Lee Spraggon

In vivo, Argonaute-bound microRNAs exist predominantly in a reservoir of low molecular weight complexes not associated with mRNA.

Significance MicroRNAs limit gene expression by recruiting a large protein complex known as the RNA-induced silencing complex (RISC) to target mRNAs. While attempting to understand physiological regulation of RISC assembly, we found that most healthy adult tissues retain a reserve of microRNAs not stably associated with target mRNA. Recruitment of microRNAs to large mRNA-containing complexes was accompanied by an increase in their ability to repress targets and was regulated in part by phosphoinositide-3 kinase–RAC-alpha serine/threonine-protein kinase–mechanistic target of rapamycin pathway-dependent enhancement of the glycine-tryptophan protein of 182 kDa protein expression. Data presented here suggest that in vivo, many expressed microRNAs exist in an inactive reserve, allowing resting cells to use microRNAs to dynamically regulate the translation of target mRNAs in their environment. MicroRNAs repress mRNA translation by guiding Argonaute proteins to partially complementary binding sites, primarily within the 3′ untranslated region (UTR) of target mRNAs. In cell lines, Argonaute-bound microRNAs exist mainly in high molecular weight RNA-induced silencing complexes (HMW-RISC) associated with target mRNA. Here we demonstrate that most adult tissues contain reservoirs of microRNAs in low molecular weight RISC (LMW-RISC) not bound to mRNA, suggesting that these microRNAs are not actively engaged in target repression. Consistent with this observation, the majority of individual microRNAs in primary T cells were enriched in LMW-RISC. During T-cell activation, signal transduction through the phosphoinositide-3 kinase–RAC-alpha serine/threonine-protein kinase–mechanistic target of rapamycin pathway increased the assembly of microRNAs into HMW-RISC, enhanced expression of the glycine-tryptophan protein of 182 kDa, an essential component of HMW-RISC, and improved the ability of microRNAs to repress partially complementary reporters, even when expression of targeting microRNAs did not increase. Overall, data presented here demonstrate that microRNA-mediated target repression in nontransformed cells depends not only on abundance of specific microRNAs, but also on regulation of RISC assembly by intracellular signaling.

Whole-genome single-cell copy number profiling from formalin-fixed paraffin-embedded samples

A substantial proportion of tumors consist of genotypically distinct subpopulations of cancer cells. This intratumor genetic heterogeneity poses a substantial challenge for the implementation of precision medicine. Single-cell genomics constitutes a powerful approach to resolve complex mixtures of cancer cells by tracing cell lineages and discovering cryptic genetic variations that would otherwise be obscured in tumor bulk analyses. Because of the chemical alterations that result from formalin fixation, single-cell genomic approaches have largely remained limited to fresh or rapidly frozen specimens. Here we describe the development and validation of a robust and accurate methodology to perform whole-genome copy-number profiling of single nuclei obtained from formalin-fixed paraffin-embedded clinical tumor samples. We applied the single-cell sequencing approach described here to study the progression from in situ to invasive breast cancer, which revealed that ductal carcinomas in situ show intratumor genetic heterogeneity at diagnosis and that these lesions may progress to invasive breast cancer through a variety of evolutionary processes.

U1 snRNP-Dependent Suppression of Polyadenylation: Physiological Role and Therapeutic Opportunities in Cancer

Pre-mRNA splicing and polyadenylation are critical steps in the maturation of eukaryotic mRNA. U1 snRNP is an essential component of the splicing machinery and participates in splice-site selection and spliceosome assembly by base-pairing to the 5′ splice site. U1 snRNP also plays an additional, nonsplicing global function in 3′ end mRNA processing; it actively suppresses the polyadenylation machinery from using early, mostly intronic polyadenylation signals which would lead to aberrant, truncated mRNAs. Thus, U1 snRNP safeguards pre-mRNA transcripts against premature polyadenylation and contributes to the regulation of alternative polyadenylation. Here, we review the role of U1 snRNP in 3′ end mRNA processing, outline the evidence that led to the recognition of its physiological, general role in inhibiting polyadenylation, and finally highlight the possibility of manipulating this U1 snRNP function for therapeutic purposes in cancer.

Mammalian GW220/TNGW1 is essential for the formation of GW/P bodies containing miRISC

Localization of the miRNA-induced silencing complex to GW/P bodies by GW220/TNGW1 may regulate the fate of target mRNAs.

YES1 amplification is a mechanism of acquired resistance to EGFR inhibitors identified by transposon mutagenesis and clinical genomics

Significance Despite high response rates to treatment with small molecule inhibitors of EGFR tyrosine kinase activity, patients with EGFR-mutant lung adenocarcinomas eventually develop resistance to these drugs. In many cases, the basis of acquired resistance remains unclear. We have used a transposon mutagenesis screen in an EGFR-mutant cell line and clinical genomic sequencing in cases of acquired resistance to identify amplification of YES1 as a targetable mechanism of resistance to EGFR inhibitors in EGFR-mutant lung cancers. In ∼30% of patients with EGFR-mutant lung adenocarcinomas whose disease progresses on EGFR inhibitors, the basis for acquired resistance remains unclear. We have integrated transposon mutagenesis screening in an EGFR-mutant cell line and clinical genomic sequencing in cases of acquired resistance to identify mechanisms of resistance to EGFR inhibitors. The most prominent candidate genes identified by insertions in or near the genes during the screen were MET, a gene whose amplification is known to mediate resistance to EGFR inhibitors, and the gene encoding the Src family kinase YES1. Cell clones with transposon insertions that activated expression of YES1 exhibited resistance to all three generations of EGFR inhibitors and sensitivity to pharmacologic and siRNA-mediated inhibition of YES1. Analysis of clinical genomic sequencing data from cases of acquired resistance to EGFR inhibitors revealed amplification of YES1 in five cases, four of which lacked any other known mechanisms of resistance. Preinhibitor samples, available for two of the five patients, lacked YES1 amplification. None of 136 postinhibitor samples had detectable amplification of other Src family kinases (SRC and FYN). YES1 amplification was also found in 2 of 17 samples from ALK fusion-positive lung cancer patients who had progressed on ALK TKIs. Taken together, our findings identify acquired amplification of YES1 as a recurrent and targetable mechanism of resistance to EGFR inhibition in EGFR-mutant lung cancers and demonstrate the utility of transposon mutagenesis in discovering clinically relevant mechanisms of drug resistance.

Abstract A21: Modeling of oncogenic chromosomal translocations of aggressive fusion-positive sarcomas by CRISPR-Cas9 genomic engineering

A prominent challenge in fusion-positive sarcoma research is that the oncogenic driver is typically a chimeric transcription factor. Although considered the disease-specific molecular drivers, these chimeric transcription factors remain challenging to target pharmacologically. An alternative approach is the elucidation of molecular pathways controlled by the oncogenic transcription factor, with the aim of identifying druggable targets. However, many fusion-driven sarcomas have a paucity of suitable genetic models, with some lacking any patient-derived cell lines. In the situations where patient-derived cell lines are available, most have been passaged for decades, potentially adding further complexity and bias. Therefore, development of faithful model systems of oncogenic chromosomal translocations is of principal importance for these aggressive translocation-driven pediatric sarcomas. Here, we describe a novel approach that combines CRISPR-Cas9 genomic editing technology, with homology-directed repair (HDR) to engineer, capture, and modulate the expression of chromosomal translocation products in a human cell line. We have applied this approach to the genetic modeling of t(11;22)(q24;q12) and t(11;22)(p13;q12), translocation products of the EWSR1 gene and its 3 fusion partners FLI1 and WT1, present in Ewing9s sarcoma and desmoplastic small round cell tumor, respectively. Our approach establishes an innovative platform for constructing isogenic and conditionally inducible biologically relevant models for a variety of sarcomas driven by chromosomal translocations. Citation Format: Lee Spraggon, Luciano Martelotto, Julija Hmeljak, Tyler Hitchman, Jiang Wang, Lu Wang, Emily Slotkin, Pang-Dian Fan, Jorge Reis-Filho, Marc Ladanyi. Modeling of oncogenic chromosomal translocations of aggressive fusion-positive sarcomas by CRISPR-Cas9 genomic engineering [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr A21.

Abstract LB-031: Generation of inducible oncogenic chromosomal translocations using CRISPR-Cas9 genomic editing

Specific, reciprocal chromosomal translocations are found in numerous cancers, typically leading to the formation and expression of fusion genes with oncogenic properties. It has been demonstrated that by inducing double-stranded breaks (DSBs) in genomic DNA, using either zinc fingers nucleases (ZF), transcription activator-like effector nucleases (TALENs), or the CRISPR-Cas9 system, it is possible to faithfully recapitulate such chromosomal translocations in human cell lines. A potential pitfall is that the induction of the chromosomal translocation generates a constitutively active oncogenic fusion product, which can be detrimental to cell growth or viability in certain heterologous cell types. To overcome these obstacles, we have combined homology directed repair (HDR) with CRISPR-Cas9 genome editing to generate conditionally inducible chromosomal translocations. As proof of principle, we have used this approach to engineer human cell lines to carry the molecular hallmark present in Ewing Sarcoma, the t(11;22)(q24;q12) translocation. Our approach allows for the de novo generation and capture of the desired chromosomal translocation in the context of a conditional allele. Whole genome sequencing (WGS) and fluorescence in situ hybridization (FISH) confirmed the accuracy of our genomic editing approach, whilst analysis of mRNA and protein levels confirmed the selective inducibility and generation of biologically functional EWSR1-FLI1 fusion oncogene. Therefore, our approach establishes an innovative platform for constructing isogenic and conditionally inducible biologically relevant models for a variety of cancers driven by chromosomal translocations. Citation Format: Lee Spraggon, Luciano Martelotto, Julija Hmeljak, Emily Slotkin, Lu Wang, Jorge Reis-Filho, Marc Ladanyi. Generation of inducible oncogenic chromosomal translocations using CRISPR-Cas9 genomic editing. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-031.

Abstract LB-238: Direct targeting of oncogenic transcription factors using next-generation oligonucleotides as a novel therapeutic strategy for translocation-positive sarcomas

Many sarcoma subtypes including Ewing sarcoma (ES), alveolar rhabdomyosarcoma (ARMS), synovial sarcoma (SS), and desmoplastic small round cell tumor (DSRCT), are driven by oncogenic chimeric transcription factors. Outcomes for these diseases remain unacceptably poor despite intensification of standard therapies. Although these translocation positive sarcomas possess well-defined oncogenic drivers, as transcription factors that lack intrinsic enzymatic activity, they have been considered undruggable. We have developed a two-tiered pre-clinical therapeutic program 1) creating and characterizing faithful in vitro and in vivo models of these rare malignancies and 2) designing next generation antisense oligonucleotides (ASOs) to directly target the oncogenic chromosomal translocations underlying these multiple sarcoma subtypes. As proof of principle, we present here the program experience with DSRCT, a rare sarcoma for which there has been an overwhelming paucity of both biological tools for pre-clinical investigation, as well as clinical therapeutic options. We have extensively characterized two new DSRCT cell lines harboring the characteristic oncogenic EWSR1-WT1 t(11;22)(p13;q12) fusion, and have used these tools to develop orthotopic xenograft models. Utilizing these accurate model systems we have designed and evaluated a series of ASO compounds directly targeting the EWSR1-WT1 translocation product. Among the series of ASOs developed, we identified D2A, a selective and potent inhibitor of EWSR1-WT1. In vitro, we have shown D2A to abolish the EWSR1-WT1 fusion product with exquisite specificity in a dose-dependent fashion. D2A similarly exhibits efficacy in vivo, with anti-tumorigenic activity in our pre-clinical models resulting in an 80% reduction in xenograft tumor growth. With the aid of authentic in vitro and in vivo modeling systems, this ASO platform has wide-ranging implications for both biological as well as therapeutic advances. Citation Format: Emily Slotkin, Lee Spraggon, Julija Hmeljak, Luciano Martelotto, Romel Somwar, Elisa de Stanchina, Luca Cartegni, Marc Ladanyi. Direct targeting of oncogenic transcription factors using next-generation oligonucleotides as a novel therapeutic strategy for translocation-positive sarcomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-238.

Abstract B26: Therapeutic targeting of sarcomas driven by EWSR1 fusion oncogenes by modulation of the fusion oncogene pre-mRNA

Many predominantly pediatric sarcomas are driven by chromosomal translocations that generate oncogenic fusion transcription factors. The Ewing sarcoma breakpoint region 1 (EWSR1) is one of the most commonly involved genes in sarcoma translocations. This gene has a large number of fusion partners, primarily associated with the pathogenesis of Ewing sarcoma, but also with other soft tissue sarcomas. Targeting the resulting aberrant oncogenic chimeric transcription factors would provide an ideal therapy, but this has approach has proved elusive with conventional small molecule approaches. We have developed an approach to employ splice-switching oligonucleotides to redirect the pre-mRNA splicing of EWSR1 fusion transcription factors. Through targeted exon skipping, and/or activation of intronic polyadenylation, these new compounds provide a targeted therapeutic approach for EWSR1 translocation positive sarcomas and other malignancies. Citation Format: Emily Slotkin, Elisa de Stanchina, Luca Cartegni, Marc Ladanyi, Lee Spraggon. Therapeutic targeting of sarcomas driven by EWSR1 fusion oncogenes by modulation of the fusion oncogene pre-mRNA. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B26.

Abstract LB-239: Induction of therapeutic soluble decoy EGFR variants by antisense manipulation of RNA processing

Activation of the receptor tyrosine kinase (RTK) signaling pathways represents a key aspect of tumorigenesis in a broad range of human cancers. Thus, targeting of pathogenic RTKs, mainly with small molecule inhibitors and antibodies, has become an attractive therapeutic approach for cancer therapy. However, resistance by multiple mechanisms, including mutational re-activation of RTK signaling, activation of alternative RTK pathways, EMT transition and others, inevitably occurs over time. We recently identified a number of novel secreted soluble decoy RTKs isoforms (sdRTKs) generated by an U1-snRNP-dependent alternative intronic polyadenylation mechanism. We therefore developed a novel antisense-based method to effectively activate the expression of these sdRTKs, both in vitro and in vivo. This leads to the generation of endogenous dominant negative variants of RTKs, which can specifically affect the signaling of pathogenic RTKs and therefore provides a novel cancer therapy approach. Employing specific antisense compounds designed to activate intronic polyA sites, we are able to induce natural truncated EGFR variants that encode its extracellular ligand-binding domain but lack the intracellular signaling domain. These compounds thus can inhibit EGFR signaling by a three fold-mechanism: 1) removal of FL receptor, 2) sequestration of ligands and 3) non-productive dimerization with residual receptors. They are therefore designed to affect signaling both of targeted cells and of bystanders. Here we will show that, using this approach, we can target EGFR signaling both in vitro and in tumors. Importantly, with this method we can bypass the emergence of resistance typically seen with targeted drug treatments. Further development of this methodology will therefore provide the basis for an exciting novel approach to targeting and inhibiting oncogenic EGFR signaling and other pathways in a variety of cancers. Citation Format: Jeong Eun Park, Lee Spraggon, Luca Cartegni. Induction of therapeutic soluble decoy EGFR variants by antisense manipulation of RNA processing. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-239.