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 siRNA 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 EGFR antagonists and clinically relevant drugs targeting proteins connected in the EGFR network, such as the kinases protein kinase C or Aurora kinase A, or the transcriptional regulator STAT3, synergized 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.

Background-Much effort has been devoted to development of cancer therapies targeting EGFR, based on its role in regulating cell growth. Small-molecule and antibody EGFR inhibitors have clinical roles based on their efficacy in a subset of cancers, generally as components of combination therapies. Many cancers are either initially resistant to EGFR inhibitors or become resistant during treatment, limiting the efficacy of these reagents.Objective/Methods-To review cellular resistance mechanisms to EGFR-targeted therapies.Results/Conclusions-The best validated of these mechanisms include activation of classic ATP-binding casette (ABC) multidrug transporters; activation or mutation of EGFR; and overexpression or activation of signaling proteins operating in relation to EGFR. We discuss current efforts and potential strategies to override these sources of resistance. We describe emerging systemsbiology-based concepts of alternative resistance to EGFR-targeted therapies, and discuss their implications for use of EGFR-targeted and other targeted therapies.

Resistance to therapies targeting the estrogen pathway remains a challenge in the treatment of estrogen-receptor positive breast cancer. To address this challenge, a systems biology approach was used. A library of siRNAs targeting an estrogen receptor- and aromatase-centered network identified 46 genes that are dispensable in estrogen-dependent MCF7 cells, but are selectively required for the survival of estrogen-independent MCF7-derived cells, and multiple additional estrogen-independent breast cancer cell lines. Integration of this information identified a tumor suppressor gene TOB1 as a critical determinant of estrogen-independent estrogen receptor-positive breast cell survival. Depletion of TOB1 selectively promoted G1 phase arrest and sensitivity to AKT and mTOR inhibitors in estrogen-independent cells but not estrogen-dependent cells. Phosphoproteomic profiles from reverse phase protein array analysis supported by mRNA profiling identified a significant signaling network reprogramming by TOB1 that differed in estrogen-sensitive and estrogen-resistant cell lines. These data support a novel function for TOB1 in mediating survival of estrogen-independent breast cancers. These studies also provide evidence for combining TOB1 inhibition and AKT/mTOR inhibition as a therapeutic strategy, with potential translational significance for the management of patients with estrogen receptor-positive breast cancers.

Monoclonal antibodies can modulate cancer cell signal transduction and recruit antitumor immune effector mechanisms – including antibody-dependent cellular cytotoxicity (ADCC). Although several clinically effective antibodies can promote ADCC, therapeutic resistance is common. We hypothesized that oncogenic signaling networks within tumor cells affect their sensitivity to ADCC. We developed a screening platform and targeted 60 genes derived from an EGFR gene network using RNA interference (RNAi) in an in vitro ADCC model system. Knockdown of GRB7, PRKCE, and ABL1 enhanced ADCC by primary and secondary screens. ABL1 knockdown also reduced cell proliferation, independent of its ADCC enhancement effects. c-Abl overexpression decreased ADCC sensitivity and rescued the effects of ABL1 knockdown. Imatinib inhibition of c-Abl kinase activity also enhanced ADCC – phenocopying ABL1 knockdown – against several EGFR-expressing head-and-neck squamous cell carcinoma (HNSCC) cell lines by ex vivo primary NK cells. Our findings suggest that combining c-Abl inhibition with ADCC-promoting antibodies, such as cetuximab, could translate into increased therapeutic efficacy of monoclonal antibodies.

Antiestrogens are the most widely administered endocrine agents for the treatment of estrogen receptor (ER)-expressing breast cancers, such as tamoxifen, fulvestrant and aromatase inhibitor. However, most initially responsive breast tumors acquire resistance and become estrogen-independent. To find potential therapeutic target(s) against drug resistance, we performed siRNA library screening studies to identify cell survival determinants in estrogen independent cell lines from 631 ER network related genes. LCC1 and LCC9 cells, derivatives of MCF-7, are estrogen-independent. LCC1 cells are sensitive to fulvestrant and tamoxifen. LCC9 cells are resistant to fulvestrant and cross-resistant to tamoxifen. A number of genes selectively support the survival of estrogen-independent tumor cells, including TOB1 (Transducer of ErbB2). TOB1 gene knockdown significantly inhibited (>50%) estrogen-independent cell growth in LCC1 and LCC9 cells, but had much less effect on the survival of MCF-7 cells (10%). These cell lines have similar levels of Tob1 protein. TOB1 knockdown did not cause apoptosis or autophagy. DNA replication was reduced and G1 to S phase transition was blocked by TOB1 knockdown in the estrogen-independent cell lines. Next, we studied cell-signaling pathways that regulate cell cycle transition. Cyclin D1 was upregulated and activated by TOB1 knockdown in estrogen-independent cells, but not in MCF-7 cells. Therefore, cyclin D1 might be involved in G1/S transition blockage induced by Tob1 inhibition. TOB1 knockdown also activated cell cycle checkpoint kinase 2 (Chk2) and induced DNA damage. Our data demonstrated that Tob1 promotes cell survival after ER+ breast cancer cells acquire estrogen-independence, which might be mediated by cyclin D1. Citation Format: Yong-Wei Zhang, Rochelle Nasto, Robert Clarke, Louis M. Weiner. TOB1 promotes cell survival in estrogen-independent estrogen receptor-positive breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2042. doi:10.1158/1538-7445.AM2013-2042

Monoclonal antibodies can induce antibody-dependent cellular cytotoxicity (ADCC) through immune effector cells, such as natural killer (NK) cells. Although clinical evidence suggests a role for ADCC in effective antibody therapy of cancer, molecular determinants of ADCC responsiveness—in oncogenic signaling networks, for example—have not been well characterized. Using an in vitro model system including an NK-like cell line (NK92-CD16V), cetuximab, and A431 cells, we screened 60 genes from an EGFR gene network by RNAi to assess for synthetic enhancement of ADCC. Primary and validation screens, and correlative characterization studies, identified four genes whose knockdown enhanced ADCC: GRB7, PRKCE, RET, and ABL1. Inhibition of Abl kinase activity enhanced ADCC, phenocopying the effects of ABL1 knockdown and providing pre-clinical evidence for combining Abl inhibitors and cetuximab. Next, we derived an ADCC-resistant cell population (A431/ADCC-R) by treating parental A431 cells with over 30 successive ADCC treatments. We describe the development, screening, and characterization of A431/ADCC-R. Our screening strategy has revealed molecular determinants of both tumor cell sensitivity and resistance to ADCC in vitro that may translate into increased monoclonal antibody efficacy in the clinic. Citation Format: Joseph C. Murray, Rochelle E. Nasto, Sandra A. Jablonski, Yong Tang, Louis M. Weiner. Molecular determinants of sensitivity and resistance to tumor-directed antibody-dependent cellular cytotoxicity (ADCC). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2847. doi:10.1158/1538-7445.AM2013-2847

<p>Development of a screening assay for modulators of ADCC.</p>

<p>Effect of various c-Abl tyrosine kinase inhibitors on ADCC.</p>