Ron Chen

Wilms tumor 1 (WT1) regulates KRAS-driven oncogenesis and senescence in mouse and human models

KRAS is one of the most frequently mutated human oncogenes. In some settings, oncogenic KRAS can trigger cellular senescence, whereas in others it produces hyperproliferation. Elucidating the mechanisms regulating these 2 drastically distinct outcomes would help identify novel therapeutic approaches in RAS-driven cancers. Using a combination of functional genomics and mouse genetics, we identified a role for the transcription factor Wilms tumor 1 (WT1) as a critical regulator of senescence and proliferation downstream of oncogenic KRAS signaling. Deletion or suppression of Wt1 led to senescence of mouse primary cells expressing physiological levels of oncogenic Kras but had no effect on wild-type cells, and Wt1 loss decreased tumor burden in a mouse model of Kras-driven lung cancer. In human lung cancer cell lines dependent on oncogenic KRAS, WT1 loss decreased proliferation and induced senescence. Furthermore, WT1 inactivation defined a gene expression signature that was prognostic of survival only in lung cancer patients exhibiting evidence of oncogenic KRAS activation. These findings reveal an unexpected role for WT1 as a key regulator of the genetic network of oncogenic KRAS and provide important insight into the mechanisms that regulate proliferation or senescence in response to oncogenic signals.

Mutations in NGLY1 cause an inherited disorder of the endoplasmic reticulum-associated degradation pathway

Purpose:The endoplasmic reticulum–associated degradation pathway is responsible for the translocation of misfolded proteins across the endoplasmic reticulum membrane into the cytosol for subsequent degradation by the proteasome. To define the phenotype associated with a novel inherited disorder of cytosolic endoplasmic reticulum–associated degradation pathway dysfunction, we studied a series of eight patients with deficiency of N-glycanase 1.Methods:Whole-genome, whole-exome, or standard Sanger sequencing techniques were employed. Retrospective chart reviews were performed in order to obtain clinical data.Results:All patients had global developmental delay, a movement disorder, and hypotonia. Other common findings included hypolacrima or alacrima (7/8), elevated liver transaminases (6/7), microcephaly (6/8), diminished reflexes (6/8), hepatocyte cytoplasmic storage material or vacuolization (5/6), and seizures (4/8). The nonsense mutation c.1201A>T (p.R401X) was the most common deleterious allele.Conclusion:NGLY1 deficiency is a novel autosomal recessive disorder of the endoplasmic reticulum–associated degradation pathway associated with neurological dysfunction, abnormal tear production, and liver disease. The majority of patients detected to date carry a specific nonsense mutation that appears to be associated with severe disease. The phenotypic spectrum is likely to enlarge as cases with a broader range of mutations are detected.Genet Med 16 10, 751–758.

A Peripheral Blood Diagnostic Test for Acute Rejection in Renal Transplantation

Monitoring of renal graft status through peripheral blood (PB) rather than invasive biopsy is important as it will lessen the risk of infection and other stresses, while reducing the costs of rejection diagnosis. Blood gene biomarker panels were discovered by microarrays at a single center and subsequently validated and cross‐validated by QPCR in the NIH SNSO1 randomized study from 12 US pediatric transplant programs. A total of 367 unique human PB samples, each paired with a graft biopsy for centralized, blinded phenotype classification, were analyzed (115 acute rejection (AR), 180 stable and 72 other causes of graft injury). Of the differentially expressed genes by microarray, Q‐PCR analysis of a five gene‐set (DUSP1, PBEF1, PSEN1, MAPK9 and NKTR) classified AR with high accuracy. A logistic regression model was built on independent training‐set (n = 47) and validated on independent test‐set (n = 198)samples, discriminating AR from STA with 91% sensitivity and 94% specificity and AR from all other non‐AR phenotypes with 91% sensitivity and 90% specificity. The 5‐gene set can diagnose AR potentially avoiding the need for invasive renal biopsy. These data support the conduct of a prospective study to validate the clinical predictive utility of this diagnostic tool.

A meta-analysis of lung cancer gene expression identifies PTK7 as a survival gene in lung adenocarcinoma

Lung cancer remains the most common cause of cancer-related death worldwide and it continues to lack effective treatment. The increasingly large and diverse public databases of lung cancer gene expression constitute a rich source of candidate oncogenic drivers and therapeutic targets. To define novel targets for lung adenocarcinoma, we conducted a large-scale meta-analysis of genes specifically overexpressed in adenocarcinoma. We identified an 11-gene signature that was overexpressed consistently in adenocarcinoma specimens relative to normal lung tissue. Six genes in this signature were specifically overexpressed in adenocarcinoma relative to other subtypes of non-small cell lung cancer (NSCLC). Among these genes was the little studied protein tyrosine kinase PTK7. Immunohistochemical analysis confirmed that PTK7 is highly expressed in primary adenocarcinoma patient samples. RNA interference-mediated attenuation of PTK7 decreased cell viability and increased apoptosis in a subset of adenocarcinoma cell lines. Further, loss of PTK7 activated the MKK7-JNK stress response pathway and impaired tumor growth in xenotransplantation assays. Our work defines PTK7 as a highly and specifically expressed gene in adenocarcinoma and a potential therapeutic target in this subset of NSCLC.

A common peripheral blood gene set for diagnosis of operational tolerance in pediatric and adult liver transplantation.

To identify biomarkers of operational tolerance in pediatric and adult liver transplant recipients, transcriptional profiles were examined from 300 samples by microarrays and Q‐PCR measurements of blood specimens from pediatric and adult liver transplant recipients and normal tissues. Tolerance‐specific genes were validated in independent samples across two different transplant programs and validated by Q‐PCR. A minimal set of 13 unique genes, highly expressed in natural killer cells (p = 0.03), were significantly expressed in both pediatric and adult liver tolerance, irrespective of different clinical and demographic confounders. The performance of this gene set by microarray in independent samples was 100% sensitivity and 83% specificity and the AUC was 0.988 for only three genes by Q‐PCR. 26% of adults and 64% of children with excellent liver allograft function, on minimal or dual immunosuppression, showed high prediction scores for tolerance. Novel peripheral transcriptional profiles can be identified in operational tolerance in pediatric and adult recipients of liver allografts, suggesting a high incidence of a pro‐tolerogenic phenotype in stable patients on chronic immunosuppression. Given the high incidence of viral infections and malignancies in liver transplant recipients, this gene set provides an important monitoring tool that can move the field toward personalized and predictive medicine in organ transplantation.

Long noncoding RNA EWSAT1-mediated gene repression facilitates Ewing sarcoma oncogenesis

Chromosomal translocation that results in fusion of the genes encoding RNA-binding protein EWS and transcription factor FLI1 (EWS-FLI1) is pathognomonic for Ewing sarcoma. EWS-FLI1 alters gene expression through mechanisms that are not completely understood. We performed RNA sequencing (RNAseq) analysis on primary pediatric human mesenchymal progenitor cells (pMPCs) expressing EWS-FLI1 in order to identify gene targets of this oncoprotein. We determined that long noncoding RNA-277 (Ewing sarcoma-associated transcript 1 [EWSAT1]) is upregulated by EWS-FLI1 in pMPCs. Inhibition of EWSAT1 expression diminished the ability of Ewing sarcoma cell lines to proliferate and form colonies in soft agar, whereas EWSAT1 inhibition had no effect on other cell types tested. Expression of EWS-FLI1 and EWSAT1 repressed gene expression, and a substantial fraction of targets that were repressed by EWS-FLI1 were also repressed by EWSAT1. Analysis of RNAseq data from primary human Ewing sarcoma further supported a role for EWSAT1 in mediating gene repression. We identified heterogeneous nuclear ribonucleoprotein (HNRNPK) as an RNA-binding protein that interacts with EWSAT1 and found a marked overlap in HNRNPK-repressed genes and those repressed by EWS-FLI1 and EWSAT1, suggesting that HNRNPK participates in EWSAT1-mediated gene repression. Together, our data reveal that EWSAT1 is a downstream target of EWS-FLI1 that facilitates the development of Ewing sarcoma via the repression of target genes.

Integration of disease-specific single nucleotide polymorphisms, expression quantitative trait loci and coexpression networks reveal novel candidate genes for type 2 diabetes

Aims/hypothesisWhile genome-wide association studies (GWASs) have been successful in identifying novel variants associated with various diseases, it has been much more difficult to determine the biological mechanisms underlying these associations. Expression quantitative trait loci (eQTL) provide another dimension to these data by associating single nucleotide polymorphisms (SNPs) with gene expression. We hypothesised that integrating SNPs known to be associated with type 2 diabetes with eQTLs and coexpression networks would enable the discovery of novel candidate genes for type 2 diabetes.MethodsWe selected 32 SNPs associated with type 2 diabetes in two or more independent GWASs. We used previously described eQTLs mapped from genotype and gene expression data collected from 1,008 morbidly obese patients to find genes with expression associated with these SNPs. We linked these genes to coexpression modules, and ranked the other genes in these modules using an inverse sum score.ResultsWe found 62 genes with expression associated with type 2 diabetes SNPs. We validated our method by linking highly ranked genes in the coexpression modules back to SNPs through a combined eQTL dataset. We showed that the eQTLs highlighted by this method are significantly enriched for association with type 2 diabetes in data from the Wellcome Trust Case Control Consortium (WTCCC, p = 0.026) and the Gene Environment Association Studies (GENEVA, p = 0.042), validating our approach. Many of the highly ranked genes are also involved in the regulation or metabolism of insulin, glucose or lipids.Conclusions/interpretationWe have devised a novel method, involving the integration of datasets of different modalities, to discover novel candidate genes for type 2 diabetes.

Two Is Better Than One: Combining IGF1R and MEK Blockade as a Promising Novel Treatment Strategy Against KRAS-Mutant Lung Cancer

A small-molecule inhibitor screen on a panel of human lung cancer cell lines has uncovered an unexpected sensitivity of cells expressing oncogenic KRAS toward insulin-like growth factor 1 receptor (IGF1R) inhibition. Combining IGF1R and MAP-ERK kinase blockade led to significant effects on viability in human non-small cell lung cancer (NSCLC) cell lines and in 2 mouse models of oncogenic KRAS-driven lung cancer. The mechanistic basis for this effect seems to be an increased baseline activation of IGF1R-mediated activation of AKT in cells that express oncogenic KRAS. The studies thus point to a novel approach for treatment of KRAS-driven NSCLC, a particularly difficult subset of patients to treat with existing approaches.

Abstract IA16: Identifying KRAS synthetic lethal interactions using systems biology and functional genomics

Our laboratory uses cross-species genomic approaches to understand the oncogenic effects of point mutations in KRAS. Using cross-species analysis of mouse models of lung cancer and human lung cancer, we have identified “KRAS signatures” of gene expression and used them to identify key drivers of gene regulation downstream of Kras. A key goal of our laboratory is to reverse engineer these oncogenic signatures to identify key transcriptional regulators which could be inhibited for anti-oncogenic therapy. Previously we used this approach to show that Wt1 is required to bypass a senescence response in cells expressing oncogenic KRAS (Vicent et al JCI 2010). We have now carried out a “second generation” synthetic lethal screen using a more comprehensive computational approach to identify “master regulators” of KRAS-driven gene expression. A focused shRNA screen in a large panel of human NSCLC lines confirmed the synthetic lethality of Wt1 in Kras-mutant tumor lines. In addition, we have identified several other likely “hits.” One of these, TCEB1, is a gene involved in regulating both elongation and the hypoxia response. We are currently carrying out further experiments to elucidate the function of TCEB1 and determine why it is required in cells expressing oncogenic Kras. Citation Format: Ron Chen, Dana Gwinn, Alejandro Sweet-Cordero. Identifying KRAS synthetic lethal interactions using systems biology and functional genomics. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr IA16. doi: 10.1158/1557-3125.RASONC14-IA16

Abstract A31: The long noncoding RNA lnc277 mediates a repressive gene signature in Ewing's sarcoma

Ewing9s sarcoma is a highly aggressive tumor characterized by a translocation between members of the FET family of RNA binding proteins and one of several ETS transcription factors, with the most common translocation being EWS-FLI1. Like many oncogenic translocations, EWS-FLI1 leads to changes in gene expression through mechanisms that are not completely understood. We performed RNA sequencing analysis on human mesenchymal progenitor cells (MPCs) expressing EWS-FLI1, which are thought to be the cell of origin of Ewing9s sarcoma. This analysis identified lnc277 as a previously uncharacterized long non-coding RNA upregulated by EWS-FLI1 in MPCs. Expression of lnc277 in Ewing9s sarcoma cell lines requires EWS-FLI1. In addition, inhibiting the expression of lnc277 diminished the ability of Ewing9s sarcoma cell lines to proliferate and form soft agar colonies. Using RNA sequencing and shRNA knockdown, we defined the transcriptional changes induced by EWS-FLI1 and lnc277. This analysis revealed that both EWS-FLI1 and lnc277 repress far more genes that they induce and that a large fraction of EWS-FLI1 repressed targets are also modulated by lnc277. Thus, lnc277 is an oncogenic long non-coding RNA downstream of EWS-FLI1 that facilitates the formation of Ewing9s sarcoma via the repression of target genes. Citation Format: Michelle Marques, David Simpson, Ron Chen, Dedeepya Vaka, Marcus Breese, Allayne Brunner, Rob West, Alejandro Sweet-Cordero. The long noncoding RNA lnc277 mediates a repressive gene signature in Ewing9s sarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A31.