Hepatotoxicity of high affinity gapmer antisense oligonucleotides is mediated by RNase H1 dependent promiscuous reduction of very long pre-mRNA transcripts.

High affinity antisense oligonucleotides (ASOs) containing bicylic modifications (BNA) such as locked nucleic acid (LNA) designed to induce target RNA cleavage have been shown to have enhanced potency along with a higher propensity to cause hepatotoxicity. In order to understand the mechanism of this hepatotoxicity, transcriptional profiles were collected from the livers of mice treated with a panel of highly efficacious hepatotoxic or non-hepatotoxic LNA ASOs. We observed highly selective transcript knockdown in mice treated with non-hepatotoxic LNA ASOs, while the levels of many unintended transcripts were reduced in mice treated with hepatotoxic LNA ASOs. This transcriptional signature was concurrent with on-target RNA reduction and preceded transaminitis. Remarkably, the mRNA transcripts commonly reduced by toxic LNA ASOs were generally not strongly associated with any particular biological process, cellular component or functional group. However, they tended to have much longer pre-mRNA transcripts. We also demonstrate that the off-target RNA knockdown and hepatotoxicity is attenuated by RNase H1 knockdown, and that this effect can be generalized to high affinity modifications beyond LNA. This suggests that for a certain set of ASOs containing high affinity modifications such as LNA, hepatotoxicity can occur as a result of unintended off-target RNase H1 dependent RNA degradation.

U1 adaptors result in reduction of multiple pre-mRNA species principally by sequestering U1snRNP.

U1 Adaptors are a recently reported novel approach for targeted reduction of mRNA transcripts. A U1 adaptor oligonucleotide comprising of a target-complimentary hybridization domain and a U1 recruitment domain, directs the U1 snRNP complex to the terminal exon of a targeted gene, subsequently inhibiting poly(A) tail addition and leading to degradation of that RNA species within the nucleus. Here, we present data demonstrating U1 adapter-mediated gene silencing can result in significant 'off-target' silencing effects as demonstrated by the reduction of multiple mRNA species that were not intended to be targeted. Our data suggest that a substantial portion of this U1 adaptor-mediated off-target mRNA reduction is the result of sequestration U1 snRNP at levels sufficient to affect splicing and processing of non-target transcripts.

A mutation-created novel intra-exonic pre-mRNA splice site causes constitutive activation of KIT in human gastrointestinal stromal tumors.

We report a new mechanism of aberrant pre-mRNA splicing resulting in constitutive activation of a mis-spliced oncoprotein (KIT) leading to malignancy (gastrointestinal stromal tumor) in contrast to loss of function of mis-spliced proteins resulting in diverse human diseases in the literature. The mechanisms of three consecutive molecular events, deletion of noncoding and coding regions encompassing the 3' authentic splice site, creation of a novel intra-exonic pre-mRNA 3' splice acceptor site leading to in-frame loss of 27 nucleotides (nine amino acids; Lys550-Lys558), and the mechanism of constitutive activation of the mis-spliced KIT are elucidated. Loss of a peptide in a critical location unleashed the protein from autoinhibition (as evidenced by three-dimensional structural analysis), causing KIT to become constitutively activated and resulting in the GIST phenotype. We also demonstrated that only one of the following two exonic splicing enhancers is sufficient for inclusion of the KIT exon 11 in vivo: AACCCATGT (nucleotides 2-10 from the 5' end, which are recognized by SC35, SRp55, and SF2/ASF) or GGTTGTTGAGG (nucleotides 27-37 from the 5' end, which are recognized by SC35 and SRp55), suggestive of exonic enhancer redundancy.

Partial Hepatectomy Induced Long Noncoding RNA Inhibits Hepatocyte Proliferation during Liver Regeneration.

Liver regeneration after partial hepatectomy (PHx) is a complex and well-orchestrated biological process in which synchronized cell proliferation is induced in response to the loss of liver mass. To define long noncoding RNAs (lncRNAs) that participate in the regulation of liver regeneration, we performed microarray analysis and identified more than 400 lncRNAs exhibiting significantly altered expression. Of these, one lncRNA, LncPHx2 (Long noncoding RNA induced by PHx 2), was highly upregulated during liver regeneration. Depletion of LncPHx2 during liver regeneration using antisense oligonucleotides led to a transient increase in hepatocyte proliferation and more rapid liver regeneration. Gene expression analysis showed that LncPHx2 depletion resulted in upregulation of mRNAs encoding proteins known to promote cell proliferation, including MCM components, DNA polymerases, histone proteins, and transcription factors. LncPHx2 interacts with the mRNAs of MCM components, making it a candidate to regulate the expression of MCMs and other genes post-transcriptionally. Collectively, our data demonstrate that LncPHx2 is a key lncRNA that participates in a negative feedback loop modulating hepatocyte proliferation through RNA-RNA interactions.

Correlation of staining for LKB1 and COX-2 in hamartomatous polyps and carcinomas from patients with Peutz-Jeghers syndrome.

Germline mutations of the LKB1 gene lead to Peutz-Jeghers syndrome (PJS), which is associated with a predisposition to gastrointestinal polyposis and cancer. In this study we tested for germline mutations of LKB1 in 11 patients with PJS from nine families and analyzed the expression patterns of the LKB1 and cyclo-oxygenase-2 (COX-2) proteins in 28 Peutz-Jeghers polyps (PJPs) and five carcinomas from these patients by immunohistochemical (IHC) analysis. In eight of those families we identified seven different mutations, which consisted of two splice site mutations, two nonsense mutations, one small in-frame deletion, one frame-shift mutation, and one silent mutation. Immunostaining revealed nuclear and cytoplasmic expression of LKB1 protein in 23 PJPs and five carcinomas, nuclear expression alone in one PJP, and loss of LKB1 protein expression in four PJPs, indicating a heterogeneous LKB1 expression pattern in PJPs. Overexpression of COX-2 was detected in 23 (82%) of 28 PJPs and in all carcinomas. Despite heterogeneity in staining of LKB1 among individuals and even among samples from the same individual, we found statistically significant correlations in staining of LKB1 relative to COX-2. These results suggest that COX-2 plays a role in tumorigenesis in PJS and may therefore be considered as a potential target for PJS chemoprevention.

Use of tree-based models to identify subgroups and increase power to detect linkage to cardiovascular disease traits.

BACKGROUND: Our goal was to identify subgroups of sib pairs from the Framingham Heart Study data set that provided higher evidence of linkage to particular candidate regions for cardiovascular disease traits. The focus of this method is not to claim identification of significant linkage to a particular locus but to show that tree models can be used to identify subgroups for use in selected sib-pair sampling schemes. RESULTS: We report results using a novel recursive partitioning procedure to identify subgroups of sib pairs with increased evidence of linkage to systolic blood pressure and other cardiovascular disease-related quantitative traits, using the Framingham Heart Study data set provided by the Genetic Analysis Workshop 13. This procedure uses a splitting rule based on Haseman-Elston regression that recursively partitions sib-pair data into homogeneous subgroups. CONCLUSIONS: Using this procedure, we identified a subgroup definition for use as a selected sib-pair sampling scheme. Using the characteristics that define the subgroup with higher evidence for linkage, we have identified an area of focus for further study.

Combining targeted therapy with immunotherapy (interferon-α): Rational, efficacy in gastrointestinal stromal tumor model and implications in other malignancies.

Imatinib revolutionized gastrointestinal stromal tumor (GIST) treatment but median-progression-free-survival of unresectable/metastatic disease is < 2 y. B-RAF(V600)-mutated-melanoma responds to vemurafenib dramatically but median-progression-free-survival is < 9 mo. Combining imatinib with immunotherapy (peginterferon α-2b) in GIST showed significant induction of antitumor immunity and highly promising clinical outcomes. This strategy warrants further testing in other malignancies.

Genomic analysis of wig-1 pathways.

BACKGROUND: Wig-1 is a transcription factor regulated by p53 that can interact with hnRNP A2/B1, RNA Helicase A, and dsRNAs, which plays an important role in RNA and protein stabilization. in vitro studies have shown that wig-1 binds p53 mRNA and stabilizes it by protecting it from deadenylation. Furthermore, p53 has been implicated as a causal factor in neurodegenerative diseases based in part on its selective regulatory function on gene expression, including genes which, in turn, also possess regulatory functions on gene expression. In this study we focused on the wig-1 transcription factor as a downstream p53 regulated gene and characterized the effects of wig-1 down regulation on gene expression in mouse liver and brain. METHODS AND RESULTS: Antisense oligonucleotides (ASOs) were identified that specifically target mouse wig-1 mRNA and produce a dose-dependent reduction in wig-1 mRNA levels in cell culture. These wig-1 ASOs produced marked reductions in wig-1 levels in liver following intraperitoneal administration and in brain tissue following ASO administration through a single striatal bolus injection in FVB and BACHD mice. Wig-1 suppression was well tolerated and resulted in the reduction of mutant Htt protein levels in BACHD mouse brain but had no effect on normal Htt protein levels nor p53 mRNA or protein levels. Expression microarray analysis was employed to determine the effects of wig-1 suppression on genome-wide expression in mouse liver and brain. Reduction of wig-1 caused both down regulation and up regulation of several genes, and a number of wig-1 regulated genes were identified that potentially links wig-1 various signaling pathways and diseases. CONCLUSION: Antisense oligonucleotides can effectively reduce wig-1 levels in mouse liver and brain, which results in specific changes in gene expression for pathways relevant to both the nervous system and cancer.

Microarray data analysis: from disarray to consolidation and consensus.

In just a few years, microarrays have gone from obscurity to being almost ubiquitous in biological research. At the same time, the statistical methodology for microarray analysis has progressed from simple visual assessments of results to a weekly deluge of papers that describe purportedly novel algorithms for analysing changes in gene expression. Although the many procedures that are available might be bewildering to biologists who wish to apply them, statistical geneticists are recognizing commonalities among the different methods. Many are special cases of more general models, and points of consensus are emerging about the general approaches that warrant use and elaboration.

Imatinib resistance in gastrointestinal stromal tumors.

Conventional chemotherapeutic drugs are ineffective in treatment of gastrointestinal stromal tumors (GISTs). Imatinib (STI571, Gleevec, Glivec; Novartis Pharmaceuticals, East Hanover, NJ), a selective inhibitor of KIT, ABL, BCR-ABL, PDGFRA, and PDGFRB, represents a new paradigm of targeted cancer therapy and has revolutionized the treatment of patients with chronic myelogenous leukemia and GISTs. Unfortunately, imatinib resistance has emerged. The reported mechanism of imatinib resistance in GISTs involves missense mutation in the kinase domain of KIT, including Thr670Ile, Tyr823Asp, and Val654Ala. The established mechanisms and potential mechanisms of imatinib resistance in GISTs, the imaging studies indicative of early development of imatinib resistance, and the management of imatinib-resistant GISTs are discussed.