Selected Alkylating Agents Can Overcome Drug Tolerance of G0-Like Tumor Cells And

Home , Nimustine

Author Manuscript Published OnlineFirst on August 18, 2017; DOI: 10.1158/1078-0432.CCR-17-1279 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

1 Selected alkylating agents can overcome drug tolerance of G0-like tumor cells and

2 eradicate BRCA1-deficient mammary tumors in mice.

3 Marina Pajic1,2,3*, Sohvi Blatter4*, Charlotte Guyader1, Maaike Gonggrijp1,5, Ariena Kersbergen1,

4 Aslı Küçükosmanoğlu1, Wendy Sol1, Rinske Drost6, Jos Jonkers6, Piet Borst1, Sven Rottenberg4,6

6 Authors’ affiliations: 1Division of Molecular Oncology, The Netherlands Cancer Institute,

7 Amsterdam, The Netherlands; 2The Kinghorn Cancer Centre, The Garvan Institute of Medical

8 Research, Sydney, Australia; 3St Vincent’s Clinical School, Faculty of Medicine, University of

9 NSW, Australia; 4Institute of Animal Pathology, University of Bern, Bern, Switzerland; 5current

10 address: GD Animal Health, Deventer, The Netherlands; 6Division of Molecular Pathology, The

11 Netherlands Cancer Institute, Amsterdam, The Netherlands

12 *These authors contributed equally to this work

14 Running title: Nimustine eradicates BRCA1-deficient tumors

15 Keywords: BRCA1, genetically engineered mouse model, alkylating chemotherapy, drug

16 tolerance, fluorescent ubiquitination-based cell cycle indicator (FUCCI)

18 Additional Information:

19 Financial support: This work was supported by grants from the Dutch Cancer Society

20 (2009-4303), the Netherlands Organization for Scientific Research (NWO-VIDI-91711302), the

21 European Research Council (ERC-CoG-681572), the Swiss National Science Foundation (project

22 grant 310030_156869) and the Swiss Cancer Research foundation (MD-PhD-3446-01-2014 to

23 SB)

24 Correspondence to: Prof. Sven Rottenberg, Institute of Animal Pathology, University of Bern,

25 Laenggassstrasse 122, 3012 Bern, Switzerland; e-mail: [email protected],

26 phone: 0041 31 631 2395; fax: 0041 31 631 2635

27 Total number of figures: 6

28 Word count text: 5192

Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 18, 2017; DOI: 10.1158/1078-0432.CCR-17-1279 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

30 ABSTRACT

31 Purpose: We aimed to characterize and target drug-tolerant BRCA1-deficient tumor cells that

32 cause residual disease and subsequent tumor relapse.

34 Experimental design: We studied responses to various mono- and bifunctional alkylating agents

35 in a genetically engineered mouse model for BRCA1/p53-mutant breast cancer. Due to the large

36 intragenic deletion of the Brca1 gene, no restoration of BRCA1 function is possible, and therefore

37 no BRCA1-dependent acquired resistance occurs. To characterize the cell cycle stage from which

38 Brca1-/-;p53-/- mammary tumors arise after cisplatin treatment, we introduced the fluorescent

39 ubiquitination-based cell cycle indicator (FUCCI) construct into the tumor cells.

41 Results: Despite repeated sensitivity to the maximum tolerated dose (MTD) of platinum drugs,

42 the Brca1-mutated mammary tumors are not eradicated, not even by a frequent dosing schedule.

43 We show that relapse comes from single nucleated cells delaying entry into S phase. Such slowly

44 cycling cells, which are present within the drug-naïve tumors, are enriched in tumor remnants.

45 Using the FUCCI construct we identified non-fluorescent G0-like cells as the population most

46 tolerant to platinum drugs. Intriguingly, these cells are more sensitive to the DNA crosslinking

47 agent nimustine resulting in an increased number of multinucleated cells that lack clonogenicity.

48 This is consistent with our in vivo finding that the nimustine MTD, among several alkylating

49 agents, is most effective in eradicating Brca1-mutated mouse mammary tumors.

51 Conclusions: Our data show that targeting G0-like cells is crucial for the eradication of

52 BRCA1/p53-deficient tumor cells. This can be achieved with selected alkylating agents such as

53 nimustine.

55 TRANSLATIONAL RELEVANCE

56 Despite the recent approval of poly(ADP-ribose) polymerase inhibitors, chemotherapy using

57 alkylating agents remains an important therapy in the treatment of high risk BRCA1-mutated

58 breast cancer. Patients often benefit from such chemotherapy, but residual disease is a major

59 clinical hurdle. The underlying mechanisms are poorly understood, in particular in patients who

60 have a relapse and respond to the same chemotherapy again. In a mouse model for BRCA1/p53-

61 deficient breast cancer targeted genetic modifications allow us to study mechanisms that result in

62 the survival of residual cells but do not provoke secondary drug resistance. In this model, we have

63 tested the MTD of various alkylating agents and found that nimustine eradicates BRCA1-deficient

64 tumors. As underlying mechanism, we show that nimustine is more efficient in killing a G0-like

65 subpopulation that we identified to be most drug-tolerant. Hence, selected alkylating agents may

66 be useful to cure patients with high risk BRCA1-mutated breast cancers.

68 INTRODUCTION

69 Although immunotherapy and more personalized treatment (1) have led to important advances in

70 the treatment of cancer, chemotherapy using alkylating agents remains a cornerstone in the

71 treatment of many tumors (2,3). The most important cellular target of alkylating agents is DNA, in

72 which bifunctional alkylators induce interstrand and/or intrastrand crosslinks (4). It is usually

73 assumed that the interstrand crosslinks are the lethal lesion, as they block DNA copying and are

74 difficult to repair. The basic explanation for the efficacy of alkylating agents has long been the fact

75 that tumor cells replicate rapidly (5). In recent years we have learned that DNA crosslinking

76 agents preferentially kill tumors that show defects in the DNA damage response (4,6,7). In

77 particular, tumors that are defective in homology-directed DNA repair due to the lack of BRCA1 or

78 BRCA2 function appear to benefit from intensive chemotherapy with alkylating agents, as shown

79 for patients with HER2-negative, high risk breast cancers that show BRCA1/2-like signatures

80 (8,9). Despite this high sensitivity, some residual cancer cells persist and lead to tumor recurrence

81 and treatment failure. The precise mechanisms underlying this residual disease are poorly

82 understood (10).

83 The current choice of DNA crosslinkers to treat breast cancer is empiric, because these have

84 been selected long before any genetic analysis of tumor sub-groups was possible. Some

85 chemotherapy regimens using alkylating agents have been developed clinically based on the

86 concept of ‘more is better’. Agents that mainly caused bone marrow toxicity were favored, since

87 their dose could be substantially escalated using peripheral blood progenitor cell transplantation

88 (PBPC-Tx). Since the nature and extent of DNA crosslinks differs between alkylating agents (3,4),

89 however, it is conceivable that tumors which cannot employ homologous recombination (HR) for

90 DNA repair show differential responses to various DNA crosslinkers.

91 We have previously shown that mammary tumors generated in the K14cre;Brca1F/F;p53F/F (KB1P)

92 mouse model for hereditary breast cancer are highly sensitive to cisplatin (11) and carboplatin, as

93 well as to the PARP inhibitor olaparib given as single agent or in combination with platinum drugs

94 (12). Despite this high sensitivity, tumors were usually not eradicated, and we could also not

95 achieve this goal by more frequent dosing of cisplatin (13), or with other cytotoxic drugs with a

96 different site of action, such as docetaxel or doxorubicin (11,14). The drug-tolerant “remnants”

97 surviving cisplatin treatment were neither enriched in tumor-initiating cells, nor in biochemical

98 defense mechanisms (13). We therefore hypothesized that the drug-tolerant residual cells

99 causing tumor relapse were stalled in the cell cycle, and we have now identified a drug-tolerant

100 tumor cell population in a G0-like cell cycle stage.

101 An important question is whether these drug-tolerant cells can be eradicated at all by drug

102 treatment. When cell lines are tested for drug response in clonogenic assays, there are usually

103 colonies coming up after treatment with doses above 10 times the IC50, indicating that there are

104 cells in the population that can survive drug doses that cannot be reached in mice or patients. To

105 test this in our model, we have investigated a range of alkylating agents. Whereas we obtained

106 cures in about half of our mice with melphalan, we were able to completely eradicate the tumors

107 with nimustine, an alkylating agent previously identified in an in vitro compound screen to

108 preferentially kill Brca2-/- mammary tumor cells (15). Eradication required the full MTD; at half the

109 MTD all tumors relapsed. This shows that the drug-tolerant G0-like cells can be eliminated, if

110 sufficient damage is inflicted.

111

112 MATERIALS AND METHODS

113

114 Mice, tumor transplantation and treatment of tumor bearing mice

115 Brca1-/-;p53-/- mouse mammary tumors were generated and transplanted into syngeneic mice as

116 described previously (11,16). In this study we used KB1P mice, which were backcrossed to an

117 FVB/N background (17). Tumor bearing mice were treated as indicated in the different

118 experiments. Melphalan (Alkeran, GlaxoSmithKline) and docetaxel (Taxotere, Sanofi-Aventis)

119 were reconstituted immediately before administration, according to the manufacturer's protocol.

120 Thiotepa (Ledertepa, Sigma) and bendamustine hydrochloride (Sigma) were dissolved with

121 physiologic salt solution (stock solution of 10mg/ml) just before injection. Nimustine hydrochloride

122 (1g; Sigma) was dissolved in 6.5ml DMSO and just before injection, diluted 25-fold with

123 physiologic salt solution. Cisplatin and carboplatin solutions ready for injection were obtained from

124 Mayne Pharma. 4-hydroxy-cyclophosphamide (4-OH-CP) was purchased from Dr. Ulf Niemeyer

125 (IIT GmbH, University of Bielefeld, Germany). For temozolomide (Temodal) a 5mg/ml dosing

126 solution was prepared using corn oil, and the freshly prepared solution was sonicated for 10-

127 15min before oral gavage. Tumor volume was calculated using caliper measurements

128 (v=length*width2*0.5). Total white blood cell numbers were measured on a haematology analyzer

129 (Becton Dickinson). All experimental procedures were approved by the Animal Ethics Committee

130 of the Netherlands Cancer Institute, Amsterdam, the Netherlands.

131

132 Immunohistochemistry

133 The following antibodies were used for immunohistochemical staining of tumors: the in-house

134 produced NKI-A59 antibody for the detection of cisplatin DNA-adducts (as described previously

135 (18)), anti-Ki67 from Abcam (ab15580, 1:3000), anti-γH2AX from Cell Signaling (#2577, 1:50),

136 and also anti-cleaved caspase 3 from Cell Signaling (#9661, 1:400). The terminal

137 deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed using the

138 ApopTag® Peroxidase In situ Apoptosis Detection Kit (Chemicon International, S7100). Antigen

139 retrieval was performed by boiling the formalin(10%)-fixed tissue samples for 15min in citrate

140 buffer (pH 6.0). Slides were incubated at 4 °C overnight with the primary antibodies, followed by

141 incubation with a biotinylated goat anti-rabbit secondary antibody (Dakocytomation, # E043201,

142 1:800 in 1% PBSA) for 30 min at room temperature. For detection, we used a standard StreptABC

143 amplified staining procedure with DAB (Dakocytomation, # K037711) and haematoxylin

144 counterstaining. Positive and negative (only secondary antibody) controls were included for each

145 slide and staining procedure. Positively labeled cells were counted in the tumor sections in 10

146 fields of 650x650µm.

147

148 Label incorporation and label retaining assay

149 5-iodo-2-deoxyuridine (IUdR) label retaining assay: 2 individual GFP-labeled

150 Brca1-/-;p53-/- mammary tumors were orthotopically transplanted into 20 wild-type FVB/N mice

151 each. After transplantation 30mg IUdR per kg was injected i.p. daily for about 3 weeks until

152 tumors reached the size of about 20mm3. 4 days after the last IUdR injection tumors were treated

153 with 100µl saline i.v. or with 6mg cisplatin per kg i.v. Tissue specimens were stained with DAPI

154 and IUdR positive cells were detected by immunofluorescence. Tumors were analyzed by

155 immunofluorescence prior to and 8 days post-treatment.

156 5-ethynyl-2’-deoxyuridine (EdU) incorporation assay: 2 individual GFP-labeled

157 Brca1-/-;p53-/- mammary tumors were orthotopically transplanted into wild-type FVB/N mice.

158 160mg EdU per kg was injected i.p. 10 days after saline or cisplatin treatment. 2h after EdU

159 injection tumors were harvested and stained with DAPI and EdU positive cells were detected by

160 immunofluorescence. For double labelling with IUdR and EdU animals were injected with IUdR as

161 described above and EdU was injected 2h before saline or cisplatin treatment. Tumors were

162 analyzed by immunofluorescence prior to and 8 days post treatment.

163

164 Cell lines

165 The Brca1-/-;p53-/- cell lines KB1P-B11 and KB1P-G3 were derived from a genetically engineered

166 mouse model for BRCA1-mutated breast cancer as previously described (17). The cell lines were

167 last authenticated by genotyping of the specific Brca1 and p53 mutations in January 2017. The

168 KB1P-B11 cell line was transduced with the vectors CSII-EF-MCS expressing the fluorescent

169 ubiquitination-based cell cycle indicator (FUCCI) mKO2-hCdt1(30/120) or mAG-hGem(1/110) (19)

170 and the packaging and envelope plasmids pCMV-R 8.2, pMDG and pRSV-Rev. After selection

171 using zeocin the cells were subcloned and sorted for green and red fluorescence subsequently to

172 produce a cell line stably expressing the FUCCI system (KB1P-B11-FUCCI). Cell lines were

173 tested every 6 months for mycoplasma contamination using MycoFluorTM Mycoplasma Detection

174 kit (Thermo Fisher).

175

176 Live cell microscopy

177 KB1P-B11 cells were plated and treated 24h later with 0.7µM cisplatin for 24h and colony

178 formation was tracked using bright field settings on the Zeiss Axiovert 200M fluorescence/live cell

179 imaging system. KB1P-B11-FUCCI cells were treated with 5-fold or 10-fold IC50 carboplatin (IC50

180 = 610nM) or nimustine (IC50 = 750nM) 2 days after seeding. The IC50 was determined using a

181 classical clonogenic assay. Cell cycle specific response of the KB1P-B11-FUCCI cells was

182 analyzed by live cell imaging for 72h.

183

184 FACS

185 To identify Ki67-negative cells KB1P-B11-FUCCI cells were grown to 100% confluency. The cells

186 were fixed and permeabilized using the BD Cytofix/Cytoperm™ kit (Cat. No. 554722) and stained

187 for Ki67 (Ki67-BV421, Clone 16A8, BioLegend, 0.2mg/ml) and DNA content (Draq5 BioLegend, 5

188 nM). The cell cycle stage of Ki67-negative KB1P-B11-FUCCI cells was analyzed using the BD

189 LSRII Flow Cytometer (Ki67-BV421 excitation: 407nm, filter: 450/50; mAG: excitation: 488nm,

190 filter: 525/50; mKO2: excitation: 561nm, filter 585/15; Draq5: excitation: 640nm, filter: 710/50).

191 To sort cells for clonogenic assays KB1P-B11 and KB1P-G3 cells were labeled with Hoechst

192 33342 (Sigma), the cell cycle stage was analyzed and cells were sorted for 2n and 4n content

193 using FACS (BD FACSAria, blue/violet laser 375nm). The cell cycle stage of KB1P-B11-FUCCI

194 cells was analyzed using a BD FACSARIA III (mAG: excitation: 488nm, filter: 530/30; mKO2:

195 excitation: 561nm, filter 582/15) and cells in different cell cycle stages (non-fluorescent (mAG-;

196 mKO2-), red (mAG-; mKO2+), double positive (mAG+; mKO2+) and green (mAG+; mKO2-)) were

197 sorted.

198

199 Colony formation assay

200 IC50 of KB1P-B11 and KB1P-G3 was determined in a short term clonogenic assay: cells were

201 treated for 24h with different drug concentrations and allowed to grow for 6 days. Cells were fixed

202 with 4% paraformaldehyde and stained with 1% Crystal Violet and the drug concentration at

203 which only half of the colonies were growing out compared to untreated cells was calculated.

204 KB1P-B11-FUCCI cells were treated with cisplatin (0.7µM), carboplatin (5µM) or nimustine (4.2

205 and 5µM) for 24h 1 day after seeding or left untreated. Non-fluorescent, red, green and double-

206 positive cells were sorted into FCS and seeded at equal numbers (5000 cells (untreated), 100’000

207 cells (treated)). Cells were fixed after 6 (untreated) or 9 (treated) days and colony formation (at

208 least 20 cells) was quantified. For long term clonogenic assay KB1P-B11 or KB1P-G3 were

209 treated with 5-fold the IC50 of nimustine or carboplatin for 24h and were allowed to grow for 3

210 weeks.

211

212 RESULTS

213

214 Why are KB1P mammary tumors not eradicated by cisplatin?

215 We have previously shown that the KB1P tumors, which contain a large irreversible deletion of

216 the Brca1 gene, do not acquire resistance to the MTD of cisplatin (11,20). Despite this inability of

217 tumors to acquire resistance, animals were usually not cured and tumors eventually relapsed,

218 even after several treatment cycles (Fig. 1A). Consistent with the absence of acquired drug

219 resistance, we did not observe a shortening in the time interval between tumor regrowth after 5

220 repeated cisplatin treatments for 12 individual KB1P tumors (Fig. 1B). Intensifying the drug

221 treatment did not prevent relapse. Although an additional cisplatin MTD treatment on day 14

222 significantly increased the time until relapse, the tumors still re-grew (13). Based on the time until

223 relapse for the day 0 and day 0+14 treatments (Fig. 1C), we estimated the surviving fraction of

224 tumor cells to be <10-8 (tumor of 200mm3 contains about 108 cells) after applying 2 cisplatin doses

225 on day 0+14 (surviving fraction = 2-growth delay/doubling time, tumor doubling time: about 3 days). If the

226 doubling time is constant and cells are randomly hit by cisplatin, a third cisplatin dose on day 28

227 (after the animals have recovered from the previous cisplatin doses) should definitely result in

228 tumor eradication. It did not, however (Fig. 1C). Spreading the additional cisplatin dose over

229 several days would be expected to help eradicate tumor cells, which are by chance not in the right

230 cell cycle stage when cisplatin is dosed, but this treatment regime did not increase tumor

231 eradication either (Supplementary Fig. S1). To test whether drug-tolerant tumor cells accumulate

232 in residual tumors we compared the cisplatin response of small drug-naïve tumors (about 20mm3)

233 with the response of remnants of the same size 14 days after surviving the initial cisplatin

234 treatment. As shown in Fig. 1D tumor remnants relapsed earlier than the naïve tumors (P=0.006).

235 This difference is even more significant given the fact that the residual tumors contain an

236 increased amount of stroma and about a third less tumor cells, as we have shown previously (13).

237 In contrast to the small tumor remnants, previous treatment does not influence the response of

238 the relapsed tumors (Fig. 1A+B), suggesting that the drug-tolerant cells only represent a small

239 fraction of the latter. Together, our data show that the tumor remnants remaining after the initial

240 cisplatin treatment are enriched in cells that are drug-tolerant, but have not acquired stable drug

241 resistance.

242

243 Residual KB1P tumors are quiescent

244 We recently showed that residual tumors are not enriched in tumor-initiating cells, which strongly

245 suggests that the tumor-initiating cells of our model do not have increased biochemical defense

246 mechanisms against cisplatin (13). The residual tumor cells are also accessible to drug: we found

247 a homogeneous distribution of the platinum-DNA adducts throughout the tumor (Supplementary

248 Fig. S2A). 10 days after cisplatin treatment several residual tumor cells appeared to have

249 removed platinum-DNA adducts, but upon re-treatment the adducts reappeared. We infer from

250 these results that the residual tumor cells of our model are accessible to the drug.

251 Another plausible mechanism for tumor cells to escape drug toxicity is to stop dividing, and to

252 enter transiently into a quiescent program. This hypothesis is supported by the substantial

253 decrease in Ki-67-positive cells in the residual tumors (Fig. 2A). To verify this result we

254 investigated EdU uptake into the tumor remnants. To distinguish tumor from stromal cells, we

255 introduced the GFP marker into our model (21) (Supplementary Fig. S2B). In drug-naïve tumors,

256 we found that many tumor cells were easily labeled with EdU. In contrast, the residual GFP-

257 positive tumor islands after cisplatin treatment hardly incorporated EdU (Supplementary Fig.

258 S3A). Hence, residual tumor cells dropped out of cycle.

259 We next investigated whether slowly cycling cells are already present in drug-naïve tumors, or

260 whether they are exclusively induced by drug-treatment. For this purpose, we performed a label-

261 retaining assay of two different GFP-labeled KB1P donor tumors using IUdR (Fig. 2B). Daily

262 injection of IUdR after orthotopic transplantation successfully labeled most tumor cells once a

263 palpable tumor (about 3-5mm in diameter) was detected (“2h after last IUdR injection”). At this

264 size the IUdR injections were stopped and 4 days later most tumor cells had lost the label (“4d

265 after last IUdR injection”). We then compared the effect of saline versus cisplatin treatment on the

266 tumors 12d after the last IUdR injection. Whereas labeled cells were rare (<1%) in the saline-

267 treated tumors (“12d after last IUdR injection; 8d after saline”), the IUdR-retaining cells were

268 enriched (about 25-35%) in the remnants of cisplatin-treated tumors (“12d after last IUdR

269 injection; 8d after cisplatin”). This shows that slowly cycling cells are present within our KB1P

270 tumor model, and these cells have an advantage in surviving the cisplatin treatment onslaught. In

271 addition, we investigated cells that went through S phase just before cisplatin or saline treatment.

272 To mark those cells, we injected EdU into mice 2 hours before cisplatin or saline treatment

273 (Supplementary Fig. S3B-D). As expected, IUdR-labelled cells did not pick up EdU

274 (Supplementary Fig. S3C). Moreover, in contrast to the quiescent IUdR-retaining cells, the

275 number of proliferating EdU-positive cells clearly decreased after cisplatin treatment

276 (Supplementary Fig. S3C+D), showing that these cells are more vulnerable.

277

278 Single nucleated G0-like KB1P cells are tolerant to platinum drugs

279 In our model we observed the presence of giant and multinucleated tumor cells in the cisplatin-

280 treated tumor remnants (Fig. 3A). We could even reproduce this phenotype in vitro in

281 Brca1-/-;p53-/- cell lines (KB1P-B11 and KB1P-G3) that we derived from a spontaneous KB1P

282 tumor. These cell lines highly mimic the spontaneous tumor in their genomic profile

283 (Supplementary Fig. S4A) and they mimic the morphology and drug response of KB1P tumors if

284 grafted back into mice (17). We found many giant and multinucleated KB1P-B11 cells within a few

285 days after treatment with 0.7µM cisplatin. In tumors or colonies that relapsed 3-4 weeks later,

286 those cells were largely absent (Fig. 3A). The formation of multinucleated cells and subsequent

287 “giant cell death” in the G1 phase of the cell cycle has been described to result from DNA

288 interstrand crosslinks (22). Using time-lapse video microscopy, we found that the multinucleated

289 cells after cisplatin treatment are doomed and eventually undergo crosslink-induced giant cell

290 death (Fig. 3B).

291 To determine from which cells relapsing colonies evolve, we backtracked colonies formed after

292 cisplatin treatment by time-lapse video microscopy. When KB1P-B11 cells were treated with

293 0.7µM cisplatin for 24h, we found that colonies were always derived from small cells with a single

294 nucleus (an example is shown in Fig. 4A). To determine the cell cycle stage from which the

295 single-nucleated cells are able to repopulate colonies, we labeled cells with Hoechst. Whereas

296 equal numbers of untreated cells (unsorted, 2n, 4n or 2n+4n) resulted in the same number of

297 colonies (Fig. 4B), only cells with a 2n DNA content, hence cells in G0 or G1, were able to form

298 new colonies after cisplatin treatment (Fig. 4C).

299 To further characterize the cell cycle stage from which Brca1-/-;p53-/- mammary tumors arise after

300 cisplatin treatment, we introduced the fluorescent ubiquitination-based cell cycle indicator

301 (FUCCI) construct (19) in KB1P-B11 cells. The cells express the fluorescent proteins mKO2-

302 hCdt1(30/120) in G1, mAG-hGem(1/110) in S/G2/M, both fluorescent proteins in G1/S transition

303 and there is a non-fluorescent stage after mitosis of the cells. Since we observed an enrichment

304 of Ki67-negative cells in the non-fluorescent pre G1 population (Fig. 5A), we called them G0-like.

305 In contrast to the cells used by Tomura et al. (23) we could not identify an increase of cells with

306 high intensities of mKO2 as potential G0 cells when cells were grown to confluency or treated with

307 drugs. This difference may be due to the use of a different promoter for the FUCCI system and

308 the different genetic background in our model. We then analyzed the colony formation capacity

309 (experimental layout in Supplementary Figure S4B) of the different cell cycle stages after

310 treatment with DNA crosslinking agents. The cell cycle stage did not influence the colony

311 formation capacity of untreated cells. If treated with cis- or carboplatin the G0-like cells had a

312 much higher colony formation capacity than cells in G1, G1/S transition or cells in S/G2/M phase

313 (Fig. 5B). The same pattern was observed with other drug concentrations than those used in Fig.

314 5B+C (data not shown). Thus, residual G0-like cells are drug-tolerant to platinum drugs.

315

316 G0-like cells are less tolerant to the alkylator nimustine and more multinucleated cells form

317 upon nimustine treatment

318 Evers et al. recently found in an in vitro screen that BRCA2/p53-deficient cells are hypersensitive

319 to the alkylator nimustine (a chloroethylnitrosourea) (15). We therefore tested whether the

320 platinum drug-tolerant G0-like KB1P cells respond differently to nimustine treatment. Intriguingly,

321 nimustine treatment resulted in a different cell cycle-dependent sensitivity pattern: the selective

322 drug tolerance of G0-like cells is not detectable when the cells are treated with nimustine at

323 concentrations that resulted in about 100 colonies (Fig. 5C and Supplementary Figure S4B).

324 Since time-lapse video microscopy revealed that multinucleation in response to DNA crosslinking

325 agents kills the BRCA1/p53-deficient tumor cells, we analyzed whether nimustine causes more

326 multinucleated cells than platinum drugs. For this purpose we first determined the carboplatin and

327 nimustine IC50 in a short-term (1 week) clonogenic assay (Supplementary Figure S5A).

328 Interestingly, filming of the KB1P-B11-FUCCI cells showed that 10-fold nimustine IC50 caused

329 more multinucleation than the corresponding carboplatin concentration (Fig. 5D left panel).

330 Moreover, the difference was most evident when multinucleation of G0-like cells was compared

331 after nimustine or carboplatin treatment (Fig. 5D right panel). Most of the multinucleated cells

332 were able to complete mitosis and underwent cell lysis in G1 (Supplementary figure S5B).

333 Consistent with the inability of multinucleated cells to form colonies, we observed in long-term (3

334 weeks) clonogenic assays that even at 5-fold IC50 concentrations some colonies grew back when

335 cells were treated with carboplatin but fewer colonies grew back with the equivalent dose of

336 nimustine (Fig. 6A).

337 Together, these data strongly suggest that in contrast to platinum drug-induced DNA crosslinks,

338 the increased amount of nimustine-induced interstrand crosslinks (24,25) cannot be removed

339 effectively in the G0-like cells. This results in multinucleated cells which subsequently die.

340

341 A single dose of the nimustine MTD eradicates KB1P tumors

342 Based on these in vitro results we tested nimustine efficacy in Brca1-/-;p53-/- (KB1P) or p53-/- (KP)

343 tumors. Since Evers et al. (15) also identified the bifunctional alkylator melphalan as an efficient

344 compound for the treatment of BRCA2/p53-deficient cells, we examined this agent in parallel in

345 our in vivo experiments. Moreover, we tested the nimustine analog, nitrogen mustard

346 bendamustine, which has recently re-emerged as chemotherapeutic treatment. While KP tumors

347 were not particularly sensitive to the MTD of melphalan (10 mg/kg i.p.), nimustine (30 mg/kg i.p.)

348 or bendamustine (40 mg/kg i.p.) (Supplementary Fig. S6A and B), all KB1P tumors in the

349 nimustine-treated, half of the melphalan-treated and 1/3 of the bendamustine-treated animals

350 appeared to be eradicated (Fig. 6B, upper panel). Dose matters, however. When we lowered the

351 nimustine dose to 75% of the MTD, half of the tumors relapsed, and all tumors re-grew when 50%

352 of the MTD was injected (Fig. 6B, lower panel). In contrast, we could not eradicate the KB1P

353 tumors using the monofunctional alkylator temozolomide (Supplementary Fig. S6C and D) and

354 most KB1P tumors relapsed after high dose monotherapy or combination therapy with

355 carboplatin, thiotepa, and cyclophosphamide (CTC) (Supplementary Fig. S7A) (8,26).

356 Unfortunately, the gut toxicity of the cocktail of alkylating agents CTC turned out to be the limiting

357 factor in mice, and we could not escalate the dose above MTD levels by mimicking bone marrow

358 reconstitution (Supplementary Fig. S7B-E and S8A). Despite the lack of eradication, relapsing

359 tumors were still sensitive to repeated treatments of monotherapy or combination therapy with

360 carboplatin, thiotepa, and cyclophosphamide (CTC) (Supplementary Fig. S8B). This pattern is

361 similar to what we found in response to cisplatin (Fig. 1A) or carboplatin (12). It supports the

362 notion that in the absence of functional BRCA1, resistance to DNA crosslinking agents given at

363 MTD levels does not evolve in our model (27).

364 To further explore the differential sensitivity of KB1P tumors to carboplatin or nimustine, we

365 investigated the drug-induced damage in situ. IHC analysis revealed that 72h after treatment,

366 more DNA damage foci (determined by γH2AX positivity), apoptotic cells (measured by cleaved

367 caspase 3 positivity) and DNA breaks (measured by TUNEL) were present in nimustine-treated

368 tumors (Fig. 6C) compared to carboplatin-treated tumors. This difference between nimustine- and

369 carboplatin-treated tumors was not detectable 24h after treatment indicating that DNA damage

370 caused by nimustine is not resolved as efficiently as damage caused by platinum drugs.

371 To investigate whether carboplatin is more effective when the drug-tolerant G0-like cells are

372 depleted, we pre-treated animals with docetaxel for 24h. This resulted in an enrichment of cells

373 arrested in M phase (Fig. 6D). When the docetaxel-pretreated animals were dosed with

374 carboplatin, we observed that 6 out of 9 animals were cured and their survival was significantly

375 increased compared with carboplatin treatment alone (Fig. 6D). This suggests that reducing the

376 G0-like fraction of tumor cells is a useful therapeutic strategy to achieve tumor eradication.

377

378 DISCUSSION

379 Using a mouse model for BRCA1-deficient breast cancer we identified a slowly cycling drug-

380 tolerant population of tumor cells, which survives the platinum drug therapy and causes tumor

381 relapse. We show that these residual tumor cells are in a G0-like cell cycle stage and can be

382 effectively targeted with the chloroethylating agent nimustine resulting in disease eradication.

383 A major difference in DNA damage between platinum drugs and nimustine is the type of DNA

384 crosslinks caused. DNA crosslinking agents bind to nucleotides either on the same DNA strand or

385 on complementary DNA strands causing intrastrand and interstrand crosslinks respectively.

386 Platinum drugs such as cisplatin or carboplatin mainly cause intrastrand crosslinks (28), whereas

387 nimustine mainly causes interstrand crosslinks (24,25).

388 By measuring DNA damage foci, apoptotic cells and DNA breaks 24h and 72h after treatment we

389 have shown that DNA damage caused by nimustine is not resolved as efficiently as damage

390 caused by platinum drugs in BRCA1-deficient tumors. The increased sensitivity of G0-like cells to

391 nimustine may be explained by the impaired DNA damage repair of the nimustine-induced

392 interstrand crosslinks. Platinum-induced intrastrand crosslinks can be removed by nucleotide

393 excision repair (NER) during the G0/G1 phase of the cell cycle independent of a sister chromatid

394 as template DNA (29). Platinum adducts linking two neighboring nucleotides on the same strand

395 bend the DNA significantly which might facilitate the recognition and repair of crosslink sites (30).

396 In contrast, only a subset of interstrand crosslinks is repaired by NER. Unresolved interstrand

397 crosslinks lead to stalled replication forks in S phase which are stabilized and repaired by the

398 Fanconi anemia (FA) pathway and HR. BRCA1 is important in further processing of the defect by

399 HR (31,32). In BRCA1-deficient tumor cells interstrand crosslink repair might therefore be

400 impaired. Unresolved interstrand crosslinks caused by nimustine may lead to stalled replication

401 forks, multinucleation and cell death, whereas the intrastrand crosslinks caused by platinum drugs

402 may be resolved by NER before the cells enter S phase.

403 Characterization of drug tolerant tumor cells using cell lines derived from the BRCA1-deficient

404 mouse model showed that relapsing colonies after cisplatin treatment are derived from single

405 nucleated 2n cells. In the Brca1-/-;p53-/- model the multinucleated cells observed after treatment

406 with DNA crosslinking agents in vivo and in vitro lacked clonogenicity and eventually died. Using

407 FUCCI-expressing Brca1-/-;p53-/- cells we found that most cells do not die from mitotic

408 catastrophe, but exit mitosis without proper segregation of sister chromatids. This event is

409 described as mitotic slippage (33) resulting in multinucleated cells. The multinucleated cells in G1

410 can still enter S phase due to the defective G1/S checkpoint in p53 deficient cells (34).

411 Nevertheless, the multinucleated cells die after several cell cycles and interestingly most of the

412 cells die in G1 and not in S phase, as described in other p53-deficient cells (33). Cell death in G1

413 must be executed via a p53-independent pathway in the Brca1-/-;p53-/- model (35). These results

414 differ from those obtained by Puig et al. (36). In their rat model tumor cells can escape cisplatin-

415 induced cell death through DNA endoreduplication and reversible polyploidy. In contrast, our

416 results are more compatible with the findings of Osawa et al. (22), who observed giant cell death

417 of multinucleated cells following treatment with DNA crosslinking agents. A difference to the rat

418 model is that the relapsing tumors in our genetically engineered model do not show increased

419 resistance to cisplatin treatment. Due to the large intragenic deletion of the Brca1 gene, no

420 restoration of BRCA1 function is possible, a resistance mechanism against platinum drugs that

421 was described earlier (37). This lack of acquired resistance we also observed in response to other

422 frequently used alkylating agents, including cyclophosphamide and thiotepa. Unresolved

423 interstrand crosslinks might therefore explain the formation of more multinucleated cells and cell

424 death after nimustine treatment compared to platinum drug treatment in the Brca1-/-;p53-/- cells.

425 The most likely explanation for the platinum drug tolerance of G0-like cells is that they have more

426 time to repair DNA crosslinks before entry into S-phase than G1 cells. We do not think that

427 stabilization of stalled replication forks plays a role, even though this stabilization is known to

428 preserve genomic integrity (38,39) and can contribute to acquired PARPi resistance in BRCA2-

429 deficient cells (40). In contrast, the BRCA1-deficient cells in our model do not acquire resistance

430 to the DNA crosslinking agents. In particular, the nimustine-induced interstrand crosslinks cannot

431 be resolved by increased fork stabilization that is sufficient to overcome PARPi-induced fork

432 stalling.

433 There is also no evidence that G0-like cells are more proficient in repairing the DNA damage.

434 RNASeq analysis of the G0-like cells did not show increased expression of NER-related genes

435 compared to G1 cells (data not shown). Nevertheless, further characterizations of the drug tolerant

436 G0-like cells may be helpful to find drug targets to specifically deplete G0-like cells before

437 treatment with platinum drugs.

438 The G0-like drug tolerant cells in our BRCA1-deficient mouse model do not appear to be identical

439 to the sub-population of reversibly drug-tolerant cells identified by Settleman and coworkers in

440 tumor cell lines treated with tyrosine kinase inhibitors (TKI’s). These cells have been called “drug-

441 tolerant persisters” (DTPs) to emphasize the analogy with drug-resistant “persisters” arising in

442 bacterial cultures treated with antibiotics (reviewed in Harms et al., 2016 (41)), even though this

443 analogy is not persuasive (42). DTPs are reported to be stalled in G1, but G0 was not separately

444 checked. A sub-fraction of DTPs can give rise to “drug-tolerant expanded persisters” that can

445 multiply in drug indefinitely (43). Various independent mechanisms appear to contribute to the

446 formation of DTPs (43–45). Resistance of DTPs is not limited to TKI’s, as some cross-resistance

447 was observed to cisplatin (43). Although superficially similar to DTPs, at the RNA level the drug-

448 tolerant, G0-like cells that we find in our mouse tumors lack the characteristic features reported for

449 DTPs, such as activated IGF-1 receptor signaling (43), elevated aldehyde dehydrogenase levels

450 (44), or high peroxiredoxin 6 (45) (data not shown). Moreover, the G0-like cells that we find in vivo

451 in our model are not very resistant, as shown by treatment with the appropriate alkylating agent

452 nimustine, in contrast to the DTPs that are more than 100-fold resistant to TKI’s. Although we also

453 find rare colonies in vitro after treating our cells with 5-fold the IC50 of nimustine, we think that

454 these rare surviving cells are probably eliminated in vivo by the intact host immune system.

455 The finding that even at 5-fold the IC50 of cisplatin or nimustine some colonies grow back in vitro

456 raised the question whether these drug-tolerant cells can be eradicated at all by drug treatment.

457 The fact that we were able to eradicate the Brca1-/-;p53-/- tumors with nimustine in vivo is

458 consistent with the results that high dose chemotherapy using alkylating agents can cure patients

459 with high risk BRCA1/2-like breast cancer (8,9). Although not possible in our model, upfront

460 mutations that restore BRCA function may be present in drug-naïve human tumors and

461 counteract tumor eradication. The fact that tumor eradication is achieved in patients with high risk

462 BRCA1/2-like breast cancer (8,9), however, supports the notion that this is not a general clinical

463 hurdle. Eradication of the mouse Brca1-/-;p53-/- tumors with nimustine is highly dose dependent

464 and this strongly suggests that the amount of damage caused is crucial for the successful

465 eradication of residual tumor cells. In our model, we did not succeed in testing the standard high-

466 dose chemotherapy used in patients due to the gut toxicity of the DNA crosslinking agents in

467 mice. Nevertheless, we infer from our data that the high-dose chemotherapy in humans also

468 results in an increased number of interstrand crosslinks of transiently dormant G0-like cells. When

469 these cells re-enter the cell cycle with persisting crosslinks, damage cannot be properly repaired

470 in the absence of BRCA1/2 function and the cells become multinucleated and eventually die.

471 Future studies aimed at identifying specific markers for these G0-like cells are crucial to

472 understand their role in drug tolerance in human cancer.

473

474 ACKNOWLEDGEMENTS

475 We thank the late Adrian Begg (The Netherlands Cancer Institute) for his help with calculating the

476 surviving fraction and Ben Floot (The Netherlands Cancer Institute) for providing the NKI-A59

477 antibody. We are also grateful to Sjoerd Rodenhuis and Sabine Linn (Antoni van Leeuwenhoek

478 hospital) for suggesting clinically relevant alkylating agents/combinations. Rob Wolthuis (VU

479 University Medical Center, Amsterdam) was very helpful in discussing cell cycle-related

480 questions. Moreover, we wish to thank Sjoerd Rodenhuis, Nora Gerhards and Paola Francica for

481 critical reading of the manuscript.

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605

606 FIGURES AND FIGURE LEGENDS

607

608 Figure 1. Responses of Brca1-/-;p53-/- mouse mammary tumors to cisplatin treatments. (A)

609 Example of the response to repeated MTD cisplatin doses (arrows) of an orthotopically

610 transplanted tumor in a mouse. When the tumor reached a volume of 200mm3 the animal was

611 treated with 6mg cisplatin per kg i.v. The same treatment was resumed each time the tumor grew

612 back to 200mm3. (B) The time interval between 5 individual treatments (time until relapse: t1-t4) is

613 shown for 12 individual Brca1-/-;p53-/- mammary tumors that were treated as depicted in (A). The

614 ANOVA gives a p>0.95 (C) Intensified cisplatin treatment. Time until relapse was analyzed after a

615 single dose of 6mg cisplatin per kg i.v. (day 0, n=18, black line)), or after repeated treatments with

616 the same dose before relapse (treatment day 0+14, n=13, red line; treatment day 0+14+28, n=9,

617 blue line). The log-rank test comparing time until relapse between single treatment (black line)

618 and treatment on day 0+14 (red line) gives a p=0.007 and a p>0.96 comparing treatment on day

619 0+14 (red line) and treatment on day 0+14+28 (blue line) (D) Treatment of cisplatin remnants and

620 drug-naïve tumors. 5 individual Brca1-/-;p53-/- donor tumors were transplanted into 9 animals each

621 to compare the time until relapse (tumor size of 20mm3 (treatment) until the tumor relapsed to a

622 size of 200mm3) for the following groups: cisplatin treatment of naïve tumors at a tumor volume of

623 20mm3 (n=15, red line); residual tumors (n=15, blue line) which were initially treated at 200mm3

624 and received a second treatment on day 14 at a size of about 20mm3; saline-treated control: black

625 line (n=15). The log-rank test gives a p=0.006 when the cisplatin response of residual tumors and

626 naïve tumors is compared.

627

628 Figure 2. Identification of slowly cycling cells in tumor remnants after cisplatin treatment (A) Ki-67

629 IHC and quantification in tumors before and 10 days after 6mg cisplatin per kg i.v. Ki-67 was also

630 quantified separately for single-nucleated cells only. Bar=50µm; graph shows mean and SD of

631 Ki67-positive cells in 5 individual tumors, ***P <0.001, ****P <0.0001 (two-tailed t-test). (B) Label-

632 retaining assay using IUdR incorporation to investigate the presence of pre-existing slowly cycling

633 cells. Outline of the experiment and treatment schedule as exemplified for a saline- (left panel)

634 and cisplatin-treated tumors (right panel) is shown. 2 individual GFP-labeled

635 Brca1-/-;p53-/- mammary tumors were orthotopically transplanted into 20 wild-type FVB/N mice

636 each. After transplantation 30mg IUdR per kg was injected i.p. daily for about 3 weeks until

637 tumors became palpable (size of about 20mm3). 4 days after the last IUdR injection tumors were

638 either treated with 100µl saline i.v. or with 6mg cisplatin per kg i.v. Before and 8 days after

639 treatment tumors were analyzed by immunofluorescence for the presence of IUdR in GFP-

640 positive tumor cells. The bar diagram shows the mean (n= 5) and SD; P values of the IUdR-

641 positive cells were determined using the two-tailed t-test.

642

643 Figure 3. Drug-induced multinucleation in KB1P tumors and Brca1-/-;p53-/- cells causing cell

644 death. (A) HE staining (in vivo) or bright field microscopy (in vitro) of multinucleated giant cells 7d

645 after 6mg cisplatin per kg i.v. (in vivo) and KB1P-B11 cells treated with 0.7µM cisplatin (in vitro)

646 and outgrowth of a tumor or colony composed of single-nucleated cells 3-4 weeks post cisplatin.

647 Quantification of multinucleated cells in untreated tumors (in vivo) or colonies (in vitro), in residual

648 disease 7 days after cisplatin treatment and in relapse (3-4 weeks post cisplatin). P value of two-

649 tailed t-test <0.0001, in vivo: n=10; in vitro: n=5-10 (B) Time-lapse microscopy reveals frequent

650 aberrant cell divisions, formation of multinucleated KB1P-B11 tumor cells and cell death following

651 treatment with 0.7µM cisplatin (CDDP). t (time) = time since start of monitoring using time-lapse

652 microscopy, time since previous division.

653

654 Figure 4. Colony formation of cisplatin-treated Brca1-/-;p53-/- cells. (A) Example of a backtracking

655 experiment. KB1P-B11 cells were treated with 0.7µM cisplatin (CDDP) for 24h, the drug was

656 subsequently removed and cells were monitored using time-lapse microscopy. A colony that

657 formed 1 week after treatment start was monitored backwards to the start of filming to visualize

658 the initial colony-forming cell. t (time) = time since start of monitoring using time-lapse microscopy,

659 time since previous division. (B) Colony formation of untreated or cisplatin-treated KB1P-B11 cells

660 in G0/G1 or G2/M. KB1P-B11 cells were left unsorted or sorted for a 2n or 4n content. Equal

661 numbers of cells (100’000) of the indicated populations was then plated to examine their

662 clonogenic capacity. (C) The experiment described in (B) was performed for 2n (G0/G1 shown in

-/- -/- 663 blue) versus 4n (G2/M shown in red) cells of Brca1 ;p53 cells (KB1P-B11 and KB1P-G3) 24h

664 after cisplatin treatment (0.7µM for KB1P-B11; 1µM for KB1P-G3).

665

666 Figure 5. Cell cycle dependent colony formation of platinum- or nimustine-treated KB1P-B11-

667 FUCCI cells. (A) The cell cycle stage in Brca1-/-;p53-/-cells stably expressing the FUCCI construct

668 was analyzed using live cell imaging or flow cytometry to distinguish four different populations:

669 Q1: mKO2+/mAG- (red); Q2: mKO2+/mAG+ (double positive); Q3: mKO2-/mAG+ (green); Q4:

670 mKO2-/mAG- (non-fluorescent). Nuclear staining using Draq5 confirms cell cycle-dependent

671 expression of the fluorescent proteins mKO2-hCdt1(30/120) (red) and mAG-hGem(1/110)

672 (green). The percentage of Ki67-negative cells in different cell cycle stages was analyzed using

673 flow cytometry and quantification is shown in the bar diagram. P value has been determined using

674 the two-tailed t-test; n=3 (B) and (C) Colony formation of untreated, cisplatin-, carboplatin- or

675 nimustine-treated cells (24h treatment) followed by sorting for cell cycle stage (G0-like: non

676 fluorescent, G1: red, G1/S: double positive, S/G2/M: green), seeding of equal number of cells per

677 cell cycle stage (untreated: 5000 cells, treated: 100’000 cells) and fixation and quantification of

678 colonies after 6 (untreated) or 9 (treated) days. Drug concentrations were chosen which result in

679 about 100 colonies per treatment condition. ****P value of two-tailed t-test <0.0001; n=3. Note

680 scale differences. (D) Quantification of multinucleated cells in all cell cycle stages (left panel) and

681 the G0-like and G1-cells analyzed separately (right panel) using time-lapse microscopy of

-/- -/- 682 Brca1 ;p53 FUCCI cells after treatment with 10 times the IC50 of carboplatin (6.1µM) or

683 nimustine (7.5µM). P values have been determined using the two-tailed t-test; n=3

684

685 Figure 6. Eradication of KB1P tumors with a single dose of the MTD of nimustine caused by

686 unresolved DNA damage (A) Colony formation capacity of KB1P-B11 and KB1P-G3 cells in a

687 long-term clonogenic assays (3 weeks) using 5 times the IC50 of carboplatin (KB1P-B11: 3.05µM;

688 KB1P-G3: 14.5µM) or nimustine (KB1P-B11: 3.75µM; KB1P-G3: 5.5µM). P values were

689 calculated using the two-tailed t-test; n=4 (B) Time until relapse of BRCA1-deficient mammary

690 carcinomas treated with the MTD of melphalan (10mg/kg i.p.), bendamustine (40mg/kg i.p.) or

691 nimustine (30mg/kg i.p.) (upper panel) or 75%, 50% and 25% of the nimustine MTD (lower panel).

692 P values were calculated using the log-rank test; n=12 (C) Carboplatin- or nimustine-induced

693 damage in KB1P tumors. Animals carrying 2 individual orthotopically transplanted KB1P tumors

694 were treated with saline, 100mg carboplatin per kg i.p. or 30mg nimustine per kg i.p. and tumors

695 were sampled 24h or 72h after treatment. Examples of the detection of γH2AX, cleaved caspase

696 3 or TUNEL (bar=100µm) are shown on the left and the data quantification on the right. Graphs

697 show the mean (n=5) and SD per group. P values were measured using the two-tailed t-test. (D)

698 Enrichment of KB1P cells in M phase 24h after docetaxel treatment. HE staining shows tumor

699 cells with condensed chromosomes in M phase (examples are indicated with red arrows) shown

700 on the left. Bar=25µm. Survival of animals carrying KB1P tumors that were left untreated (n=5),

701 treated with 25mg docetaxel per kg i.v. (n=9), 100mg carboplatin per kg i.p. (n=6) or 25mg

702 docetaxel per kg i.v. followed by 100mg carboplatin per kg i.p. 24h later (n=9) shown on the right.

703 The P value was calculated using the log-rank test.

Selected alkylating agents can overcome drug tolerance of G0 -like tumor cells and eradicate BRCA1-deficient mammary tumors in mice.

Marina Pajic, Sohvi Blatter, Charlotte GUYADER, et al.

Clin Cancer Res Published OnlineFirst August 18, 2017.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-17-1279

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