Oncogene https://doi.org/10.1038/s41388-019-0894-3 ARTICLE Cyclin D1 is a mediator of gastrointestinal stromal tumor KIT-independence Wen-Bin Ou 1,2 ● Nan Ni 1 ● Rui Zuo 1 ● Weihao Zhuang 1 ● Meijun Zhu 2 ● Anastasios Kyriazoglou 2 ● Duolin Wu 1 ● Grant Eilers 2 ● George D. Demetri 3 ● Haibo Qiu 2,4 ● Bin Li 2,5 ● Adrian Marino-Enriquez 2 ● Jonathan A. Fletcher 2 Received: 6 August 2018 / Revised: 22 February 2019 / Accepted: 3 April 2019 © The Author(s), under exclusive licence to Springer Nature Limited 2019 Abstract Oncogenic KIT or PDGFRA tyrosine kinase mutations are compelling therapeutic targets in most gastrointestinal stromal tumors (GISTs), and the KIT inhibitor, imatinib, is therefore standard of care for patients with metastatic GIST. However, some GISTs lose expression of KIT oncoproteins, and therefore become KIT-independent and are consequently resistant to KIT-inhibitor drugs. We identified distinctive biologic features in KIT-independent, imatinib-resistant GISTs as a step towards identifying drug targets in these poorly understood tumors. We developed isogenic GIST lines in which the parental forms were KIT oncoprotein-dependent, whereas sublines had loss of KIT oncoprotein expression, accompanied by markedly downregulated expression of the GIST biomarker, protein kinase C-theta (PRKCQ). Biologic mechanisms unique to KIT- independent GISTs were identified by transcriptome sequencing, qRT-PCR, immunoblotting, protein interaction studies, knockdown and expression assays, and dual-luciferase assays. Transcriptome sequencing showed that cyclin D1 expression was extremely low in two of three parental KIT-dependent GIST lines, whereas cyclin D1 expression was high in each of the KIT-independent GIST sublines. Cyclin D1 inhibition in KIT-independent GISTs had anti-proliferative and pro-apoptotic effects, associated with Rb activation and p27 upregulation. PRKCQ, but not KIT, was a negative regulator of cyclin D1 expression, whereas JUN and Hippo pathway effectors YAP and TAZ were positive regulators of cyclin D1 expression. PRKCQ, JUN, and the Hippo pathway coordinately regulate GIST cyclin D1 expression. These findings highlight the roles of PRKCQ, JUN, Hippo, and cyclin D1 as oncogenic mediators in GISTs that have converted, during TKI-therapy, to a KIT- independent state. Inhibitors of these pathways could be effective therapeutically for these now untreatable tumors. Introduction Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract [1], arising from a transformed progenitor cell in the inter- stitial cell of Cajal lineage. GISTs are heterogeneous his- tologically, but irrespective of whether spindle cell (70%), epithelioid cell (20%) or mixed type (10%) [2], they gen- erally contain oncogenic gain-of-function mutations of KIT (~85%) or PDGFRA (~5–10%) [2–4]. These receptor tyr- osine kinase oncoproteins are crucial to GIST cell trans- formation, proliferation and survival, as evidenced by the clinical successes of KIT/PDGFRA-inhibition by small molecule therapeutics [5–7]. Therefore, imatinib is standard of care in patients with inoperable GIST [8, 9], and adjuvant imatinib is used post-operatively in patients with localized GIST at high risk of recurrence [10]. Despite the dramatic clinical success of imatinib, most patients with inoperable GIST eventually experience * Wen-Bin Ou ouwenbin@tsinghua.org.cn * Jonathan A. Fletcher jfletcher@partners.org 1 Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine, College of Life Sciences and Medicine, Zhejiang Sci-Tech University, Hangzhou, Zhejiang, China 2 Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115, USA 3 Ludwig Center at Dana-Farber/Harvard Cancer Center and Department of Medical Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA 02115, USA 4 State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center, Guangzhou, China 5 Division of Oncology, Xiangya Hospital, Central South University, Changsha, China Supplementary information The online version of this article (https:// doi.org/10.1038/s41388-019-0894-3) contains supplementary material, which is available to authorized users. 1234567890();,: 1234567890();,: