Genome and Epigenome ERBB3 and IGF1R Signaling Are Required for Nrf2-Dependent Growth in KEAP1-Mutant Lung Cancer Steffan Vartanian 1 , James Lee 1 , Christiaan Klijn 2 , Florian Gnad 2 , Maria Bagniewska 1 , Gabriele Schaefer 3 , Donglu Zhang 4 , Jenille Tan 1 , Sara A. Watson 1 , Liling Liu 4 , Honglin Chen 5 , Yuxin Liang 5 , Colin Watanabe 2 , Trinna Cuellar 5 , David Kan 3 , Ryan J. Hartmaier 6 , Ted Lau 1 , Michael R. Costa 1 , Scott E. Martin 1 , Mark Merchant 3 , Benjamin Haley 5 , and David Stokoe 1 Abstract Mutations in KEAP1 and NFE2L2 (encoding the protein Nrf2) are prevalent in both adeno and squamous subtypes of non–small cell lung cancer, as well as additional tumor indications. The consequence of these mutations is stabi- lized Nrf2 and chronic induction of a battery of Nrf2 target genes. We show that knockdown of Nrf2 caused modest growth inhibition of cells growing in two-dimension, which was more pronounced in cell lines expressing mutant KEAP1. In contrast, Nrf2 knockdown caused almost com- plete regression of established KEAP1-mutant tumors in mice, with little effect on wild-type (WT) KEAP1 tumors. The strong dependency on Nrf2 could be recapitulated in certain anchorage-independent growth environments and was not prevented by excess extracellular glutathione. A CRISPR screen was used to investigate the mechanism(s) underlying this dependence. We identified alternative path- ways critical for Nrf2-dependent growth in KEAP1-mutant cell lines, including the redox proteins thioredoxin and peroxiredoxin, as well as the growth factor receptors IGF1R and ERBB3. IGF1R inhibition was effective in KEAP1- mutant cells compared with WT, especially under condi- tions of anchorage-independent growth. These results point to addiction of KEAP1-mutant tumor cells to Nrf2 and suggest that inhibition of Nrf2 or discrete druggable Nrf2 target genes such as IGF1R could be an effective therapeutic strategy for disabling these tumors. Significance: This study identifies pathways activated by Nrf2 that are important for the proliferation and tumorige- nicity of KEAP1-mutant non–small cell lung cancer. Introduction Lung cancer is the leading cause of cancer death in men and women, so new insights into driver genes for this indication are especially needed. Exome sequencing of 230 tumor/normal pairs from lung adenocarcinoma by The Cancer Genome Atlas (TCGA) consortium showed that KEAP1 was the third most mutated gene, present in 17% cases, with only TP53 and KRAS displaying higher mutation frequencies (1). KEAP1 is a sub- strate targeting protein for the Cul3 E3 ubiquitin ligase that ubiquitinates the Nrf2 transcription factor, resulting in its proteasomal degradation. NFE2L2, the gene encoding Nrf2, is also found frequently mutated across multiple human tumors, especially in squamous lung (15%; ref. 2). Mutations in Nrf2 are localized around 2 regions that interact with KEAP1, and mutations impair association with KEAP1 (3). In contrast, mutations in KEAP1 are spread throughout the gene, and may play a more complex role in affecting the interaction and ubiquitination of Nrf2 (4). The KEAP1/Nrf2 pathway plays an important role in the cellular response to reactive oxygen species (ROS). Under non- stressed conditions, KEAP1 dimers maintain low levels of Nrf2 through binding 2 regions of Nrf2 (DLG and ETGE motifs), resulting in its Cullin 3-dependent ubiquitination and degrada- tion. Upon increases in oxidative stress, key cysteine residues in KEAP1 become oxidized, changing the conformation of the KEAP1/Nrf2 complex such that Nrf2 no longer becomes ubiqui- tinated, leading to stabilization and accumulation in the cytosol and nucleus (5). Many transcriptional targets of Nrf2 have been identified, some of which counteract the cellular increases in ROS. For example, multiple components of the glutathione biosyn- thesis pathway are direct Nrf2 target genes, as well as enzymes in the pentose phosphate pathway (6), which is one of the major mechanisms generating NADPH, an important source of reducing 1 Department of Discovery Oncology. 2 Department of Bioinformatics and Computational Biology. 3 Department of Translation Oncology. 4 Department of Drug Metabolism and Pharmacokinetics. 5 Department of Molecular Biology, Genentech Inc., South San Francisco, California. 6 Foundation Medicine Inc., Cambridge, Massachusetts. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Current address for F. Gnad: Cell Signaling Technology, Danvers, Massachusetts; Current address for T. Cuellar: Gotham Therapeutics, New York, New York; Current address for D. Stokoe: Calico, South San Francisco, California. S. Vartanian and J. Lee authors contributed equally to this article. Corresponding Author: David Stokoe, Calico, 1170 Veterans Boulevard, South San Francisco, CA 94080. Phone: 650-267-7935; E-mail: dstokoe@pacbell.net Cancer Res 2019;79:4828–39 doi: 10.1158/0008-5472.CAN-18-2086 Ó2019 American Association for Cancer Research. Cancer Research Cancer Res; 79(19) October 1, 2019 4828 Downloaded from http://aacrjournals.org/cancerres/article-pdf/79/19/4828/2786257/4828.pdf by guest on 21 June 2022