Alex Chenchik

p53 cooperates with DNA methylation and a suicidal interferon response to maintain epigenetic silencing of repeats and noncoding RNAs

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.

Specific pattern of p53 phosphorylation during nitric oxide-induced cell cycle arrest

Nitric oxide (NO) is an efficient inhibitor of cell proliferation. Here we show that part of the antiproliferative activity of NO in fibroblasts is mediated through p53 signaling pathway. Cells from p53−/− knockout mice are compromised in their ability to stop dividing in the presence of NO. NO strongly induces expression of genes which are transcriptional targets of p53, and p53 is necessary for some, but not all, of the transcription activation effects of NO. Furthermore, NO strongly increases the cellular level of p53 protein. Since phosphorylation of particular residues of the p53 molecule has been correlated with its functional activity, we determined the phosphorylation pattern of p53 molecule after exposure to NO and compared it with the phosphorylation patterns that develop upon treatment with gamma-irradiation, UV light, and adriamycin. We found that NO induces a specific signature pattern of p53 phosphorylation, distinct from the patterns evoked by other inducers. This study suggests that NO activates specific signaling pathways that may partially overlap, but that do not coincide, with signaling pathways activated by other known inducers of p53 activity.

Amplification of cDNA ends using PCR suppression effect and step-out PCR.

From: Generation of cDNA libraries: Methods and protocols (Shao-Yao Ying Ed.) Humana press, pp. 41-50.

Long-Distance Effects of Insertional Mutagenesis

Background 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. Methodology/Principal Findings 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. Conclusions/Significance 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.

Optimization of a Genome-Wide Disordered Lentivector-Based Short Hairpin RNA Library

To obtain a whole genome library that suppresses the total diversity of human mRNAs, lentiviral vector constructs and a short hairpin RNA (shRNA) expression cassette were optimized. The optimization of the vector increased the virus titer in preparations by 15–20 times. A simple shRNA structure with a 21-bp stem proved to be the most effective. Lentivector-based shRNA expression constructs were obtained by using puroR, copGFP, or H-2Kk as a selectable marker. The efficiency of the optimized library was demonstrated when screening for shRNAs reactivating the tumor suppressor p53 in HeLa cells. Cells carried a reporter construct ensuring p53-responsive synthesis of a fluorescent protein, which allowed selection of cells with reactivated p53 by flow cytometry.

Peptides genetically selected for NF-κB activation cooperate with oncogene Ras and model carcinogenic role of inflammation

Significance Despite general acceptance of the link between chronic inflammation and cancer, the precise molecular mechanisms underlying the cancer-promoting effects of inflammation remain undefined. Inducible transcription factor NF-κB is the key regulator of inflammation, which is commonly deregulated in cancer cells to become constitutively active. Whether this deregulation contributes to malignant transformation was the main question addressed in this study. We isolated a series of genetic elements encoding artificial intracellular proteins capable of constitutive activation of NF-κB, named NF-κB–activating selectable peptides (NASPs), and demonstrated that all of them had carcinogenic activity in conventional cellular models. Specifically, NASPs made normal rodent cells susceptible to malignant transformation by oncogene Ras, which cannot do it on its own. This result defines chronically active NF-κB as an oncogene. 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-RasV12), NASPs allowed rodent fibroblasts to overcome H-RasV12–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.

Use of RNAi screens to uncover resistance mechanisms in cancer cells and identify synthetic lethal interactions.

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.

Screening of cell death genes with a mammalian genome-wide RNAi library

We report the construction and application of a mammalian genome-wide RNAi library. The oligodeoxynucleotides encoding approximately 200,000 shRNA sequences that targeted 47,400 human transcripts were inserted into a lentivirus vector pFIV-H1-puro, and a pool of pseudovirus particles with a complexity of approximately 200,000 were used to infect target cells. From the cells surviving apoptogenic Fas stimulation, four candidate shRNA sequences were obtained that provided resistance to Fas-induced cell death, including two shRNAs for caspase-8, an shRNA for Bid, and an shRNA for Fas. The reconstructed shRNAs with these sequences were shown to reduce expression of the respective gene products and increase survival after Fas stimulation. When similar selection was performed for tunicamycin-induced apoptosis, no shRNA strongly inhibiting tunicamycin-induced cell death was isolated, although a few reconstructed shRNAs led to a slight increase of survival. Thus, this genome-wide shRNA library proved useful for selection of genes that are involved in cell death, but some limitation was also revealed.

Construction of cDNA Libraries from Small Quantities of Total RNA Using Template Switching Catalyzed by M-MLV Reverse Transcriptase

Generation of high quality cDNA libraries with a comprehensive representation of the original mRNA population requires relatively large amounts (5–50 μg) of poly ( A)+RNA which is difficult to obtain when the amount of biological material is limited ( e.g., rare and unstable cell lines, microdissected cancer cells, biopsy materials, pathological specimens, embryonic and neuron tissues, cells in body fluids and so on). To circumvent this problem, several PCR-based technologies for amplification of cDNA from small amounts of total RNA have been described (Froussard 1993; Bertioli et al. 1994; Korneev et al. 1994). Basically, the amplification of a total cDNA population requires that universal primer binding sites are available at both cDNA ends. An arbitrary sequence can easily be imposed at the 5′ cDNA end by priming reverse transcription from the poly (A)+RNA fraction of total RNA by oligo (dT) or anchored oligo (dT) primer. Several strategies have been developed to add a determined sequence (anchor) at the 3′ end of the first-strand cDNA. These strategies include: (1) oligo (dG) or oligo (dA) tailing by terminal deoxynu- cleotidyltransferase (Bertioli et al. 1994; Korneev et al. 1994); ( 2) the use of T4 RNA ligase to covalently attach a single-stranded (ss) anchor oligonucleotide to the 3′ end of the ss cDNA (Apte and Siebert 1993); (3) the ligation of double-stranded (ds) adaptors to both ends of the ds cDNA (Frohman et al. 1988); and (4) the removal of the 7-MeGppp cap structure followed by ligation of an anchor sequence to the 5′ end of the decapped mRNA by T4 RNA ligase (Fromont-Racine et al. 1993).

Abstract A059: Targeted RNA expression profiling for biomarker discovery in complex biologic samples

New rapid and robust transcriptome-based methods for cellular characterization of the tumor microenvironment and biomarker discovery are required to improve prognosis and treatment of cancer and other diseases. However, challenges with current approaches for the above applications include high sample requirements, poor sensitivity, low dynamic range, and limited throughput. To address these limitations, we have developed the DriverMap targeted RNA expression profiling assay using a genome-wide set of 19,000 validated primer pairs that leverages the sensitivity of multiplex RT-PCR with the throughput and digital readout depth of next-generation sequencing (NGS). Starting from just 10pg (single-cell) to 100ng (10,000 cells) of total RNA is sufficient to quantify over 5 orders of magnitude variation in gene expression levels with performance similar to conventional qRT-PCR. Further, the use of gene-specific primers enables direct analysis of total RNA isolate and obviates the need for globin and rRNA depletion from whole blood samples. In this study, we present the performance of the assay for immunophenotyping of immune cells in whole blood samples from sepsis patients and assess the immune responses to complex immunomodulatory stimuli in an ex vivo model system. We will also present profiling results that demonstrate how this assay can be used to analyze the level of immune cell infiltration in tumor samples, and identify active pathways in tumor and xenograft samples. Preliminary studies demonstrate the assay’s unparalleled specificity and sensitivity, resulting in better detection of low-abundance mRNA transcripts as well as an improved cost-effectiveness for high-throughput clinical applications. Citation Format: Alex Chenchik, Costa Frangou, Mikhail Makhanov. Targeted RNA expression profiling for biomarker discovery in complex biologic samples [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A059.