<p>Supplementary Table S5. RPPA analysis of phosphorylated and total protein levels in PC9 and NCI-H1975 AZD9291 resistant populations compared to respective parental cells.</p>

Supplementary Figure Legend. Legends for Supplementary Figures S1-S6.

Supplementary Tables S1-S4. Generation of resistant cell populations (S1); Small molecule inhibitors (S2); IC50 (µM) values from cell growth inhibition assays comparing compound sensitivity between parental and resistant cell populations (S3); Genetic analysis of resistant cell populations (S4).

Supplementary Methods and References. Description of additional methods and procedures used in the study. Also includes Supplementary References.

<p>Supplementary Figures S1-S6. Comparison of genetic alterations across multiple populations resistant to AZD9291 and other EGFR TKIs (S1); Treatment of resistant populations with AZD9291 (S2); Detection and Validation of a novel NRAS E63K mutation (S3); Lysates were prepared from parental PC9 and resistant populations analysed for levels of total and phosphorylated ERK, NRAS and KRAS by western blot (S4); The novel NRAS E63K mutation is an activating mutation that when expressed enhances resistance to cell growth inhibition by gefitinib or AZD9291 in EGFRm cell lines (S5); In vitro combination of AZD9291 with selumetinib induces more profound phenotype inhibition (S6).</p>

Abstract The identification of novel oncology targets for small molecule drug discovery is becoming increasingly challenging despite 10-15% of the human genome estimated to be druggable. The literature is an important source of novel targets; however, several recent reports by pharma and academia have indicated that approximately 11-55% of published studies are irreproducible. Moreover, oncology drug attrition rates are extremely poor, with 66% of candidates in Phase III clinical trials not achieving approval. This is reflected by increasing failure rates of drug development projects in Phase II, from 72% in 2006-2007 to 82% in 2008-2009. These failures have been attributed to lack of efficacy, thus highlighting the critical requirement for new novel drug targets that demonstrate clear promise of clinical efficacy through predictive in vitro and in vivo models. Furthermore, it is important to prioritize and pursue targets that are most likely to bind a small molecule inhibitor with high infinity. We sought to utilize the wealth of unvalidated genomic data now available in the public domain by developing a bioinformatic pipeline founded on the collateral vulnerability hypothesis, which is based on the concept of synthetic lethality and exploits the co-occurrence of deletions of genes flanking tumor suppressors that are lost during the course of tumor evolution. We can then target the paralogs of these genes if together they belong to gene families with essential predicted function, thereby exploiting a vulnerability of the cancer cells with these losses. Here, we describe our industry-standard approaches to the target validation of the output generated from lung adenocarcinoma data, and present the validation and de-validation of several novel targets predicted by the collateral vulnerability pipeline, including SMARCA2, KMT2C, and SETD1B. Citation Format: H. Nikki March, Phil Chapman, Elizabeth Blaikley, Catherine A. Eberlein, Mark Cockerill, Samantha Hitchin, Ian D. Waddell, Donald Ogilvie. Discovery, validation and targeting of novel synthetic lethal interactions in academic drug discovery [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr B05.

AbstractResistance to targeted EGFR inhibitors is likely to develop in EGFR-mutant lung cancers. Early identification of innate or acquired resistance mechanisms to these agents is essential to direct development of future therapies. We describe the detection of heterogeneous mechanisms of resistance within populations of EGFR-mutant cells (PC9 and/or NCI-H1975) with acquired resistance to current and newly developed EGFR tyrosine kinase inhibitors, including AZD9291. We report the detection of NRAS mutations, including a novel E63K mutation, and a gain of copy number of WT NRAS or WT KRAS in cell populations resistant to gefitinib, afatinib, WZ4002, or AZD9291. Compared with parental cells, a number of resistant cell populations were more sensitive to inhibition by the MEK inhibitor selumetinib (AZD6244; ARRY-142886) when treated in combination with the originating EGFR inhibitor. In vitro, a combination of AZD9291 with selumetinib prevented emergence of resistance in PC9 cells and delayed resistance in NCI-H1975 cells. In vivo, concomitant dosing of AZD9291 with selumetinib caused regression of AZD9291-resistant tumors in an EGFRm/T790M transgenic model. Our data support the use of a combination of AZD9291 with a MEK inhibitor to delay or prevent resistance to AZD9291 in EGFRm and/or EGFRm/T790M tumors. Furthermore, these findings suggest that NRAS modifications in tumor samples from patients who have progressed on current or EGFR inhibitors in development may support subsequent treatment with a combination of EGFR and MEK inhibition. Cancer Res; 75(12); 2489–500. ©2015 AACR.