The Argonaute CSR-1 and its 22G-RNA cofactors are required for holocentric chromosome segregation.

RNAi-related pathways regulate diverse processes, from developmental timing to transposon silencing. Here, we show that in C. elegans the Argonaute CSR-1, the RNA-dependent RNA polymerase EGO-1, the Dicer-related helicase DRH-3, and the Tudor-domain protein EKL-1 localize to chromosomes and are required for proper chromosome segregation. In the absence of these factors chromosomes fail to align at the metaphase plate and kinetochores do not orient to opposing spindle poles. Surprisingly, the CSR-1-interacting small RNAs (22G-RNAs) are antisense to thousands of germline-expressed protein-coding genes. Nematodes assemble holocentric chromosomes in which continuous kinetochores must span the expressed domains of the genome. We show that CSR-1 interacts with chromatin at target loci but does not downregulate target mRNA or protein levels. Instead, our findings support a model in which CSR-1 complexes target protein-coding domains to promote their proper organization within the holocentric chromosomes of C. elegans.

Incorporation of aptamers in the terminal loop of shRNAs yields an effective and novel combinatorial targeting strategy.

Gene therapy by engineering patient's own blood cells to confer HIV resistance can potentially lead to a functional cure for AIDS. Toward this goal, we have previously developed an anti-HIV lentivirus vector that deploys a combination of shRNA, ribozyme and RNA decoy. To further improve this therapeutic vector against viral escape, we sought an additional reagent to target HIV integrase. Here, we report the development of a new strategy for selection and expression of aptamer for gene therapy. We developed a SELEX protocol (multi-tag SELEX) for selecting RNA aptamers against proteins with low solubility or stability, such as integrase. More importantly, we expressed these aptamers in vivo by incorporating them in the terminal loop of shRNAs. This novel strategy allowed efficient expression of the shRNA-aptamer fusions that targeted RNAs and proteins simultaneously. Expressed shRNA-aptamer fusions targeting HIV integrase or reverse transcriptase inhibited HIV replication in cell cultures. Viral inhibition was further enhanced by combining an anti-integrase aptamer with an anti-HIV Tat-Rev shRNA. This construct exhibited efficacy comparable to that of integrase inhibitor Raltegravir. Our strategy for the selection and expression of RNA aptamers can potentially extend to other gene therapy applications.

HIV-1 RRE RNA acts as an RNA silencing suppressor by competing with TRBP-bound siRNAs.

Several proteins and RNAs expressed by mammalian viruses have been reported to interfere with RNA interference (RNAi) activity. We investigated the ability of the HIV-1-encoded RNA elements Trans-Activation Response (TAR) and Rev-Response Element (RRE) to alter RNAi. MicroRNA let7-based assays showed that RRE is a potent suppressor of RNAi activity, while TAR displayed moderate RNAi suppression. We demonstrate that RRE binds to TAR-RNA Binding Protein (TRBP), an essential component of the RNA Induced Silencing Complex (RISC). The binding of TAR and RRE to TRBP displaces small interfering (si)RNAs from binding to TRBP. Several stem-deleted RRE mutants lost their ability to suppress RNAi activity, which correlated with a reduced ability to compete with siRNA-TRBP binding. A lentiviral vector expressing TAR and RRE restricted RNAi, but RNAi was restored when Rev or GagPol were coexpressed. Adenoviruses are restricted by RNAi and encode their own suppressors of RNAi, the Virus-Associated (VA) RNA elements. RRE enhanced the replication of wild-type and VA-deficient adenovirus. Our work describes RRE as a novel suppressor of RNAi that acts by competing with siRNAs rather than by disrupting the RISC. This function is masked in lentiviral vectors co-expressed with viral proteins and thus will not affect their use in gene therapy. The potent RNAi suppressive effects of RRE identified in this study could be used to enhance the expression of RNAi restricted viruses used in oncolysis such as adenoviruses.

Nuclease-free precise genome editing corrects MECP2 mutations associated with Rett syndrome.

Rett syndrome is an acquired progressive neurodevelopmental disorder caused by de novo mutations in the X-linked MECP2 gene which encodes a pleiotropic protein that functions as a global transcriptional regulator and a chromatin modifier. Rett syndrome predominantly affects heterozygous females while affected male hemizygotes rarely survive. Gene therapy of Rett syndrome has proven challenging due to a requirement for stringent regulation of expression with either over- or under-expression being toxic. Ectopic expression of MECP2 in conjunction with regulatory miRNA target sequences has achieved some success, but the durability of this approach remains unknown. Here we evaluated a nuclease-free homologous recombination (HR)-based genome editing strategy to correct mutations in the MECP2 gene. The stem cell-derived AAVHSCs have previously been shown to mediate seamless and precise HR-based genome editing. We tested the ability of HR-based genome editing to correct pathogenic mutations in Exons 3 and 4 of the MECP2 gene and restore the wild type sequence while preserving all native genomic regulatory elements associated with MECP2 expression, thus potentially addressing a significant issue in gene therapy for Rett syndrome. Moreover, since the mutations are edited directly at the level of the genome, the corrections are expected to be durable with progeny cells inheriting the edited gene. The AAVHSC MECP2 editing vector was designed to be fully homologous to the target MECP2 region and to insert a promoterless Venus reporter at the end of Exon 4. Evaluation of AAVHSC editing in a panel of Rett cell lines bearing mutations in Exons 3 and 4 demonstrated successful correction and rescue of expression of the edited MECP2 gene. Sequence analysis of edited Rett cells revealed successful and accurate correction of mutations in both Exons 3 and 4 and permitted mapping of HR crossover events. Successful correction was observed only when the mutations were flanked at both the 5' and 3' ends by crossover events, but not when both crossovers occurred either exclusively upstream or downstream of the mutation. Importantly, we concluded that pathogenic mutations were successfully corrected in every Rett line analyzed, demonstrating the therapeutic potential of HR-based genome editing.

The Role of Recombinant AAV in Precise Genome Editing.

The replication-defective, non-pathogenic, nearly ubiquitous single-stranded adeno-associated viruses (AAVs) have gained importance since their discovery about 50 years ago. Their unique life cycle and virus-cell interactions have led to the development of recombinant AAVs as ideal genetic medicine tools that have evolved into effective commercialized gene therapies. A distinctive property of AAVs is their ability to edit the genome precisely. In contrast to all current genome editing platforms, AAV exclusively utilizes the high-fidelity homologous recombination (HR) pathway and does not require exogenous nucleases for prior cleavage of genomic DNA. Together, this leads to a highly precise editing outcome that preserves genomic integrity without incorporation of indel mutations or viral sequences at the target site while also obviating the possibility of off-target genotoxicity. The stem cell-derived AAV (AAVHSCs) were found to mediate precise and efficient HR with high on-target accuracy and at high efficiencies. AAVHSC editing occurs efficiently in post-mitotic cells and tissues in vivo. Additionally, AAV also has the advantage of an intrinsic delivery mechanism. Thus, this distinctive genome editing platform holds tremendous promise for the correction of disease-associated mutations without adding to the mutational burden. This review will focus on the unique properties of direct AAV-mediated genome editing and their potential mechanisms of action.

Activation of EPHA2-ROBO1 Heterodimer by SLIT2 Attenuates Non-canonical Signaling and Proliferation in Squamous Cell Carcinomas.

The tyrosine kinase receptor ephrin receptor A2 (EPHA2) is overexpressed in lung (LSCC) and head and neck (HNSCC) squamous cell carcinomas. Although EPHA2 can inhibit tumorigenesis in a ligand-dependent fashion via phosphorylation of Y588 and Y772, it can promote tumorigenesis in a ligand-independent manner via phosphorylation of S897. Here, we show that EPHA2 and Roundabout Guidance Receptor 1 (ROBO1) interact to form a functional heterodimer. Furthermore, we show that the ROBO1 ligand Slit Guidance Ligand 2 (SLIT2) and ensartinib, an inhibitor of EPHA2, can attenuate growth of HNSCC cells and act synergistically in LSCC cells. Our results suggest that patients with LSCC and HNSCC may be stratified and treated based on their EPHA2 and ROBO1 expression patterns. Although ~73% of patients with LSCC could benefit from SLIT2+ensartinib treatment, ~41% of patients with HNSCC could be treated with either SLIT2 or ensartinib. Thus, EPHA2 and ROBO1 represent potential LSCC and HNSCC theranostics.

SRC-1 and Wnt signaling act together to specify endoderm and to control cleavage orientation in early C. elegans embryos.

In early C. elegans embryos, signaling between a posterior blastomere, P2, and a ventral blastomere, EMS, specifies endoderm and orients the division axis of the EMS cell. Although Wnt signaling contributes to this polarizing interaction, no mutants identified to date abolish P2/EMS signaling. Here, we show that two tyrosine kinase-related genes, src-1 and mes-1, are required for the accumulation of phosphotyrosine between P2 and EMS. Moreover, src-1 and mes-1 mutants strongly enhance endoderm and EMS spindle rotation defects associated with Wnt pathway mutants. SRC-1 and MES-1 signal bidirectionally to control cell fate and division orientation in both EMS and P2. Our findings suggest that Wnt and Src signaling function in parallel to control developmental outcomes within a single responding cell.

EPHA2 mutations with oncogenic characteristics in squamous cell lung cancer and malignant pleural mesothelioma.

Squamous cell carcinoma (SCC) and malignant pleural mesothelioma (MPM) are thoracic malignancies with very poor prognosis and limited treatment options. It is an established fact that most of the solid tumors have overexpression of EPHA2 receptor tyrosine kinase. EPHA2 is known to exhibit opposing roles towards cancer progression. It functions in inhibiting cancer survival and migration via a ligand and tyrosine kinase dependent signaling (Y772). Whereas it is known to promote tumor progression and cell migration through a ligand-independent signaling (S897). We analyzed the expression profile and mutational status of the ephrin receptor A2 (EPHA2) in SCC and MPM cell lines and primary patient specimens. The EPHA2 receptor was found to be either overexpressed, mutated or amplified in SCC and MPM. In particular, the EPHA2 mutants A859D and T647M were interesting to explore, A859D Y772 dead mutant exhibited lower levels of phosphorylation at Y772 compared to T647M mutant. Molecular Dynamics simulations studies suggested that differential changes in conformation might form the structural basis for differences in the level of EPHA2 activation. Consequently, A859D mutant cells exhibited increased proliferation as well as cell migration compared to controls and T647M mutant. Kinomics analysis demonstrated that the STAT3 and PDGF pathways were upregulated whereas signaling through CBL was suppressed. Considered together, the present work has uncovered the oncogenic characteristics of EPHA2 mutations in SSC and MPM reinstating the dynamics of different roles of EPHA2 in cancer. This study also suggests that a combination of doxazosin and other EPHA2 inhibitors directed to inhibit the pertinent signaling components may be a novel therapeutic strategy for MPM and Non-small cell lung cancer patients who have either EPHA2 or CBL alterations.

Inhibiting crosstalk between MET signaling and mitochondrial dynamics and morphology: a novel therapeutic approach for lung cancer and mesothelioma.

The receptor tyrosine kinase MET is frequently involved in malignant transformation and inhibiting its activity in MET-dependent cancers is associated with improved clinical outcomes. Emerging evidence also suggests that mitochondria play an essential role in tumorigenesis and Dynamin Related Protein (DRP1), a key component of the mitochondrial fission machinery, has emerged as an attractive therapeutic target. Here, we report that inhibiting MET activity with the tyrosine kinase inhibitor MGCD516 attenuates viability, migration, and invasion of non-small cell lung cancer (NSCLC) and malignant pleural mesothelioma (MPM) cell lines in vitro, and significantly retards tumor growth in vivo. Interestingly, MGCD516 treatment also results in altered mitochondrial morphology in these cell lines. Furthermore, inhibiting MET pharmacologically or knocking down its expression using siRNA, decreases DRP1 activity alluding to possible crosstalk between them in these two cancers. Consistently, a combination of MGCD516 and mdivi-1, a quinazolinone reported to inhibit mitochondrial fission, is more effective in attenuating proliferation of NSCLC and MPM cell lines than either drug alone. Considered together, the present study has uncovered a novel mechanism underlying mitochondrial regulation by MET that involves crosstalk with DRP1, and suggests that a combination therapy targeting both MET and DRP1 could be a novel strategy for NSCLC and MPM.

Analysis of the C. elegans Argonaute family reveals that distinct Argonautes act sequentially during RNAi.

Argonaute (AGO) proteins interact with small RNAs to mediate gene silencing. C. elegans contains 27 AGO genes, raising the question of what roles these genes play in RNAi and related gene-silencing pathways. Here we describe 31 deletion alleles representing all of the previously uncharacterized AGO genes. Analysis of single- and multiple-AGO mutant strains reveals functions in several pathways, including (1) chromosome segregation, (2) fertility, and (3) at least two separate steps in the RNAi pathway. We show that RDE-1 interacts with trigger-derived sense and antisense RNAs to initiate RNAi, while several other AGO proteins interact with amplified siRNAs to mediate downstream silencing. Overexpression of downstream AGOs enhances silencing, suggesting that these proteins are limiting for RNAi. Interestingly, these AGO proteins lack key residues required for mRNA cleavage. Our findings support a two-step model for RNAi, in which functionally and structurally distinct AGOs act sequentially to direct gene silencing.

The minibrain kinase homolog, mbk-2, is required for spindle positioning and asymmetric cell division in early C. elegans embryos.

In the newly fertilized Caenorhabditis elegans zygote, cytoplasmic determinants become localized asymmetrically along the anterior-posterior (A-P) axis of the embryo. The mitotic apparatus then orients so as to cleave the embryo into anterior and posterior blastomeres that differ in both size and developmental potential. Here we describe a role for MBK-2, a member of the Dyrk family of protein kinases, in asymmetric cell division in C. elegans. In mbk-2 mutants, the initial mitotic spindle is misplaced and cytoplasmic factors, including the germline-specific protein PIE-1, are mislocalized. Our findings support a model in which MBK-2 down-regulates the katanin-related protein MEI-1 to control spindle positioning and acts through distinct, as yet unknown factors, to control the localization of cytoplasmic determinants. These findings in conjunction with work from Schizosaccharomyces pombe indicate a possible conserved role for Dyrk family kinases in the regulation of spindle placement during cell division.