Mutational inactivation of mTORC1 repressor gene DEPDC5 in human gastrointestinal stromal tumors Yuzhi Panga,1, Feifei Xiea,1, Hui Caob,1, Chunmeng Wangc,d,1, Meijun Zhue, Xiaoxiao Liua, Xiaojing Lua, Tao Huangf, Yanying Sheng,KeLia, Xiaona Jiaa, Zhang Lia, Xufen Zhenga, Simin Wanga,YiHeh, Linhui Wangi, Jonathan A. Fletchere,2, and Yuexiang Wanga,2 aKey Laboratory of Tissue Microenvironment and Tumor, Shanghai Institutes for Biological Sciences–Changzheng Hospital Joint Center for Translational Medicine, Changzheng Hospital, Institutes for Translational Medicine, Chinese Academy of Sciences–Second Military Medical University, Shanghai Institute of Nutrition and Health, Shanghai Institutes for Biological Sciences, University of Chinese Academy of Sciences, Chinese Academy of Sciences, 200031 Shanghai, China; bDepartment of Gastrointestinal Surgery, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, 200127 Shanghai, China; cDepartment of Bone and Soft Tissue Sarcomas, Fudan University Shanghai Cancer Center, 200032 Shanghai, China; dDepartment of Oncology, Shanghai Medical College, Fudan University, 200032 Shanghai, China; eDepartment of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115; fShanghai Information Center for Life Sciences, Shanghai Institute of Nutrition and Health, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 200031 Shanghai, China; gDepartment of Pathology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, 200127 Shanghai, China; hDepartment of Urology, No.1 Hospital of Jiaxing, 314000 Jiaxing, China; and iDepartment of Urology, Changzheng Hospital, Second Military Medical University, 200003 Shanghai, China Edited by Kenneth W. Kinzler, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University, Baltimore, MD, and approved October 1, 2019 (received for review August 26, 2019) Gastrointestinal stromal tumors (GISTs) are the most common demonstrate that chromosome 22q-targeting DEPDC5, silenced human sarcoma and are initiated by activating mutations in the by somatic mutations, is a GIST specific tumor suppressor and a KIT or PDGFRA receptor tyrosine kinases. Chromosome 22q dele- TKI treatment response modifier. tions are well-recognized frequent abnormalities in GISTs, occur- ring in ∼50% of GISTs. These deletions are thought to contribute Results and Discussion to the pathogenesis of this disease via currently unidentified tu- Whole Exome Sequencing Identifies Recurrent Inactivating DEPDC5 mor suppressor mechanisms. Using whole exome sequencing, we Aberrations in GISTs. To identify the causative tumor suppressor DEPDC5 report recurrent genomic inactivated gene mutations in genes at chromosome 22q in GISTs, we performed whole exome GISTs (16.4%, 9 of 55 patients). The demonstration of clonal sequencing in 40 GIST patients (Dataset S1). These studies con- DEPDC5 inactivation mutations in longitudinal specimens and in firmed reported GIST genes, such as KIT (3), PDGFRA (4), RB1 multiple metastases from individual patients suggests that these CDKN2A DMD MAX SETD2 mutations have tumorigenic roles in GIST progression. DEPDC5 in- (15), (15), (16), (17), and (18) activation promotes GIST tumor growth in vitro and in nude mice. (Datasets S2 and S3). Notably, these studies revealed somatic ho- DEPDC5 reduces cell proliferation through the mTORC1-signaling mozygous inactivating genomic DEPDC5 (encoding Dishevelled, Egl- pathway and subsequently induces cell-cycle arrest. Furthermore, 10 and Pleckstrin [DEP] domain-containing protein 5) aberrations, DEPDC5 modulates the sensitivity of GIST to KIT inhibitors, and the combination therapy with mTOR inhibitor and KIT inhibitor may Significance work better in GIST patients with DEPDC5 inactivation. These find- ings of recurrent genomic alterations, together with functional Activating mutations of KIT or PDGFRA are initiating events in data, validate the DEPDC5 as a bona fide tumor suppressor con- most gastrointestinal stromal tumors (GISTs) and indeed are tributing to GIST progression and a biologically relevant target of present in micro-GISTs, which are asymptomatic subcentimeter the frequent chromosome 22q deletions. lesions found in one-third of the general population. The bio- logical underpinnings of GIST progression are poorly un- sarcoma | GIST | KIT tyrosine kinase inhibitors | DEPDC5 derstood. Chromosome 22q deletions are well-recognized abnormalities in GISTs. However, the crucial gene has been arcomas are diverse mesenchymal malignancies that account unknown. We report recurrent genomic inactivated DEPDC5 Sfor ∼20% of pediatric and 1% of adult cancers (1). Gastro- mutations in GISTs. The DEPDC5 inactivated mutations are intestinal stromal tumors (GISTs) are the most common human prognostic in that they are associated with aggressive GISTs in sarcoma (2), which are mostly initiated by activating mutations of which they promote GIST progression and reduce sensitivity to the receptor tyrosine kinase KIT (75–80%) or PDGFRA (5–10%) KIT inhibitors. (3, 4). Although sharing the same KIT/PDGFRA mutations, micro- GISTs have a limited growth potential and hence are restrained at Author contributions: J.A.F. and Y.W. designed research; Y.P., F.X., H.C., C.W., M.Z., X. Liu, the subcentimeter level. The fact that micro-GISTs are common in X. Lu, Y.S., K.L., X.J., Z.L., X.Z., S.W., and Y.W. performed research; Y.P., F.X., H.C., C.W., and J.A.F. provided samples and clinical data; Y.P., F.X., C.W., M.Z., X. Liu, X. Lu, T.H., Y.S., general individuals (found in one-third of the general population) K.L., X.J., Z.L., X.Z., S.W., Y.H., L.W., J.A.F., and Y.W. analyzed data; and Y.P. and Y.W. without clinical symptoms (5–7) indicates that additional genetic wrote the paper. alterations contribute to the progression of clinical GISTs. Chro- The authors declare no competing interest. mosome 22q deletions are frequent chromosomal abnormalities in This article is a PNAS Direct Submission. ∼ – human GISTs, occurring in 50% of GISTs (2, 8 11), and are Published under the PNAS license. thought to contribute to the pathogenesis of this disease by yet- – Data deposition: Whole exome sequencing and RNA sequencing data reported in this unidentified tumor suppressor mechanisms (2, 8 11). study have been deposited in the National Omics Data Encyclopedia (accession no. Most GISTs with activating mutations in KIT often respond to OEP000478). treatment with KIT tyrosine kinase inhibitors (TKIs), such as first- 1Y.P., F.X., H.C., and C.W. contributed equally to this work. line imatinib, second-line sunitinib and third-line regorafenib, but 2To whom correspondence may be addressed. Email: [email protected] or the magnitude of tumor regression is variable (12–14). This het- [email protected]. erogeneity in TKI response could result from genetic modifiers This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. that regulate the degree to which tumor cells are dependent upon 1073/pnas.1914542116/-/DCSupplemental. the driver kinase and the response to TKI treatment. Here we First published October 21, 2019. 22746–22753 | PNAS | November 5, 2019 | vol. 116 | no. 45 www.pnas.org/cgi/doi/10.1073/pnas.1914542116 Downloaded by guest on September 30, 2021 including nonsense mutation, frameshift mutation, and dele- have somatic homozygous DEPDC5-inactivating aberrations tions in 7 of 40 (17.5%) GIST patients (Figs. 1 A and B and Dataset in 9 GIST patients (16.4%). Of 55 patients, 31 (∼56%) har- S1). Homozygous DEPDC5 mutations were confirmed by Sanger bored chromosome 22 loss (Dataset S4). All of the 9 patients sequencing (SI Appendix,Fig.S1A), single-nucleotide polymorphism harboring genomic DEPDC5 aberrations contain chromosome 22 (SNP) arrays (SI Appendix,Fig.S1B), quantitative PCR (SI Appendix, loss (Dataset S4). Therefore, DEPDC5 aberrations are significantly Fig. S1C), and fluorescence in situ hybridization (SI Appendix,Fig. more frequent in GISTs with chromosome 22 loss compared to S1D). Somatic inactivating genomic DEPDC5 aberrations were vali- chromosome 22 normal copy number (29 vs. 0%, P = 0.01177, 2- dated in 2 of 15 (13.3%) additional GIST patients (cases 41 to 55, tailed Fisher’stest)(Dataset S4). All of the 9 patients with genomic Dataset S4). This total set of 55 GIST patients was shown to DEPDC5 aberrations have both copies of DEPCD5 inactivated MEDICAL SCIENCES Fig. 1. Genomic DEPDC5 aberrations in 40 GIST patients. (A) Whole exome sequencing identifies genomic DEPDC5 aberrations in 7 of 40 (17.5%) GIST patients. Inactivating DEPDC5 mutations were intragenic homozygous deletions (blue lines indicate deleted exons) and hemizygous nucleotide alterations. Mutations are described according to international guidelines for sequence variant nomenclature provided by the Human Genome Variation Society (http:// varnomen.hgvs.org). Annotations in blue represent the nucleotide coding sequence mutations (indicated by “c.”) whereas annotations in green represent the resultant protein sequence mutations (indicated by “p.”). (B) Integrative genome viewer images of part of chromosome 22q from matched tumor and nonneoplastic cell DNAs from the same patients, demonstrating the tumor-restricted nature of DEPDC5 mutations. Pang et al. PNAS | November 5, 2019 | vol. 116 | no. 45 | 22747 Downloaded by guest on September 30, 2021 Fig. 2. Genomic DEPDC5 aberrations are a clonal genetic event in GIST progression. (A) Multiple tumors from the same patients share the