Margret B. Einarson

Synthetic Lethal Screen of an EGFR-Centered Network to Improve Targeted Therapies

A targeted RNAi screen reveals potential targets for combination approaches to cancer treatment. Rationally Designing Combination Therapy Drug resistance is a problem in cancer treatment, making combination therapies common. However, all too often, resistance also develops to empirically developed combination therapies, or those combinations are generally cytotoxic and not selective for the cancer cells. Astsaturov et al. developed a library of candidate genes centered on the epidermal growth factor receptor (EGFR) and targeted these genes with silencing RNAs to identify candidate proteins that could be inhibited to reduce cancer cell viability in the presence of EGFR inhibitors. Cotreatment with EGFR inhibitors and clinically available drugs that inhibit the candidate proteins reduced tumor size in xenografts and cell viability of multiple cancer cell lines. These results suggest that this network-centered approach may be fruitful for development of rationally designed combination therapies. Intrinsic and acquired cellular resistance factors limit the efficacy of most targeted cancer therapeutics. Synthetic lethal screens in lower eukaryotes suggest that networks of genes closely linked to therapeutic targets would be enriched for determinants of drug resistance. We developed a protein network centered on the epidermal growth factor receptor (EGFR), which is a validated cancer therapeutic target, and used small interfering RNA screening to comparatively probe this network for proteins that regulate the effectiveness of both EGFR-targeted agents and nonspecific cytotoxic agents. We identified subnetworks of proteins influencing resistance, with putative resistance determinants enriched among proteins that interacted with proteins at the core of the network. We found that clinically relevant drugs targeting proteins connected in the EGFR network, such as protein kinase C or Aurora kinase A, or the transcriptional regulator signal transducer and activator of transcription 3 (STAT3), synergized with EGFR antagonists to reduce cell viability and tumor size, suggesting the potential for a direct path to clinical exploitation. Such a focused approach can potentially improve the coherent design of combination cancer therapies.

The docking protein HEF1 is an apoptotic mediator at focal adhesion sites.

ABSTRACT HEF1 (human enhancer of filamentation 1) is a member of a docking protein family that includes p130Cas and Efs. Through assembly of multiple protein interactions at focal adhesion sites, these proteins activate signaling cascades in response to integrin receptor binding of the extracellular matrix. The HEF1 protein is cell cycle regulated, with full-length forms cleaved in mitosis at a caspase consensus site to generate an amino-terminal 55-kDa form that localizes to the mitotic spindle. The identification of a caspase cleavage site in HEF1 led us to investigate whether HEF1 belongs to a select group of caspase substrates cleaved in apoptosis to promote the morphological changes characteristic of programmed cell death. Significantly, inducing expression of HEF1 in MCF-7 or HeLa cells causes extensive apoptosis, as assessed by multiple criteria. Endogenous HEF1 is cleaved into 65- and 55-kDa fragments and a newly detected 28-kDa form in response to the induction of apoptosis, paralleling cleavage of poly(ADP-ribose) polymerase and focal adhesion kinase (FAK); the death-promoting activity of over-expressed HEF1 is associated with production of the 28-kDa form. While the generation of the cleaved HEF1 forms is caspase dependent, the accumulation of HEF1 forms is further regulated by the proteasome, as the proteasome inhibitorsN-acetyl-l-leucinyl-l-leucinyl-l-norleucinyl and lactacystin enhance their stability. Finally, the induction of HEF1 expression also increases Jun N-terminal protein kinase (JNK) activation, and activated JNK colocalizes with HEF1, implicating this pathway in HEF1 action. Based on these results, we propose that dysregulation of HEF1 and its family members along with FAK may signal the destruction of focal adhesion sites and regulate the onset of apoptosis.

Identification of a Functional Network of Human Epigenetic Silencing Factors

Epigenetic silencing is mediated by families of factors that place, remove, read, and transmit repressive histone and DNA methylation marks on chromatin. How the roles for these functionally diverse factors are specified and integrated is the subject of intense study. To address these questions, HeLa cells harboring epigenetically silent green fluorescent protein reporter genes were interrogated with a small interference RNA library targeting 200 predicted epigenetic regulators, including potential activators, silencers, chromatin remodelers, and ancillary factors. Using this approach, individual, or combinatorial requirements for specific epigenetic silencing factors could be detected by measuring green fluorescent protein reactivation after small interference RNA-based factor knockdown. In our analyses, we identified a specific subset of 15 epigenetic factors that are candidates for participation in a functional epigenetic silencing network in human cells. These factors include histone deacetylase 1, de novo DNA methyltransferase 3A, components of the polycomb PRC1 complex (RING1 and HPH2), and the histone lysine methyltransferases KMT1E and KMT5C. Roles were also detected for two TRIM protein family members, the cohesin component Rad21, and the histone chaperone CHAF1A (CAF-1 p150). Remarkably, combinatorial knockdown of factors was not required for reactivation, indicating little functional redundancy. Consistent with this interpretation, knockdown of either KMT1E or CHAF1A resulted in a loss of multiple histone-repressive marks and concomitant gain of activation marks on the promoter during reactivation. These results reveal how functionally diverse factors may cooperate to maintain gene silencing during normal development or in disease. Furthermore, the findings suggest an avenue for discovery of new targets for epigenetic therapies.

Genome-wide siRNA screen reveals a new cellular partner of NK cell receptor KIR2DL4: heparan sulfate directly modulates KIR2DL4-mediated responses.

KIR2DL4 (CD158d) is a distinct member of the killer cell Ig-like receptor (KIR) family in human NK cells that can induce cytokine production and cytolytic activity in resting NK cells. Soluble HLA-G, normally expressed only by fetal-derived trophoblast cells, was reported to be a ligand for KIR2DL4; however, KIR2DL4 expression is not restricted to the placenta and can be found in CD56high subset of peripheral blood NK cells. We demonstrated that KIR2DL4 can interact with alternative ligand(s), expressed by cells of epithelial or fibroblast origin. A genome-wide high-throughput siRNA screen revealed that KIR2DL4 recognition of cell-surface ligand(s) is directly regulated by heparan sulfate (HS) glucosamine 3-O-sulfotransferase 3B1 (HS3ST3B1). KIR2DL4 was found to directly interact with HS/heparin, and the D0 domain of KIR2DL4 was essential for this interaction. Accordingly, exogenous HS/heparin can regulate cytokine production by KIR2DL4-expressing NK cells and HEK293T cells (HEK293T-2DL4), and induces differential localization of KIR2DL4 to rab5+ and rab7+ endosomes, thus leading to downregulation of cytokine production and degradation of the receptor. Furthermore, we showed that intimate interaction of syndecan-4 (SDC4) HS proteoglycan (HSPG) and KIR2DL4 directly affects receptor endocytosis and membrane trafficking.

An RNA Interference Lethality Screen of the Human Druggable Genome to Identify Molecular Vulnerabilities in Epithelial Ovarian Cancer

Targeted therapies have been used to combat many tumor types; however, few have effectively improved the overall survival in women with epithelial ovarian cancer, begging for a better understanding of this deadly disease and identification of essential drivers of tumorigenesis that can be targeted effectively. Therefore, we used a loss-of-function screening approach to help identify molecular vulnerabilities that may represent key points of therapeutic intervention. We employed an unbiased high-throughput lethality screen using a 24,088 siRNA library targeting over 6,000 druggable genes and studied their effects on growth and/or survival of epithelial ovarian cancer (EOC) cell lines. The top 300 “hits” affecting the viability of A1847 cells were rescreened across additional EOC cell lines and non-tumorigenic, human immortalized ovarian epithelial cell lines. Fifty-three gene candidates were found to exhibit effects in all tumorigenic cell lines tested. Extensive validation of these hits refined the list to four high quality candidates (HSPA5, NDC80, NUF2, and PTN). Mechanistic studies show that silencing of three genes leads to increased apoptosis, while HSPA5 silencing appears to alter cell growth through G1 cell cycle arrest. Furthermore, two independent gene expression studies show that NDC80, NUF2 and PTN were significantly aberrantly overexpressed in serous adenocarcinomas. Overall, our functional genomics results integrated with the genomics data provide an important unbiased avenue towards the identification of prospective therapeutic targets for drug discovery, which is an urgent and unmet clinical need for ovarian cancer.

Human Enhancer of Invasion-Cluster, a Coiled-Coil Protein Required for Passage through Mitosis

ABSTRACT In a cross-species overexpression approach, we used the pseudohyphal transition of Saccharomyces cerevisiae as a model screening system to identify human genes that regulate cell morphology and the cell cycle. Human enhancer of invasion-cluster (HEI-C), encoding a novel evolutionarily conserved coiled-coil protein, was isolated in a screen for human genes that induce agar invasion in S. cerevisiae. In human cells, HEI-C is primarily localized to the spindle during mitosis. Depletion of HEI-C in vivo with short interfering RNAs results in severe mitotic defects. Analysis by immunofluorescence, flow cytometry analysis, and videomicroscopy indicates that HEI-C-depleted cells form metaphase plates with normal timing after G2/M transition, although in many cases cells have disorganized mitotic spindles. Subsequently, severe defects occur at the metaphase-anaphase transition, characterized by a significant delay at this stage or, more commonly, cellular disintegration accompanied by the display of classic biochemical markers of apoptosis. These mitotic defects occur in spite of the fact that HEI-C-depleted cells retain functional cell cycle checkpoints, as these cells arrest normally following nocodazole or hydroxyurea treatment. These results place HEI-C as a novel regulator of spindle function and integrity during the metaphase-anaphase transition.

Compounds identified by virtual docking to a tetrameric EGFR extracellular domain can modulate Grb2 internalization

BackgroundOverexpression or mutation of the epidermal growth factor receptor (EGFR) potently enhances the growth of many solid tumors. Tumor cells frequently display resistance to mechanistically-distinct EGFR-directed therapeutic agents, making it valuable to develop therapeutics that work by additional mechanisms. Current EGFR-targeting therapeutics include antibodies targeting the extracellular domains, and small molecules inhibiting the intracellular kinase domain. Recent studies have identified a novel prone extracellular tetrameric EGFR configuration, which we identify as a potential target for drug discovery.MethodsOur focus is on the prone EGFR tetramer, which contains a novel protein-protein interface involving extracellular domain III. This EGFR tetramer is computationally targeted for stabilization by small molecule ligand binding. This study performed virtual screening of a Life Chemicals, Inc. small molecule library of 345,232 drug-like compounds against a molecular dynamics simulation of protein-protein interfaces distinct to the novel tetramer. One hundred nine chemically diverse candidate molecules were selected and evaluated using a cell-based high-content imaging screen that directly assessed induced internalization of the EGFR effector protein Grb2. Positive hits were further evaluated for influence on phosphorylation of EGFR and its effector ERK1/2.ResultsFourteen hit compounds affected internalization of Grb2, an adaptor responsive to EGFR activation. Most hits had limited effect on cell viability, and minimally influenced EGFR and ERK1/2 phosphorylation. Docked hit compound poses generally include Arg270 or neighboring residues, which are also involved in binding the effective therapeutic cetuximab, guiding further chemical optimization.ConclusionsThese data suggest that the EGFR tetrameric configuration offers a novel cancer drug target.

Human factors and pathways essential for mediating epigenetic gene silencing

Cellular identity in both normal and disease processes is determined by programmed epigenetic activation or silencing of specific gene subsets. Here, we have used human cells harboring epigenetically silent GFP-reporter genes to perform a genome-wide siRNA knockdown screen for the identification of cellular factors that are required to maintain epigenetic gene silencing. This unbiased screen interrogated 21,121 genes, and we identified and validated a set of 128 protein factors. This set showed enrichment for functional categories, and protein-protein interactions. Among this set were known epigenetic silencing factors, factors with no previously identified role in epigenetic gene silencing, as well as unstudied factors. The set included non-nuclear factors, for example, components of the integrin-adhesome. A key finding was that the E1 and E2 enzymes of the small ubiquitin-like modifier (SUMO) pathway (SAE1, SAE2/UBA2, UBC9/UBE2I) are essential for maintenance of epigenetic silencing. This work provides the first genome-wide functional view of human factors that mediate epigenetic gene silencing. The screen output identifies novel epigenetic factors, networks, and mechanisms, and provides a set of candidate targets for epigenetic therapy and cellular reprogramming.

Identification of novel inhibitors of DLK palmitoylation by High Content Screening

After axonal insult and injury, Dual leucine-zipper kinase (DLK) conveys retrograde pro-degenerative signals to neuronal cell bodies via its downstream target c-Jun N-terminal kinase (JNK). We recently reported that such signals critically require modification of DLK by the fatty acid palmitate, via a process called palmitoylation. Compounds that inhibit DLK palmitoylation could thus reduce neurodegeneration, but identifying such inhibitors requires a suitable assay. Here we report that DLK subcellular localization in non-neuronal cells is highly palmitoylation-dependent and can be used as a proxy readout to identify inhibitors of DLK palmitoylation by High Content Screening (HCS). We exploited this highly specific localization of DLK-GFP as the basis for a screen of the Prestwick Compound Library™. We found that ketoconazole, a Prestwick Library compound that most dramatically affected DLK subcellular localization in our primary screen, inhibited DLK palmitoylation in a dose-dependent manner in follow-up biochemical assays. Moeroever, ketoconazole significantly blunted phosphorylation of c-Jun in primary sensory neurons subjected to Trophic Deprivation, a well known model of DLK-dependent pro-degenerative signaling. These findings suggest that our HCS platform is capable of identifying novel inhibitors of DLK palmitoylation and signalling that may have considerable therapeutic potential.

Identification of evolutionarily conserved DNA damage response genes that alter sensitivity to cisplatin

Ovarian, head and neck, and other cancers are commonly treated with cisplatin and other DNA damaging cytotoxic agents. Altered DNA damage response (DDR) contributes to resistance of these tumors to chemotherapies, some targeted therapies, and radiation. DDR involves multiple protein complexes and signaling pathways, some of which are evolutionarily ancient and involve protein orthologs conserved from yeast to humans. To identify new regulators of cisplatin-resistance in human tumors, we integrated high throughput and curated datasets describing yeast genes that regulate sensitivity to cisplatin and/or ionizing radiation. Next, we clustered highly validated genes based on chemogenomic profiling, and then mapped orthologs of these genes in expanded genomic networks for multiple metazoans, including humans. This approach identified an enriched candidate set of genes involved in the regulation of resistance to radiation and/or cisplatin in humans. Direct functional assessment of selected candidate genes using RNA interference confirmed their activity in influencing cisplatin resistance, degree of γH2AX focus formation and ATR phosphorylation, in ovarian and head and neck cancer cell lines, suggesting impaired DDR signaling as the driving mechanism. This work enlarges the set of genes that may contribute to chemotherapy resistance and provides a new contextual resource for interpreting next generation sequencing (NGS) genomic profiling of tumors.