Daniel H. Wai

The genomic landscape of the Ewing Sarcoma family of tumors reveals recurrent STAG2 mutation.

The Ewing sarcoma family of tumors (EFT) is a group of highly malignant small round blue cell tumors occurring in children and young adults. We report here the largest genomic survey to date of 101 EFT (65 tumors and 36 cell lines). Using a combination of whole genome sequencing and targeted sequencing approaches, we discover that EFT has a very low mutational burden (0.15 mutations/Mb) but frequent deleterious mutations in the cohesin complex subunit STAG2 (21.5% tumors, 44.4% cell lines), homozygous deletion of CDKN2A (13.8% and 50%) and mutations of TP53 (6.2% and 71.9%). We additionally note an increased prevalence of the BRCA2 K3326X polymorphism in EFT patient samples (7.3%) compared to population data (OR 7.1, p = 0.006). Using whole transcriptome sequencing, we find that 11% of tumors pathologically diagnosed as EFT lack a typical EWSR1 fusion oncogene and that these tumors do not have a characteristic Ewing sarcoma gene expression signature. We identify samples harboring novel fusion genes including FUS-NCATc2 and CIC-FOXO4 that may represent distinct small round blue cell tumor variants. In an independent EFT tissue microarray cohort, we show that STAG2 loss as detected by immunohistochemistry may be associated with more advanced disease (p = 0.15) and a modest decrease in overall survival (p = 0.10). These results significantly advance our understanding of the genomic and molecular underpinnings of Ewing sarcoma and provide a foundation towards further efforts to improve diagnosis, prognosis, and precision therapeutics testing.

Prkar1a is an osteosarcoma tumor suppressor that defines a molecular subclass in mice.

Some cancers have been stratified into subclasses based on their unique involvement of specific signaling pathways. The mapping of human cancer genomes is revealing a vast number of somatic alterations; however, the identification of clinically relevant molecular tumor subclasses and their respective driver genes presents challenges. This information is key to developing more targeted and personalized cancer therapies. Here, we generate a new mouse model of genomically unstable osteosarcoma (OSA) that phenocopies the human disease. Integrative oncogenomics pinpointed cAMP-dependent protein kinase type I, alpha regulatory subunit (Prkar1a) gene deletions at 11qE1 as a recurrent genetic trait for a molecularly distinct subclass of mouse OSA featuring RANKL overexpression. Using mouse genetics, we established that Prkar1a is a bone tumor suppressor gene capable of directing subclass development and driving RANKL overexpression during OSA tumorigenesis. Finally, we uncovered evidence for a PRKAR1A-low subset of human OSA with distinct clinical behavior. Thus, tumor subclasses develop in mice and can potentially provide information toward the molecular stratification of human cancers.

Synthetic Lethality Screens Reveal RPS6 and MST1R as Modifiers of Insulin-like Growth Factor-1 Receptor Inhibitor Activity in Childhood Sarcomas

The insulin-like growth factor-1 receptor (IGF1R) is emerging as a promising therapeutic target in human cancers. In the high-risk childhood sarcomas Ewing family tumor and rhabdomyosarcoma, IGF1R-blocking antibodies show impressive antitumor activity in some but not all patients, and acquired resistance is observed. Because tumor IGF1R mutations are not described, the basis of IGF1R inhibitor resistance remains unknown. We hypothesized that compensatory signaling cascades bypassing targeted IGF1R inhibition might be involved. To test this systematically, we performed small interfering RNA (siRNA) screens in sarcoma cell lines to identify IGF1R pathway components or related protein tyrosine kinase (PTK) networks that modulate the antitumor efficacy of the BMS-536924 IGF1R kinase inhibitor. This strategy revealed (a) that sarcoma cells are exquisitely sensitive to loss of distal rather than proximal IGF1R signaling components, such as ribosomal protein S6 (RPS6); (b) that BMS-536924 fails to block RPS6 activation in resistant sarcoma cell lines; and (c) that siRNA knockdown of the macrophage-stimulating 1 receptor tyrosine kinase (MST1R; also known as RON) restores BMS-536924 efficacy, even in highly drug-resistant cell lines. We confirmed MST1R expression across a broad panel of childhood sarcomas, and found that loss of MST1R by RNA interference blocks downstream RPS6 activation when combined with BMS-536924 in vitro. These findings underscore the importance of fully understanding PTK networks for successful clinical implementation of kinase inhibitor strategies.

ERBB4 confers metastatic capacity in Ewing sarcoma

Metastatic spread is the single‐most powerful predictor of poor outcome in Ewing sarcoma (ES). Therefore targeting pathways that drive metastasis has tremendous potential to reduce the burden of disease in ES. We previously showed that activation of the ERBB4 tyrosine kinase suppresses anoikis, or detachment‐induced cell death, and induces chemoresistance in ES cell lines in vitro. We now show that ERBB4 is transcriptionally overexpressed in ES cell lines derived from chemoresistant or metastatic ES tumours. ERBB4 activates the PI3K‐Akt cascade and focal adhesion kinase (FAK), and both pathways contribute to ERBB4‐mediated activation of the Rac1 GTPase in vitro and in vivo. ERBB4 augments tumour invasion and metastasis in vivo, and these effects are blocked by ERBB4 knockdown. ERBB4 expression correlates significantly with reduced disease‐free survival, and increased expression is observed in metastatic compared to primary patient‐matched ES biopsies. Our findings identify a novel ERBB4‐PI3K‐Akt‐FAK‐Rac1 pathway associated with aggressive disease in ES. These results predict that therapeutic targeting of ERBB4, alone or in combination with cytotoxic agents, may suppress the metastatic phenotype in ES.

Diagnostic and prognostic sarcoma signatures.

Sarcomas are a diverse group of childhood and adult tumors that arise from mesenchymal tissue. In contrast to epithelial tumors, most of which are defined by a specific organ system, sarcomas can arise virtually anywhere in the body, such that their characteristic histopathology and clinical presentation form the core diagnostic criteria.Precise identification by differential diagnosis is the first element of a successful treatment, since these tumors show wide variation in response to specific therapies and misdiagnosis can lead to suboptimal therapy. However, due to overlapping histopathologic features among the sarcomas, as well as the multiple subtypes or variants within a single histologic group, pathologists and clinicians are increasingly reliant on molecular diagnostic approaches to aid in the differential diagnosis. Gene expression profiling or microarray analysis is now being used to develop expression signatures that appear to be better than histological features or any single biomarker at discriminating tumor types, identifying clinical variants, and modeling complex tumor behavior.This review examines the current progress in identifying diagnostic and prognostic expression signatures for four sarcomas: rhabdomyosarcoma, Ewing’s family of tumors, synovial sarcoma, and osteosarcoma. In this context, we discuss the current status and future potential for using expression signatures to improve tumor classification, outcome prediction, and therapeutic response in patients with these sarcomas.

Influence of RNA Labeling on Expression Profiling of MicroRNAs

Although a number of technical parameters are now being examined to optimize microRNA profiling experiments, it is unknown whether reagent or component changes to the labeling step affect starting RNA requirements or microarray performance. Human brain/lung samples were each labeled in duplicate, at 1.0, 0.5, 0.2, and 0.1 μg of total RNA, by means of two kits that use the same labeling procedure but differ in the reagent composition used to label microRNAs. Statistical measures of reliability and validity were used to evaluate microarray data. Cross-platform confirmation was accomplished using TaqMan microRNA assays. Synthetic microRNA spike-in experiments were also performed to establish the microarray signal dynamic range using the ligation-modified kit. Technical replicate correlations of signal intensity values were high using both kits, but improved with the ligation-modified assay. The drop in detection call sensitivity and miRNA gene list correlations, when using reduced amounts of standard-labeled RNA, was considerably improved with the ligation-modified kit. Microarray signal dynamic range was found to be linear across three orders of magnitude from 4.88 to 5000 attomoles. Thus, optimization of the microRNA labeling reagent can result in at least a 10-fold decrease in microarray total RNA requirements with little compromise to data quality. Clinical investigations bottlenecked by the amount of starting material may use a ligation mix modification strategy to reduce total RNA requirements.

Synergistic and antagonistic AML cell type-specific responses to 5-AZA-2’-Deoxycytidine and 1-H-D-Arabinofuranosylcytosine

Background The search for synergistic drug combinations is critical to the treatment of drug resistant cancers, such as acute myelogenous leukemia (AML), which has 50% to 60% overall survival in children and lower in adults [1]. Conventional treatments include the S phase-specific polymerase II inhibitor 1-h-D-Arabinofuranosylcytosine (ARA-C), but DNA methyltransferase inhibitors such as 5-Aza-2 -deoxycytidine (DAC) also have activity in AML, through DNA genomic demethylation and altered gene expression [2]. Although genome-wide changes in RNA expression associated with DAC and ARA-C are not fully understood [3], characterizing these responses is a critical step to increasing the efficacy of combinatorial therapies that include DAC and ARA-C.

Abstract B18: The transcribed non-coding genome modulates the biology of Ewing sarcoma family of tumors

Abstract Introduction: Ewing sarcoma family of tumors (EFT) is the second most common primary malignant bone tumor in children and adolescents. It features a characteristic chromosomal translocation; the most common being (t11;22)(q24;q12), which encodes for the chimeric EWS-FLI1 oncoprotein. The disease is highly invasive with a 30% 5-year survival rate for patients with metastatic disease. This study identifies and characterizes a non-coding locus that is unique to EFTs and mediates at least in part the role of the chimeric oncogene in EFT pathogenesis. This particular locus suggests that better understanding of the functional non-coding transcriptome of this developmental cancer can potentially assist in discovering biomarkers that improve the diagnosis and therapeutic management of the disease. Methods: Expression profiles from a panel of 500 tissue samples including over 100 EFTs, other primary tumors, and normal tissues were generated using Affymetrix Human Exon 1.0 ST microarrays. Data were analyzed using institutional software, Genetrix. RNAseq data was generated on two EFT cell lines derived from a primary tumor and its metastatic derivative. Data was validated by PCR on 33 cell lines including EFTs and other tumors. Functional studies on EFT cell lines were performed using various RNAi techniques and viral transductions. Additional characterization of the RNA was done by cellular fractionation studies and northern blots. Results: Expression microarray profiles showed unannotated transcripts to be the strongest discriminators of EFTs from all other tumor types. One such transcript, AK057037 , was the most highly expressed RNA (1.07 × 10 −20 in EFTs. In silico modeling and RNASeq analyses revealed that this transcript is a multi-exonic, presumably, long non-coding RNA (lncRNA) with a maximum predicted open reading frame of 137 amino acids that had no homology, protein domains, or characteristic motifs. Additionally, multiple variants of the transcript exist in both the nucleus and the cytoplasm. Validation studies confirmed that this lncRNA was exclusively expressed in EFTs. The EFT-specific oncoprotein, EWS-FLI1, regulates expression of this transcript by directly binding to its promoter region. Furthermore, functional data based on knocking down and overexpressing the transcript shows that AK057037 acts as an oncogene in EFT and enhances tumorigenicity by increasing the metastatic potential of the tumor cell lines. Conclusion: The study established that lncRNA AK057037 promotes EFT pathogenesis. Its exclusive presence in EFT suggests that expression of this lncRNA is tissue-specific and highly regulated. Targeting AK057037 in these tumors may help improve outcomes in children with metastatic disease. Further work on the non-coding transcriptome may reveal additional functional RNAs that contribute to the biology of this disease. Citation Format: Sheetal A. Mitra, Daniel H. Wai, William A. May, Jonathan D. Buckley, Philipp Kapranov, Robert J. Arceci, Timothy J. Triche. The transcribed non-coding genome modulates the biology of Ewing sarcoma family of tumors [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr B18.

Abstract 4350: Translating diagnostic gene expression profiles for pediatric solid tumors

BACKGROUND: Features of cancer genomics including gene expression levels can be employed to create biomarker profiles that can predict prior to therapy the diagnosis and prognosis of an individual patient. We aim to translate robust and reproducible diagnostic and prognostic profiles for pediatric solid tumors into clinical tools applicable to all tumor specimens, fresh frozen (FF) or formalin fixed (FFPE). RESULTS: We used Affymetrix GeneChip Human Exon 1.0 ST (HuEx) arrays on 24 cell lines or tumors (4 fusion-positive rhabdomyosarcoma [RMSpos], 5 fusion-negative RMS [RMSneg], 6 Ewing sarcoma family of tumors [ESFT], 5 neuroblastoma [NB], and 4 osteosarcoma [OS]) to identify a 41-feature diagnostic metagene that clearly distinguished both the original test samples and a set of validation samples. We then incorporated the 41-feature metagene into a 48-gene panel. To translate this diagnostic signature, we compared the performance of HuEx arrays with several mid-plex assay platforms including Fluidigm9s quantitative reverse-transcriptase PCR (q-RT-PCR) and NanoString9s nCounter™ Digital Analyzer by measuring gene expression on an 18-cell line panel (4 RMSpos, 4 RMSneg, 5 ESFT, 1 NB, and 4 OS). With q-RT-PCR, we observed r2 values of 0.703 ± 0.085 (RMSpos), 0.558 ± 0.138 (RMSneg), 0.689 ± 0.070 (ESFT), and 0.607 ± 0.121 (OS). nCounter reported r2 values of 0.672 ± 0.171 (RMSpos), 0.620 ± 0.143 (RMSneg), 0.675 ± 0.094 (ESFT), and 0.583 ± 0.129 (OS). Hierarchical clustering with all platforms was able to distinguish the RMSpos, ESFT, and NB cell lines; however, the RMSneg and OS cell lines were less clearly distinguished because some of the OS cell lines lacked high expression of genes characteristic of OS tumors. Cross-platform comparisons yielded good correlation between nCounter and Q-RT-PCR (average r2 = 0.710 ± 0.183). An 18-sample dilution series revealed that EWS-FLI1 type I or PAX3-FKHR transcripts were consistently detected by q-RT-PCR or nCounter even at 1:1000 RNA dilution. Among biological duplicates, q-RT-PCR reported an average r2 = 0.916 ± 0.039 while nCounter obtained average r2 = 0.983 ± 0.011 including raw cell lysates. In addition, we also compared Fluidigm9s q-RT-PCR with Applied Biosystems’ Taqman Low-Density Arrays (TLDA) on five NB patient bone marrow samples to detect residual tumor cells. We observed an average r2 = 0.929 ± 0.119 across five NB genes and one housekeeping gene. Finally, we have also successfully profiled FF vs. FFPE tumors on HuEx and are currently applying these mid-plex technologies to paired FF and FFPE samples in order to create diagnostic profiles applicable to routine FFPE material. CONCLUSIONS: Mid-plex platform can reliably distinguish bone and soft tissue sarcoma cell lines and can be used to translate the application of diagnostic signatures. These data warrant further studies to analyze FFPE samples, to compare additional mid-plex platforms, and to test potential prognostic signatures. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4350. doi:10.1158/1538-7445.AM2011-4350

Abstract 4087: Large intergenic noncoding RNAs associated with Ewing sarcoma family of tumors

BACKGROUND: Ewing sarcoma family of tumors (EFT) represents the second most common primary malignant bone tumor in children and adolescents. The majority of EFTs harbor a translocation (t11;22)(q24;q12) resulting in the expression of the EWS-FLI1 chimeric oncoprotein. We hypothesized that microarray gene expression profiling, in combination with next-generation sequencing technology, could be used to identify EFT-specific genes and transcripts including large intergenic noncoding (linc)RNAs. Next-generation sequencing, coupled with target enrichment for validation, can also identify driver mutations of metastasis and treatment resistance in high-risk cases. RESULTS: Genome-wide expression profiles of childhood sarcoma and normal tissues were analyzed using both Partek Genomics Suite as well as our own customized software, Genetrix. These are compared with Helicos single molecule sequencing data. The results show that both known genes and candidate lincRNAs strongly associate with EFT and can be used to distinguish EFT from other childhood tumors. Moreover, many lincRNAs are seen on both the poly-A selected track and on the random hexamer primed total RNA expression profile, indicating that the transcripts are multi-exonic and poly-adenylated. Importantly, regions of EFT-associated lincRNA expression may indicate regions that are deregulated by EWS-FLI1 in these tumors. We also focused on lincRNAs with differential expression in an EFT cell line pair (CHLA-9 and −10) derived, respectively, from patient-matched primary and metastatic tumors. RNA-seq determined ∼160,000 regions representing ∼16MB of genomic sequence as being at least 3-fold differentially expressed between the two cell lines. A higher threshold of at least 10-fold expression difference still revealed ∼20,000 regions representing ∼2MB of genomic sequence. In addition, the proportion of differentially-expressed intergenic transcripts was higher in CHLA-9 (18.1%) versus CHLA-10 (11.2%). Annotated transcripts with at least 10-fold expression in CHLA-10 are enriched in plasma membrane and adhesion-related functions, and this is consistent with metastatic behavior. Genes most highly expressed in CHLA-10 include ANXA5, FABP3, and HIST1H1A, whereas genes with much lower expression include CXCL14, MCTP2, and TRIM22. These genes, along with lincRNAs, may play important roles in disease progression and drug resistance. CONCLUSIONS: We have identified several large intergenic noncoding (linc)RNAs that are highly and differentially expressed by EFT. We hypothesize that these lincRNAs may be novel therapeutic targets in EFT. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4087.