Large parts of mammalian genomes are transcriptionally inactive and enriched with various classes of interspersed and tandem repeats. Here we show that the tumor suppressor protein p53 cooperates ...with DNA methylation to maintain silencing of a large portion of the mouse genome. Massive transcription of major classes of short, interspersed nuclear elements (SINEs) B1 and B2, both strands of near-centromeric satellite DNAs consisting of tandem repeats, and multiple species of noncoding RNAs was observed in p53-deficient but not in p53 wild-type mouse fibroblasts treated with the DNA demethylating agent 5-aza-2’-deoxycytidine. The abundance of these transcripts exceeded the level of β-actin mRNA by more than 150-fold. Accumulation of these transcripts, which are capable of forming double-stranded RNA (dsRNA), was accompanied by a strong, endogenous, apoptosis-inducing type I IFN response. This phenomenon, which we named “TRAIN” (for “transcription of repeats activates interferon”), was observed in spontaneous tumors in two models of cancer-prone mice, presumably reflecting naturally occurring DNA hypomethylation and p53 inactivation in cancer. These observations suggest that p53 and IFN cooperate to prevent accumulation of cells with activated repeats and provide a plausible explanation for the deregulation of IFN function frequently seen in tumors. Overall, this work reveals roles for p53 and IFN that are key for genetic stability and therefore relevant to both tumorigenesis and the evolution of species.
BackgroundMulti-omic analysis of microsamples of lancet-induced blood drops allows frequent capture and quantitation of numerous metabolites, lipids, cytokines, and proteins. Microsample-based ...transcriptome profiling would facilitate use of RNA biomarkers in diagnosis and treatment of immunotherapy patients, but a suitable method has not been available. We tested a targeted sequencing protocol for this purpose in human blood microsamples.MethodsWe tested normal blood collected either by phlebotomy into standard vacutainer tubes or by absorption of 30 uL of blood onto a Mitra device pre-treated with a RNA-stabilization reagent. We tested detection of gene expression changes by incubating anti-coagulated blood for 18 hours with endotoxin (LPS, 100 ng/ml) followed by isolation of identical volumes of blood using a standard method (Qiagen kit) or from Mitra devices air dried for 24 h to mimic a home-use scenario. RNA was extracted from the microsamplers by immersion of the tips into a high NaCL buffer containing proteinase K, NP-40 and vigorous shaking at 60°C for 30 minutes. After RNA purification, a panel of 274 immune/inflammatory genes was quantified by NGS after PCR using targeted primers following the Cellecta DriverMap protocol, which avoids counting of abundant rRNA and mitochondrial sequences. The targeted sequencing results were normalized to counts per million and used to compare results in standard vs microsamples tested in triplicate.ResultsWe found robust detection of gene expression and correlation (figure 1) using the two methods in both unstimulated (r2 = 0.94) and LPS-stimulated blood (r2 = 0.97, figure 1). Comparison of the top up-or down-regulated genes showed comparable changes in microsamples vs standard method analysis (e.g., log2 ratio of LPS-stimulated to control for CXCL10, IL1B, HMOX1, FCGR3B, respectively: 8.77 vs 8.72; 7.99 vs 7.83; -3.84 vs -4.32; -3.47 vs -3.18). The correlation was strongest for genes with high counts (>1K) but lower for the subset of low-count genes (r2 = 0.7 for <1K). The differentially expressed genes identified in both standard and microsample methods showed high overlap with DEGs reported in public datasets in similar experiments.ConclusionsRNA transcriptome profiling using targeted sequencing allows sensitive detection of gene expression levels in normal and activated blood samples. Additional testing of a whole transcriptome panel (~19K protein-coding genes) is underway and will be reported. Because microsamples can be air-dried and mailed in for analysis, this approach has great promise for easy and repeated monitoring of RNA biomarkers in immunotherapy.Abstract 165 Figure 1
BackgroundThe results of adaptive immune receptor (AIR) repertoire diversity assays can be affected by various biases from differences in conditions in the RT-PCR and NGS sequencing steps. Spike-in ...synthetic controls can be used as calibration standards to address these biasesMethodsIn this study, we synthesized near full-length BCR and TCR constructs that mimic seven different IGH, IGK, IGL, TRB, TRA, TRG and TRD genes. To test our spike-in controls, three sets of variants at different concentrations were added to the RNA samples before the reverse transcription reaction with Cellecta’s DriverMap™ Adaptive Immune Receptor (AIR) Profiling Assay. The DriverMap™ protocol uses reverse gene-specific primers with unique molecular identifiers (UMI), allowing UMI-based correction of amplification biases during data analysisResultsCalculating UMI-based correction and comparing that with spike-in controls enabled us to differentiate between real and background sequences and estimate the average sequencing error rate at 0.4%-0.8% per base, which is within the reported range of Illumina sequencing.ConclusionsThis suggests that our spike-in controls are reliable and may be used as a universal tool to correct AIR protocol biases and calculate error and mutation rates in different AIR profiling assays.
Tumors acquire alterations in oncogenes and tumor suppressor genes in an adaptive walk through the fitness landscape of tumorigenesis. However, the interactions between oncogenes and tumor suppressor ...genes that shape this landscape remain poorly resolved and cannot be revealed by human cancer genomics alone. Here, we use a multiplexed, autochthonous mouse platform to model and quantify the initiation and growth of more than one hundred genotypes of lung tumors across four oncogenic contexts: KRAS G12D, KRAS G12C, BRAF V600E, and EGFR L858R. We show that the fitness landscape is rugged-the effect of tumor suppressor inactivation often switches between beneficial and deleterious depending on the oncogenic context-and shows no evidence of diminishing-returns epistasis within variants of the same oncogene. These findings argue against a simple linear signaling relationship amongst these three oncogenes and imply a critical role for off-axis signaling in determining the fitness effects of inactivating tumor suppressors.
Chronic inflammation is associated with increased cancer risk. Furthermore, the transcription factor NF-κB, a central regulator of inflammatory responses, is constitutively active in most tumors. To ...determine whether active NF-κB inherently contributes to malignant transformation, we isolated a set of NF-κB-activating genetic elements and tested their oncogenic potential in rodent cell transformation models. Genetic elements with desired properties were isolated using biologically active selectable peptide technology, which involves functional screening of lentiviral libraries encoding 20 or 50 amino acid-long polypeptides supplemented with endoplasmic reticulum-targeting and oligomerization domains. Twelve NF-κB-activating selectable peptides (NASPs) representing specific fragments of six proteins, none of which was previously associated with NF-κB activation, were isolated from libraries of 200,000 peptides derived from 500 human extracellular proteins. Using selective knockdown of distinct components of the NF-κB pathway, we showed that the isolated NASPs act either via or upstream of TNF receptor-associated factor 6. Transduction of NASPs into mouse and rat embryo fibroblasts did not, in itself, alter their growth. However, when coexpressed with oncogenic Ras (H-Ras(V12)), NASPs allowed rodent fibroblasts to overcome H-Ras(V12)-mediated p53-dependent senescence and acquire a transformed tumorigenic phenotype. Consistent with their ability to cooperate with oncogenic Ras in cell transformation, NASP expression reduced the transactivation activity of p53. This system provides an in vitro model of NF-κB-driven carcinogenesis and suggests that the known carcinogenic effects of inflammation may be at least partially due to NF-κB-mediated abrogation of oncogene-induced senescence.
Most common systems of genetic engineering of mammalian cells are associated with insertional mutagenesis of the modified cells. Insertional mutagenesis is also a popular approach to generate random ...alterations for gene discovery projects. A better understanding of the interaction of the structural elements within an insertional mutagen and the ability of such elements to influence host genes at various distances away from the insertion site is a matter of considerable practical importance.
We observed that, in the context of a lentiviral construct, a transcript, which is initiated at an internal CMV promoter/enhancer region and incorporates a splice donor site, is able to extend past a collinear viral LTR and trap exons of host genes, while the polyadenylation signal, which is naturally present in the LTR, is spliced out. Unexpectedly, when a vector, which utilizes this phenomenon, was used to produce mutants with elevated activity of NF-κB, we found mutants, which owed their phenotype to the effect of the insert on a gene located tens or even hundreds of kilobases away from the insertion site. This effect did not result from a CMV-driven transcript, but was sensitive to functional suppression of the insert. Interestingly, despite the long-distance effect, expression of loci most closely positioned to the insert appeared unaffected.
We concluded that a polyadenylation signal in a retroviral LTR, when occurring within an intron, is an inefficient barrier against the formation of a hybrid transcript, and that a vector containing a strong enhancer may selectively affect the function of genes far away from its insertion site. These phenomena have to be considered when experimental or therapeutic transduction is performed. In particular, the long-distance effects of insertional mutagenesis bring into question the relevance of the lists of disease-associated retroviral integration targets, which did not undergo functional validation.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The mechanisms underlying tyrosine kinase inhibitor (TKI) resistance in chronic myeloid leukemia (CML) patients lacking explanatory BCR-ABL1 kinase domain mutations are incompletely understood. To ...identify mechanisms of TKI resistance that are independent of BCR-ABL1 kinase activity, we introduced a lentiviral short hairpin RNA (shRNA) library targeting ∼5000 cell signaling genes into K562R, a CML cell line with BCR-ABL1 kinase-independent TKI resistance expressing exclusively native BCR-ABL1. A customized algorithm identified genes whose shRNA-mediated knockdown markedly impaired growth of K562R cells compared with TKI-sensitive controls. Among the top candidates were 2 components of the nucleocytoplasmic transport complex, RAN and XPO1 (CRM1). shRNA-mediated RAN inhibition or treatment of cells with the XPO1 inhibitor, KPT-330 (Selinexor), increased the imatinib sensitivity of CML cell lines with kinase-independent TKI resistance. Inhibition of either RAN or XPO1 impaired colony formation of CD34+ cells from newly diagnosed and TKI-resistant CML patients in the presence of imatinib, without effects on CD34+ cells from normal cord blood or from a patient harboring the BCR-ABL1T315I mutant. These data implicate RAN in BCR-ABL1 kinase-independent imatinib resistance and show that shRNA library screens are useful to identify alternative pathways critical to drug resistance in CML.
•A function-first shRNA library screen identifies pathways involved in BCR-ABL1 kinase-independent TKI resistance.•RAN or XPO1 inhibition impairs survival of progenitors from newly diagnosed or TKI-resistant CML patients.
Advances in the fields of cancer initiating cells and high-throughput in vivo shRNA screens have highlighted a need to observe the growth of tumor cells in cancer models at the clonal level. While in ...vivo cancer cell growth heterogeneity in xenografts has been described, it has yet to be measured. Here, we tested an approach to quantify the clonal growth heterogeneity of cancer cells in subcutaneous xenograft mouse models. Using a high-throughput sequencing method, we followed the fate in vitro and in vivo of ten thousand HCT-116 cells individually tagged with a unique barcode delivered by lentiviral transduction. While growth in vitro was less homogeneous than anticipated, we still find that 95% of the final cells derived from 80% of the original cells. In xenografts, however, 95% of the retrieved barcoded cells originated from only 6% of the initially injected cells, an effect we term "clonal dominance". We observed this clonal dominance in two additional xenograft models (MDA-MB-468 and A2780(cis)) and in two different host strains (NSG and Nude). By precisely and reproducibly quantifying clonal cancer cell growth in vivo, we find that a small subset of clones accounts for the vast majority of the descendant cells, even with HCT-116, a cell line reported to lack a tumor-initiating compartment. The stochastic in vivo selection process we describe has important implications for the fields of in vivo shRNA screening and tumor initiating cells.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
RNAi loss-of-function screens, which have proven effective to identify genes functionally responsible for cellular phenotypes, can be designed to use different genetic backgrounds or altered ...environmental conditions to elucidate genetic dependencies. These sorts of screening approaches can be exploited to identify genetic targets that minimize resistance to approved drugs, and provide a basis on which to develop new targeted therapies and predict the secondary targets for combinatorial treatments. Four types of pooled short hairpin RNA (shRNA) screens, in particular, have been used to look for genetic targets that work together with known drugs or other anticancer targets, either in an additive or synergistic fashion. Each method produces results that provide a useful but limited picture of the genetic elements driving oncogenesis.
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