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ERBB3 and IGF1R Signaling Are Required for Nrf2-Dependent

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ERBB3 and IGF1R Signaling Are Required for Nrf2-Dependent Published OnlineFirst August 15, 2019; DOI: 10.1158/0008-5472.CAN-18-2086 Cancer Genome and Epigenome Research ERBB3 and IGF1R Signaling Are Required for Nrf2-Dependent Growth in KEAP1-Mutant Lung Cancer Steffan Vartanian1, James Lee1, Christiaan Klijn2, Florian Gnad2, Maria Bagniewska1, Gabriele Schaefer3, Donglu Zhang4, Jenille Tan1, Sara A. Watson1, Liling Liu4, Honglin Chen5, Yuxin Liang5, Colin Watanabe2, Trinna Cuellar5, David Kan3, Ryan J. Hartmaier6, Ted Lau1, Michael R. Costa1, Scott E. Martin1, Mark Merchant3, Benjamin Haley5, and David Stokoe1 Abstract Mutations in KEAP1 and NFE2L2 (encoding the protein underlying this dependence. We identified alternative path- Nrf2) are prevalent in both adeno and squamous subtypes ways critical for Nrf2-dependent growth in KEAP1-mutant of non–small cell lung cancer, as well as additional tumor cell lines, including the redox proteins thioredoxin and indications. The consequence of these mutations is stabi- peroxiredoxin, as well as the growth factor receptors IGF1R lized Nrf2 and chronic induction of a battery of Nrf2 target and ERBB3. IGF1R inhibition was effective in KEAP1- genes. We show that knockdown of Nrf2 caused modest mutant cells compared with WT, especially under condi- growth inhibition of cells growing in two-dimension, which tions of anchorage-independent growth. These results point was more pronounced in cell lines expressing mutant to addiction of KEAP1-mutant tumor cells to Nrf2 and KEAP1. In contrast, Nrf2 knockdown caused almost com- suggest that inhibition of Nrf2 or discrete druggable Nrf2 plete regression of established KEAP1-mutant tumors in target genes such as IGF1R could be an effective therapeutic mice, with little effect on wild-type (WT) KEAP1 tumors. strategy for disabling these tumors. The strong dependency on Nrf2 could be recapitulated in certain anchorage-independent growth environments and Significance: This study identifies pathways activated by was not prevented by excess extracellular glutathione. A Nrf2 that are important for the proliferation and tumorige- CRISPR screen was used to investigate the mechanism(s) nicity of KEAP1-mutant non–smallcelllungcancer. Introduction displaying higher mutation frequencies (1). KEAP1 is a sub- strate targeting protein for the Cul3 E3 ubiquitin ligase that Lung cancer is the leading cause of cancer death in men and ubiquitinates the Nrf2 transcription factor, resulting in its women, so new insights into driver genes for this indication are proteasomal degradation. NFE2L2, the gene encoding Nrf2, is especially needed. Exome sequencing of 230 tumor/normal also found frequently mutated across multiple human tumors, pairs from lung adenocarcinoma by The Cancer Genome Atlas especially in squamous lung (15%; ref. 2). Mutations in Nrf2 (TCGA) consortium showed that KEAP1 was the third most are localized around 2 regions that interact with KEAP1, and mutated gene, present in 17% cases, with only TP53 and KRAS 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 1Department of Discovery Oncology. 2Department of Bioinformatics and Computational Biology. 3Department of Translation Oncology. 4Department of ubiquitination of Nrf2 (4). Drug Metabolism and Pharmacokinetics. 5Department of Molecular Biology, The KEAP1/Nrf2 pathway plays an important role in the Genentech Inc., South San Francisco, California. 6Foundation Medicine Inc., cellular response to reactive oxygen species (ROS). Under non- Cambridge, Massachusetts. stressed conditions, KEAP1 dimers maintain low levels of Nrf2 Note: Supplementary data for this article are available at Cancer Research through binding 2 regions of Nrf2 (DLG and ETGE motifs), Online (http://cancerres.aacrjournals.org/). resulting in its Cullin 3-dependent ubiquitination and degrada- Current address for F. Gnad: Cell Signaling Technology, Danvers, Massachusetts; tion. Upon increases in oxidative stress, key cysteine residues in Current address for T. Cuellar: Gotham Therapeutics, New York, New York; KEAP1 become oxidized, changing the conformation of the Current address for D. Stokoe: Calico, South San Francisco, California. KEAP1/Nrf2 complex such that Nrf2 no longer becomes ubiqui- S. Vartanian and J. Lee authors contributed equally to this article. tinated, leading to stabilization and accumulation in the cytosol and nucleus (5). Many transcriptional targets of Nrf2 have been Corresponding Author: David Stokoe, Calico, 1170 Veterans Boulevard, South fi San Francisco, CA 94080. Phone: 650-267-7935; E-mail: [email protected] identi ed, some of which counteract the cellular increases in ROS. For example, multiple components of the glutathione biosyn- Cancer Res 2019;79:4828–39 thesis pathway are direct Nrf2 target genes, as well as enzymes in doi: 10.1158/0008-5472.CAN-18-2086 the pentose phosphate pathway (6), which is one of the major Ó2019 American Association for Cancer Research. mechanisms generating NADPH, an important source of reducing 4828 Cancer Res; 79(19) October 1, 2019 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst August 15, 2019; DOI: 10.1158/0008-5472.CAN-18-2086 Cellular Pathways Critical for Nrf2-Driven Proliferation power. ChIP-seq experiments in human and mouse cells have shRNA reagents are described in Supplementary Table S1. Three identified additional Nrf2 target genes under basal and stim- days after transfection, cells were split into selection media ulated conditions (7–9), thereby expanding the range of containing 2 mg/mL puromycin, and selected for 3 days. Cells biological processes known to be under Nrf2 transcriptional were then assayed for knockdown by Western blot analysis after control. However, the specific transcriptional targets of Nrf2 treatment with 500 ng/mL doxycycline for 5 days. that generate a selective advantage for cells with mutations in this pathway are not clearly defined. Some studies have siRNA-mediated depletion of Nrf2 and PRDX1 shown that supplementation with n-acetyl cysteine, a cell- Sequences of all siRNA reagents are described in Supplemen- permeable precursor of cysteine, can rescue viability defects tary Table S1. siRNA knockdown of Nrf2 and PRDX1 was con- induced by Nrf2 knockdown or knockout, under either basal ducted in 384 well plate format. Six siRNAs were tested per gene or challenged conditions (10, 11), but whether this pathway and each was tested in quadruplicate. Briefly, siRNA (20 nmol/L) represents the main survival benefit in KEAP1/Nrf2 mutant was printed to plate wells and Lipofectamine RNAiMax was added cells is not clear. In this study, we explore the consequences of in 20 mL of serum-free media. Complexes were incubated for 30 KEAP1 mutations on the requirement for Nrf2 activity under minutes at ambient temperature prior to adding cells in 20 mLof different growth environments and show that Nrf2 activity is media containing 2Â serum. Cells were incubated at 37 C for essential for growth in anchorage independent conditions. 96 hours prior to assaying with CellTiter Glo (Promega). Data Surprisingly Nrf2 dependence is uncoupled from the glutathi- were normalized on a scale set between negative (Ambion Silenc- one synthesis pathway. Rather, through a CRISPR screen, we er Select Negative Control #2, n ¼ 8) and positive (Qiagen All Stars show that the thioredoxin/peroxiredoxin/thioredoxin reduc- Cell Death Control, n ¼ 8) siRNA controls. The median activity of tase pathway is important for Nrf2-driven growth and viability. all 6 siRNAs was taken as a gene-level score. Data are shown in In addition, we find that growth factors signaling through Supplementary Table S2. IGF1R and ERBB3 are critical mediators of the growth of KEAP1-mutant cells, thus identifying multiple avenues for Western blotting, immunoprecipitation potential therapeutic intervention or biomarker discovery with- The following antibodies were used for Western blotting: Nrf2 in this mutant context. (EP1808Y) diluted 1:1,000, SLC7A11 (Cell Signaling Technolo- gy, #12691) diluted 1:1,000, actin (Cell Signaling Technology, 5125S) diluted 1:5,000, IGF1R (Cell Signaling Technology, Materials and Methods 9750S), ERBB3 (Cell Signaling Technology, 12708S), PRDX1 Cell culture and creation of doxycycline-inducible shRNA cell (Cell Signaling Technology, 5499S), TXN (Cell Signaling Tech- lines nology, 2429S), TXNRD (Cell Signaling Technology, 15140S) all All cell lines were obtained from Genentech's cell line core diluted 1:1,000, donkey anti-rabbit (NA9340, GE-Healthcare) facility gCell. STR profiles are determined for each line using the diluted 1:5,000, goat antimouse diluted 1:5,000. Monoclonal Promega PowerPlex 16 System. This is performed once and Anti-HA-agarose antibody (A2095, Sigma) was used for immu- compared with external STR profiles of cell lines (when avail- noprecipitating HA-tagged ubiquitin. Cells were grown in on able) to determine cell line ancestry. Cells are Mycoplasma tissue culture plates, lysed on ice using RIPA buffer, spun at tested before distribution. Cells were maintained in either 14,000 rpm for 20 minutes, and the supernatant diluted in RPMIorDMEMinthepresenceof10%FBS(v/v)and2 SDS-containing sample buffer. Twenty micrograms of total lysate mmol/L L-glutamine, apart from BEAS2B cells that were main- was run on either 4% to 12% or 4% to 20% Tris-glycine gels, and tained in BEGM growth media (Lonza, CC-3170). Doxycycline- transferred
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