Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes.

Pancreatic cancer is a highly lethal malignancy with few effective therapies. We performed exome sequencing and copy number analysis to define genomic aberrations in a prospectively accrued clinical cohort (n = 142) of early (stage I and II) sporadic pancreatic ductal adenocarcinoma. Detailed analysis of 99 informative tumours identified substantial heterogeneity with 2,016 non-silent mutations and 1,628 copy-number variations. We define 16 significantly mutated genes, reaffirming known mutations (KRAS, TP53, CDKN2A, SMAD4, MLL3, TGFBR2, ARID1A and SF3B1), and uncover novel mutated genes including additional genes involved in chromatin modification (EPC1 and ARID2), DNA damage repair (ATM) and other mechanisms (ZIM2, MAP2K4, NALCN, SLC16A4 and MAGEA6). Integrative analysis with in vitro functional data and animal models provided supportive evidence for potential roles for these genetic aberrations in carcinogenesis. Pathway-based analysis of recurrently mutated genes recapitulated clustering in core signalling pathways in pancreatic ductal adenocarcinoma, and identified new mutated genes in each pathway. We also identified frequent and diverse somatic aberrations in genes described traditionally as embryonic regulators of axon guidance, particularly SLIT/ROBO signalling, which was also evident in murine Sleeping Beauty transposon-mediated somatic mutagenesis models of pancreatic cancer, providing further supportive evidence for the potential involvement of axon guidance genes in pancreatic carcinogenesis.

Molecular heterogeneity of non-small cell lung carcinoma patient-derived xenografts closely reflect their primary tumors.

Availability of lung cancer models that closely mimic human tumors remains a significant gap in cancer research, as tumor cell lines and mouse models may not recapitulate the spectrum of lung cancer heterogeneity seen in patients. We aimed to establish a patient-derived tumor xenograft (PDX) resource from surgically resected non-small cell lung cancer (NSCLC). Fresh tumor tissue from surgical resection was implanted and grown in the subcutaneous pocket of non-obese severe combined immune deficient (NOD SCID) gamma mice. Subsequent passages were in NOD SCID mice. A subset of matched patient and PDX tumors and non-neoplastic lung tissues were profiled by whole exome sequencing, single nucleotide polymorphism (SNP) and methylation arrays, and phosphotyrosine (pY)-proteome by mass spectrometry. The data were compared to published NSCLC datasets of NSCLC primary and cell lines. 127 stable PDXs were established from 441 lung carcinomas representing all major histological subtypes: 52 adenocarcinomas, 62 squamous cell carcinomas, one adeno-squamous carcinoma, five sarcomatoid carcinomas, five large cell neuroendocrine carcinomas, and two small cell lung cancers. Somatic mutations, gene copy number and expression profiles, and pY-proteome landscape of 36 PDXs showed greater similarity with patient tumors than with established cell lines. Novel somatic mutations on cancer associated genes were identified but only in PDXs, likely due to selective clonal growth in the PDXs that allows detection of these low allelic frequency mutations. The results provide the strongest evidence yet that PDXs established from lung cancers closely mimic the characteristics of patient primary tumors.

Tumour evolution inferred by single-cell sequencing.

Genomic analysis provides insights into the role of copy number variation in disease, but most methods are not designed to resolve mixed populations of cells. In tumours, where genetic heterogeneity is common, very important information may be lost that would be useful for reconstructing evolutionary history. Here we show that with flow-sorted nuclei, whole genome amplification and next generation sequencing we can accurately quantify genomic copy number within an individual nucleus. We apply single-nucleus sequencing to investigate tumour population structure and evolution in two human breast cancer cases. Analysis of 100 single cells from a polygenomic tumour revealed three distinct clonal subpopulations that probably represent sequential clonal expansions. Additional analysis of 100 single cells from a monogenomic primary tumour and its liver metastasis indicated that a single clonal expansion formed the primary tumour and seeded the metastasis. In both primary tumours, we also identified an unexpectedly abundant subpopulation of genetically diverse 'pseudodiploid' cells that do not travel to the metastatic site. In contrast to gradual models of tumour progression, our data indicate that tumours grow by punctuated clonal expansions with few persistent intermediates.

Computational approaches to identify functional genetic variants in cancer genomes.

The International Cancer Genome Consortium (ICGC) aims to catalog genomic abnormalities in tumors from 50 different cancer types. Genome sequencing reveals hundreds to thousands of somatic mutations in each tumor but only a minority of these drive tumor progression. We present the result of discussions within the ICGC on how to address the challenge of identifying mutations that contribute to oncogenesis, tumor maintenance or response to therapy, and recommend computational techniques to annotate somatic variants and predict their impact on cancer phenotype.

Copy number variation analysis implicates the cell polarity gene glypican 5 as a human spina bifida candidate gene.

Neural tube defects (NTDs) are common birth defects of complex etiology. Family and population-based studies have confirmed a genetic component to NTDs. However, despite more than three decades of research, the genes involved in human NTDs remain largely unknown. We tested the hypothesis that rare copy number variants (CNVs), especially de novo germline CNVs, are a significant risk factor for NTDs. We used array-based comparative genomic hybridization (aCGH) to identify rare CNVs in 128 Caucasian and 61 Hispanic patients with non-syndromic lumbar-sacral myelomeningocele. We also performed aCGH analysis on the parents of affected individuals with rare CNVs where parental DNA was available (42 sets). Among the eight de novo CNVs that we identified, three generated copy number changes of entire genes. One large heterozygous deletion removed 27 genes, including PAX3, a known spina bifida-associated gene. A second CNV altered genes (PGPD8, ZC3H6) for which little is known regarding function or expression. A third heterozygous deletion removed GPC5 and part of GPC6, genes encoding glypicans. Glypicans are proteoglycans that modulate the activity of morphogens such as Sonic Hedgehog (SHH) and bone morphogenetic proteins (BMPs), both of which have been implicated in NTDs. Additionally, glypicans function in the planar cell polarity (PCP) pathway, and several PCP genes have been associated with NTDs. Here, we show that GPC5 orthologs are expressed in the neural tube, and that inhibiting their expression in frog and fish embryos results in NTDs. These results implicate GPC5 as a gene required for normal neural tube development.

WaveCNV: allele-specific copy number alterations in primary tumors and xenograft models from next-generation sequencing.

MOTIVATION: Copy number variations (CNVs) are a major source of genomic variability and are especially significant in cancer. Until recently microarray technologies have been used to characterize CNVs in genomes. However, advances in next-generation sequencing technology offer significant opportunities to deduce copy number directly from genome sequencing data. Unfortunately cancer genomes differ from normal genomes in several aspects that make them far less amenable to copy number detection. For example, cancer genomes are often aneuploid and an admixture of diploid/non-tumor cell fractions. Also patient-derived xenograft models can be laden with mouse contamination that strongly affects accurate assignment of copy number. Hence, there is a need to develop analytical tools that can take into account cancer-specific parameters for detecting CNVs directly from genome sequencing data. RESULTS: We have developed WaveCNV, a software package to identify copy number alterations by detecting breakpoints of CNVs using translation-invariant discrete wavelet transforms and assign digitized copy numbers to each event using next-generation sequencing data. We also assign alleles specifying the chromosomal ratio following duplication/loss. We verified copy number calls using both microarray (correlation coefficient 0.97) and quantitative polymerase chain reaction (correlation coefficient 0.94) and found them to be highly concordant. We demonstrate its utility in pancreatic primary and xenograft sequencing data. AVAILABILITY AND IMPLEMENTATION: Source code and executables are available at https://github.com/WaveCNV. The segmentation algorithm is implemented in MATLAB, and copy number assignment is implemented Perl. CONTACT: lakshmi.muthuswamy@gmail.com SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Dosage-dependent phenotypes in models of 16p11.2 lesions found in autism.

Recurrent copy number variations (CNVs) of human 16p11.2 have been associated with a variety of developmental/neurocognitive syndromes. In particular, deletion of 16p11.2 is found in patients with autism, developmental delay, and obesity. Patients with deletions or duplications have a wide range of clinical features, and siblings carrying the same deletion often have diverse symptoms. To study the consequence of 16p11.2 CNVs in a systematic manner, we used chromosome engineering to generate mice harboring deletion of the chromosomal region corresponding to 16p11.2, as well as mice harboring the reciprocal duplication. These 16p11.2 CNV models have dosage-dependent changes in gene expression, viability, brain architecture, and behavior. For each phenotype, the consequence of the deletion is more severe than that of the duplication. Of particular note is that half of the 16p11.2 deletion mice die postnatally; those that survive to adulthood are healthy and fertile, but have alterations in the hypothalamus and exhibit a "behavior trap" phenotype-a specific behavior characteristic of rodents with lateral hypothalamic and nigrostriatal lesions. These findings indicate that 16p11.2 CNVs cause brain and behavioral anomalies, providing insight into human neurodevelopmental disorders.

A Large-Scale Proteomics Resource of Circulating Extracellular Vesicles for Biomarker Discovery in Pancreatic Cancer.

Pancreatic cancer has the worst prognosis of all common tumors. Earlier cancer diagnosis could increase survival rates and better assessment of metastatic disease could improve patient care. As such, there is an urgent need to develop biomarkers to diagnose this deadly malignancy earlier. Analyzing circulating extracellular vesicles (cEVs) using 'liquid biopsies' offers an attractive approach to diagnose and monitor disease status. However, it is important to differentiate EV-associated proteins enriched in patients with pancreatic ductal adenocarcinoma (PDAC) from those with benign pancreatic diseases such as chronic pancreatitis and intraductal papillary mucinous neoplasm (IPMN). To meet this need, we combined the novel EVtrap method for highly efficient isolation of EVs from plasma and conducted proteomics analysis of samples from 124 individuals, including patients with PDAC, benign pancreatic diseases and controls. On average, 912 EV proteins were identified per 100µL of plasma. EVs containing high levels of PDCD6IP, SERPINA12 and RUVBL2 were associated with PDAC compared to the benign diseases in both discovery and validation cohorts. EVs with PSMB4, RUVBL2 and ANKAR were associated with metastasis, and those with CRP, RALB and CD55 correlated with poor clinical prognosis. Finally, we validated a 7-EV protein PDAC signature against a background of benign pancreatic diseases that yielded an 89% prediction accuracy for the diagnosis of PDAC. To our knowledge, our study represents the largest proteomics profiling of circulating EVs ever conducted in pancreatic cancer and provides a valuable open-source atlas to the scientific community with a comprehensive catalogue of novel cEVs that may assist in the development of biomarkers and improve the outcomes of patients with PDAC.

Organoid Sensitivity Correlates with Therapeutic Response in Patients with Pancreatic Cancer.

PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) remains a significant health issue. For most patients, there are no options for targeted therapy, and existing treatments are limited by toxicity. The HOPE trial (Harnessing Organoids for PErsonalized Therapy) was a pilot feasibility trial aiming to prospectively generate patient-derived organoids (PDO) from patients with PDAC and test their drug sensitivity and correlation with clinical outcomes. EXPERIMENTAL DESIGN: PDOs were established from a heterogeneous population of patients with PDAC including both basal and classical PDAC subtypes. RESULTS: A method for classifying PDOs as sensitive or resistant to chemotherapy regimens was developed to predict the clinical outcome of patients. Drug sensitivity testing on PDOs correlated with clinical responses to treatment in individual patients. CONCLUSIONS: These data support the investigation of PDOs to guide treatment in prospective interventional trials in PDAC.

Commitment and oncogene-induced plasticity of human stem cell-derived pancreatic acinar and ductal organoids.

The exocrine pancreas, consisting of ducts and acini, is the site of origin of pancreatitis and pancreatic ductal adenocarcinoma (PDAC). Our understanding of the genesis and progression of human pancreatic diseases, including PDAC, is limited because of challenges in maintaining human acinar and ductal cells in culture. Here we report induction of human pluripotent stem cells toward pancreatic ductal and acinar organoids that recapitulate properties of the neonatal exocrine pancreas. Expression of the PDAC-associated oncogene GNASR201C induces cystic growth more effectively in ductal than acinar organoids, whereas KRASG12D is more effective in modeling cancer in vivo when expressed in acinar compared with ductal organoids. KRASG12D, but not GNASR201C, induces acinar-to-ductal metaplasia-like changes in culture and in vivo. We develop a renewable source of ductal and acinar organoids for modeling exocrine development and diseases and demonstrate lineage tropism and plasticity for oncogene action in the human pancreas.

Patient-derived tumor xenograft and organoid models established from resected pancreatic, duodenal and biliary cancers.

Patient-derived xenograft (PDX) and their xenograft-derived organoid (XDO) models that recapitulate the genotypic and phenotypic landscape of patient cancers could help to advance research and lead to improved clinical management. PDX models were established from 276 pancreato-duodenal and biliary cancer resections. Initial, passage 0 (P0) engraftment rates were 59% (118/199) for pancreatic, 86% (25/29) for duodenal, and 35% (17/48) for biliary ductal tumors. Pancreatic ductal adenocarcinoma (PDAC), had a P0 engraftment rate of 62% (105/169). KRAS mutant and wild-type PDAC models were molecularly profiled, and XDO models were generated to perform initial drug response evaluations. Subsets of PDAC PDX models showed global copy number variants and gene expression profiles that were retained with serial passaging, and they showed a spectrum of somatic mutations represented in patient tumors. PDAC XDO models were established, with a success rate of 71% (10/14). Pathway activation of KRAS-MAPK in PDXs was independent of KRAS mutational status. Four wild-type KRAS models were characterized by one with EGFR (L747-P753 del), two with BRAF alterations (N486_P490del or V600E), and one with triple negative KRAS/EGFR/BRAF. Model OCIP256, characterized by BRAF (N486-P490 del), had activated phospho-ERK. A combination treatment of a pan-RAF inhibitor (LY3009120) and a MEK inhibitor (trametinib) effectively suppressed phospho-ERK and inhibited growth of OCIP256 XDO and PDX models. PDAC/duodenal adenocarcinoma have high success rates forming PDX/organoid and retaining their phenotypic and genotypic features. These models may be effective tools to evaluate novel drug combination therapies.

PDX-derived organoids model in vivo drug response and secrete biomarkers.

Patient-derived organoid models are proving to be a powerful platform for both basic and translational studies. Here we conduct a methodical analysis of pancreatic ductal adenocarcinoma (PDAC) tumor organoid drug response in paired patient-derived xenograft (PDX) and PDX-derived organoid (PXO) models grown under WNT-free culture conditions. We report a specific relationship between area under the curve value of organoid drug dose response and in vivo tumor growth, irrespective of the drug treatment. In addition, we analyzed the glycome of PDX and PXO models and demonstrate that PXOs recapitulate the in vivo glycan landscape. In addition, we identify a core set of 57 N-glycans detected in all 10 models that represent 50%-94% of the relative abundance of all N-glycans detected in each of the models. Last, we developed a secreted biomarker discovery pipeline using media supernatant of organoid cultures and identified potentially new extracellular vesicle (EV) protein markers. We validated our findings using plasma samples from patients with PDAC, benign gastrointestinal diseases, and chronic pancreatitis and discovered that 4 EV proteins are potential circulating biomarkers for PDAC. Thus, we demonstrate the utility of organoid cultures to not only model in vivo drug responses but also serve as a powerful platform for discovering clinically actionable serologic biomarkers.

RB1 deficiency in triple-negative breast cancer induces mitochondrial protein translation.

Triple-negative breast cancer (TNBC) includes basal-like and claudin-low subtypes for which no specific treatment is currently available. Although the retinoblastoma tumor-suppressor gene (RB1) is frequently lost together with TP53 in TNBC, it is not directly targetable. There is thus great interest in identifying vulnerabilities downstream of RB1 that can be therapeutically exploited. Here, we determined that combined inactivation of murine Rb and p53 in diverse mammary epithelial cells induced claudin-low-like TNBC with Met, Birc2/3-Mmp13-Yap1, and Pvt1-Myc amplifications. Gene set enrichment analysis revealed that Rb/p53-deficient tumors showed elevated expression of the mitochondrial protein translation (MPT) gene pathway relative to tumors harboring p53 deletion alone. Accordingly, bioinformatic, functional, and biochemical analyses showed that RB1-E2F complexes bind to MPT gene promoters to regulate transcription and control MPT. Additionally, a screen of US Food and Drug Administration-approved (FDA-approved) drugs identified the MPT antagonist tigecycline (TIG) as a potent inhibitor of Rb/p53-deficient tumor cell proliferation. TIG preferentially suppressed RB1-deficient TNBC cell proliferation, targeted both the bulk and cancer stem cell fraction, and strongly attenuated xenograft growth. It also cooperated with sulfasalazine, an FDA-approved inhibitor of cystine xCT antiporter, in culture and xenograft assays. Our results suggest that RB1 deficiency promotes cancer cell proliferation in part by enhancing mitochondrial function and identify TIG as a clinically approved drug for RB1-deficient TNBC.

Ductal pancreatic cancer modeling and drug screening using human pluripotent stem cell- and patient-derived tumor organoids.

There are few in vitro models of exocrine pancreas development and primary human pancreatic adenocarcinoma (PDAC). We establish three-dimensional culture conditions to induce the differentiation of human pluripotent stem cells into exocrine progenitor organoids that form ductal and acinar structures in culture and in vivo. Expression of mutant KRAS or TP53 in progenitor organoids induces mutation-specific phenotypes in culture and in vivo. Expression of TP53(R175H) induces cytosolic SOX9 localization. In patient tumors bearing TP53 mutations, SOX9 was cytoplasmic and associated with mortality. We also define culture conditions for clonal generation of tumor organoids from freshly resected PDAC. Tumor organoids maintain the differentiation status, histoarchitecture and phenotypic heterogeneity of the primary tumor and retain patient-specific physiological changes, including hypoxia, oxygen consumption, epigenetic marks and differences in sensitivity to inhibition of the histone methyltransferase EZH2. Thus, pancreatic progenitor organoids and tumor organoids can be used to model PDAC and for drug screening to identify precision therapy strategies.

Mislocalization of the cell polarity protein scribble promotes mammary tumorigenesis and is associated with basal breast cancer.

Scribble (SCRIB) localizes to cell-cell junctions and regulates establishment of epithelial cell polarity. Loss of expression of SCRIB functions as a tumor suppressor in Drosophila and mammals; conversely, overexpression of SCRIB promotes epithelial differentiation in mammals. Here, we report that SCRIB is frequently amplified, mRNA overexpressed, and protein is mislocalized from cell-cell junctions in human breast cancers. High levels of SCRIB mRNA are associated with poor clinical prognosis, identifying an unexpected role for SCRIB in breast cancer. We find that transgenic mice expressing a SCRIB mutant [Pro 305 to Leu (P305L)] that fails to localize to cell-cell junctions, under the control of the mouse mammary tumor virus long terminal repeat promoter, develop multifocal hyperplasia that progresses to highly pleomorphic and poorly differentiated tumors with basal characteristics. SCRIB interacts with phosphatase and tensin homolog (PTEN) and the expression of P305L, but not wild-type SCRIB, promotes an increase in PTEN levels in the cytosol. Overexpression of P305L, but not wild-type SCRIB, activates the Akt/mTOR/S6K signaling pathway. Human breast tumors overexpressing SCRIB have high levels of S6K but do not harbor mutations in PTEN or PIK3CA, identifying SCRIB amplification as a mechanism of activating PI3K signaling in tumors without mutations in PIK3CA or PTEN. Thus, we demonstrate that high levels of mislocalized SCRIB functions as a neomorph to promote mammary tumorigenesis by affecting subcellular localization of PTEN and activating an Akt/mTOR/S6kinase signaling pathway.

Integrated omic analysis of lung cancer reveals metabolism proteome signatures with prognostic impact.

Cancer results from processes prone to selective pressure and dysregulation acting along the sequence-to-phenotype continuum DNA → RNA → protein → disease. However, the extent to which cancer is a manifestation of the proteome is unknown. Here we present an integrated omic map representing non-small cell lung carcinoma. Dysregulated proteins not previously implicated as cancer drivers are encoded throughout the genome including, but not limited to, regions of recurrent DNA amplification/deletion. Clustering reveals signatures composed of metabolism proteins particularly highly recapitulated between patient-matched primary and xenograft tumours. Interrogation of The Cancer Genome Atlas reveals cohorts of patients with lung and other cancers that have DNA alterations in genes encoding the signatures, and this was accompanied by differences in survival. The recognition of genome and proteome alterations as related products of selective pressure driving the disease phenotype may be a general approach to uncover and group together cryptic, polygenic disease drivers.

Integrated genomic, transcriptomic, and RNA-interference analysis of genes in somatic copy number gains in pancreatic ductal adenocarcinoma.

OBJECTIVES: This study used an integrated analysis of copy number, gene expression, and RNA interference screens for identification of putative driver genes harbored in somatic copy number gains in pancreatic ductal adenocarcinoma (PDAC). METHODS: Somatic copy number gain data on 60 PDAC genomes were extracted from public data sets to identify genomic loci that are recurrently gained. Array-based data from a panel of 29 human PDAC cell lines were used to quantify associations between copy number and gene expression for the set of genes found in somatic copy number gains. The most highly correlated genes were assessed in a compendium of pooled short hairpin RNA screens on 27 of the same human PDAC cell lines. RESULTS: A catalog of 710 protein-coding and 46 RNA genes mapping to 20 recurrently gained genomic loci were identified. The gene set was further refined through stringent integration of copy number, gene expression, and RNA interference screening data to uncover 34 candidate driver genes. CONCLUSIONS: Among the candidate genes from the integrative analysis, ECT2 was found to have significantly higher essentiality in specific PDAC cell lines with genomic gains at the 3q26.3 locus, which harbors this gene, suggesting that ECT2 may play an oncogenic role in the PDAC neoplastic process.

Expression profiling during mammary epithelial cell three-dimensional morphogenesis identifies PTPRO as a novel regulator of morphogenesis and ErbB2-mediated transformation.

Identification of genes that are upregulated during mammary epithelial cell morphogenesis may reveal novel regulators of tumorigenesis. We have demonstrated that gene expression programs in mammary epithelial cells grown in monolayer cultures differ significantly from those in three-dimensional (3D) cultures. We identify a protein tyrosine phosphate, PTPRO, that was upregulated in mature MCF-10A mammary epithelial 3D structures but had low to undetectable levels in monolayer cultures. Downregulation of PTPRO by RNA interference inhibited proliferation arrest during morphogenesis. Low levels of PTPRO expression correlated with reduced survival for breast cancer patients, suggesting a tumor suppressor function. Furthermore, we showed that the receptor tyrosine kinase ErbB2/HER2 is a direct substrate of PTPRO and that loss of PTPRO increased ErbB2-induced cell proliferation and transformation, together with tyrosine phosphorylation of ErbB2. Moreover, in patients with ErbB2-positive breast tumors, low PTPRO expression correlated with poor clinical prognosis compared to ErbB2-positive patients with high levels of PTPRO. Thus, PTPRO is a novel regulator of ErbB2 signaling, a potential tumor suppressor, and a novel prognostic marker for patients with ErbB2-positive breast cancers. We have identified the protein tyrosine phosphatase PTPRO as a regulator of three-dimensional epithelial morphogenesis of mammary epithelial cells and as a regulator of ErbB2-mediated transformation. In addition, we demonstrated that ErbB2 is a direct substrate of PTPRO and that decreased expression of PTPRO predicts poor prognosis for ErbB2-positive breast cancer patients. Thus, our results identify PTPRO as a novel regulator of mammary epithelial transformation, a potential tumor suppressor, and a predictive biomarker for breast cancer.