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Crebbp Loss Drives Small Cell Lung Cancer and Increases Sensitivity to HDAC Inhibition

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Crebbp Loss Drives Small Cell Lung Cancer and Increases Sensitivity to HDAC Inhibition Published OnlineFirst September 4, 2018; DOI: 10.1158/2159-8290.CD-18-0385 RESEARCH ARTICLE Crebbp Loss Drives Small Cell Lung Cancer and Increases Sensitivity to HDAC Inhibition Deshui Jia1, Arnaud Augert1, Dong-Wook Kim2, Emily Eastwood1, Nan Wu1, Ali H. Ibrahim1, Kee-Beom Kim2, Colin T. Dunn2, Smitha P.S. Pillai3, Adi F. Gazdar4, Hamid Bolouri1, Kwon-Sik Park2, and David MacPherson1,5 Downloaded from cancerdiscovery.aacrjournals.org on September 27, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst September 4, 2018; DOI: 10.1158/2159-8290.CD-18-0385 ABSTRACT CREBBP, encoding an acetyltransferase, is among the most frequently mutated genes in small cell lung cancer (SCLC), a deadly neuroendocrine tumor type. We report acceleration of SCLC upon Crebbp inactivation in an autochthonous mouse model. Extending these observations beyond the lung, broad Crebbp deletion in mouse neuroendocrine cells cooperated with Rb1/Trp53 loss to promote neuroendocrine thyroid and pituitary carcinomas. Gene expression analyses showed that Crebbp loss results in reduced expression of tight junction and cell adhesion genes, including Cdh1, across neuroendocrine tumor types, whereas suppression of Cdh1 promoted transformation in SCLC. CDH1 and other adhesion genes exhibited reduced histone acetylation with Crebbp inactivation. Treatment with the histone deacetylase (HDAC) inhibitor Pracinostat increased histone acetylation and restored CDH1 expression. In addition, a subset of Rb1/Trp53/Crebbp- deficient SCLC exhibited exceptional responses to Pracinostatin vivo. Thus, CREBBP acts as a potent tumor suppressor in SCLC, and inactivation of CREBBP enhances responses to a targeted therapy. SIGNIFICANCE: Our findings demonstrate that CREBBP loss in SCLC reduces histone acetylation and transcription of cellular adhesion genes, while driving tumorigenesis. These effects can be partially restored by HDAC inhibition, which exhibited enhanced effectiveness in Crebbp-deleted tumors. These data provide a rationale for selectively treating CREBBP-mutant SCLC with HDAC inhibitors. Cancer Discov; 8(11); 1–16. ©2018 AACR. INTRODUCTION been shown to abrogate CREBBP-mediated histone acetyla- tion (6). CREBBP and EP300 acetylation of lysine residues on Recent identification of the genomic alterations in small histone tails neutralizes their positive charge and can increase cell lung cancer (SCLC), a deadly type of lung cancer, may chromatin accessibility. Acetylation of a specific histone resi- provide new opportunities for therapeutic intervention (1–3). due, histone H3 lysine 27 (H3K27), by CREBBP/EP300 can Critical challenges remain, however, as few of these SCLC promote transcriptional enhancer function (7), and dele- alterations are readily actionable, and a majority of them tion of Crebbp/Ep300 in mouse fibroblasts eliminates the have not been validated for their roles in disease initiation vast majority of H3K27 acetylation (8). CREBBP/EP300 also and progression (4). Along with RB1 and TP53 inactivation, acetylates nonhistone proteins, such as p53 and BCL6 (9, 10). mutations in the CREBBP and EP300 acetyltransferases are CREBBP is mutated in lymphomas, urothelial carcinoma, and among the most frequent in SCLC, appearing in 15% to 17% other human tumor types (11–13). Studies employing mouse and 5% to 13% of tumors in patients with SCLC, respectively models have demonstrated that Crebbp functions as a tumor (1, 2, 5, 6). In SCLC, deletions and truncating mutations in suppressor in leukemia and lymphoma (14–17). However, CREBBP and EP300 genes along with missense mutations in vivo evidence that Crebbp functions as a tumor suppressor in the histone acetyltransferase (HAT) domain are frequent, in solid tumors is lacking. In lymphoma, it has been posited and these occur in a mutually exclusive manner. For CREBBP, that loss of CREBBP-mediated acetylation and activation of HAT domain mutations observed in SCLC samples have p53 drives tumorigenesis (13, 17). p53-dependent mecha- nisms of tumor suppression mediated by CREBBP are likely not relevant to tumors such as SCLC that almost invari- ably harbor TP53 mutations (1). Thus, elucidating roles for 1Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, Washington. 2Department of Microbiology, Immunology, and p53-independent tumor-suppressive activities of CREBBP in Cancer Biology, University of Virginia School of Medicine, Charlottes- SCLC is important. In this study, we demonstrate p53-inde- ville, Virginia. 3Division of Comparative Medicine, Fred Hutchinson Cancer pendent Crebbp tumor-suppressor function not only in SCLC 4 Research Center, Seattle, Washington. The University of Texas South- but across multiple neuroendocrine tumor types. We report western Medical Center, Hamon Center for Therapeutic Oncology and Department of Pathology, Dallas, Texas. 5Department of Genome Sciences, CREBBP control of adhesion-related transcript expression, University of Washington, Seattle, Washington. including CDH1, encoding E-Cadherin, as contributing to Note: Supplementary data for this article are available at Cancer Discovery tumor suppression, and we identify a potential therapeutic Online (http://cancerdiscovery.aacrjournals.org/). approach for treating CREBBP-deficient SCLC. D. Jia, A. Augert, and D.-W. Kim contributed equally to this article. Corresponding Authors: David MacPherson, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue N, Seattle, WA 98109. Phone: RESULTS 206-667-6464; Fax: 206-667-2917; E-mail: [email protected]; and Kwon-Sik Park, University of Virginia School of Medicine, Charlottesville, Crebbp Mutation Promotes Tumorigenesis of VA 22908. Phone: 434-982-1947; E-mail: [email protected] Preneoplastic Neuroendocrine Cells doi: 10.1158/2159-8290.CD-18-0385 To study the potential role of Crebbp in SCLC tumor sup- ©2018 American Association for Cancer Research. pression, we mutated Crebbp in a cell-based model of early-stage NOVEMBER 2018 CANCER DISCOVERY | OF2 Downloaded from cancerdiscovery.aacrjournals.org on September 27, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst September 4, 2018; DOI: 10.1158/2159-8290.CD-18-0385 RESEARCH ARTICLE Jia et al. A B C preSC preSC preSC sgControl sgControl sgCrebbp-1 sgControl sgControl 100 sgCrebbp-1 sgCrebbp-2 sgCrebbp-1 sgCrebbp-2 50 Survival (%) Log-rank test P = 0.0015 0 0204060 80 * Days 10 D Normal lung Crebbp-deficient tumor Crebbp-deficient tumor * 5 Fold change Fold 0 (number of colony/well) -1 -2 sgControl sgControl sgCrebbp sgCrebbp E Rb1/Trp53 (n = 28) G Rb1/Trp53 H Rb1/Tr p53 Rb1/Trp53/Crebbp (n = 48) 100 CGRP; DAPI TTF-1; DAPI Median survival 444 days Median survival 50 384 days Log-rank test Tumor-free survival Tumor-free P = 0.0003 Rb1/Tr p53/Crebbp Rb1/Tr p53/Crebbp 0 0 200 400 600 800 CGRP; DAPI TTF-1; DAPI Days F Rb1/Trp53 Rb1/Trp53/Crebbp 250 CREBBP 37 β-ACTIN Figure 1. Inactivation of Crebbp accelerates SCLC in mouse models. A, Representative images of control and Crebbp-targeted preSC cells in soft agar 3 weeks after seeding of 1 × 104 cells. Two independent single-guide RNAs were employed (sgCrebbp-1, sgCrebbp-2). Bottom, quantification of colonies > 0.1 mm in diameter (n = 4). Scale bar, 0.5 mm. *, P < 0.001, Student t-test. B, Images of preSC-derived allografts, 40 days after s.c. injec- tion of cells. Scale bars, 1 cm. C, Kaplan–Meier overall survival curves of mice injected with control-preSC cells (Control, n = 6) and mice injected with Crebbp-knockout preSC cells (n = 5 each). Statistical significance was calculated using the log-rank (Mantel–Cox) test. D, Images of UCHL1-stained sections of Crebbp-mutant tumor and normal lung. Arrow points to neuroepithelial body in the airway. Scale bars, 100 μm. Representative section of Crebbp-deficient tumors stained with H&E. Scale bar, 20μ m. E, Kaplan–Meier tumor-free survival curves of Rb1/Trp53-deficient (blue,n = 28) and Rb1/ Trp53/Crebbp-mutant (red, n = 48) mice from autochthonous model infected with Ad-CGRP-Cre (day 0). Statistical significance was calculated using the log-rank (Mantel–Cox) test. F, Representative immunoblotting results of CREBBP protein levels in 5 lung tumor tissues from each cohort (Rb1/ Trp53 vs. Rb1/Trp53/Crebbp). β-actin was used as a loading control. G, Representative H&E-stained section of SCLC in each cohort (Rb1/Trp53 vs. Rb1/Trp53/Crebbp). Scale bars, 20 μm. H, Representative immunofluorescence for SCLC markers TTF-1 and CGRP in each cohort Rb1/Trp53( vs. Rb1/ Trp53/Crebbp). DAPI was used as a nuclear stain. Original magnification,× 40. SCLC that we previously described (18). Rb1/Trp53/Rbl2- tumors, with delayed kinetics (Fig. 1C). Hematoxylin–eosin deficient “preSC” cells, derived from a mouse SCLC model at (H&E) staining showing typical SCLC morphology and immu- an early stage in tumorigenesis, become fully transformed with nostaining showed the expression of UCHL1 and CGRP, mark- ectopic expression of SCLC oncogenes such as MYCL (18). Here, ers of both SCLC and normal pulmonary neuroendocrine cells we expressed Cas9 and two single-guide RNAs (sgRNA) target- (Fig. 1D; Supplementary Fig. S1C). These data support a role ing DNA sequences encoding the HAT domain of the murine for CREBBP in SCLC tumor suppression. Crebbp gene and validated loss of CREBBP protein (Supple- mentary Fig. S1A and S1B). We found that Crebbp-mutant Crebbp Inactivation Accelerates SCLC in an preSC cells formed more and individually larger colonies in soft Autochthonous Mouse Model agar compared with control preSC cells (Fig. 1A). When preSC To further investigate the contribution of Crebbp inactiva- cells were injected into the flanks of immune-compromised tion to SCLC development in vivo, we employed an autoch- mice, tumors emerged at 30 to 50 days in the sites injected with thonous model. We performed a genetic cross to incorporate the Crebbp-mutant preSC cells but not in those injected with a floxedCrebbp allele (14) into an Rb1/Trp53-deleted model control cells (Fig. 1B). Further aging of the mice injected of SCLC that develops lung tumors with histopathologic with control preSC cells showed that these cells also formed and molecular features of human SCLC (19, 20).
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