Inhibitor of Differentiation-1 Sustains Mutant KRAS-Driven Progression

Published OnlineFirst December 18, 2018; DOI: 10.1158/0008-5472.CAN-18-1479

Cancer Translational Science Research

Inhibitor of Differentiation-1 Sustains Mutant KRAS-Driven Progression, Maintenance, and Metastasis of Lung Adenocarcinoma via Regulation of a FOSL1 Network Marta Roman 1,2,Ines Lopez 2, Elisabeth Guruceaga3, Iosune Baraibar1,2, Margarita Ecay2, María Collantes2, Ernest Nadal4, Adrian Vallejo2, Silvia Cadenas2, Marta Echavarri-de Miguel1,2, Jae Hwi Jang5, Patxi San Martin-Uriz6, Laura Castro-Labrador6, Amaia Vilas-Zornoza6, David Lara-Astiaso6, Mariano Ponz-Sarvise1,2, Christian Rolfo7, Edgardo S. Santos8, Luis E. Raez9, Simona Taverna10, Carmen Behrens11, Walter Weder5, Ignacio I. Wistuba11, Silvestre Vicent2,12,13,14, and Ignacio Gil-Bazo1,2,12,13

Abstract

Because of the refractory nature of mutant KRAS lung survival. Mechanistically, Id1 was regulated by the KRAS adenocarcinoma (LUAD) to current therapies, identification oncogene through JNK, and loss of Id1 resulted in down- of new molecular targets is essential. Genes with a prognostic regulation of elements of the mitotic machinery via inhibition role in mutant KRAS LUAD have proven to be potential of the transcription factor FOSL1 and of several kinases within molecular targets for therapeutic development. Here we deter- the KRAS signaling network. Our study provides clinical, mine the clinical, functional, and mechanistic role of inhibitor functional, and mechanistic evidence underscoring Id1 as a of differentiation-1 (Id1) in mutant KRAS LUAD. Analysis of critical gene in mutant KRAS LUAD and warrants further LUAD cohorts from TCGA and SPORE showed that high studies of Id1 as a therapeutic target in patients with LUAD. expression of Id1 was a marker of poor survival in patients harboring mutant, but not wild-type KRAS. Abrogation of Id1 Significance: These findings highlight the prognostic sig- induced G2–M arrest and apoptosis in mutant KRAS LUAD nificance of the transcriptional regulator Id1 in KRAS-mutant cells. In vivo, loss of Id1 strongly impaired tumor growth and lung adenocarcinoma and provide mechanistic insight into maintenance as well as liver metastasis, resulting in improved how it controls tumor growth and metastasis.

Introduction molecular targets might address this deﬁciency. Recent data Lung cancer is the principal cause of cancer-related mortality illustrate that single genes that are markers of poor survival only (1). The estimated 5-year survival for advanced non–small cell in patients with KRAS mutations have a critical role in tumor lung cancer (NSCLC) is below 5% (2). Although some patients homeostasis (5–7). KRAS oncogene activation sustains a pro- with NSCLC, such as those with EGFR mutations and ALK or ROS- oncogenic phenotype involving multiple cancer hallmarks (8). 1 rearrangements are susceptible to tailored treatments (3), However, different types of stress, including DNA damage/repli- patients with KRAS mutations, the most frequently mutated cation stress, proteotoxic, mitotic, metabolic, or oxidative stress, oncogene (around 25%), still lack efﬁcient therapies (4). Novel may represent a cellular adaptive process in oncogene activation

1Department of Oncology, Clínica Universidad de Navarra, Pamplona, Spain. 12IdiSNA, Navarra Institute for Health Research, Pamplona, Spain. 13Centro de 2Program of Solid Tumors, Center for Applied Medical Research, University of Investigacion Biomedica en Red de Cancer (CIBERONC), Madrid, Spain. Navarra, Pamplona, Spain. 3Proteomics, Genomics and Bioinformatics Core 14Department of Pathology, Anatomy and Physiology, University of Navarra, Facility, Center for Applied Medical Research, University of Navarra, Pamplona, Pamplona, Spain. Spain. 4Thoracic Oncology Unit, Department of Medical Oncology, Catalan Institute of Oncology (ICO), L'Hospitalet de Llobregat, Barcelona, Spain. 5Klinik Note: Supplementary data for this article are available at Cancer Research fur€ Thoraxchirurgie, Universitatsspital€ Zurich,€ Zurich,€ Switzerland. 6Advanced Online (http://cancerres.aacrjournals.org/). Genomics Laboratory, Center for Applied Medical Research, University of Navarra, Pamplona, Spain. 7Phase I-Early Clinical Trials Unit, Oncology Depart- S. Vicent and I. Gil-Bazo co-shared senior authorship. ment, Antwerp University Hospital, Edegem, Belgium. 8Department of Oncol- Corresponding Author: Ignacio Gil-Bazo, Clínica Universidad de Navarra, Av. ogy, Boca Raton Regional Hospital, Boca Raton, Florida. 9Memorial Cancer Pío XII, 36, 31008 Pamplona, Spain. Phone: 0034948255400; Fax: Institute, Memorial Health Care System, Florida International University, Miami, 0034948255500; E-mail: [email protected] Florida. 10Institute of Biomedicine and Molecular Immunology (IBIM), National doi: 10.1158/0008-5472.CAN-18-1479 Research Council, Palermo, Italy. 11Translational Molecular Pathology Depart- ment, MD Anderson Cancer Center, University of Texas, Houston, Texas. 2018 American Association for Cancer Research.

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(9). Interfering with key elements sustaining cancer hallmarks was cloned into the retroviral vector pBABE-hygro (Addgene, may provide new opportunities for KRAS targeting. Some exam- plasmid #1765). FOSL1 cDNA was synthesized and cloned into ples have been provided in genetic inhibition experiments exam- the pLenti6/V5-DEST Gateway Vector (ThermoFisher Scientific, ining elements of the oxidative (10) and central carbon metab- #V49610). Wild-type and mutant (G12D) KRAS were obtained olism (11, 12), or components of the mitotic machinery (5). from the Ras Initiative (Addgene, Kit #1000000089) and cloned Unfortunately, the development of pharmacologic inhibitors has into the pLenti6-CMV (Invitrogen). Lentiviral and retroviral par- lagged behind the promising observations from these genetic ticles were produced as described earlier (16, 19). Selection inhibition studies. Thus, a deeper understanding of the molecular was carried out with Puromycin (1 mg/mL; Sigma), hygromycin mechanism involved in the regulation of stress states induced by (50 mg/mL), or blasticidin (10 mg/mL). mutant KRAS may yield novel molecular targets. Inhibitor of differentiation (Id) proteins (Id1, Id2, Id3, and Pharmacologic inhibitors Id4) are a family of highly conserved transcriptional regulators SP600125, LY294002, and SB203850 were obtained from that result both during developmental processes and in adult Sigma; BIX02189 was purchased from Tocris; trametinib, tissue homeostasis. Although Id proteins lack a DNA-binding GSK2126458 and IKK-16 were acquired from Selleckchem. domain, this family of proteins inhibits basic helix–loop–helix, ETS, and paired box (PAX) transcription factors and nontranscrip- RNA extraction and quantification tion factors of the RB family. Id proteins (mainly Id1 and Id3) are RNA extraction and analysis was done as previously published overexpressed in human cancers, including lung neoplasms, and (16). their deregulation has a direct impact on cancer initiation, maintenance, and progression (13, 14). In the largest subgroup of Western blot analysis NSCLC, lung adenocarcinoma (LUAD), high tumor expression of Western blot analysis was performed as previously described Id1 has been shown to correlate with reduced overall survival (OS; (6, 16). Antibodies used: Id1 (1:2500, #BCH-1/195-14; Bio- ref. 15). More recently, Id1 expression in cancer cells and the host check), GAPDH (1:5,000, #ab9484; Abcam), FOSL1 (1:2,000, liver microenvironment enabled liver metastasis through an #5281), and ERK1/2 (1:2,000, #9102) were Cell Signaling Tech- increased migration and colonization capacity of mouse LUAD nology, and AURKA (1:500, sc-56881), HURP (1:500, sc- cells in vivo. Furthermore, Id1 regulated some of the principal 377004), TACC3 (1:1000, sc-376900), CCNB1 (1:500, sc-245), epithelial-to-mesenchymal transition (EMT) proteins, favoring PLK1 (1:500, sc-17783), KRAS (1:200, sc-30), and b-tubulin the establishment of a premetastatic niche (16). However, there (1:2,000, sc-9104) from Santa Cruz Biotechnology. is limited evidence about the functional implications of Id1 expression in mutant KRAS LUAD. Here we provide key evidence on the clinical and functional role of Id1 in human mutant KRAS Cell viability assay and cell-cycle assay LUAD by integrating clinical information and survival outcome Cell viability and cell cycle analyses were done as previously through in vitro assays, in vivo models of progression, maintenance described (6). and metastasis, and mechanistic characterization of the effect of Id1 depletion. Apoptosis assay Apoptosis was assessed using Invitrogen Kits: an Alexa Fluor 647-conjugated Annexin V antibody in combination with 7AAD Materials and Methods and CellEvent Caspase-3/7 Green Flow Cytometry following the Cell line cultures manufacturer's instructions. Cells were acquired in FACSCanto II Human LUAD cell lines (H358, H1792, H2009, H23, H441, Cytometer (BD Biosciences) and analyzed using FlowJo software A549, HCC78, H1437, H1568, and H2126) were obtained from v9.3. ATCC. 3KT and 3KTp53kd cells were a kind gift of John Minna. The H1792.604 cell line was isolated from a liver metastasis Murine models induced by intracardiac injection of H1792 transduced with a All animal procedures were approved by the institutional reporter retroviral vector expressing thymidine kinase, GFP and Committee on Animal Research and Ethics (regional Government luciferase (17), in Rag2 / mice. All cell lines were grown accord- of Navarra) under the protocol numbers CEEA 151-14 and E2-16 ing to ATCC specifications and authenticated by the Genomics (151-14E1). For xenograft experiments, 2 106 cells (H1792.604 Unit at CIMA using short tandem repeat profiling (AmpFLSTR and H2009) infected with shRNAs to Id1 and GFP were resus- Identifiler Plus PCR Amplification Kit). Only mycoplasma neg- pended in 100 mL of DPBS and injected subcutaneously into the ative cells were used. two lower flanks of 8 weeks-old Rag2 / female mice (Harlan Laboratories). A group of mice was treated with doxycycline Silencing and overexpression plasmids diluted in the drinking water (2 mg/mL) immediately after cell Oligonucleotides for Id1 shRNA (TRCN0000019029) were: injection; a second group was administered doxycycline when the forward 50-CCGGCCTACTAGTCACCAGAGACTTCTCGAGAAG- tumor reached approximately 100 mm3. TCTCTGGTGACTAGTAGGTTTTTG-30; reverse 50-AATTCAAAAA- Beginning 1 week post-injection, tumor was measured every 3 CCTACTAGTCACCAGAGACTTCTCGAGAAGTCTCTGGTGACT- days using the 0 to 150 mm Digital caliper (DIN862, Ref 112-G, AGTAGG-30 (Sigma-Aldrich). Oligonucleotides were annealed SESA Tools) and tumor volume was calculated by the formula: and cloned into the lentiviral plasmid pLKO-Tet-On (Addgene Volume ¼ p/6 length width2. For liver colonization experi- plasmid #21915). The shRNA to KRAS cloned into the pLKO-Tet- ments, a murine model of intrasplenic injection was used (16). On was described previously (18). The pcDNA3-human Id1 (a gift Two weeks post-injection, the development of liver tumors was from Dr. Robert Benezra, MSKCC; Addgene, plasmid #16061) monitored in vivo using micro-PET (mPET).

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Id1 in Mutant KRAS Lung Adenocarcinoma

18F-FDG mPET study covariates (25). All survival analyses were performed, with R and mPET studies were performed every 2 weeks by injecting the P values <0.05 considered statistically significant. radiotracer 18-fluorodeoxyglucose (18F-FDG; 18.62 MBq 2in 80–100 mL) via tail vein to assess the development of liver Statistical analysis metastases in vivo, as previously published (16). PMOD software Sample size was based on similar experiments published by the (PMOD Technologies Ltd.) was used to analyze the data. Images group (16). To compare the two groups, samples were analyzed were expressed in standardized uptake value (SUV) units, using for normality using Shapiro–Wilk test, and variance was analyzed the formula SUV ¼ [tissue activity concentration (Bq/cm3)/ using the Levene test. Groups with nonnormal distribution were injected dose (Bq)] body weight (g). analyzed using the Mann–Whitney test; Student t test was used to analyze samples with a normal distribution. All analyses were Animal sacrifice and necropsy two-tailed. Statistical analyses were conducted with SPSS v15.0 The mice were anesthetized and euthanized by cervical dislo- software. A P value <0.05 was considered statistically significant. cation and tumors were harvested and immediately fixed in formaldehyde 3.7% to 4% pH 7 (Panreac) for 48 hours. The Results relevant parts were embedded in paraffin, and sections produced for hematoxylin–eosin staining (H&E). High Id1 expression is a marker of poor prognosis in mutant KRAS LUAD Id1 KRAS Immunohistochemistry To determine the prognostic role of in the context of fi IHC was done as previously described (6, 16) using antibodies genotype, patients of the TCGA LUAD data set (28) were strati ed Id1 to Id1 (1:1500; Biocheck), Ki67 [1:100, RM-9106 (SP6); Thermo], based on median expression as previously described (26). OS KRAS CC3 (1:100, #9661; Cell Signaling Technologies), and FOSL1 analysis of patients with LUAD harboring mutations Id1 fi (1:200, #sc-376148; Santa Cruz Biotechnology). Slides were showed that high expression identi ed patients with the worst P ¼ scanned with the Aperio Digital Scanner (Leyca) and analyzed survival outcome ( 0.0106; Fig. 1A), whereas no correlation KRAS P ¼ with ImageJ. was observed in wild-type patients ( 0.7122; Fig. 1B). Second, we explored whether these clinical findings could be extrapolated to an independent set of patients using the UT Lung RNA sequencing SPORE cohort (the University of Texas Lung Specialized Programs RNA sequencing was carried out as previously described (20). of Research Excellence) from The University of Texas MD Ander- fl Data analysis was performed using the following work ow: (i) the son Cancer Center (29). Similarly to the TCGA data set, OS of fi quality of the samples was veri ed using FastQC software; (ii) the mutant KRAS patients associated with Id1 expression (P ¼ 0.0073; alignment of reads to the human genome (hg19) was performed Supplementary Fig. S1A), and no association was observed in fi using STAR (21); (iii) gene expression quanti cation using read patients lacking KRAS mutations (P ¼ 0.451; Supplementary Fig. counts of exonic gene regions was carried out with featureCounts S1B). Furthermore, taking advantage of disease-free survival (22) and genes with read counts lower than 6 in more than 50% of (DFS) information in the UT Lung SPORE study, high Id1 expres- the samples in all studied conditions were considered as not sion was found to be an adverse marker of earlier tumor recurrence expressed; (iv) the gene annotation reference was Gencode v19 in LUAD patients with KRAS mutations (P ¼ 0.0284; Supple- (23); and (v) differential expression statistical analysis was per- mentary Fig. S1C). No correlation was found in the group of wild- > formed using R/Bioconductor (24) using a B cut off B 0 and type KRAS patients (P ¼ 0.977; Supplementary Fig. S1D). > fi logFC 1. RNAseq les can be found at GEO (GSE108491). Next, the potential correlation between Id1 expression levels and clinical–pathologic variables was assessed in the TCGA data Patient data set. No correlation was found either with stage, lymph node Two LUAD patient cohorts were used in this study: one from metastasis, or age, but Id1 high expression was associated with The Cancer Genome Atlas (TCGA; LUAD data set; n ¼ 444) and male gender (P ¼ 0.0172; Fig. 1C). Notably, complementary the second from the University of Texas (UT Lung SPORE cohort; analyses of the UT Lung SPORE data set revealed a similar lack n ¼ 151). Clinical information was available at the TCGA website of correlation of Id1 expression with stage, nodal status and age, as (https://portal.gdc.cancer.gov/) or has been described previously well as positive correlation of its high expression with male gender (25). Only normalized/processed data of coded clinical informa- (P ¼ 0.0015; Supplementary Fig. S1E). tion were made available to this study to preserve patients' The survival data prompted us to investigate Id1 expression in anonymity. the context of KRAS mutations, as well as other driver oncogenes, in the LUAD TCGA data set where this information is available. Survival analyses Id1 expression was higher in LUAD patients with KRAS mutations Survival analysis was conducted on both gene sets and on (P ¼ 0.0223; Fig. 1D), which was confirmed by association individual genes on the TCGA LUAD and the UT Lung SPORE analysis between mutations in KRAS and Id1 expression (OR cohorts as previously described (26). Log-rank test calculated the 1.7534; P ¼ 0.0179; Supplementary Table S1). Analyses of addi- statistical significance of differences observed among Kaplan– tional oncogenes mutated in LUAD revealed no positive correla- Meier curves (27). In the case of the gene sets, a summation of tion of Id1 expression with BRAF (P ¼ 0.0519), DDR2 (P ¼ 0.737), all the genes for a particular sample was calculated as previously or EML4-ALK (P ¼ 0.858). Notably, Id1 expression was lower in described (26). Patients were stratified based on the median of Id1 tumors with EGFR mutations (P ¼ 0.0002; Fig. 1D), and associ- or mean of gene set summation. Multivariate Cox proportional ation studies revealed a similar trend (OR 0.1958, P ¼ 0; Sup- hazards analysis was also performed considering the age, gender, plementary Table S1). Although the association of KRAS muta- tumor stage, Id1 expression, and STK11 mutational status as tions with high Id1 expression was independent of gender, the

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Roman et al.

A Mutant KRAS Wild-type KRAS P−value = 0.01058 B P−value = 0.71225 Low Id1 Low Id1 High Id1 High Id1 Survival 0.40.60.81.0 Survival 0.2 0.0 0.0 0.2 0.4 0.6 0.8 1.0 0 102030405060708090 0 30 60 90 120 150 180 210 240 Time (months) Time (months) Low652618137 5 3 3 3 0 Low158374211110 High 64 24 10 5 5 5 4 2 1 0 High 157 36 8 4 2 2 1 1 0

Age Gender Stage N log2FC=0.2 log2FC=0.328 C P = 0.573 P = 0.716 P = 0.11 P = 0.0172 10 12 Id1 log2(Expression) 68 681012 6 8 10 12 681012

I II III IV N0 N1 N2 LH Female Male

D log2FC= 0.383 log2FC= −0.854 log2FC= −0.046 log2FC= −0.458 log2FC= −0.105 P = 0.0223 P = 0.0002 P = 0.858 P = 0.0519 P = 0.737 FDR= 0.111 FDR= 0.00489 FDR= 0.998 FDR= 0.853 FDR= 0.999 8 9 10 11 12 log2(Expression) 7 6 7 8 9 10 11 12 13 6 7 8 9 10 11 12 6 681012 6 7 8 9 10 11 12 Wt KRAS mut Wt EGFR mut Wt EML4-ALK fus. Wt BRAF mut Wt DDR2 mut E

LUAD patients with KRAS mutation (n = 129) Id1_exp STK11 (cor=0.239 P = 0.0064) KEAP1 (cor=0.21 P = 0.0167) ATM (cor=−0.172 P = 0.0515) CDKN2A (cor=0.154 P = 0.0809) SETD2 (cor=−0.152 P = 0.0861) U2AF1 (cor=−0.152 P = 0.0861) RIT1 (cor=−0.146 P = 0.0998) MGA (cor=0.137 P = 0.1207) TP53 (cor=−0.125 P = 0.1566) SMARCA4 (cor=0.116 P = 0.191) RBM10 (cor=0.104 P = 0.24) NF1 (cor=0.068 P = 0.443) RB1 (cor=0.029 P = 0.744) ARID1A (cor=−0.024 P = 0.7888) High Id1 Low Id1

Figure 1. Id1 is a prognostic marker in KRAS-mutated LUAD TCGA data set. A and B, Kaplan–Meier plot of the OS for median Id1 expression in patients with mutant KRAS (n ¼ 129; A) and patients with wild-type (wt) KRAS (n ¼ 315; B). C, Box plots of Id1 expression levels according to N, age, and gender. D, Box plots of Id1 expression levels association with driver oncogenes (KRAS, EGFR, EML4-ALK, BRAF, DDR2). The wt group includes only patients without mutations in driver oncogenes (KRAS, EGFR, EML4-ALK, BRAF, and DDR2). E, Concurrent mutations in patients with mutant KRAS LUAD associated with Id1 expression levels. Green, Id1 high patients; red, Id1 low patients.

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Id1 in Mutant KRAS Lung Adenocarcinoma

inverse association between Id1 and EGFR mutations was driven entially cytotoxic to mutant KRAS LUAD cells, suggesting that Id1 by this clinical parameter (Supplementary Table S1). might represent a potential molecular target in patients with lung Recent evidence suggests the influence of concurrent mutations cancer with KRAS mutations. on the phenotype and survival of mutant KRAS patients (30). We To understand the preferential sensitivity of mutant KRAS explored if Id1 expression in mutant KRAS patients would be LUAD cells to Id1 decreased protein expression, we investigated affected by the mutational landscape of tumor suppressor genes. whether Id1 expression is regulated by the KRAS oncogene itself. Interestingly, high Id1 expression levels were associated with KRAS knockdown in three mutant KRAS LUAD cell lines with an mutations in STK11/LKB1 (P ¼ 0.0064) and KEAP1 (P ¼ specific shRNA led to decrease Id1 levels (Fig. 2J). Conversely, 0.0167; Fig. 1E). Thus, a multivariate survival analysis was per- exogenous overexpression of mutant KRAS in wild-type KRAS formed in mutant KRAS patients to query the dependence of Id1 LUAD cell lines increased Id1 expression (Fig. 2K). To gain insight expression on clinical and mutational parameters. The Cox pro- into the mechanisms involved in Id1 regulation, we carried out portional hazard model showed that the impact of Id1 expression pharmacologic inhibition of KRAS canonical effectors in two on patient survival is irrespective of stage, age, gender, and STK11/ mutant KRAS LUAD cell lines. Inhibition of the JNK consistently LKB1 mutations (P ¼ 0.039; HR 1.599 [1.177–2.172]). These downregulated Id1 expression in both mutant cell lines, suggest- results indicate that Id1 is a prognostic marker in KRAS-mutated ing a consistent mechanism of Id1 regulation (Fig. 2L). Therefore, LUAD and that its expression may be modulated by KRAS onco- the connection of Id1 expression to KRAS oncogene signaling gene along with commutated tumor suppressor genes. through JNK may explain the phenotypic differences observed between mutant and wild-type LUAD cells to Id1 depletion. Viability of KRAS-mutated LUAD cells relies on Id1 expression To establish the functional role of Id1 in human LUAD, we Id1 loss impairs LUAD growth and maintenance in vivo analyzed Id1 expression levels in a panel of human LUAD cell To define the role of Id1 in the in vivo setting, xenograft models lines with known KRAS status (n ¼ 12) and in immortalized were generated by injection of mutant KRAS LUAD cells normal lung epithelial cells (n ¼ 2). qPCR analyses revealed an (H1792.604 and H2009) into the dorsal flanks of immunode- upregulation of Id1 mRNA in 11 of 12 LUAD cell lines with regard ficient mice (n ¼ 10). Both cell lines carried doxycycline-inducible to nontumor cells (Fig. 2A). This result was consistent with the shRNAs targeting either GFP (control) or Id1, which were induced protein expression analyses (Fig. 2B) and with previous data from the time of cell inoculation. A significant decrease in tumor showing that Id1 is highly expressed in human LUAD compared volume was observed in mice injected with Id1-depleted with normal lung (15). H1792.604 cells compared with the control group (60.10 Next, Id1 expression was depleted in six mutant and four wild- 32.38 mm3 vs. 356.28 115.33 mm3, P ¼ 0.00021; Fig. 3A). type KRAS LUAD cell lines using an inducible short hairpin RNA More strikingly, in mice injected with H2009, Id1 knockdown (shRNA) against Id1 (Fig. 2C). A significant impairment of cell completely prevented tumor onset (P ¼ 5.38e05; Fig. 3B), growth was observed in 6 of 10 LUAD cell lines upon Id1 precluding tumors collection for further analyses. Interestingly, inhibition (H2009: 55.07 4.56%, P ¼ 3.35e09; H358: exogenous expression of Id1 after its endogenous knockdown 64.76 3.60%, P ¼ 4.78e06; H441: 59.16 3.11%, P ¼ largely rescued the deleterious phenotype in vivo, consistent with 3.00e09; H1792.604: 18.31 2.36%, P ¼ 9.06e09; A549: the in vitro data (Fig. 3C). 79.04 5.68% P ¼ 3.70e07; H23: 78.51 6.71%, P ¼ Next, IHC analysis in H1792.604-derived tumors confirmed 1.25e06; Fig. 2D). Interestingly, all LUAD cell lines harboring Id1 loss at the experiment endpoint (2.37 2.54% expression, P ¼ KRAS mutations were sensitive to Id1 depletion, and mutant KRAS 3.22e11; Fig. 3D). Complementary analyses revealed that LUAD cell lines with mutations in TRP53 and/or KEAP1 were expression of the proliferative marker Ki67 was drastically more sensitive to Id1 depletion than those with mutated STK11/ reduced in comparison with the control group (8.51 10.39% LKB1 (Fig. 2D). Notably, reconstitution of Id1 expression using an expression, P ¼ 7.21e10; Fig. 3E). These results indicate that Id1 exogenous cDNA refractory to shRNA inhibition significantly depletion is required for the formation and growth of mutant rescued the proliferative capacity of two mutant KRAS cell lines KRAS tumors in vivo. upon Id1 depletion (Fig. 2E and F; Supplementary Fig. S2A and To assess the potential role of Id1 as a therapeutic target in vivo, S2B), suggesting an on-target effect of the shRNA. These data may we used xenograft models to evaluate the effect of Id1 abrogation confirm Id1's key role in tumor cell proliferation and viability of in LUAD established tumors. Mice were subcutaneously inocu- LUAD cells, specifically in those harboring KRAS mutations. lated with either H1792.604 or H2009 human LUAD cells To further characterize the cellular mechanisms underlying the (expressing an inducible GFP or Id1 shRNA) and doxycycline was effect of Id1 decreased protein expression, cell-cycle analyses administered 15 days (in H1792.604) or 30 days (in H2009) using EdU incorporation were carried out. Id1 decreased protein post-inoculation, when macroscopic tumors reached approxi- expression in mutant KRAS LUAD cells (H1792.604, H2009, and mately 100 mm3. Id1 knockdown led to the regression of 4 of H358) induced an arrest in G2–M phase; no significant changes in 10 tumors in mice inoculated with H1792.604 cells (Fig. 3F) and cell-cycle profile were observed in wild-type KRAS cells (H1437, all tumors in mice inoculated with H2009 cells (Fig. 3G). IHC H2126, and HCC78; Fig. 2G). Also, we tested the effect of Id1 on analysis revealed effective Id1 protein reduction in H2009-derived tumor cell viability, because an induction of G2–M arrest in tumor tumors cells (4.14% 3.11, P ¼ 3.49e11; Fig. 3H), whereas cells is often accompanied by induction of apoptosis (31, 32). As tumors originated from H1792.604 cells were cystic, contained a anticipated, a significant apoptosis increase, measured by liquid content and could not be processed for histologic analysis. Annexin V staining and caspase 3/7 activation, was observed in In addition, we investigated the cellular mechanisms regulated as mutant KRAS cells, whereas no changes in cell viability were a consequence of Id1 absence in established tumors. First, the lack found in wild-type KRAS LUAD cells (Fig. 2H and I; Supplemen- of Id1 protein expression again correlated with a decrease in Ki67 tary Table S2). These results indicate that Id1 depletion is prefer- protein levels (8.98 6.21%, P ¼ 4.29E11; Fig. 3I). Second, an

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Id1 Fold change (shID1/shGFP) H1568 0 1 2 3 4 5 eei the in gene mutant , H1568 MUT , D, H2009 H358 P < *** WT Published OnlineFirst December 18, 2018; DOI: 10.1158/0008-5472.CAN-18-1479

Id1 in Mutant KRAS Lung Adenocarcinoma

A F H1792.604 H1792.604 500 1,000 *** TET GFPsh ) TET GFPsh *** 300 ) 3 3 TET Id1sh 400 TET Id1sh *** 800 ** DOXY 200 300 600 (%) *** 200 400 100

Tumor volume (mm DOXY *** 100 *** Tumor volume (mm 200 0 *** Tumor change -30 0 0 -60 -100 0 7 4 0 7 4 1 10 1 18 21 25 10 1 18 2 Time (days) Time (days) TET GFPsh TET Id1sh B G H2009 H2009 300 200 TET GFPsh *** TET GFPsh ** 300 ) 3 ) TET Id1sh TET Id1sh *** 3 150 DOXY ** 200 *** 200 *** 100 100 100 *** change (%) DOXY *** 50 r

Tumor volume (mm ****** *** Tumor volume (mm 0 ****** Tumo * ** * -30 0 0 -60 0 7 0 4 5 5 0 7 7 5 8 2 5 1 14 17 21 2 28 31 3 38 42 4 49 -100 10 14 1 21 24 28 31 3 3 4 4 49 Time (days) Time (days) TET GFPsh TET Id1sh C H I H1792.604 H2009 (tumor maintenance) 300 H2009 (tumor maintenance) 30 40 %) TET GFPsh %) ( s l

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0 F

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30 e 30 *** 1.5 tive cells 20 20 i 1.0 os p 10 TET_GFPsh 10 TET_GFPsh 0.5 TET_GFPsh sp3 Id1 positive cells (%) a *** Ki67 positive c 0 0 C 0.0 h sh sh 1 Ps 1 F G _Id _ ET TET TET_Id 1sh TET_Id T TET_Id1sh

TET_GFPsh TET_Id1sh TET_Id1sh TET_GFPsh

Figure 3. Detrimental effect of Id1 loss in mutant KRAS LUAD progression and maintenance. A, Tumor volume of xenografts from H1792.604 cells expressing an inducible GFP or Id1 shRNA (n ¼ 10). B, Tumor volume of xenografts from H2009 cells expressing an inducible GFP or Id1 shRNA (n ¼ 10). C, Tumor volume of xenografts from H1792.604 (n ¼ 8) after exogenous expression of an Id1 shRNA-resistant cDNA. D, IHC and quantification of Id1 expression in representative sections of tumors in A. E, IHC and quantification of Ki67 expression in representative sections of the same tumors as in D. F, Tumor volume of xenografts from H1792.604 cells expressing an inducible GFP or Id1 shRNA (n ¼ 10). Analysis of tumor change from samples at the end of each experiment. G, Tumor volume of xenografts from H2009 cells expressing an inducible GFP or Id1 shRNA (n ¼ 10). Analysis of tumor change from samples at the end of each experiment. H, I,andJ, IHC and quantification of Id1 (H), Ki67 (I), and caspase-3 (J) expression in representative sections of tumors in G. , P < 0.05, , P < 0.01, , P < 0.001. Scale bar, 500 mm.

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Roman et al.

increased in the percentage of apoptotic cells was found in those nontreated mice showed massive macroscopic liver colonization tumors in which Id1 was knocked-down (1.740.80%, P ¼ whereas mice with Id1 knockdown did not present any gross 0.026; Fig. 3J). Both the in vitro and the in vivo findings strongly macroscopic tumor burden. Id1 loss was achieved in tumors support an important role for Id1 in LUAD development and derived from H1792.604 cells where the shRNA was induced by maintenance in mutant KRAS-driven LUAD. doxycycline, whereas its expression was unaffected in the two control groups (Fig. 4A). Id1 depletion impairs tumor progression and increases survival Next, we investigated the impact of Id1 depletionontumor in a clinically relevant metastasis model progression and survival by mPET scans. Mice were analyzed by To explore Id1's role in LUAD tumorigenesis, a humanized mPET at 2, 4, 6, and 8 weeks after cell inoculation. No18F-FDG model of cancer colonization to the liver, a primary site of mPET uptake was observed for either groups at 2 weeks, metastasis in patients with LUAD (33) was tested. H1792.604 whereas 4 weeks after injection of Id1-expressing tumor cells, KRAS mutant cells expressing a doxycycline-inducible Id1 shRNA control mice (n ¼ 10) showed high metabolic uptake reflecting as well as the parental cell line were directly inoculated in the massive hepatic tumor infiltration. Mice inoculated with Id1- spleen of immunodeficient mice (n ¼ 5) and treated with doxy- depleted cells (n ¼ 15) did not present any evident liver tumor cycline in drinking water from the time of cell inoculation. As an lesion. At that time, the SUVmax50 value obtained for the additional control group, H1792.604 cells transduced with the control group was 1.86 0.51, compared with 0.71 0.09 Id1 shRNA were intrasplenically injected into mice that received (P ¼ 0.007) for the Id1-inhibited group (Fig. 4B). In addition, no doxycycline. Twenty-five days post-injection, the control and control mice had to be sacrificed after an average of 26.67 days

AB Ctrl Doxy IHC Id1 (5X) TET_Id1sh Ctrl TET_Id1sh Doxy TET_Id1sh

** lrtC 100 2.5

80 2.0 * Ctrl 60 (With Doxy) 1.5

1.0 1sh 40 ** dI_

% of tumor tissue 20 0.5 SUVmax50 (mPET #2)

TET

0 0.0 H1792.604 TET Id1sh TET Id1sh H1792.604 2 Weeks 4 Weeks 6 Weeks 8 Weeks (with Doxy) (without Doxy) (mPET #1) (mPET #2) (mPET #3) (mPET #4)

C D H1792.604 (liver colonization)E H1792.604 (liver colonization)

100 50 40 90 80 40 30 70 *** 60 30 50 Ctrl 20 *** Ctrl 40 20 30

Id1 positive cells (%) 10

Mice survival (%) Ctrl 20 10 Ki67 positive cells TET_Id1sh 10 *** 0 0 0 TET_ld1sh TET_Id1sh 0 102030405060708090100 Ctrl Time (days) Ctrl TET_Id1sh TET_Id1sh

Figure 4. Id1 loss impairs liver colonization of KRAS-mutated LUAD in vivo. A, Bar chart of the quantification of tumor size in a humanized model of cancer colonization to the liver (n ¼ 15) using H1792.604 parental cells and shRNA to Id1 cells treated or nontreated with doxycycline. IHC to detect Id1 expression in representative sections of tumors. B, SUVmax50 analysis and representative mPET images in the different experimental groups from A treated with doxycycline. C, Comparison between H1792.604 Ctrl and with a shRNA to Id1 with doxycycline treatment. D, IHC and quantification of Id1 expression in representative sections of tumors in B. E, IHC and quantification of Ki67 expression in representative sections of the same tumors as in D. , P < 0.05, , P < 0.01, , P < 0.001. Scale bar, 500 mm.

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Id1 in Mutant KRAS Lung Adenocarcinoma

Mutant KRAS Wild-type KRAS A P−value=0.00046 B P−value=0.05393 1.0 1.0 Id1i-down sig_Low Id1i-down sig_Low Id1i-down sig_High Id1i-down sig_High 0.8 0.6 0.8

Figure 5. Overall survival Id1 regulates a gene signature that Overall survival predicts poor prognosis of mutant 0.2 0.4 KRAS LUAD patients. A, Survival analysis of LUAD patients with 0.0 KRAS mutation (TCGA data set) 0.0 0.2 0.4 0.6 0 102030405060708090 0 30 60 90 120 150 180 210 240 stratiﬁed by mean expression of an Time (months) Time (months) Id1 signature (n ¼ 129 patients). Low733019159 8 6 4 3 0 Low 166 35 5 1 1 1 1 1 0 B, Survival analysis of LUAD High562093321110 High 149 38 7 5 2 2 1 1 0 patients with KRAS wild-type C 0.1 D H1792.

ﬁ tne H2009 H358 (TCGA data set) strati ed by 0.0 604 Id1 -0.1

expression of an signature erocs mhcirnE -0.2 shRNA GFPId1 GFPId1 GFP Id1 (n ¼ 315 patients). C, GSEA of a -0.3 -0.4 FOSL1 signature on the Id1i-down -0.5 FOSL1 -0.6 signature. D, Protein analysis by AURKA Western blot analysis of FOSL1 and HURP

tsildeknaR downstream proteins, AURKA, 5.0 HURP, TACC3, PLK1, and CCNB1, cir 2.5 TACC3

tem using three mutant LUAD cells 0.0 -2.5 PLK1 transduced with an shRNA control 0 2,500 5,000 7,500 10,000 12,500 (GFP) and an shRNA to Id1 CCNB1 Enrichment score (ES): -0.629 (H1792.604, H2009, H358). E, IHC Normalized enrichment score (NES): -2.081 β-Tubulina and quantification of FOSL1 FDR q-value 0.0 expression in representative FWER P-Value 0.0 sections of the same tumors as in E H1792.604 (tumor initiation) F H2009 (tumor maintenance) G H1792.604 (liver colonization) Fig. 3D. F, IHC and quantification of 60 60 30 FOSL1 expression in representative sections of the same tumors as in Fig. 3H. G, IHC and quantification of 40 40 20 FOSL1 expression in representative sections of the same tumors as in *** TET_GFPsh TET_GFPsh Ctrl Fig. 4D. H, Western blot analysis of 20 20 10 Id1 in cell lysates from H1792.604 *** FOSL1 positive cells (%) FOSL1 positive cells (%) and H2009 transduced with a FOSL1 positive cells(%) *** shRNA control (GFP), a shRNA to 0 0 0 Id1 Id1 and a shRNA to together with TET_Id1sh TET_Id1sh Ctrl TET_Id1sh a cDNA overexpressing FOSL1. I, Cell proliferation assay (MTS) of TET_Id1sh TET_Id1sh TET_Id1sh TET_GFPsh the same H1792.604 cells as in G for TET_GFPsh 5 days and repeated three times. H I J J, Tumor volume of xenografts from H1792.604 H2009 H1792.604 300 H1792.604 (n ¼ 8) after exogenous 150 TET_GFPsh GFPsh expression of FOSL1 once Id1 was FOSL1 FOSL1 TET_Id1sh Id 1sh ) 3 depleted. , P < 0.001. Scale bar, TET_Id 1sh FOSL1 OE Id 1sh FOSL1 OE *** GAPDH β-Tubulina 200 500 mm. 100 *** *** Id1sh Id1sh *** GFPsh GFPsh 100 50 *** Tumor volume (mm Cell viability (% of control)

Id1sh FOSL1 O.E. 0 Id1sh FOSL1 O.E. 0 0 7 0 4 7 H1792.604 1 1 1 21 Time (days)

due to the gross tumor masses and the poor animals condition. in this clinically relevant model reduced liver colonization Conversely, mice carrying Id1-depleted tumors did not show significantly improving mice survival. liver tumor foci until 8 weeks post-inoculation, and only 4miceweresacrificed before the end of the experiment at day An Id1 signature enriched in E2F targets and elements of the 100 (P ¼ 1.10E06; Fig. 4C). As previously observed in vivo, G2–M cell-cycle phase these changes were associated with a significantly lower expres- To investigate the molecular mechanisms involved in the Id1- sion of Id1 (0.73 1.71%; P ¼ 2.31e10), Ki67 (6.15 loss phenotype, we performed global transcriptomic profiling 11.32%; P ¼ 2.29e07), respectively (Fig. 4D–E). Thus, Id1 loss using RNAseq on mutant KRAS cells (H1792.604) expressing a

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Roman et al.

control or an Id1 shRNA. Id1-loss cells led to upregulation of 413 genes and downregulation of 362 genes (B>0 and logFC>1) (Supplementary Table S3). Since the list of downregulated genes would a priori involve components of the prooncogenic Id1- dependent phenotype, we focused on those genes for subsequent studies (Id1-inhibition down or "Id1i-down" signature). Given our findings reporting Id1 expression is a marker of survival exclusively in mutant KRAS LUAD patients, we wanted to see if the Id1i-down signature would have any clinical implications. Notably, high expression of this signature identified the group of patients with the poorest survival within the KRAS mutatedgroup(Fig.5A).However,expressionofthe Id1i-down signature was not associated with OS in the group of patients lacking mutations in KRAS (Fig.5B).Next,considering the observations in the LUAD TCGA data set showing asignificant correlation between high Id1 expression and the presence of KEAP1 and LKB1 mutations, we tested the Id1i-

down signature against a human KEAP1 signature recently M checkpoint, as in progression through the cell division cycle – described (34). Gene set enrichment analysis (GSEA) revealed 2 a positive enrichment of the Id1i-down signature into the KEAP1 signature (Supplementary Fig. S3A), providing molecular evidence that Id1 may upregulate the expression of a gene ning late response to estrogen set commonly expressed in a subset of mutant KRAS patients fi with concurrent KEAP1 mutations. Then, we tested the Id1i-down signature against the Molec- consisting of iron and porphyrin) and erythroblast differentiation ular Signatures Data Base (MSigDB) to investigate the molecular features related to this signature. Analysis of the HALL- MARK gene sets in MSigDB identified E2F target genes as the 04 Gametes (sperm), as in spermatogenesis 05 A subgroup opf genes regulated by MYC- version 1 07 Genes important for mitotic spindle assembly 0303 Genes involved in metabolism of heme (a cofactor Genes upregulated by STAT5 in response to IL2 stimulation value Description 04 Genes de 25 Genes encoding cell cycle related targets of E2F transcription factors 24 Genes involved in the G 04 Genes upregulated through activation of mTORC1 Complex 04 Genes encoding proteins involved in glycolysis and gluconeogenesis fi most signi cantly enriched feature (Supplementary Fig. S3B). q This finding was highly interesting in that Id1 can stabilize expression of the transcription factor E2F1 in tumor cells (35). In fact, decreased expression of E2F1 upon Id1 knockdown and 05 4.08E 08 3.47E 04 2.34E 04 2.34E 05 5.61E 05 5.61E 25 8.16E 05 1.30E 06 2.09E rescue of E2F1 levels after ectopic expression of a shRNA- 26 6.81E insensitive Id1 construct were observed in mutant KRAS LUAD value FDR cells (Supplementary Fig. S3C). In addition, the Id1i-down signature was also enriched in gene sets representative of G2–M checkpoint, mitotic spindle, MYC kP

Id1 / 0.035 5.50E 0.06 2.08E 0.05 1.68E 0.035 5.50E 0.045 1.30E 0.04 8.98E 0.052 4.90E 0.13 3.26E 0.04 8.98E 0.135 1.36E targets, glycolysis, and mTORC1 signaling, suggesting con- k trols relevant molecular mechanisms for cell homeostasis (Table 1). Next, analysis of the GO gene sets showed a similar enrichment of features related to G2–M cell cycle (cell-cycle process, mitotic cell cycle, organelle ﬁssion, cell division, and mitotic nuclear division) and related processes (Supplementary 7 9 8 7 12 10 7 26 8 27 Table S4). Notably, these cellular features were consistent with No. genes in overlap (k) the G2–M arrest observed in mutant KRAS cells upon Id1 knockdown.

FOSL1 partially mediates Id1 loss phenotype in mutant KRAS 200 200 200 No. genes in LUAD gene set (K) Mutant KRAS cancer cells are subjected to high levels of mitotic stress (5). An arrest in G2–M phase and an induction of apoptosis, similarly to that observed in Id1-depleted cells, has been recently described in mutant KRAS LUAD cell lines upon inhibition of FOSL1 (6). The FOSL1-knockdown phenotype involved the downregulation of elements of the G2–M transition network such as AURKA and its coactivators HURP and TACC3, as well as PLK1 and CCNB1. Thus, we investigated the potential correlation between Id1 and FOSL1 by comparing the Id1i-down Id1i-down signature enrichment in Hallmark gene sets from MSigDB signature with a previously described FOSL1-loss gene signature. GSEA (36) showed a signiﬁcant negative enrichment of the Table 1. Gene set name HALLMARK_ESTROGEN_RESPONSE 200 HALLMARK_HEME_METABOLISM 200 HALLMARK_SPERMATOGENESISHALLMARK_GLYCOLYSIS HALLMARK_MTORC1_SIGNALING 135 HALLMARK__IL2_STAT5_SIGNALING 200 200 HALLMARK_MITOTIC_SPINDLEHallmark_MYC_TARGETS_V1 200 HALLMARK_E2F_TARGETS FOSL1-loss signature on the Id1i-down signature (Fig. 5C), HALLMARK_G2M_CHECKPOINT 200

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Id1 in Mutant KRAS Lung Adenocarcinoma

including many genes involved in mitotic progression, suggesting To investigate whether FOSL1 played any functional role that Id1 and FOSL1 converge on the regulation of a common set downstream of Id1, an exogenous cDNA was expressed in two of genes. The second most differentially expressed gene in different mutant KRAS LUAD cell lines (Fig. 5H). Forced FOSL1 the leading edge of the enrichment analysis was FOSL1 itself expression rescued the Id1-silenced phenotype in vitro in both cell (Supplementary Table S5), suggesting FOSL1 functions down- lines (Fig. 5I; Supplementary Fig. S3E). Moreover, the Id1 knock- stream of Id1. down rescue effect was largely recapitulated in vivo (Fig. 5J). To validate the RNA-seq data, mRNA and protein analyses of FOSL1 reconstitution recovered expression of the FOSL1 targets FOSL1 expression levels and some of its transcriptional target AURKA, HURP, TACC3, PLK1, and CCNB1 (Supplementary Fig. genes were performed in three mutant KRAS LUAD cell lines in the S3F), suggesting a potential involvement of these proteins in the context of Id1 loss. A signiﬁcant reduction in FOSL1 as well as the rescue effect. Thus, FOSL1 is a critical mediator of the Id1-depen- targets AURKA, HURP, TACC3, PLK1, and CCNB1 was found in dent pro-oncogenic phenotype in mutant KRAS cells. all cell lines (Fig. 5D). However, Id1 knockdown in wild-type KRAS LUAD cell lines did not consistently decrease either FOSL1 Id1 regulates multiple kinases downstream of oncogenic KRAS or its downstream-regulated proteins (Supplementary Fig. S3D). FOSL1 expression has been previously shown to be regulated Then, to determine this regulation in vivo, FOSL1 expression was by kinases within the KRAS oncogene network in mutant KRAS also assessed in different xenograft models of tumor growth, LUAD cell lines, including ERK1/2, ERK5, JNK, and p38 (6). The maintenance, and metastasis. Of note, a drastic decrease in FOSL1 RNA-seq analyses revealed that MAPK1 (ERK2) was downregu- expression was also seen in the three experimental settings lated upon Id1 inhibition, suggesting an additional link of Id1 to (Fig. 5E, F and G). These results suggest the regulation of FOSL1 the KRAS pathway. This ﬁnding was validated at the protein level by Id1 as a robust molecular mechanism in the context of KRAS in two different cell lines (Supplementary Fig. S4A). Furthermore, mutations in LUAD. a series of kinases reported to function within the KRAS oncogene

ACB H1792.604 H1792 200 200 Id1 shRNA Id1 OE 150 150 TNK2

MAPK1 AURKA 100 100 GRK6 PAK1 RPS6KA4 Log ( P value) 50 50

0 0 Fold change (relative to GFPsh) Fold change (relative to GFPsh) 1 4 6 1 4 A 1 6 1 Id K K Id1 K KA AK1 R NK2 P KA RK NK2 0 5 10 15 20 25 6 P T A 6 PAK G T S G S AURKA M P AUR MAPK −4 −2 0 2 4 6 RP R Fold change Genes Genes

D KRAS

RAF1 JNK1 RAC1 JNK2 MEK1 MEK2

ERK1 PAK1 Id1 ERK2

Genes regulated upon Id1 knockdown

FOSL1 Regulation reported in this study

Regulation reported in the literature

TNK2 GRK6 RSK-B AURKA

Figure 6. Id1 controls the expression of various kinases. A, Volcano plot of H1792.604 cells expressing a GFP shRNA versus an Id1 shRNA. B, mRNA analysis by qPCR for indicated genes using H1792.604 cells with a shRNA to Id1 and a shRNA to Id1 together with a cDNA overexpressing Id1. Data are relative to cells expressing a GFP shRNA. C, mRNA analysis by qPCR for the same genes as in A using H1792 cells with a shRNA to FOSL1. Data are relative to H1792 cells expressing a GFP shRNA. D, Working model for the regulation of Id1 and its target genes in the context of a KRAS oncogene network.

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network, such as PAK1, RPS6KA4 (RSK-B), or AURKA, appeared sion rescued the Id1-loss phenotype in vitro and in vivo.Our downregulated upon Id1 knockdown and rescued by ectopic observations provide further evidence for interfering with ele- expression of Id1 (Fig. 6A). These results were validated in inde- ments of the mitotic machinery as a means to treat KRAS- pendent RNA samples by qPCR and in a different mutant KRAS mutated tumors. Along these lines, different combinatorial LUAD cell line (Fig. 6B; Supplementary Fig. S4B). A second set of strategies incorporating pharmacologic inhibitors against pro- kinases not previously linked to mutant KRAS, GRK6, or TNK2 teins involved in mitotic progression, such as AURKA, PLK1, were also reduced at the mRNA level upon Id1 decreased protein and WEE1, have been recently shown to trigger tumor regres- expression (Fig. 6B; Supplementary Fig. S4B). Furthermore, sion of KRAS-mutated LUAD (6, 40, 41). expression of some of these kinases was strictly dependent on On the other hand, our experimental data showed that other Id1, since FOSL1 inhibition did not decrease mRNA levels transcriptional targets to those of the ERK–FOSL1–AURKA axis (Fig. 6C). Thus, Id1 influences cancer cell signaling in KRAS are regulated by Id1, suggesting that additional target genes mutations through expression regulation of several protein beyond the scope of this study could also play a role in the kinases, in part through the action of FOSL1, explaining the high context of mutant KRAS LUAD. Indeed, other kinases impli- sensitivity of mutant KRAS LUAD cells to Id1 loss (Fig. 6D). cated in KRAS oncogene biology, including PAK1 and RPS6KA4 (RSK-B), were found to be regulated by Id1. These kinases function downstream of KRAS canonical effector pathways, Discussion including the RAF-MEK–ERK and the Rac1–PAK pathways Here we describe Id1 as a critical gene in human KRAS mutant (6, 42, 43). Indeed, PAK1 inhibition impaired homeostasis of LUAD. Id1 plays a role in human NSCLC progression, invasion, mutant KRAS NSCLC cells (44). Moreover, in addition to and metastasis under external stimuli, irrespective of the under- featuresrelatedtoG2–M phase and mitotic cell cycle, the GSEA lying mutations in driver oncogenes (37). However, the implica- analyses revealed that the Id1i-down signature contained genes tions of Id1 regulation under endogenous expression of the KRAS involved in glycolysis or the mTOR pathway. Furthermore, oncogene are unknown. Clinically, we found that Id1 expression genes expressed in KEAP1 mutations, covering functional path- may represent a marker of disease outcome specifically in the ways distinct to FOSL1 target genes, were also enriched in Id1- subgroup of patients with mutant KRAS tumors in two large regulated genes. Therefore, functional validation of additional independent data sets correlating high expression with the worst Id1 target genes may unveil novel vulnerabilities in mutant OS. The classical pathologic factors in NSCLC prognosis imply KRAS LUAD and may provide further explanation of the critical that larger tumors and/or lymph node infiltration show worse role of Id1 in these tumors and its potential link to the presence prognosis. However, Id1 expression did not correlate either with of concurrent mutations in tumor suppressor genes. the stage or the presence of lymph node metastasis, and multi- The possibility of targeting Id1 in human cancer is supported by variate survival analyses confirmed Id1 expression as an indepen- previous data attempting to inhibit Id1 in vivo. Two different dent prognostic marker. Notably, Id1 expression positively asso- groups reported that Id1 inhibition via antisense molecules ciated with male gender and inversely with EGFR mutations. This (45) or peptide aptamers (46) recapitulated the effect of genetic could be explained by differences in tobacco exposure between Id1 knockout in mice and demonstrated a significant antitumor males and females, especially because EGFR mutations generally effect. Overall, our results strongly support the inhibition of Id1, occur in females with low tobacco-consumption habits (38). This particularly via pharmacological inhibitors, as a potential thera- observation supports Pillai and colleagues findings showing Id1 peutic strategy for the molecularly more prevalent LUAD patient expression in NSCLC cell lines after nicotine and EGF exposure subgroup in which clinically no targeted therapies are yet (37). available. More importantly, those clinical observations extended to the KRAS functional setting, as mutant LUAD were remarkably sen- Disclosure of Potential Conflicts of Interest Id1 in vitro in vivo Id1 sitive to loss and , indicating that may be a C. Rolfo has received speakers bureau honoraria from MSD, Guardant relevant molecular target in these otherwise targeted therapy- Health, and Novartis, and is a consultant/advisory board member of Mylan. orphan tumors. The consequences of Id1 depletion on mutant No potential conflicts of interest were disclosed by the other authors. KRAS LUAD may in part be explained by Id1 intersection with the KRAS oncogene signaling pathway through several means. On the Authors' Contributions Id1 one hand, controlled the expression of the transcription factor Conception and design: I. Baraibar, L.E. Raez, S. Vicent, I. Gil-Bazo FOSL1. One potential mechanism explaining FOSL1 regulation Development of methodology: M. Roman, I. Lopez, I. Baraibar, D. Lara-Astiaso, by Id1 is through ERK2. FOSL1 was found to be regulated by L.E. Raez, I.I. Wistuba, S. Vicent, I. Gil-Bazo multiple kinases downstream of KRAS, including ERK1/2 in Acquisition of data (provided animals, acquired and managed patients, mutant KRAS LUAD (6). In addition, FOSL1 expression was provided facilities, etc.): M. Roman, I. Lopez, I. Baraibar, M. Ecay, M. Collantes, recently reported to be controlled by endogenous ERK2 and not M. Echavarri-de Miguel, J.H. Jang, D. Lara-Astiaso, M. Ponz-Sarvise, L.E. Raez, S. Taverna, C. Behrens, W. Weder, I.I. Wistuba, S. Vicent ERK1 during the process of melanoma and lung cancer drug Analysis and interpretation of data (e.g., statistical analysis, biostatistics, addiction to different targeted therapies (39). At the functional computational analysis): M. Roman, I. Lopez, E. Guruceaga, I. Baraibar, M. level, FOSL1 inhibition has been recently described as a vulner- Collantes, E. Nadal, M. Ponz-Sarvise, C. Rolfo, E.S. Santos, S. Vicent, I. Gil-Bazo ability in LUAD and pancreatic cancer driven by the KRAS onco- Writing, review, and/or revision of the manuscript: M. Roman, I. Baraibar, gene (6). In this regard, we saw that Id1 knockdown in mutant M. Ecay, M. Collantes, E. Nadal, M. Ponz-Sarvise, C. Rolfo, E.S. Santos, L.E. Raez, KRAS LUAD cell lines recapitulated the G –M phase arrest pre- S. Taverna, W. Weder, I.I. Wistuba, S. Vicent, I. Gil-Bazo 2 Administrative, technical, or material support (i.e., reporting or organizing viously observed upon FOSL1 inhibition, which was accompa- data, constructing databases): M. Roman, I. Lopez, A. Vallejo, S. Cadenas, nied by the downregulation of FOSL1 target genes that are part of S. Vicent the mitotic machinery. More importantly, FOSL1 overexpres- Study supervision: M. Roman, L.E. Raez, S. Vicent, I. Gil-Bazo

636 Cancer Res; 79(3) February 1, 2019 Cancer Research

Id1 in Mutant KRAS Lung Adenocarcinoma

Other (RNAseq library preparation and sequencing): P.S. Martin-Uriz donation by Maria Eugenia Burgos de la Iglesia's family. S. Vicent and I. Gil-Bazo Other (bioinformatics support): L. Castro-Labrador were supported by a grant (RD12/0036/0040) from Red Tematica de Other (RNAseq experiments): A. Vilas-Zornoza Investigacion Cooperativa en Cancer, Instituto de Salud Carlos III, Spanish Ministry of Economy and Competitiveness & European Regional Development Acknowledgments Fund "Una manera de hacer Europa" and cofunded by FEDER funds/European We thank Mr. David Carpenter for editorial assistance with this manuscript. Regional Development Fund (ERDF). I. Gil-Bazo was also supported by two M. Roman was supported by ADA and FPU15/00173 fellowships. M. Echavarri- grants from Instituto de Salud Carlos III (PI11/00976 and PI15/02223). de Miguel was funded by a fellowship from Asociacion Espanola~ Contra el Cancer (AECC). I.I. Wistuba was supported by the NIH/NCI through The The costs of publication of this article were defrayed in part by the payment of University of Texas Lung Specialized Programs of Research Excellence grant page charges. This article must therefore be hereby marked advertisement in (P50CA70907). S. Vicent was supported by the Spanish Ministry of Economy accordance with 18 U.S.C. Section 1734 solely to indicate this fact. and Competitiveness (MINECO, SAF2013-46423-R, and SAF2017-89944-R), the European Commission (to S. Vicent, 618312 KRASmiR FP7-PEOPLE-2013- CIG), the Worldwide Cancer Research (16-0224), the Fundacion La Caixa-FIMA Received May 14, 2018; revised October 29, 2018; accepted December 11, agreement, the Asociacion de Novelda de ayuda a personas con cancer and a 2018; published ﬁrst December 18, 2018.

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638 Cancer Res; 79(3) February 1, 2019 Cancer Research

Inhibitor of Differentiation-1 Sustains Mutant KRAS-Driven Progression, Maintenance, and Metastasis of Lung Adenocarcinoma via Regulation of a FOSL1 Network

Marta Román, Inés López, Elisabeth Guruceaga, et al.

Cancer Res 2019;79:625-638. Published OnlineFirst December 18, 2018.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-18-1479

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