The muscleblind-like (Mbnl) family of RNA-binding proteins plays important roles in muscle and eye development and in myotonic dystrophy (DM), in which expanded CUG or CCUG repeats functionally ...deplete Mbnl proteins. We identified transcriptome-wide functional and biophysical targets of Mbnl proteins in brain, heart, muscle, and myoblasts by using RNA-seq and CLIP-seq approaches. This analysis identified several hundred splicing events whose regulation depended on Mbnl function in a pattern indicating functional interchangeability between Mbnl1 and Mbnl2. A nucleotide resolution RNA map associated repression or activation of exon splicing with Mbnl binding near either 3′ splice site or near the downstream 5′ splice site, respectively. Transcriptomic analysis of subcellular compartments uncovered a global role for Mbnls in regulating localization of mRNAs in both mouse and Drosophila cells, and Mbnl-dependent translation and protein secretion were observed for a subset of mRNAs with Mbnl-dependent localization. These findings hold several new implications for DM pathogenesis.
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► A nucleotide resolution RNA map of the splicing regulatory activity of Mbnl1 ► Functional interchangeability of Mbnl1 and Mbnl2 in splicing regulation ► A global role for Mbnls in regulating mRNA localization in mouse and fly ► Mbnl-dependent regulation of translation and protein secretion of Mbnl-bound mRNAs
Muscleblinds are a family of conserved RNA-binding proteins that are important in development and myotonic dystrophy. In addition to regulating alternative splicing of hundreds of exons, muscleblinds directly bind 3′ ends of mRNAs to regulate their subcellular localization and translation.
RNA binding proteins of the conserved CUGBP1, Elav-like factor (CELF) family contribute to heart and skeletal muscle development and are implicated in myotonic dystrophy (DM). To understand their ...genome-wide functions, we analyzed the transcriptome dynamics following induction of CELF1 or CELF2 in adult mouse heart and of CELF1 in muscle by RNA-seq, complemented by crosslinking/immunoprecipitation-sequencing (CLIP-seq) analysis of mouse cells and tissues to distinguish direct from indirect regulatory targets. We identified hundreds of mRNAs bound in their 3' UTRs by both CELF1 and the developmentally induced MBNL1 protein, a threefold greater overlap in target messages than expected, including messages involved in development and cell differentiation. The extent of 3' UTR binding by CELF1 and MBNL1 predicted the degree of mRNA repression or stabilization, respectively, following CELF1 induction. However, CELF1's RNA binding specificity in vitro was not detectably altered by coincubation with recombinant MBNL1. These findings support a model in which CELF and MBNL proteins bind independently to mRNAs but functionally compete to specify down-regulation or localization/stabilization, respectively, of hundreds of mRNA targets. Expression of many alternative 3' UTR isoforms was altered following CELF1 induction, with 3' UTR binding associated with down-regulation of isoforms and genes. The splicing of hundreds of alternative exons was oppositely regulated by these proteins, confirming an additional layer of regulatory antagonism previously observed in a handful of cases. The regulatory relationships between CELFs and MBNLs in control of both mRNA abundance and splicing appear to have evolved to enhance developmental transitions in major classes of heart and muscle genes.
The use of targeted therapeutics directed against BRAF(V600)-mutant metastatic melanoma improves progression-free survival in many patients; however, acquired drug resistance remains a major medical ...challenge. By far, the most common clinical resistance mechanism involves reactivation of the MAPK (RAF/MEK/ERK) pathway by a variety of mechanisms. Thus, targeting ERK itself has emerged as an attractive therapeutic concept, and several ERK inhibitors have entered clinical trials. We sought to preemptively determine mutations in ERK1/2 that confer resistance to either ERK inhibitors or combined RAF/MEK inhibition in BRAF(V600)-mutant melanoma. Using a random mutagenesis screen, we identified multiple point mutations in ERK1 (MAPK3) and ERK2 (MAPK1) that could confer resistance to ERK or RAF/MEK inhibitors. ERK inhibitor-resistant alleles were sensitive to RAF/MEK inhibitors and vice versa, suggesting that the future development of alternating RAF/MEK and ERK inhibitor regimens might help circumvent resistance to these agents.
Most patients with BRAF(V600)-mutant metastatic melanoma develop resistance to selective RAF kinase inhibitors. The spectrum of clinical genetic resistance mechanisms to RAF inhibitors and options ...for salvage therapy are incompletely understood. We performed whole-exome sequencing on formalin-fixed, paraffin-embedded tumors from 45 patients with BRAF(V600)-mutant metastatic melanoma who received vemurafenib or dabrafenib monotherapy. Genetic alterations in known or putative RAF inhibitor resistance genes were observed in 23 of 45 patients (51%). Besides previously characterized alterations, we discovered a "long tail" of new mitogen-activated protein kinase (MAPK) pathway alterations (MAP2K2, MITF) that confer RAF inhibitor resistance. In three cases, multiple resistance gene alterations were observed within the same tumor biopsy. Overall, RAF inhibitor therapy leads to diverse clinical genetic resistance mechanisms, mostly involving MAPK pathway reactivation. Novel therapeutic combinations may be needed to achieve durable clinical control of BRAF(V600)-mutant melanoma. Integrating clinical genomics with preclinical screens may model subsequent resistance studies.
Treatment of BRAF-mutant melanoma with combined dabrafenib and trametinib, which target RAF and the downstream MAP-ERK kinase (MEK)1 and MEK2 kinases, respectively, improves progression-free survival ...and response rates compared with dabrafenib monotherapy. Mechanisms of clinical resistance to combined RAF/MEK inhibition are unknown. We performed whole-exome sequencing (WES) and whole-transcriptome sequencing (RNA-seq) on pretreatment and drug-resistant tumors from five patients with acquired resistance to dabrafenib/trametinib. In three of these patients, we identified additional mitogen-activated protein kinase (MAPK) pathway alterations in the resistant tumor that were not detected in the pretreatment tumor, including a novel activating mutation in MEK2 (MEK2(Q60P)). MEK2(Q60P) conferred resistance to combined RAF/MEK inhibition in vitro, but remained sensitive to inhibition of the downstream kinase extracellular signal-regulated kinase (ERK). The continued MAPK signaling-based resistance identified in these patients suggests that alternative dosing of current agents, more potent RAF/MEK inhibitors, and/or inhibition of the downstream kinase ERK may be needed for durable control of BRAF-mutant melanoma.
To further our understanding of the somatic genetic basis of uveal melanoma, we sequenced the protein-coding regions of 52 primary tumors and 3 liver metastases together with paired normal DNA. Known ...recurrent mutations were identified in GNAQ, GNA11, BAP1, EIF1AX, and SF3B1. The role of mutated EIF1AX was tested using loss of function approaches including viability and translational efficiency assays. Knockdown of both wild type and mutant EIF1AX was lethal to uveal melanoma cells. We probed the function of N-terminal tail EIF1AX mutations by performing RNA sequencing of polysome-associated transcripts in cells expressing endogenous wild type or mutant EIF1AX. Ribosome occupancy of the global translational apparatus was sensitive to suppression of wild type but not mutant EIF1AX. Together, these studies suggest that cells expressing mutant EIF1AX may exhibit aberrant translational regulation, which may provide clonal selective advantage in the subset of uveal melanoma that harbors this mutation.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Whole-exome sequencing of metastatic castration-resistant prostate cancer (mCRPC) reveals that 5% to 7% of tumors harbor promyelocytic leukemia zinc finger (PLZF) protein homozygous deletions. PLZF ...is a canonical androgen-regulated putative tumor suppressor gene whose expression is inhibited by androgen deprivation therapy (ADT). Here, we demonstrate that knockdown of PLZF expression promotes a CRPC and enzalutamide-resistant phenotype in prostate cancer cells. Reintroduction of PLZF expression is sufficient to reverse androgen-independent growth mediated by PLZF depletion. PLZF loss enhances CRPC tumor growth in a xenograft model. Bioinformatic analysis of the PLZF cistrome shows that PLZF negatively regulates multiple pathways, including the MAPK pathway. Accordingly, our data support an oncogenic program activated by ADT. This acquired mechanism together with the finding of genetic loss in CRPC implicates PLZF inactivation as a mechanism promoting ADT resistance and the CRPC phenotype.
Objectives To examine mortality and revision rates among patients with osteoarthritis undergoing hip arthroplasty and to compare these rates between patients undergoing cemented or uncemented ...procedures and to compare outcomes between men undergoing stemmed total hip replacements and Birmingham hip resurfacing.Design Cohort study.Setting National Joint Registry.Population About 275 000 patient records.Main outcome measures Hip arthroplasty procedures were linked to the time to any subsequent mortality or revision (implant failure). Flexible parametric survival analysis methods were used to analyse time to mortality and also time to revision. Comparisons between procedure groups were adjusted for age, sex, American Society of Anesthesiologists (ASA) grade, and complexity.Results As there were large baseline differences in the characteristics of patients receiving cemented, uncemented, or resurfacing procedures, unadjusted comparisons are inappropriate. Multivariable survival analyses identified a higher mortality rate for patients undergoing cemented compared with uncemented total hip replacement (adjusted hazard ratio 1.11, 95% confidence interval 1.07 to 1.16); conversely, there was a lower revision rate with cemented procedures (0.53, 0.50 to 0.57). These translate to small predicted differences in population averaged absolute survival probability at all time points. For example, compared with the uncemented group, at eight years after surgery the predicted probability of death in the cemented group was 0.013 higher (0.007 to 0.019) and the predicted probability of revision was 0.015 lower (0.012 to 0.017). In multivariable analyses restricted to men, there was a higher mortality rate in the cemented group and the uncemented group compared with the Birmingham hip resurfacing group. In terms of revision, the Birmingham hip resurfacings had a similar revision rate to uncemented total hip replacements. Both uncemented total hip replacements and Birmingham hip resurfacings had a higher revision rate than cemented total hip replacements.Conclusions There is a small but significant increased risk of revision with uncemented rather than cemented total hip replacement, and a small but significant increased risk of death with cemented procedures. It is not known whether these are causal relations or caused by residual confounding. Compared with uncemented and cemented total hip replacements, Birmingham hip resurfacing has a significantly lower risk of death in men of all ages. Previously, only adjusted analyses of hip implant revision rates have been used to recommend and justify use of cheaper cemented total hip implants. Our investigations additionally consider mortality rates and suggest a potentially higher mortality rate with cemented total hip replacements, which merits further investigation.
Implementing cancer precision medicine in the clinic requires assessing the therapeutic relevance of genomic alterations. A main challenge is the systematic interpretation of whole-exome sequencing ...(WES) data for clinical care.
One hundred sixty-five adults with metastatic colorectal and lung adenocarcinomas were prospectively enrolled in the CanSeq study. WES was performed on DNA extracted from formalin-fixed paraffin-embedded tumor biopsy samples and matched blood samples. Somatic and germ-line alterations were ranked according to therapeutic or clinical relevance. Results were interpreted using an integrated somatic and germ-line framework and returned in accordance with patient preferences.
At the time of this analysis, WES had been performed and results returned to the clinical team for 165 participants. Of 768 curated somatic alterations, only 31% were associated with clinical evidence and 69% with preclinical or inferential evidence. Of 806 curated germ-line variants, 5% were clinically relevant and 56% were classified as variants of unknown significance. The variant review and decision-making processes were effective when the process was changed from that of a Molecular Tumor Board to a protocol-based approach.
The development of novel interpretive and decision-support tools that draw from scientific and clinical evidence will be crucial for the success of cancer precision medicine in WES studies.Genet Med advance online publication 26 January 2017.
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