Project description:Single Gland Whole-exome sequencing: building on our prior description of multi-region WES of colorectal tumors and targeted single gland sequencing (E-MTAB-2247), we performed WES of multiple single glands from different sides (right: A and left: B) of two tumors in this study (tumor O and U) on the illumina platform using the Agilent SureSelect 2.0 or illumina Nextera Rapid Capture Exome kit (SureSelect or NRCE, as indicated in the naming of fastq files). Colorectal Cancer Xenograft Whole-exome sequencing: The HCT116 and LoVo Mismatch-Repair-deficient colorectal adenocarcinoma cell lines were obtained from the ATCC and cultured under standard conditions. For both cell lines, a single âfoundingâ cell was cloned and expanded in vitro to ~6M cells. Two aliquots of ~1M cells were subcutaneously injected into opposite flanks (right and left) of a nude mouse and tumors allowed to reach a size of ~1B cells (1cm3) before the animal was sacrificed. Tumor tissue was collected separately from the right and left lesions and DNA was extracted for WES using the illumina TruSeq Exome kit or Nextera Rapid Capture Exome expanded Kits (Truseq or NRCEe), as was DNA from the first passage population (a polyclonal tissue culture for HCT116 and a polyclonal xenograft sample for LoVo), which were employed as a control to study mutation accumulation in culture and post xenotransplantation.
Project description:Blood samples from patients with myeloid malignancies were analyzed using whole exome sequencing (WES). Data set from genotyping by microarray of the same samples has been deposited in ArrayExpress under accession number E-MTAB-1845 (https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-1845/).
Project description:The mechanisms underlying hepatoblastoma are not well defined. To address this, we generated transcriptomic profiles of normal, background, and hepatoblastoma liver samples from patients aged 0.01 months to 6 years, using RNA-sequencing. Hepatoblasoma was histologically confirmed. Here we focus on the elevation of stem cell markers and the loss of tumor suppressor proteins leading to the development of hepatoblastoma in very young children.
Project description:To determine the influence of in vivo tumor growth and antitumor immune responses on the generation of tumor neoepitopes, we performed whole exome sequencing (WES) on the mT3-2D cell line, WT tumors and SCID tumors
Project description:Hepatoblastoma is a primitive liver cancer occurring mainly in infants with defined molecular alterations driving its progression, which is difficult to model in vivo. Here we present a new animal model for hepatoblastoma on the chick chorioallantoic membrane (CAM), which recapitulates relevant features of hepatoblastoma in patients. Expression of classic tumor-associated proteins such as β-catenin, EpCAM and CK19 was maintained in acini-like organized tumors on CAM, as was synthesis of AFP, a tumor marker used for monitoring patient response. RNA sequencing revealed an unexpected molecular evolution of hepatoblastoma cells on the CAM, with significant deregulation of more than 6000 genes including more than half of all HOX genes. Bioinformatic analysis could clearly distinguish between tumor cell-expressed genes and chick host genes, thereby shedding new light on the complex interactions taking place during experimental hepatoblastoma progression. Importantly, human tumor suppressive ribosomal genes were downregulated after implantation, whereas mitochondrial genes encoding for anti-apoptotic peptides were strongly induced in vivo. Meprin-1α expression was increased during evolution of CAM tumors and confirmed by immunohistochemistry. Cisplatin, a commonly used chemotherapeutic agent for hepatoblastoma, showed significant anti-tumoral effects in this model. Our results broaden the understanding of the molecular adaptation process of human cancer cells to the microenvironment and might help to elaborate novel therapeutic concepts for the treatment of this pediatric liver tumor.
Project description:For many years, immortalized cell lines have been used as model systems for cancer research. Cell line panels were established for basic research and drug development, but did not cover the full spectrum of leukemia and lymphoma. Therefore, we now developed a novel panel (LL-100), 100 cell lines covering 22 entities of human leukemia and lymphoma including T-cell, B-cell and myeloid malignancies. Importantly, all cell lines are unequivocally authenticated and assigned to the correct tissue. Cell line samples were proven to be free of mycoplasma and virus contamination. Whole exome sequencing (WES) and RNA sequencing (RNA-seq) of the hundred authenticated leukemia-lymphoma cell lines were conducted with a uniform methodology to complement existing data on these publicly available cell lines. This part captures WES. This data set will be useful for understanding the function of oncogenes and tumor suppressor genes and to develop targeted therapies.
Project description:Hepatoblastoma remains one of the most difficult childhood tumors to treat and is alarmingly understudied. Over half of patients initially present with locally advanced or metastatic disease and the prognosis for this cohort remains dismal. In addition, many of these children have disease that is resistant to standard therapies and will require novel and targeted therapies to effectively treat or manage their disease. We previously demonstrated that Proviral Insertion site in Maloney murine leukemia virus (PIM) kinases, specifically PIM3, are overexpressed in human hepatoblastoma cells and function to promote tumorigenesis. We aimed to use CRISPR/Cas9 gene editing technology with dual gRNAs to introduce large inactivating deletions in the PIM3 gene and achieve stable PIM3 knockout (KO) in the human hepatoblastoma cell line, HuH6. PIM3 KO of hepatoblastoma cells led to significantly decreased proliferation, viability, and motility, inhibited cell-cycle progression, decreased tumor growth in a xenograft murine model, and increased animal survival. Analysis of RNA sequencing data revealed that PIM3 KO downregulated expression of pro-migratory and pro-invasive genes and upregulated expression of genes involved in apoptosis and differentiation. Furthermore, PIM3 KO decreased hepatoblastoma cancer cell stemness as evidenced by decreased tumorsphere formation, decreased mRNA abundance of stemness markers, and decreased cell surface expression of CD133, a marker of hepatoblastoma stem cell-like cancer cells. Reintroduction of PIM3 into PIM3 KO cells rescued the malignant phenotype. These findings emphasize the role of PIM3 in promoting hepatoblastoma tumorigenesis and provide evidence that targeting PIM3 may offer a novel therapeutic approach for children with hepatoblastoma.
Project description:Carbonic anhydrase 9 (CAIX) is a transmembrane metalloenzyme whose main function is to maintain the acid-base balance of the cell and hypoxic condition induces its expression. Solid tumors often exhibit regions with low oxygen content in areas where the blood supply is insufficient. CAIX is overexpressed in multiple solid tumors, including hepatoblastoma, the most frequent childhood liver malignancy. SLC-0111 is a commercially available CAIX inhibitor that is currently passed Phase I clinical trial on solid tumors. In this study we assessed the impact of SLC-0111 on transcriptomic changes of HUH6 hepatoblastoma cell line cells in both normoxic and hypoxic condition.
Project description:Lack of relevant disease animal models and cell lines hampers our understanding of hepatoblastoma and identification of therapeutic targets. We report a liver-specific MYC-driven hepatoblastoma murine model that faithfully recapitulates the pathological features of mixed fetal and embryonic hepatoblastoma, with transcriptomics resembling the high-risk gene signatures of human disease. Single-cell RNA-sequencing (scRNA-seq) and spatiotranscriptomics identify a subpopulation of hepatoblastoma cells with high levels of adult hemoglobin genes. After deriving a cell line from the mouse model, we mapped the cancer dependency genes using CRISPR-Cas9 and identified druggable targets shared with human hepatoblastoma (i.e., CDK7, CDK9, PRMT1, PRMT5). Our screen also discovered oncogenes and tumor suppressive genes in hepatoblastoma that engage multiple cancer signaling pathways. Chemotherapy is the mainstay of human hepatoblastoma. Genetic map of doxorubicin response by CRISPR-Cas9 identified modifiers whose loss-of-function synergizes (PRKDC) and antagonizes (Polycomb repressive complex 2) the effect of chemotherapy. Combination of PRKDC inhibitor and chemotherapy greatly enhances therapeutic efficacy. Our studies have provided useful disease models and potential therapeutic targets of hepatoblastoma.