Kaja Kostyrko

High-level transgene expression by homologous recombination-mediated gene transfer

Gene transfer and expression in eukaryotes is often limited by a number of stably maintained gene copies and by epigenetic silencing effects. Silencing may be limited by the use of epigenetic regulatory sequences such as matrix attachment regions (MAR). Here, we show that successive transfections of MAR-containing vectors allow a synergistic increase of transgene expression. This finding is partly explained by an increased entry into the cell nuclei and genomic integration of the DNA, an effect that requires both the MAR element and iterative transfections. Fluorescence in situ hybridization analysis often showed single integration events, indicating that DNAs introduced in successive transfections could recombine. High expression was also linked to the cell division cycle, so that nuclear transport of the DNA occurs when homologous recombination is most active. Use of cells deficient in either non-homologous end-joining or homologous recombination suggested that efficient integration and expression may require homologous recombination-based genomic integration of MAR-containing plasmids and the lack of epigenetic silencing events associated with tandem gene copies. We conclude that MAR elements may promote homologous recombination, and that cells and vectors can be engineered to take advantage of this property to mediate highly efficient gene transfer and expression.

STAT2/IRF9 directs a prolonged ISGF3-like transcriptional response and antiviral activity in the absence of STAT1.

Evidence is accumulating for the existence of a signal transducer and activator of transcription 2 (STAT2)/interferon regulatory factor 9 (IRF9)-dependent, STAT1-independent interferon alpha (IFNα) signalling pathway. However, no detailed insight exists into the genome-wide transcriptional regulation and the biological implications of STAT2/IRF9-dependent IFNα signalling as compared with interferon-stimulated gene factor 3 (ISGF3). In STAT1-defeicient U3C cells stably overexpressing human STAT2 (hST2-U3C) and STAT1-deficient murine embryonic fibroblast cells stably overexpressing mouse STAT2 (mST2-MS1KO) we observed that the IFNα-induced expression of 2′-5′-oligoadenylate synthase 2 (OAS2) and interferon-induced protein with tetratricopeptide repeats 1 (Ifit1) correlated with the kinetics of STAT2 phosphorylation, and the presence of a STAT2/IRF9 complex requiring STAT2 phosphorylation and the STAT2 transactivation domain. Subsequent microarray analysis of IFNα-treated wild-type (WT) and STAT1 KO cells overexpressing STAT2 extended our observations and identified ∼120 known antiviral ISRE-containing interferon-stimulated genes (ISGs) commonly up-regulated by STAT2/IRF9 and ISGF3. The STAT2/IRF9-directed expression profile of these IFN-stimulated genes (ISGs) was prolonged as compared with the early and transient response mediated by ISGF3. In addition, we identified a group of ‘STAT2/IRF9-specific’ ISGs, whose response to IFNα was ISGF3-independent. Finally, STAT2/IRF9 was able to trigger an antiviral response upon encephalomyocarditis virus (EMCV) and vesicular stomatitis Indiana virus (VSV). Our results further prove that IFNα-activated STAT2/IRF9 induces a prolonged ISGF3-like transcriptome and generates an antiviral response in the absence of STAT1. Moreover, the existence of ‘STAT2/IRF9-specific’ target genes predicts a novel role of STAT2 in IFNα signalling.

The unique role of STAT2 in constitutive and IFN-induced transcription and antiviral responses

In the canonical pathway of IFN-I-mediated signaling, phosphorylation of STAT1 and STAT2 leads to heterodimerization and interaction with IRF9. This complex, also known as IFN-stimulated gene factor 3 (ISGF3), then translocates into the nucleus and binds the IFN-I-stimulated response element (ISRE) leading to the activation of transcription of over 300 interferon stimulated genes (ISGs). In addition, STAT1 homodimers [known as γ-activated factor (GAF)] are formed and translocate to the nucleus, where they target genes containing the γ-activated sequence (GAS). The primary function of ISGF3 is to mediate a rapid and robust IFN-I activated response by regulating transient transcription of antiviral ISGs. This requires the quick assembly of ISGF3 from its pre-existing components STAT1, STAT2 and IRF9 and transport to the nucleus to bind ISRE-containing ISGs. The exact events that take place in formation, nuclear translocation and DNA-binding of active ISGF3 are still not clear. Over the years many studies have provided evidence for the existence of a multitude of alternative STAT2-containing (ISRE or GAS-binding) complexes involved in IFN-I signaling, emphasizing the importance of STAT2 in the regulation of specific IFN-I-induced transcriptional programs, independent of its involvement in the classical ISGF3 complex. This review describes the unique role of STAT2 in differential complex formation of unphosphorylated and phosphorylated ISGF3 components that direct constitutive and IFN-I-stimulated transcriptional responses. In addition, we highlight the existence of a STAT1-independent IFN-I signaling pathway, where STAT2/IRF9 can potentially substitute for the role of ISGF3 and offer a back-up response against viral infection.

Assays for DNA double-strand break repair by microhomology-based end-joining repair mechanisms

DNA double stranded breaks (DSBs) are one of the most deleterious types of DNA lesions. The main pathways responsible for repairing these breaks in eukaryotic cells are homologous recombination (HR) and non-homologous end-joining (NHEJ). However, a third group of still poorly characterized DSB repair pathways, collectively termed microhomology-mediated end-joining (MMEJ), relies on short homologies for the end-joining process. Here, we constructed GFP reporter assays to characterize and distinguish MMEJ variant pathways, namely the simple MMEJ and the DNA synthesis-dependent (SD)-MMEJ mechanisms. Transfection of these assay vectors in Chinese hamster ovary (CHO) cells and characterization of the repaired DNA sequences indicated that while simple MMEJ is able to mediate relatively efficient DSB repair if longer microhomologies are present, the majority of DSBs were repaired using the highly error-prone SD-MMEJ pathway. To validate the involvement of DNA synthesis in the repair process, siRNA knock-down of different genes proposed to play a role in MMEJ were performed, revealing that the knock-down of DNA polymerase θ inhibited DNA end resection and repair through simple MMEJ, thus favoring the other repair pathway. Overall, we conclude that this approach provides a convenient assay to study MMEJ-related DNA repair pathways.

A role for homologous recombination proteins in cell cycle regulation

Eukaryotic cells respond to DNA breaks, especially double-stranded breaks (DSBs), by activating the DNA damage response (DDR), which encompasses DNA repair and cell cycle checkpoint signaling. The DNA damage signal is transmitted to the checkpoint machinery by a network of specialized DNA damage-recognizing and signal-transducing molecules. However, recent evidence suggests that DNA repair proteins themselves may also directly contribute to the checkpoint control. Here, we investigated the role of homologous recombination (HR) proteins in normal cell cycle regulation in the absence of exogenous DNA damage. For this purpose, we used Chinese Hamster Ovary (CHO) cells expressing the Fluorescent ubiquitination-based cell cycle indicators (Fucci). Systematic siRNA-mediated knockdown of HR genes in these cells demonstrated that the lack of several of these factors alters cell cycle distribution, albeit differentially. The knock-down of MDC1, Rad51 and Brca1 caused the cells to arrest in the G2 phase, suggesting that they may be required for the G2/M transition. In contrast, inhibition of the other HR factors, including several Rad51 paralogs and Rad50, led to the arrest in the G1/G0 phase. Moreover, reduced expression of Rad51B, Rad51C, CtIP and Rad50 induced entry into a quiescent G0-like phase. In conclusion, the lack of many HR factors may lead to cell cycle checkpoint activation, even in the absence of exogenous DNA damage, indicating that these proteins may play an essential role both in DNA repair and checkpoint signaling.

MAR-Mediated transgene integration into permissive chromatin and increased expression by recombination pathway engineering

Untargeted plasmid integration into mammalian cell genomes remains a poorly understood and inefficient process. The formation of plasmid concatemers and their genomic integration has been ascribed either to non‐homologous end‐joining (NHEJ) or homologous recombination (HR) DNA repair pathways. However, a direct involvement of these pathways has remained unclear. Here, we show that the silencing of many HR factors enhanced plasmid concatemer formation and stable expression of the gene of interest in Chinese hamster ovary (CHO) cells, while the inhibition of NHEJ had no effect. However, genomic integration was decreased by the silencing of specific HR components, such as Rad51, and DNA synthesis‐dependent microhomology‐mediated end‐joining (SD‐MMEJ) activities. Genome‐wide analysis of the integration loci and junction sequences validated the prevalent use of the SD‐MMEJ pathway for transgene integration close to cellular genes, an effect shared with matrix attachment region (MAR) DNA elements that stimulate plasmid integration and expression. Overall, we conclude that SD‐MMEJ is the main mechanism driving the illegitimate genomic integration of foreign DNA in CHO cells, and we provide a recombination engineering approach that increases transgene integration and recombinant protein expression in these cells. Biotechnol. Bioeng. 2017;114: 384–396.

Inhibition of activin signaling in lung adenocarcinoma increases the therapeutic index of platinum chemotherapy

Inhibition of activin signaling enhances the efficacy and safety of platinum chemotherapy in lung adenocarcinoma models. Blocking activin actively treats cancer Platinum-based chemotherapy is a mainstay of treatment for lung cancer, but resistance to this therapy is a common problem, as are dose-limiting side effects, particularly kidney toxicity. To search for mechanisms that may contribute to treatment resistance, Marini et al. performed a whole-genome RNA interference screen and identified the activin pathway, which can be targeted. The authors demonstrated that inhibition of this pathway using a small molecule or a protein called follistatin can offer a dual benefit in that it potentiates the effects of platinum drugs in mouse models of cancer and also protects the animals from kidney damage. These findings suggest that activin inhibitors could be a valuable addition to platinum chemotherapy, enhancing the efficacy of treatment while also allowing the use of higher doses or longer periods of drug exposure. Resistance to platinum chemotherapy is a long-standing problem in the management of lung adenocarcinoma. Using a whole-genome synthetic lethal RNA interference screen, we identified activin signaling as a critical mediator of innate platinum resistance. The transforming growth factor–β (TGFβ) superfamily ligands activin A and growth differentiation factor 11 (GDF11) mediated resistance via their cognate receptors through TGFβ-activated kinase 1 (TAK1), rather than through the SMAD family of transcription factors. Inhibition of activin receptor signaling or blockade of activin A and GDF11 by the endogenous protein follistatin overcame this resistance. Consistent with the role of activin signaling in acute renal injury, both therapeutic interventions attenuated acute cisplatin-induced nephrotoxicity, its major dose-limiting side effect. This cancer-specific enhancement of platinum-induced cell death has the potential to dramatically improve the safety and efficacy of chemotherapy in lung cancer patients.

SPOT-007 Identifying novel combinatorial synthetic lethal vulnerabilities in kras-driven lung cancer

Introduction KRAS is one of the most frequently mutated genes in human cancer, but the efforts to target KRAS directly have thus far been unsuccessful, highlighting the need for alternative approaches. One promising strategy is to target KRAS through synthetic lethality. However, KRAS activates multiple effector pathways, suggesting that targeting one gene may not be sufficient to fully inhibit KRAS-driven oncogenesis. Therefore, targeting combinations of genes that together are synthetic lethal with KRAS may constitute a better therapeutic approach. Material and methods To discover novel combinatorial KRAS synthetic lethal genes, we used affinity purification/mass spectrometry (AP/MS), to systematically identify KRAS interacting proteins and construct a detailed map of protein-protein interactions centred on KRAS. Based on this network we designed a CRISPR library targeting pairwise combinations of KRAS-interacting genes. Using this library we simultaneously knocked-out pairs of 119 genes in two KRAS-driven non-small cell lung cancer (NSCLC) cell lines expressing Cas9. Knock-out of several gene pairs synergistically impaired growth of these cells, while the knock-out of each of the genes alone had no or little effect. We chose 20 most promising targets for further screening in vitro and in vivo in a panel of 5 KRAS mutant and 4 wild type NSCLC cell lines. We also selected six gene pairs that had the most synergistic effect on growth for individual validation in Cas9-expressing NSCLC cells and normal human bronchial epithelial cells (HBECs). The cells were cultured in 3D, which was shown to more faithfully recapitulate important aspects of cancer biology than cells grown as monolayers. Results and discussions Out of the six gene combinations, the simultaneous knock-out of one pair of genes, Rap1GDS1 and RhoA, selectively impaired sphere growth of KRAS-dependent lung cancer cells but had little effect on the growth of KRAS-independent cells or HBECs. Moreover the individual knock-out of these genes had no effect on 3D growth in any of the cell lines, suggesting that only the combination of these two genes is synthetically lethal with KRAS. We are now performing further validation in vivo. Finally, human relevance will be determined using patient-derived xenograft (PDX) models. Conclusion Combinatorial inhibition of Rap1GDS1 and RhoA appears to be synthetically lethal with mutated KRAS and therefore these genes may constitute attractive drug targets for the treatment of KRAS-dependent NSCLC and other KRAS-driven cancers.

Abstract PR12: A combined protein-protein interaction and genetic interaction map defines new and critical Kras effectors in non-small cell lung cancer

Despite intensive study, no drugs in clinical use specifically target KRAS-mutant tumors. Uncharacterized feedback mechanisms and parallel pathways have stymied the treatment of KRAS-mutant tumors with Raf and PI3K inhibitors, and the KRas protein itself does not easily accommodate binding of small-molecule inhibitors. These challenges demand more systematic and quantitative characterization of the physical and genetic relationships between Ras regulators and effectors. To that end, we used tandem affinity purification of Kras, Hras and Nras, their activated alleles and key proteins with known regulatory (GEFs, GAPs) or effector (Raf, RalGDS, RIN1/2) roles in both 293 cells and A549 NSCLC cells to generate a high-confidence protein-protein interaction (PPI) network. This map of 220 proteins and 1,400 physical interactions was used to design an sgRNA library with 10 guides/gene. This library was screened in Cas9-expressing A549 cells and grown for 14 days before analysis for dropout or enhanced representation of sgRNAs. Approximately 120 genes showed positive or negative growth effects. PPIs and genetic interactions (GIs) were cross-referenced with public PPI data and TCGA patient data to assemble a combined physical PPI and genetic map informed by cancer mutations. This map suggests many hypotheses for PPIs critical for growth control. This set was used to construct a sgRNA library covering 120 genes of probable relevance to the Ras pathway with ~60 “safe harbor” control sgRNAs. This library was screened in a two-cassette sgRNA system testing 14K pairwise genetic effects to identify quantitative changes in growth in A549 and H23 NSCLC lines. This screen showed >100 genetic interactions, which in conjunction with PPIs, identify coupling between the Raf/MEK/ERK kinase, Ral and Rap GTPase, RNA processing, and cell adhesion pathways. The screen identified new candidate effector pathways for cell adhesion, RNA processing, Rap GTPase regulation, and protein processing, including the RADIL, RGL, and RIN Kras effectors. Validation focused using the synthetic lethal interactions observed in the sgRNA screen to predict drug combinations showing drug synergy in A549 and H23 cells. Using 11-point dose titrations and isobologram analysis of drug combinations, we see strong synergy among PI3 kinase, Raf, and Erk inhibitors in these cells. Using the recently described Kras G12C inhibitor, expressed in H23 cells, we have validated that sgRNA deletion of the the key Kras effector for specific pathways including cell adhesion (RADIL), growth signaling (RAF), and endocytosis/ macropinocytosis (RIN) are affected and that use of the Kras inhibitor ARS-853 shows much reduced effects on specific Kras effector pathways in cells deleted for these effectors. These systematic data underscore the limitations of our current understanding of Kras-driven cancers, revealing new genetic vulnerabilities and target candidates. This abstract is also being presented as Poster A28. Citation Format: Marcus R. Kelly, Kyuho Han, Nancie Mooney, Edwin Jeng, Kaja Kostyrko, Alejandro Sweet-Cordero, Michael Bassik, Peter K. Jackson. A combined protein-protein interaction and genetic interaction map defines new and critical Kras effectors in non-small cell lung cancer [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr PR12.