Compensatory ErbB3/c-Src signaling enhances carcinoma cell survival to ionizing radiation

EGFR and ErbB2 are two members of the ErbB family of receptor Tyr Kinases identified as therapeutic targets for treating carcinomas. Breast carcinoma cells express different complements and variable proportions of ErbB receptor Tyr kinases, which activate unique and redundant signaling cascades that are essential for cell survival. Previously it was shown that a COOH-terminal truncation mutant of the EGFR (EGFR-CD533) blocks EGFR dependent signals and radiosensitizes breast carcinoma cells. In this study the effects of EGFR-CD533 and an analogous truncation mutant of ErbB2 (ErbB2-CD572) on ErbB receptor family dimerization and signaling are further investigated. Using adenoviral vectors in breast carcinoma cell lines with variable ErbB expression profiles, we demonstrate different effects for each deletion mutant. EGFR-CD533 blocks ligand stimulation of EGFR, ErbB2, and ErbB4, but is associated with a compensatory Tyr kinase activity resulting in phosphorylation of ErbB3. In contrast, ErbB2-CD572 produces a weaker, non-specific pattern of ErbB receptor family inhibition, based upon the ErbB expression pattern of the cell type. Investigation of the compensatory Tyr kinase activity associated with EGFR-CD533 expression identified an ErbB3/c-Src signaling pathway that regulates expression of anti-apoptotic Bcl family proteins. This signaling is active in the T47D cell line, which inherently over-express ErbB3, absent in MDA-MB231 cells, which have low ErbB3 expression levels, and is restored in a MDA-MB231 cell line engineered to over-express ErbB3. Furthermore we demonstrate that ErbB3/c-Src signaling is radio-protective, and that its elimination through pharmacologic inhibition of c-Src enhances radiation-induced apoptosis. In summary, these studies identify a novel ErbB3/c-Src survival signal and point to ErbB3 expression levels as an important variable in therapeutic targeting of ErbB receptors in breast carcinoma cells.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44234/1/10549_2005_Article_9023.pd

Oligosaccharyltransferase Inhibition Induces Senescence in RTK-Driven Tumor Cells

Asparagine (N)-linked glycosylation is a protein modification critical for glycoprotein folding, stability, and cellular localization. To identify small molecules that inhibit new targets in this biosynthetic pathway, we initiated a cell-based high throughput screen and lead compound optimization campaign that delivered a cell permeable inhibitor (NGI-1). NGI-1 targets the oligosaccharyltransferase (OST), a hetero-oligomeric enzyme that exists in multiple isoforms and transfers oligosaccharides to recipient proteins. In non-small cell lung cancer cells NGI-1 blocks cell surface localization and signaling of the EGFR glycoprotein, but selectively arrests proliferation in only those cell lines that are dependent on EGFR (or FGFR) for survival. In these cell lines OST inhibition causes cell cycle arrest accompanied by induction of p21, autofluorescence, and changes in cell morphology; all hallmarks of senescence. These results identify OST inhibition as a potential therapeutic approach for treating receptor tyrosine kinase-dependent tumors and provides a chemical probe for reversibly regulating N-linked glycosylation in mammalian cells

Abstract 3981: Targeting SRP receptor-beta reduces protein glycosylation and cell growth in lung cancer

Abstract PURPOSE: Lung cancer is a major cause of morbidity and mortality world wide. Signaling receptors, adhesion molecules, and surface antigens that drive lung cancer progression are regulated by N-glycosylation, a post-translational protein modification initiated in the ER. Strategies for inhibition of N-glycosylation have recently been refined through the discovery of small molecules that partially disrupt glycan transfer. Here we screened for the target of a novel small molecule that reduces protein glycosylation with the goal of identifying new therapeutic targets for lung cancer. METHODS: Pooled results from chemical library HTS efforts identified hit compounds with activity. Counter-screening was performed in A549 cells using a fluorescence-based reporter (Halo1N) that detects aberrant N-glycosylation. The cellular targets for one class of inhibitors were evaluated by combining a whole-genome CRISPR-Cas9 screen with FACS sorting for loss of pharmacologic function. Significant changes in gRNA between populations was determined by MAGeCK analysis. The interaction between a compound and its target was determined by CETSA, and mechanism of action was verified by gene knockout and rescue experiments. Glycosylation status of glycoproteins were measured by western blot and cell compartment fluorescence. Predicted compound binding sites on the protein were mutated using site-directed mutagenesis. RESULTS: The lead tool compound (CP-9) has an IC50 in uM range and was used as a probe for target identification. CP-9 activates Halo-1N fluorescence, and whole genome CRISPR-Cas9 screening was used to identify gRNAs associated with loss of CP-9 activity. MAGeCK analysis demonstrated significant enrichment of genes associated with the translocon and ribosome (FDR P &amp;lt; 0.01) in the non-fluorescent population. We tested components of the signal recognition particle-receptor complex for direct interactions using the CETSA and demonstrate CP-9 binding to SR-β but not SR-α. Knockout experiments confirm that inhibition of glycosylation by CP-9 is dependent on SR-β expression, and CP-9’s effect on glycosylation and was rescued by SR-β re-expression. Mutated SR-β diminished CP-9’s effects on glycosylation and reduced SR-β thermal stabilization. CP-9 disrupted an interaction between SR-β/SR-α and the ribosome-translocon-OST complex suggesting a mechanism of action. Interestingly, a transmembrane domain mutant (ΔTM) of SR-β reduced the CP-9 effect and glycoprotein expression. ΔTM-SR-β also significantly reduced cell proliferation in A459 lung adenocarcinoma cells. CONCLUSION: CP-9 partially inhibits N-glycosylation by interfering with SR-β/SR-α and the translocon complex, leading to the disruption of protein import and glycosylation in the ER. Transmembrane domain mutants of SR-β reduce lung cancer cell growth and suggest this protein as a potential target for lung cancer drug development in the future. Citation Format: Chatchai Phoomak, Joseph N. Contessa. Targeting SRP receptor-beta reduces protein glycosylation and cell growth in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3981.</jats:p

Abstract 4998: Novel N-glycosilation inhibitor blocks proliferation of EGFR-dependent NSCLC

Abstract Purpose: Receptor Tyrosine Kinase (RTK) are transmembrane glycoproteins that are known to drive cellular proliferation in non-small cell lung cancer (NSCLC). Asparagine (N) linked glycosylation (NLG) is an endoplasmic reticulum (ER) post-translational modification common to RTKs that is crucial for receptor stability, localization, and activation. We hypothesized that blockade of NLG would be a novel strategy for reducing RTK signaling in NSCLC and therefore initiated a small molecule screening program to identify novel inhibitors of this biosynthetic process that have the potential for clinical translation. Experimental Procedures: The ER-LucT NLG reporter was used to perform a cell-based high throughput screen of 358,301 compounds and identified a novel small molecule with NLG inhibitory activity (NGI-1). Effects on protein localization were determined using confocal microscopy and by surface biotin labeling. Cell proliferation was assessed with MTT assays over 5 days of drug treatment and cell cycle analysis was performed by flow cytometry. Senescence was determined by autofluorescence and p21 and p53 protein levels measured by western blot. Results: NGI-1 is a first in class inhibitor of the oligosaccharyltransferase that is cell permeable and has a submicromolar IC50. This small molecule partially inhibits NLG and blocks epidermal growth factor receptor (EGFR) trafficking to the cell surface. Partially glycosylated receptors are retained in the secretory pathway as demonstrated by colocalization with the ER marker calreticulin. The loss of EGFR cell surface expression correlated with a significant reduction of EGFR phosphorylation and downstream signaling through MAPK and AKT. After 48hrs, NGI-1 treatment reduced proliferation of EGFR kinase domain mutant cell lines (PC9, HCC827, H3255) by &amp;gt;90% (p&amp;lt;0.001) but did not significantly block proliferation of KRAS mutant A549 cells or normal fibroblasts, suggesting a preferential effect on RTK addicted NSCLC. Cell cycle analysis also showed a differential effect on G1 arrest for EGFR addicted cell lines relative to controls. This proliferative block was shown to correlate with increased levels of p53 phosphorylation and increased expression of p21, as well as accumulation of lipofuscin and cell autofluorescence. Together these data suggest that NGI-1 induces a senescence-like phenotype in EGFR addicted NSCLC. Similar results were obtained in PC9 cells with T790M mutation, demonstrating the potential advantage of this approach for overcoming TKI acquired resistance in NSCLC. Conclusion: This study suggests that partial inhibition of NLG with NGI-1 is a novel approach for blocking proliferation of RTK dependent NSCLC. Citation Format: Cecilia Lopez Sambrooks, Joseph N. Contessa. Novel N-glycosilation inhibitor blocks proliferation of EGFR-dependent NSCLC. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4998. doi:10.1158/1538-7445.AM2015-4998</jats:p

Abstract 3053: Inhibition of heregulin-mediated ErbB3 signaling as a radiosensitization therapy for head and neck cancers

Abstract PURPOSE: EGFR signaling confers resistance to radiation therapy (RT) and is a validated target in head and neck squamous cell carcinoma (HNSCC). Inhibition of EGFR function in combination with RT improves local control and overall survival in this patient population, however, the mechanisms of resistance to combined treatment with RT + cetuximab are incompletely understood. We sought to develop cell line models of true cetuximab resistance and to define the molecular characteristics of these tumor cells with respect to radiation sensitivity. EXPERIMENTAL PROCEDURES: A431 and FaDu cell lines with resistance to cetuximab were generated by two methods: (i) exposure to increasing doses of cetuximab over 14 weeks or (ii) single cell clonal selection prior to identical cetuximab exposure regimens or for untreated controls. Receptor tyrosine kinase activity and dependent downstream signaling were assessed by phospho-blot analysis. Proliferation was determined with MTT. Radiosensitivity was determined through clonogenic survival analysis. The EFM-19 cell line was used as a functional indicator of heregulin family ligand expression. The efficacy of cetuximab was assessed in xenografts grown in nude mice. RESULTS: Cell line cultures exposed to increasing doses of cetuximab achieved only partial resistance in vivo. The process of clonal selection allowed us to identify distinct cell populations: (a) cetuximab-sensitive clones from untreated controls, (b) partial-cetuximab resistant clones from untreated controls, and (c) cetuximab-resistant clones from cell lines exposed to increasing doses. Partial- and cetuximab-resistant clones showed an increase of phospho-ErbB3, AKT, and Met. These clones were also more resistant to ionizing radiation. We also tested whether KTN3379, a monoclonal antibody that inhibits ligand-dependent and independent ErbB3 activation, affected cell proliferation and survival. After 5 days, KTN3379 + cetuximab reduced proliferation of cetuximab-resistant clones but did not further blocked partial- and sensitive-clones compared to cetuximab alone. This corresponded to a significant reduction of ErbB3/Akt (and Met) mediated by KTN3379. Using the EFM-19 cell model, which lacks endogenous heregulin, we demonstrate that cetuximab resistant cells upregulate heregulin family ligand expression and that KTN3379 effectively blocks this autocrine survival signaling. CONCLUSION: Tumor cell heterogeneity obscures the identification of operative resistance mechanisms in vitro. Autocrine ligand activation of ErbB3 is a mechanism for therapeutic resistance to cetuximab and may also cause radioresistance. Cross-resistance to both therapeutic modalities identifies HRG and ErbB3 as attractive therapeutic targets for improving delivery of radiation therapy for HNSCC. Citation Format: Marta Baro, Josep Balart, Joseph N. Contessa. Inhibition of heregulin-mediated ErbB3 signaling as a radiosensitization therapy for head and neck cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3053.</jats:p

Abstract 3312: A CRISPR-Cas9 screen in meningioma reveals GCN2/ATF4 is a novel target that enhances the response to radiation

Abstract Purpose: Meningioma is a common primary brain tumor in adults and accounts for ~ 36% of all adult brain tumors. Therapy for patients with meningioma is individualized and comprised of a combination of surgery and/or radiotherapy. Chemotherapy is not effective and there is no approved targeted treatment. Therefore, novel therapeutic approaches are warranted. We used CRISPR screening-based technology to uncover novel mechanisms of radiation resistance that may be therapeutically relevant in meningioma. Methods: A patient-derived meningioma cell line with TRAF7/KLF4 mutations (Tm77) was transduced with a Human Kinome CRISPR KnockOut (KO) library containing 3,052 RNA guides (sgRNA) targeting 763 kinase genes involved in cancer therapy. Following radiation, cells were collected (400x coverage) and analyzed using next-generation sequencing (NGS). MAGeCK analysis was used to evaluate sgRNA enrichment and depletion. Radiation survival was determined by clonogenic analysis. Kinase activity and downstream signaling were assessed by phospho-blot analysis. Cell cycle analysis was performed by flow cytometry and DNA damage was assessed by gH2AX immunofluorescence. Results: Analysis of NGS results shows that after radiation ATM and DNA-PK sgRNAs were depleted (p&amp;lt;0.001; FDR&amp;lt;0.001), suggesting that the inhibition of either induces radiosensitivity. These genes, together with ATR, are involved in the DNA damage response induced by external stress such as radiation. ATM (AZD1930) and ATR (AZD6738) inhibitors were used to test the screening results. While AZD1390 reduces ATM signaling, gH2AX foci, and radiation survival with a dose enhancement ratio (DER) at 2Gy of 2; AZD6738 did not induce any changes in signaling, gH2AX foci, or radiosensitivity. These results indicate that inhibition of ATM can regulate radiation response in meningioma and thus validated the screening results. The CRISPR-Cas9 KO screen also identified a potential novel druggable target, GCN2. In the NGS analysis, GCN2 sgRNAs were enriched (p&amp;lt;0.001; FDR&amp;lt;0.005), and in agreement with this finding GCN2-KO enhanced radiation survival with a DER at 2Gy of 1.4. In contrast, halofuginone, a natural product that activates GCN2 reduced radiation survival in Tm77 with a DER at 2Gy of 1.25 through activation of ATF4 and cell cycle arrest. These effects were not observed in GCN2-KO cell lines. In addition, the proteasome inhibitor, bortezomib, also activates ATF4 signaling, causes cell cycle arrest, and radiosensitization with a DER at 2Gy of 1.3. Conclusion: CRISPR-Cas9 KO screening provides a novel and unbiased approach to identify cellular targets that modify the response to radiation. These results suggests that the activation of ATF4 radiosensitizes meningioma. The evidence provided in this study supports further basic and preclinical research on approaches for activating GCN2 in combination with radiation therapy in meningioma. Citation Format: Marta Baro, Murat Gunel, Jennifer Moliterno, Joseph N. Contessa. A CRISPR-Cas9 screen in meningioma reveals GCN2/ATF4 is a novel target that enhances the response to radiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3312.</jats:p