Lee P. Lim

A microRNA component of the p53 tumour suppressor network

A global decrease in microRNA (miRNA) levels is often observed in human cancers, indicating that small RNAs may have an intrinsic function in tumour suppression. To identify miRNA components of tumour suppressor pathways, we compared miRNA expression profiles of wild-type and p53-deficient cells. Here we describe a family of miRNAs, miR-34a–c, whose expression reflected p53 status. Genes encoding miRNAs in the miR-34 family are direct transcriptional targets of p53, whose induction by DNA damage and oncogenic stress depends on p53 both in vitro and in vivo. Ectopic expression of miR-34 induces cell cycle arrest in both primary and tumour-derived cell lines, which is consistent with the observed ability of miR-34 to downregulate a programme of genes promoting cell cycle progression. The p53 network suppresses tumour formation through the coordinated activation of multiple transcriptional targets, and miR-34 may act in concert with other effectors to inhibit inappropriate cell proliferation.

Global Survey of Genomic Imprinting by Transcriptome Sequencing

Genomic imprinting restricts gene expression to a paternal or maternal allele. To date, approximately 90 imprinted transcripts have been identified in mouse, of which the majority were detected after intense interrogation of clusters of imprinted genes identified by phenotype-driven assays in mice with uniparental disomies [1]. Here we use selective priming and parallel sequencing to measure allelic bias in whole transcriptomes. By distinguishing parent-of-origin bias from strain-specific bias in embryos derived from a reciprocal cross of mice, we constructed a genome-wide map of imprinted transcription. This map was able to objectively locate over 80% of known imprinted loci and allowed the detection and confirmation of six novel imprinted genes. Even in the intensely studied embryonic day 9.5 developmental stage that we analyzed, more than half of all imprinted single-nucleotide polymorphisms did not overlap previously discovered imprinted transcripts; a large fraction of these represent novel noncoding RNAs within known imprinted loci. For example, a previously unnoticed, maternally expressed antisense transcript was mapped within the Grb10 locus. This study demonstrates the feasibility of using transcriptome sequencing for mapping of imprinted gene expression in physiologically normal animals. Such an approach will allow researchers to study imprinting without restricting themselves to individual loci or specific transcripts.

microRNAs regulate human embryonic stem cell division

RNA interference-mediated suppression of DICER and DROSHA in human embryonic stem cells (hESCs) attenuates cell proliferation, supporting a role for an intact microRNA (miRNA) pathway in the control of hESC cell division. Normal cell growth can be partially restored by introduction of the mature miRNAs miR-195 and miR-372. These miRNAs regulate two tumor suppressor genes, respectively: WEE1, which encodes a negative G2/M kinase modulator of the cycB/CDK complex and CDKN1A, which encodes p21, a cycE/CDK cyclin dependent kinase inhibitor that regulates the G1/S transition. We show that in wild-type hESCs, WEE1 levels control the rate of hESC division, whereas p21 levels must be maintained at a low level for hESC division to proceed. These data support a model for hESC cell cycle control in which miRNAs regulate negative cell cycle modulators at two phases of the cell cycle to ensure proper replenishment of the stem cell population. Supplemental information can be found here.

Bias-Corrected Targeted Next-Generation Sequencing for Rapid, Multiplexed Detection of Actionable Alterations in Cell-Free DNA from Advanced Lung Cancer Patients

Purpose: Tumor genotyping is a powerful tool for guiding non–small cell lung cancer (NSCLC) care; however, comprehensive tumor genotyping can be logistically cumbersome. To facilitate genotyping, we developed a next-generation sequencing (NGS) assay using a desktop sequencer to detect actionable mutations and rearrangements in cell-free plasma DNA (cfDNA). Experimental Design: An NGS panel was developed targeting 11 driver oncogenes found in NSCLC. Targeted NGS was performed using a novel methodology that maximizes on-target reads, and minimizes artifact, and was validated on DNA dilutions derived from cell lines. Plasma NGS was then blindly performed on 48 patients with advanced, progressive NSCLC and a known tumor genotype, and explored in two patients with incomplete tumor genotyping. Results: NGS could identify mutations present in DNA dilutions at ≥0.4% allelic frequency with 100% sensitivity/specificity. Plasma NGS detected a broad range of driver and resistance mutations, including ALK, ROS1, and RET rearrangements, HER2 insertions, and MET amplification, with 100% specificity. Sensitivity was 77% across 62 known driver and resistance mutations from the 48 cases; in 29 cases with common EGFR and KRAS mutations, sensitivity was similar to droplet digital PCR. In two cases with incomplete tumor genotyping, plasma NGS rapidly identified a novel EGFR exon 19 deletion and a missed case of MET amplification. Conclusions: Blinded to tumor genotype, this plasma NGS approach detected a broad range of targetable genomic alterations in NSCLC with no false positives including complex mutations like rearrangements and unexpected resistance mutations such as EGFR C797S. Through use of widely available vacutainers and a desktop sequencing platform, this assay has the potential to be implemented broadly for patient care and translational research. Clin Cancer Res; 22(4); 915–22. ©2015 AACR. See related commentary by Tsui and Berger, p. 790

Single-stranded microRNA mimics.

miRNAs are ∼22-nt RNAs that bind to the Argonaute family of proteins and have important regulatory roles in plants and animals. Here, we show that miRNAs exhibit targeting activity in cells when delivered as single strands that are 5-phosphorylated and that contain 2-fluoro ribose modifications. Length preferences, chemical modification sensitivity, and genome-wide seed-based targeting all suggest that this activity is Ago-based. Activity could be enhanced by annealing of segmented passenger strands containing non-nucleic acid spacers. Furthermore, screening of randomly generated sequences identified pyrimidine rich 3 cassette sequences that increased single strand activity. These results provide an initial step in the development of single-stranded miRNA mimics for therapeutic use.

Persistence of seed-based activity following segmentation of a microRNA guide strand.

microRNAs are ∼ 22 nucleotide regulatory RNAs that are processed into duplexes from hairpin structures and incorporated into Argonaute proteins. Here, we show that a nick in the middle of the guide strand of an miRNA sequence allows for seed-based targeting characteristic of miRNA activity. Insertion of an inverted abasic, a dye, or a small gap between the two segments still permits target knockdown. While activity from the seed region of the segmented miRNA is apparent, activity from the 3 half of the guide strand is impaired, suggesting that an intact guide backbone is required for contribution from the 3 half. miRNA activity was also observed following nicking of a miRNA precursor. These results illustrate a structural flexibility in miRNA duplexes and may have applications in the design of miRNA mimetics.

Longitudinal Cell-Free DNA Analysis in Patients with Small Cell Lung Cancer Reveals Dynamic Insights into Treatment Efficacy and Disease Relapse

Introduction Patients with SCLC have a poor prognosis and limited treatment options. Because access to longitudinal tumor samples is very limited in patients with this disease, we chose to focus our studies on the characterization of plasma cell‐free DNA (cfDNA) for rapid, noninvasive monitoring of disease burden. Methods We developed a liquid biopsy assay that quantifies somatic variants in cfDNA. The assay detects single nucleotide variants, copy number alterations, and insertions or deletions in 14 genes that are frequently mutated in SCLC, including tumor protein p53 gene (TP53), retinoblastoma 1 gene (RB1), BRAF, KIT proto‐oncogene receptor tyrosine kinase gene (KIT), notch 1 gene (NOTCH1), notch 2 gene (NOTCH2), notch 3 gene (NOTCH3), notch 4 gene (NOTCH4), phosphatidylinositol‐4,5‐bisphosphate 3‐kinase catalytic subunit alpha gene (PIK3CA), phosphatase and tensin homolog gene (PTEN), fibroblast growth factor receptor 1 gene (FGFR1), v‐myc avian myelocytomatosis viral oncogene homolog gene (MYC), v‐myc avian myelocytomatosis viral oncogene lung carcinoma derived homolog gene (MYCL1), and v‐myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog gene (MYCN). Results Over the course of 26 months of peripheral blood collection, we examined 140 plasma samples from 27 patients. We detected disease‐associated mutations in 85% of patient samples with mutant allele frequencies ranging from 0.1% to 87%. In our cohort, 59% of the patients had extensive‐stage disease, and the most common mutations occurred in TP53 (70%) and RB1 (52%). In addition to mutations in TP53 and RB1, we detected alterations in 10 additional genes in our patient population (PTEN, NOTCH1, NOTCH2, NOTCH3, NOTCH4, MYC, MYCL1, PIK3CA, KIT, and BRAF). The observed allele frequencies and copy number alterations tracked closely with treatment responses. Notably, in several cases analysis of cfDNA provided evidence of disease relapse before conventional imaging. Conclusions These results suggest that liquid biopsies are readily applicable in patients with SCLC and can potentially provide improved monitoring of disease burden, depth of response to treatment, and timely warning of disease relapse in patients with this disease.