Results from the high throughput drug screen in the non-isogenic OCCC cell line panel, see also Figure 1

siRNA library gene list for dasatinib resistance screen, see also figure 5

MS Stats output from dasatinib bead proteomics, see also Figure 3

Results from the dasatinib target siRNA screen, see also Figure S6

ARID1A mutation status in OCCC panel determined by whole exome sequencing

MS Stats output from dasatinib bead proteomics, see also Figure 3

Pearson's correlation and Z prime values for OCCC panel demonstrating robust reproducibility and dynamic range from the high-throughput drug screen

Additional compounds included in the isogenic high-throughput drug screen

Results from the siRNA kinase and tumour suppressor dasatinib resistance screen, see also Figure 5

Pearson's correlation and Z prime values for isogenic cell lines demonstrating robust reproducibility and dynamic range from the high-throughput drug screen, see also Figure 1

Figure legends for Supplementary figures and supplementary methods

Supplementary Figure 1 ARID1A mutations in the panel of OCCC tumour cell line models with corresponding protein expression. Supplementary Figure 2 Un-cropped western blots from main and supplementary figures. Supplementary Figure 3 ARID1A selective effects from the high throughput drug screen. Supplementary Figure 4 Inhibitors of the PI3K/mTOR signalling pathway in the panel of OCCC cell lines and ARID1A selectivity. Supplementary Figure 5 Dasatinib is a synthetic lethal drug in ARID1A mutant OCCC tumour cell line models - see also Figure 2. Supplementary Figure 6 Dasatinib sensitivity in ARID1A mutant isogenic HCT116 colorectal tumour cell line model. Supplementary Figure 7 Dasatinib siRNA screen results. Supplementary Figure 8 Apoptosis assay in four OCCC cell line models, see also Figure 4. Supplementary Figure 9 Dasatinib sensitivity in ARID1A mutant OCCC is dependent upon G1/S checkpoint effectors, see also Figure 5. Supplementary Figure 10 Determining the optimal method of dasatinib delivery in vivo.

One of the requirements for tumor development is blood supply, most often driven by hypoxia-induced angiogenesis. Hypoxia induces the stabilization of hypoxiainducible factor-1 alpha (HIF-1 alpha), which induces expression of an angiogenic factor, vascular endothelial growth factor (VEGF). The purpose of this study is to validate a new screening platform combined with orthogonal assays to rapidly identify HIF-1 inhibitors and to evaluate the effectiveness of approved drugs on modulating HIF-1 signaling.We generated an endogenous HIF-1 alpha-NanoLuc luciferase reporter allele in the human HCT116 colon cancer cell line using genome editing and screened a panel of small interfering RNAs (siRNAs) to 960 druggable targets and approximately 2,500 drugs on a quantitative high-throughput screening (qHTS) platform. Selected compounds were further investigated with secondary assays to confirm their anti-HIF activity and to study their mode of action. The qHTS assay identified over 300 drugs that inhibited HIF-1 alpha-NanoLuc expression. The siRNA screening results supported the effectiveness of several target-specific inhibitors. Moreover, the identified HIF-1 inhibitors, such as mycophenolate mofetil, niclosamide, and trametinib, were able to suppress cancer cell proliferation and angiogenesis. Our study indicates that blocking the mitogen-activated protein kinase (MAPK) and phosphoinositol 3-kinase (PI3K) pathways effectively inhibits hypoxia-induced HIF-1 alpha accumulation and HIF-1 alpha transactivation and that proteasome inhibitors induce accumulation and decrease transcriptional activity of HIF-1 alpha. These findings underline the importance of developing a battery of robust assay platforms and confirmation studies that focus on endogenous protein targets so that only relevant and reliable data will be taken into pre-clinical and clinical studies.

Abstract New targeted approaches to ovarian clear cell carcinomas (OCCC) are needed, given the limited treatment options in this disease and the poor response to standard chemotherapy. Using a series of high-throughput cell-based drug screens in OCCC tumor cell models, we have identified a synthetic lethal (SL) interaction between the kinase inhibitor dasatinib and a key driver in OCCC, ARID1A mutation. Imposing ARID1A deficiency upon a variety of human or mouse cells induced dasatinib sensitivity, both in vitro and in vivo, suggesting that this is a robust synthetic lethal interaction. The sensitivity of ARID1A-deficient cells to dasatinib was associated with G1–S cell-cycle arrest and was dependent upon both p21 and Rb. Using focused siRNA screens and kinase profiling, we showed that ARID1A-mutant OCCC tumor cells are addicted to the dasatinib target YES1. This suggests that dasatinib merits investigation for the treatment of patients with ARID1A-mutant OCCC. Mol Cancer Ther; 15(7); 1472–84. ©2016 AACR.

Abstract We have previously reported using cell lines generated with Horizon Discovery's rAAV-based GENESIS™ gene editing platform to establish a comprehensive list of isogenic cancer models for in vivo compound screening, with mutations in a wide variety of genes including KRAS, PIK3CA, PTEN, IDH1 and IDH2, and p53. These isogenic tumor models comprise pairs of cell lines which share the same genetic background, differing only by the mutation of interest, and therefore allow definitive studies of specific genetic variances to be performed. In the current study we developed a DualXenoTM method where isogenic pairs of a colorectal cell line, one of each pair with a KRAS mutation, were inoculated simultaneously in the two flanks of the same mouse. The tumors were then treated with EGFR targeted therapeutics to address the question of resistant phenotypes elicited by different KRAS mutations. This design allows direct comparison of wild type and mutant isogenic pairs for treatment responses that are associated with the defined genetic variations. Our results demonstrated that tumors harboring the G12V mutation were resistant to both Cetuximab and Erlotinib treatment, while tumors harboring the G13D mutation remained sensitive to both agents. This is in consistent with clinical findings (De Roock, et al. JAMA 2010, 304(16), pp 1812), and our own findings with PDX mouse clinical trials in colon cancer, suggesting that the KRAS G13D mutation may establish a different signaling network to other KRAS mutations, and that colon cancer patients with the mutation should not be excluded from the EGFR targeted therapies. Citation Format: Yanmei Sun, Songling Zhang, Nan Li, Holly Astley, Rebecca Foster, Christine Schofield, Chris Chris Torrance, Jinying Ning, Qian Shi. X-MAN™ isogenic DualXenoTM models with KRAS mutation predicts the effect of anti-EGFR agents. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3136. doi:10.1158/1538-7445.AM2014-3136

Abstract The explosion of knowledge regarding the genetic underpinnings of human cancer heralds a new era of targeted therapy. To date, in vivo cell based screening has proven a useful tool in almost all drug development programs. Cells used in such screens are usually harvested from cancer patients that harbor the specific mutation of interest, but these cells almost invariably contain many other additional mutations making it difficult to ascertain the specific functions of molecules being screened. Thus the lack of true control cells hampers the development of new cancer therapeutics. Using cell lines generated with Horizon Discovery's proprietary rAAV-based GENESIS™ gene editing platform, we have established a comprehensive range of isogenic cancer models for our in vivo compound screening program to service our clients in academia and industry, with mutations in a wide variety of genes including KRAS, PIK3CA, PTEN, IDH1 and p53. These isogenic tumor models comprise pairs of cell lines which share the same genetic background, differing only by the mutation of interest and therefore allowing definitive studies of specific genetic variances to be performed. The same isogenic pairs of lines can be used for in vitro and in vivo experiments to ensure continuity and relevance of results. We have carried out intensive validation of these models in vivo to ensure the lines generate robust tumor growth in mice and the matched tumors differ only in the genetic composition of target gene. In a POC study, colon cancer cell lines with the KRAS G13D mutation have been demonstrated to respond to Cetuximab treatment, consistent with recent findings in the clinic. These results challenge the current clinical practice of only using Cetuximab as a therapeutic for KRAS wild type patients, and provide the basis for expanding the usage of the drug to benefit more patients. In summary, we have established a series of reliable in vivo isogenic models with precise and highly specific genetic modifications as predictors of clinical effect. They will provide a valuable tool in the drug discovery and development arena to drive forward personalized medicine by enabling novel target validation, expansion of the target population for existing therapeutics, and definition of patient responsive genotypes. Citation Format: Yanmei Sun, Songling Zhang, Holly Astley, Rebecca Foster, Christine Schofield, Chris Torrance, Jinying Ning, Taiping Chen, Qian Shi. Utilization of in vivo human isogenic cancer models in the new era of targeted therapies. [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 2782. doi:10.1158/1538-7445.AM2013-2782

Successful drug development in oncology requires a deeper understanding of the functional consequences of the diverse genetic changes observed in human cancers. For example, responses to epidermal growth factor receptor (EGFR) inhibitors are observed in patients whose tumors express EGFR alleles with activating mutations, rather than in tumors overexpressing EGFR. Furthermore, antibodies against EGFR are ineffective in tumors bearing certain activating alleles of KRAS. Horizon Discovery has used its proprietary rAAV gene engineering technology to generate isogenic cell lines covering a range of mutations commonly found in cancer patients. Use of a non-tumorigenic ‘clean’ cell line background such as MCF10A allows specific evaluation of the mutations without any confounding factors due to the presence of other genetic alterations. Mutations introduced into cancer cell line backgrounds allow the contextual evaluation of a cancer related gene. Here we describe the use of isogenic cell line panels as powerful tools for investigating sensitivity and resistance markers to cancer therapeutics. Some 50% of human tumors exhibit p53 loss or inactivation. To investigate how p53 loss in combination with other common cancer-driving mutations may influence therapeutic responses, we have generated a suite of MCF10A isogenic cell lines covering some of the major cancer genotypes, either in isolation or on a TP53 (-/-) background. These genotypes include EGFR (delE746-A750/+), EGFR (L858R/+), KRAS (G12V/+), BRAF (V600E/+), BRAF (V600K/+) and PIK3CA (H1047R/+). Thus, we have been able to investigate the interaction effects of discrete mutations in molecularly defined, but more tumor-like cell models. One data highlight arose from the profiling of the EGFR mutant panel using small molecule EGFR inhibitors; in isolation, the introduction of common activating EGFR mutations L858R or deletion of E746-A750 led to increased sensitivity, recapitulating clinical findings. However, combining EGFR mutation with loss of p53 further enhanced the cell response. Through systematic profiling of this panel to targeted therapeutic agents such as gefinitib, selumetinib, vemurafenib, and pictilisib, we have identified interesting differential sensitivities, which can be directly attributable to introduction of a given mutation. Results such as these can enable better patient stratification for anticancer agents, and allow incorporation of molecular markers into clinical trial design for personalised therapeutic regimens. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A148. Citation Format: Annette S. Little, Jessica Hunt, David Hughes, Ruth Feltell, Daniel Gitterman, Rachel Leah, Holly Astley, Ramu Mangena, Kyla Grimshaw, Christopher Torrance. Modeling patient responses to targeted therapy with rAAV mediated gene editing. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A148.

Abstract To allow promoter activity and protein dynamics to be studied at endogenous levels for the first time, Horizon Discovery has developed suites of X-MAN™ reporter cell lines incorporating NanoLuc™ and HaloTag® technologies from Promega. By creating these innovative reporters, we have removed the need for either exogenous plasmid based overexpression studies, or use of surrogate markers of activity, both of which can yield artefactual data. Horizon's proprietary rAAV-based GENESIS™ gene editing platform has been used to introduce reporter genes (NanoLuc™ or HaloTag®) into several specific chromosomal loci (including HIF1A, cMYC, β-Catenin and NRF2) either as endogenous promoter fusions or in-frame protein fusions. Our extensive expertise in cell line engineering means that the technology can be rapidly applied to virtually any gene of interest. NanoLuc™ produces high intensity luminescence enabling accurate quantification of gene expression even at low endogenous expression levels. HaloTag® is a multifunctional protein reporter which can be used for many applications including intracellular fluorescent imaging of live cells in real time. Validation experiments, including kinetic measurements and treatment with compounds or conditions that modulate transcription or protein expression, reveal robust and reproducible results for all reporter cell lines and demonstrate their value in a wide variety of applications, from pathway analysis to high throughput screening platforms. To further demonstrate the utility of the X-MAN™ reporter cell lines in HTS-screening applications, we used the HCT116 HIF1A NanoLuc™ protein reporter line in a multiplexed siRNA library screen against 960 ‘druggable’ targets, under both normoxic and reduced oxygen conditions. As expected, many known regulators of HIF1A were identified, such as AKT, PDK1 and cRaf, showing once more the robust nature of the reporter system. Several novel regulators were also identified highlighting the value of the reporter cell lines for rapid identification of key regulators of endogenous proteins. In conclusion, we have used the combination of NanoLuc™, HaloTag® and the GENESIS™ gene editing platform to generate highly sensitive reporter cell lines that are capable of registering physiological levels of gene transcription and protein activity/localization in live cells. These reporter technologies can be used for a wide range of applications and provide an exciting new tool for biologically relevant drug discovery. Citation Format: Holly Astley, Suzanne Grooby, Jo Francis, Sue Griffin, Annette Little, Hélène Benink, Jeff Kelly, Rebecca Foster. X-MAN™ reporter cell lines: Enabling the study of endogenous promoter activity and protein dynamics. [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 5522. doi:10.1158/1538-7445.AM2013-5522

Mutations in the genes encoding isocitrate dehydrogenase 1 and 2 (IDH1/2) occur in a variety of tumor types, resulting in production of the proposed oncometabolite, 2-hydroxyglutarate (2-HG). How mutant IDH and 2-HG alter signaling pathways to promote cancer, however, remains unclear. Additionally, there exist relatively few cell lines with IDH mutations. To examine the effect of endogenous IDH mutations and 2-HG, we created a panel of isogenic epithelial cell lines with either wild-type IDH1/2 or clinically relevant IDH1/2 mutations. Differences were noted in the ability of IDH mutations to cause robust 2-HG accumulation. IDH1/2 mutants that produce high levels of 2-HG cause an epithelial-mesenchymal transition (EMT)-like phenotype, characterized by changes in EMT-related gene expression and cellular morphology. 2-HG is sufficient to recapitulate aspects of this phenotype in the absence of an IDH mutation. In the cells types examined, mutant IDH-induced EMT is dependent on up-regulation of the transcription factor ZEB1 and down-regulation of the miR-200 family of microRNAs. Furthermore, sustained knockdown of IDH1 in IDH1 R132H mutant cells is sufficient to reverse many characteristics of EMT, demonstrating that continued expression of mutant IDH is required to maintain this phenotype. These results suggest mutant IDH proteins can reversibly deregulate discrete signaling pathways that contribute to tumorigenesis.