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Cooperativity between mutations in tyrosine kinases and in hematopoietic transcription factors in AML
Abstract
In summary, our understanding of the genetics of human leukemia has provided the tools that we require to develop rationally designed targeted molecular therapies. The available evidence indicates that AML is the consequence of collaboration between at least two broad classes of mutations. Class I mutations confer a proliferative and/or survival advantage without affecting differentiation, whereas class II mutations serve primarily to impair hematopoietic differentiation. We have proof-of-principle that each of these classes of mutations can be targeted with small molecules. Examples include specific inhibition of BCR/ABL by ST1471 in treatment of CML and CML blast crisis, and induction of differentiation in promyelocytic leukemias by use of all-trans retinoic acid to override the dominant negative block in differentiation induced by the PML/RARalpha fusion proteins. The combination of continued discovery and characterization of AML disease alleles with design, development and clinical testing of targeted therapies provides real hope for improved outcome in AML, and perhaps in other hematologic malignancies and solid tumors.
... Currently it is possible to comprehensively identify genetic mutations, including single base changes, deletions, insertions and genomic rearrangements in early events for AML/MDS development. 1 As a hypothetical model, the molecular pathogenesis of AML requires cooperating mutations of several genes such as early mutation, class-I and class-II mutations. 1 Early mutations are present in clinically silent preleukemic cells, and involve genes for nucleolar ribonucleoprotein, nucleophosmin (NPM1) and the RNA splicing machinery. ...
... 1 As a hypothetical model, the molecular pathogenesis of AML requires cooperating mutations of several genes such as early mutation, class-I and class-II mutations. 1 Early mutations are present in clinically silent preleukemic cells, and involve genes for nucleolar ribonucleoprotein, nucleophosmin (NPM1) and the RNA splicing machinery. 2,3 Class-I and class-II mutations are mainly present in leukemic cells. 1 Class-I mutations constitutively activate genes in the kinase signaling pathways, including FLT3, c-KIT, c-FMS and RAS, which enhance the proliferation of leukemic cells, whereas class-II mutations inactivate hematopoietic transcription factors such as AML1, ASXL, TET2 and IDH, which are associated with epigenetic regulation of gene expression. ...
... 2,3 Class-I and class-II mutations are mainly present in leukemic cells. 1 Class-I mutations constitutively activate genes in the kinase signaling pathways, including FLT3, c-KIT, c-FMS and RAS, which enhance the proliferation of leukemic cells, whereas class-II mutations inactivate hematopoietic transcription factors such as AML1, ASXL, TET2 and IDH, which are associated with epigenetic regulation of gene expression. 4 Although combined genetic abnormalities are likely essential for the development of AML/ MDS, aberrant function and chromosomal abnormality of human bone marrow (BM) stromal cells could be involved MDS development and subsequent secondary AML. 5 Interestingly, impaired microRNA biogenesis in human BM stromal cells from MDS patients, with Dicer1 and Drosha gene and protein downregulation, correlated with abnormal gene and microRNA expression. ...
Aberrant reactivation of hedgehog (Hh) signaling has been described in a wide variety of human cancers including cancer stem cells. However, involvement of the Hh-signaling system in the bone marrow (BM) microenvironment during the development of myeloid neoplasms is unknown. In this study, we assessed the expression of Hh-related genes in primary human CD34(+) cells, CD34(+) blastic cells and BM stromal cells. Both Indian Hh (Ihh) and its signal transducer, smoothened (SMO), were expressed in CD34(+) acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS)-derived cells. However, Ihh expression was relatively low in BM stromal cells. Remarkably, expression of the intrinsic Hh-signaling inhibitor, human Hh-interacting protein (HHIP) in AML/MDS-derived stromal cells was markedly lower than in healthy donor-derived stromal cells. Moreover, HHIP expression levels in BM stromal cells highly correlated with their supporting activity for SMO(+) leukemic cells. Knockdown of HHIP gene in stromal cells increased their supporting activity although control cells marginally supported SMO(+) leukemic cell proliferation. The demethylating agent, 5-aza-2'-deoxycytidine rescued HHIP expression via demethylation of HHIP gene and reduced the leukemic cell-supporting activity of AML/MDS-derived stromal cells. This indicates that suppression of stromal HHIP could be associated with the proliferation of AML/MDS cells.
... 21 Genetic abnormalities in AML Available evidence indicates that AML is the consequence of collaboration between at least two different types of pathological genetic events. 22,23 Type I abnormalities confer a proliferative and/or survival advantage of the leukemic cell while type II abnormalities/mutations primarily serve to impair hematopoietic differentiation. 24,25 This two-hit process leads to maturation arrest, proliferation advantage and the development of leukemia. ...
... To correlate the results of the drug analyses to biological and clinical parameters such as dose, age, sex, renal and hepatic functions, DS, response to induction therapy and outcome 22 Material and methods ...
... Limitless expansion is achieved through an endless sequence of cell division cycles and abrogation of restriction points. An accepted model of leukemogenesis suggests that two major classes of mutations cooperate to transform HPCs [4]. Class I mutations confer the ability of limitless growth and class II mutations impair hematopoietic differentiation [4]. ...
... An accepted model of leukemogenesis suggests that two major classes of mutations cooperate to transform HPCs [4]. Class I mutations confer the ability of limitless growth and class II mutations impair hematopoietic differentiation [4]. In addition, a third class of mutations (class III mutations) which hits epigenetic modifiers and hence alters protein synthesis in favor of proteins with oncogenic characteristics has recently come into focus [126]. ...
Acute myeloid leukemia (AML) is the result of a multistep transforming process of hematopoietic precursor cells (HPCs) which enables them to proceed through limitless numbers of cell cycles and to become resistant to cell death. Increased proliferation renders these cells vulnerable to acquiring mutations and may favor leukemic transformation. Here, we review how deregulated cell cycle control contributes to increased proliferation in AML and favors genomic instability, a prerequisite to confer selective advantages to particular clones in order to adapt and independently proliferate in the presence of a changing microenvironment. We discuss the connection between differentiation and proliferation with regard to leukemogenesis and outline the impact of specific alterations on response to therapy. Finally, we present examples, how a better understanding of cell cycle regulation and deregulation has already led to new promising therapeutic strategies.
... 2 of 8 The clinically significant BRAF missense mutations are predominantly located in the tyrosine kinase domain, between exon 11 and exon 15, particularly within the glycinerich loop and activation segment, respectively. Among these mutations, the BRAF V600E mutation accounts for 80% of BRAF mutations identified in human cancers [7]. A recent study by Ping et al. indicated the absence of BRAF mutations involving exon 15 in acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), and myeloproliferative neoplasms (MPN) within a cohort of 578 patients with myeloid neoplasms [4]. ...
BRAF mutations are rare in myeloid neoplasms and are reported to be associated with poor treatment outcomes. The purpose of our study is to characterize BRAF mutations in myeloid neoplasms using a next-generation sequencing (NGS) panel based on the experiences of a single cancer center. We conducted a retrospective review of patients with myeloid neoplasms who underwent the HopeSeq studies between January 2018 and September 2023. A total of 14 patients with myeloid neoplasms carrying BRAF mutations were included in our cohort. The clinical, pathological, and molecular features of these patients were investigated. Our study indicates that BRAF mutations are rare in myeloid neoplasms, constituting only 0.53% (14/2632) of all myeloid neoplasm cases, with the most common BRAF mutation being BRAF V600E (4/14; 28.6%). Interestingly, we observed that six out of seven patients with acute myeloid leukemia (AML) exhibited AML with monocytic differentiation, and all the patients with AML exhibited an extremely poor prognosis compared to those without BRAF mutations. TET2 (5/14; 35.7%), ASXL1 (4/14; 28.6%), and JAK2 (4/14; 28.6%) were the three most frequently co-mutated genes in these patients. Moreover, we noted concurrent KMT2A gene rearrangement with BRAF mutations in three patients with AML (3/7; 42.9%). Our study suggests that although BRAF mutations are rare in myeloid neoplasms, they play a crucial role in the pathogenesis of specific AML subtypes. Furthermore, RAS pathway alterations, including BRAF mutations, are associated with KMT2A gene rearrangement in AML. However, these findings warrant further validation in larger studies.
... AML is characterized not only by the differentiation blockade of AML cells but also by the uncontrolled clonal expansion of immature myeloid cells [118]. Notably, many gene alterations affecting leukemogenesis, progression, and prognosis in AML cells may be enhanced by overproliferation. ...
Acute myeloid leukemia (AML) is a heterogeneously malignant disorder resulting in poor prognosis. Ubiquitination, a major post-translational modification (PTM), plays an essential role in regulating various cellular processes and determining cell fate. Despite these initial insights, the precise role of ubiquitination in AML pathogenesis and treatment remains largely unknown. In order to address this knowledge gap, we explore the relationship between ubiquitination and AML from the perspectives of signal transduction, cell differentiation, and cell cycle control; and try to find out how this relationship can be utilized to inform new therapeutic strategies for AML patients.
... Cell cycle control contributes to increased proliferation in AML and favors genomic instability. Additionally, hyperproliferation bears an enhanced risk of genetic damage and induces leukemogenesis [82]. Apoptosis, which is a programmed cell death mechanism, is critical for the development and homeostasis of the hematopoietic system. ...
Before the advent of next-generation sequencing, research on acute myeloid leukemia (AML) mostly centered on protein-coding genes. In recent years, breakthroughs in RNA sequencing technologies and whole transcriptome analysis have led to the discovery that approximately 97.5% of the human genome is transcribed into non-coding RNAs (ncRNAs). This paradigm shift has led to an explosion of research interest in different classes of non-coding RNAs, such as circular RNAs (circRNAs) as well as non-coding untranslated regions (UTRs) of protein-coding messenger RNAs. The critical roles of circRNAs and UTRs in AML pathogenesis have become increasingly apparent. In this review, we discuss the cellular mechanisms of circRNAs and summarize recent studies that reveal their biological roles in AML. Furthermore, we also review the contribution of 3′UTRs to disease progression. Finally, we discuss the potential of circRNAs and 3′UTRs as new biomarkers for disease stratification and/or the prediction of treatment response and targets for the development of RNA-directed therapeutic applications.
... Apart from being the master regulators of the HSCs, these transcription factors are also involved in the development of other organs, such as heart and vascular systems as well as in diseases. Consequently, any mutation/dysfunction of hematopoietic transcription factors in the lymphoid cells has been shown to cause diseases like leukemia, failed embryonic, and immune system development (Deguchi and Gilliland, 2002;Yang and Karsenty, 2002;Koschmieder et al., 2005;Rosenbauer and Tenen, 2007;Mullighan et al., 2009). Furthermore, mutations in these transcription factors resulted in cardiovascular diseases (CVDs) like coronary artery disease, cardiac hypertrophy, and congenital heart diseases involving conduction abnormalities and cardiac malformations (Schott et al., 1998;Ikeda et al., 2002;Bhagavatula et al., 2004;Akazawa and Komuro, 2005;Kohli et al., 2011;Ang et al., 2016). ...
Transcription factors as multifaceted modulators of gene expression that play a central role in cell proliferation, differentiation, lineage commitment, and disease progression. They interact among themselves and create complex spatiotemporal gene regulatory networks that modulate hematopoiesis, cardiogenesis, and conditional differentiation of hematopoietic stem cells into cells of cardiovascular lineage. Additionally, bone marrow-derived stem cells potentially contribute to the cardiovascular cell population and have shown potential as a therapeutic approach to treat cardiovascular diseases. However, the underlying regulatory mechanisms are currently debatable. This review focuses on some key transcription factors and associated epigenetic modifications that modulate the maintenance and differentiation of hematopoietic stem cells and cardiac progenitor cells. In addition to this, we aim to summarize different potential clinical therapeutic approaches in cardiac regeneration therapy and recent discoveries in stem cell-based transplantation.
... Here we present findings that are based on an in depth analysis of interactomes and related networks, which provides insights exceeding those provided by purely genetic informations. This will modify current concepts of pathogenesis of AML, mainly based on the two hit model postulating cooperation between class I and class II mutations which are considered independent in their contribution to leukemia induction (Deguchi and Gilliland, 2002). ...
Acute myeloid leukemias (AML) are characterized by recurrent genomic alterations, often in transcriptional regulators, which form the basis on which current prognostication and therapeutic intervention is overlaid. In AML transformation can often be attributed to single chromosomal aberrations encoding oncogenes, such as t(15;17)-PML/RARα or t(6;9)-DEK/CAN but it is unclear how these aberrant transcription factors drive leukemic signaling and influence cellular responses to targeted therapies. Here we show that by using a novel “subtractive interaction proteomics” approach, the high risk AML-inducing oncogene t(6;9)-DEK/CAN directly activates signaling pathways that are driven by the ABL1, AKT/mTOR, and SRC family kinases. The interplay of these signaling pathways creates a network with nodes that are credible candidates for combinatorial therapeutic interventions. These results reveal specific interdependencies between nuclear oncogenes and cancer signaling pathways thus providing a foundation for the design of therapeutic strategies to better address the complexity of cancer signaling.
... Here we present findings that are based on an in depth analysis of interactomes and related networks, which provides insights exceeding those provided by purely genetic informations. This will modify current concepts of pathogenesis of AML, mainly based on the two hit model postulating cooperation between class I and class II mutations which are considered independent in their contribution to leukemia induction (Deguchi and Gilliland, 2002). ...
Acute myeloid leukemias (AML) are characterized by recurrent genomic alterations, often in transcriptional regulators, which form the basis on which current prognostication and therapeutic intervention is overlaid. In AML transformation can often be attributed to single chromosomal aberrations encoding oncogenes, such as t(15;17)-PML/RARα or t(6;9)-DEK/CAN but it is unclear how these aberrant transcription factors drive leukemic signaling and influence cellular responses to targeted therapies. Here we show that by using a novel “subtractive interaction proteomics” approach, the high risk AML-inducing oncogene t(6;9)-DEK/CAN directly activates signaling pathways that are driven by the ABL1, AKT/mTOR, and SRC family kinases. The interplay of these signaling pathways creates a network with nodes that are credible candidates for combinatorial therapeutic interventions. These results reveal specific interdependencies between nuclear oncogenes and cancer signaling pathways thus providing a foundation for the design of therapeutic strategies to better address the complexity of cancer signaling.
Graphical Abstract
... AML is a highly heterogeneous leukemia associated with excessive progenitor cell proliferation and a differentiation block for cell-cycle arrest. AML is often caused by karyotypic abnormalities, including chromosomal translocations, deletions and inversions (3,4). Etiological factors driving AML development remain unclear, but lifestyle and environmental exposures, including obesity and smoking, are reported to be associated with the disease (5). ...
Acute myeloid leukemia (AML) is a common myelogenous malignancy in adults that is often characterized by disease relapse. The pathophysiological mechanism of AML has not yet been elucidated. The present study aimed to identify the crucial microRNAs (miRNAs/miRs) and target genes in AML, and to uncover the potential oncogenic mechanism of AML. miRNA and mRNA expression-profiling microarray datasets were downloaded from the Gene Expression Omnibus database. Differential expression analysis was performed and a regulatory network between miRNAs and target genes was constructed. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were used to predict the biological functions of the differentially expressed genes. Reverse transcription-quantitative polymerase chain reaction analysis was employed to verify the expression levels of miRNAs and target genes in AML patient samples. A total of 86 differentially expressed miRNAs and 468 differentially expressed mRNAs between AML and healthy blood samples were identified. In total, 47 miRNAs and 401 mRNAs were found to be upregulated, and 39 miRNAs and 67 mRNAs were found to be downregulated in AML. A total of 223 miRNA-target genes pairs were subjected to the construction of a regulatory network. Differentially expressed target genes were significantly enriched in the Wnt signaling pathway (hsa04310), melanogenesis (hsa04916) and pathways in cancer (hsa05200). Significantly differentially expressed miRNAs and genes, including hsa-miR-155, hsa-miR-192, annexin A2 (ANXA2), frizzled class receptor 3 (FZD3), and pleomorphic adenoma gene 1 (PLAG1), may serve essential roles in AML oncogenesis. Overall, hsa-miR-155, hsa-miR-192, ANXA2, FZD3 and PLAG1 may be associated with the development of AML via the involvement of the Wnt signaling pathway, melanogenesis and other cancer-associated signaling pathways.
... Many AML-specific oncogenic mutations and epigenetic alterations have been identified and their ability to cause leukemia has been analyzed in primary and human xenograft mouse models. A model has been suggested that at least two mutations from different complementation classes have to accumulate in a myeloid progenitor cell to cause AML [4]. One of these mutations is thought to cause deregulation of transcriptional programs needed for the orchestration of myeloid differentiation. ...
Study protocol version 2.0 (amended protocol).
(PDF)
... Many AML-specific oncogenic mutations and epigenetic alterations have been identified and their ability to cause leukemia has been analyzed in primary and human xenograft mouse models. A model has been suggested that at least two mutations from different complementation classes have to accumulate in a myeloid progenitor cell to cause AML [4]. One of these mutations is thought to cause deregulation of transcriptional programs needed for the orchestration of myeloid differentiation. ...
Study protocol version 1.0.
(PDF)
... 1,2 Este modelo propuesto por Gillilland en 2001, conocido como teoría de los dos "hits", supone que la mayor parte de las leucemias agudas son la consecuencia de al menos dos mutaciones. [3][4][5] La identificación de estas modificaciones genéticas requiere tecnología y metodologías que no son convencionales en los laboratorios de diagnóstico y para muchas de ellas aún no se conoce su asociación con alguna característica clínica de valor diagnóstico o pronóstico. ...
La leucemia mieloide aguda es un grupo de enfermedades fenotípica y genéticamente heterogéneas, originadas por la acumulación de mutaciones en una célula madre hematopoyética o célula progenito- ra. De manera tradicional, los estudios citogenéticos se han utilizado para estrati car a los pacientes con leucemia mieloide aguda en tres categorías: favorable, intermedio y desfavorable. Sin embargo, la estrati cación pronóstica y la decisión del tratamiento para los pacientes que tienen cariotipo normal muestra di cultades debido a la alta heterogeneidad clínica. Recientemente, la identi cación de mutaciones genéticas adicionales a los marcadores clásicos permitió generar nuevas entidades en el grupo de leucemia mieloide aguda respecto al tratamiento y seguimiento de esta enfermedad. En 2008, la Organización Mundial de la Salud introdujo las mutaciones en NPM1 y C/EBPα como entidades dentro del grupo de leucemia mieloide aguda con anormalidades genéticas recurrentes, mientras que las mutaciones en el gen FLT3 se han descrito como de mal pronóstico en los pacientes portadores. El conocimiento de estas mutaciones no sólo apoya el seguimiento de esta enfermedad, sino que abre la posibilidad de generar nuevas estrategias de tratamiento.
... According to the model proposed by Deguchi & Gilliland (2002), development of AML is the consequence of collaboration between at least two broad classes of mutations: (i) class I mutations which result in constitutively activated tyrosine kinases (gain of function) and confer a proliferative and/or survival advantage without affecting differentiation - Mutations of RUNX1 are common in therapy-related myelodysplasia following therapy with alkylating agents and are significantly associated with deletion or loss of chromosome arm 7q and with a subsequent leukemic transformation. FLT3 mutation and trisomy 21 are thought to be second hits in AML with RUNX1 Mutations (Osato M., 2004;Christiansen et al., 2004). ...
... The CCAAT/enhancer binding protein alfa (CEBPA) is a member of the leucine zipper (LZ) family of transcription factors, which in physiological conditions induces both growth arrest and terminal granulocytic differentiation by activation of myeloid gene expression [1][2][3]. Members of this family exert these functions by different domains: two transactivation domains (TAD1 and TAD2) for transcription activation; the basic region (BR) domain for DNA-binding and a leucine zipper domain (LZ) mediating homo-or heterodimerization [4]. Intronless CEBPA gene encodes mRNA, that is translated into 42 kDa (p42) and 30 kDa (p30) CEBPA proteins from different translation sites, with p42 representing the main physiological product of CEBPA. ...
... The CCAAT/enhancer binding protein alfa (CEBPA) is a member of the leucine zipper (LZ) family of transcription factors, which in physiological conditions induces both growth arrest and terminal granulocytic differentiation by activation of myeloid gene expression [1][2][3]. Members of this family exert these functions by different domains: two transactivation domains (TAD1 and TAD2) for transcription activation; the basic region (BR) domain for DNA-binding and a leucine zipper domain (LZ) mediating homo-or heterodimerization [4]. Intronless CEBPA gene encodes mRNA, that is translated into 42 kDa (p42) and 30 kDa (p30) CEBPA proteins from different translation sites, with p42 representing the main physiological product of CEBPA. ...
... Les LAM sont caractérisées par un blocage de différenciation ainsi qu'un avantage de survie et de prolifération des cellules tumorales. Un modèle de coopération oncogénique d'au moins deux évènements successifs a été décrit afin d'expliquer le processus de leucémogenèse (Deguchi and Gilliland, 2002). Dans ce modèle, le premier évènement correspond à une anomalie génétique perturbant la fonction de facteurs de transcription, aboutissant au blocage de la différenciation observé (mutations de classe II). ...
Acute myeloid leukaemias (AML) are heterogeneous diseases associated with poor prognosis. In AML, aberrant activation of many signaling pathways enhances proliferation and survival of leukemic blast cells. Understanding the mechanisms underlying survival of tumoral cells should allow the development of targeted therapies. The oncogenic kinase mTOR belongs to two distinct multimeric complexes. MTORC1 that controls protein translation, is constitutively activated in most of primary blast cells at AML diagnosis, while mTORC2 is constitutively activated in about half of AML samples. In AML, some phosphorylation events of the translational repressor 4E-BP1, are resistant to allosteric inhibitors of mTORC1 including rapamycin and its analogs. These first generation inhibitors of mTORC1 have only few effects on AML and do not induce significant apoptosis in vitro. I have tested a second generation mTOR kinase inhibitor active on both mTORC1 and mTORC2 complexes. In vitro, AZD8055 blocked mTORC1 and mTORC2 signaling, including 4E-BP1 rapamycin resistant phosphorylation events and protein synthesis. This compound decreased AML blast cells proliferation and cell cycle progression, reduced the clonogenic growth of leukemic progenitors and induced caspase-dependant apoptosis in leukemic cells but not in normal immature CD34+ cells. Finally, AZD8055 reduced tumor growth and improved survival in xenografted mouse model. In the second part of this work, I have studied the regulation of mTORC1 by amino acids in AML. In mammalian cells, activation of mTORC1 requires the presence of glutamine and leucine acting together via two membrane transporters, SLC1A5 and SLC7A5/SLC3A2. I showed that glutamine deprivation, obtained by L-asparaginase glutaminase activity or specific alpha-MEM use, inhibited mTORC1 and induced apoptosis in AML cell lines and primary AML blasts. L-asparaginase also inhibited protein synthesis and I have observed a correlation between the functional effects of L-asparaginase and its glutaminase activity. L-asparaginase induced an up-regulation of glutamine synthase (GS) protein and shRNA-induced GS inhibition increased L-asparaginase-dependant apoptosis in the MV4-11 AML cell line. I have also studied the effects of SLC1A5 inhibition with an inducible shRNA expressed in MOLM14 cells. Inhibition of this high afffinity transporter for glutamine blocked mTORC1 stimulation by leucine and glutamine after deprivation and induced apoptosis in MOLM-14 cell line. SLC1A5 inhibition reduced tumor growth and improved survival in transplanted mice
... Gene cooperations Previously, an association and possible cooperation between muta-tions of genes involved in signal transduction in the tyrosine kinase re-ceptor RAS-BRAF pathway (class I mutations) and mutations of genes for hematopoietic transcription factors (class II mutations) was demon-strated. 32,33 If considering the NPM1 gene as a transcription factor, as it is DNA binding and has not been shown involved in signal transduction, in total 33 of our 140 patients with t-MDS or t-AML presented class I mutations and 58 patients presented class II mutations. An association between class I and class II mutations was observed in 24 patients (χ 2 = 15.78, ...
Alternative genetic pathways for t-MDS and t-AML were previously suggested based on characteristic chromosome aberrations identical with those observed in de novo MDS and AML. The recurrent balanced translocations and inversions of these diseases in most cases result in chimeric rearrangement and inactivation of genes for hematopoietic transcription factors (class II mutations) which disturb cellular differen-tiation. Recently, activating point mutations or internal tandem duplications of genes for signal transduction in the receptor tyrosine kinase - RAS/BRAF pathway (class I mutations) have gained interest in de novo MDS and AML. A synergism between class I and class II mutations in the development of AML has been suggested. This hypothesis is now supported by our investigations of 140 unselected patients with t-MDS or t-AML for class I and class II mutations. A clustering of class I mutations in the different genetic pathways support the model for leukemic transformation.
... Disruption of this balance can result in pathologies characterised by an overproduction of progenitor cells and/or a lack of differentiation into mature cell lineages. Importantly it has been demonstrated in mouse models that a combination of a differentiation suppression signal along with a growth factor, proliferation/survival signal, can reconstitute leukaemia (Deguchi and Gilliland 2002). This requirement for cooperating lesions is a reflection of the multi-step process that is characteristic of leukaemia and many other cancers. ...
... It is well known that driver mutations may be caused by multiple pathways, such as cooccurring and possibly cooperative. For example, acute myeloid leukemia is caused by CBF translocations and kinase mutations [28]. So, we emphasize that assumption of mutual exclusivity occurs only in the same driver pathway. ...
With the development of next-generation DNA sequencing technologies, large-scale cancer genomics projects can be implemented to help researchers to identify driver genes, driver mutations, and driver pathways, which promote cancer proliferation in large numbers of cancer patients. Hence, one of the remaining challenges is to distinguish functional mutations vital for cancer development, and filter out the unfunctional and random "passenger mutations." In this study, we introduce a modified method to solve the so-called maximum weight submatrix problem which is used to identify mutated driver pathways in cancer. The problem is based on two combinatorial properties, that is, coverage and exclusivity. Particularly, we enhance an integrative model which combines gene mutation and expression data. The experimental results on simulated data show that, compared with the other methods, our method is more efficient. Finally, we apply the proposed method on two real biological datasets. The results show that our proposed method is also applicable in real practice.
... PML/RARalpha) producing a differentiation block, and the mutation of signaling molecules (e. g. FLT3) providing a proliferative or survival signal [49]. ...
FLT3, a member of the class III receptor tyrosine kinases (RTKs), is preferentially expressed on the cell surface of hematopoietic progenitors, and the ligand of FLT3 (FL) is expressed as a membrane-bound or soluble form by bone marrow stroma cells. It has been disclosed that FL-FLT3 interaction plays an important role in the maintenance, proliferation and differentiation of hematopoiesis. FLT3 is also expressed in a high proportion of acute myeloid leukemia (AML) and B-lineage acute lymphoblastic leukemia cells. Activating mutations of FLT3 are the most frequent genetic lesions in AML, and AML patients with FLT3 mutations have a worse prognosis than those with normal FLT3. Exploring the mechanism by which FLT3 mutations cause autoactivation and uncontrolled signaling might lead to a better understanding of how FLT3 becomes oncogenic and provide insights for the development of new drugs.
... Mutations in FLT3 confer a proliferative advantage and are referred to as 'class I mutations'. In addition to class I mutations, leukemogenesis requires further genetic lesions such as mutations that lead to blocked hematopoietic differentiation ('class II mutations') (Deguchi and Gilliland, 2002;Ishikawa et al., 2009;Cancer Genome Atlas Research Network, 2013). Another group of class I mutations are activating mutations in RAS genes (Fröhling et al., 2005;Tyner et al., 2009). ...
FLT3 ITD (FMS-like tyrosine kinase 3 with internal tandem duplication) is an important oncoprotein in Acute Myeloid Leukemia (AML). Owing to its constitutive kinase activity FLT3 ITD accumulates partially at endomembranes, a feature shared with other disease-associated, mutated receptor tyrosine kinases. Since Ras proteins also transit through endomembranes we have investigated the possible existence of an intracellular FLT3 ITD/Ras signaling pathway by comparing Ras signaling of FLT3 ITD with that of wild-type FLT3. Ligand stimulation activated both K- and N-Ras in cells expressing wild-type FLT3. Life-cell Ras-GTP imaging revealed ligand-induced Ras activation at the plasma membrane (PM). FLT3 ITD dependent constitutive activation of K-Ras and N-Ras was also observed primarily at the PM, supporting the view that the PM-resident pool of FLT3 ITD engaged the Ras/Erk pathway in AML cells. Accordingly, specific interference with FLT3 ITD/Ras signaling at the PM using PM-restricted dominant negative K-RasS17N potently inhibited cell proliferation and promoted apoptosis, corroborating that Ras signalling is crucial for FLT3 ITD dependent cell transformation and confirming that FLT3 ITD addresses PM-bound Ras despite its pronounced mislocalization to endomembranes.
... It is well known that cancer genomes harbor driver mutations in multiple pathways, and the exclusivity assumption does not preclude the presence of such co-occurring, and possibly cooperative, driver mutations, examples of which are known [14,15]. Indeed, current estimates of the number of driver mutations and number of mutated pathways in a cancer genome are remarkably similar (≈ 10-15 [16,17]) suggesting that the assumption of approximately one driver mutation per pathway is not too strong of an assumption. ...
Background
Cancer sequencing projects are now measuring somatic mutations in large numbers of cancer genomes. A key challenge in interpreting these data is to distinguish driver mutations, mutations important for cancer development, from passenger mutations that have accumulated in somatic cells but without functional consequences. A common approach to identify genes harboring driver mutations is a single gene test that identifies individual genes that are recurrently mutated in a significant number of cancer genomes. However, the power of this test is reduced by: (1) the necessity of estimating the background mutation rate (BMR) for each gene; (2) the mutational heterogeneity in most cancers meaning that groups of genes (e.g. pathways), rather than single genes, are the primary target of mutations.
Results
We investigate the problem of discovering driver pathways, groups of genes containing driver mutations, directly from cancer mutation data and without prior knowledge of pathways or other interactions between genes. We introduce two generative models of somatic mutations in cancer and study the algorithmic complexity of discovering driver pathways in both models. We show that a single gene test for driver genes is highly sensitive to the estimate of the BMR. In contrast, we show that an algorithmic approach that maximizes a straightforward measure of the mutational properties of a driver pathway successfully discovers these groups of genes without an estimate of the BMR. Moreover, this approach is also successful in the case when the observed frequencies of passenger and driver mutations are indistinguishable, a situation where single gene tests fail.
Conclusions
Accurate estimation of the BMR is a challenging task. Thus, methods that do not require an estimate of the BMR, such as the ones we provide here, can give increased power for the discovery of driver genes.
... This review focuses on aspects relevant when individuals with cancer ask their physician about factors in the working environment that might be related to their blood disease and for hematologists seeking updated knowledge on benzeneinduced malignancies of the blood and blood-forming organs . Leukemogenesis is a multistep process that is believed to include a combination of mutated signal transduction and perturbed transcription factors [1,2] . The multifactorial origin of most types of cancer, including malignancies of the blood and blood-forming organs, creates difficulty in determining the contribution of single agents. ...
Benzene, an aromatic hydrocarbon that is a natural component of crude oil and natural gas, is toxic to the blood and blood-forming organs. Epidemiological studies have established an association between benzene exposure and acute myeloid leukemia, and increasing evidence also indicates a possible association between benzene and multiple myeloma. A specific benzene-associated myelodysplastic syndrome has also been suggested. Chronic hematotoxic effects of ben-zene exposure, including reduced lymphocyte, neutrophil and platelet counts in peripheral blood, have been detected at occupational exposure below a level that had previously been considered not to cause any health effects. Whether these abnormalities represent bone marrow damage and/or initial events in the development of a true neoplastic disease is not known. Together with a reported nonlinear relationship between benzene exposure and the level of various metabolites, favoring production of biologically reactive quinones at exposure below 1 part per million, these observations suggest that benzene even at low exposure levels may contribute to the risk of acute myeloid leukemia or myelodysplastic syndrome, especially among genetically susceptible individuals.
... It is well known that cancer genomes harbor driver mutations in multiple pathways, and the exclusivity assumption does not preclude the presence of such co-occurring, and possibly cooperative, driver mutations, examples of which are known [14,15]. Indeed, current estimates of the number of driver mutations and number of mutated pathways in a cancer genome are remarkably similar (≈ 10-15 [16,17]) suggesting that the assumption of approximately one driver mutation per pathway is not too strong of an assumption. ...
Cancer sequencing projects are now measuring somatic mutations in large numbers of cancer genomes. A key challenge in interpreting
these data is to distinguish driver mutations, mutations important for cancer development, from passenger mutations that have accumulated in somatic cells but without functional consequences. A common approach to identify genes
harboring driver mutations is a single gene test that identifies individual genes that are mutated in a significant number of cancer genomes. However, the power of this test
is reduced by the mutational heterogeneity in most cancer genomes and by the necessity of estimating the background mutation rate (BMR). We investigate the problem of discovering driver pathways, groups of genes containing driver mutations, directly from cancer mutation data and without prior knowledge of pathways
or other interactions between genes. We introduce two generative models of somatic mutations in cancer and study the algorithmic
complexity of discovering driver pathways in both models. We show that a single gene test for driver genes is highly sensitive
to the estimate of the BMR. In contrast, we show that an algorithmic approach that maximizes a straightforward measure of
the mutational properties of a driver pathway successfully discovers these groups of genes without an estimate of the BMR.
Moreover, this approach is also successful in the case when the observed frequencies of passenger and driver mutations are
indistinguishable, a situation where single gene tests fail.
... Another possibility is that this type of mutation may reflect specific mechanisms of selection or generation of this mutation in the presence of trisomy 21 (Rainis et al., 2003). According some evidences the arising of AL is due to the cooperation between one class of mutations which interferes with differentiation (class II mutations) and another class which confers a proliferative advantage to cells (class I mutations) (Deguchi & Gilliland, 2002). It has been shown that high level expression of exogenous GATA-1 lacking the N-terminus induced differentiation rather than decreased the aberrant growth of GATA1-null megakaryocytes (Kuhl et al., 2005;Muntean & Crispino, 2005). ...
... An accepted paradigm is that both cell intrinsic as well as extrinsic factors contribute to leukemia formation and its progression [4]. The cell intrinsic mutation theory for example suggests that at least two types of 'collaborative' hits are required to cause leukemia: one that confers a survival advantage for the cell (affecting for example a kinase gene), and a second defect resulting in a block in hematopoietic differentiation (exemplified by core binding factor) [5]. It is also widely accepted that during in leukemia, leukemic stem cells lodge into normal stem cell niches in the BM and initiate a crosstalk with their surrounding tissue, which might result initiation of the disease, repression of normal HSC functions, alter the lineage differentiation of leukemic cells or regulate the response to drugs67891011. ...
The molecular and cellular mechanisms of the age-associated increase in the incidence of acute myeloid leukemia (AML) remain poorly understood. Multiple studies support that the bone marrow (BM) microenvironment has an important influence on leukemia progression. Given that the BM niche itself undergoes extensive functional changes during lifetime, we hypothesized that one mechanism for the age-associated increase in leukemia incidence might be that an aged niche promotes leukemia progression. The most frequent genetic alteration in AML is the t(8;21) translocation, resulting in the expression of the AML1-ETO fusion protein. Expression of the fusion protein in hematopoietic cells results in mice in a myeloproliferative disorder. Testing the role of the age of the niche on leukemia progression, we performed both transplantation and in vitro co-culture experiments. Aged animals transplanted with AML1-ETO positive HSCs presented with a significant increase in the frequency of AML-ETO positive early progenitor cells in BM as well as an increased immature myeloid cell load in blood compared to young recipients. These findings suggest that an aged BM microenvironment allows a relative better expansion of pre-leukemic stem and immature myeloid cells and thus imply that the aged microenvironment plays a role in the elevated incidence of age-associated leukemia.
Childhood leukemia is mostly a ‘‘developmental accident’’ during fetal hematopoiesis and may require multiple prenatal and postnatal ‘‘hits’’. The World Health Organization defines transient leukemia of Down syndrome as increased peripheral blood blasts cells in neonates with Down syndrome and classifies this type of leukemia as a separate entity. Although it was shown that Down syndrome predisposes children to myeloid leukemia, neither the nature of the predisposition nor the associated genetic lesions have been defined. Acute myeloid leukemia of Down syndrome is a unique disease characterized by a long pre-leukemic, myelodysplastic phase, unusual chromosomal findings and a high cure rate. In the present manuscript, we present a comprehensive review of the literature about clinical and biological findings of transient leukemia of Down syndrome and link them with the genetic discoveries in the field. We address the manuscript to the pediatric generalist and especially to the next generation of pediatric hematologists.
Accumulating evidence suggests that dysregulation of Dishevelled-Axin domain-containing 1 (DIXDC1) is involved in the progression and development of various cancers. However, little is known about the relevance of DIXDC1 in acute myeloid leukemia (AML). In this study, we aimed to investigate the expression status and potential biological function of DIXDC1 in AML. Our results showed that DIXDC1 expression was highly upregulated in AML cell lines and primary AML blasts compared with normal blasts. Knockdown of DIXDC1 by siRNA-mediated gene silencing significantly inhibited proliferation, induced cell cycle arrest, and promoted apoptosis of AML cells in vitro. By contrast, DIXDC1 overexpression promoted proliferation, accelerated cell cycle progression, and reduced apoptosis of AML cells. Moreover, we found that DIXDC1 knockdown decreased the expression of β-catenin and restricted the activation of Wnt signaling. In addition, DIXDC1 knockdown decreased the expression of Wnt/β-catenin target genes, including cyclin D1 and c-myc, while DIXDC1 overexpression had the opposite effect. Notably, β-catenin knockdown partially reversed the oncogenic effect of DIXDC1 in AML cells. Taken together, these results demonstrate that DIXDC1 promotes the growth of AML cells, possibly through upregulating the Wnt/β-catenin signaling pathway. Our study suggests that DIXDC1 may serve as a potential therapeutic target for the treatment of AML.
La duplication interne en tandem au sein du gène du Fms-like tyrosine kinase 3 (FLT3) est l’une des mutations les plus fréquemment observées dans les leucémies aiguës myéloblastiques (LAM). Elle est corrélée à un mauvais pronostic. Des inhibiteurs de tyrosine kinase anti-FLT3 (FLT3-ITK) sont en cours de développement mais les premiers essais cliniques ont été décevants. Les rémissions sont de courte durée, et si une clairance leucémique sanguine est observée, la LAM persiste au sein de la moelle osseuse. Dans ce travail, nous avons démontré que les cytokines activatrices de STAT5, telles que l’interleukine-3 et la thrombopoïétine, et les basses pressions en oxygène, telles que celles observées au sein de la niche hématopoïétique augmentent l’expression et l’activité du récepteur tyrosine kinase AXL qui protège les cellules de LAM FLT3-ITD de l’apoptose induite par le FLT3-ITK quizartinib (AC220). Nous avons démontré dans un modèle murin que les cellules de LAM FLT3-ITD « knock-down » pour AXL sont plus sensibles au quizartinib, et que cette différence se révèle spécifiquement dans un modèle de prise de greffe hématopoïétique. La combinaison de stratégies inhibitrices du FLT3-ITD et d’AXL permettra d’améliorer l’efficacité des FLT3-ITK en atteignant la fraction de cellules responsable des rechutes, nichée dans son microenvironnement. A l’issue, nous avons démontré que le gilteritinib (ASP2215), double FLT3/AXL-ITK est plus efficace que le quizartinib pour atteindre ces cellules leucémiques médullaires. Enfin, nous avons démontré que la combinaison d’un anticorps monoclonal anti-AXL avec un FLT3-ITK ou de la cytarabine était une stratégie thérapeutique prometteuse dans les LAM FLT3-ITD ou sauvage.
Inherited bone marrow failure syndromes (IBMFS) are a heterogeneous group of genetic disorders characterized by bone marrow failure, congenital anomalies, and increased risk of malignant disease. IBMFS may affect all blood cell lineages, causing clinical symptoms similar to aplastic anemia, or they may be restricted to one or two blood cell lineages with symptoms specific to the affected cell lineage. Early and accurate diagnosis of the disease is important, as there are implications for management and long-term follow-up. However, diagnosis is often difficult due to the wide varieties of clinical presentation. Recent advances in our understanding of IBMFS have largely come from the identification of the causative genes and investigations of their pathways. In this chapter, advances in the pathobiology and clinical management of two representative diseases, Diamond-Blackfan anemia and Fanconi’s anemia, will be described.
Trisomy 21, the genetic hallmark of Down syndrome (DS), is the most frequent human chromosomal abnormality. In neonates with DS, about 5–10% develop transient abnormal myelopoiesis (TAM). Almost all cases of TAM have mutations in GATA1. In most cases, it resolves spontaneously within 3 months. However, early death occurs in about 20% of the cases. Furthermore, approximately 20% of TAM patients develop myeloid leukemia of DS (ML-DS) within 4 years of life. Human tumors have been shown to progress by the accumulation of genetic abnormalities. The malignant progression from TAM to ML-DS offers a unique model to study the stepwise development of cancer pathogenesis. Recent studies have provided fascinating insights into the pathogenesis of TAM, details that may provide insight not only into DS leukemia but also contribute to our understanding of the pathogenesis of other leukemias.
Hematopoietic stem cells (HSCs), which are responsible for producing all blood cell types, first appear in the early stage of embryonic development and transit through several different tissues, including the yolk sac, aorta-gonad-mesonephros (AGM) region, placenta, and fetal liver, before colonizing in the bone marrow where they reside throughout the individual’s life. HSCs, characterized by the ability to self-renew and generate all types of blood cells, are supported by their specific environment called niches and depend on many developmental signaling pathways, molecules, and cytokines for their generation, maintenance, and expansion. Any disruption in this well-balanced system may cause aberrant HSC production, leading to malignant hematopoiesis. Leukemic stem cells (LSCs), originally identified using xenograft models of acute myeloid leukemia (AML), are a distinct cell population that can initiate leukemia in immunodeficient mice. LSCs are thought to emerge from HSCs or hematopoietic progenitors after obtaining multiple genetic changes that provide aberrant growth advantage and self-renewal ability. The emergence of LSCs is a multi-step event, including genetic diversification and clonal selection, resulting in genetic heterogeneity among leukemic cells. LSCs generally exist in the immature CD34+CD38− leukemic population in most cases of AML and share some features with normal HSCs. However, recent studies have shown that in acute lymphoblastic leukemia (ALL), LSCs exist in B-lineage-committed progenitors expressing CD19. In contrast to that in AML, in which LSCs generate leukemic cells in a hierarchical order with LSCs at the top, leukemia propagation in ALL is better explained by a stochastic model.
Acute myeloid leukemia (AML) constitutes a group of diseases heterogeneous with regard to clinical course, response to therapy as well as genetic features that contribute to disease pathogenesis, progression, and outcome. Numerous molecular lesions found in AML patients serve as prognostic and predictive factors; some of these markers have been included in the latest WHO classification. The analysis of cryptic genomic changes and alterations of gene expression is particularly important in cytogenetically normal AML patients. NPM1 and biallelic CEBPA mutations are favorable prognostic factors, while MLL and FLT3 duplications, WT1, IDH1/IDH2, KIT, TET2, and DNMT3A mutations, as well as overexpression of BAALC, ERG, or MN1 have an adverse prognostic impact. Aside from aberrations involving structural genes, mutations of microRNAs, copy number alterations, and altered methylation of gene promoter regions also influence the development and progression of AML.
New-generation high-throughput technologies, including next-generation sequencing technology, have been extensively applied to solve biological problems. As a result, large cancer genomics projects such as the Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium are producing large amount of rich and diverse data in multiple cancer types.. The identification of mutated driver genes and driver pathways from these data is a significant challenge. Genome aberrations in cancer cells can be divided into two types: random ‘passenger mutation’ and functional ‘driver mutation’. In this paper, we introduced a Multi-objective Optimization model based on a Genetic Algorithm (MOGA) to solve the maximum weight submatrix problem, which can be employed to identify driver genes and driver pathways promoting cancer proliferation. The maximum weight submatrix problem defined to find mutated driver pathways is based on two specific properties, i.e., high coverage and high exclusivity. The multi-objective optimization model can adjust the trade-off between high coverage and high exclusivity. We proposed an integrative model by combining gene expression data and mutation data to improve the performance of the MOGA algorithm in a biological context.
In this paper, we described current knowledge of molecular aberrations in haematological disorders (myeloproliferative disorders, acute and chronic leukemia) - its detection usefulness at diagnosis and during the therapy. These rearrangements such as translocations, mutations and changes in gene expression level, lead to disregulation of proliferation, differentiation and apoptosis pathways. Some of them are risk factors and have therapeutic significance and others are used in monitoring of minimal residual disease. Detection of ABL gene mutations in patients with chronic myeloid leukaemia resistant to standard therapy with tyrosine kinase inhibitors can result in treatment modification. Molecular methods used for haematopoietic chimerism analysis are important diagnostic tools used for engraftment assessment of both: sex - matched and mismatched allogeneic haematopoietic stem cells.
Acute myeloid leukemia (AML) is a genetically heterogeneous disorder of the hematopoietic system marked by maturation arrest and proliferation of leukemic cells in the bone marrow. Although most patients can be induced into a remission with initial chemotherapy, the majority is destined to relapse, with most relapsed patients eventually succumbing to their disease. The reservoir for relapse has been postulated to be a pool of "leukemic stem cells" (LSC), which have the capability to self-renew, giving rise to both new LSCs and non-self renewing progeny to recapitulate the full phenotypic and functional diversity of the primary leukemia. It is thought that LSCs resist chemotherapy by multiple mechanisms including increased quiescence, the expression of multi-drug resistance pumps, and residence in a protective bone marrow (BM) microenvironment. To date, direct evidence that LSCs represent the chemo-resistant population in AML is limited, and much work remains to more fully characterize this largely conceptual cell type in the pathogenesis of treatment resistance and disease relapse.
Leukemia is a hematopoietic cancer that is characterized by the abnormal differentiation and proliferation of hematopoietic cells. It is ranked 7th by death rate among cancer types in USA, even though it is not one of the top 10 cancers by incidence (USCS, 2010). This indicates an urgent need for more effective treatment strategies. In order to design the new ways of prevention and treatment of leukemia, it is important to understand the molecular mechanisms involved in development of the disease.
In this study, we investigated mechanisms involved in the development of acute myeloid leukemia (AML) that is associated with CBF fusion genes. The RUNX1 and CBFB genes that encode subunits of a transcriptional regulator complex CBF, are mutated in a subset (20 – 25%) of AML cases. As a result of these mutations, fusion genes called CBFB-MYH11 and RUNX1-ETO arise. The chimeric proteins encoded by the fusion genes provide block in proliferation for myeloid progenitors, but are not sufficient for AML development. Genetic studies have indicated that activation of cytokine receptor signaling is a major oncogenic pathway that cooperates in leukemia development. The main goal of my work was to determine a role of two factors that regulate cytokine signaling activity, the microRNA cluster miR-17-92 and the thrombopoietin receptor MPL, in their potential cooperation with the CBF fusions in AML development.
We determined that the miR-17-92 miRNA cluster cooperates with Cbfb-MYH11 in AML development in a mouse model of human CBFB-MYH11 AML. We found that the miR-17-92 cluster downregulates Pten and activates the PI3K/Akt pathway in the leukemic blasts. We also demonstrated that miR-17-92 provides an anti-apoptotic effect in the leukemic cells, but does not seem to affect proliferation. The anti-apoptotic effect was mainly due to activity of miR-17 and miR-20a, but not miR-19a and miR-19b.
Our second study demonstrated that wild type Mpl cooperated with RUNX1-ETO fusion in development of AML in mice. Mpl induced PI3K/Akt, Ras/Raf/Erk and Jak2/Stat5 signaling pathways in the AML cells. We showed that PIK3/Akt pathway plays a role in AML development both in vitro and in vivo by increasing survival of leukemic cells. The levels of MPL transcript in the AML samples correlated with their response to thrombopoietin (THPO). Moreover, we demonstrated that MPL provides pro-proliferative effect for the leukemic cells, and that the effect can be abrogated with inhibitors of PI3K/AKT and MEK/ERK pathways.
Taken together, these data confirm important roles for the PI3K/AKT and RAS/RAF/MEK pathways in the pathogenesis of AML, identifies two novel genes that can serve as secondary mutations in CBF fusions-associated AML, and in general expands our knowledge of mechanisms of leukemogenesis.
Introduction The secondary genetic changes other than the PML-RARA fusion gene may contribute to the acute promyelocytic leukemogenesis. Chromosomal alterations and mutation of FLT3 tyrosine kinase receptor gene are the frequent genetic alterations in acute myeloid leukemia (AML). However, the prognostic significance of FLT3 mutations in acute promyelocytic leukemia (APL) is not firmly established. Methods and materials: Cytogenetic analysis of 45 APL patient's BM samples, FLT3 ITD screening by fragment length analysis and FLT3 D835 mutation by melting curve analysis in 23 APL samples was screened in this study. Results: Cytogenetic study showed 14.3% trisomy 8 and 17.1% chromosomal abnormalities other than t(15;17). About 13% of the patients had FLT3 internal tandem duplications (ITDs), and 26% had D835 point mutation. FLT3 ITD mutation was associated with higher white blood cell (WBC) count at presentation and poor prognosis. Conclusion: As the PML-RARA is not sufficient to develop APL, we assume FLT3 mutations and additional chromosomal alterations we found in this APL series may cooperate with PML-RARA in APL development.
Acute myeloid leukemia (AML) is a clFinically and biologically heterogeneous group of neoplasms found in both the adult and pediatric populations. Many of the mutations that underlie AML pathogenesis have been elucidated and include both large-scale genomic events such as chromosomal additions, deletions, and translocations, as well as small-scale point mutations in tumorigenic genes. As patient prognosis and therapeutic decisions are largely determined by these genetic events, multifaceted genetic analysis of tissue sample from AML patients is required for proper diagnosis. Thus, an understanding of both the genetic events associated with AML and the testing modalities that assess these aberrations is essential for appropriate sample evaluation. Additionally, recognition of the molecular differences between adult and pediatric AML may provide insights into critical pathways involved in AML pathogenesis.
Lymphohematopoietic neoplasia are one of the most common types of cancer induced by therapeutic and environmental agents. Of the more than 100 human carcinogens identified by the International Agency for Research on Cancer, approximately 25% induce leukemias or lymphomas. The objective of this review is to provide an introduction into the origins and mechanisms underlying lymphohematopoietic cancers induced by xenobiotics in humans with an emphasis on acute myeloid leukemia, and discuss the implications of this information for risk assessment. Among the agents causing lymphohematopoietic cancers, a number of patterns were observed. Most physical and chemical leukemia-inducing agents such as the therapeutic alkylating agents, topoisomerase II inhibitors, and ionizing radiation induce mainly acute myeloid leukemia through DNA-damaging mechanisms that result in either gene or chromosomal mutations. In contrast, biological agents and a few immunosuppressive chemicals induce primarily lymphoid neoplasms through mechanisms that involve alterations in immune response. Among the environmental agents examined, benzene was clearly associated with acute myeloid leukemia in humans, with increasing but still limited evidence for an association with lymphoid neoplasms. Ethylene oxide and 1-3-butadiene were linked primarily to lymphoid cancers. Although the association between formaldehyde and leukemia remains controversial, several recent evaluations have indicated a potential link between formaldehyde and acute myeloid leukemia. The four environmental agents examined in detail were all genotoxic, inducing gene mutations, chromosomal alterations, and/or micronuclei in vivo. Although it is clear that rapid progress has been made in recent years in our understanding of leukemogenesis, many questions remain for future research regarding chemically induced leukemias and lymphomas, including the mechanisms by which the environmental agents reviewed here induce these diseases and the risks associated with exposures to such agents.
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AML (Acute myeloid leukemia) is a form of blood cancer where growth of myeloid cells occurs in the bone marrow. The prognosis is poor in general for many reasons. One is the presence of leukaemia-specific recognition markers such as FLT3 (fms-like tyrosine kinase 3). Another name of FLT3 is stem cell tyrosine kinase-1 (STK1), which is known to take part in proliferation, differentiation and apoptosis of hematopoietic cells, usually being present on haemopoietic progenitor cells in the bone marrow. FLT3 act as an independent prognostic factor for AML. Although a vast literature is available about the association of FLT3 with AML there still is a need of a brief up to date overview which draw a clear picture about this association and their effect on overall survival.
This chapter provides updated information on the myelodysplastic syndromes (MDS) and the myelodysplastic/myeloproliferative
neoplasms (MDS/MPN). The different biological features are illustrated in their variable clinical presentation, morphologic
characteristics, molecular genetic findings, and flow cytometric anomalies. The diagnostic criteria recommended by the International
Working Group (IWG) and disease classification by World Health Organization (WHO) are incorporated into the discussion of
diagnostic approaches to MDS and MDS/MPD. Specifically, the significance and diagnostic challenges of some unusual forms of
MDS and MDS/MPD are discussed, including MDS with fibrosis, hypoplastic MDS, MDS with a PNH clone, MDS with minimal dysplasia,
MDS with erythroid predominance or aplasia, and morphological assessment following treatment.
KeywordsMyelodysplastic syndrome (MDS), minimal diagnostic criteria–Myelodysplastic syndrome, international working group–Abnormal localization of immature precursors (ALIP)–Bone marrow, dysplastic features–Ringed sideroblasts, definition–Myelodysplastic syndrome, cytogenetic findings–Myelodysplastic syndrome, flow cytometry–Myelodysplastic syndrome, international prognostic scoring system (IPSS)–Myelodysplastic syndrome, immune-mediated–Myelodysplastic/myeloproliferative neoplasms (MDS/MPN)–Idiopathic cytopenia with uncertain (undetermined) significance (ICUS)–Paroxysmal nocturnal hemoglobinuria (PNH)–Aplastic anemia–Myelodysplastic syndrome, hypocellular–Myelodysplastic syndrome, fibrotic–Chronic myelomonocytic leukemia (CMML)–Chronic myeloid leukemia, atypical (aCML)–Refractory anemia with ring sideroblasts and marked thrombocytosis (RARS-T)
The secondary genetic changes other than the promyelocytic leukemia-retinoic acid receptor (PML-RARA) fusion gene may contribute to the acute promyelocytic leukemogenesis. Chromosomal alterations and mutation of FLT3 (FMS-like tyrosine kinase 3) tyrosine kinase receptor are the frequent genetic alterations in acute myeloid leukemia. However, the prognostic significance of FLT3 mutations in acute promyelocytic leukemia (APL) is not firmly established.
In this study, the chromosomal abnormalities were analyzed by bone marrow cytogenetic in 45 APL patients and FLT3 internal tandem duplications (ITD) screening by fragment length analysis and FLT3 D835 mutation by melting curve analysis were screened in 23 APL samples.
Cytogenetic study showed 14.3% trisomy 8 and 17.1% chromosomal abnormalities other than t(15;17). About 13% of the patients had FLT3 ITD, and 26% had D835 point mutation. FLT3 ITD mutation was associated with higher white blood cell count at presentation and poor prognosis.
The PML-RARA translocation alone may not be sufficient to induce leukemia. Therefore, we assume that FLT3 mutations and the other genetic and chromosomal alterations may cooperate with PML-RARA in the development of APL disease.
Sustained clinical cytopenia is a frequent laboratory finding in ambulatory and hospitalized patients. For pathologists and hematopathologists who examine the bone marrow (BM), a diagnosis of cytopenia secondary to an infiltrative BM process or acute leukemia can be readily established based on morphologic evaluation and flow cytometry immunophenotyping. However, it can be more challenging to establish a diagnosis of myelodysplastic syndrome (MDS). In this article, the practical approaches for establishing or excluding a diagnosis of MDS (especially low-grade MDS) in patients with clinical cytopenia are discussed along with the current diagnostic recommendations provided by the World Health Organization and the International Working Group for MDS.
Chronic myelogenous leukemia (CML), a malignancy of a hematopoietic stem cell, is caused by the Bcr-Abl tyrosine kinase. STI571(formerly CGP 57148B), an Abl tyrosine kinase inhibitor, has specific in vitro antileukemic activity against Bcr-Abl–positive cells and is currently in Phase II clinical trials. As it is likely that resistance to a single agent would be observed, combinations of STI571 with other antileukemic agents have been evaluated for activity against Bcr-Abl–positive cell lines and in colony-forming assays in vitro. The specific antileukemic agents tested included several agents currently used for the treatment of CML: interferon-alpha (IFN), hydroxyurea (HU), daunorubicin (DNR), and cytosine arabinoside (Ara-C). In proliferation assays that use Bcr-Abl–expressing cells lines, the combination of STI571 with IFN, DNR, and Ara-C showed additive or synergistic effects, whereas the combination of STI571 and HU demonstrated antagonistic effects. However, in colony-forming assays that use CML patient samples, all combinations showed increased antiproliferative effects as compared with STI571 alone. These data indicate that combinations of STI571 with IFN, DNR, or Ara-C may be more useful than STI571 alone in the treatment of CML and suggest consideration of clinical trials of these combinations.
The chromosomal translocation, t(8;21), is found frequently in acute myeloid leukemia (AML) with maturation (FAB-M2). We have previously mapped the translocation breakpoints of t(8;21) in a specific intron of the AML1 gene on chromosome 21. In this study, we cloned cDNAs synthesized from a cell line carrying t(8;21) by reverse transcription polymerase chain reaction (RT-PCR) using an AML1-specific primer. The analysis of the cDNAs structure has led to the identification of the fusion of AML1 with a gene named MTG8 on chromosome 8, which seems to be identical to ETO. Northern analysis using MTG8 (ETO) probes detected 7.8-kb and 6.2-kb RNAs and several minor RNAs in the cell line with t(8;21), but failed to detect any transcripts in a cell line without t(8;21). A set of primers were designed to detect the AML1/MTG8(ETO) fusion by PCR. The PCR amplified identical products in all 6 patients and one cell line with t(8;21), suggesting that the AML1/MTG8(ETO) fusion is a constant feature associated with t(8;21) and the junctions of the AML1/MTG8(ETO) fusion are restricted in a unique site. Because the PCR detection of the AML1/MTG8(ETO) fusion at the RNA level is highly sensitive, it can be used as a sensitive method for diagnosis and detection of minimal residual disease in t(8;21) leukemia.
The CBFβ subunit is the non-DNA-binding subunit of the heterodimeric core-binding factor (CBF). CBFβ associates with DNA-binding CBFα subunits and increases their affinity for DNA. Genes encoding the CBFβ subunit (CBFB) and one of the CBFα subunits (CBFA2, otherwise known as AML1) are the most frequent targets of chromosomal translocations in acute leukemias in humans. We and others previously demonstrated that homozygous disruption of the mouse Cbfa2 (AML1) gene results in embryonic lethality at midgestation due to hemorrhaging in the central nervous system and blocks fetal liver hematopoiesis. Here we demonstrate that homozygous mutation of the Cbfb gene results in the same phenotype. Our results demonstrate that the CBFβ subunit is required for CBFα2 function in vivo.
FLT3 is a member of receptor tyrosine kinases expressed in leukemia cells, as well as in hematopoietic stem cells. Recently, a somatic alteration of the FLT3 gene was found in acute myeloid leukemia, as an internal tandem duplication (FLT3/ITD) which caused elongation of the juxtamembrane (JM) domain of FLT3. Here we characterized the FLT3/ITD and investigated its clinical significance in acute promyelocytic leukemia (APL). Seventy-four newly diagnosed patients with APL, who were treated with the same protocol in a multi-institutional study, were studied for the FLT3/ITD. Genomic and message sequences of the FLT3 gene were amplified by means of polymerase chain reaction (PCR), and elongated PCR products were sequenced. Fifteen patients (20.3%) had FLT3/ITD, all of which were transcribed in frame. Location of the duplicated fragments (six to 30 amino acids) varied from patient to patient. However, they always contained either Y591 or Y599, but the tyrosine kinase domain was not significantly affected. This finding implied that signal transduction of FLT3 is amplified by the duplication. Clinically, the presence of FLT3/ITD was related to high peripheral white blood cell counts as well as peripheral leukemia cell counts (P < 0.0001), high LDH level (P = 0.04), and low fibrinogen concentration (P = 0.04). These data suggest that FLT3/ITD plays a significant role in progression of APL.
Nuclear receptor corepressor (CoR)-histone deacetylase (HDAC) complex recruitment is indispensable for the biological activities
of the retinoic acid receptor fusion proteins of acute promyelocytic leukemias. We report here that ETO (eight-twenty-one
or MTG8), which is fused to the acute myelogenous leukemia 1 (AML1) transcription factor in t(8;21) AML, interacts via its
zinc finger region with a conserved domain of the corepressors N-CoR and SMRT and recruits HDAC in vivo. The fusion protein
AML1-ETO retains the ability of ETO to form stable complexes with N-CoR/SMRT and HDAC. Deletion of the ETO C terminus abolishes
CoR binding and HDAC recruitment and severely impairs the ability of AML1-ETO to inhibit differentiation of hematopoietic
precursors. These data indicate that formation of a stable complex with CoR–HDAC is crucial to the activation of the leukemogenic
potential of AML1 by ETO and suggest that aberrant recruitment of corepressor complexes is a general mechanism of leukemogenesis.
t(8;21) is one of the most frequent translocations associated with acute myeloid leukemia. It produces a chimeric protein,
acute myeloid leukemia-1 (AML-1)–eight-twenty-one (ETO), that contains the amino-terminal DNA binding domain of the AML-1
transcriptional regulator fused to nearly all of ETO. Here we demonstrate that ETO interacts with the nuclear receptor corepressor
N-CoR, the mSin3 corepressors, and histone deacetylases. Endogenous ETO also cosediments on sucrose gradients with mSin3A,
N-CoR, and histone deacetylases, suggesting that it is a component of one or more corepressor complexes. Deletion mutagenesis
indicates that ETO interacts with mSin3A independently of its association with N-CoR. Single amino acid mutations that impair
the ability of ETO to interact with the central portion of N-CoR affect the ability of the t(8;21) fusion protein to repress
transcription. Finally, AML-1/ETO associates with histone deacetylase activity and a histone deacetylase inhibitor impairs
the ability of the fusion protein to repress transcription. Thus, t(8;21) fuses a component of a corepressor complex to AML-1
to repress transcription.
We have developed a restriction map of the chromosome 21 breakpoint region involved in t(8;21)(q22;q22.3) acute myelogenous leukemia (AML) and have isolated a genomic junction clone containing chromosome 8 and 21 material. Using probes from these regions, rearrangements have been identified in each of nine cases of t(8;21) AML examined. In addition, we have isolated cDNA clones from a t(8;21) AML cDNA library that contain fused sequences from chromosome 8 and 21. The chromosome 8 component, referred to as ETO (for eight twenty-one), is encoded over a large genomic region, as suggested by the analysis of corresponding yeast artificial chromosomes (YACs). The DNA sequence of the chromosome 21 portion of the fusion transcript is derived from the normal AML1 gene. A striking similarity (67% identity over 387 bp, with a corresponding 69% amino acid identity) was detected between AML1 and the Drosophila segmentation gene, runt. The critical consequence of the translocation is the juxtaposition of 5' sequences of AML1 to 3' sequences of ETO, oriented telomere to centromere on the der(8) chromosome.
The chromosome translocation forming the hybrid bcr-abl gene is thought to be the initiating event in chronic myeloid leukaemia (CML) and some cases of acute lymphoblastic leukaemia. To assess the impact of bcr-abl upon haemopoiesis, lethally irradiated mice were reconstituted with bone marrow cells enriched for cycling stem cells and infected with a bcr-abl bearing retrovirus. The mice developed several fatal diseases with abnormal accumulations of macrophage, erythroid, mast and lymphoid cells, and marked strain differences in disease distribution and kinetics. Some mice exhibited more than one neoplastic cell type and, in some instances, these were clonally related, indicating that a progenitor or stem cell had been transformed. While classical CML was not observed, the macrophage tumours were accompanied by a mild CML-like syndrome, probably due to myeloid growth factor production by tumour cells. The erythroid and mast cell diseases were rarely transplantable, in contrast to the macrophage tumours and lymphomas, but all disease types displayed limited clonality. These results establish that bcr-abl confers a proliferative advantage on diverse haemopoietic cells but complete transformation probably involves additional genetic changes.
Although acute promyelocytic leukemias (APLs) are consistently associated with a reciprocal chromosome 15;17 translocation, the gene(s) directly affected by the breakpoints have never been isolated. The chromosome 17 breakpoint maps to near the retinoic acid receptor alpha (RAR alpha) locus. Investigation of 20 APLs and a large series of other neoplastic patients and normal controls revealed RAR alpha gene rearrangements and aberrant transcripts only in the APL cases. These findings suggest that the RAR alpha gene is involved in the APL chromosome 17 breakpoint, is implicated in leukemogenesis, and could be used as a marker for identifying leukemic promyelocytes.
Analysis of a growing number of chromosomal translocations in human tumors have shown that they frequently result in gene fusions encoding chimeric proteins. We have characterized the recurrent t(12;21)(p12;q22) translocation present in human B-lineage acute leukemias. This translocation fused two genes, tel and AML1, that have previously been described in chromosomal translocations specific for myeloid malignancies. These two genes therefore belong to an increasing number of human genes that are involved in a variety of hematopoietic malignant disorders and can be rearranged with numerous partners. Interestingly, in these acute leukemias, deletion of the other tel allele from the normal chromosome 12 was associated with the tel rearrangement, whereas both tel alleles were present in the chronic leukemias bearing a t(5;12) that we have tested.
The AML-1/CBF beta transcription factor complex is targeted by both the t(8;21) and the inv(16) chromosomal alterations, which
are frequently observed in acute myelogenous leukemia. AML-1 is a site-specific DNA-binding protein that recognizes the enhancer
core motif TGTGGT. The t(8;21) translocation fuses the first 177 amino acids of AML-1 to MTG8 (also known as ETO), generating
a chimeric protein that retains the DNA-binding domain of AML-1. Analysis of endogenous AML-1 DNA-binding complexes suggested
the presence of at least two AML-1 isoforms. Accordingly, we screened a human B-cell cDNA library and isolated a larger, potentially
alternatively spliced, form of AML1, termed AML1B. AML-1B is a protein of 53 kDa that binds to a consensus AML-1-binding site
and complexes with CBF beta. Subcellular fractionation experiments demonstrated that both AML-1 and AML-1/ETO are efficiently
extracted from the nucleus under ionic conditions but that AML-1B is localized to a salt-resistant nuclear compartment. Analysis
of the transcriptional activities of AML-1, AML-1B, and AML-1/ETO demonstrated that only AML-1B activates transcription from
the T-cell receptor beta enhancer. Mixing experiments indicated that AML-1/ETO can efficiently block AML-1B-dependent transcriptional
activation, suggesting that the t(8;21) translocation creates a dominant interfering protein.
In Philadelphia chromosome-positive human leukemias, the c-abl proto-oncogene on chromosome 9 becomes fused to the bcr gene
on chromosome 22, and chimeric Bcr-Abl proteins are produced. The fused Bcr sequences activate the tyrosine kinase, actin-binding,
and transforming functions of Abl. Activation of the Abl transforming function has been shown to require two distinct domains
of Bcr: domain 1 (Bcr amino acids 1 to 63) and domain 2 (Bcr amino acids 176 to 242). The amino acid sequence of domain 1
indicates that it may be a coiled-coil oligomerization domain. We show here that domain 1 of Bcr forms a homotetramer. Tetramerization
of Bcr-Abl through Bcr domain 1 correlates with activation of the tyrosine kinase and F-actin-binding functions of Abl. Disruption
of the coiled coil by insertional mutagenesis inactivates the oligomerization function as well as the ability of Bcr-Abl to
transform Rat-1 fibroblasts or to abrogate interleukin-3 dependence in lymphoid cells. These results strongly suggest that
Bcr-Abl oligomers are the active entities in transformation.
The t(3;21)(q26;q22) translocation, which is one of the consistent chromosomal abnormalities found in blastic crisis of chronic myelocytic leukemia (CML), is thought to play an important role in the leukemic progression of CML to an acute blastic crisis phase. The AML1 gene, which is located at the translocation breakpoint of the t(8;21)(q22;q22) translocation found in acute myelocytic leukemia, was also rearranged by the t(3;21)(q26;q22) translocation. Screening of a cDNA library of the t(3;21)-carrying leukemic cell line cells (SKH1) resulted in the isolation of two potentially complete AML1-EVI-1 chimeric cDNAs of 6 kb. Two species of AML1-EVI-1 fusion transcripts of 8.2 and 7.0 kb were detected in SKH1 cells. These cells expressed the 180 kDa AML1-EVI-1 fusion protein containing an N-terminal half of AML1 including a runt homology domain which is fused to the entire zinc finger EVI-1 protein. The AML1-EVI-1 fusion transcript was consistent in all three cases of the t(3;21)-carrying leukemia examined by RNA-based PCR. These findings strongly suggest that the t(3;21) translocation results in the formation of a new class of chimeric transcription factor which could contribute to the leukemic progression of CML through interference with cell growth and differentiation.
The t(8;21) translocation is one of the most frequent chromosome abnormalities in acute myeloid leukemia. It has been shown that the t(8;21) breakpoints on chromosome 21 cluster within a single specific intron of the AML1 gene, which is highly homologous to the Drosophila segmentation gene runt. Here we report that this translocation juxtaposes the AML1 gene with a novel gene, named MTG8, on chromosome 8, resulting in the synthesis of an AML1-MTG8 fusion transcript. The fusion protein predicted by the AML1-MTG8 transcript consists of the runt homology region of AML1 and the most part of MTG8, which contains putative zinc finger DNA binding motifs and proline-rich regions constituting a characteristic feature of transcription factors. The MTG8 gene is not expressed in normal hematopoietic cells, whereas AML1 is expressed at high levels. Our results indicate that the production of chimeric AML1-MTG8 protein, probably a chimeric transcription factor, may contribute to myeloid leukemogenesis.
The chromosomal translocation, t(8;21), is found frequently in acute myeloid leukemia (AML) with maturation (FAB-M2). We have previously mapped the translocation breakpoints of t(8;21) in a specific intron of the AML1 gene on chromosome 21. In this study, we cloned cDNAs synthesized from a cell line carrying t(8;21) by reverse transcription polymerase chain reaction (RT-PCR) using an AML1-specific primer. The analysis of the cDNAs structure has led to the identification of the fusion of AML1 with a gene named MTG8 on chromosome 8, which seems to be identical to ETO. Northern analysis using MTG8 (ETO) probes detected 7.8-kb and 6.2-kb RNAs and several minor RNAs in the cell line with t(8;21), but failed to detect any transcripts in a cell line without t(8;21). A set of primers were designed to detect the AML1/MTG8(ETO) fusion by PCR. The PCR amplified identical products in all 6 patients and one cell line with t(8;21), suggesting that the AML1/MTG8(ETO) fusion is a constant feature associated with t(8;21) and the junctions of the AML1/MTG8(ETO) fusion are restricted in a unique site. Because the PCR detection of the AML1/MTG8(ETO) fusion at the RNA level is highly sensitive, it can be used as a sensitive method for diagnosis and detection of minimal residual disease in t(8;21) leukemia.
Translocations involving chromosome band 11q23 are very frequent in both acute lymphoblastic and acute myeloid leukemias and are the most common genetic alteration in infants with leukemia. In all age groups and all phenotypes of leukemia, an 11q23 translocation carries a poor prognosis. A major question has been whether one or several genes on band 11q23 are implicated in these leukemias. Previously, we identified the chromosomal breakpoint region in leukemias with the common 11q23 translocations and subsequently cloned a gene named MLL that spans the 11q23 breakpoint.
We isolated a 0.74-kb BamHI fragment from a complementary DAN (cDNA) clone of the MLL gene. To determine the incidence of MLL rearrangements in patients with 11q23 abnormalities, we analyzed DNA from 61 patients with acute leukemia, 3 cell lines derived from such patients, and 20 patients with non-Hodgkin's lymphoma and 11q23 aberrations.
The 0.74-kb cDNA probe detected DNA rearrangements in the MLL gene in 58 of the patients with leukemia, in the 3 cell lines, and in 3 of the patients with lymphoma. All the breaks occurred in an 8.3-kb breakpoint cluster region within the MLL gene. The probe identified DNA rearrangements in all 48 patients with the five common 11q23 translocations involving chromosomes 4, 6, 9, and 19, as well as in 16 patients with uncommon 11q23 aberrations. Twenty-one different chromosomal breakpoints involving the MLL gene were detected.
MLL gene rearrangements were detected with a single probe and a single restriction-enzyme digest in all DNA samples from patients with the common 11q23 translocations as well as in 16 patients or cell lines with other 11q23 anomalies. The ability to detect an MLL gene rearrangement rapidly and reliably, especially in patients with limited material for cytogenetic analysis, should make it possible to identify patients who have a poor prognosis and therefore require aggressive chemotherapy or marrow transplantation.
Expression of Hoxa7 and Hoxa9 is activated by proviral integration in BXH2 murine myeloid leukaemias. This result, combined with the mapping of the HOXA locus to human chromosome 7p15, suggested that one of the HOXA genes might be involved in the t(7;11)(p15;p15) translocation found in some human myeloid leukaemia patients. Here we show that in three patients with t(7;11), the chromosome rearrangement creates a genomic fusion between the HOXA9 gene and the nucleoporin gene NUP98 on chromosome 11p15. The translocation produces an invariant chimaeric NUP98/HOXA9 transcript containing the amino terminal half of NUP98 fused in frame to HOXA9. These studies identify HOXA9 as an important human myeloid leukaemia gene and suggest an important role for nucleoporins in human myeloid leukaemia given that a second nucleoporin, NUP214, has also been implicated in human myeloid leukaemia.
Chromosomal abnormalities of band 8p11 are associated with a distinct subtype of acute myeloid leukemia with French-American-British M4/5 morphology and prominent erythrophagocytosis by the blast cells. This subtype is usually associated with the t(8;16)(p11;p13), a translocation that has recently been shown to result in a fusion between the MOZ and CBP genes. We have cloned the inv(8)(p11q13), an abnormality associated with the same leukemia phenotype, and found a novel fusion between MOZ and the nuclear receptor transcriptional coactivatorTIF2/GRIP-1/NCoA-2. This gene has not previously been implicated in the pathogenesis of leukemia or other malignancies. MOZ-TIF2 retains the histone acetyltransferase homology domains of both proteins and also the CBP binding domain of TIF2. We speculate that the apparently identical leukemia cell phenotype observed in cases with the t(8;16) and the inv(8) arises by recruitment of CBP by MOZ-TIF2, resulting in modulation of the transcriptional activity of target genes by a mechanism involving abnormal histone acetylation.
Chromosomal abnormalities in acute leukemia have led to the discovery of many genes involved in normal hematopoiesis and in malignant transformation. We have identified the fusion partners in an inv(8)(p11q13) from a patient with acute mixed lineage leukemia. We show by fluorescence in situ hybridization (FISH) analysis, Southern blotting, and reverse transcriptase-polymerase chain reaction (RT-PCR) that the genes for MOZ, monocytic leukemiazinc finger protein, and TIF2,transcriptional intermediary factor 2, are involved in the inv(8)(p11q13). We demonstrate that the inversion creates a fusion between the 5′ end of MOZ mRNA and the 3′ end of TIF2 mRNA maintaining the translational frame of the protein. The predicted fusion protein contains the zinc finger domains, the nuclear localization domains, the histone acetyltransferase (HAT) domain, and a portion of the acidic domain ofMOZ, coupled to the CREB-binding protein (CBP) interaction domain and the activation domains of TIF2. The breakpoint is distinct from the breakpoint in the t(8;16)(p11;p13) translocation in acute monocytic leukemia with erythrophagocytosis that fuses MOZ with CBP. The reciprocalTIF2-MOZ fusion gene is not expressed, perhaps as a result of a deletion near the chromosome 8 centromere. TheMOZ-TIF2 fusion is one of a new family of chromosomal rearrangements that associate HAT activity, transcriptional coactivation, and acute leukemia.
© 1998 by The American Society of Hematology.
The TEL/PDGFβR fusion protein is expressed as the consequence of a recurring t(5;12) translocation associated with chronic myelomonocytic leukemia (CMML). Unlike other activated protein tyrosine kinases associated with hematopoietic malignancies, TEL/PDGFβR is invariably associated with a myeloid leukemia phenotype in humans. To test the transforming properties of TEL/PDGFβR in vivo, and to analyze the basis for myeloid lineage specificity in humans, we constructed transgenic mice with TEL/PDGFβR expression driven by a lymphoid-specific immunoglobulin enhancer-promoter cassette. These mice developed lymphoblastic lymphomas of both T and B lineage, demonstrating that TEL/PDGFβR is a transforming protein in vivo, and that the transforming ability of this fusion is not inherently restricted to the myeloid lineage. Treatment of TEL/PDGFβR transgenic animals with a protein tyrosine kinase inhibitor with in vitro activity against PDGFβR (CGP57148) resulted in suppression of disease and a prolongation of survival. A therapeutic benefit was apparent both in animals treated before the development of overt clonal disease and in animals transplanted with clonal tumor cells. These results suggest that small-molecule tyrosine kinase inhibitors may be effective treatment for activated tyrosine kinase–mediated malignancies both early in the course of disease and after the development of additional transforming mutations.
We have developed a restriction map of the chromosome 21 breakpoint region involved in t(8;21)(q22;q22.3) acute myelogenous leukemia (AML) and have isolated a genomic junction clone containing chromosome 8 and 21 material. Using probes from these regions, rearrangements have been identified in each of nine cases of t(8;21) AML examined. In addition, we have isolated cDNA clones from a t(8;21) AML cDNA library that contain fused sequences from chromosome 8 and 21. The chromosome 8 component, referred to as ETO (for eight twenty-one), is encoded over a large genomic region, as suggested by the analysis of corresponding yeast artificial chromosomes (YACs). The DNA sequence of the chromosome 21 portion of the fusion transcript is derived from the normal AML1 gene. A striking similarity (67% identity over 387 bp, with a corresponding 69% amino acid identity) was detected between AML1 and the Drosophila segmentation gene, runt. The critical consequence of the translocation is the juxtaposition of 5′ sequences of AML1 to 3′ sequences of ETO, oriented telomere to centromere on the der(8) chromosome.
The AML1 gene encodes a DNA-binding protein that contains the runt domain and is the most frequent target of translocations associated with human leukemias. Here, point mutations of the AML1 gene, V105ter (single-letter amino acid code) and R139G, (single-letter amino acid codes) were identified in 2 cases of myelodysplastic syndrome (MDS) by means of the reverse transcriptase–polymerase chain reaction single-strand conformation polymorphism method. Both mutations are present in the region encoding the runt domain of AML1 and cause loss of the DNA-binding ability of the resultant products. Of these mutants, V105ter has also lost the ability to heterodimerize with polyomavirus enhancer binding protein 2/core binding factor β (PEBP2β/CBFβ). On the other hand, the R139G mutant acts as a dominant negative inhibitor by competing with wild-type AML1 for interaction with PEBP2β/CBFβ. This study is the first report that describes mutations of AML1 in patients with MDS and the mechanism whereby the mutant acts as a dominant negative inhibitor of wild-type AML1.
Somatic mutations of the receptor tyrosine kinase Flt3 consisting of internal tandem duplications (ITD) occur in 20% of patients with acute myeloid leukemia. They are associated with a poor prognosis of the disease. In this study, we characterized the oncogenic potential and signaling properties of Flt3 mutations. We constructed chimeric molecules that consisted of the murine Flt3 backbone and a 510-base pair human Flt3 fragment, which contained either 4 different ITD mutants or the wild-type coding sequence. Flt3 isoforms containing ITD mutations (Flt3-ITD) induced factor-independent growth and resistance to radiation-induced apoptosis in 32D cells. Cells containing Flt3-ITD, but not those containing wild-type Flt3 (Flt3-WT), formed colonies in methylcellulose. Injection of 32D/Flt3-ITD induced rapid development of a leukemia-type disease in syngeneic mice. Flt3-ITD mutations exhibited constitutive autophosphorylation of the immature form of the Flt3 receptor. Analysis of the involved signal transduction pathways revealed that Flt3-ITD only slightly activated the MAP kinases Erk1 and 2 and the protein kinase B (Akt) in the absence of ligand and retained ligand-induced activation of these enzymes. However, Flt3-ITD led to strong factor-independent activation of STAT5. The relative importance of the STAT5 and Ras pathways for ITD-induced colony formation was assessed by transfection of dominant negative (dn) forms of these proteins: transfection of dnSTAT5 inhibited colony formation by 50%. Despite its weak constitutive activation by Flt3-ITD, dnRas also strongly inhibited Flt3-ITD–mediated colony formation. Taken together, Flt3-ITD mutations induce factor-independent growth and leukemogenesis of 32D cells that are mediated by the Ras and STAT5 pathways.
CGP 57148 is a compound of the 2-phenylaminopyrimidine class that selectively inhibits the tyrosine kinase activity of the ABL and the platelet-derived growth factor receptor (PDGFR) protein tyrosine kinases. We previously showed that CGP 57148 selectively kills p210BCR-ABL–expressing cells. To extend these observations, we evaluated the ability of CGP 57148 to inhibit other activated ABL tyrosine kinases, including p185BCR-ABL and TEL-ABL. In cell-based assays of ABL tyrosine phosphorylation, inhibition of ABL kinase activity was observed at concentrations similar to that reported for p210BCR-ABL. Consistent with the in vitro profile of this compound, the growth of cells expressing activated ABL protein tyrosine kinases was inhibited in the absence of exogenous growth factor. Growth inhibition was also observed with a p185BCR-ABL–positive acute lymphocytic leukemia (ALL) cell line generated from a Philadelphia chromosome–positive ALL patient. As CGP 57148 inhibits the PDGFR kinase, we also showed that cells expressing an activated PDGFR tyrosine kinase, TEL-PDGFR, are sensitive to this compound. Thus, this compound may be useful for the treatment of a variety of BCR-ABL–positive leukemias and for treatment of the subset of chronic myelomonocytic leukemia patients with a TEL-PDGFR fusion protein.
Chromosomal abnormalities in acute leukemia have led to the discovery of many genes involved in normal hematopoiesis and in malignant transformation. We have identified the fusion partners in an inv(8)(p11q13) from a patient with acute mixed lineage leukemia. We show by fluorescence in situ hybridization (FISH) analysis, Southern blotting, and reverse transcriptase-polymerase chain reaction (RT-PCR) that the genes for MOZ, monocytic leukemiazinc finger protein, and TIF2,transcriptional intermediary factor 2, are involved in the inv(8)(p11q13). We demonstrate that the inversion creates a fusion between the 5′ end of MOZ mRNA and the 3′ end of TIF2 mRNA maintaining the translational frame of the protein. The predicted fusion protein contains the zinc finger domains, the nuclear localization domains, the histone acetyltransferase (HAT) domain, and a portion of the acidic domain ofMOZ, coupled to the CREB-binding protein (CBP) interaction domain and the activation domains of TIF2. The breakpoint is distinct from the breakpoint in the t(8;16)(p11;p13) translocation in acute monocytic leukemia with erythrophagocytosis that fuses MOZ with CBP. The reciprocalTIF2-MOZ fusion gene is not expressed, perhaps as a result of a deletion near the chromosome 8 centromere. TheMOZ-TIF2 fusion is one of a new family of chromosomal rearrangements that associate HAT activity, transcriptional coactivation, and acute leukemia.
© 1998 by The American Society of Hematology.
The recurrent translocation t(11; 16)(q23; p13) has been reported to be associated with therapy-related acute leukemia. The MLL gene involved in other 11q23 abnormalities was also rearranged by this translocation. We analyzed two patients with myelodysplastic syndrome with t(11; 16) and showed that the MLL gene on 11q23 was fused with CREB-binding protein (CBP) gene on 16p13 in these patients. The CBP gene encodes a transcriptional adaptor/coactivator protein and it is mutated in patients with Rubinstein-Taybi syndrome. The CBP gene is also involved in acute myeloid leukemia (AML) with t(8; 16)(p11; p13). In-frame MLL-CBP fusion transcripts combine the MLL AT-hook motifs and DNA methyltransferase homology region with a largely intact CBP. Our results combined with the finding of the MOZ-CBP fusion in t(8; 16)-AML suggest that the CBP gene may be associated with leukemogenesis through translocations.
The AML1 gene encoding the DNA-binding alpha-subunit in the Runt domain family of heterodimeric transcription factors has been noted for its frequent involvement in chromosomal translocations associated with leukemia. Using reverse transcriptase-polymerase chain reaction (RT-PCR) combined with nonisotopic RNase cleavage assay (NIRCA), we found point mutations of the AML1 gene in 8 of 160 leukemia patients: silent mutations, heterozygous missense mutations, and biallelic nonsense or frameshift mutations in 2, 4, and 2 cases, respectively. The mutations were all clustered within the punt domain. Missense mutations identified in 3 patients showed neither DNA binding nor transactivation, although being active in heterodimerization. These defective missense mutants may be relevant to the predisposition or progression of leukemia. On the other hand, the biallelic nonsense mutants encoding truncated AML1 proteins lost almost all functions examined and may play a role in leukemogenesis leading to acute myeloblastic leukemia. (C) 1999 by The American Society of Hematology.
The P210bcr/abl protein is associated with virtually every case of human chronic myelogenous leukemia. Unlike the related P160gag/v-abl oncogene product of Abelson murine leukemia virus, P210bcr/abl does not transform NIH 3T3 fibroblasts. To assess whether P210bcr/abl might transform hematopoietic cell types, retroviral constructs encoding P210bcr/abl were used to infect the bone marrow-derived interleukin 3-dependent Ba/F3 cell line. As for P160gag/v-abl, cell lines expressing P210bcr/abl were growth factor independent and tumorigenic in nude mice. No evidence for autocrine production of interleukin 3 by factor-independent cell lines was found. These experiments establish that P210bcr/abl can transform hematopoietic cell types to tumorigenicity.
Although acute promyelocytic leukemias (APLs) are consistently associated with a reciprocal chromosome 15;17 translocation, the gene(s) directly affected by the breakpoints have never been isolated. The chromosome 17 breakpoint maps to near the retinoic acid receptor alpha (RAR alpha) locus. Investigation of 20 APLs and a large series of other neoplastic patients and normal controls revealed RAR alpha gene rearrangements and aberrant transcripts only in the APL cases. These findings suggest that the RAR alpha gene is involved in the APL chromosome 17 breakpoint, is implicated in leukemogenesis, and could be used as a marker for identifying leukemic promyelocytes.
Chronic myelomonocytic leukemia (CMML) is a myelodysplastic syndrome characterized by abnormal clonal myeloid proliferation, and by progression to acute myelogenous leukemia (AML). A recently recognized subgroup of CMML has a t(5;12) (q33;p13) balanced translocation. Fluorescence in situ hybridization (FISH) localized the translocation breakpoint near the CSF1 receptor (CSF1R) locus on chromosome 5q. Pulsed-field gel electrophoresis confirmed rearrangements near CSF1R, but involvement of CSF1R itself was excluded. Southern blotting showed a rearrangement within the closely linked PDGF receptor β (PDGFRβ) gene. Ribonuclease protection assays localized the translocation breakpoint to nucleotide 1766 in PDGFRβ RNA. Anchored PCR was used to identify the chromosome 12 fusion partner, a novel ets-like protein, tel. Tel contains a highly conserved carboxy terminal ets-like DNA-binding domain, and an amino terminal domain with a predicted helix-loop-helix (HLH) secondary structure. The consequence of the t(5;12) translocation is fusion of the tel HLH domain to the PDGFRβ transmembrane and tyrosine kinase domains. The tel HLH domain may contribute a dimerization motif which serves to constitutively activate PDGFRβ tyrosine kinase activity. The tel-PDGFRβ fusion demonstrates the oncogenic potential of PDGFRβ, and may provide a paradigm for early events in the pathogenesis of AML.
ObjectiveTEL/PDGFβR is a tyrosine kinase fusion protein associated with the pathogenesis of chronic myelomonocytic leukemia. The following experiments were undertaken to understand the mechanisms whereby TEL/PDGFβR transforms cells.
The transcription factor C/EBPα (for CCAAT/enhancer binding protein-α; encoded by the gene CEBPA) is crucial for the differentiation of granulocytes. Conditional expression of C/EBPα triggers neutrophilic differentiation, and no mature granulocytes are observed in Cebpa-mutant mice. Here we identify heterozygous mutations in CEBPA in ten patients with acute myeloid leukemia (AML). We found that five mutations in the amino terminus truncate the full-length protein, but did not affect a 30-kD protein initiated further downstream. The mutant proteins block wild-type C/EBPα DNA binding and transactivation of granulocyte target genes in a dominant-negative manner, and fails to induce granulocytic differentiation. Ours is the first report of CEBPA mutations in human neoplasia, and such mutations are likely to induce the differentiation block found in AML.
The t(7;11)(p15;p15) translocation is a recurrent aberration observed in acute myeloblastic leukaemia (AML) and chronic myelogenous leukaemia (CML). It has been shown that the NUP98 gene at 11p15 is fused with the HOXA9 gene at 7p15 in AML with t(7;11). We report the first case with CML expressing the NUP98/HOXA9 fusion transcript. A 27-year-old Japanese man was initially diagnosed as in the chronic phase of Philadelphia-positive CML. At the diagnosis of myeloid blast crisis, the karyotype evolved to 46, XY, t(7;11)(p15;p15), t(9;22)(q34;q11). Reverse transcriptase polymerase chain reaction identified the NUP98/HOXA9 transcript, suggesting that the NUP98/HOXA9 fusion protein could play a critical role in the progression to blast crisis.
The involvement of 11q23-balanced translocations in acute leukemia after treatment with drugs that inhibit the function of DNA topoisomerase II (topo II) is being recognized with increasing frequency. We and others have shown that the gene at 11q23 that is involved in all of these treatment-related leukemias is MLL (also called ALL1, Htrx, and HRX). In general, the translocations in these leukemias are the same as those occurring in de novo leukemia [eg, t(9;11), t(11;19), and t(4;11)], with the treatment-related leukemias accounting for no more than 5% to 10% of any particular translocation type. We have cloned the t(11;16)(q23;p13.3) and have shown that it involves MLL and CBP (CREB binding protein). The CBP gene was recently identified as a partner gene in the t(8;16) that occurs in acute myelomonocytic leukemia (AML-M4) de novo and rarely in treatment-related acute myeloid leukemia. We have studied eight t(11;16) patients, all of whom had prior therapy with drugs targetting topo II with fluorescence in situ hybridization (FISH) using a probe for MLL and a cosmid contig covering the CBP gene. Both probes were split in all eight patients and the two derivative (der) chromosomes were each labeled with both probes. Use of an approximately 100-kb PAC located at the breakpoint of chromosome 16 from one patient revealed some variability in the breakpoint because it was on the der(16) in three patients, on the der(11) in another, and split in four others. We assume that the critical fusion gene is 5'MLL/3'CBP. Our series of patients is unusual because three of them presented with a myelodysplastic syndrome (MDS) most similar to chronic myelomonocytic leukemia (CMMoL) and one other had dyserythropoiesis; MDS is rarely seen in 11q23 translocations either de novo or with t-AML. Using FISH and these same probes to analyze the lineage of bone marrow cells from one patient with CMMoL, we showed that all the mature monocytes contained the fusion genes as did some of the granulocytes and erythroblasts; none of the lymphocytes contained the fusion gene. The function of MLL is not well understood, but many domains could target the MLL protein to particular chromatin complexes. CBP is an adapter protein that is involved in regulating transcription. It is also involved in histone acetylation, which is thought to contribute to an increased level of gene expression. The fusion gene could alter the CBP protein such that it is constitutively active; alternatively, it could modify the chromatin-association functions of MLL.
The t(7;11)(p15;p15) translocation is a recurrent chromosomal abnormality associated primarily with acute myeloid leukaemia (FAB M2 and M4). We present here the molecular definition of this translocation. On chromosome 7 positional cloning revealed the consistent rearrangement of the HOXA9 gene, which encodes a class I homeodomain protein potentially involved in myeloid differentiation. On chromosome 11 the translocation targets the human homologue of NUP98, a member of the GLFG nucleoporin family. Chimaeric messages spliced over the breakpoint fuse the GLFG repeat domains of NUP98 in-frame to the HOXA9 homeobox. The predicted NUP98-HOXA9 fusion protein may promote leukaemogenesis through inhibition of HOXA9-mediated terminal differentiation and/or aberrant nucleocytoplasmic transport.
The TEL (ETV6)-AML1 (CBFA2) gene fusion is the most common reciprocal chromosomal rearrangement in childhood cancer occurring in approximately 25% of the most predominant subtype of leukemia- common acute lymphoblastic leukemia. The TEL-AML1 genomic sequence has been characterized in a pair of monozygotic twins diagnosed at ages 3 years, 6 months and 4 years, 10 months with common acute lymphoblastic leukemia. The twin leukemic DNA shared the same unique (or clonotypic) but nonconstitutive TEL-AML1 fusion sequence. The most plausible explanation for this finding is a single cell origin of the TEL-AML fusion in one fetus in utero, probably as a leukemia-initiating mutation, followed by intraplacental metastasis of clonal progeny to the other twin. Clonal identity is further supported by the finding that the leukemic cells in the two twins shared an identical rearranged IGH allele. These data have implications for the etiology and natural history of childhood leukemia.
Some acute lymphocytic leukaemias, particularly those in young children, are associated with a t(4;11)(q21;q23) reciprocal translocation. We have cloned the translocation breakpoint on chromosome 11q23 and isolated corresponding RNA transcripts from this region. The translocation occurs within a cluster of Alu repetitive elements located within an intron of a gene that gives rise to 11.5 (kb) transcript spanning the translocation breakpoint. The 11.5 kb transcript encodes a protein that is highly homologous to the Drosophila trithorax gene, a developmental regulator. An analysis of a series of leukaemic patients carrying t(4;11) and t(9;11) translocations indicate that the majority of breakpoints in infant leukaemias lie within a 5 kb region.
We have identified a human homolog of the Drosophila trithorax protein that is structurally altered by 11q23 translocations in acute leukemias. Human trithorax (HRX) is a predicted 431 kd protein containing two potential DNA-binding motifs consisting of zinc fingers conserved with the fly protein and nonconserved amino-terminal "AT hook" motifs related to the DNA-binding motifs in HMG proteins. 11q23 translocations disrupt the HRX gene between these two motifs, and in a t(11;19)-carrying cell line fusion transcripts are expressed from both derivative chromosomes. The more abundant derivative 11 transcript codes for a chimeric protein containing the AT hook motifs fused to a previously undescribed protein (ENL) from chromosome 19. These data suggest a novel role for a trithorax-homologous protein in multilineage human leukemias that may be mediated by DNA binding within the minor groove at AT-rich sites, implicated to play an important role in bacterial IHF-, yeast datin-, and mammalian HMG-mediated gene activation.
We have previously shown that the t(15;17) translocation specifically associated with acute promyelocytic leukemia (APL) fuses the retinoic acid receptor alpha (RAR alpha) locus to an as yet unknown gene, initially called myl and now renamed PML. We report here that this gene product contains a novel zinc finger motif common to several DNA-binding proteins. The PML-RAR alpha mRNA encodes a predicted 106 kd chimeric protein containing most of the PML sequences fused to a large part of RAR alpha, including its DNA- and hormone-binding domains. In transient expression assays, the hybrid protein exhibits altered transactivating properties if compared with the wild-type RAR alpha progenitor. Identical PML-RAR alpha fusion points are found in several patients. These observations suggest that in APL, the t(15;17) translocation generates an RAR mutant that could contribute to leukemogenesis through interference with promyelocytic differentiation.
A unique mRNA produced in leukemic cells from a t(15;17) acute promyelocytic leukemia (APL) patient encodes a fusion protein between the retinoic acid receptor alpha (RAR alpha) and a myeloid gene product called PML. PML contains a cysteine-rich region present in a new family of apparent DNA-binding proteins that includes a regulator of the interleukin-2 receptor gene (Rpt-1) and the recombination-activating gene product (RAG-1). Accordingly, PML may represent a novel transcription factor or recombinase. The aberrant PML-RAR fusion product, while typically retinoic acid responsive, displays both cell type- and promoter-specific differences from the wild-type RAR alpha. Because patients with APL can be induced into remission with high dose RA therapy, we propose that the nonliganded PML-RAR protein is a new class of dominant negative oncogene product. Treatment with RA would not only relieve this inhibition, but the activated PML-RAR protein may actually promote myelocyte differentiation.
The t(8;21)(q22;q22) translocation is a non-random chromosomal abnormality frequently found in patients with acute myeloid leukemia (AML) with maturation (M2 subtype). We report here the cloning of a gene, named AML1, on chromosome 21 that was found to be rearranged in the leukemic cell DNAs from t(8;21) AML patients. The breakpoints in 16 out of 21 patients were clustered within a limited region of AML1, and detailed analysis in 3 patients revealed that the breakpoints occurred in the same intron of the gene. Sequencing of cDNA clones identified a long open reading frame encoding a 250-amino acid protein. Northern blot analysis detected four constant mRNA species in t(8;21) leukemic and normal cells; the largest species was more abundant in the leukemic cells than in normal cells. In addition, two mRNA species limited to the leukemic cells were found. These findings indicate that the AML1 gene may be involved in neoplastic transformation of AML with the t(8;21) translocation.
A reciprocal chromosomal translocation, t(15;17)(q22;q11.2-12), is characteristic of acute promyelocytic leukemia (APL) of French-American-British (FAB) subtype M3, and is not associated with any other human malignancy. The non-random pattern of the APL translocations suggests that specific genes on chromosomes 15 and 17 are somehow altered or deregulated as a consequence of the rearrangement. Translocation breakpoints in APL patients provide physical landmarks that suggest an approach to isolating the APL gene(s). Genetic and physical maps constructed for the APL breakpoint region on chromosome 17 have indicated that two fully-linked DNA markers, defining loci for THRA1 and D17S80, map to opposite sides of an APL breakpoint yet reside on a common 350-kb Clal fragment. Cosmid-walking experiments to clone this APL breakpoint have revealed a 38-kilobase deletion on chromosome 17. Studies in additional APL patients have shown that the breakpoint region on chromosome 17 spans at least 80 kilobases.
The v-abl oncogene of the Abelson murine leukemia virus (A-MuLV) is known to efficiently transform NIH/3T3 fibroblasts in
vitro and to cause an acute lymphosarcoma in susceptible murine hosts. The role of its relative, the bcr/abl gene product,
in the etiology of human chronic myelogenous leukemia (CML) remains speculative. To assess the transforming properties of
the bcr/abl gene product, complementary DNA clones encoding the CML-specific P210 bcr/abl protein were expressed in NIH/3T3
fibroblasts. In contrast to the v-abl oncogene product P160, the P210 bcr/abl gene product did not transform NIH/3T3 cells.
Cell lines were isolated that expressed high levels of the P210 bcr/abl protein but were morphologically normal. During the
course of these experiments, a transforming recombinant of bcr/abl was isolated which fuses gag determinants derived from
helper virus to the NH2-terminus of the bcr/abl protein. This suggests that a property of viral gag sequences, probably myristylation-dependent
membrane localization, must be provided to bcr/abl for it to transform fibroblasts.
The P210bcr/abl protein is associated with virtually every case of human chronic myelogenous leukemia. Unlike the related P160gag/v-abl oncogene product of Abelson murine leukemia virus, P210bcr/abl does not transform NIH 3T3 fibroblasts. To assess whether P210bcr/abl might transform hematopoietic cell types, retroviral constructs encoding P210bcr/abl were used to infect the bone marrow-derived interleukin 3-dependent Ba/F3 cell line. As for P160gag/v-abl, cell lines expressing P210bcr/abl were growth factor independent and tumorigenic in nude mice. No evidence for autocrine production of interleukin 3 by factor-independent cell lines was found. These experiments establish that P210bcr/abl can transform hematopoietic cell types to tumorigenicity.
Rearrangements involving chromosome band 11q23 are very common in acute leukaemia, both lymphoblastic and myeloid (monoblastic), and are less common in lymphoma. Although several different genes have been cloned from translocation breakpoints, the great majority of translocations involve the MLL (myeloid-lymphoid leukaemia) gene. The MLL gene has several different names, ALL1, Htrx, HRX; the central part of the gene codes for multiple zinc fingers which show strong homology to the Drosophila trithorax gene. MLL is involved in four common translocations as well as in 25 uncommon or rare translocations, insertions and deletions. The translocation breakpoints occur within an 8.3 kb region which can be detected with a 0.74 kb cDNA probe. Twenty-five percent of patients have a deletion 3' of the breakpoint which includes the zinc finger region. Patients who previously received drugs that inhibit topoisomerase II often develop acute leukaemia with translocations involving 11q23. These translocations break MLL in the same 8.3 kb region. In the three breakpoints cloned to date, the translocation has led to a fusion gene on the derivative 11 chromosome with a chimaeric transcript, consisting of 5' MLL and the 3' segment of the other gene. Although transcripts were also cloned from the other derivative chromosome, all the evidence indicates that the critical fusion gene is on the derivative 11 chromosome. The molecular dissection of these rearrangements will provide insights into the biology of MLL and into the interaction of MLL with topoisomerase II inhibitors. In addition, this research has provided DNA probes that will be important for diagnosis and for monitoring patients during the course of their disease.
Chronic myelomonocytic leukemia (CMML) is a myelodysplastic syndrome characterized by abnormal clonal myeloid proliferation and by progression to acute myelogenous leukemia (AML). CMML thus offers an opportunity to study early genetic events in the transition to AML. A recently recognized subgroup of CMML has a t(5;12)(q33;p13) balanced translocation. We report that the consequence of the t(5;12) translocation is expression of a fusion transcript in which the tyrosine kinase domain of the platelet-derived growth factor receptor beta (PDGFR beta) on chromosome 5 is coupled to a novel ets-like gene, tel, on chromosome 12. The tel-PDGFR beta fusion demonstrates the oncogenic potential of PDGFR beta and may provide a paradigm for early events in the pathogenesis of AML.
The pericentric inversion of chromosome 16 [inv(16)(p13q22)] is a characteristic karyotypic abnormality associated with acute
myeloid leukemia, most commonly of the M4Eo subtype. The 16p and 16q breakpoints were pinpointed by yeast artificial chromosome
and cosmid cloning, and the two genes involved in this inversion were identified. On 16q the inversion occurred near the end
of the coding region for CBF beta, also known as PEBP2 beta, a subunit of a heterodimeric transcription factor regulating
genes expressed in T cells; on 16p a smooth muscle myosin heavy chain (SMMHC) gene (MYH11) was interrupted. In six of six
inv(16) patient samples tested, an in-frame fusion messenger RNA was demonstrated that connected the first 165 amino acids
of CBF beta with the tail region of SMMHC. The repeated coiled coil of SMMHC may result in dimerization of the CBF beta fusion
protein, which in turn would lead to alterations in transcriptional regulation and contribute to leukemic transformation.
In the 8;21 translocation, the AML1 gene, located at chromosome band 21q22, is translocated to chromosome 8 (q22), where it is fused to the ETO gene and transcribed as a chimeric gene. AML1 is the human homolog of the recently cloned mouse gene pebp2 alpha B, homologous to the DNA binding alpha subunit of the polyoma enhancer factor pebp2. AML1 is also involved in a translocation with chromosome 3 that is seen in patients with therapy-related acute myeloid leukemia and myelodysplastic syndrome and in chronic myelogenous leukemia in blast crisis. We have isolated a fusion cDNA clone from a t(3;21) library derived from a patient with therapy-related myelodysplastic syndrome; this clone contains sequences from AML1 and from EAP, which we have now localized to band 3q26. EAP has previously been characterized as a highly expressed small nuclear protein of 128 residues (EBER 1) associated with Epstein-Barr virus small RNA. The fusion clone contains the DNA binding 5' part of AML1 that is fused to ETO in the t(8;21) and, in addition, at least one other exon. The translocation replaces the last nine codons of AML1 with the last 96 codons of EAP. The fusion does not maintain the correct reading frame of EAP and may not lead to a functional chimeric protein.
The AML1-CBF beta transcription factor is the most frequent target of chromosomal rearrangements in human leukemia. To investigate its normal function, we generated mice lacking AML1. Embryos with homozygous mutations in AML1 showed normal morphogenesis and yolk sac-derived erythropoiesis, but lacked fetal liver hematopoiesis and died around E12.5. Sequentially targeted AML1-/-es cell retained their capacity to differentiate into primitive erythroid cells in vitro; however, no myeloid or erythroid progenitors of definitive hematopoietic origin were detected in either the yolk sac or fetal livers of mutant embryos. Moreover, this hematopoietic defect was intrinsic to the stem cells in that AML1-/-ES cells failed to contribute to hematopoiesis in chimeric animals. These results suggest that AML1-regulated target genes are essential for definitive hematopoiesis of all lineages.