Genome-Wide Functional Screening Identifies CDC37 As a Crucial HSP90-Cofactor for KIT Oncogenic Expression in Gastrointestinal S

SHORT COMMUNICATION Genome-wide functional screening identiﬁes CDC37 as a crucial HSP90-cofactor for KIT oncogenic expression in gastrointestinal stromal tumors

A Marin˜ o-Enrı´quez1,2, W-B Ou1,3, G Cowley4, B Luo4, AH Jonker1, M Mayeda1, M Okamoto4, G Eilers1, JT Czaplinski5, E Sicinska5, Y Wang1, T Taguchi6, GD Demetri7, DE Root4 and JA Fletcher1

Most gastrointestinal stromal tumors (GISTs) contain KIT or PDGFRA kinase gain-of-function mutations, and therefore respond clinically to imatinib and other tyrosine kinase inhibitor (TKI) therapies. However, clinical progression subsequently results from selection of TKI-resistant clones, typically containing secondary mutations in the KIT kinase domain, which can be heterogeneous between and within GIST metastases in a given patient. TKI-resistant KIT oncoproteins require HSP90 chaperoning and are potently inactivated by HSP90 inhibitors, but clinical applications in GIST patients are constrained by the toxicity resulting from concomitant inactivation of various other HSP90 client proteins, beyond KIT and PDGFRA. To identify novel targets responsible for KIT oncoprotein function, we performed parallel genome-scale short hairpin RNA (shRNA)-mediated gene knockdowns in KIT-mutant GIST-T1 and GIST882. GIST cells were infected with a lentiviral shRNA pooled library targeting 11 194 human genes, and allowed to proliferate for 5–7 weeks, at which point assessment of relative hairpin abundance identiﬁed the HSP90 cofactor, CDC37, as one of the top six GIST-speciﬁc essential genes. Validations in treatment-naive (GIST-T1, GIST882) vs imatinib-resistant GISTs (GIST48, GIST430) demonstrated that: (1) CDC37 interacts with oncogenic KIT; (2) CDC37 regulates expression and activation of KIT and downstream signaling intermediates in GIST; and (3) unlike direct HSP90 inhibition, CDC37 knockdown accomplishes prolonged KIT inhibition (420 days) in GIST. These studies highlight CDC37 as a key biologic vulnerability in both imatinib-sensitive and imatinib- resistant GIST. CDC37 targeting is expected to be selective for KIT/PDGFRA and a subset of other HSP90 clients, and thereby represents a promising strategy for inactivating the myriad KIT/PDGFRA oncoproteins in TKI-resistant GIST patients.

Oncogene (2014) 33, 1872–1876; doi:10.1038/onc.2013.127; published online 15 April 2013 Keywords: CDC37; HSP90; GIST; targeted therapy; functional genomics; shRNA library

INTRODUCTION loop regions are the most frequent.7 Less common imatinib Gastrointestinal stromal tumor (GIST) is the most common resistance mechanisms include KIT gene ampliﬁcation, and 8,9 mesenchymal tumor of the gastrointestinal tract. Gain-of-function activation of kinases downstream of KIT/PDGFRA. Notably, at mutation of the receptor tyrosine kinases KIT or PDGFRA is a time of progression on imatinib there can be substantial crucial oncogenic event in most GISTs, detectable in 85–90% of heterogeneity in these molecular resistance mechanisms within cases.1,2 Therapeutic targeting of the KIT/PDGFRA oncoproteins and between metastases in an individual patient. The multikinase with imatinib enables clinical responses in 80% of inoperable GIST inhibitor sunitinib is the only currently approved therapy for patients3 and has also improved progression-free survival as an advanced GIST following resistance to imatinib; although sunitinib adjuvant treatment postoperatively in patients with high-risk is a potent inhibitor of imatinib resistance caused by mutations in localized GIST.4 the KIT ATP-binding pocket, this therapy is less effective against Although dramatic therapeutic responses to imatinib are the imatinib resistance mutations affecting the KIT kinase activation norm in patients with inoperable GIST, clinical complete responses loop.10 Therefore, treatment of the entire spectrum of imatinib are rare (p5%) and up to 90% of responding patients eventually resistance mutations, particularly those encoded by KIT exons 17 develop secondary resistance, with median time to progression of and 18, as well as PDGFRA exon 18 remains an urgent unmet 2 years with ﬁrst-line tyrosine kinase inhibitor (TKI) therapy.5,6 medical need in GIST. Clinical progression during imatinib therapy occurs typically at The extreme dependence of GIST cells on KIT/PDGFRA multiple metastatic sites because of a variety of molecular imatinib activation is a striking example of oncogene addiction,11 in resistance mechanisms of which KIT/PDGFRA kinase domain which adaptations are required to optimize and stabilize secondary mutations in the ATP-binding pocket or activation the essential KIT/PDGFRA oncoproteins, creating secondary

1Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA; 2Departamento de Anatomıá Patolo´ gica, Hospital Universitario La Paz, Fundacioń para la Investigacioń Biome´dica FIBHULP, Universidad Autońoma de Madrid, IdiPAZ, Madrid, Spain; 3Zhejiang Provincial Key Laboratory of Applied Enzymology, Yangtze Delta Region Institute of Tsinghua University, Jiaxing, Zhejiang, China; 4RNAi Platform, Broad Institute of Harvard and MIT, Cambridge, MA, USA; 5Center for Molecular Oncologic Pathology, Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA; 6Department of Human Health and Medical Science, Graduate School of Kuroshio Science, Kochi University, Nankoku, Kochi, Japan and 7Center for Sarcoma and Bone Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA. Correspondence: Dr A Marinõ-Enrı´quez or Dr JA Fletcher, Department of Pathology, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115, USA. E-mail: [email protected] or jfl[email protected] Received 10 October 2012; revised 15 February 2013; accepted 26 February 2013; published online 15 April 2013 CDC37 is essential for KIT signaling in GIST A Marinõ-Enrı´quez et al 1873 dependencies on proteins that are requisite for KIT/PDGFRA addition, the GIST882 and GIST-T1 replicates clustered next to transforming activity. One such biologic dependency is HSP90 each other when compared with a reference data set of 12 chaperoning, required for folding, localization and stabilization of publicly available cancer cell line data sets,15 as well as after the mutant KIT/PDGFRA oncoproteins in GIST.12 Preclinical linkage analysis in the context of additional unpublished sarcoma validations have shown compelling responses to HSP90 cell lines (Supplementary Figure 1). A comprehensive list of the inhibition in GIST, in vitro and in vivo: after HSP90 inhibition by 11 194 ranked genes, along with annotation for the shRNA clones, a variety of compounds, KIT oncoproteins are rapidly degraded, is provided in Supplementary Table 1. with antiproliferative and pro-apopotic consequences.12,13 However, clinical translation of HSP90 targeting has been challenging, presumably because inhibition of HSP90 also CDC37 is essential for GIST cell survival targets HSP90-dependent, non-oncogenic, client proteins In the pooled proliferation screens, cells carrying shRNAs that limiting the tolerance to chronic and potent HSP90 inhibition. targeted proliferation-essential genes were depleted from the cell These considerations, together with issues relating to trial design population over time. Scored according to the second best- and patient selection criteria, might explain why initial clinical scoring shRNA within each hairpin set, 25 out of 56 genes ranked trials of ansamycin-analog HSP90 inhibition in GIST—although in the top 0.5% of the distribution for both GIST882 and GIST-T1 showing evidence of biological activity—have had low response (Table 1, left column). Of these 25 genes, 19 were also within the 15 rates accompanied by toxicity.14 To identify candidate targets that top 0.5% in at least 8 of 12 comparison non-GIST cancer lines, might have greater specificity for KIT-mutant GIST, we undertook a and were thus identified as ‘commonly essential’ genes not genome-scale functional screen in which stable gene knockdowns specific to GIST (Figure 1a). These genes belonged to functional were achieved by RNA interference. categories known to be essential in cancer cell lines: regulation of mRNA splicing and processing, protein translation, and ribosome and proteasome structure and function. The other six genes were selectively essential for the two GIST cell lines vs the other RESULTS AND DISCUSSION lines (bold italic font, Table 1 left column): five of these encode shRNA pooled library screen performance in GIST-T1 and GIST882 mRNA processing proteins, whereas the remaining gene, CDC37, cells encodes an HSP90 cofactor. CDC37 is known to coordinate Reproducibility of short hairpin RNA (shRNA) enrichment and HSP90 chaperoning activity for a subset of HSP90 client depletion profiles was evaluated across the experimental repli- proteins, including several kinases,17,18 by mechanisms involving cates, as a quality control. The GIST882 and GIST-T1 screen replicates clustered closely within each cell line by both unsupervised and consensus clustering of shRNA depletion and enrichment profiles, attesting to the robustness of the screens. In

Table 1. Top 0.5% essential genes according to the second best- scoring hairpin in GIST882 and GIST-T1 (n ¼ 56 for each)

GIST882 and GIST-T1 GIST882 only GIST-T1 only

CDC37 UBC PSMC4 POLE DYNC1H1 EFTUD2 RPS18 VCP PSMA2 PSMA3 HNRPK PRPF3 SFRS3 PABPN1 NCBP2 EIF3S10 SNRPD1 ABCB7 RPS13 PSMC1 EIF3S3 RPS29 FRAP1 ATPBD1C RPL23A AFG3L2 RPAP1 PSMB2 LOC375133 RBM8A RPL5 PSMA6 DDX48 RPS9 EIF3S5 RNPS1 RPS17 TSG101 COPS2 RPL31 PTPRCAP RPS10 Figure 1. Primary shRNA pooled screen. Development and applica- ARCN1 TUBB MLXIP tions of the 54K lentiviral shRNA pooled library from the RNAi PHB2 RPS19 ATP1A1 Consortium (TRC) have been described previously.16 In brief, GIST RPS7 KARS POLR2F cells were infected with a pool of 54 020 viruses targeting 11 194 RPS27A SNRPD2 RUVBL2 genes and subjected to puromycin selection. Replicates of 20 million RPS8 PSMA1 NUP205 RPS15A RPS14 MKI67IP infected GIST-T1 and GIST882 cells were established after the PSMD1 HNRPU RAN infections and allowed to proliferate independently for 6–7 weeks. U2AF2 RPS6 RPS3A Genomic DNA was isolated from final harvests of cultured cells for CHD4 RPS3 RPS4X shRNA amplification and massively parallel sequencing as described AQR ASCC3L1 RPS11 previously.16 The 54 020 shRNAs were ranked by their relative NHP2L1 RPL34 RPL7 NDUFA4L2 RPL6 depletion from the cell pool, and the corresponding 11 194 genes RPSA EIF5B were then scored according to the rank of the second-most depleted USP39 EIF2S2 shRNA (out of B5 shRNAs targeting each gene), using the GENE-E PHB SNRPE program (http://www.broadinstitute.org/cancer/software/GENE-E/ EIF1AX HSPE1 download.html). (a) Most of the top 0.5% essential genes for TPR U2AF1 GIST882 and GIST-T1 were commonly essential genes, based on their Abbreviation: GIST, gastrointestinal stromal tumor. Genes in bold font ranks in at least 8 of 12 non-GIST cancer cell lines of various lineages. (upper fields) scored top 0.5% in the gene distribution in GIST lines but not However, six genes, including CDC37, were selectively essential in in 12 non-GIST reference cancer cell lines of various lineages (described by GIST882 and GIST-T1 compared with the non-GIST lines. (b) Gene Luo et al.15). Genes in regular font (lower fields) scored top 0.5% in both ranks (red) and shRNA ranks (black) corresponding to CDC37, KIT and GIST and the 12 non-GIST reference set. ETV1 in GIST882 cells. Essential genes (oncogenes) rank on the top of the distribution.

& 2014 Macmillan Publishers Limited Oncogene (2014) 1872 – 1876 CDC37 is essential for KIT signaling in GIST A Marin˜o-Enrı´quez et al 1874 CDC37 homodimerization, CDC37-HSP90 heterodimerization and chaperoning in GIST. The KIT oncogenic driver and the GIST- the formation of CDC37-kinase-HSP90 complexes.19 These lineage-related transcription factor ETV1 also scored as essential observations suggest that CDC37 targeting might be a selective genes in these primary screens and serve as positive controls approach to derailing HSP90-mediated KIT oncoprotein (Figure 1b). In GIST-T1 cells, only one out of the ﬁve shRNAs targeting KIT was highly depleted, so KIT did not rank highly in the essential genes list; however, subsequent experiments showed that only the strongly depleted shRNA was highly effective at suppressing KIT in these cells (B70% knockdown) whereas the other four shRNAs produced o30% knockdown of KIT (Supplementary Figure 2).

CDC37 interacts with KIT in GIST, maintaining KIT expression and cell survival CDC37 expression was demonstrated by immunoblotting in the Figure 2. CDC37 expression and CDC37-KIT co-immunoprecipita- GIST cell lines (Figure 2). Interaction between CDC37 and KIT in tions in GIST cell lines, demonstrating CDC37:KIT interaction. Whole- these GISTs was demonstrated by co-immunoprecipitations using cell lysates, electrophoresis and immunoblotting were carried out as KIT and CDC37 antibodies in KIT-positive GIST882 and GIST-T1 described previously.20 cells, with KIT-negative GIST48B cells serving as a negative control

Figure 3. Persistent inhibition of KIT oncoprotein expression and phosphorylation, and decreased downstream signaling pathway activation in KIT- dependent GIST cells on shRNA-mediated CDC37 knockdown. (a) Lentiviral constructs encoding shRNA-specific sequences targeting CDC37 transcripts on the pLKO.1puro backbone were selected from the RNAi Consortium (TRC) library (TRCN0000116632: clone 1; and TRCN0000116633: clone 2; the TRC website is http://www.broadinstitute.org/rnai/trc/lib). Lentivirus preparations and lentiviral infections were performed as described previously.23 Cells were lysed for immunoblot analysis at 4, 10 and 20 days post-infection; day 10 is shown here. Whole-cell lysates, electrophoresis and immunoblotting were carried out as described previously.20 (b) shRNA-mediated CDC37 knockdown inhibits viability of GISTs, including those resistant to imatinib (GIST430) and to the ansamycin-type HSP90-inhibitor 17-AAG (GIST882/AAG and GIST-T1/AAG). For cell viability evaluations by bright field microscopy, cell images were obtained using SPOT software (SPOT Imaging Solutions, Sterling Heights, MI, USA) and an Eclipse TE2000-5 inverted microscope (Nikon, Japan). For viability evaluations by ATP-based CellTiter-Glo luminescent assay (Promega, Madison, WI, USA) (c), luciferase-catalyzed luciferin/ATP reactions were measured with a Veritas Microplate Luminometer (Turner Biosystems, Sunnyvale, CA, USA) and normalized to the day 0 and pLKO control reads. (d) Increase in the number of cells in sub-G0 phase of the cell cycle (flow cytometry analysis of propidium iodide-stained DNA content as previously described),12 10 days after lentiviral infection and puromycin selection.

Oncogene (2014) 1872 – 1876 & 2014 Macmillan Publishers Limited CDC37 is essential for KIT signaling in GIST A Marin˜o-Enrı´quez et al 1875 (Figure 2). CDC37 shRNA-mediated knockdowns resulted in 490% these responses were also seen in GIST lines resistant to imatinib reduction of KIT expression and activation in the KIT-dependent (GIST430) or to the ansamycin HSP90 inhibitor 17-AAG (GIST-T1/ GIST882, GIST430 and GIST-T1 lines (Figure 3a). KIT inhibition was AAG and GIST882/AAG). CDC37 knockdown induced an increased associated with inactivation of downstream growth and survival sub-G0 cell cycle peak, consistent with a pro-apoptotic effect signaling intermediates, including AKT. By contrast, AKT was not (Figure 3d). inhibited by CDC37 knockdown in the KIT-negative cell line shRNA-mediated CDC37 knockdown in GIST-T1 cells resulted in GIST48B, suggesting that the observed inhibition of downstream decreased tumor growth in vivo, with decreased tumor volume, signaling pathways in GIST882, GIST430 and GIST-T1 was KIT overall decreased cellularity and decreased mitotic activity in dependent (Figure 3a). CDC37 knockdown, unlike direct HSP90 mouse xenografts (Figure 4). inhibition,21 resulted in persistent inhibition of KIT expression for 420 days, indicating that GIST cells have few compensatory pathways for CDC37 function. The above-mentioned biochemical Celastrol does not enable selective CDC37:HSP90 pharmacologic responses to CDC37 knockdown were accompanied by decreased inhibition GIST proliferation and survival as assessed by bright ﬁeld Preclinical pharmacologic validations were attempted using the microscopy and CellTiter-Glo assays (Figures 3b and c). Notably, HSP90:CDC37 interface inhibitor celastrol.22 However, celastrol

Figure 4. shRNA-mediated CDC37 knockdown inhibits growth of GIST xenografts in mice. Athymic nude mice were injected subcutaneously with GIST-T1 cells expressing CDC37-targeting shRNA in one flank (shRNA1: TRCN0000116632; shRNA 2: TRCN0000116633; n ¼ 3 each) and empty pLKO.1 lentiviral vector in the other flank (n ¼ 3). In all, 2 Â 106 infected cells on puromycin selection were resuspended in BD Matrigel and implanted subcutaneously at each injection site. Tumor volume was evaluated weekly. Mice were killed by CO2 inhalation and necropsied 6 weeks after injection. (a, b) Tumors were resected and photographed, demonstrating significantly decreased tumor growth on CDC37 knockdown. (c) Western blot confirming inhibition of CDC37 expression in GIST-T1 cells infected with CDC37 shRNA1 and shRNA2, compared with pLKO.1 lentiviral vector. (d) Histologic evaluation of formalin-fixed and paraffin-embedded samples after hematoxylin and eosin staining demonstrates sparsely cellular areas with no mitotic activity in CDC37-knockdown GIST xenografts, in comparison with highly cellular and mitotically active xenografts of pLKO.1 lentiviral-infected cells.

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Merck, ZioPharm, N-of-One, Champions Biotechnology, Blueprint Medicines; he is a 15 Luo B, Cheung HW, Subramanian A, Sharifnia T, Okamoto M, Yang X et al. Highly member of the Scientific Advisory Boards of ZioPharm, Kolltan Pharmaceuticals, N-of- parallel identification of essential genes in cancer cells. Proc Natl Acad Sci USA One, and Blueprint Medicines; and he holds minor equity stakes at N-of-One, 2008; 105: 20380–20385. Champions Biotechnology, Kolltan Pharmaceuticals and Blueprint Medicines. 16 Cheung HW, Cowley GS, Weir BA, Boehm JS, Rusin S, Scott JA et al. Systematic Novartis, Pfizer, Sanofi-Aventis, Glaxo-Smith-Kline, Johnson & Johnson, Merck, and investigation of genetic vulnerabilities across cancer cell lines reveals lineage-specific Amgen support clinical trials in the Center for Sarcoma and Bone Oncology, Dana- dependencies in ovarian cancer. Proc Natl Acad Sci USA 2011; 108: 12372–12377. Farber Cancer Institute. Dr Jonathan A Fletcher has consulting arrangements with 17 Vaughan CK, Mollapour M, Smith JR, Truman A, Hu B, Good VM et al. Hsp90- Novartis, Pfizer and Deciphera Pharmaceuticals. None of these relationships dependent activation of protein kinases is regulated by chaperone-targeted constitute a conflict of interest for this work. The remaining authors declare no dephosphorylation of CDC37. Mol Cell 2008; 31: 886–895. conflict of interest. 18 Smith JR, Workman P. Targeting CDC37: an alternative, kinase-directed strategy for disruption of oncogenic chaperoning. Cell Cycle 2009; 8: 362–372. 19 Xu W, Mollapour M, Prodromou C, Wang S, Scroggins BT, Palchick Z et al. Dynamic ACKNOWLEDGEMENTS tyrosine phosphorylation modulates cycling of the HSP90-P50(CDC37)-AHA1 Adriań Marinõ-Enrı´quez, Wen-Bin Ou, Yuexiang Wang, Jonathan A Fletcher and chaperone machine. Mol Cell 2012; 47: 434–443. George D Demetri, are supported by the GI SPORE 1P50CA12703-05, Virginia 20 Rubin BP, Singer S, Tsao C, Duensing A, Lux ML, Ruiz R et al. KIT activation is a and Daniel K Ludwig Trust for Cancer Research, Paul’s Posse and Team Cesarini of the ubiquitous feature of gastrointestinal stromal tumors. Cancer Res 2001; 61: 8118–8121. Pan Mass Challenge, LifeRaft Group and the GIST Cancer Research Fund. Adriań 21 Chandarlapaty S, Sawai A, Ye Q, Scott A, Silinski M, Huang K et al. SNX2112, a Marinõ-Enrı´quez is also supported by a Sarcoma Alliance for Research Through synthetic heat shock protein 90 inhibitor, has potent antitumor activity against Collaboration (SARC) Career Development Award. Wen-Bin Ou is also supported by HER kinase-dependent cancers. Clin Cancer Res 2008; 14: 240–248. Qianjiang Talents Project of Zhejiang (2012R10079), a grant from the Science and 22 Zhang T, Li Y, Yu Y, Zou P, Jiang Y, Sun D. Characterization of celastrol to inhibit Technology Bureau of Jiaxing, Zhejiang (2012AY1039) and the Major Science and HSP90 and CDC37 interaction. J Biol Chem 2009; 284: 35381–35389. Technology Special Project of Zhejiang Province (2012C03007-4). The RNAi 23 Lee CH, Ou WB, Marino-Enriquez A, Zhu M, Mayeda M, Wang Y et al. 14-3-3 fusion Consortium (TRC) supported the development of pooled screening methods used oncogenes in high-grade endometrial stromal sarcoma. Proc Natl Acad Sci USA for these screens. 2012; 17: 929–934.

Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)

Oncogene (2014) 1872 – 1876 & 2014 Macmillan Publishers Limited