Summary RNA interference is a powerful tool for studying gene function, however, the reproducible generation of RNAi transgenic mice remains a significant limitation. By combining optimized fluorescence-coupled miR30-based shRNAs with high efficiency ES cell targeting, we developed a fast, scalable pipeline for the production of shRNA transgenic mice. Using this system, we generated eight tet-regulated shRNA transgenic lines targeting Firefly and Renilla luciferases, Oct4 and tumor suppressors p53, p16INK4a, p19ARF and APC and demonstrate potent gene silencing and GFP-tracked knockdown in a broad range of tissues in vivo. Further, using an shRNA targeting APC, we illustrate how this approach can identify predicted phenotypes and also unknown functions for a well-studied gene. In addition, through regulated gene silencing we validate APC/Wnt and p19ARF as potential therapeutic targets in T cell acute lymphoblastic leukemia/lymphoma and lung adenocarcinoma, respectively. This system provides a cost-effective and scalable platform for the production of RNAi transgenic mice targeting any mammalian gene.

The epidermal growth-factor receptor (EGFR) tyrosine kinase inhibitor erlotinib has been proven to be highly effective in the treatment of nonsmall cell lung cancer (NSCLC) harboring oncogenic EGFR mutations. The majority of patients, however, will eventually develop resistance and succumb to the disease. Recent studies have identified secondary mutations in the EGFR (EGFR T790M) and amplification of the N-Methyl-N′-nitro-N-nitroso-guanidine (MNNG) HOS transforming gene (MET) oncogene as two principal mechanisms of acquired resistance. Although they can account for approximately 50% of acquired resistance cases together, in the remaining 50%, the mechanism remains unknown. In NSCLC-derived cell lines and early-stage tumors before erlotinib treatment, we have uncovered the existence of a subpopulation of cells that are intrinsically resistant to erlotinib and display features suggestive of epithelial-to-mesenchymal transition (EMT). We showed that activation of TGF-β–mediated signaling was sufficient to induce these phenotypes. In particular, we determined that an increased TGF-β–dependent IL-6 secretion unleashed previously addicted lung tumor cells from their EGFR dependency. Because IL-6 and TGF-β are prominently produced during inflammatory response, we used a mouse model system to determine whether inflammation might impair erlotinib sensitivity. Indeed, induction of inflammation not only stimulated IL-6 secretion but was sufficient to decrease the tumor response to erlotinib. Our data, thus, argue that both tumor cell-autonomous mechanisms and/or activation of the tumor microenvironment could contribute to primary and acquired erlotinib resistance, and as such, treatments based on EGFR inhibition may not be sufficient for the effective treatment of lung-cancer patients harboring mutant EGFR.

Oncogene-induced senescence is a potent barrier to tumorigenesis that limits cellular expansion following certain oncogenic events. Senescent cells display a repressive chromatin configuration thought to stably silence proliferation-promoting genes, while simultaneously activating an unusual form of immune surveillance involving a secretory program referred to as the senescence-associated secretory phenotype (SASP). Here we demonstrate that senescence also involves a global remodeling of the enhancer landscape with recruitment of the chromatin reader BRD4 to newly activated super-enhancers adjacent to key SASP genes. Transcriptional profiling and functional studies indicate that BRD4 is required for the SASP and downstream paracrine signaling. Consequently, BRD4 inhibition disrupts immune cell mediated targeting and elimination of premalignant senescent cells in vitro and in vivo. Our results identify a critical role for BRD4-bound super-enhancers in senescence immune surveillance and in the proper execution of a tumor-suppressive program.

Background: More than 300 million people carry a diagnosis of asthma, with data to suggest that they are at a higher risk for infection or adverse outcomes from severe acute respiratory syndrome coronavirus 2. Asthma is remarkably heterogeneous, and it is currently unclear how patient-intrinsic factors may relate to coronavirus disease 2019. Objective: We sought to identify and characterize subsets of patients with asthma at increased risk for severe acute respiratory syndrome coronavirus 2 infection. Methods: Participants from 2 large asthma cohorts were stratified using clinically relevant parameters to identify factors related to angiotensin-converting enzyme-2 (ACE2) expression within bronchial epithelium. ACE-2-correlated gene signatures were used to interrogate publicly available databases to identify upstream signaling events and novel therapeutic targets. Results: Stratifying by type 2 inflammatory biomarkers, we identified subjects who demonstrated low peripheral blood eosinophils accompanied by increased expression of the severe acute respiratory syndrome coronavirus 2 receptor ACE2 in bronchial epithelium. Genes highly correlated with ACE2 overlapped with type 1 and 2 IFN signatures, normally induced by viral infections. T-cell recruitment and activation within bronchoalveolar lavage cells of ACE2-high subjects was reciprocally increased. These patients demonstrated characteristics corresponding to risk factors for severe coronavirus disease 2019, including male sex, history of hypertension, low peripheral blood, and elevated bronchoalveolar lavage lymphocytes. Conclusions: ACE2 expression is linked to upregulation of viral response genes in a subset of type 2-low patients with asthma with characteristics resembling known risk factors for severe coronavirus disease 2019. Therapies targeting the IFN family and T-cell-activating factors may therefore be of benefit in a subset of patients.

We have identified 7SK RNA to be enriched in nuclear speckles. Knock-down of 7SK results in the mislocalization of nuclear speckle constituents, and the transcriptional up-regulation of a reporter gene locus. 7SK RNA transiently associates with the locus upon transcriptional down-regulation correlating with the displacement of pTEF-b.

Although much is known about the underlying mechanisms of p53 activity and regulation, the factors that influence the diversity and duration of p53 responses are not well understood. Here we describe a unique mode of p53 regulation involving alternative splicing of the TP53 gene. We found that the use of an alternative 3′ splice site in intron 6 generates a unique p53 isoform, dubbed p53Ψ. At the molecular level, p53Ψ is unable to bind to DNA and does not transactivate canonical p53 target genes. However, like certain p53 gain-offunction mutants, p53Ψ attenuates the expression of E-cadherin, induces expression of markers of the epithelial-mesenchymal transition, and enhances the motility and invasive capacity of cells through a unique mechanism involving the regulation of cyclophilin D activity, a component of the mitochondrial inner pore permeability. Hence, we propose that p53Ψ encodes a separation-of-function isoform that, although lacking canonical p53 tumor suppressor/transcriptional activities, is able to induce a prometastatic program in a transcriptionally independent manner. reactive oxygen species | cancer A n evolutionarily conserved transcription factor, p53 has origins that can be traced back to the early metazoans, ∼700 Mya (1). This transcription factor plays a critical role in regulating many fundamental aspects of reversible and irreversible cellular stress responses, genome surveillance, and suppression of oncogenic transformation (1). In response to strong cellular stresses such as DNA damage or oncogenic signals, p53 regulates the expression of a large cohort of genes that affect cell cycle arrest, senescence, and apoptosis (1). Recent work has uncovered additional roles for p53 under basal physiological conditions, such as regulation of development, reproduction, metabolism, and self-renewal capacity (2-5). However, the factors that influence the diversity and duration of p53 responses are not well understood. Here we describe a unique mode of p53 regulation that involves alternative splicing of the TP53 gene. We found that the use of an alternative 3′ splice site in intron 6 generates a previously uncharacterized p53 isoform that we named p53Ψ. Interestingly, this isoform is highly expressed in cells characterized by a CD44 high /CD24 low immune type. At the molecular level, p53Ψ lacks major portions of the DNA-binding domain, the nuclear localization sequence, and the tetramerization domain, features that are normally present in full-length p53 (p53FL). Consequently, this isoform proved incapable of sequence-specific DNA binding and transactivation of canonical p53 target genes. However, expression of the p53Ψ isoform attenuated the expression of E-cadherin, induced expression of markers associated with epithelial-mesenchymal transition (EMT), and enhanced the motility and invasive capacity of normal and malignant cells. Consistent with a role of these features in enhancing the prometastatic capabilities of cells, we observed that in patients with early-stage nonsmall cell lung carcinoma (...

While the term asthma has long been known to describe heterogeneous groupings of patients, only recently have data evolved which enable a molecular understanding of the clinical differences. The evolution of transcriptomics (and other ‘omics platforms) and improved statistical analyses in combination with large clinical cohorts opened the door for molecular characterization of pathobiologic processes associated with a range of asthma patients. When linked with data from animal models and clinical trials of targeted biologic therapies, emerging distinctions arose between patients with and without elevations in type 2 immune and inflammatory pathways, leading to the confirmation of a broad categorization of type 2-Hi asthma. Differences in the ratios, sources, and location of type 2 cytokines and their relation to additional immune pathway activation appear to distinguish several different (sub)molecular phenotypes, and perhaps endotypes of type 2-Hi asthma, which respond differently to broad and targeted anti-inflammatory therapies. Asthma in the absence of type 2 inflammation is much less well defined, without clear biomarkers, but is generally linked with poor responses to corticosteroids. Integration of “big data” from large cohorts, over time, using machine learning approaches, combined with validation and iterative learning in animal (and human) model systems is needed to identify the biomarkers and tightly defined molecular phenotypes/endotypes required to fulfill the promise of precision medicine.

Asthma is an immune-mediated heterogeneous disease. Current type-2 biomarkers provide limited understanding of underlying pathobiology or therapeutic guidance. Using a multi-omics approach, Camiolo et al. identify two clusters of severe asthma patients with similar type-2 biomarkers but with strikingly distinct immune profiles and associated biological pathways.