We describe the landscape of somatic genomic alterations based on multidimensional and comprehensive characterization of more than 500 glioblastoma tumors (GBMs). We identify several novel mutated ...genes as well as complex rearrangements of signature receptors, including EGFR and PDGFRA. TERT promoter mutations are shown to correlate with elevated mRNA expression, supporting a role in telomerase reactivation. Correlative analyses confirm that the survival advantage of the proneural subtype is conferred by the G-CIMP phenotype, and MGMT DNA methylation may be a predictive biomarker for treatment response only in classical subtype GBM. Integrative analysis of genomic and proteomic profiles challenges the notion of therapeutic inhibition of a pathway as an alternative to inhibition of the target itself. These data will facilitate the discovery of therapeutic and diagnostic target candidates, the validation of research and clinical observations and the generation of unanticipated hypotheses that can advance our molecular understanding of this lethal cancer.
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•Exome DNA sequencing in 291 glioblastomas, 42 with whole-genome sequencing•RNA sequencing of 164 glioblastomas identifies recurrent gene rearrangements•Copy-number, DNA methylation, protein, mRNA, and miRNA expression profiles of 543 GBMs•Integrated analysis of somatic alterations, molecular subtypes and affected pathways
Integration of a large sample size of glioblastoma tumors with multidimensional ‘omic characterization and clinical annotation provides new insights into driver mutations, distinctions between subtypes, and therapeutic options.
Lung squamous cell carcinoma is a common type of lung cancer, causing approximately 400,000 deaths per year worldwide. Genomic alterations in squamous cell lung cancers have not been comprehensively ...characterized, and no molecularly targeted agents have been specifically developed for its treatment. As part of The Cancer Genome Atlas, here we profile 178 lung squamous cell carcinomas to provide a comprehensive landscape of genomic and epigenomic alterations. We show that the tumour type is characterized by complex genomic alterations, with a mean of 360 exonic mutations, 165 genomic rearrangements, and 323 segments of copy number alteration per tumour. We find statistically recurrent mutations in 11 genes, including mutation of TP53 in nearly all specimens. Previously unreported loss-of-function mutations are seen in the HLA-A class I major histocompatibility gene. Significantly altered pathways included NFE2L2 and KEAP1 in 34%, squamous differentiation genes in 44%, phosphatidylinositol-3-OH kinase pathway genes in 47%, and CDKN2A and RB1 in 72% of tumours. We identified a potential therapeutic target in most tumours, offering new avenues of investigation for the treatment of squamous cell lung cancers.
Medulloblastomas are the most common malignant brain tumours in children. Identifying and understanding the genetic events that drive these tumours is critical for the development of more effective ...diagnostic, prognostic and therapeutic strategies. Recently, our group and others described distinct molecular subtypes of medulloblastoma on the basis of transcriptional and copy number profiles. Here we use whole-exome hybrid capture and deep sequencing to identify somatic mutations across the coding regions of 92 primary medulloblastoma/normal pairs. Overall, medulloblastomas have low mutation rates consistent with other paediatric tumours, with a median of 0.35 non-silent mutations per megabase. We identified twelve genes mutated at statistically significant frequencies, including previously known mutated genes in medulloblastoma such as CTNNB1, PTCH1, MLL2, SMARCA4 and TP53. Recurrent somatic mutations were newly identified in an RNA helicase gene, DDX3X, often concurrent with CTNNB1 mutations, and in the nuclear co-repressor (N-CoR) complex genes GPS2, BCOR and LDB1. We show that mutant DDX3X potentiates transactivation of a TCF promoter and enhances cell viability in combination with mutant, but not wild-type, β-catenin. Together, our study reveals the alteration of WNT, hedgehog, histone methyltransferase and now N-CoR pathways across medulloblastomas and within specific subtypes of this disease, and nominates the RNA helicase DDX3X as a component of pathogenic β-catenin signalling in medulloblastoma.
We performed massively parallel sequencing of paired tumor/normal samples from 203 multiple myeloma (MM) patients and identified significantly mutated genes and copy number alterations and discovered ...putative tumor suppressor genes by determining homozygous deletions and loss of heterozygosity. We observed frequent mutations in KRAS (particularly in previously treated patients), NRAS, BRAF, FAM46C, TP53, and DIS3 (particularly in nonhyperdiploid MM). Mutations were often present in subclonal populations, and multiple mutations within the same pathway (e.g., KRAS, NRAS, and BRAF) were observed in the same patient. In vitro modeling predicts only partial treatment efficacy of targeting subclonal mutations, and even growth promotion of nonmutated subclones in some cases. These results emphasize the importance of heterogeneity analysis for treatment decisions.
•Deep sequencing reveals significant genetic events in multiple myeloma•Intratumor genetic heterogeneity is common in multiple myeloma•Recurrent mutations can occur either early or late in the evolution of a tumor•Genetic heterogeneity in cancer may limit effectiveness of targeted therapy
The incidence of esophageal adenocarcinoma (EAC) has risen 600% over the last 30 years. With a 5-year survival rate of ~15%, the identification of new therapeutic targets for EAC is greatly ...important. We analyze the mutation spectra from whole-exome sequencing of 149 EAC tumor-normal pairs, 15 of which have also been subjected to whole-genome sequencing. We identify a mutational signature defined by a high prevalence of A>C transversions at AA dinucleotides. Statistical analysis of exome data identified 26 significantly mutated genes. Of these genes, five (TP53, CDKN2A, SMAD4, ARID1A and PIK3CA) have previously been implicated in EAC. The new significantly mutated genes include chromatin-modifying factors and candidate contributors SPG20, TLR4, ELMO1 and DOCK2. Functional analyses of EAC-derived mutations in ELMO1 identifies increased cellular invasion. Therefore, we suggest the potential activation of the RAC1 pathway as a contributor to EAC tumorigenesis.
Spiny mice (Acomys cahirinus) are terrestrial mammals that evolved unique scar-free regenerative wound-healing properties. Myofibroblasts (MFs) are the major scar-forming cell type in skin. We found ...that following traumatic injury to ear pinnae, MFs appeared rapidly in both Acomys and mouse yet persisted only in mouse. The timing of MF loss in Acomys correlated with wound closure, blastema differentiation, and nuclear localization of the Hippo pathway target protein Yap. Experiments in vitro revealed an accelerated PP2A-dependent dephosphorylation activity that maintained nuclear Yap in Acomys dermal fibroblasts (DFs) and was not detected in mouse or human DFs. Treatment of Acomys in vivo with the nuclear Yap-TEAD inhibitor verteporfin prolonged MF persistence and converted tissue regeneration to fibrosis. Forced Yap activity prevented and rescued TGF-β1-induced human MF formation in vitro. These results suggest that Acomys evolved modifications of Yap activity and MF fate important for scar-free regenerative wound healing in vivo.
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•Ear injury-activated MFs are transient in Acomys but persist in Mus•Acomys MFs in vitro resist maturation via rapid Yap dephosphorylation•Blocking Acomys Yap-TEAD interaction in vivo inhibits regeneration, promotes fibrosis•MF phenotypes in spiny mice may find application for novel anti-fibrotic therapies
Brewer et al. report that following ear pinnae injury, myofibroblasts (MFs) appear rapidly in both Acomys (spiny mice) and Mus but persist only in Mus; Acomys MFs in vitro resist TGF-β1-stimulated maturation via accelerated PP2A-dependent Yap phosphatase activity. Blocking Yap-TEAD interaction in vivo alters blastemal cell fate, inhibits regeneration, and promotes fibrosis.
Campylobacteriosis as a disease, is of huge significance to the human population worldwide. It remains the number one cause of human zoonosis within the EU with over 245,000 cases of ...campylobacteriosis reported in 2018 with many more cases thought to have gone unreported. Campylobacter is of particular significance for the poultry industry due to the fact chickens are a significant reservoir for Campylobacter spp. and ultimately cause a food safety risk should chicken meat be mishandled or improperly cooked. As such there has been a huge focus on Campylobacter reduction across the EU which has seen measures introduced to educate consumers on poultry meat handling and cooking as well as to force poultry producers to reduce levels within the supply chain. Within the UK, the Foods Standards Agency (FSA) have pressurised retailers to reduce Campylobacter loading on chicken products within their stores. This in turn has resulted in significant efforts to reduce levels of Campylobacter along the supply chain, particularly on farm. To date a fail-safe method of excluding Campylobacter from commercial UK and Irish poultry farms remains elusive. This body of research is unique in that it was done in conjunction with the commercial poultry industry, subsequently allowing access to a greater depth of metadata associated with Campylobacter prevalence on farm. Questions that were off interest to this work were i) the understanding of prevalence at a UK level and associated risk factors ii) the effect of Campylobacter on chicken health and performance at commercial farm level iii) investigate the chicken gut microbiome over time and how this may influence Campylobacter appearance and performance; and iv) assess the impact of farming production system parameters on the chicken microbiome, its influence on Campylobacter and performance. Our results are the first to analyse Campylobacter prevalence on farm during the period 2014 - 2016, during which Campylobacter reduction was a clear focus for the industry. We identified a year on year reduction in Campylobacter positive flocks from 60% in 2014 to 46% in 2016. Furthermore, we identified key risk factors associated with Campylobacter for UK poultry farmers. Unsurprisingly seasonality was a clear risk but other factors such as those in relation to farm infrastructure were identified and will ultimately inform the industry on best practice for the future. This research also sets out clearly the impact Campylobacter can have on flock performance, providing a clear incentive for farmers to invest and adhere to best practice biosecurity. Nonetheless, it is recognised that biosecurity alone will not prevent Campylobacter colonisation and further understanding of the chicken gut microbiome, how it interacts with Campylobacter and how commercial farm practices influence this, is required. Our chicken gut microbiome analysis clearly shows rapidly increasing microbial diversity up to day 12 with variation observed both in terms of genera and abundance, before a stabilization of the microbial diversity after day 20. In particular, we identified a shift from competitive to environmental drivers of microbial community from days 12 to 20 creating a window of opportunity whereby Campylobacter can appear. Additionally, we demonstrated for the first time how different production systems influence chicken microbial communities and as such could ultimately lead to improved performance and better intervention strategies against Campylobacter within the food chain.
Treatment of BRAF-mutant melanoma with combined dabrafenib and trametinib, which target RAF and the downstream MAP-ERK kinase (MEK)1 and MEK2 kinases, respectively, improves progression-free survival ...and response rates compared with dabrafenib monotherapy. Mechanisms of clinical resistance to combined RAF/MEK inhibition are unknown. We performed whole-exome sequencing (WES) and whole-transcriptome sequencing (RNA-seq) on pretreatment and drug-resistant tumors from five patients with acquired resistance to dabrafenib/trametinib. In three of these patients, we identified additional mitogen-activated protein kinase (MAPK) pathway alterations in the resistant tumor that were not detected in the pretreatment tumor, including a novel activating mutation in MEK2 (MEK2(Q60P)). MEK2(Q60P) conferred resistance to combined RAF/MEK inhibition in vitro, but remained sensitive to inhibition of the downstream kinase extracellular signal-regulated kinase (ERK). The continued MAPK signaling-based resistance identified in these patients suggests that alternative dosing of current agents, more potent RAF/MEK inhibitors, and/or inhibition of the downstream kinase ERK may be needed for durable control of BRAF-mutant melanoma.
High-throughput sequencing technology enables population-level surveys of human genomic variation. Here, we examine the joint allele frequency distributions across continental human populations and ...present an approach for combining complementary aspects of whole-genome, low-coverage data and targeted high-coverage data. We apply this approach to data generated by the pilot phase of the Thousand Genomes Project, including whole-genome 2-4x coverage data for 179 samples from HapMap European, Asian, and African panels as well as high-coverage target sequencing of the exons of 800 genes from 697 individuals in seven populations. We use the site frequency spectra obtained from these data to infer demographic parameters for an Out-of-Africa model for populations of African, European, and Asian descent and to predict, by a jackknife-based approach, the amount of genetic diversity that will be discovered as sample sizes are increased. We predict that the number of discovered nonsynonymous coding variants will reach 100,000 in each population after
Abstract
Hematopoietic stem and progenitor cells (HSPCs) reside in niches that provide regulatory signals for their function. Perturbations such as acute leukemia induce cellular and molecular ...insults to the niche to support disease progression. Mutation of the MYC oncogene family is a frequent event leading to leukemogenesis. We developed a new zebrafish model of acute erythroid leukemia (AEL) by overexpression of human CMYC under the blood specific promotor draculin (drl). Analyses of drl:CMYC marrows demonstrated a significant expansion of progenitors and a decrease of erythroid, lymphoid and myeloid mature cells (fc=4.8, -4.5, -3.3, -6.5; p<0.000001). RNA-Sequencing of drl:CMYC marrows revealed an upregulation of the erythroid master regulator gata1a (fc=1.4, p=0.01) and fetal hemoglobins hbbe1.1/2 (fc=4.7, 2.9; p=0.0004). Primary and secondary transplantation of drl:CMYC marrows resulted in engraftment and disease propagation (7/7; 17/18). We used the cellular barcoding GESTALT technique to uniquely barcode single cells using CRISPR-CAS9 during embryonic development. We induced a round of barcoding at the one-cell stage and another one at 28 hours post-fertilization, the time of HSC birth. We injected these GESTALT embryos with drl:CMYC to induce AEL, barcode HSPCs and their niche to perform clone tracing. The number of HSPC clones was decreased by half compared to controls (p=0.008) indicative of a clonal expansion of the disease. We performed barcode and single-cell transcriptome profiling of flk1:GFP+ niche endothelial cells and found a significant decrease in the number of endothelial cells clones (fc= -3.5, p<0.05) paired with the identification of a novel AEL venous endothelial population upregulating 99 genes (fc>1; p<0.05) suggestive of angiogenesis likely supporting leukemogenesis. We sorted cxcl12a:dsRed+ niche stromal cells and found that AEL marrows have significantly less stromal clones (fc= -2.1, p<0.01) that are selectively amplified (>20% of the stromal compartment). We hypothesized that AEL expands a subset of stromal cells to promote disease progression and scRNA-Seq of 3,263 cxcl12a:dsRed+ stromal cells revealed an increased fraction of lepr+ mesenchymal stromal cells (MSCs, 66 vs 24% in controls). We hypothesized that AEL progenitors secrete a signal that can specifically remodel the marrow niche and we mined our scRNA-Sequencing data for a candidate ligand significantly upregulated in AEL cells compared to control HSPCs (fc>1; p<0.05) and paired receptors expressed on more than 20% of AEL associated endothelial and/or stromal cells. We identified the peptide hormone apelin expressed by the leukemia and tested if apelin alone could remodel the normal marrow niche endothelial and stromal cells by overexpressing apelin under the drl promotor. Adult marrow analyses revealed a decrease in the absolute number of stromal cells per marrow (fc= -2.2, p<0.001). Together our data support a model in which leukemia induces a clonal expansion of HSPC niche clones via apelin signaling to promote disease progression.
Citation Format: Chloé S Baron, Serine Avagyan, Song Yang, Aaron McKenna, Leonard Zon. Cellular barcoding of the leukemic niche reveals an apelin-mediated clonal expansion of niche endothelial and mesenchymal stromal cell clones abstract. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A27.
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