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 substrate 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 2019 American Association for Cancer Research. mechanisms generating NADPH, an important source of reducing

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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 2 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 to polyvinylidene difluoride membranes using the inducible cell lines were maintained as above but with 10% Tet- iBlot transfer apparatus (Life Technologies). Primary and second- system approved FBS (Clontech, 631101). All cell lines were ary antibodies were diluted in 3% dried milk or 5% BSA, and cultured in a humidified incubator at 37 C/5%CO2 unless chemiluminescence detected using a fluorchem HD2 system otherwise noted, and discarded or replaced following culture (ProteinSimple). Precision Plus molecular weight markers were for 3 months. from Bio-Rad. Nrf2- and nontargeting shRNA pINDUCER 10 lentiviral expression vectors were cotransfected with pCMV-VSVG and pCMV- Two-dimensional and three-dimensional viability and dR8.9 plasmids into HEK293T cells using Lipofectamine 2000 confluency assays reagent (Life Technologies, #11668). Viral supernatants were For two-dimensional (2D) assays, cells were seeded at 2,000 collected after 48 hours and were used with 8 mg/mL of polybrene cells/well in 96W plates, treated with 500 ng/mL doxycycline or reagent (Millipore, #TR-1003-G) to infect cell lines. Stably trans- the appropriate compound, and viability was assessed using formed cell line pools were generated by placing the transduced CellTiter-Glo reagent (Promega, #G7572), which measures cells under 2 mg/mL puromycin selection for a minimum of cellular ATP levels as a surrogate for cell number and growth, 2 weeks. although changes in cell metabolism could also contribute to For all additional targets, A549 or H460 cells were seeded this assay. Cell confluency was examined using the IncuCyte in 6-well plates and grown to 50% confluence in media Live Cell Imaging system (Essen Bioscience). For soft agar containing Tet-free FBS. The cells were then cotransfected assays, a base layer of 0.8% agar (in RPMI) was dispensed into with 250 ng of PiggyBac transposase expression plasmid (pBO, each well of a 12W plate (1 mL base layer per well), and Transposagen) and 750 ng of a PiggyBac transposon plasmid allowedtosolidifyat4C. Cells were then seeded in 1 mL containing a dox-inducible shRNA cassette (pBInducer- 0.4% agar (in RPMI) on top of the base layer and allowed to OD_tRFP_miRE, Genentech), using Lipofectamine 3000 trans- solidify. Finally, 1 mL of medium was added on top of the cell fection reagent (Thermo Fisher Scientific). Sequences of all layer and plates were carried in a 37 Cincubator/5%CO2.

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Compounds and/or doxycycline were added to the plates in the Measurement of glutathione levels final 1 mL of medium, at 3 concentration to account for Cells were seeded onto 6-well dishes overnight at 37C, 5% the total volume of agar and medium. Fresh medium and CO2 using complete growth medium (RPMI1640 þ 10% FBS þ compound were added as needed. Once colonies reached 1% L-glu) to nearly 100% confluent. appropriate size for quantitation, photos were taken and the After cells in 6-well dish were washed twice with cold 1 colonies were stained by adding 100 mL12mmol/LMTTper PBS,0.5mLof0.5Nperchloricacidwasaddedtoeachwell. well, and allowed to stain at 37C. Colonies were then imaged/ The cells were scraped off and the contents were transferred to quantified via Gelcount. 1.5 mL eppendorf tubes. Additional 0.5 mL perchloric acid was added to each well to recover any remaining contents and Lung tumor xenograft models combined. The samples were sonicated for 30 seconds in water Eleven- to 12-week-old female C.B-17 SCID.beige mice sonication bath, then centrifuged at 10,000 rpm at 4Cfor5 (Charles River Laboratories) were subcutaneously inoculated in minutes, and the supernatants were stored at 80Cbefore the right lateral flank with 10 106 A549 cells stably transduced analysis. with respective shRNA vectors in 100 mL Hank's Balanced Salt Cells were also harvested by trypsin and then counted on Vicell Solution/Matrigel (BD Biosciences) or with 10 106 shRNA- to give estimated cell counts and average diameter cell size for expressing H441 cells in 100 mL Hank's Balanced Salt Solution subsequent calculations. per mouse. All experiments using mice were approved by the Samples were analyzed by LC/MS-MS. The chromatography was Genentech Institutional Animal Care and Use Committee performed on Shimadzu Nexera on a 100 4.6 mm, 3 m,Hypercard (IACUC). When tumor volume reached approximately 150 to column. The sample injection volume was 1 to 3 mL. The com- 250 mm3, mice were randomized to receive drinking water pounds were eluted by a linear gradient of 0.1% formic acid to containing 1 mg/mL doxycycline (in 5% sucrose) or no doxycy- acetonitrile containing 0.1% formic acid in 5 minutes. Mass cline (5% sucrose alone) ad libitum. The doxycycline was replaced spectrometry analysis used AB Sciex 5500 in a positive mode. 3 times a week and the sucrose replaced once a week. Tumor The MRM transitions were 308/179 for glutathione and 311/182 volumes were determined using digital calipers (Fred V. Fowler for isotopically labeled glutathione as international standard. Company, Inc.) using the formula (L W W)/2 and plotted as mean tumor volume (mm3) SEM. Tumor growth inhibition ROS measurements (%TGI) was calculated as the percentage of the area under the A549 cells were reverse transfected with Nrf2 or Control fitted curve (AUC) for the respective dose group per day in relation nontargeting siGenome pools (Dharmacon) using Dharmafect to the vehicle, such that %TGI ¼ 100 1 (AUC treatment/day)/ 3 reagent (Dharmacon). Two days posttransfection, cells (AUC vehicle/day). In a separate study, mice with 150 to 250 mm3 were treated with 50 mmol/L erastin (Sigma-Aldrich, #E7781), tumors were dosed with 1 mg/mL doxycycline for 5 days before 1 mmol/L glutathione, 50 mmol/L tert-butyl hydrogen peroxide the tumors were excised and analyzed by Western blotting for or DMSO control for 12 hours, then labeled with 4 mmol/L DCF Nrf2 levels. All individuals participating in animal care and use are (InVitrogen, #C6827 or Abcam, ab113851) for 30 minutes as required to undergo training by the institution's veterinary staff. per manufacturer protocol. Then, cells were removed from Any procedures, including handling, dosing, and sample collec- plates and analyzed with a FACS Caliber 4 instrument or a tion mandates training and validation of proficiency under the fluorescent plate reader. direction of the veterinary staff prior to performing procedures in experimental in vivo studies. All animals were dosed and moni- CRISPR-modified xenografts tored according to guidelines from the IACUC on study protocols A549.pLENTI6.3-Cas9 cells were transduced with individual approved by Genentech's Laboratory Animal Resource Commit- sgRNA expressing lentiviral particles, selected for stable inte- tee at Genentech, Inc. gration using 2 mg/mL puromycin for 5 days, expanded, and injected subcutaneously into the left side flank of Nu/Nu mice RNA-seq at 5 106 cells per mouse. Primary xenografts were measured A549 and H441 cells were plated overnight and then treated via caliper. Fifteen and 30 days after transplantation, 7 mice with a final concentration of 0.5 mg/mL doxycycline for 24 or respectively were sacrificed and primary tumors were manually 48 hours. Cells were harvested and total RNA isolated as previ- dissected. Primary tumors were flash frozen in liquid nitrogen. ously described (12). Samples were prepared and sequenced All animal work was performed with approval by the IACUC at using MiSeq on an Illumina instrument. Genentech Inc. RNA-seq data were aligned to the human reference genome (GRCh37/hg19) using GSNAP version 2013-10-10 (13) (para- meters: -M 2 -n 10 -B 2 -i 1 -N 1 -w 200000 -E 1 –pairmax-rna Results ¼ 200000). Gene expression levels for RefSeq genes were KEAP1-mutant lung cancer cells are selectively addicted to quantified based on counts and RPKM (reads per kilobase of Nrf2 target and million reads sequenced) values. We previously used a panel of wild-type (WT) and mutant KEAP1 lung cancer cell lines to define a Nrf2 gene signa- Cystine uptake assays ture(15).Herewewishedtoexploretheconsequencesof 0 Cystine L-[3,3 -14C] (Perkin-Elmer, #NEC845050UC) was used KEAP1 mutations on Nrf2 levels and activity using a panel of in uptake experiments in A549 cells as previously described (14). A lung cancer cell lines. Blotting for Nrf2 levels in KEAP1 and total of 1 10e6 cells/well were plated in 6-well plates. Following Nrf2 mutant cell lines showed a strong increase in both the treatments with either doxycycline (0.5 mg/mL) for 48 hours cytosol and the nucleus compared with WT lines (Supplemen- and/or 50 mmol/L erastin (Sigma-Aldrich, #E7781) for 12 hours. tary Fig. S1A), which was independent of changes in Nrf2

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A Nrf2 Nrf2 Nrf2 Nrf2 Nrf2 Nrf2 sh1 sh3 sh10 sh1 sh3 sh10 dox - + - + - + dox - + - + - + Nrf2 Nrf2 A549 Calu6 β-Actin β-Actin Nrf2 Nrf2 H441 β-Actin H460 β-Actin Nrf2 Nrf2 KEAP1 H1437 Calu1 β-Actin KEAP1 β-Actin WT mut Nrf2 Nrf2 H1355 H1048 β-Actin β-Actin Nrf2 Nrf2 H2122 H1838 β-Actin β-Actin Nrf2 H2228 Nrf2 β-Actin Nrf2 BEAS2B mut β-Actin Nrf2 β-Actin EBC1 CD B Filled symbols - NTCs P = 0.01 Open symbols - Nrf2 shRNAs P = 0.04 100 100 ***** ** ) ) ) 100

50 50 50 Cell ATP levels Cell ATP levels Cell ATP levels normalized to NTCs ( normalized to NTCs normalized to NTCs ( ( 0 0 0

A549 H460 H441 H1437H1355H2122 EBC1H2228 H1048 Calu1 Calu6 KEAP1 mut KEAP1 WT KEAP1 mut KEAP1 WT BEAS2B 5% Sucrose 1 mg/mL dox E S D S D S D

Nrf2 Nrf2 * *

5% Sucrose *** 400 5% Sucrose F 800 1 mg/mL Doxycycline 1 mg/mL Doxycycline

) *** )

3 ** 3 300 600 *** ***

*** 400 200 **

200 100 Tumor volume (mm Tumor volume (mm 0 0 0 102030 0 5 10 15 20 25 Time (days) Time (days) A549 – KEAP1-mutant H441 – KEAP1 WT

Figure 1. Nrf2 knockdown decreases ATP levels of mutant KEAP1 cell lines. A, The indicated cell lines were infected with lentiviruses expressing three independent Nrf2 shRNA sequences (sh1, sh3, sh10) and, following puromycin selection, were incubated for 48 hours 500 ng/mL doxycycline. Nrf2 levels were detected by Western blotting. B, The cell lines shown in A were incubated 500 ng/mL doxycycline for 7 days and then cell ATP levels assessed using CellTiter-Glo. Each circle represents a unique gRNA and is the average of 6 technical replicates, and values were normalized to the average percentage viability (as assessed using CellTiter-Glo assays to measure cellular ATP content) of three independent NTCs þ dox. The average value for the three independent target shRNAs is shown by the boxplot. C, The averages from the three target shRNAs in B for each WT and mutant KEAP1 cell line are plotted, and significance was calculated using a Student t test. D, The ATP levels of 39 lung cancer cell lines following treatment with Nrf2 siRNA relative to NTC treatment. Cells are grouped by KEAP1 genotype. Significance was calculated using a Student t test. E, Mice were implanted with A549 or H441 cells expressing Nrf2 sh10. When tumors reached 200 mm3, 1 mg/mL doxycycline (D) or 5% sucrose (S) was added to the drinking water. Five days later, tumor extracts were Western blotted for Nrf2. The asterisk indicates a putative nonspecific band that is seen using the Nrf2 antibody in some cell lines. F, Mice were implanted with A549 or H441 cell lines expressing Nrf2sh10. When tumors reached 200 mm3, mice were randomized into groups of 10, and either 1 mg/mL doxycycline or 5% sucrose added to the drinking water. Tumors were measured over a 28-day period. Error bars, SEM (n ¼ 10). Differences between doxycycline and sucrose treated animals were assessed using Student t test. , P < 0.05; , P < 0.01; , P < 0.001.

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mRNA (Supplementary Fig. S1B). These suggested posttrans- down. Similar negative results were seen with the more cell lational regulation of Nrf2 by KEAP1. Indeed, phosphorylated permeable glutathione precursor N-acetyl cysteine (Supplemen- Nrf2 was detected (e.g., Supplementary Fig. S1C), and ubiqui- tary Fig. S6D). Exogenously added reduced glutathione was able tinated Nrf2 could be detected in some WT KEAP1 cell lines to reduce reactive oxygen levels, as measured by dichlorofluor- following proteasomal inhibition, but not mutant cell lines escein staining (Fig. 2D). (Supplementary Fig. S1D and S1E). We next wanted to examine the consequences of loss of Nrf2 Induction of ROS is not sufficient to decrease viability of across WT and mutant KEAP1 and Nrf2 cell lines. We therefore KEAP1-mutant lung cancer cells established stable cell lines expressing 3 independent Nrf2 To explore the effects of the glutathione pathway in Nrf2 shRNAs under the control of doxycycline, as well as 3 indepen- responses in more detail, we monitored the expression and dent nontargeting controls (NTCs and full blots shown in Sup- activity of the xCT glutamate/cysteine antiporter, 1 of the rate- plementary Figs. S2 and S3). Fig. 1A shows that these Nrf2 shRNAs limiting steps in glutathione synthesis. As expected from a direct were effective at reducing Nrf2 protein levels in 5 mutant KEAP1, 2 Nrf2 target gene (15–17), SLC7A11 expression was reduced mutant Nrf2, and 5 WT lung cancer cell lines, as well as in following Nrf2 knockdown (Fig. 3A), causing a decrease in cystine immortalized but nontransformed lung epithelial BEAS2B cells. uptake (Fig. 3B). Concurrent with this, Nrf2 knockdown also Upon doxycycline addition, ATP levels (a surrogate for viability) caused a large increase in ROS levels (Fig. 3C). To determine of most cell lines were decreased to varying extents, with the whether inhibition of SLC7A11 expression and cystine uptake mutant KEAP1 cell lines generally resulting in larger decreases contributed to decreased viability following Nrf2 knockdown, we (Fig. 1B), a consequence that was statistically significant (Fig. 1C). inhibited xCT function using erastin (18). This compound inhib- Knockdown of Nrf2 by siRNA in a larger panel of lung cancer cell ited cystine uptake (Fig. 3B), and induced large increases in lines confirmed a genotype-dependent effect (Fig. 1D; Supple- oxidative stress (Fig. 3C). However, this was not sufficient to mentary Table S2). decrease the viability of the KEAP1-mutant cell line A549 We next determined the consequence of Nrf2 knockdown in (Fig. 3D), or indeed most KEAP1-mutant cell lines (Supplemen- tumor xenografts. The KEAP1-mutant A549 cell line and the tary Fig. S7A). The combination of erastin and Nrf2 knockdown KEAP1 WT H441 cell lines expressing dox-inducible Nrf2 shRNAs did result in a dramatic decrease in cell viability however were implanted into the flanks of female SCID mice. Fig. 1E shows (Fig. 3D). Similarly, the glutathione synthase inhibitor BSO or the effective knockdown of Nrf2 in doxycycline treated mice in the glutaminase inhibitor BPTES also did not display preferential both tumors (full blots shown in Supplementary Fig. S4). Fig. 1F toxicity for KEAP1-mutant cell lines (Supplementary Fig. S7B and shows that Nrf2 knockdown in the KEAP1-mutant A549 line S7C). Therefore, supplementation with glutathione is not suffi- shows a dramatic effect on tumor growth, resulting in complete cient to rescue viability effects induced by Nrf2 knockdown, nor is tumor regression in 5/10 tumors. In contrast, the effect on depletion of glutathione sufficient to decrease viability of KEAP1- H441 growth was more modest, showing a 37% reduction in mutant cell lines. tumor growth, with all animals displaying maintained tumor burden. A Nrf2 target gene CRISPR screen reveals pathways important for viability Nrf2 requirement depends on growth conditions and is To understand the potential importance of pathways activated independent of glutathione metabolism due to Nrf2 activation/KEAP1 loss, we performed a pooled, To understand the dramatic contrast, we observed between the lentiviral CRISPR screen using a library of genes either decreased effects of Nrf2 knockdown on tumor propagation in xenografts in expression upon Nrf2 knockdown in A549 cells, and/or versus growth on plastic we tested several additional cell culture elevated in a panel of KEAP1-mutant lung cancer cell lines environments that might underlie differences between these relative to Keap1 WT (Supplementary Table S3). As distinct conditions. Nrf2 knockdown in cells grown in low adherence consequences were observed following Nrf2 knockdown in 2D, plates, low oxygen (0.5%), or a combination of both, showed three-dimensional (3D), and xenograft growth conditions, we similar consequences to cells grown on plastic (Supplementary performed the screen in KEAP1-mutant A549 cells under all 3 Fig. S5A). In contrast, the growth of KEAP1-mutant cell lines upon environments to determine whether discrete dependencies could Nrf2 knockdown was severely compromised when cultured in be found. The full data for the screen is in Supplementary soft agar (Fig. 2A and B, NTC treatments shown in Supplementary Table S4. Fig. 4A–C show the 15-day time point for all 3 condi- Fig. S5B), on micropatterned plastic films ("Scivax," Supplemen- tions, illustrating that all 3 screens performed similarly, with tary Fig. S6A and S6B), or in methyl cellulose (Supplementary gRNAs representing only a small number of genes showing Fig. S6C). We therefore used growth in soft agar to characterize the significant drop-out (Table 1). NFE2L2 and its binding partner consequences of Nrf2 knockdown in more detail. Although MAFG were among the most significantly depleted genes, showing knockdown of Nrf2 completely abolished colony formation in that the screen performed as expected. The pentose phosphate 3 KEAP1-mutant cell lines, it had almost no effect in H1048 and pathway genes PGD, G6PD, and TKT, known Nrf2 target H441, 2 WT KEAP1 lung cancer cell lines (Fig. 2A and B). We also genes (19), also showed strong drop-out. However, these genes addressed the role of the glutathione pathway in the response to show depletion in many cell lines in CRISPR screens (20), so they Nrf2 knockdown, as this pathway has previously been shown to are unlikely to mediate the strong genotype-dependency of Nrf2 mediate survival properties facilitated by high Nrf2 activity (10). knockdown. The other strong hits in the screen were 2 growth Knockdown of Nrf2 decreased reduced glutathione levels factor receptors, IGF1R and ERBB3, and 3 components of a redox (Fig. 2C). Although addition of reduced glutathione generally signaling relay, PRDX1, TXN, and TXNRD1 (21). These latter increased the ability of all tested cell lines to form colonies in soft 3 genes were selected for inclusion in this CRISPR library due to agar, it was unable to rescue the consequences of Nrf2 knock- their elevated expression in KEAP1-mutant lung cancer cell lines,

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P A -dox,-GSH +dox,-GSH -dox,+GSH +dox+GSH C P < 0.0001 = 0.0004 1.2 A549 1 0.8

H1437 0.6

mol/L/mL cell) 0.4 μ 0.2 H460 GSH ( 0 A549 A549 H1437 H1437 -dox +dox -dox +dox H1048

H441

Calu6

B 1,400 1,200 3,000 945A 7341H 064H -dox,-GSH 1,200 1,000 2,500 +dox,-GSH 1,000 800 2,000 -dox,+GSH 800 600 1,500 +dox,+GSH 600 400 1,000 Colonies 400 200 200 500 0 0 0 NTCsh1 NTCsh2 Nrf2sh1 Nrf2sh10 NTCsh1 NTCsh2 Nrf2sh1 Nrf2sh10 NTCsh1 NTCsh2 Nrf2sh1 Nrf2sh10 12,000 800 3,000 8401H 144H 6ulaC 700 10,000 2,500 600 8,000 500 2,000 6,000 400 1,500 300

Colonies 4,000 1,000 200 2,000 100 500 0 0 0

D 12,000 siNTC 10,000 siNrf2 8,000 6,000 4,000 2,000 ROS (DCF RFU) 0

No Trx TBHP

GSH 0.1GSH mmol/L 1.0 mmol/L GSH 10.0 mmol/L

TBHP TBHP+ GSH +TBHP 0.1 GSH mmol/L + 1.0 GSH mmol/L 10.0 mmol/L

Figure 2. Requirement for Nrf2 under anchorage independent growth is independent of exogenously added reduced glutathione. A, The indicated cell lines stably expressing Nrf2sh1 were plated in soft agar and treated with either vehicle, 500 ng/mL doxycycline (dox), or 1 mmol/L reduced glutathione (GSH) as indicated for the duration of the experiment. Representative areas of the plate were photographed. B, Quantiﬁcation of the colonies shown in A from biological triplicate wells. Error bars represent SD from triplicate wells, and P values are from a Student t test. C, GSH levels were measured in A549 and H1437 cells in the absence or presence of 500 ng/mL doxycycline. Error bars, SD (n ¼ 4). D, ROS levels in A549 cells under the conditions shown as measured using 20,70- dichlorodihydroﬂuorescein diacetate (H2DCFDA). Error bars, SD from triplicate wells.

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A Hr post dox 0 8 24 48 72 96 156 Nrf2sh10-dox SLC7A11 β-Actin

Nrf2sh10+dox SLC7A11

β-Actin B 60,000 cont Erastin 10 μmol/L μ 40,000 Erastin 50 mol/L Erastin 250 μmol/L +dox 20,000 Erastin 10 μmol/L+dox Erastin 50 μmol/L+dox Erastin 250 μmol/L+dox 14C-cystine uptake 14C-cystine 0 -dox+dox -dox +dox NTC0 Nrf2sh10 C D 100 4,000,000

80 3,000,000 0 ng/mL dox 60 50 ng/mL dox CTNhs 2,000,000 150 ng/mL dox 40 600 ng/mL dox

0 ng/mL dox 01hs2frN

20 Cell ATP levels 1,000,000 50 ng/mLdox

Normalized to mode 150 ng/mL dox 0 units) (luminescence 600 ng/mL dox 102 103 104 105 0 ROS (DCF) 0.0001 0.001 0.01 0.1 1 10 100 Erastin (μmol/L) Cells only Cells+DCF Cells+DCF+Nrf2sh10 Cells+DCF+Erastin Cells+DCF+Nrf2sh10+Erastin

Figure 3. Effects of Nrf2 knockdown on SLC7A11 expression and cystine uptake. A, A549 cells expressing Nrf2 sh10 were treated with vehicle or 500 ng/mL doxycycline (dox) for the indicated time points and were Western blotted using SLC7A11 and actin antibodies. B, A549 cells expressing NTC1 or Nrf2 sh10 were incubated with vehicle or doxycycline for 48 hours, then incubated with 0.5 mCi 14C-Cystine for 20 minutes. Four hundred mmol/L cold cystine and 400 mmol/L erastin were added at uptake (cold cystine) or 20 hours before uptake (erastin) as controls. Cells were lysed and intracellular cystine was measured by liquid scintillation counting. C, ROS levels were measured using H2DCFDA in A549 cells subjected to Nrf2 siRNA, erastin (50 mmol/L), or both for 24 hours. D, A549 cells expressing NTC1 or Nrf2 sh10 were treated with the indicated concentrations of erastin for 72 hours, in the absence or presence of the indicated concentration of doxycycline, and cell ATP levels were measured using CellTiter-Glo.

as well as their decreased expression following Nrf2 knockdown the most effective 2 for further study (Supplementary Fig. S9A). in KEAP1-mutant A549 cells (Supplementary Table S3; Supple- Despite effective knockdown, reducing the expression of these mentary Fig. S8A–S8F). ERBB3 was selected due to its decreased proteins had minimal consequences on cell proliferation, either expression following Nrf2 knockdown (although its expression in 5-day CellTiter-Glo assays, 12-day clonogenic growth assays, or was also elevated in KEAP1-mutant lung cancer cell lines (Sup- 14-day soft agar assays (Supplementary Figs. S9B–S9E). siRNA plementary Fig. S8G, S8H) and IGF1R was selected due to its knockdown of PRDX1 also showed minimal effect on growth in a elevated expression in KEAP1-mutant cell lines (although its 5-day CellTiter-Glo assay in a panel of 39 lung cancer cell lines expression was also decreased following Nrf2 knockdown (Sup- (Supplementary Fig. S9F; Supplementary Table S2). One differ- plementary Fig. S8I, S8J). ence between these validation assays and the CRISPR screen is that We created stable A549 cell lines expressing 7 independent dox- the screen is performed under competitive growth conditions regulated hairpins against PRDX1, TXN, and TXNRD1, and chose where the majority of the neighboring cells do not lack these

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Cellular Pathways Critical for Nrf2-Driven Proliferation

A B 1 PRDX1 1 2D TXNRD1 -4 -3 -2IGF1R -1 TXN MAFG -1 TXNRD1 2D NFE2L2 ERBB3-2 -4 -3 -2 -1 1 MAFG PRDX1 -3 IGF1R xeno TXN -1 C 1 ERBB3 PRDX1 NFE2L2 3D 3- 2- 1- 1 -2 TKT IGF1R TXN -1 MAFG NFE2L2 -3 ERBB3 -2 3D -3 D CTN 01hs2frN xeno

E 50 120

45 100 40

35 80 NTC_av 30 NTC_av PRDX1sh3 PRDX1sh3 PRDX1sh7 PRDX1sh7 25 60 TXNsh1 TXNsh1 20 TXNsh2 TXNsh2 TXNRD1sh5

Total confluence (%) TXNRD1sh5

Red confluence (%) TXNRD1sh7 40 15 Nrf2sh10 TXNRD1sh7 Nrf2sh10 10 20 5

0 0 136 154 172 190 208 236 254 136 154 172 190 208 236 254 sruoH sruoH

Figure 4. TXN, PRDX1, and TXNRD1 are required for KEAP1-mutant lung cancer cells under competitive growth conditions. A, A549 cells were infected with lentivirus (0.3 MOI at 1,000 coverage) expressing a gRNA library comprising 481 Nrf2/KEAP1 target genes and 37 control genes. Puromycin-resistant cells were then plated into 2D plastic tissue culture ﬂasks, grown in methyl cellulose, or implanted into nude mice. After various time points, cells were collected, genomic DNA was

isolated and PCR amplified, and gRNAs identified by Next Gen sequencing. Average gRNA expression per gene (log2-fold change relative to 2D, day 0) at the 15-day time point for 2D and 3D growth is shown. B and C, Same as A, but plots for 2D vs. xenograft and 3D vs. xenograft are displayed. D, Parental A549 cells were plated 1:1 with A549 cells expressing either NTC or Nrf2sh10 shRNAs and treated with 500 ng/mL doxycycline. Cells were imaged for total and red cell confluence 5 days later. E, Parental A549 cells were mixed 1:1 with A549 cells expressing the indicated shRNAs and treated with 500 ng/mL doxycycline. Cells were split after 5 days and red (left) and total (right) cell confluence were quantified over the following 5 days.

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Vartanian et al.

Table 1. All genes whose aggregate gRNAs showed a signiﬁcant (P < 0.05) reduction in abundance at the 15-day time-point in any 1 of the 3 growth conditions Gene symbol Gene name log2FC 2D log2FC 3D log2FC xeno PRDX1 Peroxiredoxin 1 −3.42 −0.597 −0.0625 TXN Thioredoxin −2.44 −0.873 −0.766 PGD Phosphogluconate dehydrogenase −2.43 −1.19 −0.478 −2.29 −1.81 −1.97 NFE2L2 Nuclear factor, erythroid 2-like 2 −1.48 −0.762 −0.351 IGF1R Insulin-like growth factor 1 receptor −1.15 −0.0168 −0.064 TXNRD1 Thioredoxin reductase 1 −1.1 −0.0677 −0.229 G6PD Glucose-6-phosphate dehydrogenase MAFG v-maf avian musculoaponeurotic ﬁbrosarcoma oncogene homolog G −1.08 −0.46 −1.07 −1.05 −0.276 −0.43 TKT Transketolase ERBB3 v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 3 −0.882 −1.51 −1.98 MAPT Microtubule-associated protein tau −0.839 −0.932 −0.927 KNTC1 Kinetochore associated 1 −0.764 −0.214 −0.366 AK4 Adenylate kinase 4 −0.723 −0.904 −0.33 NFKBID Nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, delta −0.64 −0.74 −0.827 SQSTM1 Sequestosome 1 −0.595 −0.702 −0.18 IGLON5 IgLON family member 5 −0.471 −0.38 −0.472 GPD1 Glycerol-3-phosphate dehydrogenase 1 (soluble) −0.411 −0.652 −0.482 SLCO2B1 Solute carrier organic anion transporter family, member 2B1 −0.401 −0.324 −0.723 −0.239 −1.83 −0.136 NAMPT Nicotinamide phosphoribosyltransferase −0.221 −0.643 −0.923 HNF4A Hepatocyte nuclear factor 4, alpha NOTE: Genes are ranked by their log-fold change in the 2D condition. The top 10 genes are colored by biological associations.

proteins. Therefore, we tested whether knockdown of these genes not show strong effects in 5-day CellTiter-Glo assays on plastic could reduce growth of cells when cultured together with parental (Supplementary Fig. S14A). However, knockdown of IGF1R A549 cells. Induction of shRNA expression is accompanied by showed substantial inhibition of colony growth in soft agar expression of RFP in these vectors (22). The area of red expressing (Fig. 5B; Supplementary Fig. S14B). We next tested the potent cells can therefore be used as an indicator for shRNA expressing and selective IGF1R small molecule inhibitor OSI-906/linsiti- cells, and measured independently from total cell area. As a nib (25). Similar to IGF1R knockdown, this compound showed positive control, Fig. 4D shows that induction of Nrf2 shRNA little effect on ATP levels when tested in 3 WT and 3 mutant reduces the red area compared with the negative control NTC KEAP1 lung cancer cell lines grown in 2D (Supplementary shRNA. A modest reduction in competitive proliferation could be Fig. S14C). However, this compound was very potent at inhi- detected for PRDX1, TXN, and TXNRD1 shRNAs 5 days following biting colony growth of A549 cells in soft agar (IC50 20 nmol/ induction (Supplementary Fig. S10A), but this was more dramatic L). Moreover, when tested against a large panel of lung cancer when these cells were split and then monitored for a further 5 days cell lines, there was a selective effect of this compound inhibit- (Fig. 4E). A similar effect was also seen in H460 cells; little effect of ing colony growth of KEAP1-mutant cell lines (Fig. 5C and D). PRDX1, TXN, or TXNRD1 knockdown on cellular ATP levels, This is true for cells cultured both in the presence (Fig. 5C and clonogenic, or anchorage-independent growth (Supplementary D) and absence (Supplementary Fig. S14D and S14E) of Figs. S10B–S10E), but decreased representation of knockdown reduced glutathione. A similar selective effect on KEAP1- cells in competitive growth assays (Supplementary Figs. S11A). mutant cell lines when grown under anchorage independent The disconnect between competitive and noncompetitive growth conditions was also seen with an independent IGF1R inhibitor was also confirmed using a completely orthogonal method to NVP-AEW541 (Supplementary Fig. S14F). Interrogation of the induce gene ablation, using gRNAs against the target genes of Broad Institute's Project Achilles genome-wide shRNA knock- interest (Supplementary Fig. S11B–S11D). down and CRISPR datasets support the relationship between KEAP1-mutant lung cancer and sensitivity to IGF1R (Fig. 5E). KEAP1-mutant lung cancer cells are selectively dependent on Moreover, the cell lines that were most sensitive to linsitinib IGF1R signaling for anchorage-independent growth treatment were also the most sensitive to IGF1R depletion Another class of hits were the growth factor receptors ERBB3 (Supplementary Fig. S14G). In support of this, some KEAP1- and IGF1R. ERBB3 knockdown had minimal effects on the ATP mutant cell lines [A549 (26, 27), NCI-H838, NCI-H322 (28)] levels of A549 cells grown in 2D, or the colony number seen in have been shown to be sensitive to IGF1R inhibition in vivo. 3D environments (Supplementary Fig. S12A–S12C). Consis- Therefore, dependence on IGF1R signaling may represent a tent with this, the use of 2 independent ERBB3 inhibitory feature of KEAP1-mutant lung cancers. antibodies (23) also had no effect on A549 ATP levels in 2D or colony growth in 3D conditions (Supplementary Fig. S13A and S13B). However, we could demonstrate the requirement Discussion for ERBB3 in A549 tumor propagation in a xenograft model It is important to identify the spectrum of genes regulated (Supplementary Fig. S13C), confirming previous observa- by transcription factors that act as oncogenes. Several studies tions (24). Administration of the YW57.88.5 antibody showed have performed this analysis for Nrf2, predominantly using a 65% reduction in tumor growth, with no effect on body ChIP-seq and RNAseq-based approaches (7–9). Although mod- weight (Supplementary Fig. S13C). ulation of glutathione synthesis is clearly a well-documented and Effective inducible shRNAs against IGF1R were designed and important consequence of Nrf2 activity, we were surprised to find established in A549 cells (Fig. 5A). Knockdown of IGF1R did that it does not seem critical for the oncogenic functions of Nrf2 in

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Cellular Pathways Critical for Nrf2-Driven Proliferation

A Par sh1 sh2 sh3 sh4 sh5 sh6 sh7 Dox − − + − + − + − + − + − + − + IGF1R

β-Actin B 2,500 D P = 0.001 NS 200

2,000 NS 150 P < 0.0001 P < 0.0001 1,500 −Dox 100 +Dox

Colonies 1,000 linsitinib AUC 50 500

0 0 NTC1 NTC2 IGF1Rsh2 IGF1Rsh4

KEAP1 mut KEAP1 WT C E H650 P H441 = 0.017 P Calu-6 = 0.003 0.2 5 KNS-62 100 H1048 H1299 0.0 Calu-3

A427 score z HCC827 −0.2 0 H460 50 H1437 −0.4 H322T MOR Demeter H1355 −0.6 LXF-289 −5 Ceres gene effect score Ceres gene effect Colony number (% DMSO) Rerf-LC-MS 0 A549 −0.8 0.001 0.01 0.1 1 linsitinib (μmol/L)

WT KEAP1 WT KEAP1 MUT KEAP1 MUT KEAP1

Figure 5. IGF1R signaling is selectively required for proliferation of KEAP1-mutant cells in anchorage independent growth. A, Seven independent doxycycline (Dox)-inducible shRNA's targeting IGF1R were transfected into A549 cells using the PiggyBac system and selected with 2 mg/mL puromycin. Western blots were performed 72 hours following 5 daysoftreatmentwith500ng/mLdoxycycline.B, A549 cells expressing NTC or IGF1R shRNAs were plated into soft agar, then treated 500 ng/mL doxycycline for 24 hours. Colonies were stained with MTT and counted after 14 days. Error bars, SD from triplicate wells. P values were calculated using Student t test. C, The indicated cell lines were grown in softagarinthepresenceof1mmol/L reduced glutathione and treated with either vehicle (0.1% DMSO) or increasing concentrations of the IGF1R inhibitor linsitinib. Colonies were stained with MTT and counted when colonies reached sufﬁcient sizes for imaging (10–15 days). KEAP1 WT and mutant cell lines are colored black and red respectively. Error bars, SD of triplicate wells. D, AUCs of data from C were calculated and P values calculated using a Student t test. E, Dependencies of lung cancer cell lines by KEAP1 mutation/deletion following IGF1R deletion (left), or knockdown (right). Data were downloaded from Depmap.org (18Q3 release), and P values are from a Student t test. NS, nonsigniﬁcant.

the context of KEAP1 mutations described in this study. Neither inhibitors did not show a preferential effect on KEAP1-mutant excess reduced glutathione nor its precursor N-acetyl cysteine cell lines, despite causing large increases in cellular ROS levels. In could rescue the consequences of Nrf2 knockdown on 2D or addition, several glutathione pathway genes present in the Nrf2 3D growth. Similarly, glutathione synthesis or cystine uptake target gene library, did not show any drop-out in any of the

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Vartanian et al.

conditions used in the CRISPR screen. The glutaminase inhibitor scription itself, as IGF1R mRNA is elevated in KEAP1-mutant BPTES did show a trend for more activity in KEAP1-mutant lung cell lines (Supplementary Fig. S8J) as well as primary tumors cancer cell lines (Supplementary Fig. S7B), consistent with greater (Supplementary Fig. S15D). Validation studies showed that activity of glutaminase inhibitors reported in KEAP1-deleted there was a much stronger requirement for IGF1R in 3D versus mouse lung tumor cells (29, 30). Interestingly, genetic screens 2D growth conditions. Similar observations in response to have generated conflicting data on the interaction between GLS growth factor signaling pathway inhibition have been made and KEAP1 mutations in lung cancer cell lines, with CRISPR recently (31), and more dramatic consequences of IGF1R screens showing no interaction, whereas shRNA screens showing expression have also been observed in 3D versus 2D environ- increased dependency (Supplementary Fig. S15A, data from ments in breast cancer cell lines (32). Nevertheless, IGF1R DepMap.org). Whether this represents a genuine discrepancy gRNAs were effectively depleted in the 2D arm of the CRISPR between knockout versus knockdown, or a reflection of increased screen, pointing to sensitivity of this type of screening number of cell lines in the knockdown study providing a higher approach. Although there was no expectation that hits from powered analysis, remains to be seen. This increased requirement this screen performed only in a KEAP1-mutant background for glutaminase activity may be to elevate glutamate levels to would show selectivity for KEAP1-mutant cells, IGF1R does support increased activity of the SLC7A11 glutamate/cysteine appear to show this property. Although IGF1R inhibitors have antiporter, which is highly expressed in KEAP1-deleted lung shown promise in clinical trials, progress has been hampered tumor cells (15). due to lack of robust biomarkers (33). It is possible that Proteins involved in a protein oxidation relay emerged as incorporating analysis of KEAP1 and Nrf2 mutation status, in strong hits in the CRISPR screen of Nrf2/KEAP1 target genes. addition to measuring IGF1R levels may help in identifying Peroxiredoxin and thioredoxin are thought to modulate elec- patients who might benefitmostfromthistypeoftherapy. tron flow from cellular oxidants such as hydrogen peroxide to downstream signaling proteins, thereby regulating their activ- Disclosure of Potential Conflicts of Interest ities (21). Exactly which target proteins showing differential C. Klijn has ownership interest (including patents) in Roche. G. Schaefer has oxidation by peroxiredoxin/thioredoxin are important for ownership interest (including patents) in Roche. Ryan J. Hartmaier is a senior KEAP1/Nrf2-dependent tumor growth is an important avenue scientist at Foundation Medicine, is an associate director at AstraZeneca; and has ownership interest (including patents) in Foundation Medicine and Astra- for future investigation. Proteins involved in translation have Zeneca. S.E. Martin reports receiving an other commercial research support been shown to be differentially oxidized in a Nrf2-dependent from Genentech/Roche and has ownership interest (including patents) in manner (10). Genentech/Roche. M. Merchant is an associate director at Roche/Genentech Interestingly, although peroxiredoxin, thioredoxin, and thior- and has ownership interest (including patents) in Roche/Genentech. No poten- edoxin reductase were strong hits from the CRISPR screen, their tial conflicts of interest were disclosed by the other authors. effects on the growth properties of several cell lines following knockdown or knockout were inconsequential or very modest Authors' Contributions (Supplementary Figs. S9 and S10). In contrast, when cells deplet- Conception and design: J. Lee, M. Bagniewska, D. Stokoe Development of methodology: S. Vartanian, J. Lee, M. Bagniewska, D. Zhang, ed of these proteins were grown in the presence of parental cells (a T. Cuellar, T. Lau, M.R. Costa, B. Haley, D. Stokoe condition that more closely mimics that of the screens), the effects Acquisition of data (provided animals, acquired and managed patients, on cell proliferation were more dramatic (Fig. 4; Supplementary provided facilities, etc.): S. Vartanian, J. Lee, D. Zhang, J. Tan, S.A. Watson, Figs. S10 and S11). It is not clear what underlies this discrepancy; it Y. Liang, D. Kan, R.J. Hartmaier, T. Lau, M.R. Costa, M. Merchant could be that competitive growth is a more sensitive assay that can Analysis and interpretation of data (e.g., statistical analysis, biostatistics, visualize modest effects on cell proliferation, or there might be computational analysis): S. Vartanian, J. Lee, C. Klijn, F. Gnad, L. Liu, C. Watanabe, R.J. Hartmaier, T. Lau, S.E. Martin, M. Merchant, D. Stokoe some cellular communication between knockdown and parental Writing, review, and/or revision of the manuscript: S. Vartanian, J. Lee, cells that remains to be understood. Therefore, although PRDX1, M. Bagniewska, G. Schaefer, L. Liu, D. Kan, M.R. Costa, D. Stokoe TXN, and TXNRD1 validate as hits in the CRISPR screen using Administrative, technical, or material support (i.e., reporting or organizing competitive growth assays, it is unclear whether inhibition of data, constructing databases): J. Lee, M. Bagniewska, G. Schaefer, H. Chen, these targets would be effective in tumors where mutations in B. Haley, D. Stokoe KEAP1 are generally clonal. Study supervision: D. Stokoe The requirement of KEAP1-mutant cells for growth factor signals such as ERBB3 and IGF1R was somewhat unexpected. Acknowledgments However, it is reminiscent of a recent study showing that We would like to thank the Genentech DNA/RNA sequencing, gCell (cell culture), and gCSI (drug screening) core facilities and Yuxin Liang and increased EGF signaling is also a consequence of Nrf2 activi- Honglin Chen for packaging the virus library. We would also like to thank ty (10). Interestingly, interrogation of a large number of human Shiva Malek and Jeff Settleman for helpful advice throughout this study. lung adenocarcinomas showed that EGFR mutations were the Funding for this study was supplied by Genentech Inc. and Foundation most significantly mutually exclusive genetic alteration in Medicine Inc. KEAP1 mutated tumors (Supplementary Fig. S15B, S15C). This is consistent with the notion that KEAP1-mutant tumors can The costs of publication of this article were defrayed in part by the supply their own growth factors, obviating the need for acti- payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate vating EGFR mutations. In the case of EGF, this appeared to be this fact. through Nrf2-mediated posttranslational cleavage of EGF from its precursor through the activity of ADAM10 (10). For IGF1R, Received July 8, 2018; revised November 7, 2018; accepted August 6, 2019; the dependency may arise at least in part from IGFR1 tran- published first August 15, 2019.

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www.aacrjournals.org Cancer Res; 79(19) October 1, 2019 4839

ERBB3 and IGF1R Signaling Are Required for Nrf2-Dependent Growth in KEAP1-Mutant Lung Cancer

Steffan Vartanian, James Lee, Christiaan Klijn, et al.

Cancer Res 2019;79:4828-4839. Published OnlineFirst August 15, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-18-2086

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