Shana Claeys

Upregulation of MAPK Negative Feedback Regulators and RET in Mutant ALK Neuroblastoma: Implications for Targeted Treatment

Purpose: Activating ALK mutations are present in almost 10% of primary neuroblastomas and mark patients for treatment with small-molecule ALK inhibitors in clinical trials. However, recent studies have shown that multiple mechanisms drive resistance to these molecular therapies. We anticipated that detailed mapping of the oncogenic ALK-driven signaling in neuroblastoma can aid to identify potential fragile nodes as additional targets for combination therapies. Experimental Design: To achieve this goal, transcriptome profiling was performed in neuroblastoma cell lines with the ALKF1174L or ALKR1275Q hotspot mutations, ALK amplification, or wild-type ALK following pharmacologic inhibition of ALK using four different compounds. Next, we performed cross-species genomic analyses to identify commonly transcriptionally perturbed genes in MYCN/ALKF1174L double transgenic versus MYCN transgenic mouse tumors as compared with the mutant ALK-driven transcriptome in human neuroblastomas. Results: A 77-gene ALK signature was established and successfully validated in primary neuroblastoma samples, in a neuroblastoma cell line with ALKF1174L and ALKR1275Q regulable overexpression constructs and in other ALKomas. In addition to the previously established PI3K/AKT/mTOR, MAPK/ERK, and MYC/MYCN signaling branches, we identified that mutant ALK drives a strong upregulation of MAPK negative feedback regulators and upregulates RET and RET-driven sympathetic neuronal markers of the cholinergic lineage. Conclusions: We provide important novel insights into the transcriptional consequences and the complexity of mutant ALK signaling in this aggressive pediatric tumor. The negative feedback loop of MAPK pathway inhibitors may affect novel ALK inhibition therapies, whereas mutant ALK induced RET signaling can offer novel opportunities for testing ALK-RET oriented molecular combination therapies. Clin Cancer Res; 21(14); 3327–39. ©2015 AACR.

A high-throughput 3 ' UTR reporter screening identifies microRNA interactomes of cancer genes

Introduction Despite the established contribution of deregulated microRNA (miRNA) function to carcinogenesis, relatively few miRNA-cancer gene interactions have been validated, making it difficult to appreciate the true complexity of miRNA-cancer gene regulatory networks. Results In this effort, we identify miRNA interactomes of 17 well-established cancer genes, involved in various cancer types, through a miRNome-wide 3’ UTR reporter screening. Using a novel and performant strategy for high-throughput screening data analysis, we identify 390 interactions, quadrupling the size of the known miRNA interactome for the cancer genes under investigation. Clear enrichments of established and predicted interactions underscore the validity of the interactome data set. Interactomes appear to be primarily driven by canonical binding site interactions. Nonetheless, non-canonical binding sites, such as offset 6mer and seed-mismatched or G:U wobble sites, also have regulatory activity, albeit clearly less pronounced. Furthermore, we observe enhanced regulation in the presence of 3’ supplementary pairing for both canonical and non-canonical binding sites. Conclusions Altogether, the cancer gene-miRNA interactome data set represents a unique resource that will aid in the unraveling of regulatory miRNA networks and the dynamic regulation of key protein-coding cancer genes. In addition, it uncovers aspects of the functional miRNA binding site’s architecture and the relative contributions of different binding site types.