Acute myeloid leukemia (AML) with an FLT3 internal tandem duplication (FLT3-ITD) mutation is an aggressive hematologic malignancy with a grave prognosis. To identify the mutational spectrum associated with relapse, whole-exome sequencing was performed on 13 matched diagnosis, relapse, and remission trios followed by targeted sequencing of 299 genes in 67 FLT3-ITD patients. The FLT3-ITD genome has an average of 13 mutations per sample, similar to other AML subtypes, which is a low mutation rate compared with that in solid tumors. Recurrent mutations occur in genes related to DNA methylation, chromatin, histone methylation, myeloid transcription factors, signaling, adhesion, cohesin complex, and the spliceosome. Their pattern of mutual exclusivity and cooperation among mutated genes suggests that these genes have a strong biological relationship. In addition, we identified mutations in previously unappreciated genes such as MLL3, NSD1, FAT1, FAT4, and IDH3B. Mutations in 9 genes were observed in the relapse-specific phase. DNMT3A mutations are the most stable mutations, and this DNMT3A-transformed clone can be present even in morphologic complete remissions. Of note, all AML matched trio samples shared at least 1 genomic alteration at diagnosis and relapse, suggesting common ancestral clones. Two types of clonal evolution occur at relapse: either the founder clone recurs or a subclone of the founder clone escapes from induction chemotherapy and expands at relapse by acquiring new mutations. Relapse-specific mutations displayed an increase in transversions. Functional assays demonstrated that both MLL3 and FAT1 exert tumor-suppressor activity in the FLT3-ITD subtype. An inhibitor of XPO1 synergized with standard AML induction chemotherapy to inhibit FLT3-ITD growth. This study clearly shows that FLT3-ITD AML requires additional driver genetic alterations in addition to FLT3-ITD alone.
Pituitary tumor-transforming gene (PTTG) is a recently characterized oncogene whose expression product contains a transcriptional activation domain at the C terminus. To understand the mechanisms involved in PTTG biological functions, we used yeast two-hybrid screening to identify proteins that interact with PTTG. This study reports the isolation and characterization of a novel PTTG-binding factor (PBF). PBF contains an open reading frame of 179 amino acids with a predicted molecular mass of 22 kDa. In Northern blot analyses, PBF mRNA was ubiquitously expressed in human tissues. Glutathione S-transferase pull-down and co-immunoprecipitation assays demonstrate that PBF interacts specifically with PTTG under both in vitro and in vivo conditions. The PTTG binding domain in PBF was located within the C-terminal 30-amino acid region that contain a nuclear localization signal. Immunofluorescence and subcellular fractionation studies showed that PTTG is predominantly expressed in the cytoplasm with partial nuclear localization, whereas PBF is localized both in the cytoplasm and the nucleus. The interaction between PBF and PTTG facilitated PTTG translocation from the cytoplasm to the nucleus. Furthermore, PBF is required for transcriptional activation of basic fibroblast growth factor by PTTG. In summary, we have characterized a novel PTTG-binding protein that facilitates PTTG nuclear translocation and potentiates its transcriptional activation function. Pituitary tumor-transforming gene (PTTG)1 was isolated by its differential mRNA expression in rat pituitary tumor cells (1). Overexpression of PTTG induces cell transformation and generates tumors in nude mice (1). Several human PTTG homologues have since been cloned (2-5). High level expression of PTTG mRNA in multiple types of tumors as well as in carcinoma cell lines (2-7) suggests that PTTG may be involved in tumorigenesis of many tissues in addition to the pituitary. In normal adult tissues, PTTG mRNA expression is restricted to a small number of tissues, including testis, thymus, and placenta (1-3).The mechanisms involved in PTTG biological function are largely unknown. To elucidate the biological function of PTTG, we have used a yeast two-hybrid system to identify proteins that associate with PTTG (8). This report shows that the ribosomal protein S10 and a novel human homologue of the bacterial heat-shock protein, DnaJ (HSJ2), interact specifically with PTTG under both in vitro and in vivo conditions (8). Association of PTTG with these proteins indicates that PTTG may link to the ribosome and is involved in the regulation of translation (8). In addition, during the rat spermatogenic cycle, PTTG mRNA is expressed stage specifically in only spermatocytes and spermatids, suggesting that PTTG may play a role in rat spermatogenesis (8). The C-terminal portion of human PTTG was shown to function as a transcriptional activator when fused to a heterologous DNA binding domain (2). In NIH3T3 cells that overexpress PTTG, increased expression of bFGF mRNA and protein was obs...
Acute promyelocytic leukemia (APL) is a subtype of myeloid leukemia characterized by differentiation block at the promyelocyte stage. Besides the presence of chromosomal rearrangement t(15;17), leading to the formation of PML-RARA (promyelocytic leukemia-retinoic acid receptor alpha) fusion, other genetic alterations have also been implicated in APL. Here, we performed comprehensive mutational analysis of primary and relapse APL to identify somatic alterations, which cooperate with PML-RARA in the pathogenesis of APL. We explored the mutational landscape using whole-exome (n=12) and subsequent targeted sequencing of 398 genes in 153 primary and 69 relapse APL. Both primary and relapse APL harbored an average of eight non-silent somatic mutations per exome. We observed recurrent alterations of FLT3, WT1, NRAS and KRAS in the newly diagnosed APL, whereas mutations in other genes commonly mutated in myeloid leukemia were rarely detected. The molecular signature of APL relapse was characterized by emergence of frequent mutations in PML and RARA genes. Our sequencing data also demonstrates incidence of loss-of-function mutations in previously unidentified genes, ARID1B and ARID1A, both of which encode for key components of the SWI/SNF complex. We show that knockdown of ARID1B in APL cell line, NB4, results in large-scale activation of gene expression and reduced in vitro differentiation potential.
We investigated the oncogenic role of SETDB1 focusing on non-small cell lung cancer (NSCLC) having high expression of this protein. A total of 387 lung cancer cases were examined by immunohistochemistry, 72% of NSCLC samples were positive for SETDB1 staining, compared to 46% samples of normal bronchial epithelium (106 cases) (p<0.0001). Percent positive cells and intensity of staining increased significantly with increased grade of disease. Forced expression of SETDB1 in NSCLC cell lines enhanced their clonogenic growth in vitro and markedly increased tumor size in a murine xenograft model; while silencing (shRNA) SETDB1 in NSCLC cells slowed their proliferation. SETDB1 positively stimulated activity of the WNT/β-catenin pathway and diminished P53 expression resulting in enhanced NSCLC growth in vitro and in vivo. Our finding suggests therapeutic targeting SETDB1 may benefit patients whose tumors express high levels of SETDB1.
Partial tandem-duplication of MLL (MLL-PTD) characterizes acute myeloid leukemia (AML) patients often with a poor prognosis. To understand the order of occurrence of MLL-PTD in relation to other major AML mutations and to identify novel mutations that may be present in this unique AML molecular subtype, exome and targeted sequencing was performed on 85 MLL-PTD AML samples using HiSeq-2000. Genes involved in the cohesin complex (STAG2), a splicing factor (U2AF1) and a poorly studied gene, MGA were recurrently mutated while NPM1, one of the most frequently mutated AML gene, was not mutated in MLL-PTD patients. Interestingly, clonality analysis suggests that IDH2/1, DNMT3A, U2AF1 and TET2 mutations are clonal and occur early, and MLL-PTD likely arises after these initial mutations. Conversely, proliferative mutations (FLT3, RAS) typically appear later, are largely subclonal and tend to be unstable. This study provides important insights for understanding the relative importance of different mutations for defining a targeted therapeutic strategy for MLL-PTD AML patients.
Current chemotherapy of advanced non-small cell lung cancer (NSCLC) produces only a modest increase in survival time. New approaches are needed for this disease. The development of lung cancer is associated with silencing tumor suppressor genes that can occur not only by deletion or mutation, but also by epigenetic changes including histone deacetylation of key lysines. Histone deacetylase inhibitor (HDACI) increases histone acetylation, resulting in DNA with a more open chromatin that favors transcription. We found that the HDACI, suberoylanilide hydroxamic acid (SAHA), suppressed cell growth of five non-small cell lung cancer cell lines in a dose-dependent manner (50% growth inhibition ≈2 μM). Cell cycle assay by fluorescence-activated cell sorting (FACS) demonstrated that SAHA induced a significant G0-G1 growth arrest of NSCLC cells. Protein assay by Western blot analysis showed that SAHA induced expression of p21 WAF1. These results demonstrated that administration of SAHA may be a novel approach to the treatment of non-small cell lung cancer.
To our knowledge, this is the first report investigating the effect of LAMC2 on cell growth, cell cycle, migration, invasion, and EGFR signaling in ATC cells, suggesting that LAMC2 may be a potential therapeutic target for the treatment of ATC.
Current standard of care for patients with pediatric acute lymphoblastic leukemia (ALL) is mainly effective, with high remission rates after treatment. However, the genetic perturbations that give rise to this disease remain largely undefined, limiting the ability to address resistant tumors or develop less toxic targeted therapies. Here we report the use of next generation sequencing to interrogate the genetic and pathogenic mechanisms of 240 pediatric ALL cases with their matched remission samples. Commonly mutated genes fell into several categories, including RAS/receptor tyrosine kinases, epigenetic regulators, transcription factors involved in lineage commitment and the p53/cell cycle pathway. Unique recurrent mutational hotspots were observed in epigenetic regulators CREBBP (R1446C/H), WHSC1 (E1099K) and the tyrosine kinase FLT3 (K663R, N676K). The mutant WHSC1 was established as a gain-of-function oncogene, while the epigenetic regulator ARID1A and transcription factor CTCF were functionally identified as potential tumor suppressors. Analysis of 28 diagnosis/relapse trio patients plus 10 relapse cases revealed four evolutionary paths and uncovered the ordering of acquisition of mutations in these patients. This study provides a detailed mutational portrait of pediatric ALL and gives insights into the molecular pathogenesis of this disease.
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