Oncogenic K-Ras suppresses global miRNA function.

K-Ras frequently acquires gain-of-function mutations (K-RasG12D being the most common) that trigger significant transcriptomic and proteomic changes to drive tumorigenesis. Nevertheless, oncogenic K-Ras-induced dysregulation of post-transcriptional regulators such as microRNAs (miRNAs) during oncogenesis is poorly understood. Here, we report that K-RasG12D promotes global suppression of miRNA activity, resulting in the upregulation of hundreds of targets. We constructed a comprehensive profile of physiological miRNA targets in mouse colonic epithelium and tumors expressing K-RasG12D using Halo-enhanced Argonaute pull-down. Combining this with parallel datasets of chromatin accessibility, transcriptome, and proteome, we uncovered that K-RasG12D suppressed the expression of Csnk1a1 and Csnk2a1, subsequently decreasing Ago2 phosphorylation at Ser825/829/832/835. Hypo-phosphorylated Ago2 increased binding to mRNAs while reducing its activity to repress miRNA targets. Our findings connect a potent regulatory mechanism of global miRNA activity to K-Ras in a pathophysiological context and provide a mechanistic link between oncogenic K-Ras and the post-transcriptional upregulation of miRNA targets.

High-Resolution In Vivo Identification of miRNA Targets by Halo-Enhanced Ago2 Pull-Down.

The identification of microRNA (miRNA) targets by Ago2 crosslinking-immunoprecipitation (CLIP) methods has provided major insights into the biology of this important class of non-coding RNAs. However, these methods are technically challenging and not easily applicable to an in vivo setting. To overcome these limitations and facilitate the investigation of miRNA functions in vivo, we have developed a method based on a genetically engineered mouse harboring a conditional Halo-Ago2 allele expressed from the endogenous Ago2 locus. By using a resin conjugated to the HaloTag ligand, Ago2-miRNA-mRNA complexes can be purified from cells and tissues expressing the endogenous Halo-Ago2 allele. We demonstrate the reproducibility and sensitivity of this method in mouse embryonic stem cells, developing embryos, adult tissues, and autochthonous mouse models of human brain and lung cancers. This method and the datasets we have generated will facilitate the characterization of miRNA-mRNA networks in vivo under physiological and pathological conditions.

Zurletrectinib is a next-generation TRK inhibitor with strong intracranial activity against NTRK fusion-positive tumours with on-target resistance to first-generation agents.

BACKGROUND: While NTRK fusion-positive cancers can be exquisitely sensitive to first-generation TRK inhibitors, resistance inevitably occurs, mediated in many cases by acquired NTRK mutations. Next-generation inhibitors (e.g., selitrectinib, repotrectinib) maintain activity against these TRK mutant tumors; however, there are no next-generation TRK inhibitors approved by the FDA and select trials have stopped treating patients. Thus, the identification of novel, potent and specific next-generation TRK inhibitors is a high priority. METHODS: In silico modeling and in vitro kinase assays were performed on TRK wild type (WT) and TRK mutant kinases. Cell viability and clonogenic assays as well as western blots were performed on human primary and murine engineered NTRK fusion-positive TRK WT and mutant cell models. Finally, zurletrectinib was tested in vivo in human xenografts and murine orthotopic glioma models harboring TRK-resistant mutations. RESULTS: In vitro kinase and in cell-based assays showed that zurletrectinib, while displaying similar potency against TRKA, TRKB, and TRKC WT kinases, was more active than other FDA approved or clinically tested 1st- (larotrectinib) and next-generation (selitrectinib and repotrectinib) TRK inhibitors against most TRK inhibitor resistance mutations (13 out of 18). Similarly, zurletrectinib inhibited tumor growth in vivo in sub-cute xenograft models derived from NTRK fusion-positive cells at a dose 30 times lower when compared to selitrectinib. Computational modeling suggests this stronger activity to be the consequence of augmented binding affinity of zurletrectinib for TRK kinases. When compared to selitrectinib and repotrectinib, zurletrectinib showed increased brain penetration in rats 0.5 and 2 h following a single oral administration. Consistently, zurletrectinib significantly improved the survival of mice harboring orthotopic NTRK fusion-positive, TRK-mutant gliomas (median survival = 41.5, 66.5, and 104 days for selitrectinib, repotrectinib, and zurletrectinib respectively; P < 0.05). CONCLUSION: Our data identifies zurletrectinib as a novel, highly potent next-generation TRK inhibitor with stronger in vivo brain penetration and intracranial activity than other next-generation agents.

MicroRNAs and cancer: short RNAs go a long way.

MicroRNAs (miRNAs) may be important regulators of gene expression. By modulating oncogenic and tumor suppressor pathways they could, in principle, contribute to tumorigenesis. Consistent with this hypothesis, recurrent genetic and epigenetic alterations of individual miRNAs are found in some tumors. Functional studies are now elucidating the mechanism of action of putative oncogenic and tumor suppressor miRNAs.

Profiling the patient with inflammatory bowel disease in the relationship between physical activity and partner/social network status: A post hoc patient-tailored analysis of the "BE-FIT-IBD" study.

INTRODUCTION: Normal quality of life is an ultimate target in the therapeutic approach to inflammatory bowel diseases (IBD), encompassing Crohn's disease (CD) and ulcerative colitis (UC) in the context of which regular physical activity (PA) is often a chimeric parameter that is not standardized in terms of quality/quantity. The study aimed to profile a sample of IBD patients about the relationship between PA-partner status and social network support. PATIENTS AND METHODS: A post hoc analysis of the "BE-FIT-IBD" study was set up by stratifying the data of PA with that of partner status and the support that the patient's social network (i.e., relatives, friends) provided in inciting the patient to practice regular PA. RESULTS: In the 219 patients included, there was a greater tendency for patients with stable partners to view the risk of reactivation/worsening of IBD as a barrier to conducting regular PA (p<0.0001). Single patients considered PA more as a protective factor (p=0.045). Patients without a PA-supporting social network retained IBD-related treatment as a PA barrier (p=0.016) and PA as a risk for IBD complications (p=0.01), with less confidence that PA could improve the course of IBD (p<0.001). Rectal syndrome was an IBD-related barrier more represented in patients with PA-deterring social network (p<0.0001). CONCLUSIONS: These factors are potential targets for recovering the IBD patient's adherence to regular PA.

Local Anesthetic Systemic Toxicity Joint Management in the Prehospital Environment: A Case Report.

Local anesthetic systemic toxicity (LAST) is a potentially life-threatening complication that may occur after local anesthetic injection. After reaching the systemic circulation, cardiovascular and central nervous system derangements may appear, with potentially fatal complications if left untreated. The pillars for LAST treatment are advanced life support measures, airway and seizure management, and a 20% lipid emulsion intravenous administration. When occurring in the prehospital setting, LAST is difficult to recognize, mostly because of its features overlapping with other acute conditions. Prompt treatment is also challenging because lipid emulsion may not be routinely carried on emergency vehicles. This article reports a case of LAST occurring in a dental ambulatory located in a remote location within the Italian Alps in which effective communication among different components of the same regional health care system (dispatch center, prehospital teams, and hospital network) led to fast lipid emulsion retrieval en route and on-site toxicity resolution. This case can inspire future operational changes, such as antidote networks available to prehospital emergency medicine crews, avoiding unnecessary deployment of antidotes on ambulances or helicopters, which is difficult to preserve without increasing management costs. However, to be established, such a network would need protocols to facilitate antidote retrieval, training focused on toxidromes recognition, and improved communication skills among different professionals involved in prehospital emergency medicine.

Targeted deletion reveals essential and overlapping functions of the miR-17 through 92 family of miRNA clusters.

miR-17 approximately 92, miR-106b approximately 25, and miR-106a approximately 363 belong to a family of highly conserved miRNA clusters. Amplification and overexpression of miR-1792 is observed in human cancers, and its oncogenic properties have been confirmed in a mouse model of B cell lymphoma. Here we show that mice deficient for miR-17 approximately 92 die shortly after birth with lung hypoplasia and a ventricular septal defect. The miR-17 approximately 92 cluster is also essential for B cell development. Absence of miR-17 approximately 92 leads to increased levels of the proapoptotic protein Bim and inhibits B cell development at the pro-B to pre-B transition. Furthermore, while ablation of miR-106b approximately 25 or miR-106a approximately 363 has no obvious phenotypic consequences, compound mutant embryos lacking both miR-106b approximately 25 and miR-17 approximately 92 die at midgestation. These results provide key insights into the physiologic functions of this family of microRNAs and suggest a link between the oncogenic properties of miR-17 approximately 92 and its functions during B lymphopoiesis and lung development.

Numerical modeling of a hybrid hollow-core fiber for enhanced mid-infrared guidance.

We propose a novel design of hollow-core fiber for enhanced light guidance in the mid-infrared. The structure combines an arrangement of non-touching antiresonant elements in the air core with a multilayer glass/polymer structure in the fiber's cladding. Through numerical modeling, we demonstrate that the combination of antiresonant/inhibited-coupling and photonic bandgap guidance mechanisms can decrease the optical loss of a tubular antiresonant fiber by more than one order of magnitude. More specifically, our simulations demonstrate losses of the HE11 mode in the few dB/km level, which can be tuned through mid-infrared wavelengths (5 µm-10.6 µm) by carefully optimizing the structural parameters of both structures. We also show that the hybrid hollow-core fiber design is more robust to bend-induced loss than an equivalent tubular antiresonant fiber or a Bragg/OmniGuide fiber. As a result, if successfully fabricated, the hybrid hollow-core fiber will offer low-loss, high beam-quality, effectively single-mode operation, and low bending losses, potentially solving many of the problems that affect all known mid-infrared fiber types.

Extruded tellurite antiresonant hollow core fiber for Mid-IR operation.

We report the first extruded tellurite antiresonant hollow core fibers (HC-ARFs) aimed at the delivery of mid-infrared (Mid-IR) laser radiation. The preform extrusion fabrication process allowed us to obtain preforms with non-touching capillaries in a single step, hence minimizing thermal cycles. The fibers were fabricated from in-house synthetized tellurite glass (containing Zn, Ba and K oxides) and co-drawn with a fluorinated ethylene propylene (FEP) polymer outer layer to improve their mechanical properties and protect the glass from humidity. The fabricated HC-ARFs transmit in the Mid-IR spectral range from 4.9 to 6 µm. We measured losses of ∼8.2, 4.8 and 6.4 dB/m at 5 µm, 5.6 µm and 5.8 µm, respectively in two different fibers. These losses, which are dominated by leakage mostly arising from a non-uniform membrane thickness, represent the lowest attenuation reported for a tellurite-based HC-ARF to date. The fibers present good beam quality and an M2 factor of 1.2. Modelling suggests that by improving the uniformity in the capillary membrane thickness losses down to 0.05 dB/m at 5.4 µm should be possible, making this solution attractive, for example, for beam delivery from a CO laser.

In vivo engineering of oncogenic chromosomal rearrangements with the CRISPR/Cas9 system.

Chromosomal rearrangements have a central role in the pathogenesis of human cancers and often result in the expression of therapeutically actionable gene fusions. A recently discovered example is a fusion between the genes echinoderm microtubule-associated protein like 4 (EML4) and anaplastic lymphoma kinase (ALK), generated by an inversion on the short arm of chromosome 2: inv(2)(p21p23). The EML4-ALK oncogene is detected in a subset of human non-small cell lung cancers (NSCLC) and is clinically relevant because it confers sensitivity to ALK inhibitors. Despite their importance, modelling such genetic events in mice has proven challenging and requires complex manipulation of the germ line. Here we describe an efficient method to induce specific chromosomal rearrangements in vivo using viral-mediated delivery of the CRISPR/Cas9 system to somatic cells of adult animals. We apply it to generate a mouse model of Eml4-Alk-driven lung cancer. The resulting tumours invariably harbour the Eml4-Alk inversion, express the Eml4-Alk fusion gene, display histopathological and molecular features typical of ALK(+) human NSCLCs, and respond to treatment with ALK inhibitors. The general strategy described here substantially expands our ability to model human cancers in mice and potentially in other organisms.

Cancer diagnosis and immunotherapy in the age of CRISPR.

The explosion in genome editing technologies that has occurred in the past decade has revolutionized cancer research and promises to improve cancer diagnosis and therapy. Ongoing efforts include engineering of chimeric antigen receptor-T cells using clustered regularly interspaced short palindromic repeats (CRISPR) to generate a safer, more effective therapy with improved performance in immunologically "cold" tumors, as well as clever adaptations of CRISPR enzymes to allow fast, simple, and sensitive detection of specific nucleotide sequences. While still in their infancy, CRISPR-based cancer therapeutics and diagnostics are developing at an impressive speed and it is likely they will soon impact clinical practice. Here, we summarize their history and the most recent developments.

NORAD: Defender of the Genome.

Long non-coding RNAs (lncRNAs) are a fascinating, but still largely uncharacterized, class of genes. A recent paper by the Mendell group identifies NORAD, a novel lncRNA that is regulated in response to DNA damage and plays a key role in maintaining genome integrity by modulating the activity the RNA binding proteins PUM2 and PUM1.

Flexible Mid-IR fiber bundle for thermal imaging of inaccessible areas.

We present a flexible coherent Mid-Infrared (Mid-IR) fiber bundle for thermal imaging made of 1200 Ge30As13Se32Te25 glass cores embedded in a Fluorinated Ethylene Propylene (FEP) polymer cladding. The high index contrast between the chalcogenide glass and the polymer cladding helps minimizing inter-pixel cross-talk, while the low Young's modulus of the polymer cladding gives the bundle good flexibility despite its millimeter scale outer diameter. The delivery of high contrast and high spatial resolution thermal images of a human hand through a 62.5 cm long bundle indicates its excellent imaging potential.

A new role for miR-182 in DNA repair.

A role for miRNAs in modulating cellular responses to DNA damage and chemotherapy is emerging. In this issue of Molecular Cell, Moskwa and colleagues propose a novel function for miR-182 in the posttranscriptional regulation of BRCA1 expression and in DNA repair (Moskwa et al., 2011).

miR than meets the eye.

Retinoblastoma is a rare pediatric cancer that has served as a paradigm to investigate the mechanisms of tumorigenesis. In this issue of Genes & Development, Conkrite and colleagues (pp. 1734-1745) found high levels of the miR-17~92 and miR-106b-25 microRNAs in primary retinoblastomas and show that overexpression of miR-17~92 accelerates retinoblastoma development in mice by promoting proliferation, in part by reducing expression of the cell cycle inhibitor p21. These experiments identify the RB/miR-17~92/p21 axis as a critical regulator of retinoblastoma tumorigenesis and potentially many other cancers.

School Gardens in the United States: Current Barriers to Integration and Sustainability.

OBJECTIVES: To elucidate details about the barriers (time, funding, staffing, and space) to integrating and sustaining school gardens. METHODS: A total of 99 school gardeners from 15 states participated in an online survey in June 2017. The 29-item survey contained qualitative and quantitative items that we analyzed using descriptive statistics and inductive content analysis. RESULTS: In order of greatest to least barrier, gardeners ranked time, staff, funding, curriculum, and space. Time for classes to use the garden (66% of respondents) and time for staff training (62%) were the most frequently listed time-related challenges. Respondents also reported low engagement within the school community. An overall lack of funding was the most common funding-related barrier, and gardeners were unaware of how to obtain more funding. CONCLUSIONS: We identified 3 aspects of school gardens as opportunities to address time- and staff-related issues: strengthening of garden committees, professional development, and community outreach. Better channels are needed to disseminate funding opportunities within schools and to communicate with communities at large. Ultimately, doing so will strengthen existing school gardens as a vehicle to promote dietary, physical, and social health within communities.

In vivo, Argonaute-bound microRNAs exist predominantly in a reservoir of low molecular weight complexes not associated with mRNA.

MicroRNAs repress mRNA translation by guiding Argonaute proteins to partially complementary binding sites, primarily within the 3' untranslated region (UTR) of target mRNAs. In cell lines, Argonaute-bound microRNAs exist mainly in high molecular weight RNA-induced silencing complexes (HMW-RISC) associated with target mRNA. Here we demonstrate that most adult tissues contain reservoirs of microRNAs in low molecular weight RISC (LMW-RISC) not bound to mRNA, suggesting that these microRNAs are not actively engaged in target repression. Consistent with this observation, the majority of individual microRNAs in primary T cells were enriched in LMW-RISC. During T-cell activation, signal transduction through the phosphoinositide-3 kinase-RAC-alpha serine/threonine-protein kinase-mechanistic target of rapamycin pathway increased the assembly of microRNAs into HMW-RISC, enhanced expression of the glycine-tryptophan protein of 182 kDa, an essential component of HMW-RISC, and improved the ability of microRNAs to repress partially complementary reporters, even when expression of targeting microRNAs did not increase. Overall, data presented here demonstrate that microRNA-mediated target repression in nontransformed cells depends not only on abundance of specific microRNAs, but also on regulation of RISC assembly by intracellular signaling.

Genetic dissection of the miR-17~92 cluster of microRNAs in Myc-induced B-cell lymphomas.

The miR-17 approximately 92 cluster is frequently amplified or overexpressed in human cancers and has emerged as the prototypical oncogenic polycistron microRNA (miRNA). miR-17 approximately 92 is a direct transcriptional target of c-Myc, and experiments in a mouse model of B-cell lymphomas have shown cooperation between these two oncogenes. However, both the molecular mechanism underlying this cooperation and the individual miRNAs that are responsible for it are unknown. By using a conditional knockout allele of miR-17 approximately 92, we show here that sustained expression of endogenous miR-17 approximately 92 is required to suppress apoptosis in Myc-driven B-cell lymphomas. Furthermore, we show that among the six miRNAs that are encoded by miR-17 approximately 92, miR-19a and miR-19b are absolutely required and largely sufficient to recapitulate the oncogenic properties of the entire cluster. Finally, by combining computational target prediction, gene expression profiling, and an in vitro screening strategy, we identify a subset of miR-19 targets that mediate its prosurvival activity.

The present and future of genome editing in cancer research.

The widespread use of high-throughput genome sequencing methods is profoundly changing the way we understand, classify, and treat human cancers. To make sense of the deluge of sequencing data generated in the clinic, more effective and rapid assessments of the functional relevance of newly discovered cancer-associated mutations are urgently needed. In this review, we discuss how genome editing technologies are responding to this major challenge. Largely focusing on CRISPR-based methods, we will highlight their potential to accelerate discovery, discuss their current limitations, and speculate about future applications.