[show abstract][hide abstract] ABSTRACT: Epigenetic dysregulation is an emerging hallmark of cancers. We developed a high-information-content mass spectrometry approach to profile global histone modifications in human cancers. When applied to 115 lines from the Cancer Cell Line Encyclopedia, this approach identified distinct molecular chromatin signatures. One signature was characterized by increased histone 3 lysine 36 (H3K36) dimethylation, exhibited by several lines harboring translocations in NSD2, which encodes a methyltransferase. A previously unknown NSD2 p.Glu1099Lys (p.E1099K) variant was identified in nontranslocated acute lymphoblastic leukemia (ALL) cell lines sharing this signature. Ectopic expression of the variant induced a chromatin signature characteristic of NSD2 hyperactivation and promoted transformation. NSD2 knockdown selectively inhibited the proliferation of NSD2-mutant lines and impaired the in vivo growth of an NSD2-mutant ALL xenograft. Sequencing analysis of >1,000 pediatric cancer genomes identified the NSD2 p.E1099K alteration in 14% of t(12;21) ETV6-RUNX1-containing ALLs. These findings identify NSD2 as a potential therapeutic target for pediatric ALL and provide a general framework for the functional annotation of cancer epigenomes.
[show abstract][hide abstract] ABSTRACT: Somatic alterations of the lymphoid transcription factor gene PAX5 (also known as BSAP) are a hallmark of B cell precursor acute lymphoblastic leukemia (B-ALL), but inherited mutations of PAX5 have not previously been described. Here we report a new heterozygous germline variant, c.547G>A (p.Gly183Ser), affecting the octapeptide domain of PAX5 that was found to segregate with disease in two unrelated kindreds with autosomal dominant B-ALL. Leukemic cells from all affected individuals in both families exhibited 9p deletion, with loss of heterozygosity and retention of the mutant PAX5 allele at 9p13. Two additional sporadic ALL cases with 9p loss harbored somatic PAX5 substitutions affecting Gly183. Functional and gene expression analysis of the PAX5 mutation demonstrated that it had significantly reduced transcriptional activity. These data extend the role of PAX5 alterations in the pathogenesis of pre-B cell ALL and implicate PAX5 in a new syndrome of susceptibility to pre-B cell neoplasia.
[show abstract][hide abstract] ABSTRACT: Background: Ribonucleotide reductase catalyzes an essential step in the cellular production of deoxyribonucleotide triphosphates and has been associated with clinical outcome in cancer patients receiving nucleoside analog-based chemotherapy. Materials & methods: In the current study, we sequenced the genes RRM1 and RRM2 in genomic DNA from HapMap cell lines with European (Utah residents with northern and western European ancestry [CEU]; n = 90) or African (Yoruba people in Ibadan, Nigeria [YRI]; n = 90) ancestry. Results: We identified 44 genetic variants including eight coding SNPs in RRM1 and 15 SNPs including one coding SNP in RRM2. RRM1 and RRM2 mRNA expression levels were significantly correlated with each other in both CEU and YRI lymphoblast cell lines, and in leukemic blasts from acute myeloid leukemia (AML) patients (AML97, n = 89; AML02, n = 187). Additionally, RRM1 expression was higher among patient features indicative of a high relapse hazard. We evaluated SNPs within the RRM1 and RRM2 genes in the HapMap lymphoblast cell lines from CEU and YRI panels for association with expression and cytarabine chemosensitivity. SNPs of potential significance were further evaluated in AML patients. RRM1 SNPs rs1042919 (which occurs in linkage disequilbrium with multiple other SNPs) and promoter SNP rs1561876 were associated with intracellular 1-β-d-arabinofuranosyl-CTP levels, response after remission induction therapy, risk of relapse and overall survival in AML patients receiving cytarabine and cladribine. Conclusion: These results suggest that SNPs within ribonucleotide reductase might be helpful predictive markers of response to nucleoside analogs and should be further validated in larger cohorts. Original submitted 2 April 2013; Revision submitted 8 July 2013.
[show abstract][hide abstract] ABSTRACT: Acute lymphoblastic leukemia (ALL) is the commonest childhood malignancy and is characterized by recurring structural genetic alterations. Previous studies of DNA methylation suggest epigenetic alterations may also be important, but an integrated genome-wide analysis of genetic and epigenetic alterations in ALL has not been performed. We analyzed 137 B-lineage and 30 T-lineage childhood ALL cases using microarray analysis of DNA copy number alterations and gene expression, and genome-wide cytosine methylation profiling using the HpaII tiny fragment enrichment by ligation-mediated PCR (HELP) assay. We found that the different genetic subtypes of ALL are characterized by distinct DNA methylation signatures that exhibit significant correlation with gene expression profiles. We also identified an epigenetic signature common to all cases, with correlation to gene expression in 65% of these genes, suggesting that a core set of epigenetically deregulated genes is central to the initiation or maintenance of lymphoid transformation. Finally, we identified aberrant methylation in multiple genes also targeted by recurring DNA copy number alterations in ALL, suggesting that these genes are inactivated far more frequently than suggested by structural genomic analyses alone. Together, these results demonstrate subtype- and disease-specific alterations in cytosine methylation in ALL that influence transcriptional activity, and are likely to exert a key role in leukemogenesis.
The Journal of clinical investigation 06/2013; · 15.39 Impact Factor
[show abstract][hide abstract] ABSTRACT: Biochemical properties of Ras oncoproteins and their transforming ability strongly support a dominant mechanism of action in tumorigenesis. However, genetic studies unexpectedly suggested that wild-type (WT) Ras exerts tumor suppressor activity. Expressing oncogenic NrasG12D in the hematopoietic compartment of mice induces an aggressive myeloproliferative neoplasm (MPN) that is exacerbated in homozygous mutant animals. Here we show that increased NrasG12D gene dosage, but not inactivation of WT Nras, underlies the aggressive in vivo behavior of NrasG12D/G12D hematopoietic cells. Modulating N-RasG12D dosage had discrete effects on myeloid progenitor growth, signal transduction, and sensitivity to MEK inhibition. Furthermore, enforced WT N-Ras expression neither suppressed the growth of Nras mutant cells nor inhibited myeloid transformation by exogenous NrasG12D. Importantly NRAS expression in increased in human cancer cell lines with NRAS mutations. These data have therapeutic implications and support reconsidering the proposed tumor suppressor activity of WT Ras in other cancers.
[show abstract][hide abstract] ABSTRACT: Reversing the aberrant biochemical output of oncogenic Ras proteins is one of the great challenges in cancer therapeutics; however, it is uncertain which Ras effectors are required for tumor initiation and maintenance. To address this question, we expressed oncogenic K-Ras(D12) proteins with "second site" amino acid substitutions that impair PI3 kinase/Akt or Raf/MEK/ERK activation in bone marrow cells and transplanted them into recipient mice. In spite of attenuated signaling properties, defective K-Ras oncoproteins initiated aggressive clonal T lineage acute lymphoblastic leukemia (T-ALL). Murine T-ALLs expressing "second site" mutant proteins restored full oncogenic Ras activity through diverse mechanisms, which included acquiring novel somatic "third site" Kras(D12) mutations and silencing PTEN. T-ALL cell lines lacking PTEN had elevated levels of phosphorylated Akt, a gene expression pattern similar to human early T precursor ALL, and were resistant to the potent and selective MEK inhibitor PD0325901. Our data demonstrating strong selective pressure to overcome defective activation of PI3 kinase/Akt and Raf/MEK/ERK implicate both Ras effector pathways as drivers of aberrant growth in T-ALL, and further suggest that leukemia cells wlll deploy multiple mechanisms to develop resistance to targeted inhibitors in vivo.
[show abstract][hide abstract] ABSTRACT: LGGs arise most frequently in children and young adults and are the most common pediatric central nervous system (CNS) neoplasms 1–3 . Although LGGs grow slowly, those that cannot be surgically resected cause considerable morbidity and premature death. Current adjuvant therapies with irradiation and pharmaceuticals extend survival but contribute to morbidity; thus, there is an urgent need for targeted therapeutics in patients with inoperable disease 1,3–10 . Studies of pediatric LGGs and related LGGNTs have implicated abnormalities of the extracellular signal–regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway in oncogenesis 11–14 , but detailed knowledge of driver mutations in these diverse tumors is lacking. Up to 15% of children with the hereditary tumor syndrome neurofibromatosis type 1 (NF-1) develop pilocytic astrocytoma, the most common type of LGG 15,16 . Neurofibromin 1, the protein product of the NF1 tumor suppressor gene, is a negative regulator of RAS in the MAPK/ERK pathway 17 . NF-1–related LGGs account for less than 15% of pediatric LGGs; however, almost all sporadic cerebellar pilocytic astrocytomas show MAPK/ERK pathway activation in association with a KIAA1549-BRAF fusion gene, which results in the expression of BRAF protein that lacks its autoinhibitory domain and is constitu-tively active 11,12 . Other mechanisms activating the MAPK/ERK path-way in LGGs are comparatively rare and include RAF1 fusion genes and BRAF V600E or KRAS mutations, although the BRAF V600E muta-tion is present in approximately 15% of LGGNTs 11,12,18–21 . Although nearly all World Health Organization (WHO) grade I LGGs from the intracranial posterior fossa harbor one of the above mutations, they occur less frequently in supratentorial grade I LGGs and rarely in dif-fuse grade II tumors 21,22 . Notably, the genetics of inoperable diseases, Whole-genome sequencing identifies genetic alterations in pediatric low-grade gliomas
[show abstract][hide abstract] ABSTRACT: The most common pediatric brain tumors are low-grade gliomas (LGGs). We used whole-genome sequencing to identify multiple new genetic alterations involving BRAF, RAF1, FGFR1, MYB, MYBL1 and genes with histone-related functions, including H3F3A and ATRX, in 39 LGGs and low-grade glioneuronal tumors (LGGNTs). Only a single non-silent somatic alteration was detected in 24 of 39 (62%) tumors. Intragenic duplications of the portion of FGFR1 encoding the tyrosine kinase domain (TKD) and rearrangements of MYB were recurrent and mutually exclusive in 53% of grade II diffuse LGGs. Transplantation of Trp53-null neonatal astrocytes expressing FGFR1 with the duplication involving the TKD into the brains of nude mice generated high-grade astrocytomas with short latency and 1 100% penetrance. FGFR1 with the duplication induced FGFR1 1 autophosphorylation and upregulation of the MAPK/ERK and PI3K pathways, which could be blocked by specific inhibitors. Focusing on the therapeutically challenging diffuse LGGs, our study of 1 151 1 tumors has discovered genetic alterations and potential therapeutic targets across
the entire range of pediatric LGGs and LGGNTs.
[show abstract][hide abstract] ABSTRACT: LIM domain Only 2 (Lmo2) is frequently deregulated in sporadic and gene therapy-induced acute T-cell lymphoblastic leukemia (T-ALL) where its overexpression is an important initiating mutational event. In transgenic and retroviral mouse models, Lmo2 expression can be enforced in multiple hematopoietic lineages but leukemia only arises from T cells. These data suggest that Lmo2 confers clonal growth advantage in T-cell progenitors. We analyzed proliferation, differentiation, and cell death in CD2-Lmo2 transgenic thymic progenitor cells to understand the cellular effects of enforced Lmo2 expression. Most impressively, Lmo2 transgenic T-cell progenitor cells were blocked in differentiation, quiescent, and immortalized in vitro on OP9-DL1 stromal cells. These cellular effects were concordant with a transcriptional signature in Lmo2 transgenic T-cell progenitor cells that is also present in hematopoietic stem cells and Early T-cell Precursor ALL. These results are significant in light of the crucial role of Lmo2 in the maintenance of the hematopoietic stem cell. The cellular effects and transcriptional effects have implications for LMO2-dependent leukemogenesis and the treatment of LMO2-induced T-ALL.
[show abstract][hide abstract] ABSTRACT: The genetic basis of hypodiploid acute lymphoblastic leukemia (ALL), a subtype of ALL characterized by aneuploidy and poor outcome, is unknown. Genomic profiling of 124 hypodiploid ALL cases, including whole-genome and exome sequencing of 40 cases, identified two subtypes that differ in the severity of aneuploidy, transcriptional profiles and submicroscopic genetic alterations. Near-haploid ALL with 24-31 chromosomes harbor alterations targeting receptor tyrosine kinase signaling and Ras signaling (71%) and the lymphoid transcription factor gene IKZF3 (encoding AIOLOS; 13%). In contrast, low-hypodiploid ALL with 32-39 chromosomes are characterized by alterations in TP53 (91.2%) that are commonly present in nontumor cells, IKZF2 (encoding HELIOS; 53%) and RB1 (41%). Both near-haploid and low-hypodiploid leukemic cells show activation of Ras-signaling and phosphoinositide 3-kinase (PI3K)-signaling pathways and are sensitive to PI3K inhibitors, indicating that these drugs should be explored as a new therapeutic strategy for this aggressive form of leukemia.
[show abstract][hide abstract] ABSTRACT: BACKGROUND: Telomeres are the protective arrays of tandem TTAGGG sequence and associated proteins at the termini of chromosomes. Telomeres shorten at each cell division due to the end-replication problem and are maintained above a critical threshold in malignant cancer cells to prevent cellular senescence or apoptosis. With the recent advances in massive parallel sequencing, assessing telomere content in the context of other cancer genomic aberrations becomes an attractive possibility. We present the first comprehensive analysis of telomeric DNA content change in tumors using whole-genome sequencing data from 235 pediatric cancers. RESULTS: To measure telomeric DNA content, we counted telomeric reads containing TTAGGGx4 or CCCTAAx4 and normalized to the average genomic coverage. Changes in telomeric DNA content in tumor genomes were clustered using a Bayesian Information Criterion to determine loss, no change, or gain. Using this approach, we found that the pattern of telomeric DNA alteration varies dramatically across the landscape of pediatric malignancies: telomere gain was found in 32% of solid tumors, 4% of brain tumors and 0% of hematopoietic malignancies. The results were validated by three independent experimental approaches and reveal significant association of telomere gain with the frequency of somatic sequence mutations and structural variations.. CONCLUSIONS: Telomere DNA content measurement using whole-genome sequencing data is a reliable approach that can generate useful insights into the landscape of the cancer genome. Measuring the change in telomeric DNA during malignant progression is likely to be a useful metric when considering telomeres in the context of the whole genome.
[show abstract][hide abstract] ABSTRACT: One of the most rapidly evolving genes in humans, PRDM9, is a key determinant of the distribution of meiotic recombination events. Mutations in this meiotic-specific gene have previously been associated with male infertility in humans and recent studies suggest that PRDM9 may be involved in pathological genomic rearrangements. By studying genomes from families with children affected by B-cell precursor acute lymphoblastic leukemia (B-ALL), we characterized meiotic recombination patterns within a family with two siblings having hyperdiploid childhood ALL and observed unusual localization of maternal recombination events. The mother of the family carries a rare PRDM9 allele, potentially explaining the unusual patterns found. From exomes sequenced in 44 additional parents of children affected with B-ALL, we discovered a substantial and significant excess of rare allelic forms of PRDM9. The rare PRDM9 alleles are transmitted to the affected children in half the cases, nonetheless there remains a significant excess of rare alleles among patients relative to controls. We successfully replicated this latter observation in an independent cohort of 50 children with B-ALL, where we found an excess of rare PRDM9 alleles in aneuploid and infant B-ALL patients. PRDM9 variability in humans is thought to influence genomic instability, and these data support a potential role for PRDM9 variation in risk of acquiring aneuploidies or genomic rearrangements associated with childhood leukemogenesis.
[show abstract][hide abstract] ABSTRACT: One recently identified subtype of pediatric B-precursor acute lymphoblastic leukemia (ALL) has been termed BCR-ABL1-like or Ph-like due to similarity of the gene expression profile (GEP) to BCR-ABL1 positive ALL suggesting the presence of lesions activating tyrosine kinases, frequent alteration of IKZF1, and poor outcome. Prior studies demonstrated that about half of these patients had genomic lesions leading to CRLF2 overexpression, with half of such cases harboring somatic mutations in the Janus kinases JAK1 and JAK2. To determine whether mutations in other tyrosine kinases might also occur in ALL, we sequenced the tyrosine kinome and downstream signaling genes in 45 high risk pediatric ALL cases with either a Ph-like GEP or other alterations suggestive of activated kinase signaling. Aside from JAK mutations and one FLT3 mutation, no somatic mutations were found in any other tyrosine kinases, suggesting that alternative mechanisms are responsible for activated kinase signaling in high risk ALL.