Integrative modeling uncovers p21-driven drug resistance and prioritizes therapies for PIK3CA-mutant breast cancer.

Utility of PI3Kα inhibitors like BYL719 is limited by the acquisition of genetic and non-genetic mechanisms of resistance which cause disease recurrence. Several combination therapies based on PI3K inhibition have been proposed, but a way to systematically prioritize them for breast cancer treatment is still missing. By integrating published and in-house studies, we have developed in silico models that quantitatively capture dynamics of PI3K signaling at the network-level under a BYL719-sensitive versus BYL719 resistant-cell state. Computational predictions show that signal rewiring to alternative components of the PI3K pathway promote resistance to BYL719 and identify PDK1 as the most effective co-target with PI3Kα rescuing sensitivity of resistant cells to BYL719. To explore whether PI3K pathway-independent mechanisms further contribute to BYL719 resistance, we performed phosphoproteomics and found that selection of high levels of the cell cycle regulator p21 unexpectedly promoted drug resistance in T47D cells. Functionally, high p21 levels favored repair of BYL719-induced DNA damage and bypass of the associated cellular senescence. Importantly, targeted inhibition of the check-point inhibitor CHK1 with MK-8776 effectively caused death of p21-high T47D cells, thus establishing a new vulnerability of BYL719-resistant breast cancer cells. Together, our integrated studies uncover hidden molecular mediators causing resistance to PI3Kα inhibition and provide a framework to prioritize combination therapies for PI3K-mutant breast cancer.

Single-nuclei and bulk-tissue gene-expression analysis of pheochromocytoma and paraganglioma links disease subtypes with tumor microenvironment.

Pheochromocytomas (PC) and paragangliomas (PG) are rare neuroendocrine tumors associated with autonomic nerves. Here we use single-nuclei RNA-seq and bulk-tissue gene-expression data to characterize the cellular composition of PCPG and normal adrenal tissues, refine tumor gene-expression subtypes and make clinical and genotypic associations. We confirm seven PCPG gene-expression subtypes with significant genotype and clinical associations. Tumors with mutations in VHL, SDH-encoding genes (SDHx) or MAML3-fusions are characterized by hypoxia-inducible factor signaling and neoangiogenesis. PCPG have few infiltrating lymphocytes but abundant macrophages. While neoplastic cells transcriptionally resemble mature chromaffin cells, early chromaffin and neuroblast markers are also features of some PCPG subtypes. The gene-expression profile of metastatic SDHx-related PCPG indicates these tumors have elevated cellular proliferation and a lower number of non-neoplastic Schwann-cell-like cells, while GPR139 is a potential theranostic target. Our findings therefore clarify the diverse transcriptional programs and cellular composition of PCPG and identify biomarkers of potential clinical significance.

Epigenetic Activation of Plasmacytoid DCs Drives IFNAR-Dependent Therapeutic Differentiation of AML.

Pharmacologic inhibition of epigenetic enzymes can have therapeutic benefit against hematologic malignancies. In addition to affecting tumor cell growth and proliferation, these epigenetic agents may induce antitumor immunity. Here, we discovered a novel immunoregulatory mechanism through inhibition of histone deacetylases (HDAC). In models of acute myeloid leukemia (AML), leukemia cell differentiation and therapeutic benefit mediated by the HDAC inhibitor (HDACi) panobinostat required activation of the type I interferon (IFN) pathway. Plasmacytoid dendritic cells (pDC) produced type I IFN after panobinostat treatment, through transcriptional activation of IFN genes concomitant with increased H3K27 acetylation at these loci. Depletion of pDCs abrogated panobinostat-mediated induction of type I IFN signaling in leukemia cells and impaired therapeutic efficacy, whereas combined treatment with panobinostat and IFNα improved outcomes in preclinical models. These discoveries offer a new therapeutic approach for AML and demonstrate that epigenetic rewiring of pDCs enhances antitumor immunity, opening the possibility of exploiting this approach for immunotherapies. SIGNIFICANCE: We demonstrate that HDACis induce terminal differentiation of AML through epigenetic remodeling of pDCs, resulting in production of type I IFN that is important for the therapeutic effects of HDACis. The study demonstrates the important functional interplay between the immune system and leukemias in response to HDAC inhibition. This article is highlighted in the In This Issue feature, p. 1397.

PTPN2 Regulates the Interferon Signaling and Endoplasmic Reticulum Stress Response in Pancreatic ß-Cells in Autoimmune Diabetes.

Type 1 diabetes (T1D) results from autoimmune destruction of ß-cells in the pancreas. Protein tyrosine phosphatases (PTPs) are candidate genes for T1D and play a key role in autoimmune disease development and ß-cell dysfunction. Here, we assessed the global protein and individual PTP profiles in the pancreas from nonobese mice with early-onset diabetes (NOD) mice treated with an anti-CD3 monoclonal antibody and interleukin-1 receptor antagonist. The treatment reversed hyperglycemia, and we observed enhanced expression of PTPN2, a PTP family member and T1D candidate gene, and endoplasmic reticulum (ER) chaperones in the pancreatic islets. To address the functional role of PTPN2 in ß-cells, we generated PTPN2-deficient human stem cell-derived ß-like and EndoC-ßH1 cells. Mechanistically, we demonstrated that PTPN2 inactivation in ß-cells exacerbates type I and type II interferon signaling networks and the potential progression toward autoimmunity. Moreover, we established the capacity of PTPN2 to positively modulate the Ca2+-dependent unfolded protein response and ER stress outcome in ß-cells. Adenovirus-induced overexpression of PTPN2 partially protected from ER stress-induced ß-cell death. Our results postulate PTPN2 as a key protective factor in ß-cells during inflammation and ER stress in autoimmune diabetes.

γδ T Cells in Merkel Cell Carcinomas Have a Proinflammatory Profile Prognostic of Patient Survival.

Merkel cell carcinomas (MCC) are immunogenic skin cancers associated with viral infection or UV mutagenesis. To study T-cell infiltrates in MCC, we analyzed 58 MCC lesions from 39 patients using multiplex-IHC/immunofluorescence (m-IHC/IF). CD4+ or CD8+ T cells comprised the majority of infiltrating T lymphocytes in most tumors. However, almost half of the tumors harbored prominent CD4/CD8 double-negative (DN) T-cell infiltrates (>20% DN T cells), and in 12% of cases, DN T cells represented the majority of T cells. Flow cytometric analysis of single-cell suspensions from fresh tumors identified DN T cells as predominantly Vδ2- γδ T cells. In the context of γδ T-cell inflammation, these cells expressed PD-1 and LAG3, which is consistent with a suppressed or exhausted phenotype, and CD103, which indicates tissue residency. Furthermore, single-cell RNA sequencing (scRNA-seq) identified a transcriptional profile of γδ T cells suggestive of proinflammatory potential. T-cell receptor (TCR) analysis confirmed clonal expansion of Vδ1 and Vδ3 clonotypes, and functional studies using cloned γδ TCRs demonstrated restriction of these for CD1c and MR1 antigen-presenting molecules. On the basis of a 13-gene γδ T-cell signature derived from scRNA-seq analysis, gene-set enrichment on bulk RNA-seq data showed a positive correlation between enrichment scores and DN T-cell infiltrates. An improved disease-specific survival was evident for patients with high enrichment scores, and complete responses to anti-PD-1/PD-L1 treatment were observed in three of four cases with high enrichment scores. Thus, γδ T-cell infiltration may serve as a prognostic biomarker and should be explored for therapeutic interventions.See related Spotlight on p. 600.

Macrophages provide a transient muscle stem cell niche via NAMPT secretion.

Skeletal muscle regenerates through the activation of resident stem cells. Termed satellite cells, these normally quiescent cells are induced to proliferate by wound-derived signals1. Identifying the source and nature of these cues has been hampered by an inability to visualize the complex cell interactions that occur within the wound. Here we use muscle injury models in zebrafish to systematically capture the interactions between satellite cells and the innate immune system after injury, in real time, throughout the repair process. This analysis revealed that a specific subset of macrophages 'dwell' within the injury, establishing a transient but obligate niche for stem cell proliferation. Single-cell profiling identified proliferative signals that are secreted by dwelling macrophages, which include the cytokine nicotinamide phosphoribosyltransferase (Nampt, which is also known as visfatin or PBEF in humans). Nampt secretion from the macrophage niche is required for muscle regeneration, acting through the C-C motif chemokine receptor type 5 (Ccr5), which is expressed on muscle stem cells. This analysis shows that in addition to their ability to modulate the immune response, specific macrophage populations also provide a transient stem-cell-activating niche, directly supplying proliferation-inducing cues that govern the repair process that is mediated by muscle stem cells. This study demonstrates that macrophage-derived niche signals for muscle stem cells, such as NAMPT, can be applied as new therapeutic modalities for skeletal muscle injury and disease.

DDX5 plays essential transcriptional and post-transcriptional roles in the maintenance and function of spermatogonia.

Mammalian spermatogenesis is sustained by mitotic germ cells with self-renewal potential known as undifferentiated spermatogonia. Maintenance of undifferentiated spermatogonia and spermatogenesis is dependent on tightly co-ordinated transcriptional and post-transcriptional mechanisms. The RNA helicase DDX5 is expressed by spermatogonia but roles in spermatogenesis are unexplored. Using an inducible knockout mouse model, we characterise an essential role for DDX5 in spermatogonial maintenance and show that Ddx5 is indispensable for male fertility. We demonstrate that DDX5 regulates appropriate splicing of key genes necessary for spermatogenesis. Moreover, DDX5 regulates expression of cell cycle genes in undifferentiated spermatogonia post-transcriptionally and is required for cell proliferation and survival. DDX5 can also act as a transcriptional co-activator and we demonstrate that DDX5 interacts with PLZF, a transcription factor required for germline maintenance, to co-regulate select target genes. Combined, our data reveal a critical multifunctional role for DDX5 in regulating gene expression programmes and activity of undifferentiated spermatogonia.

Deep multi-region whole-genome sequencing reveals heterogeneity and gene-by-environment interactions in treatment-naive, metastatic lung cancer.

Our understanding of genomic heterogeneity in lung cancer is largely based on the analysis of early-stage surgical specimens. Here we used endoscopic sampling of paired primary and intrathoracic metastatic tumors from 11 lung cancer patients to map genomic heterogeneity inoperable lung cancer with deep whole-genome sequencing. Intra-patient heterogeneity in driver or targetable mutations was predominantly in the form of copy number gain. Private mutation signatures, including patterns consistent with defects in homologous recombination, were highly variable both within and between patients. Irrespective of histotype, we observed a smaller than expected number of private mutations, suggesting that ancestral clones accumulated large mutation burdens immediately prior to metastasis. Single-region whole-genome sequencing of from 20 patients showed that tumors in ever-smokers with the strongest tobacco signatures were associated with germline variants in genes implicated in the repair of cigarette-induced DNA damage. Our results suggest that lung cancer precursors in ever-smokers accumulate large numbers of mutations prior to the formation of frank malignancy followed by rapid metastatic spread. In advanced lung cancer, germline variants in DNA repair genes may interact with the airway environment to influence the pattern of founder mutations, whereas similar interactions with the tumor microenvironment may play a role in the acquisition of mutations following metastasis.

Inhibition of activin signaling in lung adenocarcinoma increases the therapeutic index of platinum chemotherapy.

Resistance to platinum chemotherapy is a long-standing problem in the management of lung adenocarcinoma. Using a whole-genome synthetic lethal RNA interference screen, we identified activin signaling as a critical mediator of innate platinum resistance. The transforming growth factor-ß (TGFß) superfamily ligands activin A and growth differentiation factor 11 (GDF11) mediated resistance via their cognate receptors through TGFß-activated kinase 1 (TAK1), rather than through the SMAD family of transcription factors. Inhibition of activin receptor signaling or blockade of activin A and GDF11 by the endogenous protein follistatin overcame this resistance. Consistent with the role of activin signaling in acute renal injury, both therapeutic interventions attenuated acute cisplatin-induced nephrotoxicity, its major dose-limiting side effect. This cancer-specific enhancement of platinum-induced cell death has the potential to dramatically improve the safety and efficacy of chemotherapy in lung cancer patients.

Identification of dynamic undifferentiated cell states within the male germline.

The role of stem cells in tissue maintenance is appreciated and hierarchical models of stem cell self-renewal and differentiation often proposed. Stem cell activity in the male germline is restricted to undifferentiated A-type spermatogonia (Aundiff); however, only a fraction of this population act as stem cells in undisturbed testis and Aundiff hierarchy remains contentious. Through newly developed compound reporter mice, here we define molecular signatures of self-renewing and differentiation-primed adult Aundiff fractions and dissect Aundiff heterogeneity by single-cell analysis. We uncover an unappreciated population within the self-renewing Aundiff fraction marked by expression of embryonic patterning genes and homeodomain transcription factor PDX1. Importantly, we find that PDX1 marks a population with potent stem cell capacity unique to mature, homeostatic testis and demonstrate dynamic interconversion between PDX1+ and PDX1- Aundiff states upon transplant and culture. We conclude that Aundiff exist in a series of dynamic cell states with distinct function and provide evidence that stability of such states is dictated by niche-derived cues.

The tumor suppressor Hic1 maintains chromosomal stability independent of Tp53.

Hypermethylated-in-Cancer 1 (Hic1) is a tumor suppressor gene frequently inactivated by epigenetic silencing and loss-of-heterozygosity in a broad range of cancers. Loss of HIC1, a sequence-specific zinc finger transcriptional repressor, results in deregulation of genes that promote a malignant phenotype in a lineage-specific manner. In particular, upregulation of the HIC1 target gene SIRT1, a histone deacetylase, can promote tumor growth by inactivating TP53. An alternate line of evidence suggests that HIC1 can promote the repair of DNA double strand breaks through an interaction with MTA1, a component of the nucleosome remodeling and deacetylase (NuRD) complex. Using a conditional knockout mouse model of tumor initiation, we now show that inactivation of Hic1 results in cell cycle arrest, premature senescence, chromosomal instability and spontaneous transformation in vitro. This phenocopies the effects of deleting Brca1, a component of the homologous recombination DNA repair pathway, in mouse embryonic fibroblasts. These effects did not appear to be mediated by deregulation of Hic1 target gene expression or loss of Tp53 function, and rather support a role for Hic1 in maintaining genome integrity during sustained replicative stress. Loss of Hic1 function also cooperated with activation of oncogenic KRas in the adult airway epithelium of mice, resulting in the formation of highly pleomorphic adenocarcinomas with a micropapillary phenotype in vivo. These results suggest that loss of Hic1 expression in the early stages of tumor formation may contribute to malignant transformation through the acquisition of chromosomal instability.

New Monoclonal Antibodies to Defined Cell Surface Proteins on Human Pluripotent Stem Cells.

The study and application of human pluripotent stem cells (hPSCs) will be enhanced by the availability of well-characterized monoclonal antibodies (mAbs) detecting cell-surface epitopes. Here, we report generation of seven new mAbs that detect cell surface proteins present on live and fixed human ES cells (hESCs) and human iPS cells (hiPSCs), confirming our previous prediction that these proteins were present on the cell surface of hPSCs. The mAbs all show a high correlation with POU5F1 (OCT4) expression and other hPSC surface markers (TRA-160 and SSEA-4) in hPSC cultures and detect rare OCT4 positive cells in differentiated cell cultures. These mAbs are immunoreactive to cell surface protein epitopes on both primed and naive state hPSCs, providing useful research tools to investigate the cellular mechanisms underlying human pluripotency and states of cellular reprogramming. In addition, we report that subsets of the seven new mAbs are also immunoreactive to human bone marrow-derived mesenchymal stem cells (MSCs), normal human breast subsets and both normal and tumorigenic colorectal cell populations. The mAbs reported here should accelerate the investigation of the nature of pluripotency, and enable development of robust cell separation and tracing technologies to enrich or deplete for hPSCs and other human stem and somatic cell types. Stem Cells 2017;35:626-640.

Low-Dose Histone Deacetylase Inhibitor Treatment Leads to Tumor Growth Arrest and Multi-Lineage Differentiation of Malignant Rhabdoid Tumors.

PURPOSE: Malignant rhabdoid tumor (MRT) and atypical teratoid rhabdoid tumors (ATRT) are rare aggressive undifferentiated tumors primarily affecting the kidney and CNS of infants and young children. MRT are almost exclusively characterized by homozygous deletion or inactivation of the chromatin remodeling gene SMARCB1 SMARCB1 protein loss leads to direct impairment of chromatin remodeling and we have previously reported a role for this protein in histone acetylation. This provided the rationale for investigating the therapeutic potential of histone deactylase inhibitors (HDACi) in MRT. EXPERIMENTAL DESIGN: Whereas previously HDACis have been used at doses and schedules that induce cytotoxicity, in the current studies we have tested the hypothesis, both in vitro and in vivo, that sustained treatment of human MRT with low-dose HDACi can lead to sustained cell growth arrest and differentiation. RESULTS: Sustained low-dose panobinostat (LBH589) treatment led to changes in cellular morphology associated with a marked increase in the induction of neural, renal, and osteoblast differentiation pathways. Genome-wide transcriptional profiling highlighted differential gene expression supporting multilineage differentiation. Using mouse xenograft models, sustained low-dose LBH589 treatment caused tumor growth arrest associated with tumor calcification detectable by X-ray imaging. Histological analysis of LBH589-treated tumors revealed significant regions of ossification, confirmed by Alizarin Red staining. Immunohistochemical analysis showed increased TUJ1 and PAX2 staining suggestive of neuronal and renal differentiation, respectively. CONCLUSIONS: Low-dose HDACi treatment can terminally differentiate MRT tumor cells and reduce their ability to self-renew. The use of low-dose HDACi as a novel therapeutic approach warrants further investigation. Clin Cancer Res; 22(14); 3560-70. ©2016 AACR.

A Synthetic Lethal Interaction between Glutathione Synthesis and Mitochondrial Reactive Oxygen Species Provides a Tumor-Specific Vulnerability Dependent on STAT3.

Increased production of mitochondrion-derived reactive oxygen species (ROS) is characteristic of a metabolic shift observed during malignant transformation. While the exact sources and roles of ROS in tumorigenesis remain to be defined, it has become clear that maintaining redox balance is critical for cancer cell proliferation and survival and, as such, may represent a vulnerability that can be exploited therapeutically. STAT3, a latent cytosolic transcription factor activated by diverse cytokines and growth factors, has been shown to exhibit an additional, nontranscriptional function in mitochondria, including modulation of electron transport chain activity. In particular, malignant transformation by Ras oncogenes exploits mitochondrial STAT3 functions. We used mass spectrometry-based metabolomics profiling to explore the biochemical basis for the STAT3 dependence of Ras transformation. We identified the gamma-glutamyl cycle, the production of glutathione, and the regulation of ROS as a mitochondrion-STAT3-dependent pathway in Ras-transformed cells. Experimental inhibition of key enzymes in the glutathione cycle resulted in the depletion of glutathione, accumulation of ROS, oxidative DNA damage, and cell death in an oncogenic Ras- and mitochondrial STAT3-dependent manner. These data uncover a synthetic lethal interaction involving glutathione production and mitochondrial ROS regulation in Ras-transformed cells that is governed by mitochondrial STAT3 and might be exploited therapeutically.

IL-37 requires the receptors IL-18Rα and IL-1R8 (SIGIRR) to carry out its multifaceted anti-inflammatory program upon innate signal transduction.

Interleukin 37 (IL-37) and IL-1R8 (SIGIRR or TIR8) are anti-inflammatory orphan members of the IL-1 ligand family and IL-1 receptor family, respectively. Here we demonstrate formation and function of the endogenous ligand-receptor complex IL-37-IL-1R8-IL-18Rα. The tripartite complex assembled rapidly on the surface of peripheral blood mononuclear cells upon stimulation with lipopolysaccharide. Silencing of IL-1R8 or IL-18Rα impaired the anti-inflammatory activity of IL-37. Whereas mice with transgenic expression of IL-37 (IL-37tg mice) with intact IL-1R8 were protected from endotoxemia, IL-1R8-deficient IL-37tg mice were not. Proteomic and transcriptomic investigations revealed that IL-37 used IL-1R8 to harness the anti-inflammatory properties of the signaling molecules Mer, PTEN, STAT3 and p62(dok) and to inhibit the kinases Fyn and TAK1 and the transcription factor NF-κB, as well as mitogen-activated protein kinases. Furthermore, IL-37-IL-1R8 exerted a pseudo-starvational effect on the metabolic checkpoint kinase mTOR. IL-37 thus bound to IL-18Rα and exploited IL-1R8 to activate a multifaceted intracellular anti-inflammatory program.

BTB-ZF transcriptional regulator PLZF modifies chromatin to restrain inflammatory signaling programs.

Inflammation is critical for host defense, but without appropriate control, it can cause chronic disease or even provoke fatal responses. Here we identify a mechanism that limits the inflammatory response. Probing the responses of macrophages to the key sensory Toll-like receptors, we identify that the Broad-complex, Tramtrack and Bric-a-brac/poxvirus and zinc finger (BTB/POZ), transcriptional regulator promyelocytic leukemia zinc finger (PLZF) limits the expression of inflammatory gene products. In accord with this finding, PLZF-deficient animals express higher levels of potent inflammatory cytokines and mount exaggerated inflammatory responses to infectious stimuli. Temporal quantitation of inflammatory gene transcripts shows increased gene induction in the absence of PLZF. Genome-wide analysis of histone modifications distinguish that PLZF establishes basal activity states of early response genes to maintain immune homeostasis and limit damaging inflammation. We show that PLZF stabilizes a corepressor complex that encompasses histone deacetylase activity to control chromatin. Together with our previous demonstration that PLZF promotes the antiviral response, these results suggest a strategy that could realize one of the major goals of immune therapy to retain immune resistance to pathogens while curbing damaging inflammation.

Genomic characterisation of small cell lung cancer patient-derived xenografts generated from endobronchial ultrasound-guided transbronchial needle aspiration specimens.

Patient-derived xenograft (PDX) models generated from surgical specimens are gaining popularity as preclinical models of cancer. However, establishment of PDX lines from small cell lung cancer (SCLC) patients is difficult due to very limited amount of available biopsy material. We asked whether SCLC cells obtained from endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) could generate PDX lines that maintained the phenotypic and genetic characteristics of the primary tumor. Following successful EBUS-TBNA sampling for diagnostic purposes, we obtained an extra sample for cytologic analysis and implantation into the flanks of immunodeficient mice. Animals were monitored for engraftment for up to 6 months. Histopathologic and immunohistochemical analysis, and targeted next-generation re-sequencing, were then performed in both the primary sample and the derivative PDX line. A total of 12 patients were enrolled in the study. EBUS-TBNA aspirates yielded large numbers of viable tumor cells sufficient to inject between 18,750 and 1,487,000 cells per flank, and to yield microgram quantities of high-quality DNA. Of these, samples from 10 patients generated xenografts (engraftment rate 83%) with a mean latency of 104 days (range 63-188). All but one maintained a typical SCLC phenotype that closely matched the original sample. Identical mutations that are characteristic of SCLC were identified in both the primary sample and xenograft line. EBUS-TBNA has the potential to be a powerful tool in the development of new targeting strategies for SCLC patients by providing large numbers of viable tumor cells suitable for both xenografting and complex genomic analysis.

Seminoma and embryonal carcinoma footprints identified by analysis of integrated genome-wide epigenetic and expression profiles of germ cell cancer cell lines.

BACKGROUND: Originating from Primordial Germ Cells/gonocytes and developing via a precursor lesion called Carcinoma In Situ (CIS), Germ Cell Cancers (GCC) are the most common cancer in young men, subdivided in seminoma (SE) and non-seminoma (NS). During physiological germ cell formation/maturation, epigenetic processes guard homeostasis by regulating the accessibility of the DNA to facilitate transcription. Epigenetic deregulation through genetic and environmental parameters (i.e. genvironment) could disrupt embryonic germ cell development, resulting in delayed or blocked maturation. This potentially facilitates the formation of CIS and progression to invasive GCC. Therefore, determining the epigenetic and functional genomic landscape in GCC cell lines could provide insight into the pathophysiology and etiology of GCC and provide guidance for targeted functional experiments. RESULTS: This study aims at identifying epigenetic footprints in SE and EC cell lines in genome-wide profiles by studying the interaction between gene expression, DNA CpG methylation and histone modifications, and their function in the pathophysiology and etiology of GCC. Two well characterized GCC-derived cell lines were compared, one representative for SE (TCam-2) and the other for EC (NCCIT). Data were acquired using the Illumina HumanHT-12-v4 (gene expression) and HumanMethylation450 BeadChip (methylation) microarrays as well as ChIP-sequencing (activating histone modifications (H3K4me3, H3K27ac)). Results indicate known germ cell markers not only to be differentiating between SE and NS at the expression level, but also in the epigenetic landscape. CONCLUSION: The overall similarity between TCam-2/NCCIT support an erased embryonic germ cell arrested in early gonadal development as common cell of origin although the exact developmental stage from which the tumor cells are derived might differ. Indeed, subtle difference in the (integrated) epigenetic and expression profiles indicate TCam-2 to exhibit a more germ cell-like profile, whereas NCCIT shows a more pluripotent phenotype. The results provide insight into the functional genome in GCC cell lines.

Next-generation sequence analysis of cancer xenograft models.

Next-generation sequencing (NGS) studies in cancer are limited by the amount, quality and purity of tissue samples. In this situation, primary xenografts have proven useful preclinical models. However, the presence of mouse-derived stromal cells represents a technical challenge to their use in NGS studies. We examined this problem in an established primary xenograft model of small cell lung cancer (SCLC), a malignancy often diagnosed from small biopsy or needle aspirate samples. Using an in silico strategy that assign reads according to species-of-origin, we prospectively compared NGS data from primary xenograft models with matched cell lines and with published datasets. We show here that low-coverage whole-genome analysis demonstrated remarkable concordance between published genome data and internal controls, despite the presence of mouse genomic DNA. Exome capture sequencing revealed that this enrichment procedure was highly species-specific, with less than 4% of reads aligning to the mouse genome. Human-specific expression profiling with RNA-Seq replicated array-based gene expression experiments, whereas mouse-specific transcript profiles correlated with published datasets from human cancer stroma. We conclude that primary xenografts represent a useful platform for complex NGS analysis in cancer research for tumours with limited sample resources, or those with prominent stromal cell populations.