoma. Both mutations were associated with enhanced translation, suggesting that loss-of-uORF-mediated translational induction of the downstream main protein coding sequence may have contributed to carcinogenesis. Computational analysis of whole exome sequencing datasets of 464 colon adenocarcinomas subsequently revealed another 53 non-recurrent somatic mutations functionally deleting 22 uORF initiation and 31 uORF termination codons, respectively. These data provide evidence for somatic mutations affecting uORF initiation and termination codons in human cancer. The insufficient coverage of uORF regions in current whole exome sequencing datasets demands for future genome-wide analyses to ultimately define the contribution of uORF-mediated translational deregulation in oncogenesis.

Human hepatocellular carcinomas (HCCs) expressing the biliary/hepatic progenitor cell marker keratin 19 (K19) have been linked with a poor prognosis and exhibit an increase in platelet-derived growth factor receptor α (PDGFRα) and laminin beta 1 (LAMB1) expression. PDGFRα has been reported to induce de novo synthesis of LAMB1 protein in a Sjogren syndrome antigen B (La/SSB)-dependent manner in a murine metastasis model. However, the role of this cascade in human HCC remains unclear. This study focused on the functional role of the PDGFRα-La/SSB-LAMB1 pathway and its molecular link to K19 expression in human HCC. In surgical HCC specimens from a cohort of 136 patients, PDGFRα expression correlated with K19 expression, microvascular invasion and metastatic spread. In addition, PDGFRα expression in pre-operative needle biopsy specimens predicted poor overall survival during a 5-year follow-up period. Consecutive histological staining demonstrated that the signaling components of the PDGFRα-La/SSB-LAMB1 pathway were strongly expressed at the invasive front. K19-positive HCC cells displayed high levels of α2β1 integrin (ITG) receptor, both in vitro and in vivo. In vitro activation of PDGFRα signaling triggered the translocation of nuclear La/SSB into the cytoplasm, enhanced the protein synthesis of LAMB1 by activating its internal ribosome entry site, which in turn led to increased secretion of laminin-111. This effect was abrogated by the PDGFRα-specific inhibitor crenolanib. Importantly LAMB1 stimulated ITG-dependent focal adhesion kinase/Src proto-oncogene non-receptor tyrosine kinase signaling. It also promoted the ITG-specific downstream target Rho-associated coiled-coil containing protein kinase 2, induced K19 expression in an autocrine manner, invadopodia formation and cell invasion. Finally, we showed that the knockdown of LAMB1 or K19 in subcutaneous xenograft mouse models resulted in significant loss of cells invading the surrounding stromal tissue and reduced HepG2 colonization into lung and liver after tail vein injection. The PDGFRα-LAMB1 pathway supports tumor progression at the invasive front of human HCC through K19 expression.

Retroviral gene therapy offers immense potential to treat many genetic diseases and has already shown efficacy in clinical trials. However, retroviral vector mediated genotoxicity remains a major challenge and clinically relevant approaches to reduce integration near genes and proto-oncogenes are needed. Foamy retroviral vectors have several advantages over gammaretroviral and lentiviral vectors including a potentially safer integration profile and a lower propensity to activate nearby genes. Here we successfully retargeted foamy retroviral vectors away from genes and into satellite regions enriched for trimethylated histone H3 at lysine 9 by modifying the foamy virus Gag and Pol proteins. Retargeted foamy retroviral vectors integrated near genes and proto-oncogenes less often (p < 0.001) than controls. Importantly, retargeted foamy retroviral vectors can be produced at high, clinically relevant titers (>10 7 transducing units/ml), and unlike other reported retargeting approaches engineered target cells are not needed to achieve retargeting. As proof of principle for use in the clinic we show efficient transduction and retargeting in human cord blood CD34 + cells. The modified Gag and Pol helper constructs we describe will allow any investigator to simply use these helper plasmids during vector production to retarget therapeutic foamy retroviral vectors.

Relapse of chronic myeloid leukemia (CML) is triggered by stem cells with a reconstituting capacity similar to that of hematopoietic stem cells (HSCs) and CML stem cells are a source of resistance in drug therapy with tyrosine kinase inhibitors (TKIs). Ecotropic viral integration site 1 (EVI1), a key transcription factor in HSC regulation, is known to predict poor outcomes in myeloid malignancies, however, incapability of prospective isolation of EVI1-high leukemic cells precludes the functional evaluation of intraindividual EVI1-high cells. Introduction of CML into Evi1-internal ribosomal entry site (IRES)-green fluorescent protein (GFP) knock-in mice, a versatile HSC-reporter strain, enables us to separate Evi1-high CML cells from the individual. Evi1-IRES-GFP allele models of CML in chronic phase (CML-CP), by retroviral overexpression of BCR-ABL and by crossing BCR-ABL transgenic mice, revealed that Evi1 is predominantly enriched in the stem cell fraction and associated with an enhanced proliferative as well as a leukemia-initiating capacity and that Evi1-high CML-CP cells exhibit resistance to TKIs. Overexpressing BCR-ABL and NUP98-HOXA9 in Evi1-IRES-GFP knock-in mice to model CML in blast crisis (CML-BC), in which Evi1-high cells turned to be a major population as opposed to a minor population in CML-CP models, showed that Evi1-high CML-BC cells have a greater potential to recapitulate the disease and appear resistant to TKIs. Furthermore, given that Evi1 heterozygosity ameliorates CML-CP and CML-BC development and that the combination of Evi1 and BCR-ABL causes acute myeloid leukemia resembling CML-BC, Evi1 could regulate CML development as a potent driver. In addition, in human CML-CP cases, we show that EVI1 is highly expressed in stem cell-enriched CD34+CD38-CD90+ fraction at single-cell level. This is the first report to clarify directly that Evi1-high leukemic cells themselves possess the superior potential to Evi1-low cells in oncogenic self-renewal, which highlights the role of Evi1 as a valuable and a functional marker of CML stem cells.

Activation of NRas signaling is frequently found in human myeloid leukemia and can be induced by activating mutations as well as by mutations in receptors or signaling molecules upstream of NRas. To study NRas-induced leukemogenesis, we retrovirally overexpressed wild-type NRas in a murine bone marrow transplantation (BMT) model in C57BL/6J mice. Overexpression of wild-type NRas caused myelomonocytic leukemias ∼3 months after BMT in the majority of mice. A subset of mice (30%) developed malignant histiocytosis similar to mice that received mutationally activated NRas(G12D)-expressing bone marrow. Aberrant Ras signaling was demonstrated in cells expressing mutationally active or wild-type NRas, as increased activation of Erk and Akt was observed in both models. However, more NRas(G12D) were found to be in the activated, GTP-bound state in comparison with wild-type NRas. Consistent with observations reported for primary human myelomonocytic leukemia cells, Stat5 activation was also detected in murine leukemic cells. Furthermore, clonal evolution was detected in NRas wild-type-induced leukemias, including expansion of clones containing activating vector insertions in known oncogenes, such as Evi1 and Prdm16. In vitro cooperation of NRas and Evi1 improved long-term expansion of primary murine bone marrow cells. Evi1-positive cells upregulated Bcl-2 and may, therefore, provide anti-apoptotic signals that collaborate with the NRas-induced proliferative effects. As activation of Evi1 has been shown to coincide with NRAS mutations in human acute myeloid leukemia, our murine model recapitulates crucial events in human leukemogenesis.

Although the significance of cathepsin G (CTSG) in host defense has been intensively investigated, little is known about its potential roles in granulopoiesis or leukemogenesis. We report here that CTSG is directly targeted and suppressed by AML1-ETO in t(8;21) acute myeloid leukemia (AML). Luciferase assays demonstrate that the CTSG promoter is strongly transactivated by AML1 and the AML1-dependent transactivation is suppressed by AML1-ETO. We also define a novel regulatory mechanism by which AML1-ETO-mediated transrepression requires both AML1-ETO and AML1 binding at adjacent sites, instead of the replacement of AML1 by AML1-ETO, and wild-type AML1 binding is a prerequisite for the repressive effect caused by AML1-ETO. Further evidence shows that CTSG, as a hematopoietic serine protease, can degrade AML1-ETO both in vitro and in vivo. Restoration of CTSG induces partial differentiation, growth inhibition and apoptosis in AML1-ETO-positive cells. In addition to t(8;21) AML, CTSG downregulation is observed in AML patients with other cytogenetic/genetic abnormalities that potentially interrupt normal AML1 function, that is, inv(16) and EVI1 overexpression. Thus, the targeting and suppression of CTSG by AML1-ETO in t(8;21) AML may provide a mechanism for leukemia cells to escape from the intracellular surveillance system by preventing degradation of foreign proteins.

B-cell leukemia 3 (Bcl-3) is a member of the inhibitor of κB family, which regulates a wide range of biological processes by functioning as a transcriptional activator or as a repressor of target genes. As high levels of Bcl-3 expression and activation have been detected in different types of human cancer, Bcl-3 has been labeled a proto-oncogene. Our study uncovered a markedly upregulated Bcl-3 expression in human prostate cancer (PCa), where inflammatory cell infiltration was observed. Elevated Bcl-3 expression in PCa was dependent on the proinflammatory cytokine interleukin-6-mediated STAT3 activation. Microarray analyses, using Bcl-3 knockdown in PCa cells, identified the inhibitor of DNA-binding (Id) family of helix-loop-helix proteins as potential Bcl-3-regulated genes. Bcl-3 knockdown reduced the abundance of Id-1 and Id-2 proteins and boosted PCa cells to be more receptive to undergoing apoptosis following treatment with anticancer drug. Our data imply that inactivation of Bcl-3 may lead to sensitization of cancer cells to chemotherapeutic drug-induced apoptosis, thus suggesting a potential therapeutic strategy in PCa treatment.

Fusion proteins containing the amino terminus of mixed lineage leukemia (MLL) are common in acute lymphoblastic leukemia (ALL) due to translocations. The MLL-AF4 fusion protein is generated by the translocation t(4;11)(q21;q23), and t(4;11)-positive ALL patients (MLL-AF4 ALL), have a notoriously poorer prognosis compared with patients with other MLL-associated leukemias. The detailed role of this fusion protein in leukemogenesis is not well understood. MicroRNAs (miRNAs) targeting the AF4 3' untranslated regions may modulate MLL-AF4 fusion protein levels, raising the question of whether regulation of these miRNAs are involved in the progression of MLL-AF4 ALL. In this study, we show that miR-143 was identified as a regulator of MLL-AF4 expression in MLL-AF4 ALL samples. Restoration of miR-143 in MLL-AF4-positive RS4;11 and MV4-11 cells induced apoptosis, negatively contributing to leukemia cell growth by reducing MLL-AF4 fusion protein levels. Furthermore, miR-143 was epigenetically repressed by promoter hypermethylation in MLL-AF4-positive primary blasts and cell lines, but not in normal bone marrow cells and MLL-AF4-negative primary blasts, which was directly associated with expression of the MLL-AF4 oncogene. This is the first study to show that miR-143 functions as a tumor suppressor in MLL-AF4 B-cell ALL. These data reveal the therapeutic promise of upregulating miR-143 expression for MLL-AF4 B-cell ALL.

It has long been assumed that DNA sequences and corresponding RNA transcripts are almost identical; a recent discovery, however, revealed widespread RNA-DNA differences (RDDs), which represent a largely unexplored aspect of human genome variation. It has been speculated that RDDs can affect disease susceptibility and manifestations; however, almost nothing is known about how RDDs are related to disease. Here, we show that RDDs are rarer in proto-oncogenes than in tumor suppressor genes; the number of RDDs in coding exons, but not in 3'UTR and 5'UTR, is significantly lower in the former than the latter, and this trend is especially pronounced in non-synonymous RDDs, i.e., those cause amino acid changes. A potential mechanism is that, unlike proto-oncogenes, the requirement of tumor suppressor genes to have both alleles affected to cause tumor 'buffers' these genes to tolerate more RDDs.

Breast cancer is a major cause of cancer-related deaths in American women; therefore, the identification of novel breast cancer-related molecules for the discovery of new markers and drug targets remains essential. The human DEK gene, which encodes a chromatin-binding protein and DNA topology regulator, is upregulated in many types of cancer. DEK has been implicated as an oncogene in breast cancer based on mRNA expression studies, but its functional significance in breast cancer growth and progression has not yet been tested directly. We demonstrate that DEK is highly expressed in breast cancer cells compared with normal tissue, and functionally important for cellular growth, invasion and mammosphere formation. DEK overexpression in non-tumorigenic MCF10A cells resulted in increased growth and motility, with a concomitant downregulation of E-cadherin. Conversely, DEK knockdown in MCF7 and MDA-MB-468 breast cancer cells resulted in decreased growth and motility with upregulation of E-cadherin. The use of DEK-proficient and -deficient breast cancer cells in orthotopic xenografts provided further in vivo evidence that DEK contributes to tumor growth. Activation of the β-catenin signaling pathway is important for normal and cancer stem cell character, growth and metastasis. We show that DEK expression stimulated, and DEK knockdown repressed β-catenin nuclear translocation and activity. Importantly, the expression of constitutively active β-catenin rescued breast cancer invasion defects of DEK knockdown cells. Together, our data indicate that DEK expression stimulates the growth, stem cell character and motility of breast cancer cells, and that DEK-dependent cellular invasion occurs at least in part via β-catenin activation.

Proto-oncogenes encode signaling molecular switches regulating cellular homeostasis in metazoans, and can be converted to oncogenes by gain-of-function mutations. To address the molecular basis for development of the regulatory system of proto-oncogenes during evolution, we screened for ancestral proto-oncogenes from the unicellular choanoflagellate Monosiga ovata by monitoring their transforming activities, and isolated a Pak gene ortholog encoding a serine/threonine kinase as a 'primitive oncogene'. We also cloned Pak orthologs from fungi and the multicellular sponge Ephydatia fluviatilis, and compared their regulatory features with that of M. ovata Pak (MoPak). MoPak is constitutively active and induces cell transformation in mammalian fibroblasts, although the Pak orthologs from multicellular animals are strictly regulated. Analyses of Pak mutants revealed that structural alteration of the auto-inhibitory domain (AID) of MoPak confers higher constitutive kinase activity, as well as greater binding ability to Rho family GTPases than the multicellular Paks, and this structural alteration is responsible for cell transformation and disruption of multicellular tissue organization. These results show that maturation of AID function was required for the development of the strict regulatory system of the Pak proto-oncogene, and suggest a potential link between the establishment of the regulatory system of proto-oncogenes and metazoan evolution.

Mutant forms of the c-ABL gene are well known to be involved in hematopoietic malignancies such as chronic myeloid leukemia (CML). CML patients possess a fused BCR-ABL gene that activates the Abl tyrosine kinase domain within Bcr-Abl. In general fusion proteins that cause oligomerization of Abl lead to activation of its tyrosine kinase activity. In this review, we highlight recent discoveries indicating that the activated c-Abl tyrosine kinase, not as a fusion protein, plays an important role in malignant solid tumors of lung and breast.

PIKE-A (phosphoinositide 3-kinases (PI 3)-kinase enhancer) is a ubiquitously expressed GTPase, which binds to and enhances protein kinase B (Akt) kinase activity in a guanine nucleotide-dependent manner. PIKE-A is one of the components of the CDK4 amplicon that is amplified in numerous human cancers. However, whether PIKE-A itself can mediate cell transformation, proliferation and migration remains unknown. Here, we show that PIKE-A is overexpressed in various human cancer samples, escalates U87MG glioblastoma invasion and provokes NIH3T3 cell transformation. Overexpression of wild-type (WT) PIKE-A enhances NIH3T3 and U87MG cell growth, which is further increased by cancer cell-derived PIKE-A active mutants. In contrast, both the dominant-negative mutant and the phosphoinositide lipids interaction-defective mutant antagonize cell proliferation. Moreover, PIKE-A and its active and inactive mutants similarly enhance or antagonize U87MG cell survival and invasion, and their ability to do so is coupled with the catalytic effect they have on Akt activation. Furthermore, PIKE-A WT and its active mutants significantly elicit NIH3T3 cell transformation. Thus, our findings support the concept that PIKE-A acts as a proto-oncogene, promoting cell transformation through Akt activation.

Retroviral insertion mutagenesis screens in mice are powerful tools for efficient identification of oncogenic mutations in an in vivo setting. Many oncogenes identified in these screens have also been shown to play a causal role in the development of human cancers. Sequencing and annotation of the mouse genome, along with recent improvements in insertion site cloning has greatly facilitated identification of oncogenic events in retrovirus-induced tumours. In this review, we discuss the features of retroviral insertion mutagenesis screens, covering the mechanisms by which retroviral insertions mutate cellular genes, the practical aspects of insertion site cloning, the identification and analysis of common insertion sites, and finally we address the potential for use of somatic insertional mutagens in the study of nonhaematopoietic and nonmammary tumour types.

Mixed lineage leukemia (MLL) fusion proteins are derived from translocations at 11q23 that occur in aggressive subtypes of leukemia. As a consequence, MLL is joined to different unrelated proteins to form oncogenic transcription factors. Here we demonstrate a direct interaction between several nuclear MLL fusion partners and present evidence for a role of these proteins in histone binding. In two-hybrid studies, ENL interacted with AF4 and AF5q31 as well as with a fragment of AF10. A structure-function analysis revealed that the AF4/AF5q31/AF10 binding domain in ENL coincided with the C-terminus that is essential for transformation by MLL-ENL. The ENL/AF4 association was corroborated by GST-pulldown experiments and by mutual coprecipitation. Both proteins colocalized in vivo in a nuclear speckled pattern. Moreover, AF4 and ENL coeluted on sizing columns together with the known ENL binding partner Polycomb3, suggesting the presence of a multiprotein complex. The overexpression of ENL alone activated a reporter construct and a mutational screen indicated the conserved YEATS domain as essential for this function. Overlay and pulldown-assays finally showed a specific and YEATS domain-dependent association of ENL with histones H3 and H1. In summary, our studies support a common role for nuclear MLL fusion partners in chromatin biology.

The human AF4 (ALL-1 fused gene on chromosome 4) gene (4q11) is recurrently involved in reciprocal translocations to the MLL (mixed lineage leukemia) gene (11q23), correlated with high-risk acute lymphoblastic leukemia (ALL) in infants and early childhood. The t(4;11) translocation is one of the most frequent MLL translocations known today. In general, MLL translocations are the result of an illegitimate recombination process leading to reciprocal fusions of unrelated translocation partner (TP) genes with the MLL gene. Owing to the constant presence of the derivative (11) product, it was hypothesised that only MLL.TP fusion genes are responsible for the leukemogenic process. This concept has been successfully tested for some known MLL fusions, while other MLL fusions failed. Here, we demonstrate growth-transforming potential of AF4 wild-type and the AF4.MLL fusion protein. The underlying oncogenic mechanism involves the two E3 ubiquitin ligases SIAH1 and SIAH2, the N-terminal portion of AF4 and the protection of the AF4.MLL fusion protein against proteosomal degradation.

The AML1/EVI-1 chimeric gene is generated by the t(3;21)(q26;q22) translocation and plays a pivotal role in progression of hematopoietic stem cell malignancies such as chronic myelocytic leukemia and myelodysplastic syndrome. In AML1/EVI-1, an N-terminal half of AML1 including a runt homology domain is fused to the entire zinc-finger EVI-1 protein. AML1 is essential for hematopoietic cell development in fetal liver and its lineage-specific differentiation in adult. In contrast, EVI-1 is barely expressed in normal hematopoietic cells, but it is overexpressed in chronic myelocytic leukemia in blastic crisis and myelodysplastic syndrome-derived leukemia. There are at least four mechanisms identified in AML1/EVI-1 fusion protein that possibly lead into malignant transformation of hematopoietic stem cells. Firstly, AML1/EVI-1 exerts dominant-negative effects over AML1-induced transcriptional activation. Although target genes repressed by AML1/EVI-1 are still not known, binding competition to a specific DNA sequence and histone deacetylase recruitment through a co-repressor CtBP in EVI-1 part are conceivable underlying mechanisms for the dominant-negative effects. Secondly, AML1/EVI-1 interferes with TGF beta signaling and antagonizes the growth-inhibitory effects of TGF beta. The first zinc-finger domain of EVI-1 associates with Smad3, a TGF beta signal transducer, and represses its transcriptional activity by recruiting histone deacetylase through CtBP that interacts with EVI-1. Thirdly, AML1/EVI-1 blocks JNK activity and prevents stress-induced apoptosis. AML1/EVI-1 associates with JNK through the first zinc-finger domain of EVI-1 and disturbs the association between JNK and its substrates. Lastly, AML1/EVI-1 enhances AP-1 activity by activating the c-Fos promoter depending on the second zinc-finger domain of EVI-1, and promotes cell proliferation. All these functions cooperatively contribute to the malignant transformation of the hematopoietic stem cells by AML1/EVI-1.

We have previously shown that BCR/ABL, a fusion protein generated by the t(9;22)(q34;q11) translocation found in the vast majority of chronic myelogenous leukemia (CML), cooperates with AML1/MDS1/EVI1 (AME), a fusion transcription factor generated by a t(3;21)(q26;q22) translocation identified as a secondary mutation in some cases of CML during the blast phase (CML-BC), in the rapid induction of an acute myelogenous leukemia (AML) in mice. In this study, we evaluated the leukemogenic potential of EVI1-, MDS1/EVI1- and AML1-related oncoproteins (AML1Delta, AML1/MDS1). We found that ectopic expression of either EVI1 or MDS1/EVI1 impaired hematopoiesis. However, neither EVI1 nor MDS1/EVI1 was sufficient for inducing AML in mice, although EVI1 did induce some hematologic neoplasia other than AML with a low efficiency. In addition, unlike AME, none of the EVI1- or AML1-related oncoproteins examined were capable of fully cooperating with BCR/ABL in the induction of AML. The results indicate that both the AML1 and EVI1 oncogenic components are required for the leukemogenic potential of AME and for the cooperation of AME and BCR/ABL in the induction of AML.

PLAG1 is a proto-oncogene whose ectopic expression can trigger the development of pleomorphic adenomas of the salivary glands and of lipoblastomas. As PLAG1 is a transcription factor, able to activate transcription through the binding to the consensus sequence GRGGC(N)(6-8)GGG, its ectopic expression presumably results in the deregulation of target genes, leading to uncontrolled cell proliferation. The identification of PLAG1 target genes is therefore a crucial step in understanding the molecular mechanisms involved in PLAG1-induced tumorigenesis. To this end, we analysed the changes in gene expression caused by the conditional induction of PLAG1 expression in fetal kidney 293 cell lines. Using oligonucleotide microarray analyses of about 12 000 genes, we consistently identified 47 genes induced and 12 genes repressed by PLAG1. One of the largest classes identified as upregulated PLAG1 targets consists of growth factors such as the insulin-like growth factor II and the cytokine-like factor 1. The in silico search for PLAG1 consensus sequences in the promoter of the upregulated genes reveals that a large proportion of them harbor several copies of the PLAG1-binding motif, suggesting that they represent direct PLAG1 targets. Our approach was complemented by the comparison of the expression profiles of pleomorphic adenomas induced by PLAG1 versus normal salivary glands. Concordance between these two sets of experiments pinpointed 12 genes that were significantly and consistently upregulated in pleomorphic adenomas and in PLAG1-expressing cells, identifying them as putative PLAG1 targets in these tumors.

Few t(9;11) translocations in DNA topoisomerase II inhibitor-related leukemias have been studied in detail and the DNA damage mechanism remains controversial. We characterized the der(11) and der(9) genomic breakpoint junctions in a case of AML following etoposide and doxorubicin. Etoposide-, etoposide metabolite- and doxorubicin-induced DNA topoisomerase II cleavage was examined in normal homologues of the MLL and AF-9 breakpoint sequences using an in vitro assay. Induction of DNA topoisomerase II cleavage complexes in CEM and K562 cell lines was investigated using an in vivo complex of enzyme assay. The translocation occurred between identical 5'-TATTA-3' sequences in MLL intron 8 and AF-9 intron 5 without the gain or loss of bases. The 5'-TATTA-3' sequences were reciprocally cleaved by DNA topoisomerase II in the presence of etoposide, etoposide catechol or etoposide quinone, creating homologous 4-base 5' overhangs that would anneal to form both breakpoint junctions without any processing. der(11) and der(4) translocation breakpoints in a treatment-related ALL at the same site in MLL are consistent with a damage hotspot. Etoposide and both etoposide metabolites induced DNA topoisomerase II cleavage complexes in the hematopoietic cell lines. These results favor the model in which the chromosomal breakage leading to MLL translocations in DNA topoisomerase II inhibitor-related leukemias is a consequence of DNA topoisomerase II cleavage.

Mammary epithelial cells are embedded in a unique extracellular environment to which adipocytes and other stromal cells contribute. Mammary epithelial cells are critically dependent on this milieu for survival. However, it remains unknown which adipocyte-secreted factors are required for the survival of the mammary epithelia and what role these adipokines play in the process of ductal carcinoma tumorigenesis. Here, we take a systematic molecular approach to investigate the multiple ways adipocytes and adipokines can uniquely influence the characteristics and phenotypic behavior of malignant breast ductal epithelial cells. Microarray analysis and luciferase reporter assays indicate that adipokines specifically induce several transcriptional programs involved in promoting tumorigenesis, including increased cell proliferation (IGF2, FOS, JUN, cyclin D1), invasive potential (MMP1, ATF3), survival (A20, NFkappaB), and angiogenesis. One of the key changes in the transformed ductal epithelial cells associated with the cell cycle involves the induction of NFkappaB (five-fold) and cyclin D1 (three-fold). We show that by regulating the transcription of these molecules, the synergistic activity of adipocyte-derived factors can potentiate MCF-7 cell proliferation. Furthermore, compared to other stromal cell-secreted factors, the full complement of adipokines shows an unparalleled ability to promote increased cell motility, migration, and the capacity for angiogenesis. Adipocyte-secreted factors can affect tumorigenesis by increasing the stabilization of pro-oncogenic factors such as beta-catenin and CDK6 as a result of a reduction in the gene expression of their inhibitors (i.e. p18). An in vivo coinjection system using 3T3-L1 adipocytes and SUM159PT cells effectively recapitulates the host-tumor interactions in primary tumors. Type VI collagen, a soluble extracellular matrix protein abundantly expressed in adipocytes, is further upregulated in adipocytes during tumorigenesis. It promotes GSK3beta phosphorylation, beta-catenin stabilization, and increased beta-catenin activity in breast cancer cells and may critically contribute towards tumorigenesis when not counterbalanced by other factors.

The human mixed lineage leukemia (MLL) gene is involved in about 50 different chromosomal translocations, associated with the disease phenotype of acute leukemia. However, the normal function of MLL is less understood. Homozygous knockouts of murine Mll were embryonal lethal, while heterozygous disruption led to aberrant hox gene expression associated with skeletal malformations, growth retardation, and impaired hematopoiesis. To understand MLL functions on the molecular level, gene expression profiling experiments were performed with a pair of murine cell lines (MLL(+/+) and MLL(-/-)). Microarray hybridization experiments revealed 197 potential target genes that are differentially expressed, providing new and important clues about MLL functions.

We showed that the LAF4 gene on 2q11.2-12 was fused to the MLL gene on 11q23 in a pediatric patient with CD10 positive acute lymphoblastic leukemia (ALL) having t(2;11)(q11;q23). The LAF4 gene, which encodes a lymphoid nuclear protein of 1227 amino acids with transactivation potential, is thought to have a role in early lymphoid development. The LAF4 protein was homologous to AF4 and AF5q31 proteins that are fused to MLL in infant early pre-B ALL and the breakpoint of LAF4 was located within the region homologous to the transactivation domain of AF4 and AF5q31. Expression of the 8.5-kb LAF4 transcript was detected in the adult heart, brain, and placenta and in the fetal brain. LAF4 expression was found to be higher in ALL cell lines than in AML and Epstein-Barr virus-transformed B-lymphocyte cell lines. These findings suggest that LAF4, AF4 and AF5q31 might define a new family particularly involved in the pathogenesis of 11q23-associated ALL.

The MLL gene at chromosome 11q23 is frequently rearranged in acute leukemia. Here we report the identification of a new MLL fusion partner in the case of an infant with AML-M4 and a t(11;17)(q23;q21) translocation. Fluorescence in situ hybridization (FISH) and RT-PCR analyses indicated a rearrangement of the MLL gene, but no fusion with previously identified MLL fusion partners at 17q, such as AF17 or MSF. Rapid amplification of cDNA ends (RACE) revealed an in-frame fusion of MLL to LASP1, a gene that is amplified and overexpressed in breast cancer. Retroviral transduction of myeloid progenitors demonstrated that MLL/LASP1 is the fourth known fusion of MLL with a cytoplasmic protein that has no in vitro transformation capability, thus establishing a potential subgroup among the MLL fusion proteins.

We examined the MLL translocation in two cases of infant AML with X chromosome disruption. The G-banded karyotype in the first case suggested t(X;3)(q22;p21)ins(X;11)(q22;q13q25). Southern blot analysis showed one MLL rearrangement. Panhandle PCR approaches were used to identify the MLL fusion transcript and MLL genomic breakpoint junction. SEPTIN6 from chromosome band Xq24 was the partner gene of MLL. MLL exon 7 was joined in-frame to SEPTIN6 exon 2 in the fusion transcript. The MLL genomic breakpoint was in intron 7; the SEPTIN6 genomic breakpoint was in intron 1. Spectral karyotyping revealed a complex rearrangement disrupting band 11q23. FISH with a probe for MLL confirmed MLL involvement and showed that the MLL-SEPTIN6 junction was on the der(X). The MLL genomic breakpoint was a functional DNA topoisomerase II cleavage site in an in vitro assay. In the second case, the karyotype revealed t(X;11)(q22;q23). Southern blot analysis showed two MLL rearrangements. cDNA panhandle PCR detected a transcript fusing MLL exon 8 in-frame to SEPTIN6 exon 2. MLL and SEPTIN6 are vulnerable to damage to form recurrent translocations in infant AML. Identification of SEPTIN6 and the SEPTIN family members hCDCrel and MSF as partner genes of MLL suggests a common pathway to leukaemogenesis.

AML1/Evi-1 is a chimeric protein that is derived from t(3;21), found in blastic transformation of chronic myelogenous leukemia. It is composed of the N-terminal AML1 portion with the DNA-binding Runt domain and the C-terminal Evi-1 portion. It has been shown to dominantly repress AML1-induced transactivation. The mechanism for it has been mainly attributed to competition with AML1 for the DNA-binding and for the interaction with PEBP2beta (CBFbeta), a partner protein which heterodimerizes with AML1. It was recently found that Evi-1 interacts with C-terminal binding protein (CtBP) to repress TGFbeta-induced transactivation. Here, we demonstrate that AML1/Evi-1 interacts with CtBP in SKH1 cells, a leukemic cell line which endogenously overexpresses AML1/Evi-1 and that AML1/Evi-1 requires the interaction with CtBP to repress AML1-induced transactivation. The association with CtBP is also required when AML1/Evi-1 blocks myeloid differentiation of 32Dcl3 cells induced by granulocyte colony-stimulating factor. Taken together, it is suggested that one of the mechanisms for AML1/Evi-1-associated leukemogenesis should be an aberrant recruitment of a corepressor complex by the chimeric protein.

The development of acute myelogenous leukemia (AML), which is characterized by a block of myeloid differentiation, is a multi-step process that involves several genetic abnormalities, but the molecular mechanisms by which these genetic alterations cooperate in leukemogenesis are poorly understood. The human chronic myelogenous leukemia (CML) is a model for multi-step leukemogenesis. BCR-ABL, a constitutively active tyrosine kinase, is a fusion protein generated by the t(9;22)(q34;q11) translocation found in the vast majority of CML patients. BCR-ABL efficiently induces a myeloproliferative disorder (MPD) in mice, but progression to CML blast phase requires additional mutations. The AML1/MDS1/EVI1 (AME) transcription factor fusion protein, is a product of the human t(3;21)(q26;q22) translocation found as a secondary mutation in some cases of CML during the blast phase. We have previously shown that AME can induce an AML in mice but with a greatly extended latency, suggesting a requirement for additional mutations. Here we demonstrate that AME alone does not block myeloid differentiation in vivo during the 4-month pre-leukemia stage, yet co-expression of BCR-ABL and AME in mice can block myeloid differentiation and rapidly induce an AML. Our results suggest that block of myeloid differentiation and induction of AML involves cooperation between mutations that dysregulate protein tyrosine kinase signaling and those that disrupt hematopoietic gene transcription.

Development of strategies for prevention of breast cancer development requires an understanding of the effects of mammary oncogenes on mammary cells at early stages in neoplastic transformation. As mammary oncogenes wnt-1 and int-2 affect different signal transduction pathways, we investigated their effects on established mouse mammary epithelial cell lines (MMECLs) reflecting early stages in tumorigenesis. Normal interactions between beta-catenin and E-cadherin were abrogated in all three immortalized MMECLs and the cells lacked beta-catenin-mediated transactivation activity, detectable using a reporter assay, suggesting that alterations in cell adhesion may be very early events in mammary tumorigenesis. Immortalized FSK4 and EL12 cells and hyperplastic TM3 cells were stably transfected with expression vectors encoding wnt-1 or int-2 or the control vector, and drug-selected pooled cells from each line were confirmed by reverse transcription-polymerase chain reaction to express the transfected oncogene; this expression persisted in the cells analysed in vitro and in vivo. Resultant phenotypic changes depended both on the oncogene and the target mammary cell line. In FSK4 cells, expression of wnt-1 or int-2 resulted in proliferative changes in vitro, including reduced contact inhibition, increased beta-catenin expression, and decreased p53 transcriptional activity, but neither oncogene conferred upon those cells the ability to produce tumors in vivo. EL12 cells were highly refractory to the effects of both oncogenes, with the only measurable changes being increased E-cadherin levels induced by both oncogenes and increased proliferation of the int-2-transfected cells in the absence of serum. Parental TM3 cells were phenotypically similar to wnt-1- or int-2-transfected FSK4 cells and displayed an increased rate of proliferation in vitro and markedly increased tumorigenicity in vivo following transfection with int-2 but not with wnt-1. These results suggest that wnt-1 signaling is redundant in the hyperplastic TM3 cells and indicate that wnt-1-induced effects in the immortalized FSK4 and EL12 cells were not sufficient to mediate a tumorigenic phenotype. This study showed that the wnt-1 and int-2 oncogenes have similar but distinguishable effects on immortalized MMECLs and that the genetic background of the mammary cells greatly influences the consequences of oncogene expression at early stages of cell transformation.

The MLL (Mixed Lineage Leukemia) gene is a common target for chromosomal translocations associated with human acute leukemias. These translocations result in a gain of MLL function by generating novel chimeric proteins containing the amino-terminus of MLL fused in-frame with one of 30 distinct partner proteins. Structure/function studies using an in vitro myeloid progenitor immortalization assay have revealed that at least four nuclear partner proteins contribute transcriptional effector properties to MLL to produce a range of chimeric transcription factors with leukemogenic potential. Mouse models suggest that expression of an MLL fusion protein is necessary but not sufficient for leukemogenesis. Interestingly, whilst all MLL fusion proteins tested so far phenocopy each other with respect to in vitro immortalization, the latency period required for the onset of acute leukemia in vivo is variable and partner protein dependent. We discuss potential mechanisms that may account for the ability of distinct MLL fusion proteins to promote short or long latency leukemogenesis.

Genomic instability promotes tumorigenesis and can occur through various mechanisms, including defective segregation of chromosomes or inactivation of DNA mismatch repair. Although B-cell lymphomas are associated with chromosomal translocations that deregulate oncogene expression, a mechanism for genome-wide instability during lymphomagenesis has not been described. During B-cell development, the immunoglobulin variable (V) region genes are subject to somatic hypermutation in germinal-centre B cells. Here we report that an aberrant hypermutation activity targets multiple loci, including the proto-oncogenes PIM1, MYC, RhoH/TTF (ARHH) and PAX5, in more than 50% of diffuse large-cell lymphomas (DLCLs), which are tumours derived from germinal centres. Mutations are distributed in the 5' untranslated or coding sequences, are independent of chromosomal translocations, and share features typical of V-region-associated somatic hypermutation. In contrast to mutations in V regions, however, these mutations are not detectable in normal germinal-centre B cells or in other germinal-centre-derived lymphomas, suggesting a DLCL-associated malfunction of somatic hypermutation. Intriguingly, the four hypermutable genes are susceptible to chromosomal translocations in the same region, consistent with a role for hypermutation in generating translocations by DNA double-strand breaks. By mutating multiple genes, and possibly by favouring chromosomal translocations, aberrant hypermutation may represent the major contributor to lymphomagenesis.

Polycomb group (PcG) proteins assemble to form large multiprotein complexes involved in gene silencing. Evidence suggests that PcG complexes are heterogeneous with respect to both protein composition and specific function. MPc3 is a recently described mouse Polycomb (Pc) protein that shares structural homology with at least two other Pc proteins, M33 and MPc2. All three Pc proteins bind another PcG protein, RING1, through a conserved carboxy-terminal C-box motif. Here, data are presented demonstrating that MPc3 also interacts with AF9, a transcriptional activator implicated in the development of acute leukemias. The carboxy-terminus of AF9 is fused to the MLL protein in leukemias characterized by t(9;11)(p22;q23) chromosomal translocations. Importantly, it is the carboxy-terminus of AF9 to which MPc3 binds. The AF9 binding site of MPc3 maps to a central, non-conserved, region of the polypeptide sequence. In contrast to MPc3, data indicate that the Pc protein M33 does not interact with AF9. This finding suggests a potentially unique role for MPc3 in linking a PcG silencing complex to a transcriptional activator protein.