Nucleases that cleave unique genomic sequences in living cells can be used for targeted gene editing and mutagenesis. Here we develop a strategy for generating such reagents based on transcription activator-like effector (TALE) proteins from Xanthomonas. We identify TALE truncation variants that efficiently cleave DNA when linked to the catalytic domain of FokI and use these nucleases to generate discrete edits or small deletions within endogenous human NTF3 and CCR5 genes at efficiencies of up to 25%. We further show that designed TALEs can regulate endogenous mammalian genes. These studies demonstrate the effective application of designed TALE transcription factors and nucleases for the targeted regulation and modification of endogenous genes.

Pancreatic cancers arise predominantly from ductal epithelial cells of the exocrine pancreas and are of the ductal adenocarcinoma histological subtype (PDAC). PDAC is an aggressive disease associated with a poor clinical prognosis, weakly effective therapeutic options, and a lack of early detection methods. Furthermore, the genetic and phenotypic heterogeneity of PDAC complicates efforts to identify universally efficacious therapies. PDACs commonly harbor activating mutations in the KRAS oncogene, which is a potent driver of tumor initiation and maintenance. Inactivating mutations in tumor suppressor genes such as CDKN2A/p16, TP53 and SMAD4 cooperate with KRAS mutations to cause aggressive PDAC tumor growth. PDAC can be classified into 3-4 molecular subtypes by global gene expression profiling. These subtypes can be distinguished by distinct molecular and phenotypic characteristics. This chapter will provide an overview of the current knowledge of PDAC pathogenesis at the genetic and molecular level as well as novel therapeutic opportunities to treat this highly aggressive disease.

Zinc-finger nucleases (ZFNs) drive highly efficient genome editing by generating a site-specific DNA double-strand break (DSB) at a predetermined site in the genome. Subsequent repair of this break via the nonhomologous end-joining (NHEJ) or homology-directed repair (HDR) pathways results in targeted gene disruption or gene addition, respectively. Here, we report that ZFNs can be engineered to induce a site-specific DNA single-strand break (SSB) or nick. Using the CCR5-specific ZFNs as a model system, we show that introduction of a nick at this target site stimulates gene addition using a homologous donor template but fails to induce significant levels of the small insertions and deletions (indels) characteristic of repair via NHEJ. Gene addition by these CCR5-targeted zinc finger nickases (ZFNickases) occurs in both transformed and primary human cells at efficiencies of up to ∼1%–8%. Interestingly, ZFNickases targeting the AAVS1 “safe harbor” locus revealed similar in vitro nicking activity, a marked reduction of indels characteristic of NHEJ, but stimulated far lower levels of gene addition—suggesting that other, yet to be identified mediators of nick-induced gene targeting exist. Introduction of site-specific nicks at distinct endogenous loci provide an important tool for the study of DNA repair. Moreover, the potential for a SSB to direct repair pathway choice (i.e., HDR but not NHEJ) may prove advantageous for certain therapeutic applications such as the targeted correction of human disease-causing mutations.

HIV-1-infected individuals can harbor viral isolates that can use CCR5, as well as CXCR4, for viral entry. To genetically engineer HIV-1 resistance in CD4(+) T cells, we assessed whether transient, adenovirus delivered zinc-finger nuclease (ZFN) disruption of genomic cxcr4 or stable lentiviral expression of short hairpin RNAs (shRNAs) targeting CXCR4 mRNAs provides durable resistance to HIV-1 challenge. ZFN-modification of cxcr4 in CD4(+) T cells was found to be superior to cell integrated lentivirus-expressing CXCR4 targeting shRNAs when CD4(+) T cells were challenged with HIV-1s that utilizes CXCR4 for entry. Cxcr4 disruption in CD4(+) T cells was found to be stable, conferred resistance, and provided for continued cell enrichment during HIV-1 infection in tissue culture and, in vivo, in peripheral blood mononuclear cell transplanted NSG mice. Moreover, HIV-1-infected mice with engrafted cxcr4 ZFN-modified CD4(+) T cells demonstrated lower viral levels in contrast to mice engrafted with unmodified CD4(+) T cells. These findings provide evidence that ZFN-mediated disruption of cxcr4 provides a selective advantage to CD4(+) T cells during HIV-1 infection.

HIV-1 entry can be inhibited by soluble peptides from the gp41 heptad repeat-2 (HR2) domain that interfere with formation of the 6-helix bundle during fusion. Inhibition has also been seen when these peptides are conjugated to anchoring molecules and over-expressed on the cell surface. We hypothesized that potent anti-HIV activity could be achieved if a 34 amino acid peptide from HR2 (C34) were brought to the site of virus-cell interactions by conjugation to the amino termini of HIV-1 coreceptors CCR5 or CXCR4. C34-conjugated coreceptors were expressed on the surface of T cell lines and primary CD4 T cells, retained the ability to mediate chemotaxis in response to cognate chemokines, and were highly resistant to HIV-1 utilization for entry. Notably, C34-conjugated CCR5 and CXCR4 each exhibited potent and broad inhibition of HIV-1 isolates from diverse clades irrespective of tropism (i.e., each could inhibit R5, X4 and dual-tropic isolates). This inhibition was highly specific and dependent on positioning of the peptide, as HIV-1 infection was poorly inhibited when C34 was conjugated to the amino terminus of CD4. C34-conjugated coreceptors could also inhibit HIV-1 isolates that were resistant to the soluble HR2 peptide inhibitor, enfuvirtide. When introduced into primary cells, CD4 T cells expressing C34-conjugated coreceptors exhibited physiologic responses to T cell activation while inhibiting diverse HIV-1 isolates, and cells containing C34-conjugated CXCR4 expanded during HIV-1 infection in vitro and in a humanized mouse model. Notably, the C34-conjugated peptide exerted greater HIV-1 inhibition when conjugated to CXCR4 than to CCR5. Thus, antiviral effects of HR2 peptides can be specifically directed to the site of viral entry where they provide potent and broad inhibition of HIV-1. This approach to engineer HIV-1 resistance in functional CD4 T cells may provide a novel cell-based therapeutic for controlling HIV infection in humans.

KRAS mutant non-small cell lung cancer (NSCLC) may be classified into epithelial or mesenchymal subtypes. Mesenchymal NSCLCs and associated “KM” cell lines are generally less responsive than their epithelial counterparts to inhibition of the RAS pathway; identifying alternative networks that promote mesenchymal NSCLC survival may lead to more effective treatments for this subtype. Non-coding microRNA (miRNA) signatures can stratify tumors into diverse molecular subtypes. By regulating numerous targets in cancer-associated signaling pathways, miRNAs can function as tumor suppressors or oncogenes. In particular, some miRNAs regulate the epithelial-mesenchymal transition (EMT). Here, we derived an EMT-related miRNA signature by profiling the abundance of miRNAs in a panel of epithelial (KE) or mesenchymal (KM) KRAS mutant NSCLC cell lines. This signature revealed a number of silenced or suppressed miRNAs in KM cell lines, including members of the miR-200 family, which can promote tumor suppression by inhibiting EMT. Reconstituting KM cells with one of these miRNAs, miR-124, disrupted autophagy and decreased cell survival by suppressing the abundance of p62, an adaptor for selective autophagy and regulator of the transcription factor NF-κB. Suppression of p62 by miR-124 correlated with reduced abundance of the autophagy activator Beclin 1, the ubiquitin ligase TRAF6 and the NF-κB subunit RELA/p65. Abundance of miR-124 inversely correlated with expression of BECN1 and TRAF6 in patient NSCLC samples. These findings identify a role for miR-124 in regulating cell survival networks in a specific subtype of KRAS mutant NSCLC cell lines, which might lead to improved subtype-selective therapeutic strategies for patients.

Background KRAS mutant non-small cell lung cancers (NSCLC) are molecularly and histologically diverse. Epithelial-like cells are more KRAS dependent, whereas mesenchymal-like cells are less KRAS dependent. These two subtypes are designated KE (epithelial) and KM (mesenchymal), respectively. A KE versus KM subtype transcriptional signature reveals specific modes of KRAS dependent survival signaling in the KE subtype. This KRAS dependency signature is significantly enriched with predicted microRNA (miRNA) target genes of miR-205 and miR-34b/c. MiRNAs can function as tumor suppressors by coordinately regulating multiple oncogenic signaling pathways. The role of deregulated miRNA function in mediating the survival of KM versus KE NSCLC cells has not been investigated to date. Methods Differential miRNA expression in KRAS mutant cell lines was determined using Taqman low-density qPCR arrays (TLDA). Functional miRNA reconstitution experiments of downregulated miRNAs were performed in a panel of KM cell lines. Effects on apoptosis and autophagy were performed by Western blotting, immunofluorescence and live cell microscopy and caspase assays. The molecular targets of miR-124 were computationally identified by TargetScan or miRWalk and experimentally verified using 3’UTR luciferase-based assays. Functional rescue of miRNA-dependent cell viability defects was determined by ectopic predicted target gene expression. Results Comparison of KE to KM cells yielded a KE-KM miRNA subtype classifier/signature. This signature revealed a number of silenced or suppressed miRNAs in KM cell lines, including members of the miR-200 family. MiR-200 and miR-205 reconstitution in KM cells modulated epithelial plasticity by Zeb1 protein suppression and increased E-cadherin levels. Reconstitution of miR-124, miR-625 and miR-518-3p in KM cells caused pronounced loss of cell viability. Furthermore, miR-124 caused autolysosome maturation defects. We identified SQSTM1/p62, TRAF6 and RELA/p65 as key predicted targets of miR-124. MiR-124 reconstitution in KM cells caused decreased p62, TRAF6 and p65 protein levels. The effect of miR-124 on p62 expression was verified using a Luciferase-p62-3’UTR reporter construct. Overexpression of p62 in KM cells rescued the cell viability defects caused by miR-124. Conclusion These studies implicate miR-124 as a context-dependent tumor suppressor miRNA in KM subtype cells. MiR-124 directly suppresses expression of SQSTM1/p62 to promote defects in autolysosome maturation. In parallel, miR-124 suppresses RELA/ p65 and in some cases, TRAF6, to alter expression levels of several cytokines. Thus, miR-124 coordinately regulates autophagy and inflammation to disrupt the finely-tuned balance between pro and anti-inflammatory signals, resulting in cytotoxic effects in a specific subtype of mesenchymal-like KRAS mutant NSCLC cells. Citation Format: Anita K. Mehta, Kevin Hua, William Whipple, Mihn-Thuy Nguyen, Rushika M. Perera, Johanns Haybaeck, Joanne Weidhass, Jeffrey Settleman, Anurag Singh. MiR-124 suppresses p62 and p65/NFkB to regulate autophagy, inflammation and cell death in KRAS mutant mesenchymal NSCLC cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2524. doi:10.1158/1538-7445.AM2017-2524

Submission• Abstract-GSI-Mehta-Anita.doc Would you like your abstract to be considered for an oral presentation (students and post docs only)? YesA KRAS dependency microRNA signature reveals a p62/Sqstm1-centered autophagy network regulated by mir-124.