Harnessing synthetic lethality to predict the response to cancer treatment.

While synthetic lethality (SL) holds promise in developing effective cancer therapies, SL candidates found via experimental screens often have limited translational value. Here we present a data-driven approach, ISLE (identification of clinically relevant synthetic lethality), that mines TCGA cohort to identify the most likely clinically relevant SL interactions (cSLi) from a given candidate set of lab-screened SLi. We first validate ISLE via a benchmark of large-scale drug response screens and by predicting drug efficacy in mouse xenograft models. We then experimentally test a select set of predicted cSLi via new screening experiments, validating their predicted context-specific sensitivity in hypoxic vs normoxic conditions and demonstrating cSLi's utility in predicting synergistic drug combinations. We show that cSLi can successfully predict patients' drug treatment response and provide patient stratification signatures. ISLE thus complements existing actionable mutation-based methods for precision cancer therapy, offering an opportunity to expand its scope to the whole genome.

Recapitulating the clinical scenario of BRCA-associated pancreatic cancer in pre-clinical models.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies. BRCA-associated PDAC comprises a clinically relevant subtype. A portion of these patients are highly susceptible to DNA damaging therapeutics, however, responses are heterogeneous and clinical resistance evolves. We have developed unique patient-derived xenograft (PDX) models from metastatic lesions of germline BRCA-mutated patients obtained at distinct time points; before treatment and at progression. Thus, closely mimicking clinical scenarios, to further investigate treatment naïve and resistant patients. DNA was isolated from six BRCA-mutated PDXs and classified by whole-genome sequencing to stable-genome or homologous recombination deficient (HRD)-genome. The sensitivity to DNA-damaging agents was evaluated in vivo in three BRCA-associated PDAC PDXs models: (1) HRD-genome naïve to treatments; (2) stable-genome naïve to treatment; (3) HRD-genome resistant to treatment. Correlation between disease course at tissue acquisition and response to PARP inhibitor (PARPi)/platinum was demonstrated in PDXs in vivo. Only the HRD-genome PDX, naïve to treatment, was sensitive to PARP inhibitor/cisplatin treatments. Our results demonstrate heterogeneous responses to DNA damaging agents/PARPi in BRCA-associated PDX thus reflecting the wide clinical spectrum. An HRD-genome PDX generated from a naïve to treatment biopsy was sensitive to platinum/PARPi whereas no benefit was observed in treating a HRD-genome PDXs generated from a patient that had acquired resistance nor stable-genome PDXs.

Pancreatic cancer ascites xenograft-an expeditious model mirroring advanced therapeutic resistant disease.

Pancreatic ductal adenocarcinoma has limited treatment options. There is an urgent need for developing appropriate pre-clinical models recapitulating metastatic disease, the most common clinical scenario at presentation. Ascites accumulation occurs in up to 20-30% of patients with pancreatic cancer; this milieu represents a highly cellular research resource of metastatic peritoneal spread. In this study, we utilized pancreatic ascites/pleural effusion cancer cells to establish patient derived xenografts.Ascites/pleural effusion-patient derived xenografts were established from twelve independent cases. Xenografts were serially passed in nude mice and tissue bio-specimen banking has been established. Histopathology of emergent tumors demonstrates poorly to moderately differentiated, glandular and mucin producing tumors, mirroring morphology of primary pancreatic cancer tumors. Whole genome sequencing of six patient derived xenografts samples demonstrates common mutations and structural variations similar to those reported in primary pancreatic cancer. Xenograft tumors were dissociated to single-cells and in-vitro drug sensitivity screen assays demonstrated chemo-resistance, correlating with patient clinical scenarios, thus serving as a platform for clinically relevant translational research.Therefore, establishment of this novel ascites/pleural effusion patient derived xenograft model, with extensive histopathology and genomic characterization, opens an opportunity for the study of advanced aggressive pancreatic cancer. Characterization of metastatic disease and mechanisms of resistance to therapeutics may lead to the development of novel drug combinations.

GeneAnalytics: An Integrative Gene Set Analysis Tool for Next Generation Sequencing, RNAseq and Microarray Data.

Postgenomics data are produced in large volumes by life sciences and clinical applications of novel omics diagnostics and therapeutics for precision medicine. To move from "data-to-knowledge-to-innovation," a crucial missing step in the current era is, however, our limited understanding of biological and clinical contexts associated with data. Prominent among the emerging remedies to this challenge are the gene set enrichment tools. This study reports on GeneAnalytics™ ( geneanalytics.genecards.org ), a comprehensive and easy-to-apply gene set analysis tool for rapid contextualization of expression patterns and functional signatures embedded in the postgenomics Big Data domains, such as Next Generation Sequencing (NGS), RNAseq, and microarray experiments. GeneAnalytics' differentiating features include in-depth evidence-based scoring algorithms, an intuitive user interface and proprietary unified data. GeneAnalytics employs the LifeMap Science's GeneCards suite, including the GeneCards®--the human gene database; the MalaCards-the human diseases database; and the PathCards--the biological pathways database. Expression-based analysis in GeneAnalytics relies on the LifeMap Discovery®--the embryonic development and stem cells database, which includes manually curated expression data for normal and diseased tissues, enabling advanced matching algorithm for gene-tissue association. This assists in evaluating differentiation protocols and discovering biomarkers for tissues and cells. Results are directly linked to gene, disease, or cell "cards" in the GeneCards suite. Future developments aim to enhance the GeneAnalytics algorithm as well as visualizations, employing varied graphical display items. Such attributes make GeneAnalytics a broadly applicable postgenomics data analyses and interpretation tool for translation of data to knowledge-based innovation in various Big Data fields such as precision medicine, ecogenomics, nutrigenomics, pharmacogenomics, vaccinomics, and others yet to emerge on the postgenomics horizon.

Cell-based therapy approaches: the hope for incurable diseases.

Cell therapies aim to repair the mechanisms underlying disease initiation and progression, achieved through trophic effect or by cell replacement. Multiple cell types can be utilized in such therapies, including stem, progenitor or primary cells. This review covers the current state of cell therapies designed for the prominent disorders, including cardiovascular, neurological (Parkinson's disease, amyotrophic lateral sclerosis, stroke, spinal cord injury), autoimmune (Type 1 diabetes, multiple sclerosis, Crohn's disease), ophthalmologic, renal, liver and skeletal (osteoarthritis) diseases. Various cell therapies have reached advanced clinical trial phases with potential marketing approvals in the near future, many of which are based on mesenchymal stem cells. Advances in pluripotent stem cell research hold great promise for regenerative medicine. The information presented in this review is based on the analysis of the cell therapy collection detailed in LifeMap Discovery(®) (LifeMap Sciences Inc., USA) the database of embryonic development, stem cell research and regenerative medicine.

Reactivation of NCAM1 defines a subpopulation of human adult kidney epithelial cells with clonogenic and stem/progenitor properties.

The nephron is composed of a monolayer of epithelial cells that make up its various compartments. In development, these cells begin as mesenchyme. NCAM1, abundant in the mesenchyme and early nephron lineage, ceases to express in mature kidney epithelia. We show that, once placed in culture and released from quiescence, adult human kidney epithelial cells (hKEpCs), uniformly positive for CD24/CD133, re-express NCAM1 in a specific cell subset that attains a stem/progenitor state. Immunosorted NCAM1(+) cells overexpressed early nephron progenitor markers (PAX2, SALL1, SIX2, WT1) and acquired a mesenchymal fate, indicated by high vimentim and reduced E-cadherin levels. Gene expression and microarray analysis disclosed both a proximal tubular origin of these cells and molecules regulating epithelial-mesenchymal transition. NCAM1(+) cells generated clonal progeny when cultured in the presence of fetal kidney conditioned medium, differentiated along mesenchymal lineages but retained the unique propensity to generate epithelial kidney spheres and produce epithelial renal tissue on single-cell grafting in chick CAM and mouse. Depletion of NCAM1(+) cells from hKEpCs abrogated stemness traits in vitro. Eliminating these cells during the regenerative response that follows glycerol-induced acute tubular necrosis worsened peak renal injury in vivo. Thus, higher clone-forming and developmental capacities characterize a distinct subset of adult kidney-derived cells. The ability to influence an endogenous regenerative response via NCAM1 targeting may lead to novel therapeutics for renal diseases.

LifeMap Discovery™: the embryonic development, stem cells, and regenerative medicine research portal.

LifeMap Discovery™ provides investigators with an integrated database of embryonic development, stem cell biology and regenerative medicine. The hand-curated reconstruction of cell ontology with stem cell biology; including molecular, cellular, anatomical and disease-related information, provides efficient and easy-to-use, searchable research tools. The database collates in vivo and in vitro gene expression and guides translation from in vitro data to the clinical utility, and thus can be utilized as a powerful tool for research and discovery in stem cell biology, developmental biology, disease mechanisms and therapeutic discovery. LifeMap Discovery is freely available to academic nonprofit institutions at http://discovery.lifemapsc.com.

Chromatin-modifying agents reactivate embryonic renal stem/progenitor genes in human adult kidney epithelial cells but abrogate dedifferentiation and stemness.

Recent studies have suggested that epigenetic modulation with chromatin-modifying agents can induce stemness and dedifferentiation and increase developmental plasticity. For instance, valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, has been shown to promote self-renewal/expansion of hematopoietic stem cells and facilitate the generation of induced pluripotent stem cells (iPSCs). Previously, we observed that downregulation of embryonic renal stem/progenitor genes in the adult kidney was associated, at least in part, with epigenetic silencing. Therefore, we hypothesized that VPA may alter the expression of these genes and reprogram mature human adult kidney epithelial cells (hKEpCs) to a stem/progenitor-like state. Here, using quantitative RT-PCR and flow cytometry [fluorescence-activated cell sorting (FACS)] analysis, we show in VPA-treated primary cultures of human adult and fetal kidney significant reinduction of the renal stem/progenitor markers SIX2, OSR1, SALL1, NCAM, and PSA-NCAM. Robust SIX2 mRNA re-expression was confirmed at the protein level by western blot and was associated with epigenetic changes of the histones at multiple sites of the SIX2 promoter leading to gene activation, significantly increased acetylation of histones H4, and methylation of lysine 4 on H3. Furthermore, we could demonstrate synergistic effects of VPA and Wnt antagonists on SIX2 and also OSR1 reinduction. Nevertheless, VPA resulted in upregulation of E-CADHERIN and reduction in VIMENTIN, preventing the skewing of hKEpCs towards a more replicative mesenchymal state required for clonogenic expansion and acquisition of stem cell characters, altogether inducing cell senescence at early passages. These results demonstrating that chromatin-modifying agents prevent dedifferentiation of hKEpCs have important clinical implications as they may limit ex-vivo self-renewal/expansion and possibly the in vivo renal regenerative capacity initiated by dedifferentiation.

A rapid in vivo assay system for analyzing the organogenetic capacity of human kidney cells.

Transplantation of human kidney-derived cells is a potential therapeutic modality for promoting regeneration of diseased renal tissue. However, assays that determine the ability of candidate populations for renal cell therapy to undergo appropriate differentiation and morphogenesis are limited. We report here a rapid and humane assay for characterizing tubulogenic potency utilizing the well-established chorioallantoic membrane CAM) of the chick embryo. Adult human kidney-derived cells expanded in monolayer were suspended in Matrigel and grafted onto the CAM. After a week, grafts were assessed histologically. Strikingly, many of the renal cells self-organized into tubular structures. Host blood vessels penetrated and presumably fed the grafts. Immuno- and histochemical staining revealed that tubular structures were epithelial, but not blood vessels. Some of the cells both within and outside the tubules were dividing. Analysis for markers of proximal and distal renal tubules revealed that grafts contained individual cells of a proximal tubular phenotype and many tubules of distal tubule character. Our results demonstrate that the chick CAM is a useful xenograft system for screening for differentiation and morphogenesis in cells with potential use in renal regenerative medicine.

Kidney spheroids recapitulate tubular organoids leading to enhanced tubulogenic potency of human kidney-derived cells.

Cell-based approaches utilizing autologous human renal cells require their isolation, expansion in vitro, and reintroduction back into the host for renal tissue regeneration. Nevertheless, human kidney epithelial cells (hKEpCs) lose their phenotype, dedifferentiate, and assume the appearance of fibroblasts after relatively few passages in culture. We hypothesized that growth conditions may influence hKEpC phenotype and function. hKEpCs retrieved from human nephrectomy tissue samples showed the ability to reproducibly form kidney spheres when grown in suspension culture developed in nonadherent conditions. Genetic labeling and time-lapse microscopy indicated, at least in part, the aggregation of hKEpCs into 3D spheroids rather than formation of pure clonally expanded spheres. Characterization of hKEpC spheroids by real-time polymerase chain reaction and FACS analysis showed upregulation of some renal developmental and "stemness" markers compared with monolayer and mostly an EpCAM(+)CD24(+)CD133(+)CD44(+) spheroid cell phenotype. Oligonucleotide microarrays, which were used to identify global transcriptional changes accompanying spheroid formation, showed predominantly upregulation of cell matrix/cell contact molecules and cellular biogenesis processes and downregulation of cell cycle, growth, and locomotion. Accordingly, hKEpC spheroids slowly proliferated as indicated by low Ki-67 staining, but when grafted in low cell numbers onto the chorioallantoic membrane (CAM) of the chick embryo, they exclusively reconstituted various renal tubular epithelia. Moreover, efficient generation of kidney spheroids was observed after long-term monolayer culture resulting in reestablishment of tubulogenic capacity upon CAM grafting. Thus, generation of a tubular organoid in hKEpC spheroids may provide a functional benefit for kidney-derived cells in vivo.

Expression of stem cell markers in the human fetal kidney.

In the human fetal kidney (HFK) self-renewing stem cells residing in the metanephric mesenchyme (MM)/blastema are induced to form all cell types of the nephron till 34(th) week of gestation. Definition of useful markers is crucial for the identification of HFK stem cells. Because wilms' tumor, a pediatric renal cancer, initiates from retention of renal stem cells, we hypothesized that surface antigens previously up-regulated in microarrays of both HFK and blastema-enriched stem-like wilms' tumor xenografts (NCAM, ACVRIIB, DLK1/PREF, GPR39, FZD7, FZD2, NTRK2) are likely to be relevant markers. Comprehensive profiling of these putative and of additional stem cell markers (CD34, CD133, c-Kit, CD90, CD105, CD24) in mid-gestation HFK was performed using immunostaining and FACS in conjunction with EpCAM, an epithelial surface marker that is absent from the MM and increases along nephron differentiation and hence can be separated into negative, dim or bright fractions. No marker was specifically localized to the MM. Nevertheless, FZD7 and NTRK2 were preferentially localized to the MM and emerging tubules (<10% of HFK cells) and were mostly present within the EpCAM(neg) and EpCAM(dim) fractions, indicating putative stem/progenitor markers. In contrast, single markers such as CD24 and CD133 as well as double-positive CD24(+)CD133(+) cells comprise >50% of HFK cells and predominantly co-express EpCAM(bright), indicating they are mostly markers of differentiation. Furthermore, localization of NCAM exclusively in the MM and in its nephron progenitor derivatives but also in stroma and the expression pattern of significantly elevated renal stem/progenitor genes Six2, Wt1, Cited1, and Sall1 in NCAM(+)EpCAM(-) and to a lesser extent in NCAM(+)EpCAM(+) fractions confirmed regional identity of cells and assisted us in pinpointing the presence of subpopulations that are putative MM-derived progenitor cells (NCAM(+)EpCAM(+)FZD7(+)), MM stem cells (NCAM(+)EpCAM(-)FZD7(+)) or both (NCAM(+)FZD7(+)). These results and concepts provide a framework for developing cell selection strategies for human renal cell-based therapies.