The LIN28B-IMP1 post-transcriptional regulon has opposing effects on oncogenic signaling in the intestine.

RNA-binding proteins (RBPs) are expressed broadly during both development and malignant transformation, yet their mechanistic roles in epithelial homeostasis or as drivers of tumor initiation and progression are incompletely understood. Here we describe a novel interplay between RBPs LIN28B and IMP1 in intestinal epithelial cells. Ribosome profiling and RNA sequencing identified IMP1 as a principle node for gene expression regulation downstream from LIN28B In vitro and in vivo data demonstrate that epithelial IMP1 loss increases expression of WNT target genes and enhances LIN28B-mediated intestinal tumorigenesis, which was reversed when we overexpressed IMP1 independently in vivo. Furthermore, IMP1 loss in wild-type or LIN28B-overexpressing mice enhances the regenerative response to irradiation. Together, our data provide new evidence for the opposing effects of the LIN28B-IMP1 axis on post-transcriptional regulation of canonical WNT signaling, with implications in intestinal homeostasis, regeneration and tumorigenesis.

Single mammalian cells compensate for differences in cellular volume and DNA copy number through independent global transcriptional mechanisms.

Individual mammalian cells exhibit large variability in cellular volume, even with the same absolute DNA content, and so must compensate for differences in DNA concentration in order to maintain constant concentration of gene expression products. Using single-molecule counting and computational image analysis, we show that transcript abundance correlates with cellular volume at the single-cell level due to increased global transcription in larger cells. Cell fusion experiments establish that increased cellular content itself can directly increase transcription. Quantitative analysis shows that this mechanism measures the ratio of cellular volume to DNA content, most likely through sequestration of a transcriptional factor to DNA. Analysis of transcriptional bursts reveals a separate mechanism for gene dosage compensation after DNA replication that enables proper transcriptional output during early and late S phase. Our results provide a framework for quantitatively understanding the relationships among DNA content, cell size, and gene expression variability in single cells.

Global analysis of the RNA-protein interaction and RNA secondary structure landscapes of the Arabidopsis nucleus.

Posttranscriptional regulation in eukaryotes requires cis- and trans-acting features and factors including RNA secondary structure and RNA-binding proteins (RBPs). However, a comprehensive view of the structural and RBP interaction landscape of nuclear RNAs has yet to be compiled for any organism. Here, we use our ribonuclease-mediated structure and RBP-binding site mapping approaches to globally profile these features in Arabidopsis seedling nuclei in vivo. We reveal anticorrelated patterns of secondary structure and RBP binding throughout nuclear mRNAs that demarcate sites of alternative splicing and polyadenylation. We also uncover a collection of protein-bound sequence motifs, and identify their structural contexts, co-occurrences in transcripts encoding functionally related proteins, and interactions with putative RBPs. Finally, using these motifs, we find that the chloroplast RBP CP29A also interacts with nuclear mRNAs. In total, we provide a simultaneous view of the RNA secondary structure and RBP interaction landscapes in a eukaryotic nucleus.

Safety, pharmacokinetic, pharmacodynamic and clinical activity of molibresib for the treatment of nuclear protein in testis carcinoma and other cancers: Results of a Phase I/II open-label, dose escalation study.

Molibresib is an orally bioavailable, selective, small molecule BET protein inhibitor. Results from a first time in human study in solid tumors resulted in the selection of a 75 mg once daily dose of the besylate formulation of molibresib as the recommended Phase 2 dose (RP2D). Here we present the results of Part 2 of our study, investigating safety, pharmacokinetics, pharmacodynamics and clinical activity of molibresib at the RP2D for nuclear protein in testis carcinoma (NC), small cell lung cancer, castration-resistant prostate cancer (CRPC), triple-negative breast cancer, estrogen receptor-positive breast cancer and gastrointestinal stromal tumor. The primary safety endpoints were incidence of adverse events (AEs) and serious AEs; the primary efficacy endpoint was overall response rate. Secondary endpoints included plasma concentrations and gene set enrichment analysis (GSEA). Molibresib 75 mg once daily demonstrated no unexpected toxicities. The most common treatment-related AEs (any grade) were thrombocytopenia (64%), nausea (43%) and decreased appetite (37%); 83% of patients required dose interruptions and 29% required dose reductions due to AEs. Antitumor activity was observed in NC and CRPC (one confirmed partial response each, with observed reductions in tumor size), although predefined clinically meaningful response rates were not met for any tumor type. Total active moiety median plasma concentrations after single and repeated administration were similar across tumor cohorts. GSEA revealed that gene expression changes with molibresib varied by patient, response status and tumor type. Investigations into combinatorial approaches that use BET inhibition to eliminate resistance to other targeted therapies are warranted.

A Phase I/II Open-Label Study of Molibresib for the Treatment of Relapsed/Refractory Hematologic Malignancies.

PURPOSE: Molibresib is a selective, small molecule inhibitor of the bromodomain and extra-terminal (BET) protein family. This was an open-label, two-part, Phase I/II study investigating molibresib monotherapy for the treatment of hematological malignancies (NCT01943851). PATIENTS AND METHODS: Part 1 (dose escalation) determined the recommended Phase 2 dose (RP2D) of molibresib in patients with acute myeloid leukemia (AML), Non-Hodgkin lymphoma (NHL), or multiple myeloma. Part 2 (dose expansion) investigated the safety and efficacy of molibresib at the RP2D in patients with relapsed/refractory myelodysplastic syndrome (MDS; as well as AML evolved from antecedent MDS) or cutaneous T-cell lymphoma (CTCL). The primary endpoint in Part 1 was safety and the primary endpoint in Part 2 was objective response rate (ORR). RESULTS: There were 111 patients enrolled (87 in Part 1, 24 in Part 2). Molibresib RP2Ds of 75 mg daily (for MDS) and 60 mg daily (for CTCL) were selected. Most common Grade 3+ adverse events included thrombocytopenia (37%), anemia (15%), and febrile neutropenia (15%). Six patients achieved complete responses [3 in Part 1 (2 AML, 1 NHL), 3 in Part 2 (MDS)], and 7 patients achieved partial responses [6 in Part 1 (4 AML, 2 NHL), 1 in Part 2 (MDS)]. The ORRs for Part 1, Part 2, and the total study population were 10% [95% confidence interval (CI), 4.8-18.7], 25% (95% CI, 7.3-52.4), and 13% (95% CI, 6.9-20.6), respectively. CONCLUSIONS: While antitumor activity was observed with molibresib, use was limited by gastrointestinal and thrombocytopenia toxicities. Investigations of molibresib as part of combination regimens may be warranted.

Discovery of a first-in-class reversible DNMT1-selective inhibitor with improved tolerability and efficacy in acute myeloid leukemia.

DNA methylation, a key epigenetic driver of transcriptional silencing, is universally dysregulated in cancer. Reversal of DNA methylation by hypomethylating agents, such as the cytidine analogs decitabine or azacytidine, has demonstrated clinical benefit in hematologic malignancies. These nucleoside analogs are incorporated into replicating DNA where they inhibit DNA cytosine methyltransferases DNMT1, DNMT3A and DNMT3B through irreversible covalent interactions. These agents induce notable toxicity to normal blood cells thus limiting their clinical doses. Herein we report the discovery of GSK3685032, a potent first-in-class DNMT1-selective inhibitor that was shown via crystallographic studies to compete with the active-site loop of DNMT1 for penetration into hemi-methylated DNA between two CpG base pairs. GSK3685032 induces robust loss of DNA methylation, transcriptional activation and cancer cell growth inhibition in vitro. Due to improved in vivo tolerability compared with decitabine, GSK3685032 yields superior tumor regression and survival mouse models of acute myeloid leukemia.

Differential Regulation of LET-7 by LIN28B Isoform-Specific Functions.

The RNA-binding protein LIN28B plays an important role in development, stem cell biology, and tumorigenesis. LIN28B has two isoforms: the LIN28B-long and -short isoforms. Although studies have revealed the functions of the LIN28B-long isoform in tumorigenesis, the role of the LIN28B-short isoform remains unclear and represents a major gap in the field. The LIN28B-long and -short isoforms are expressed in a subset of human colorectal cancers and adjacent normal colonic mucosa, respectively. To elucidate the functional and mechanistic aspects of these isoforms, colorectal cancer cells (Caco-2 and LoVo) were generated to either express no LIN28B or the -short or -long isoform. Interestingly, the long isoform suppressed LET-7 expression and activated canonical RAS/ERK signaling, whereas the short isoform did not. The LIN28B-long isoform-expressing cells demonstrated increased drug resistance to 5-fluorouracil and cisplatin through the upregulation of ERCC1, a DNA repair gene, in a LET-7-dependent manner. The LIN28B-short isoform preserved its ability to bind pre-let-7, without inhibiting the maturation of LET-7, and competed with the LIN28B-long isoform for binding to pre-let-7 Coexpression of the short isoform in the LIN28B-long isoform-expressing cells rescued the phenotypes induced by the LIN28B-long isoform.Implications: This study demonstrates the differential antagonistic functions of the LIN28B-short isoform against the LIN28B-long isoform through an inability to degrade LET-7, which leads to the novel premise that the short isoform may serve to counterbalance the long isoform during normal colonic epithelial homeostasis, but its downregulation during colonic carcinogenesis may reveal the protumorigenic effects of the long isoform. Mol Cancer Res; 16(3); 403-16. ©2018 AACR.

MEK inhibitors overcome resistance to BET inhibition across a number of solid and hematologic cancers.

BET inhibitors exhibit broad activity in cancer models, making predictive biomarkers challenging to define. Here we investigate the biomarkers of activity of the clinical BET inhibitor GSK525762 (I-BET; I-BET762) across cancer cell lines and demonstrate that KRAS mutations are novel resistance biomarkers. This finding led us to combine BET with RAS pathway inhibition using MEK inhibitors to overcome resistance, which resulted in synergistic effects on growth and survival in RAS pathway mutant models as well as a subset of cell lines lacking RAS pathway mutations. GSK525762 treatment up-regulated p-ERK1/2 levels in both RAS pathway wild-type and mutant cell lines, suggesting that MEK/ERK pathway activation may also be a mechanism of adaptive BET inhibitor resistance. Importantly, gene expression studies demonstrated that the BET/MEK combination uniquely sustains down-regulation of genes associated with mitosis, leading to prolonged growth arrest that is not observed with either single agent therapy. These studies highlight a potential to enhance the clinical benefit of BET and MEK inhibitors and provide a strong rationale for clinical evaluation of BET/MEK combination therapies in cancer.

RNA structure, binding, and coordination in Arabidopsis.

From the moment of transcription, up through degradation, each RNA transcript is bound by an ever-changing cohort of RNA binding proteins. The binding of these proteins is regulated by both the primary RNA sequence, as well as the intramolecular RNA folding, or secondary structure, of the transcript. Thus, RNA secondary structure regulates many post-transcriptional processes. With the advent of next generation sequencing, several techniques have been developed to generate global landscapes of both RNA-protein interactions and RNA secondary structure. In this review, we describe the current state of the field detailing techniques to globally interrogate RNA secondary structure and/or RNA-protein interaction sites, as well as our current understanding of these features in the transcriptome of the model plant Arabidopsis thaliana. WIREs RNA 2017, 8:e1426. doi: 10.1002/wrna.1426 For further resources related to this article, please visit the WIREs website.

A Global View of RNA-Protein Interactions Identifies Post-transcriptional Regulators of Root Hair Cell Fate.

The Arabidopsis thaliana root epidermis is comprised of two cell types, hair and nonhair cells, which differentiate from the same precursor. Although the transcriptional programs regulating these events are well studied, post-transcriptional factors functioning in this cell fate decision are mostly unknown. Here, we globally identify RNA-protein interactions and RNA secondary structure in hair and nonhair cell nuclei. This analysis reveals distinct structural and protein binding patterns across both transcriptomes, allowing identification of differential RNA binding protein (RBP) recognition sites. Using these sequences, we identify two RBPs that regulate hair cell development. Specifically, we find that SERRATE functions in a microRNA-dependent manner to inhibit hair cell fate, while also terminating growth of root hairs mostly independent of microRNA biogenesis. In addition, we show that GLYCINE-RICH PROTEIN 8 promotes hair cell fate while alleviating phosphate starvation stress. In total, this global analysis reveals post-transcriptional regulators of plant root epidermal cell fate.

Protein Interaction Profile Sequencing (PIP-seq).

Every eukaryotic RNA transcript undergoes extensive post-transcriptional processing from the moment of transcription up through degradation. This regulation is performed by a distinct cohort of RNA-binding proteins which recognize their target transcript by both its primary sequence and secondary structure. Here, we describe protein interaction profile sequencing (PIP-seq), a technique that uses ribonuclease-based footprinting followed by high-throughput sequencing to globally assess both protein-bound RNA sequences and RNA secondary structure. PIP-seq utilizes single- and double-stranded RNA-specific nucleases in the absence of proteins to infer RNA secondary structure. These libraries are also compared to samples that undergo nuclease digestion in the presence of proteins in order to find enriched protein-bound sequences. Combined, these four libraries provide a comprehensive, transcriptome-wide view of RNA secondary structure and RNA protein interaction sites from a single experimental technique. © 2016 by John Wiley & Sons, Inc.

The Conservation and Function of RNA Secondary Structure in Plants.

RNA transcripts fold into secondary structures via intricate patterns of base pairing. These secondary structures impart catalytic, ligand binding, and scaffolding functions to a wide array of RNAs, forming a critical node of biological regulation. Among their many functions, RNA structural elements modulate epigenetic marks, alter mRNA stability and translation, regulate alternative splicing, transduce signals, and scaffold large macromolecular complexes. Thus, the study of RNA secondary structure is critical to understanding the function and regulation of RNA transcripts. Here, we review the origins, form, and function of RNA secondary structure, focusing on plants. We then provide an overview of methods for probing secondary structure, from physical methods such as X-ray crystallography and nuclear magnetic resonance (NMR) imaging to chemical and nuclease probing methods. Combining these latter methods with high-throughput sequencing has enabled them to scale across whole transcriptomes, yielding tremendous new insights into the form and function of RNA secondary structure.

Transcriptome-wide measurement of plant RNA secondary structure.

RNAs fold into intricate and precise secondary structures. These structural patterns regulate multiple steps of the RNA lifecycle, while also conferring catalytic and scaffolding functions to certain transcripts. Therefore, a full understanding of RNA posttranscriptional regulation requires a comprehensive picture of secondary structure. Here, we review several high throughput sequencing-based methods to globally survey plant RNA secondary structure. These methods are more accurate than computational prediction, and more scalable than physical techniques such as crystallography. We note hurdles to reliably measuring secondary structure, including RNA-binding proteins, RNA base modifications, and intramolecular duplexes. Finally, we survey the functional knowledge that has been gleaned from each of these methods, and identify some unanswered questions that remain.

Minor introns are embedded molecular switches regulated by highly unstable U6atac snRNA.

Eukaryotes have two types of spliceosomes, comprised of either major (U1, U2, U4, U5, U6) or minor (U11, U12, U4atac, U6atac; <1%) snRNPs. The high conservation of minor introns, typically one amidst many major introns in several hundred genes, despite their poor splicing, has been a long-standing enigma. Here, we discovered that the low abundance minor spliceosome's catalytic snRNP, U6atac, is strikingly unstable (t½<2 hr). We show that U6atac level depends on both RNA polymerases II and III and can be rapidly increased by cell stress-activated kinase p38MAPK, which stabilizes it, enhancing mRNA expression of hundreds of minor intron-containing genes that are otherwise suppressed by limiting U6atac. Furthermore, p38MAPK-dependent U6atac modulation can control minor intron-containing tumor suppressor PTEN expression and cytokine production. We propose that minor introns are embedded molecular switches regulated by U6atac abundance, providing a novel post-transcriptional gene expression mechanism and a rationale for the minor spliceosome's evolutionary conservation. DOI:http://dx.doi.org/10.7554/eLife.00780.001.