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Author Manuscript Published OnlineFirst on May 27, 2021; DOI: 10.1158/1535-7163.MCT-20-1125 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Ureshino et al. Silylation of DAC for MDS and AML.

1 Original article

2 Silylation of deoxynucleotide analog yields an orally available drug

3 with anti-leukemia effects

5 Hiroshi Ureshino1,2†, Yuki Kurahashi1†, Tatsuro Watanabe1, Satoshi Yamashita3, 6 Kazuharu Kamachi1,2, Yuta Yamamoto1, Yuki Fukuda-Kurahashi1, Nao 7 Yoshida-Sakai1,2, Naoko Hattori3, Yoshihiro Hayashi4, Atsushi Kawaguchi5, Kaoru 8 Tohyama6, Seiji Okada7, Hironori Harada4, Toshikazu Ushijima3, and Shinya Kimura1, 9 2* 10

11 † These authors equally contributed to this work.

13 1Department of Drug Discovery and Biomedical Sciences, Faculty of Medicine, Saga 14 University, Saga, Japan; 2Division of Hematology, Respiratory Medicine and Oncology, 15 Department of Internal Medicine, Faculty of Medicine, Saga University, Saga, Japan; 16 3Division of Epigenomics, National Cancer Center Research Institute, Tokyo, Japan; 17 4Laboratory of Oncology, School of Life Sciences, Tokyo University of Pharmacy and 18 Life Sciences, Tokyo, Japan; 5Center for Comprehensive Community Medicine, Faculty 19 of Medicine Saga University, Saga, Japan; 6Department of Laboratory Medicine, 20 Kawasaki Medical School, Kurashiki, Japan; 7Division of Hematopoiesis, Joint 21 Research Center for Human Retrovirus Infection, Kumamoto, Japan.

23 *Corresponding author: Shinya Kimura, MD, PhD

24 5-1-1 Nabeshima, Saga 849-8501, Japan

25 Phone: +81-952-34-2366, Fax: +81-952-34-2017

26 E-mail: [email protected] 1

Downloaded from mct.aacrjournals.org on September 24, 2021. © 2021 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 27, 2021; DOI: 10.1158/1535-7163.MCT-20-1125 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Ureshino et al. Silylation of DAC for MDS and AML.

27 Conflict-of-interest disclosure: This work was supported by Ohara Pharmaceutical.

29 Running title: Silylation of DAC for MDS and AML

30 Main text: 4115 words

31 Abstract: 248 words

32 Figures: 6

33 References: 50

34 Supplementary figure, 16; Tables, 4; method, 1

35 Supplementary figure only for reviewers: 1

Ureshino et al. Silylation of DAC for MDS and AML.

37 Abstract

38 DNA methyltransferase inhibitors have improved the prognosis of myelodysplastic

39 syndrome (MDS) and acute myeloid leukemia (AML). However, because these agents

40 are easily degraded by cytidine deaminase (CDA), they must be administered

41 intravenously or subcutaneously. Recently, two orally bioavailable DNA

42 methyltransferase inhibitors, CC-486 and ASTX727, were approved. In previous work,

43 we developed 5-O-trialkylsilylated decitabines that resist degradation by CDA.

44 However, the effects of silylation of a deoxynucleotide analog and enzymatic cleavage

45 of silylation have not been fully elucidated. Enteric administration of OR21 in a

46 cynomolgus monkey model led to high plasma concentrations and hypomethylation,

47 and in a mouse model, oral administration of enteric-coated OR21 led to high plasma

48 concentrations. The drug became biologically active after release of decitabine (DAC)

49 from OR21 following removal of the 5'-O-trisilylate substituent. Toxicities were

50 tolerable and lower than those of DAC. Transcriptome and methylome analysis of MDS

51 and AML cell lines revealed that OR21 increased expression of genes associated with

52 tumor suppression, cell differentiation, and immune system processes by altering

53 regional promoter methylation, indicating that these pathways play pivotal roles in the

54 action of hypomethylating agents. OR21 induced cell differentiation via upregulation of

55 the late cell differentiation drivers CEBPE and GATA-1. Thus, silylation of a

56 deoxynucleotide analog can confer oral bioavailability without new toxicities. Both in

57 vivo and in vitro, OR21 exerted anti-leukemia effects, and had a better safety profile

Ureshino et al. Silylation of DAC for MDS and AML.

58 than DAC. Together, our findings indicate that OR21 is a promising candidate drug for

59 phase 1 study as an alternative to azacitidine or decitabine.

60 Keywords: silylation, hypomethylating, orally available, OR-2100, myelodysplastic

61 syndrome, acute myeloid leukemia

Ureshino et al. Silylation of DAC for MDS and AML.

63 Introduction

64 Myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) are

65 characterized by impaired differentiation of hematopoietic stem cells (HSCs) or

66 hematopoietic progenitor cells (1,2). Somatic mutations and aberrant DNA

67 hypermethylation that silence cancer-regulating genes (e.g., tumor-suppressor genes) in

68 these cells (3) are involved in pathogenesis and progression of MDS and AML (4–8).

69 Epigenetic alterations lead to reduced hypermethylation, which can in turn cause

70 re-expression of silenced tumor-regulating genes. These alterations represent novel

71 therapeutic targets for both of these cancers (9).

72 Although the clinically available DNA methyltransferase (DNMT) inhibitors

73 azacitidine (AZA) and decitabine (DAC) have improved the prognosis of MDS and

74 AML (10,11), they are easily degraded by cytidine deaminase (CDA), limiting their

75 bioavailability after oral administration; consequently, they must be administered

76 intravenously or subcutaneously. The efficacy and safety of oral AZA or DAC, as well

77 as the combination of these drugs with CDA inhibitors, has been evaluated in clinical

78 trials (12–14), resulting in the approval of CC-486 (oral AZA) and ASTX727 (DAC

79 plus cedazuridine, a CDA inhibitor) (13,14).

80 Resistance to CDA is crucial for achieving elevated plasma concentrations of these

81 drugs, which would in turn increase efficacy (15). Silylation of glycoproteins is known

82 to influence their stabilities and therapeutic effects (16,17); however, the effects of

83 silylation of deoxynucleotide analogs and enzymatic cleavage of silylation have not

84 been fully elucidated. In previous work, we developed 5-O-trialkylsilylated DACs that 5

Ureshino et al. Silylation of DAC for MDS and AML.

85 were resistant to CDA and exerted anti-cancer effects against solid tumors and adult

86 T-cell leukemia/lymphoma (18,19). In this study, we assessed the oral bioavailability

87 and efficacy of 5-O-trialkylsilylated DAC for treatment of MDS and AML both in vitro

88 and in vivo.

90 Materials and methods

91 Reagents

92 5-O-trialkylsilylated DAC (OR2100, 2003, 2007, 2008, 2009, 2010, 2102, 2103, 2104,

93 and 2201) was obtained from Ohara Pharmaceutical (Shiga, Japan). AZA and DAC

94 were purchased from Sigma-Aldrich (St. Louis, MO, USA). All reagents were dissolved

95 in dimethylsulfoxide (DMSO) and stored at -20°C.

97 Pharmacokinetic (PK) studies in cynomolgus monkeys and mice

98 All procedures were performed at NISSEI BILIS. The study was compliant with Ohara

99 Pharmaceutical policies on bioethics in animal work, and all procedures were compliant

100 with the relevant national and international guidelines for the care and use of

101 experimental animals. Procedures for PK studies are described in Supplementary

102 methods.

103

104 Cell lines and cultures

105 The MDS-L cell line was derived from the non-leukemic phase of a patient with MDS

106 with refractory anemia-ringed sideroblasts (20). SKM1 cells (derived from the blast 6

Ureshino et al. Silylation of DAC for MDS and AML.

107 cells of a patient with MDS) were purchased from the Japanese Collection of Research

108 Bioresources Cell Bank (Osaka, Japan). HL60, THP-1, KG1a, and Kasumi-1 cell lines

109 (derived from AML patients) were purchased from the American Type Culture

110 Collection (Rockville, MD, USA). AZA-resistant HL60 (HL60R) cells were established.

111 Detailed cell culture methods are described in Supplementary methods.

112

113 Primary patient samples

114 Primary samples were obtained from patients with MDS or AML. Experiments were

115 approved by the Institutional Review Board of Saga University, and all procedures

116 involving human participants were undertaken in accordance with the principles of the

117 Declaration of Helsinki.

118

119 Western blot analysis

120 Western blot analyses were performed to assess the protein levels of DNMT1, UCK1,

121 UCK2, and DCK. Procedures are described in Supplementary methods.

122

123 Pyrosequencing assay of long interspersed nucleotide element-1 (LINE-1) and the

124 globin promoter methylations

125 Pyrosequencing assays of LINE-1 methylation were performed to assess global

126 methylation (19). Procedures are described in Supplementary methods. The

127 methylation status of -globin promoter status was assessed in samples (peripheral

Ureshino et al. Silylation of DAC for MDS and AML.

128 blood) sequentially collected on day 0, 1, 5, 8, 12, 15, 19, 22, 26, and 29 after initiation

129 of 5 day duodenal administration of 10.9 μmol/kg OR21, as previously described (21).

130

131 Cell proliferation

132 Cell proliferation was evaluated by CCK-8 assay (Dojindo, Kumamoto, Japan) and

133 half-maximal inhibitory concentration values (IC50) were determined by non-linear

134 regression using the CalcuSyn software (Biosoft, Cambridge, UK). Procedures for cell

135 proliferation analyses are described in Supplementary methods.

136

137 Flow cytometry

138 Antibodies used for flow cytometry are listed in Supplementary methods. Stained

139 samples were assessed using a FACSVerse cytometer (BD Biosciences, San Jose, CA,

140 USA), and data were analyzed using the FlowJo software (Tree Star, Ashland, OR,

141 USA).

142

143 Genome-wide DNA methylation analysis

144 Genome-wide DNA methylation analysis was performed using Infinium Human

145 Methylation EPIC Bead Chips (Infinium EPIC; Illumina; San Diego, CA, USA) as

146 previously described (18). -values ranged from 0 (unmethylated) to 1 (fully

147 methylated). DNA methylation data were deposited in GEO under accession number

148 GSE148314.

149 8

Ureshino et al. Silylation of DAC for MDS and AML.

150 Gene expression microarray analyses

151 Procedures for gene expression microarray analyses are described in Supplementary

152 methods. Microarray data were deposited at GEO under accession number

153 GSE141677.

154 Mice

155 All animal experiments were approved by the Institutional Review Board of Saga

156 University and performed according to the university’s institutional guidelines. BALB/c

157 Rag-2/JAK3 double-deficient (BRJ) mice (22) were housed in a specific pathogen-free

158 barrier facility; BRJ mice were used for xenograft transplantation experiments. BRJ

159 mice received twice-weekly administration of vehicle (1% DMSO), DAC, or OR21

160 after xenotransplantation. Procedures for animal experiments are described in

161 Supplementary methods.

162

163 Statistical analysis

164 All statistical analyses were performed using the EZR software package (Saitama

165 Medical Center, Jichi Medical University) (23). P < 0.05 was considered statistically

166 significant.

167

Ureshino et al. Silylation of DAC for MDS and AML.

168 Results

169

170 5-O-trialkylsilylated DACs have appropriate log P values for oral bioavailability

171 and hypomethylating effects in the MDS cell line.

172 In this study, we developed 5-O-trialkylsilylated DACs (Figure S1A). Oral

173 absorbability is assessed according to the log P value, defined as the ratio of a

174 chemical’s concentrations in the octanol vs. aqueous phase of a two-phase octanol/water

175 system (24). The log P values of 5-O-trialkylsilylated DACs were +1.64 to +5.20; the

176 appropriate log P value for oral bioavailability is +0 to +3. Determination of LINE-1

177 methylation levels by pyrosequencing, which measures methylation (the ratio of T to

178 C) at each CpG position within a sequence after bisulfite treatment, yields a

179 representative picture of global genomic DNA methylation (25,26). Treatment with

180 100 nM OR2100 (OR21), 2003, 2008, 2009, 2102, or 2201 significantly decreased

181 LINE-1 methylation levels in MDS-L (Figure S1B). Based on these drug screening

182 experiments, we defined OR21 as a promising 5-O-trialkylsilylated DAC, as in previous

183 reports (18,19).

184

185 Structure of OR21

186 OR21, a DAC prodrug, is an oligonucleotide consisting of DAC nucleoside

187 5'-O-trisilylate, which is protected from degradation by CDA (Figure. 1A). The log P

188 value of OR21 is +2.14, indicating likely oral availability. The stability of the base

Ureshino et al. Silylation of DAC for MDS and AML.

189 moiety of OR21 is almost the same (half-life, 20 h) as that of DAC in an aqueous

190 environment (pH 6.0–7.5), and the in vitro stabilities of AZA and DAC are also similar

191 (27).

192

193 Pharmacokinetics and pharmacodynamics of OR21 in cynomolgus monkeys

194 Initially, we investigated the pharmacokinetics of OR21 in six cynomolgus monkeys.

195 Sequential plasma samples were taken from monkeys given the prodrug, and plasma

196 drug concentrations were measured over time. OR21 is easily degraded by gastric acid

197 because of its chemical structure, but direct duodenal administration of OR21 to

198 cynomolgus monkeys resulted in absorption of the drug and subsequent release of DAC.

199 After administration of 6.6 µmol/kg OR21, the area under the plasma drug

200 concentration–time curve (AUC) was 0.30 (± 0.18) µM•h, and after administration of

201 6.6 µmol/kg DAC, the AUC was 0.01 (± 0.01) µM•h (Figure 2A, B). A higher dose of

202 DAC did not result in enteric bioavailability (Figure S2). Thus, OR21 exhibited higher

203 enteric absorbability than DAC. We next evaluated the pharmacodynamics of OR21.

204 After intravenous administration (i.v.) of 10.9 µmol/kg DAC, the AUC was 0.49

205 (±0.17) µM•hr, and after duodenal administration (d.a.) of 10.9 µmol/kg OR21, the

206 AUC was 0.25 (± 0.14) µM•hr (n=4 monkeys) (Figure 2C). After 5 day intravenous

207 administration of 10.9 μmol/kg DAC and duodenal administration of 10.9 μmol/kg

208 OR21, -globin promoter methylation (average three CpG sites at -53, +6, +17) levels

209 (Cont, 88.3 ± 1.35%) was significantly reduced on day 8 (DAC, 66.5 ± 2.52%, P=0.02;

Ureshino et al. Silylation of DAC for MDS and AML.

210 OR21, 79.5±4.50%, P=0.02) (Figure 2D). In this setting, the demethylating effect of

211 DAC (i.v.) was stronger than that of OR21 (d.a.), possibly due to differences in AUC:

212 9.0 mg/kg (26.3 μmol/kg) OR21 (d.a.) achieved an AUC of 0.45 ± 0.23 µM•hr,

213 comparable to the AUC of 1.67 mg/kg DAC (i.v.) (Figure S2). These results indicated

214 that OR21 has both enteral absorbability and hypomethylating effects. We next

215 evaluated the in vitro and vivo efficacy of OR21 for AML and MDS. (At that time, the

216 appropriately-sized enteric-coated OR21 for administration in vivo was not available, as

217 the enteric-coated OR21 developed for monkeys was too large to administer to mice).

218

219 OR21 induces global DNA hypomethylation by depleting DNMT1

220 Hypomethylating agents (AZA and DAC) deplete DNMT1. Consistent with this,

221 DNMT1 levels were markedly lower in two MDS (MDS-L, SKM1) and two AML

222 (HL60, THP-1) cell lines treated with OR21 (100 or 500 nM) than in untreated cells

223 (Figure 1B and Figure S3A–C). LINE-1 methylation levels were higher in both

224 MDS-L and SKM1 cells (88.9% and 82.7%, respectively). Treatment with 100 nM

225 OR21 significantly decreased LINE-1 methylation levels in MDS-L [74.2% (P < 0.01)]

226 and SKM1 [66.9% (P = 0.01)] cells (Figure 1C); the effect was equivalent to that of

227 100 nM DAC (75.2% reduction in MDS-L cells and 66.9% in SKM1 cells). By contrast,

228 100 nM AZA did not decrease the LINE-1 methylation level in MDS-L; consistent with

229 a previous report, 100 nM of AZA had weak demethylating effects relative to DAC or

230 OR21 (19). LINE-1 regions were also hypermethylated in AML cell lines (KG1a,

Ureshino et al. Silylation of DAC for MDS and AML.

231 Kasumi-1, HL60, and THP-1); in this case, 500 nM OR21 tended to induce global

232 hypomethylation (Figure 1D). These results indicate that OR21 induces global

233 genomic DNA hypomethylation in MDS and AML cell lines by depleting DNMT1, and

234 that the effect is equivalent to that of DAC.

235

236 OR21 inhibits growth and induces apoptosis in MDS and AML cells

237 OR21 inhibited growth of both MDS and AML cells in a dose-dependent manner

238 (Figure S4A); the effect was equivalent to that of DAC. The dose-response curves and

239 IC50 values for all three agents after 72 h of treatment are shown in Table S1. OR21

240 induced apoptosis of MDS and AML cells, again in a dose-dependent manner (Figure

241 S4B). In addition, OR21 induced apoptosis of primary MDS and AML cells (Figure

242 S4C). These results indicate that OR21 exerts anti-tumor effects against MDS and AML

243 cells.

244

245 Via regional demethylation, OR21 upregulates expression of genes associated with

246 immune system processes, tumor suppression, and cell differentiation

247 Vehicle-treated HL60 and MDS-L cells shared a high proportion of hypermethylated

248 and hypomethylated sites, and OR21-treated cells had reduced hypermethylation

249 values, indicating that OR21 induced DNA demethylation (Figure 3A, B). In the

250 three cell lines (HL60, SKM1, and MDS-L), a heatmap comparing log2 fold changes in

251 gene expression following OR21 treatment revealed several enriched genes, which

252 played roles in immune system processes (e.g., PRAME and CT45A), tumor suppression 13

Ureshino et al. Silylation of DAC for MDS and AML.

253 (e.g., MT1E, F, TGF and ALOX5AP), and cell differentiation (e.g., EVI2A, B, and

254 TYROBO) (Figure 3C, Figure S5A–C). These genes were demethylated to a greater

255 extent than others (Figure 3D–F, Figure S6A, B), indicating that their expression

256 levels were regulated by promoter DNA methylation. The demethylating effect of 500

257 nM OR21 was similar to that of 500 nM AZA (Figure S7A–C). (OR21 is a prodrug of

258 DAC; thus the demethylating effect and gene expression profiles of OR21-treated cells

259 were very similar to those of DAC-treated cells, as previously described (18). AZA was

260 used as a control). MDS and AML are characterized by impaired cell differentiation of

261 HSCs and hematopoietic progenitor cells (1); therefore, induction of cell differentiation

262 is a potential therapeutic strategy for both forms of cancer.

263 OR21 induces cell differentiation via upregulation of the late cell differentiation

264 drivers CEBPE and GATA-1

265 CD11b is defined as a myeloid cell differentiation marker, whereas immature cells

266 expressed HLA-DR. Hence, we used these surface markers to assess the potential of

267 OR21 to induce cell differentiation in MDS or AML cells. OR21 upregulated

268 expression of CD11b and downregulated expression of HLA-DR in MDS-L cells

269 (Figure S8A). SKM1 and HL60 cells also exhibited upregulation of CD11b after OR21

270 treatment (Figure 4A and S8B). These cells also underwent morphological changes,

271 including a reduced nuclear–cytoplasmic ratio, larger cell size, and higher numbers of

272 multi-lobed nuclei (Figure 4B and S8C), indicating that OR21 induced cell

273 differentiation, particularly granulocytic differentiation. Hence, we used real-time

Ureshino et al. Silylation of DAC for MDS and AML.

274 quantitative reverse transcription–PCR to identify the transcription factors responsible

275 for cell differentiation. OR21 upregulated expression of the CEBPE and GATA-1

276 mRNAs in MDS-L and SKM1 cells (Figure 4C and S8D). These results indicate that

277 OR21 induces cell differentiation, possibly by upregulating expression of the late cell

278 differentiation drivers CEBPE and GATA-1.

279 OR21 prolongs survival of MDS and AML model mice

280 Next, we used a mouse xenograft model to evaluate the anti-MDS efficacy of OR21.

281 For this purpose, we intravenously injected SKM1 cells into BALB/c Rag-2/JAK3

282 double-deficient (BRJ) mice. Starting on day 7 post-transplantation, the mice were

283 treated twice weekly with vehicle OR21 (Figure 5A). On day 28, the number of human

284 CD45-positive cells in peripheral blood (PB) was lower in OR21-treated mice than in

285 vehicle-treated mice [vehicle, 6.40%; OR21, 0.31% (P = 0.0597); Figure 5B]. In

286 addition, OR21 prolonged survival [median overall survival; vehicle, 29 days; OR21,

287 NR at day 53 (P = 0.0246); Figure 5C]. All OR21-treated mice survived with

288 engraftment of SKM1 cells at day 53 (Figure S9).

289 Next, we used a mouse xenograft model to evaluate the anti-AML efficacy of

290 OR21. In these experiments, BRJ mice were injected intravenously with HL60 cells and

291 treated twice weekly with vehicle, DAC (1.0 mg/kg), or OR21(2.7 mg/kg) starting on

292 day 5 post-transplantation (Figure 5D). On day 37, the number of human

293 CD45-positive cells in PB was significantly lower in OR21-treated mice than in

294 vehicle-treated mice [vehicle, 2.96%; DAC, 1.67% (P = 0.48); OR21, 0.75% (P=0.049)], 15

Ureshino et al. Silylation of DAC for MDS and AML.

295 and the OR21-treated mice exhibited prolonged survival [median overall survival;

296 vehicle, 44 days; DAC, 46.5 days (P = 0.164); OR21, 49 days (P = 0.005); Figure 5E,

297 F]. In addition, OR21 induced significant hypomethylation of LINE-1 regions in bone

298 marrow cells [vehicle, 83.7%; DAC, 56.3% (P < 0.01%); OR21, 63.0% (P < 0.01%)].

299 OR21-treated mice did not develop anemia, whereas DAC-treated mice were anemic at

300 the time of sacrifice [hemoglobin levels: vehicle, 17.5 (± 1.20) g/dL; DAC, 15.6 (±

301 1.22) g/dL; OR21, 17.1(± 0.31) g/dL; Figure 5G, H]. OR21 (5.4 mg/kg) prolonged

302 survival, whereas a high dose of DAC (2.0 mg/kg) did not [median overall survival;

303 vehicle, 48 days; DAC, 32 days (P = 0.040); OR21, 57 days (P < 0.001); Figure 5I].

304 Notably, DAC-treated mice died earlier than those in the other groups, possibly due to

305 severe toxicity relative to the vehicle. These results suggest that high dose of OR21 was

306 safer and more efficacious than a high dose of DAC. Significant cell differentiation was

307 observed in mice exposed to late treatment (from day +28) with OR21 (vehicle, 43.3%;

308 DAC, 75.7%, p=0.229; OR21, 65.5%; P = 0.038; Figure 5J). These results confirmed

309 the anti-tumor effects of OR21 in the MDS and AML mouse models.

310

311 OR21 is effective against AZA-resistant AML cells in vitro and in vivo

312 Patients with MDS or AML who fail to respond to AZA treatment have limited

313 therapeutic options, and their prognosis is extremely poor. Hence, we investigated the

314 efficacy of OR21 against AZA-resistant AML. AZA-resistant HL60 (HL60R) cells

315 survived in the presence of 10 µM AZA. However, OR21 depleted DNMT1 protein,

Ureshino et al. Silylation of DAC for MDS and AML.

316 inhibited growth, and induced apoptosis and differentiation in these cells to a similar

317 extent as DAC and more effectively than AZA in vitro (Figure S10A–C and S11).

318 OR21 also significantly prolonged survival in the HL60R xenograft model (Figure

319 S10D, p=0.0188); by contrast, DAC-treated mice exhibited severe toxicity (Figure

320 S12A, B).

321

322 Mechanism of AZA resistance

323 To investigate the mechanism underlying AZA resistance, we compared the protein

324 levels of nucleoside-metabolizing enzymes between HL60 and HL60R. HL60R had

325 lower levels of uridine-cytidine kinase 2 (UCK2) (Figure S13A). However, UCK2

326 mRNA levels and promoter methylation in proximal regions did not differ

327 significantly between the two cell lines (Figure S13B, C). These observations

328 suggested that depletion of UCK2 was associated with AZA resistance, but that the

329 underlying mechanisms might not involve transcriptional regulation via altered

330 methylation. The majority of downregulated genes in HL60R were involved in immune

331 system processes or mitochondrial pathways (Figure S13D and Table S3). HL60

332 methylome analysis revealed that HL60R cells were much more demethylated than

333 HL60 (Figure S13E); meanwhile, the downregulated genes were less demethylated

334 than the upregulated genes (Figure S13F, G). Pathway analysis of genes upregulated

335 >2.0-fold revealed that genes associated with cell adhesion were upregulated in HL60R

336 cells (Figure S14A, B). Among them, the chemokine receptor CXCR4 (Figure S14C,

337 D) was upregulated at the mRNA and protein levels in HL60R. HL60R xenotransplant 17

Ureshino et al. Silylation of DAC for MDS and AML.

338 mice developed extramedullary disease (Figure S14E), suggesting that increases in cell

339 adhesion activity are associated with elevated malignant potential. The changes in

340 HL60R may be the consequence of transcriptional regulation via altered methylation.

341

342 The toxicity of OR21 may be lower than that of DAC, even though both drugs have

343 the same AUC

344 We monitored the long-term administration of OR21 in BALB/c mice (not

345 immunodeficient) treated with a high (2.5 mg/kg of DAC or 7.5 mg/kg of OR21) or low

346 (1.25 mg/kg of DAC or 3.7 mg/kg of OR21) dose of the indicated drug. Although the

347 plasma AUCs for high-dose DAC and OR21 were approximately equal, high-dose DAC

348 resulted in greater hematopoietic toxicity, resulting in severe anemia and early death

349 (Figure 6A, B). By contrast, mice treated with a high dose of OR21 developed anemia

350 by Day 29, but recovered by Day 57 while still on the drug (Figure 6B). In addition,

351 these mice did not die early: all were still alive after 80 days, whereas all mice receiving

352 high-dose DAC died within 80 days. Neither DAC- nor OR21-treated mice exhibited

353 signs of lethal toxicity in response to low-dose DAC or OR21; however, DAC-treated

354 mice were more likely than OR21-treated mice to develop anemia (Figure 6C).

355 Together, these results indicate that OR21 may be less toxic than DAC, resulting in

356 improved tolerability even at high doses.

357

Ureshino et al. Silylation of DAC for MDS and AML.

358 Enteric-coated OR21 exhibited favorable absorbability in an oral administration

359 mouse model

360 Finally, we developed enteric-coated OR21 with the goal of making the drug resistant to

361 gastric acid. We investigated the PK of enteric-coated OR21 in five ICR mice.

362 Sequential plasma samples were taken from mice given the prodrug, and plasma drug

363 concentrations were measured over time. Oral administration of enteric-coated OR21 to

364 mice resulted in absorption of the drug and subsequent release of DAC. After

365 administration of 5 mg of enteric-coated OR21, the AUC of DAC (released from OR21)

366 was 1.06 (± 0.28) µM/h (Figure S15). Thus, enteric-coated OR21 exhibited high oral

367 absorbability in a mouse model.

368

369 Discussion

370 The findings of this study reveal that OR21 is a promising hypomethylating agent that

371 can be administered orally and exerts anti-tumor effects against MDS and AML both in

372 vitro and in vivo. In addition, OR21 is less toxic than DAC. DAC and OR21 had similar

373 effects in vitro in terms of hypomethylation, cell growth inhibition, and apoptosis

374 induction; these effects were distinct from those of AZA. These differences are

375 consistent with previous results showing that AZA is incorporated predominantly into

376 RNA, whereas DAC is incorporated predominantly into DNA (27). Hence, the in vitro

377 activity of OR21, which was triggered by release of DAC from OR21 after removal of

378 the 5'-O-trisilylate substituent, might be due to incorporation of DNA rather than RNA.

Ureshino et al. Silylation of DAC for MDS and AML.

379 Resistance to CDA is crucial for achieving elevated plasma concentrations of

380 DNMT inhibitors. Guadecitabine, which is resistant to CDA and exerts stronger

381 hypomethylating activity than AZA (28), has a favorable survival outcome in patients

382 with MDS and AML who are not responsive to AZA (15). Nevertheless, guadecitabine

383 is not orally absorbable (29). By contrast, the recently approved agents CC-486 (13) and

384 ASTX727 (14) can be orally absorbed. Silylation of DAC confers resistance to CDA

385 (18); accordingly, OR21 was enterically bioavailable in cynomolgus monkey, and

386 enteric-coated OR21 was orally bioavailable in a mouse mice model. In addition,

387 silylation of DAC and enzymatic cleavage of silylation did not result in new toxicities

388 in vivo. It is worth noting that ASTEX727 consists of two compounds, DAC and

389 cedazuridine, a CDA inhibitor, whereas OR21 is a single compound (DAC prodrug).

390 Consequently, OR21 has enormous advantages in terms of production, i.e., it is easier

391 and cheaper to manufacture. We have now prepared gelatin capsules filled with

392 granules of enteric-coated OR21, and we are currently performing PK studies of this

393 formulation in cynomolgus monkeys to determine the dose for a first-in-human clinical

394 trial. Because formulation is a very important issue related to future development, we

395 prefer not to present detailed PK data until the first in-human clinical trial is completed.

396 The preliminary results suggest that OR21 could be used as an oral hypomethylating

397 agent.

398 OR21-induced global DNA hypomethylation in MDS and AML cells could

399 upregulate expression of silenced genes associated with tumor suppression, cell

400 differentiation, and immune system processes. Among the genes that were upregulated 20

Ureshino et al. Silylation of DAC for MDS and AML.

401 after OR21 treatment, TGF is an important tumor-suppressor gene in hematological

402 malignancies that affects immune system process and cell differentiation (30);

403 accordingly, TGF may play an important role in the anti-tumor effects of OR21.

404 Although it is unclear how immune system process pathways are involved in the

405 anti-tumor effects of OR21, these pathways were downregulated in AZA-resistant HL60

406 cells (31), suggesting that immune pathways play important roles in the anti-tumor

407 effects of hypomethylating agents used to treat AML. Moreover, upregulation of genes

408 associated with immune system processes and tumor suppression could alter

409 immunogenicity or tumor recognition, thereby sensitizing various tumors (32,33).

410 Immune checkpoint inhibitors used to treat melanoma (34), lung cancer (35), or other

411 solid tumors (36,37) also exhibit anti-tumor effects in patients with MDS or AML

412 (38,39). To optimize promising combination therapeutic strategies involving OR21,

413 subsequent administration of immune checkpoint inhibitors may improve efficacy

414 against MDS or AML.

415 AZA-resistant HL60R cells exhibited downregulation of the UCK2 protein without

416 alteration of UCK2 promoter methylation or mRNA levels (40,41). UCK2 is a pivotal

417 uridine-metabolizing enzyme (42). Furthermore, alteration of promoter methylation

418 resulted in upregulation of genes related to cell adhesion in AZA-resistant HL60R cells,

419 and OR21 overcame AZA resistance. Patients with MDS or AML who fail to respond

420 to AZA treatment have limited therapeutic options, and their prognosis is extremely

421 poor (43,44). We showed that OR21 was effective agaist AZA-resistant AML in vitro

422 and in vivo, whereas DAC exerted a high degree of toxicity in vivo. These results 21

Ureshino et al. Silylation of DAC for MDS and AML.

423 suggest that OR21 may yield clinically favorable outcomes in patients with MDS or

424 AML who do not respond to AZA.

425 Patients with MDS or AML exhibit altered HSC differentiation (1,2), and AML

426 patients with low CEBPE gene expression level had significantly worse survival

427 outcomes (Figure S16); therefore, induction of appropriate cell differentiation is a

428 potential therapeutic strategy for MDS and AML (45,46). In three different cell lines,

429 OR21 upregulated genes associated with cell differentiation, resulting in induction of

430 myeloid cell differentiation. Induction of global DNA hypomethylation triggered

431 upregulation of the silenced key differentiation drivers CEBPE and GATA-1, which are

432 inactivated in MDS and AML (47,48), thereby triggering cell differentiation (49). OR21

433 induced anti-tumor effects in vivo by promoting both hypomethylation and cell

434 differentiation. The effects were similar to those of DAC, although OR21 did not induce

435 severe toxicities in mice. Long-term administration experiments confirmed that OR21

436 was less toxic than DAC at both high and low doses. Although it is not clear why the

437 toxicity of OR21 is lower than that of DAC, one possible explanation is that OR21

438 achieves a lower peak plasma concentration: the peak plasma concentration of DAC in

439 mice was 0.43 µM after intraperitoneal administration of 1.59 µM/kg OR21, vs. 0.64

440 µM after administration of 0.88 µM/kg DAC (calculated as an AUC equivalent to that

441 of released DAC, which had an AUC of 0.38 µM/h, Table S4) (18).

442 Although the safety of cleaved sialylated DAC has not been fully elucidated, we

443 confirmed that OR21 is less toxic, possibly due to differences in peak plasma

444 concentration and AUC profiles relative to DAC administration. Meanwhile, the 22

Ureshino et al. Silylation of DAC for MDS and AML.

445 efficacy of OR21 is mainly due to the release of DAC from OR21 after removal of the

446 5'-O-trisilylate substituent. Therefore, OR21 represents an orally bioavailable drug with

447 anti-tumor effects against MDS and AML similar to those of DAC, and is therefore a

448 potentially suitable treatment for patients with MDS and AML (10,50).

449 In conclusion, silylation of deoxynucleotide analog can provide oral bioavailability

450 without new toxicities. OR21 exhibits high oral availability and exerts strong anti-tumor

451 effects against MDS and AML both in vitro and in vivo; moreover, it is less toxic than

452 DAC. We anticipate that the efficacy and safety of OR21 for treatment of MDS and

453 AML will be verified in planned early-phase clinical trials.

454

455 Contributors

456 HU, YK, YS, KK, and YY collected and analyzed the data, performed the experiments,

457 and wrote the manuscript. TW, Y F-K, NY, NH, HH, KT, HH, SO, TU, and SK

458 contributed to the research design and edited the manuscript. AK performed statistical

459 analysis. TU and SK made substantial contributions to the study conception and design.

460 HU and SK critically reviewed the drafts. All authors approved the final version.

461

462 Conflict-of-interest disclosure: This work was supported by Ohara Pharmaceutical.

463

464 Acknowledgments: none

465

Ureshino et al. Silylation of DAC for MDS and AML.

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Ureshino et al. Silylation of DAC for MDS and AML.

606 Figure legends

607

608 Figure 1. Structure of OR21 and its effects on methylation.

609 Structure of 5’-O-triethylsilyl-2’-deoxy-5-azacytidine (OR2100, OR21) and decitabine

610 (DAC) (A). Agents with a log P value of +1.0 – +5.9 are expected to be absorbed after

611 oral administration. Analysis of protein expression in MDS-L and HL60 cells treated

612 with azacytidine (AZA), DAC, or OR21 (B). LINE-1 methylation analysis in MDS-L

613 and SKM1 cells, as determined by bisulfite pyrosequencing (C). MDS-L and SKM1

614 cells were strongly hypermethylated (88.9% and 82.7%, respectively). Treatment with

615 100 nM DAC or OR21 significantly decreased LINE-1 methylation levels in MDS-L

616 [AZA, 88.8% (P = 0.867); DAC, 75.2% (P < 0.01); OR21, 74.2% (P < 0.01)] and

617 SKM1 cells [AZA, 72.1% (P < 0.01); DAC, 58.4% (P < 0.01); OR21, 66.9% (P=0.01)]

618 (mean ± SD; n = 3). LINE-1 methylation analysis in AML cell lines treated with 500

619 nM OR21. AML cell lines were also hypermethylated (KG1a, 89.1%; Kasumi-1,

620 90.7%; HL60, 83.7; THP-1, 69.0%) (D). Treatment with OR21 tended to induce

621 hypomethylation [KG1a, 82.4% (P=0.091); Kasumi-1, 78.6% (P = 0.128); HL60, 76.0%

622 (P = 0.089); THP-1, 64.6% (P = 0.243)] (mean ± SD; n = 3).

623 Figure 2. Pharmacokinetics and pharmacodynamics of OR21

624 Sequential plasma samples were taken from monkeys given the prodrug, and plasma

625 drug concentrations were measured over time. After administration of 6.6 µmol/kg

626 OR21, the area under the plasma drug concentration–time curve (AUC) was 0.30 (±

Ureshino et al. Silylation of DAC for MDS and AML.

627 0.18) µM•h (A). After administration of 6.6 µmol/kg DAC, the AUC was 0.01 (± 0.01)

628 µM•h (B). After administration of 10.9 µmol/kg DAC, the AUC was 0.49 (±0.17) µM•

629 hr and after administration of 10.9 µmol/kg OR21, the AUC was 0.25 (± 0.14) µM•hr

630 (C). After 5 day intravenous administration (iv) of 10.9 μmol/kg DAC and duodenal

631 administration (d.a.) of 10.9 μmol/kg OR21, the γ-globin promoter methylation

632 (average three CpG sites at -53, +6, and +17) levels (Cont, 88.3±1.35%) were

633 significantly reduced at day 8 (DAC, 66.5 ± 2.52%, P=0.02; OR21, 79.5±4.50%,

634 P=0.02) (D). *P < 0.05.

635

636 Figure 3. Genes differentially expressed after OR21 treatment due to alteration of

637 promoter DNA methylation.

638 Methylome analysis revealed that OR21-treated HL60 and MDS-L cells had reduced

639 hypermethylation  values (A, B). Heatmap comparing log2 fold changes in gene

640 expression among three cell lines (HL60, SKM1, and MDS-L) following treatment with

641 OR21 (C). Analysis revealed enrichment of genes involved in immune system processes

642 (including cancer testis antigen, e.g., PRAME, TGF, and CT45A), tumor suppression

643 (e.g., MT1E, F, TGF, and ALOX5AP), and cell differentiation (e.g., EVI2A, B, TGF

644 and TYROBO). (D, E) Each of these genes were demethylated after OR21 treatment (D).

645 The -values of each gene decreased (paired t-test, E) **P < 0.01. The delta -values

646 (OR21-treated minus vehicle-treated) of HL60 and MDS-L cells were below zero (F).

647

648 Figure 4. OR21 induces cell differentiation in MDS and AML cells. 30

Ureshino et al. Silylation of DAC for MDS and AML.

649 Flow cytometry revealed elevated expression of CD11b (granulocytic differentiation

650 marker) in MDS and SKM1 cell lines (MDS-L) following 96 h treatment with 100 nM

651 azacytidine (AZA), decitabine (DAC), or OR21 (mean ± SD; n = 3) (A). OR21 induced

652 morphologic changes consistent with granulocytic differentiation (i.e., reduced nuclear–

653 cytoplasmic ratio, larger cell size, and higher numbers of multi-lobed nuclei) in MDS-L

654 and SKM1 cells. May–Giemsa staining of cytospin preparations at 96 h is shown. (B)

655 Levels of CEBPA, CEBPB, PU.1, GATA1, and CEBPE mRNAs were measured by

656 quantitative reverse transcription–PCR. Upregulation of CEBPE and GATA-1 was

657 observed in MDS-L and SKM1 cells treated with DAC or OR21 (mean ± SD; n = 3) (C).

658 *P < 0.05, **P < 0.01.

659

660 Figure 5. SKM1 and HL60 xenograft mouse model experiments.

661 Schedule of SKM1 cell xenograft experiments using BALB/c Rag-2/JAK3

662 double-deficient (BRJ) mice (A). Day 28: flow cytometry of hCD45-positive cells in

663 mice treated with vehicle or OR21 (B). Kaplan–Meier survival curves showing

664 cumulative survival of mice treated with vehicle or OR21 (n = 3 per cohort) (C).

665 Median overall survival time was 29 days (vehicle) (P=0.0246); NR = not reached.

666 Statistical analysis was performed using the log-rank test. Schedule of HL60 cell

667 xenograft experiments using BRJ mice (D). Day 37: flow cytometry of hCD45-positive

668 cells in mice treated with vehicle (n = 5), decitabine (DAC) (n = 4), or OR21 (n = 4) (E).

669 Kaplan–Meier survival curves showing cumulative survival of mice treated with vehicle,

670 DAC, or OR21 (F). Median overall survival times were 44 days (vehicle) (n = 10), 46.5 31

Ureshino et al. Silylation of DAC for MDS and AML.

671 days (DAC, n = 8, P=0.164), and 49 days (OR21, n = 10, P=0.005). Statistical analysis

672 was performed using the log-rank test. DAC-treated mice were more likely to develop

673 anemia than OR21-treated mice (G). Hemoglobin (Hb) levels were 17.5 g/dL (vehicle, n

674 = 8), 15.6 g/dL (DAC, n = 6, P = 0.092), and 17.1 g/dL (OR21, n = 7, P = 1.0). LINE-1

675 methylation in bone marrow cells decreased after treatment with DAC or OR21 (n = 6

676 per cohort) (H). Mice treated with a higher dose of DAC (2.0 mg/kg) did not live longer,

677 whereas survival was prolonged in mice treated with OR21 (5.4 mg/kg) [median overall

678 survival; vehicle, 48 days; DAC, 32 days (P = 0.040); OR21, 57 days (P < 0.001)] (I).

679 HL60 xenograft mice exposed to late treatment with OR21 (Day +28) had more

680 CD11b-positive cells in peripheral blood (PB) on Days +35 to +40, as determined by

681 flow cytometry (n = 4 per cohort) (vehicle, 43.3%; DAC, 75.7%, p=0.229; OR21,

682 65.5%; P = 0.038) (J).

683

684 Figure 6. In vivo safety profile of OR21.

685 (A) Kaplan–Meier survival curves showing cumulative survival of mice treated with

686 vehicle, decitabine (DAC; 2.5 mg/kg), or OR21 (7.5 mg/kg) (n = 6 per cohort). (B)

687 DAC (2.5 mg/kg) exerted high levels of toxicity, causing severe anemia by Days +29

688 and +57. Mice treated with OR21 (7.5 mg/kg) also developed anemia by Day +29, but

689 recovered by Day +57 while still on the drug (n = 6 per cohort). (C) Among mice

690 exposed to low-dose DAC (1.25 mg/kg) or OR21 (3.75 mg/kg) until Day +113, those

691 exposed to DAC were more likely to develop anemia (n = 6 per cohort). WBC, white

692 blood cell count; Hb, hemoglobin; platelet count, PLT. 32

Figure 1 Cont AZA DAC *OR12 OR21 (100 nM) 5’-O-Triethylsilyl-2’-deoxy-5-azacytidine Decitabine B A (OR2100, OR21) DNMT1

β-actin MDS-L

Cont AZA DAC OR21 (500 nM)

DNMT1 HL60 β-actin Log P = + 2.14 Log P = - 0.32

P=0.091 P=0.006 D P=0.128 C P=0.026 P=0.01 90.0

P=0.089 P=0.867 P<0.01 80.0 P<0.01 P=0.243 80.0 80.0 70.0 methylation (%) methylation

methylation (%) methylation 60.0 60.0 60.0 LINE-1 50.0 LINE-1 40.0 40.0 (500 nM) Cont AZA DAC OR21 Cont AZA DAC OR21 (100 nM) (100 nM) Downloaded from mct.aacrjournals.orgMDS-L on September 24, 2021. © 2021 AmericanSKM1 Association for Cancer KG1a Kasumi-1 HL60 THP-1 Research. Author Manuscript Published OnlineFirst on May 27, 2021; DOI: 10.1158/1535-7163.MCT-20-1125 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Figure 2 A B OR21 2.25 mg/kg (6.6 µmol/kg) DAC 1.0 mg/kg (6.6 µmol/kg)

0.600 0.600 (µM) (µM)

0.400 0.400

AUC 0.30±0.18 uM･hr concentration 0.200 concentration 0.200

AUC 0.01±0.01 uM･hr Plasma 0.000 Plasma 0.000 0 1 2 3 0 1 2 3 OR21 or DAC OR21 3.75 mg/kg d.a. C D 90.0 DAC and OR21 10.9 µmol/kg

) 0.800

μM 80.0

0.600 DAC 1.67 mg/kg i.v. * AUC 0.49±0.17 uM･hr 70.0 * DAC 1.67 mg/kg i.v. 0.400 OR21 3.75 mg/kg d.a. AUC 0.25±0.14 uM･hr 60.0 0.200 HgF CpG Methylation (%) CpG Methylation HgF Plasma concentration ( concentration Plasma 0.000 50.0 0 10 20 30 0 1 2 3 Time (hr) Day Downloaded from mct.aacrjournals.org on September 24, 2021. © 2021 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 27, 2021; DOI: 10.1158/1535-7163.MCT-20-1125 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Figure 3 HL60 B

A MDS-L

Control Control

OR-treated OR-treated Gene frequency Gene Gene frequency Gene

β value C β value Cancer antigen HL60 HL60 MDS-L 1 D 1 ** 1 **

0.75 0.75

Cont

L 0.5 0.5 0.5 value value HL60 Cont HL60 SKM1 Cont SKM1 HL60 OR21 HL60 MDS- β MDS-LOR21 SKM1 OR21 SKM1

β 0.25 0.25

0 0 0 Cont OR21 Cont OR21

MDS-L F 0.00 1

-0.05

value

0.5 -0.10 value delta β delta

β -0.15

0 HL60 MDS-L Downloaded from mct.aacrjournals.org on September 24, 2021. © 2021 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 27, 2021; DOI: 10.1158/1535-7163.MCT-20-1125 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Figure 4 MDSL SKM1 A MDSL SKM1 B 80 ** 40 **

60 30 ** ** ** Cont Cont 20 40

%CD11b 10

0 0

OR21 OR21 MDSL SKM1 C Cont AZA DAC OR21 Cont AZA DAC OR21

0.88 1.08 1.44 0.99 0.79 0.94 CEBPA 1.00 CEBPA 1.00 (±0.21) (±0.67) (±0.34) (±0.42) (±0.33) (±0.46)

1.08 0.86 1.20 0.95 2.32 1.31 CEBPB 1.00 CEBPB 1.00 (±1.00) (±0.79) (±0.89) (±0.40) (±1.21) (±0.58)

0.94 0.82 1.24 1.02 0.72 0.62 PU.1 1.00 PU.1 1.00 (±0.02) (±0.21) (±0.44) (±0.25) (±0.09) (±0.17)

1.58 3.04 5.10 3.99 118.73 42.31 GATA1 1.00 GATA1 1.00 (±0.47) (±1.90) (±3.32) (±1.89) (±70.18) (±15.80)

4.74 6.81 6.27 1.74 2.80 2.82 CEBPE 1.00 CEBPE 1.00 (±3.38) (±4.21) (±1.73) (±0.82) (±1.18) (±1.42)

8 80

2 )

SKM1 5×106 Vehicle 10 18

)

( a **

cells injection OR21 2.7 mg/kg ip start l d

% 17 60 (

( 60

a L

8 *

/ a

16 v

6 u

v n

b 40 r

i 40 S

15 H

u OR21 (n=3) 5

days S DAC (n=6) 0 7 4 4 14 D Vehicle (n=3) 20 20 OR21(n=6)

C 2 h

13 P=0.0246 Vehicle (n=6) % 0 0 12 0 t n 1 o 2 0 10 20 30 53 Vehicle DAC OR21 0 30 40 50 60 C R Author Manuscript Published OnlineFirst on May 27, 2021; DOI: 10.1158/1535-7163.MCT-20-1125 O days FigureAuthor manuscripts 5 F ihaveg ubeenr peere reviewed5 and accepted for publication but have not yet been edited. days Figure 5 P=1.0 D P=1.0 E F P=0.038 G Vehicle P=0.229 B C G HL60I 5×106 I 100 P=0.049 100 P<0.01 A A B A C B 100 C D 100 PD=0A.C0 912.0 mg/kg E H 85 P<0.01 90 100 100 P=0.092 cells injectioVehiclen n.s. J HL60 5×106 OR21 2.7 mg/kg ip start P=0.0597 DAC 1.0 mg/kg 80 P=0.0597 12 P=0.0597 80 12 80

) cells injection19

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) 19 OR21 2.7 mg/kg ip start 80

80 )

8 80 80 8

80 n

7 2

6 )

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P=0.0246 % 50 % % 0 0 0 12 0 50 0 0 0 0 12 0 0 t 1 0 t n 2 n 1 o 0 10 20 30 53 Vehicle DAC OR21 0 30 40 50 60 o 2 0 C 35V0 Rehic40le1 0 D45AC2 0 50O 3R02551 53 0 35 VVe4eh0hiciclele 45DDAACC50 OO5RR52211 0 V3e5hicle40DA0C45 OR502130 55 40 50 VeVhe6ich0liecle DDAACC OORR211 Vehicle DAC OR21 C R O days days O days days days days days Downloaded from mct.aacrjournals.org on September 24, 2021. © 2021 American Association for Cancer days D E Research. F P=0.038 D E Vehicle F P=0.038 P=0.229 Vehicle HL60 5×106 P=0.049 100 P<0.01 P=0.229 × 6 DAC 1.0 mg/kg H 85 90 HL60 5 10 DAC 1.0 mg/kg P=0.049 100 H 85 P<0.01 P<0.9001 J cells injection OR21 2.7 mg/kg ip start n.s. P<0.01 J cells injection OR21 2.7 mg/kg ip start n.s. 80 80

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% 0 50 0 0 0 Vehicle DAC OR21 0 35 40 45 50 55 Vehicle DAC OR21 Vehicle DAC OR21 Vehicle DAC OR21 0 35 40 45 50 55 Vehicle DAC OR21 Vehicle DAC OR21 days days Author Manuscript Published OnlineFirst on May 27, 2021; DOI: 10.1158/1535-7163.MCT-20-1125 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Figure 6 Day 29 Day 57 P = 0.093 P < 0.01 A 100 B P < 0.01 P=0.317 P < 0.01 P = 0.293 80 17.5 17.5 P=0.108 15.0 15.0 (%)

60 12.5 12.5

40 g/dL 10.0 10.0 Vehicle Survival

P<0.001 Hb 7.5 7.5 20 OR21 7.5 mg/kg DAC 2.5 mg/kg 5.0 5.0

0 2.5 2.5 Vehicle DAC OR21 Vehicle DAC OR21 0.0 1.0 2.0 3.0 2.5 7.5 2.5 7.5 months

C Day 113 (DAC 1.25 mg/kg, OR21 3.75 mg/kg) P=0.353 P=0.792 P=0.876 P=0.119 P=0.565 P=0.924 P=0.794 80.0 17.0 P=0.074 125.0 P=0.070

60.0 100.0 μL μL

/ / 4

2 15.0 75.0 10 10 g/dL

40.0 × ×

Hb 50.0 13.0 PLT PLT

WBC 20.0 25.0

0.0 11.0 0.0 Vehicle DAC OR21 Vehicle DAC OR21 Vehicle DAC OR21

Silylation of deoxynucleotide analog yields an orally available drug with anti-leukemia effects

Hiroshi Ureshino, Yuki Kurahashi, Tatsuro Watanabe, et al.

Mol Cancer Ther Published OnlineFirst May 27, 2021.

Updated version Access the most recent version of this article at: doi:10.1158/1535-7163.MCT-20-1125

Supplementary Access the most recent supplemental material at: Material http://mct.aacrjournals.org/content/suppl/2021/05/27/1535-7163.MCT-20-1125.DC1

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet Manuscript been edited.

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