Orthoxenografts of Testicular Germ Cell Tumors Demonstrate Genomic 2 Changes Associated with Cisplatin Resistance and Identify PDMP As a Re- 3 Sensitizing Agent

Author Manuscript Published OnlineFirst on April 4, 2018; DOI: 10.1158/1078-0432.CCR-17-1898 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

1 Orthoxenografts of testicular germ cell tumors demonstrate genomic 2 changes associated with cisplatin resistance and identify PDMP as a re- 3 sensitizing agent

1,2& 3,12& 1 1 4 Josep M. Piulats , August Vidal , Francisco J García-Rodríguez , Clara Muñoz , Marga 5 Nadal 1, Catia Moutinho 4, María Martínez-Iniesta 1, Josefina Mora 5, Agnés Figueras 1, Elisabet 6 Guinó 6, Laura Padullés 1, Àlvaro Aytés 1, David G. Molleví 1, Sara Puertas 1, Carmen Martínez- 7 Fernández8, Wilmar Castillo 1, Merce Juliachs 1, Victor Moreno 6, Purificación Muñoz 4, Milica 7 1 3 4 2 8 Stefanovic , Miguel A. Pujana , Enric Condom , Manel Esteller , Josep R. Germà , Gabriel 1 1,10 7 1,9 2 9 Capella , Lourdes Farré , Albert Morales , Francesc Viñals , Xavier García-del-Muro , 10 Julián Cerón 8,11 and Alberto Villanueva 1,11,12. 11 1. Program Against Cancer Therapeutic Resistance (ProCURE), Catalan Institute of Oncology (ICO), 12 Bellvitge Institute for Biomedical Research (IDIBELL), Oncobell Program, L’Hospitalet del 13 Llobregat, Barcelona 08908, Catalonia, Spain. 14 2. Department of Medical Oncology, Catalan Institute of Oncology – IDIBELL. 15 3. Department of Pathology, Hospital Universitari de Bellvitge – IDIBELL. CIBERONC. 16 4. Cancer Epigenetics and Cell Biology Program (PEBC), Catalan Institute of Oncology –IDIBELL 17 5. Department of Biochemistry, Hospital de Sant Pau, 08025 Barcelona, Spain. 18 6. Bioinformatic Unit, Catalan Institute of Oncology – IDIBELL. 19 7. Department of Cell Death and Proliferation, Instituto de Investigaciones Biomédicas de Barcelona 20 (IIBB-CSIC), IDIBAPS, Barcelona, Catalonia, Spain. 21 8. Modelling Human Diseases in C. elegans. Genes, Disease and Therapy Program – IDIBELL. 22 9. Departament de Ciències Fisiològiques II, Universitat de Barcelona, Avda Feixa Llarga S/N 08907 23 L'Hospitalet de Llobregat, Barcelona, Spain. 24 10. Laboratory of Experimental Pathology (LAPEX), Gonçalo Moniz Research Center, Oswaldo Cruz 25 Foundation (CPQGM/FIOCRUZ), Salvador, Bahia, Brazil; National Institute of Science and 26 Technology of Tropical Diseases (INCT/DT), Salvador, Brazil. 27 11. C. elegans Core Facilty-IDIBELL. 28 12. Xenopat S.L., Business Bioincubator, Bellvitge Health Science Campus, 08907 L’Hospitalet de 29 Llobregat, Barcelona, Spain. 30 31 Running title: Orthoxenografts to identify targets for cisplatin resistance 32 Key words: Germ cell tumor, orthoxenografts, cisplatin resistance, glucosylceramide synthase, 33 PDMP. 34 Additional information: 35 & Authors contributed equally to this work 36 Financial support: Several authors are grateful recipients of predoctoral fellowships: JMP from the 37 AECC and FJG-R from the Instituto De Salut Carlos III (ISCIII). This study was supported by grants 38 from the Spanish Ministry of Economy and Competitiveness (SAF2002-02265 and FIS: BFU2007- 39 67123; PI10-0222, PI13-01339, PI16/01898) to AV, PI15-00895 to JC, SAF2013-46063R to FV, 40 PI030264 to XGM, Fundació La Marató TV3 (051430) to FV and XGM, Generalitat de Catalunya 41 (2014SGR364) to AV and FV, FIS09/0059 to AM, confunded by FEDER funds/ European 42 Regional Development Fund (ERDF)-a way to Build Europe. A. Vidal received a BAE11/00073 43 grant. 44 Corresponding authors: Julian Ceron PhD. Genes, Disease and Therapy Program – IDIBELL. Avda 45 Gran Via s/n km 2.7 L’Hospitalet de Llobregat, 08907 Barcelona, Spain. Phone: 34-93 260 7251; 46 Fax: 34-93 260 7466; [email protected]. Alberto Villanueva PhD. Laboratory of Translational 47 Research. ICO-IDIBELL, Hospital Duran i Reynals, Avda Gran Via s/n km 2.7 L’Hospitalet de 48 Llobregat, 08907 Barcelona, Spain. Phone: 34-93260 7952; Fax: 34-93 260 7466; 49 [email protected]

Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 4, 2018; DOI: 10.1158/1078-0432.CCR-17-1898 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

50 Disclosure of Potential Conflicts of Interest: A.Villanueva and A.Vidal are founders and 51 shareholders of Xenopat S.L (Barcelona, Spain). No potential conflicts of interest were disclosed by 52 the other authors. 53 Word count: 5.339; Number of figures and tables: 5 54 TRASLATIONAL RELEVANCE 55

56 Cisplatin-based cytotoxic chemotherapy is the mainstay of the treatment of several

57 types of neoplasias being the acquired resistance a major clinical limitation for patient’s

58 survival. Orthoxenografts are the most advanced ex vivo platforms to investigate the

59 efficiency of drugs in a personalized manner but in this study we also demonstrated that

60 are valuable tools to identify prognosis markers and novel re-sensitizing therapeutic

61 approaches for the treatment of cisplatin refractory tumors. As proof-of-principle, in this

62 study we validate our approach demonstrating that presence of the 9q32-q33.1 gain is

63 associated with poor risk defined by shorter overall survival (OS) and that genetic or

64 pharmacological inhibition of glucosylceramide synthase (GCS) activity is efficient to

65 re-sensitize testicular and epithelial ovarian tumors refractory to cisplatin.

80 ABSTRACT

81 Purpose: To investigate the genetic basis of cisplatin resistance since efficacy of

82 cisplatin-based chemotherapy in the treatment of distinct malignancies is often

83 hampered by intrinsic or acquired drug resistance of tumor cells.

84 Experimental design: We produced 14 orthoxenograft transplanting human

85 nonseminomatous (NSE) testicular germ cell tumors (TGCTs) to mice, keeping the

86 primary tumor features in terms of genotype, phenotype and sensitivity to cisplatin.

87 Chromosomal and genetic alterations were evaluated in matched cisplatin-sensitive and

88 their counterpart orthoxenografts that developed resistance to cisplatin in nude mice.

89 Results: Comparative genomic hybridization analyses of four matched orthoxenografts

90 identified recurrent chromosomal rearrangements across cisplatin-resistant tumors in

91 three of them, showing gains at 9q32-q33.1 region. We found a clinical correlation

92 between the presence of 9q32-q33.1 gains in cisplatin refractory patients and poorer

93 overall survival in metastatic germ cell tumors. We studied the expression profile of the

94 sixty genes located at that genomic region. POLE3 and AKNA were the only two genes

95 deregulated in resistant tumors harboring the 9q32-q33.1 gain. Moreover, other four

96 genes (GCS, ZNF883, CTR1 and FLJ31713) were deregulated in all five resistant

97 tumors independently of the 9q32-q33.1 amplification. RT-PCRs in tumors and

98 functional analyses in C. elegans indicate that the influence of 9q32-q33.1 genes in

99 cisplatin resistance can be driven by either up- or down-regulation. We focused on

100 glucosylceramide synthase (GCS) to demonstrate that the GCS inhibitor DL-threo-

101 PDMP re-sensitizes cisplatin-resistant germline-derived orthoxenografts to cisplatin.

102 Conclusions: Orthoxenografts can be used preclinically not only to test the efficiency

103 of drugs but also to identify prognosis markers and gene alterations acting as drivers of

104 the acquired cisplatin resistance.

105 INTRODUCTION

106 Testicular germ cell tumors (TGCTs) of adolescent and young adults are the most

107 common malignancy in young men (1–3). They can be classified as seminomas (SEs)

108 (originated in epithelium of the seminiferous tubules), which represent around 40% of

109 cases, and nonseminomas (NSEs)(60%). Seminomas are radio- and chemo-sensitive

110 tumors highly curable at all stages. With the exception of teratomas, NSEs are also

111 highly sensitive to cisplatin-based chemotherapy and, when combined with surgery,

112 patients achieve high cure rates (4). In contrast with most advanced solid tumors,

113 approximately 80-90% of metastatic TGCTs will achieve complete cure after standard

114 doses of cisplatin (CDDP) chemotherapy (4) . Nevertheless, 10-15% of patients die due

115 to cisplatin refractoriness and the absence of alternative effective re-sensitizing

116 therapies (5–7). Such high success in treating advanced testicular cancer has limited the

117 number of studies addressing the treatment failure in refractory patients (8, 9).

118 In TGCTs, cisplatin resistance has been attributed to diverse cellular mechanisms (10–

119 12), although the molecular details underlying treatment failure in refractory patients

120 remains obscure (13–17). Patient derived orthotopic xenografts, named PDOX or

121 orthoxenografts, are relevant preclinical animal models that phenocopy human tumor

122 properties (18,19). We have previously used TGCT orthoxenografts to explore novel

123 therapeutic approaches for refractory TGCTs (20,21). In the present study, the genetic

124 basis of acquired cisplatin resistance was investigated by Comparative Genomic

125 Hybridization (CGH) in a collection of matched cisplatin sensitive and resistant NSE

126 tumors that were implanted orthotopically in nude mice. We studied genome

127 amplifications and further investigated the 9q32-q33.1 region to identify cisplatin

128 resistance-related genes. We found a clinical correlation between the presence of the

129 9q32-q33.1 gain and poor risk defined by shorter OS. We also found gene expression

130 alterations within the 9q32-q33.1 region that were associated with cisplatin resistance

131 but not necessarily with the 9q32-q33.1 gain. Finally, we used a drug to inhibit one of

132 the recurrently upregulated genes in cisplatin refractory tumors, the glucosylceramide

133 synthase (GCS), and observed that tumors were re-sensitized allowing the maintenance

134 of the cisplatin-based therapy.

135 MATERIAL AND METHODS

136

137 Human primary testicular germ cell tumor implantation and perpetuation in nude

138 mice.

139 To generate the collection of TGCT othoxenografts, fresh surgical specimens of 62

140 human GCTs were implanted in nude mice. Twenty-two tumors were classified as pure

141 SEs, and 40 as NSE (21 as pure and 19 as mixed tumors containing different

142 proportions of SE and NSE components). From the 40 NSE, 14 tumors were

143 perpetuated (35%), 10 derived from pure nonseminomas (three choriocarcinomas

144 (CHs), four embryonal carcinomas (ECs) and three yolk sac tumor (YS) and four from

145 mixed primary tumors). Five orthoxenografts were derived from several extragonadal

146 tumor locations, and in four cases from patients treated previously with cisplatin based-

147 chemotherapy (Table S1). None of the 22 implanted pure gonadal seminomas (SEs)

148 grew in nude mice. Of the mixed tumors, comprising both SE and NSE components,

149 only the NSEs grew in mice. Orthotopic implantation procedure of human tumors was

150 performed as previously reported by our group (21) and briefly described in the

151 Supplementary Data. Immunohistochemistry characterization is also described in the

152 Supplementary Data. All patients gave written consent to participate in the study. The

153 Institutional Ethics Committees approved the study protocol, and the animal

154 experimental design was approved by the IDIBELL animal facility committee

155 (AAALAC - Unit 1155). All experiments were performed in accordance with the

156 guidelines for Ethical Conduct in the Care and Use of Animals as stated in The

157 International Guiding Principles for Biomedical Research Involving Animals,

158 developed by the Council for International Organizations of Medical Sciences

159 (CIOMS).

160

161 Generation in mice of refractory engrafted NSEs to cisplatin treatment

162 Five selected engrafted tumors, TGT1X, TGT12X, TGT21XB, TGT34X and TGT38X,

163 from patients without prior exposure to cisplatin, were allowed to grow until intra-

164 abdominal palpable masses were noted. Animals were administered with cisplatin i.v. at

165 a dose of 2 mg/kg for 3 consecutive weeks (days 0, 7 and 14) (cycle#1 of treatment).

166 Post-cisplatin relapse tumors were harvested, prepared as previously described, and

167 engrafted in new animals. This process was repeated up to five times by treating tumor-

168 bearing mice with stepwise increasing doses of cisplatin: cycle#2, 3 mg/kg; cycle#3, 3.5

169 mg/kg; cycle#4, 4 mg/kg; and cycle#5, 5 mg/kg, as we described for ovarian tumors

170 (22). When mice were treated at doses higher than 3.5 mg/kg, the signs of cisplatin-

171 induced toxicity were ameliorated by administration of saline containing 5% glucose for

172 2 days. Dynamic CDDP responses were evaluated after assessing β-hCG and/or AFP

173 serum levels, as described in Supplementary Data.

174

175 Analysis of point mutations and genomic imbalances.

176 The presence of point mutations in a panel of selected cancer-related genes and

177 microsatellite instability (MSI) were compared between sensitive and resistant paired

178 orthoxenografts. Procedures are described in detail in the Supplementary Data.

179

180 Whole genome analysis by NimbleGen CGH arrays

181 The CGH oligonucleotide array was carried out by NimbleGen Systems, Inc., at their

182 facility in Wisconsin (USA). Array design descriptions were: 2006-07-

183 27_HG18_WG_CGH, single array CGH design for whole human genome (hg18; NCBI

184 Build 36). Methods of DNA labeling array construction, hybridization, array

185 normalization and data analysis have been described in detail by Seltzer et al. (23).

186

187 Patients

188 Eighty-eight consecutive patients diagnosed with metastatic germ cell tumors and

189 treated at the Institut Català d’Oncologia between 1989 and 2004 were initially included

190 in this study. Thirteen cases were not evaluated because of the lack of paraffin-

191 embedded tissue blocks. Patient demographics and clinical characteristics of the 75

192 patients finally evaluated are listed in Table S3. Sixty-three patients (84%) had NSE

193 tumors and 12 (16%) had SE tumors. Four patients presented with mediastinal

194 extragonadal disease. Sixty per cent of the patients were classed as having a good

195 prognosis, 19% as having an intermediate prognosis and 21% as being of poor

196 prognosis according to the IGCCCG categorization. Twenty-four patients were

197 considered resistant, defined by progression or relapse despite adequate first-line

198 chemotherapy treatment. Cases with mature teratoma only in the resected post-

199 chemotherapy mass and without posterior tumor relapse were considered sensitive.

200 Tumor samples from primary tumors and/or resected metastases obtained before

201 chemotherapy were included in a newly generated TMA, as described (24). FISH

202 analysis was described in Supplementary Material.

203

204 Quantitative gene and miRNA analysis

205 Total RNA was extracted using Trizol (Invitrogen, San Diego, CA), following the

206 manufacturer’s instructions, and reverse-transcribed to cDNA. Quantitative RNA and

207 miRNA analyses were performed as described in the Supplementary Material.

208

209 Cell culture, transfection and in vitro gene overexpression and shRNAi knockdown

210 experiments

211 The human NSE cell lines SuSaS (from teratocarcinoma origen), and its matched

212 SuSaR (“R” for CDDP-resistant derived cell line) were growth for different experiments

213 as described (25). For overexpression experiments, SuSaS cells were transfected with

214 plasmid pCMV6-XL5-GCS containing the whole GCS human cDNA from Origene

215 (SC118052; Rockville, USA). Knockdown experiments were realized in SuSaR with

216 four pre-designed small hairpin RNAs (shRNA) for the human GCS gene from Qiagen

217 (KH02376P; Manchester, UK) that were transfected with the jetPrime transfection kit

218 (Polyplus, Strasbourg), following manufacturer instructions. GCS expression levels was

219 analyzed by Western blot at 24, 48, 72 and 96 hours post-transfection by anti-GCS

220 (1/1000)(ProteinTech, Chicago, USA) using as a control the anti-β-actin-HRP antibody

221 (1/20000)(Sigma, St. Louis, USA). The chosen time to perform the experiments was 48

222 hours.

223

224 In vitro determination of drug resistance assays

225 Cisplatin (1mg/ml) dissolved in NaCl (TEVA, North Wales, USA), and DL-threo-

226 PDMP (Sigma, St. Louis, USA) in dimethyl sulfoxide (DMSO) at a final concentration

227 of 59 mM were assessed. Cell viability was determined by MTT assay. Briefly, 1 x 103

228 cells were plated onto 96-well plates, after 4 hours of transfection, fresh medium was

229 added and cells were treated for 48 hours with different drugs concentration ranged

230 from 0 to 20 µg/ml doses. MTT was added at a final concentration of 0.1% and after 2.5

231 hours of incubation (37ºC, 5% CO2), metabolic product formazan was dissolved in

232 DMSO and the absorbance measured at 570 nm. Prism Software (La Jolla, USA) was

233 used to calculate drugs half maximal inhibitory concentration (IC50).

234

235 Determination of GCS activity

236 Tumor samples were homogenized in lysis buffer (Tris-HCl 10 mM, EDTA 1mM, 0.1%

237 Triton X-100 at ph 7.4) and centrifuged at 600g for 5 minutes. GCS activity was

238 determined from NBD-C6-ceramide and UDP-glucose, the conversion product

239 separated by TLC with chloroform/methanol/32% ammonia (70:30:5, v/v), and

240 quantified by densitometry (Préférence/DVS, Sebia) as described previously (26).

241 Briefly, for each assay 200 µg of protein extract was suspended in reaction buffer (5

242 mM MgCl2, 5 mM MnCl2, and 1 mM EDTA in 50 mM HEPES, pH 7.2) and the

243 substrate mixture containing 10 µM NBD-C6-ceramide and 250 µM UDP-glucose.

244 After a 30 min incubation at 37°C, reactions were terminated by adding 2.5 ml of

245 chloroform/methanol (2:1, v/v), the samples were centrifugated (1000 x g, 5 min), the

246 lower phases dried under nitrogen and subjected to TLC by using

247 chloroform/methanol/32% ammonia (70:30:5, v/v) as the mobile phase.

248

249 Evaluation of in vivo responses of cisplatin-refractory orthoxenografts to treatment

250 with DL-threo-PMDP.

251 Tumors were implanted in mouse testicle and when homogeneous tumor sizes were

252 detected, animals were randomized to four treatment groups (n = 6-8 mice/group): (i)

253 vehicle, (ii) cisplatin (3.5 mg/kg), (iii) DL-threo-PDMP(D-threo-1-phenyl-2-

254 decanoylamino-3-morpholino-1-propanol hydrochloride) (Santa Cruz), 50 mg/kg

255 dissolved in 5% of Tween 80 - 0.85% NaCl and (iv) DL-threo-PDMP+cisplatin (50

256 mg/kg+3.5 mg/kg). Cisplatin was i.v. administered once a week for three consecutive

257 weeks (days 0, 7 and 14), while DL-threo-PDMP was administered daily by

258 intraperitoneal injection over the 21-day period and mice were sacrificed on day 22 of

259 treatment. In combined treatments, PDMP was administered one hour before cisplatin

260 treatment. Ovarian orthoxenograft 17 model (OVA17X) was generated from a

261 cisplatin-sensitive human serous epithelial ovarian tumor by ortotopic implantation (in

262 the mouse ovary) in nude mice, as previously described (22). Cisplatin-resistant Ovarian

263 orthoxenograft 17 (OVA17XR) was derived from OVA17X by iterative treatment

264 cycles with increasing doses of cisplatin, as described above for TGCT tumors. For the

265 in vivo treatment with DL-threo-PMDP, OVA17XR was grown and implanted in the

266 mouse ovary of several animals. When homogeneous tumor sizes were detected they

267 were randomized to four treatment groups (n = 6 mice/group) and treated as described.

268

269 Statistical analysis

270 For the clinicopathological features, P values were calculated using the X2 test.

271 Survival curves were estimated using the Kaplan-Meier method, and differences

272 between individual curves were evaluated by multivariate Cox proportional hazards

273 regression modeling. Analyses were adjusted for pathological diagnostic classification.

274 Hazard ratios (HRs) and 95% confidence intervals (CIs) were calculated. Likelihood

275 ratio tests were used to assess the prognostic value of genomic amplification of 9q32-

276 q33.1 by FISH in the TMA of metastatic GCTs. Values of P<0.05 were considered

277 significant.

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289

290 RESULTS

291

292 Establisment of orthoxenografts of TGCT NSE tumors

293 To study cisplatin resistance in TGCTs, our laboratory has compiled a valuable

294 collection (n=14) of both cisplatin sensitive and refractory TGCT NSE orthoxenografts

295 (Supplementary Table S1). These orthoxenografts grew on mouse testicles as a big

296 solid mass, displaying a strong correlation with their corresponding primary tumors in

297 terms of histological appearance and expression of cellular markers (Fig. 1A and 1B).

298 These tumors were kept stable throughout serial passages and, as occur in patients, the

299 secreted beta subunit of human chorionic gonadotropin (β-hCG) and/or alpha-fetoprotein

300 (AFP) were detected in mouse serum as surrogate markers of tumor growth (27,28)

301 (Supplementary Table S1).

302 Orthoxenografts reproduce in mice some of the patterns of dissemination observed in

303 humans with the presence of retroperitoneal lymph nodes, lungs and liver metastasis.

304 Rare brain metastases were not detected in mice. Distant from what happens in humans

305 in two tumors we have detected presence of peritoneal implants (Supplementary Table

306 S1).

307

308 Orthoxenografts of NSE recapitulate the responses to cisplatin treatment in

309 humans.

310 We studied the pattern of responses to chemotherapy for nine orthoxenografts. Mice

311 were treated with low (2 mg/kg) and high (5 mg/kg) doses of cisplatin, and their short-

312 and long-term responses were evaluated. All tumors had a good short-term response to

313 low doses of cisplatin, as indicated by a significant reduction in tumor weight in eight

314 cases and complete response in the tumor TGT21BX (Supplementary Fig. 1). A good

315 correlation between tumor weight and reduction or absence of serum β-hCG and/or AFP

316 levels was found, supporting its use as a dynamic surrogate marker of treatment

317 efficacy. Differences among tumor weight and serum markers observed in TGT21AX

318 after treatment can be explained by the predominance of a teratoma with a few

319 microscopic islands of viable cells (Supplementary Fig. 1C). Administration of higher

320 doses of cisplatin (5 mg/kg) was associated with a better response in all cases

321 (Supplementary Fig. 1). Additionally, there was a complete response in tumor

322 TGT21AX (Supplementary Fig. 1C).

323 To investigate long-term cisplatin responses, a subgroup of the treated mice was kept

324 alive post-chemotherapy until tumor regrowth was observed. Tumors regrew in eight

325 out nine cases, over a period of 15 to 135 days, independently of the cisplatin dose in

326 most instances (Supplementary Fig. 1). In TGT39X both treatments yielded a long and

327 sustained response, as was confirmed by constant levels of AFP over a latency period of

328 90 days (Supplementary Fig. 1A). Histological analysis of relapsed masses

329 demonstrated the presence of a viable tumor in most cases, and the maintenance of the

330 tumor heterogeneity, observed in mixed non-treated tumors (Supplementary Fig. 2).

331 As observed in patients, cisplatin induced increasing teratoma differentiation in

332 TGT21AX (Supplementary Fig. 1C, low panel and Supplementary Fig. 2).

333

334 Development of matched models of cisplatin refractoriness

335 To investigate cisplatin resistance against the same genetic background (sensitive vs.

336 resistant) we developed several cisplatin refractory tumor models. Thus, from this

337 collection of fourteen tumors, to develop this study we selected five that were not

338 exposed to cisplatin before implantation. This subset includes a yolk sac tumor

339 (TGT1X), two embryonal carcinomas (TGT12X and TGT34X), a choriocarcinoma

340 (TGT38X) and a mixed tumor (TGT21BX) (Supplementary Table S1). Through five

341 iterative cycles of treatment in different mice, and applying increasing doses of cisplatin

342 through the cycles, we generated orthoxenografts with acquired resistance in vivo

343 (named TGT1XR, TGT12XR, TGT21BXR, TGT34XR and TGT38XR) (Fig. 1C).

344 During the process of resistance acquisition, a progressive shortened time lag between

345 tumor treatment and tumor regrowth was noted, and the mice to mice passage time

346 stabilized after five cycles of treatment in all cases (Fig. 1D). To evaluate the resistance

347 to cisplatin in these transplanted tumors, paired short-term response assays between

348 untreated (TGTX) and resistant (TGTXR) tumors at cycle #5 were performed (Fig. 1E).

349 High levels of resistance were observed in all tumors at both low (2 mg/kg) and high (5

350 mg/kg) cisplatin doses. Finally, supporting the experimental value of this collection of

351 paired sensitive/resistant orthoxenografts, we observed similar histological pattern

352 between original and cisplatin-resistant tumors (Supplementary Fig. 2).

353

354

355 Recurrent chromosomal imbalances were associated with acquired cisplatin

356 resistance

357 In the DNA, cisplatin induces interstrand crosslinks and monoaducts that cause

358 mutations and genomic instability (11,29). Many of these alterations on the cisplatin-

359 exposed DNA cannot be repaired and cause cellular lethality but some may be selected

360 and promote the cellular resistance to cisplatin. Thus, we investigated in paired TGCT

361 orthoxenografts (sensitive versus resistant) with the same genetic background whether

362 the acquisition of cisplatin resistance was associated with the selection of specific

363 genomic imbalances or mutations in a panel of selected genes. First, we did not found

364 mutations in a subset of cancer-related genes including K-ras, b-raf, PI3KCA, EGFR, c-

365 Kit, PDGFRα and β, p15, p16 and SMAD4 or changes in the MSI status in resistant

366 engrafted tumors. Then, we investigate chromosomal rearrangements, using array-based

367 Comparative Genomic Hybridization (CGH), in four paired untreated parental engrafted

368 tumors and their resistant counterparts (TGT1XR was later tested for 9q32-q33.1

369 amplification only). Few recurrent genomic changes were consistently detected in

370 distinct resistant tumors when compared with their paired sensitive orthoxenograft (Fig.

371 2A). Particularly, gains at 9q were found in three out of four cases being 9q32-q33.1 the

372 smallest common gain (5.1 M bp containing 60 genes) between three resistant tumors

373 (Fig. 2B). In addition, gains at 15q23-q24.1 and 15q26.3 were identified in two tumors,

374 and the loss of the Xp22.33 region was identified in three of four tumors

375 (Supplementary Fig. 3). All these four genomic regions are hot spots to search for

376 genes involved in the acquired resistance to cisplatin. In this study we focused our

377 attention on studying the 9q32-q33.1 region.

378

379 Amplification at 9q32-q33.1 is associated with an increased risk of death in

380 advanced TGCT patients

381 To evaluate the clinical relevance of our results in orthoxenografts, gains at 9q32-q33.1

382 were studied by FISH in a human tissue microarray (TMA) including series of tumors

383 from 75 patients with metastatic TGCTs (63 NSEs and 12 SEs) homogeneously treated

384 with cisplatin-based chemotherapy in our hospital (Fig. 2C, Supplementary Table S2).

385 Amplification at 9q32-q33.1 was identified in 18 of 75 (24%) cases, including 16 NSEs

386 (5 CEs, 2 CHs, 1 YS, 2 TEs and 6 mixed tumors) and two pure SEs (Table 1). Analysis

387 of overall survival (OS) showed that amplification at the 9q32-q33.1 region was

388 associated with a 2.79-fold greater risk of death in patients with metastatic GCTs (p =

389 0.036; hazard ratio (HR) = 2.79; 95% confidence interval (CI) = 1.11–7.0) (Table 1 and

390 Fig. 2D). A higher risk of death associated to this genetic amplification was revealed

391 when considering only patients with NSE (n = 63) (p = 0.026; HR = 3.03; 95% CI =

392 1.18–7.76), but there was no difference in those with SE (p=0.54) (Table 1). OS

393 subgroup analyses in NSE patients (presence of amplification vs WT) showed a trend

394 towards for good and intermediate risk groups alone; the relationship was statistically

395 significant when we analyzed the two groups together (p = 0.014; HR = 5.16; 95% CI =

396 1.47-18.12) (Table 1). This genetic amplification was also associated with shorter

397 progression-free survival (PFS) (p = 0.043; HR = 2.46; 95% CI = 1.07–5.63) (Table 1

398 and Fig. 2D) and such correlation was significant even when the NSE group alone was

399 analyzed (p = 0.024, HR = 2.8, 95% CI = 1.19–6.57).

400 Moreover, there was a trend for tumors harboring the 9q32-q33.1 amplification to have

401 a worse cisplatin response (Supplementary Table S3). Fifty percent of tumors with the

402 amplification (9 out 18) were considered resistant to first-line chemotherapy compared

403 with 26.3% (15 out 57) of tumors without it (p = 0.060). Up to 27.8% of tumors with

404 the 9q32-q33.1 amplification did not achieve a tumor marker complete response or

405 progressed during first-line treatment (p = 0.007) (Supplementary Table S4).

406

407 Identification of 9q32-q33.1 genes whose expression is associated to tumor

408 response to cisplatin

409 Next, to identify which genes within 9q32-q33.1 region could be related to cisplatin

410 resistance, we performed quantitative PCR (qPCR) to study the expression levels of 60

411 genes and two miRNAs in the five paired sensitive/resistant engrafted tumors (Fig. 3A).

412 In order to better represent in our study resistant tumors without the 9q32-q33.1

413 amplification, we included the pair TGT1X/TGT1XR in the gene expression profiling

414 of this region to get a panel of three with and two without the 9q32-q33.1 gain. By

415 qPCR we found that thirty-seven 9q32-q33.1 genes, and the two microRNAs, were

416 expressed in these five TGCTs (Fig. 3A, B). From this gene expression analyses we

417 observed that only two genes, POLE3 and AKNA, were differently regulated in relation

418 to the presence or absence of 9q32-q33.1 amplification. Interestingly, POLE3 was

419 upregulated in resistant tumors with 9q32-q33.1 gains but downregulated in the two

420 others without this genetic alteration. AKNA was downregulated in resistant tumors

421 with 9q32-q33.1 gains but not altered in the other two tumors without this alteration.

422 Moreover, these results also indicate that a chromosomal gain does not necessarily

423 mean gains of functions. Moreover, another five genes (UGCG (also known as GCS),

424 ZNF883, CTR1, ATP6V1G1 and FLJ31713) were consistently up or downregulated in

425 all five resistant tumors independently of the 9q32-q33.1 amplification.

426 Therefore, we found that gene expression changes at the 9q32-q33.1 region in resistant

427 tumors were not necessarily correlated with the presence of the amplification,

428 suggesting the coexistence of other mechanisms modifying gene expression that confer

429 resistance to cisplatin.

430 The influence of genetic changes on resistant tumors is complex and multifactorial as

431 reflected by the fact that the functional network (obtained from the web-based tool

432 STRING) for 34 genes differentially expressed in these resistant tumors was rather poor

433 (30), indicating a low functional relation between these genes. Importantly, the six

434 genes showing deregulation in resistant tumors do not show any functional link

435 (Supplementary Fig. 4).

436

437 Some gene functions involved in cisplatin response are conserved in C. elegans.

438 We wondered whether the 9q32-q33.1 genes deregulated in resistant tumors were acting

439 in the same manner in the response to cisplatin. Cisplatin has a broad mode of action

440 being also toxic to eukaryotic cells of model organisms as C. elegans (31). We found

441 that three of the five genes deregulated in resistant tumors (GCS, CTR1 and

442 ATP6V1G1) were conserved in C. elegans. Through an automated toxicity assay we

443 found that vha-10/ATP6V1G1(RNAi) animals were sensitive to cisplatin

444 (Supplementary Fig. 5). GCS present three paralogs in C. elegans and we needed to

445 inactivate two of them at the time to produce sensitivity to cisplatin. On the contrary,

446 worms treated with CTR1/F27C1.2(RNAi) were resistant to cisplatin exposure, as

447 expected from the inactivation of the cooper transporter that is involved in cellular

448 intake of cisplatin (Supplementary Fig 5).

449 We conclude that 9q32-q33.1 genes deregulated in the resistant tumors can either be up-

450 or down-regulated, and provide either resistance or sensitivity to cisplatin. Moreover,

451 the mechanisms of response seem to be conserved through evolution rather than be

452 specific of human cells or tumors.

453

454 DL-threo-PDMP, a competitive inhibitor of glucosylceramide synthase (GCS), re-

455 sensitizes refractory TGCT and epithelial ovarian cancer orthoxenografts to

456 cisplatin

457 As a proof-of-concept of our approach to search novel therapeutic strategies for

458 overcoming cisplatin resistance, we decided to dig into the therapeutic value of one of

459 these genes/proteins at the preclinical level. GCS was chosen on the grounds that: (i)

460 displays increased mRNA expression in all cisplatin refractory orthoxenografts, and (ii)

461 there are specific inhibitors for GCS available, some of which are currently in clinical

462 use for other pathologies (32,33). First, NSE testicular germ cell line SuSaS and its

463 paired cisplatin resistant SuSaR were used as cellular models to corroborate the

464 functional relationship among GCS expression/activity and cisplatin resistance in

465 TGCTs. Significant differences among protein levels of GCS were observed between

466 both cell lines, being more abundant in resistant cells (Fig. 4A, upper panel).

467 Transfected SuSaS cells overexpressing GCS (Fig. 4A, lower panel) displayed a

468 significant cisplatin-resistance increase (5-fold) (Fig. 4B); while shRNAi knockdown of

469 the endogenous GSC gene (70% of inhibition) in SuSaR cells correlates with a partial

470 (57.6%) cisplatin re-sensitization (Fig. 4B). Likewise, the treatment of SuSaR cells with

471 the specific GCS inhibitor DL-threo-PDMP (PDMP) mimics this cisplatin sensitization

472 (44.8%) (Fig. 4C). Effect mediated by a significant increase in the intracellular levels of

473 ceramide for combined cisplatin+PDMP treatment (Fig. 4D). Thus, we demonstrated

474 that impaired GCS expression/activity in vitro resensitizes a cisplatin-resistant NSE cell

475 line newly to cisplatin treatment.

476 Interestingly, cisplatin-refractory engrafted tumors exhibited an increase in GCS activity

477 (Fig. 4D). Then, we treated TGT1XR and TGT38XR daily for 21 days with PDMP. As

478 a single agent PDMP did not produce a significant response with respect to the vehicle-

479 treated animals, and no significant differences were observed among individual PDMP

480 and cisplatin treatments (Fig. 4E). Nevertheless, both tumors experienced significant

481 tumor weight reductions (TGT38XR, 73.5% and TGT1XR, 42.8%) for combined

482 PDMP+cisplatin treatment (Fig. 4E).

483

484 Finally, we asked whether the identified association among GCS and cisplatin

485 resistance happens in other tumors commonly treated with cisplatin. Thus, GCS

486 expression/activity was also determined in a panel of five paired cases of sensitive and

487 cisplatin-resistant orthoxenografts of epithelial ovarian cancer (EOC) generated in our

488 lab following the same described approach (22). In 4 of 5 (83.3%) serous tumors a

489 median increase of 52.5% ± 9.4 GCS activity was observed in the resistant

490 orthoxenografts respect to its paired sensitive tumors (Fig. 4F). Furthermore, PDMP

491 treatment of OVA17XR, having high levels of GCS activity, has a cisplatin re-

492 sensitizing effect (Fig. 4G)(tumor weight reduction of 76,5% in combined

493 cisplatin+PDMP treatment).

494 Together, the GCS inhibitor PDMP re-sensitizes cisplatin-refractory orthoxenografts to

495 cisplatin treatment, providing a promising therapeutic opportunity for treatment of

496 refractory cases; being a strong preclinical rationale for further clinical trials.

497

498 PDMP-induced tumor response is mediated by proapoptotic features in germ cell

499 tumors and ovarian cancer

500 Next, we investigated whether tumor response mechanisms induced by PDMP

501 associated with proapoptotic induction features. Thus, apoptotic drug induction was

502 assessed in TGT1XR, TGT38XR and OVA17XR by immunodetection in paraffin-

503 embedded tissues of caspase-3, an early and specific apoptotic marker. In the three

504 orthoxenografts no significant differences for the proapoptotic induced effect was

505 observed for the single treatments respect to vehicle group (Fig. 4H). Whereas for

506 combined PDMP+cisplatin treatment significant increase in the apoptosis levels was

507 observed in the three tumors respect to cisplatin group (TGT1XR 2.7-fold (p=0.0016);

508 TGT38XR 2.81-fold (p=0.0011) and for OVA17XR 2.06-fold (p=0.0002).

509 DISCUSSION

510

511 Establishment of advanced preclinical models phenocopying patients’ primary tumor

512 features in terms of phenotype, genotype, and response to chemotherapy is a basic step

513 on the way to identifying novel therapeutic targets and for testing anti-tumoral

514 treatments. Here we report the generation of an important collection of NSE TGCTs

515 engrafted orthotopically in nude mice, keeping features of primary tumors including

516 sensitivity to cisplatin. In our study, we have used five of those original tumors and their

517 corresponding orthoxenografts that developed cisplatin resistance in vivo. Our

518 methodology to induce cisplatin resistant tumors includes exposure to an increasing

519 concentration of cisplatin through five cycles. Such cisplatin treatment does not occur in

520 the clinic, but in our hands was previously very efficient to generate cisplatin-resistant

521 orthotopic tumors derived from epithelial ovarian cancer (22). Engrafting patient tumor

522 tissues orthotopically into immune-deficient mice (termed "orthoxenografts or PDOX")

523 are state-of-the-art preclinical models that may contribute to reduce the high rate of

524 failure in translating preclinical results to patients. Among the orthoxenografts used in

525 this study, we previously reported TGT1X, TGT38X and also its paired resistant

526 TGT38XR as tools to support the preclinical value of sunitinib and lapatinib in TGCT

527 treatments (20,21,34). Likewise, other orthoxenografts of epithelial ovarian cancer and

528 lung cancer has been used to evaluate potential patients’ treatments (22,35,36).

529

530 In 1998, Rao et al provided the first evidence of chromosomal amplification associated

531 with cisplatin resistance by comparing unpaired GCTs obtained from relapse-free

532 patients with chemotherapy-resistant tumors (37). Differently, our study compared

533 resistant and sensitive tumors with the same genetic background and we identified

534 fewer recurrent genomic changes across the different refractory tumors. Still, both

535 studies found amplifications in similar regions at chromosomes 9 and 15. Other studies

536 have identified distinct chromosomal imbalances upon cisplatin exposure suggesting

537 that gains and losses of chromosomal regions are genome instability events whose

538 specific influence in cisplatin resistance acquisition needs to be unraveled. Our study

539 suggests that these genomic imbalances do not have a common impact on gene

540 activities but are hot spots to find genes involved in the response to cisplatin. Thus,

541 GCS and CTR-1/-2 genes are both deregulated in distinct manner (upregulated and

542 downregulated respectively) in all five resistant tumors independently of the 9q32-

543 q33.1, indicating that these genes are major drivers of resistance to cisplatin and

544 therefore genomic imbalance of their loci would favor tumor grow in presence of

545 cisplatin. However we still found a modest clinical correlation between the presence of

546 9q32-q33.1 gains in tumors and a poor risk defined by shorter OS. Here we demonstrate

547 that the presence of the 9q32-q33.1 amplification is associated with increased risk of

548 progression and death in one of the largest cohort of patients with metastatic GCTs, of

549 whom, 32% are truly refractory to cisplatin treatment. Thus, determining the presence

550 of this amplification can be especially helpful in the good/intermediate prognostic

551 groups and may allow clinicians to include them under more aggressive protocols, or to

552 offer alternative drug treatments. Although it is a single retrospective analysis, it is

553 important to highlight its relevance given the difficulty to obtain representative TGCT

554 series that include patients with a poor prognosis, and refractory tumors.

555

556 Regarding specific alterations in gene expression, few genes have been associated with

557 cisplatin resistance in TGCT. Thus, low incidence of mutations in KRAS, AKT1,

558 PIK3CA, and HRAS were exclusively identified in resistant GCTs cases, while

559 FGFR3 mutations occurred with equal frequency in both sensitive and resistant cases

560 (14). Controversy exist about the presence of the b-raf (V600E) mutation in some

561 refractory NSE (14,38). The whole exome sequence of 42 TGCTs (including SE an

562 NSE, but only some were refractory tumors) pointed few recurrent genetic changes,

563 identifying mutations in the XRCC2 , which is a gene strongly implicated in defining

564 cisplatin resistance (39). Recently, also by exome sequencing, TP53 pathway alterations

565 including MDM2 amplifications has been described exclusively in patients with

566 cisplatin-resistant tumors and they were particularly prevalent among primary

567 mediastinal nonseminomas (17). Here we point to six genes within the 9q32-q33.1

568 region, being two of them (POLE3 and AKNA) specifically deregulated in resistant

569 tumors carrying the 9q32-q33.1 gain. POLE3 is a subunit of the DNA polymerase

570 epsilon that binds DNA in a sequence-independent manner and is part of the CHRAC

571 chromatic-remodeling complex (40) . AKNA encodes an AT-hook transcription factor

572 (41). The role of these two DNA binding proteins in the response to cisplatin should be

573 studied in the future, but its distinct deregulation in cisplatin refractory tumors (POLE3

574 is upregulated whereas AKNA is downregulated) indicate that their mechanisms of

575 action in response to cisplatin exposure may be different. One explanation for the

576 distinct types of deregulation of genes in 9q32-q33.1 is the frequent amplification of

577 one parental chromosome (of part of it) with loss of the other parental chromosome that

578 led to loss-of-heterozygosity (LOH) in GCTs but not in other tumor types (42).

579 Other four genes displayed a deregulated expression in cisplatin-resistant ortoxenografts

580 but such deregulation was not associated to the presence of the 9q32-q33.1 gain. One of

581 them is CTR1, which encodes a cooper transporter that have previously been associated

582 to cellular mechanisms of resistance to cisplatin (33,34). ZNF883 encodes a zinc finger

583 protein that may be involved in transcriptional regulation and FLJ31713 is an

584 uncharacterized protein. Finally, we experimentally confirmed the fourth gene, the

585 glucosylceramide synthase (GCS), as a target to resensitize tumor refractory to cisplatin.

586

587 Targeting GCS, due to its central role in the glycosphingolipid synthesis pathway, has

588 emerged as a novel approach for treating metabolic diseases such as Gaucher, Niemam-

589 Pick and diabetes. In this context, several GCS inhibitors are in clinical use or under

590 development, including Miglustat, PDMP and EXEL-0346 among others (43–45) .

591 Recently, it has been reported that GCS inhibition improved sorafenib effectiveness in

592 vitro and in vivo in experimental hepatocellular carcinoma, recovering drug sensitivity

593 of sorafenib-resistant tumors in mice (46). Thus, the therapeutical value of GCS

594 inhibitors as tool to re-sensitize cell to drugs may not be only restricted to cisplatin.

595 We have demonstrated the relevance of GCS activity as a biological mechanism that

596 mediates tumor cell protection against cisplatin exposure, and they denoted the

597 significance of sphingolipid metabolism through cisplatin-induced tumor cell death.

598 Thus, we hypothesize that PDMP or other GCS inhibitors, blocking the conversion of

599 ceramide to glucosylceramide, should open an important therapeutic window in patients

600 with refractory tumors by exploring the influence of ceramide pools in cisplatin-induced

601 cell-death. Our preclinical results in advanced refractory cisplatin orthoxenografts of

602 both GCTs and EOCs tumor models demonstrate that PDMP re-sensitizes to cisplatin

603 treatment, providing a firm preclinical rationale of drug repositioning and for

604 developing further clinical trials in the field. Interestingly, the association among GCS

605 activity and cisplatin resistance has recently been reported also for head and neck cancer

606 (47), pointing to a broader usage of GCS inhibitors to treat tumors refractory to

607 cisplatin. Given the rather unspecific mechanisms of action of cisplatin we believe that

608 strategies to re-sensitize cisplatin resistant TGCT orthoxenografts may help to improve

609 the treatment of other tumors types that are unsuccessfully treated with cisplatin (8).

610 In summary, we report the generation of cisplatin-refractory orthoxenografts of germ

611 cell tumors as preclinical models and demonstrate that this preclinical platform have a

612 great potential to better design future trials for the treatment of patients with cisplatin

613 resistant/refractory tumors.

614 615 ACKNOWLEDGEMENTS 616 We thank the staff of the Animal Core Facility of IDIBELL for mouse care and 617 maintenance. 618

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Table 1 Analysis of 9q32-q33.1 amplification in metastatic germ cell tumors

Overall Survival Progression-Free Survival

n % HR 95% CI P HR 95% CI P

Chromosome copy number at 9q31-q32.1 (n=75)

WT * * 57 76 1 0.036 1 0.043

Amplification 18 24 2.79 (1.11 - 7.0) 2.46 (1.07 - 5.63)

Stratified analysis

Pathological classification

Nonseminoma (n=63) * * WT 47 74.6 1 0.026 1 0.024

Amplification 16 25.4 3.03 (1.18 - 7.76) 2.8 (1.19 - 6.57)

Seminoma (n=12) WT 10 83.3 1 (0 - Inf) 0.54 1 (0 - Inf) 0.38 Amplification 2 16.7 0 0

IGCCCG classification

NSE with good risk (n=33)

WT 27 81.8 1 0.096 1 0.22

Amplification 6 18.2 5.89 (0.82 - 42.52) 3.29 (0.55 - 19.71)

NSE with intermediate risk (n=14) WT 10 71.4 1 0.15 1 0.28 Amplification 4 28.6 3.41 (0.68 - 17.02) 2.33 (0.52 - 10.44)

NSE with poor risk (n=16) WT 10 62.5 1 0.88 1 0.30 Amplification 6 37.5 0.9 (0.21 - 3.79) 2 (0.55 - 7.21)

Grouping NSE according to good and intermediate risk (n=47) WT 37 78.7 1 0.014 * 1 Amplification 10 21.3 5.16 (1.47 - 18.12) 3.28 (1.03 - 10.37) 0.056 Abbreviations: WT, No amplification at 9q32-33.1; HR, hazard ratio; CI, confidence interval. * P values are from multivariate Cox models adjusted for pathological diagnostic classification.

FIGURE LEGENDS

Figure 1. Generation of testicular germ cell tumor orthoxenografts and development of paired cisplatin refractory tumors (A) Macroscopic appearance and hematoxylin and eosin (H&E) staining of mice with orthoxenografts (left, TGT1X pure yolk sac, right, TGT12X pure embryonal carcinoma). (B) OCT4 protein immunostaining. High levels of nuclear staining were only identified in the embryonal component (EC) of pure TGT21BX mixed tumors. Absence of protein expression is noted in TGT1X, pure yolk sac tumor (YS). Seminoma (SE) was used as a positive control. (C) Generation of engrafted tumors refractory to cisplatin treatment combines: (i) repetitive cisplatin treatments (one cycle: three doses of cisplatin administered by intravenous (i.v.) injection for three consecutive weeks, on days 0, 7 and 14) for five separate and independent cycles; and (ii), doses of cisplatin were increased after each cycle of treatment ranging from 2 mg/kg (first cycle) to 5 (fifth cycle). (D) The time-lag between tumor treatment and tumor regrowth decreased as the different treatment cycles occurred. Tumors at cycle #5 of treatment (arrow) were used to assess the response to chemotherapy. (E) Comparative short-term cisplatin response assays for paired non-treated vs. cisplatin-resistant tumor. Mice were treated with low (2 mg/kg) and high (5 mg/kg) doses of cisplatin. TGT21BX and TGT34X showed a complete response at high doses. ##, for paired TGT34X vs. TGT34XR, only the response to the low dose was assessed. *, P<0.05.

Figure 2. Recurrent gains at 9q in refractory tumors. (A) Chromosomal rearrangements related to acquired cisplatin resistance were identified when compared four paired untreated parental engrafted tumors and their resistant counterparts. Whole-genome mapping was performed by oligonucleotide array CGH analysis (60 kbp window averaging) and visually depicted with the SignalMap graphical interface tool from Nimblegen Systems. (B) The 9q21.11-q33.3 gain region (arrow) was identified in TGT12XR and TGT21BXR, while in tumor TGT38XR there was a smaller overlapping region of 5.1 Mbp at 9q32-q33.1 (arrowhead). (C) Representative FISH analyses of the copy number of 9q32-q33.1 in human metastatic CGT samples contained in the TMA. Interphase FISH with RP11-582I20 (red) and RP11-616C16 (green) probes. Panel 1: absence of amplification characterized by two red and two green signals in all interphase nuclei. Panels 2 and 3: amplification of the

region. (D) Kaplan–Meier plots by status of 9q32-q33.1 gains. Left panel, overall survival (OS), and right panel, progression-free survival (PFS). P-values are those from multivariate Cox proportional hazards regression models, controlling for the pathological diagnostic classification.

Figure 3. Differential profiling expression patterns of the 60 genes and two miRNAs annoted on 9q32-q33.1 region determined by qPCR. (A) Results are presented as changes in the expression levels in cisplatin refractory tumors relative to the untreated tumors, grouped by 9q32-q33.1 gain status. No expression changes (in grey), underexpression in resistant tumors (in green), overexpression in resistant tumors (in red) and lack of expression in engrafted tumors (in white). (B) Graphs showing qPCR experiments for relevant genes. For each gene, normalized gene expression (left graph), and the expression ratio among refractory vs. sensitive tumors (right graph) are shown. Reactions were performed in triplicate and all data were normalized with endogenous control gene (β-actin).

Figure 4. Glucosylceramide synthase (GCS) activity and cisplatin resensitization by DL-threo-PDMP (PDMP) in cisplatin resistant cell lines and orthoxenografts. (A) (A) Differential protein expression levels of GCS in SuSaS and paired SuSaR determined by Western blot (left panel) and GCS transient overexpression in SuSaS cells after transfection with by pCMV6-GCS and shRNAi knockdown in SuSaR cells with a mix of four pre-designed small hairpin RNAs (shRNAs) for the human GCS (right panel). (B) Cisplatin response of SuSaS cells overexpressing GCS and SuSaR cells with GCS silenced. Each curve represents the average of values from at least three independent experiments and cell proliferation was measured by MTT assay. (C) Dose response curves for SuSaS and SuSaR treated with cisplatin and with 30 μM of PDMP (IC50 (μM):SuSaS (29.93 ± 0.006) and SuSaR (28.73 ± 0.054)(left panel). Generation of ceramide was determined in SuSaR cells after treatment with cisplatin, PDMP and for combined cisplatin+PDMP drugs (right panel). (D) Activity of GCS in five paired sensitive vs. refractory engrafted testicular germ cell tumors. (E) Responses of cisplatin- refractory engrafted TGT1XR and TGT38XR tumors to treatment with the GCS inhibitor DL-threo-PMDP. (F) GCS activity was also determined in six paired sensitive vs. refractory orthoxenografts of epithelial ovarian cancer (EOC). Increase levels of

glucosylceramide were detected in 5 out 6 resistant cases. (G) Response of cisplatin- refractory engrafted OVA17XR orthoxenograft (serous tumor) treated with the GCS inhibitor DL-threo-PMDP. (H) Apoptosis was evaluated by immunostaining of Caspase 3 in parafﬁn sections of residual tumor masses on day 21 of single cisplatin, PDMP and combined cisplatin+ treatments of TGT1XR, TGT38XR and OVA17XR tumors (22). TGTX, untreated testicular germ cell tumor; TGTXR, cisplatin-refractory testicular germ cell tumor; OVAX, epithelial ovarian tumor; OVAXR, cisplatin-refractory epithelial ovarian tumor; *, P<0.05.

A TGT1X TGT12X B FIGURE 1 CONTROL TGT1X TGT14X TGT17X TGT21BX

X200 X200 X200 X200 X200

X200 X200

X400 X400 X400 X400 X400 X400 X400 SE YS EC CH EC and YS C Cycle #1 of treatment E 7 TGT1X vs. TGT1XR 8 TGT12X vs. TGT12XR 8 TGT21BX vs. TGT21BXR

6 7 7

6 5 6 ) ) ) 5 5 4 * Three doses of 2 mg/kg of cisplatin 4 4 administered by i.v at days 0, 7 and 15 3 3 3 2 * 2 2 D TGT1XR ( gr Tumor weight ( gr Tumor weight ( gr Tumor weight 160 1 * TGT12XR 1 1 * TGT21BXR 140 0 0 0 TGT34XR TGT1X TGT1XR TGT12X TGT12XR TGT21BX TGT21BXR and and TGT38XR 120 ## TGT34X vs. TGT34XR TGT38X vs. TGT38XR 8 6

100 7 5 Primary xenografts 6 ) 80 ) 4 Control (n=10) 5 2 mg/kg CDDP (n=10) 60 5 mg/kg CDDP (n=10) 4 3 Cisplatin refractory xenografts 40 3 lag between between lag treatment 2

- Control (n=10) Tumor weight ( gr Tumor weight 2 ( gr Tumor weight 2 mg/kg CDDP (n=10) 20 * 5 mg/kg CDDP (n=10)

1 tumor passage tumor (days) Time 1 * 0 0 0 Downloaded1 from2 3 clincancerres.aacrjournals.org4 5 6 7 8 on September 29, 2021.TGT34X © 2018 TGT34XRAmerican AssociationTGT38X for TGT38XR Cancer Research. Author Manuscript Published OnlineFirst on April 4, 2018; DOI: 10.1158/1078-0432.CCR-17-1898 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

A FIGURE 2

a Size Number of Tumor Histology of Chromosome Status Scores Region d B b c (Mbp) genes resistant tumor region

TGT12R EC 9q21.11-q33.3 gain + 0.31 67.950 K - 123.690 K 55.7 502

15q23-q24.1 gain + 0.36 69.690 K - 71.430 K 1.74 26

15q26.3 gain + 0.37 97.770 K - 98.310 K 0.54 6

Xp22.33 loss - 0.29 30 K - 2.730 K 2.70 25 TGT12X

TGT21BR YS, EC and 5p15.33-p15.2 gain + 0.30 90 K - 15.090 K 15.0 94 CH

9q21.11-q33.3 gain + 0.39 70.170 K - 123.690 K 53.5 461 TGT12XR

15q23-q24.1 gain + 0.39 69.690 K - 71.370 K 1.68 25

15q24.3 gain + 0.30 74.970 K - 75.750 K 0.78 9

15q26.3 gain + 0.41 97.830 K - 98.310 K 0.48 6 TGT21BX

Chromosome 21 gain + 0.28 0 K - 47.000 K 47 386

Xp22.33 loss - 0.45 30 K - 2.730 K 2.70 25 TGT21BXR Xp22.2 loss - 0.42 9.510 K - 9.870 K 0.36 4

TGT34R EC No changes

TGT38R CH 9q32-q33.1 gain + 0.35 112.950 K - 118.050 K 5.10 60 TGT38X 16p11.2-p11.1 gain + 0.34 34.350 K - 34.590 K 0.24 11

20q13.13-q13.2 gain + 0.25 49.050 K- 49.770 K 0.72 5

Xp22.23 loss - 0.41 30 K - 2.730 K 2.70 25

a NimbleGen microarray data processed at 60 kbp average window. All the chromosomal gains or losses TGT38XR had a log2 score ≥0.30 or ≤0.30. No microarray data were available for TGT1R, a pure YS. b Tumor histology: YS, yolk sac tumor, EC, embryonal carcinoma, CH, choriocarcinoma. c Genomic changes were consistently detected in chemoresistant tumors at cycles #3 and #5 of chemotherapy. d Location of genes in each region was based on build 36.3 from NCI. C D Chrm 9 1.0 1.0

9q32-q33.1 0.8 0.8

1 2 3 0.6 0.6

0.4 0.4 P = 0.043

P = 0.036 Progression Survival ProbabilitySurvival 0.2 0.2 WT WT Amplification Amplification 0.0 0.0 0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14 Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2018 AmericanTime Association (years) for Time (years) Cancer Research. Author Manuscript Published OnlineFirst on April 4, 2018; DOI: 10.1158/1078-0432.CCR-17-1898 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

A B FIGURE 3

2,5E+02 EDG2 4 2,0E+02 FLJ31713 60 24 23 22 21 13 12 12 13 21 22 31 32 33 34 9p 9p 9p 9p 9p 9p 9q 9q 9q 9q 9q 9q 9q 9q 2,0E+02 1,6E+02 50 3 9q32-q33.1 40 1,5E+02 1,2E+02 2 30 1,0E+02 8,0E+01 20 Gene expression Gene

1 expression Gene TGT21BX 5,0E+01 4,0E+01 TGT12X TGT34X TGT38X 10 TGT1X vs. vs. vs. vs. vs. Expression ratio (resistant/sensitive) (resistant/sensitive) ratio Expression 0,0E+00 (resistant/sensitive) ratio Expression 0 0,0E+00 0 TGT1 TGT1 TGT1 TGT1 TGT34 TGT12 TGT21 TGT38 TGT34 TGT12 TGT21 TGT38 TGT34 TGT12 TGT21 TGT38 TGT34 TGT12 TGT21 TGT38 TGT1XR TGT12XR TGT34XR TGT38XR TGT21BXR Amplification at 9q - - + + + 2,5E+02 UGCG 7 6,0E+01 AKNA 1,5 EDG2 112675875 2,0E+02 5,0E+01 OR2K2 113129584 5 KIAA0368 113162794 4,0E+01 1,0 1,5E+02 ZNF483 113327260 3,0E+01 LTB4DH 113365073 3 1,0E+02 C9orf29 113404932 2,0E+01 0,5 Gene expression Gene

DNAJC25 113433453 expression Gene 5,0E+01 LOC552891 113433487 1 1,0E+01 GNG10 113463682 Expression ratio (resistant/sensitive) (resistant/sensitive) ratio Expression 0,0E+00 (resistant/sensitive) ratio Expression 0,0E+00 0 C9orf84 113488722 TGT1 TGT1 TGT1 UGCG 113699027 TGT1 TGT34 TGT12 TGT21 TGT38 TGT34 TGT12 TGT21 TGT38 TGT34 TGT12 TGT21 TGT38 miRNA4688 TGT34 TGT12 TGT21 TGT38 113842882 SUSD1 3,0E+02 ZNF883 6 3,5E+02 ATP6V1G1 6 LOC100129332 113975623 ROD1 114020536 5 3,0E+02 5 EPF5 114083089 2,0E+02 4 2,5E+02 LOC644996 114164716 4 HSDL2 114182172 2,0E+02 3 C9orf147 114286618 3 1,5E+02 KIAA1958 114289069 2 Gene expression Gene 1,0E+02 2 C9orf80 114488607 1,0E+02 Gene expression Gene 114552955 1 SNX30 5,0E+01 1 SLC46A2 114681021 Expression ratio (resistant/sensitive) (resistant/sensitive) ratio Expression 0,0E+00 (resistant/sensitive) ratio Expression 0 0,0E+00 0 LOC100129193 114761235 TGT1 ZNF883 114799221 TGT1 TGT1 TGT1 TGT21 TGT34 TGT21 TGT34 TGT12 TGT21 TGT38 TGT12 TGT38 TGT34 TGT12 TGT38 ZFP37 114843995 TGT34 TGT12 TGT21 TGT38 LOC100128385 114906824 1,5E+02 1,0 C9orf109 114914042 CTR2 2,4E+02 TNC 25 4,0E-02 SLC31A2 (CTR2) 114953059 1,2E+02 2,0E+02 FKBP15 114967621 20 115023689 3,0E-02 SLC31A1 (CTR1) 1,6E+02 9,0E+01 CDC26 115069109 15 0,5 PRPF4 115077795 1,2E+02 2,0E-02 6,0E+01 RNF183 115099194 10

Gene expression Gene 8,0E+01 WDR31 115117751 Gene expression Gene 3,0E+01 1,0E-02 BSPRY 115151633 4,0E+01 5 HDHD3 115175519 Expression ratio (resistant/sensitive) (resistant/sensitive) ratio Expression

0 (resistant/sensitive) ratio Expression ALAD55 115188413 0,0E+00 0,0E+00 0,0E+00 0

POLE3 115209342 TGT1 TGT1 TGT1 TGT1 TGT34 TGT34 TGT12 TGT21 TGT38 TGT34 TGT12 TGT21 TGT38 TGT34 TGT12 TGT21 TGT38 C9orf43 115212843 TGT34 TGT12 TGT21 TGT38 RGS3 115246832 FLJ31713 115418339 3,0E+02 CTR1 1,0 1,6E+02 PAPPA LOC100132609 115652788 9 2,5E+02 115678383 ZNF618 1,2E+02 300 7 AMBP 115862228 2,0E+02 KIF12 115893739 8,0E+01 200 COL27A1 115958052 1,5E+02 0,5 5 miRNA455 1,0E+02 ORM1 116125157 3 Gene expression Gene 100 Gene expression Gene 4,0E+01 116131890 ORM2 5,0E+01 AKNA 116136250 1 Expression ratio (resistant/sensitive) (resistant/sensitive) ratio Expression 0,0E+00 0 0,0E+00 (resistant/sensitive) ratio Expression 0 DFNB31 116204181 LOC100131877 116293663 TGT1 TGT1 TGT1 TGT1 TGT1 TGT34 TGT34 TGT12 TGT21 TGT38 TGT34 TGT12 TGT21 TGT38 ATP6V1G1 116389815 TGT34 TGT12 TGT21 TGT38 TGT34 TGT12 TGT21 TGT38 C9orf91 116413527 17 LOC100129633 116456401 2,0E+02 POLE3 TGTX (Non-treated tumor) TNFSF15 116591421 15 LOC645266 116647695 1,6E+02 13 TGTXR (Cisplatin resistant tumor) TNFSF8 116704945 11 TNC 116822626 1,2E+02 9 DEC1 116943918 7 PAPPA 117955892 8,0E+01

Gene expression Gene 5 No expression in TGTXs 4,0E+01 3 No changes in the expression ratio among 1 paired cisplatin resistant vs. non-treated tumor 0,0E+00 (resistant/sensitive) ratio Expression 0 Over-expression in cisplatin resistant vs. paired TGT1 TGT1 TGT34 TGT12 TGT21 TGT38 non-treated tumor TGT34 TGT12 TGT21 TGT38 Under-expression in cisplatin resistant vs. paired non-treated tumorDownloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2018 American Association for Cancer Research. Β - E A actin

Tumor weight (gr) GSC Β 0 1 2 3 4 - actin GSC Cisplatin Cisplatin PDMP+ Vehicle Vehicle SuSaS pCMV6-XLS PDMP PDMP TGT1XR TGT1XR Cisplatin Vehicle pCMV6-XLS-GCS SuSaS Downloaded from * * Author manuscriptshavebeenpeerreviewedandacceptedforpublicationbutnotyetedited. SuSaR SuSaR

TGT1XR TGT1XR Sh control PDMP+cisplatin PDMP Author ManuscriptPublishedOnlineFirstonApril4,2018;DOI:10.1158/1078-0432.CCR-17-1898 4 52 2 Kda Kda

TGT38XR Sh GCS 4 52 2 Kda Kda B B * * clincancerres.aacrjournals.org Cell viability (%) 100 20 40 60 80 0 10 SuSaR SuSaR SuSaS SuSaS F F - 4 Glucer Cisplatin pCMV6 pCMV6 shGCS shcontrol GCS activity (A.U./mg prot.) Cer 10 10 15 20 25 30 - 0 0 5 2 - - orthoxenografts orthoxenografts OVA1X XLS XLS 10 (Log µg/ml) (Log Non - GCS 0 (OVAX) OVA1XR -treated -treated Cancer Research. OVA9X IC50(µg) 10 OVA47X and OVA47XR and OVA47X OVA17X OVA11XR and OVA11X 1.73 3.00 1.62 0.30 OVA9X and OVA9XR and OVA9X OVA1XR and OVA1X

OVA11X 0.23 0.24 0.22 0.13

10 and and OVA17XR SD

OVA11XR 4 orthoxenografts orthoxenografts C Resistant Resistant (OVAXR) OVA17X

OVA17XR Cell viability (%) 100 20 40 60 80 0 SuSaR SuSaS SuSaR SuSaS

OVA47X 10 - 4

OVA47XR µM30 + PDMP + PDMPµM30 Cisplatin 10 - 2 10 (Log µg/ml) (Log G Tumor weight (gr) 0 IC50(µg) 1.22 0.49 2.72 0.46 10 0 1 2 3 2

OVA17XR OVA17XR 0.16 0.22 0.17 0.01

PDMP+cisplatin Cisplatin Vehicle PDMP SD

Ceramide levels (A.U./mg protein ) 12,5 17,5 7,5 2,5 10 15 0 5 * *

Control

Cisplatin

H PDMP * NºNº of of apoptotic apoptoticcells bybyfieldfield Cisplatin+PDMP 10,0 15,0 20,0 25,0 30,0 35,0 40,0 45,0 50,0 10 2 3 4 5 D 0,0 5,0 0 0 0 0 0 GCS activity (A.U./mg prot.) 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 TGT1XR CDDP Vehicle orthoxenografts * Non ( TGT1X TGT12X TGT38X TGT21BX TGT34X TGTX) - treated TGT38XR and and TGT1XR and and TGT12XR and and TGT38XR and and TGT34XR 1 * PDMP+CDDP PDMP and and TGT21BXR FIGURE 4 4 FIGURE orthoxenografts TGT17XR

Resistant ( TGTXR

* ) PDMP CDDP CNT PDMP+CDDP PDMP CDDP CNT Series5 PDMP+CDDP PDMP CDDP CNT Author Manuscript Published OnlineFirst on April 4, 2018; DOI: 10.1158/1078-0432.CCR-17-1898 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Orthoxenografts of testicular germ cell tumors demonstrate genomic changes associated with cisplatin resistance and identify PDMP as a re-sensitizing agent

Josep Maria Piulats, August Vidal, Francisco J García-Rodríguez, et al.

Clin Cancer Res Published OnlineFirst April 4, 2018.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-17-1898

Supplementary Access the most recent supplemental material at: Material http://clincancerres.aacrjournals.org/content/suppl/2018/04/04/1078-0432.CCR-17-1898.DC1

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