Activation of the Lysosome-Associated Membrane Protein LAMP5 by DOT1L

Author Manuscript Published OnlineFirst on January 16, 2019; DOI: 10.1158/1078-0432.CCR-18-1474 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

1 Title: Activation of the lysosome-associated membrane protein LAMP5 by DOT1L

2 serves as a bodyguard for MLL fusion oncoproteins to evade degradation in leukemia

4 Authors: Wen-Tao Wang1*, Cai Han1*, Yu-Meng Sun1*, Zhen-Hua Chen1, Ke Fang1,

5 Wei Huang1, Lin-Yu Sun1, Zhan-Cheng Zeng1, Xue-Qun Luo2, Yue-Qin Chen1#

6 *WTW, CH and YMS equally contributed to the study

8 Affiliation:

9 1. MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory for

10 Biocontrol, Sun Yat-sen University, Guangzhou 510275, China

11 2. The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, 510080, China

13 Running Title: LAMP5 regulates MLL fusion protein degradation

15 Keywords: MLL leukemia, LAMP5, DOT1L, autophagic degradation，survival

16 #Corresponding authors:

17 School of Life Science, Sun Yat-sen University, Guangzhou 510275, P. R. China

18 Phone: 86-20-84112739 Fax: 86-20-84036551

19 E-mail: [email protected] to Y.-Q. Chen

20 Conflict of interests

21 The authors have declared that no conflict of interest exists. 1

Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on January 16, 2019; DOI: 10.1158/1078-0432.CCR-18-1474 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

22 Statement of translational relevance

23 Due to the development of resistance and high relapse rates with currently available

24 therapies, mixed-lineage leukemia (MLL leukemia) is frequently associated with a

25 poor prognosis; therefore, novel therapeutic targets are urgently needed. Herein, we

26 demonstrate, for the first time, that suppressing the novel autophagic suppressor

27 LAMP5 promotes the autophagic degradation of MLL fusion proteins and inhibits

28 MLL leukemia progression in both an animal model and primary cells. Notably,

29 suppressing LAMP5 expression effectively released MLL fusions from its defense

30 system and extended survival in vivo. This effect was particularly strong when

31 combining DOT1L inhibitors and LAMP5 knockdown. The finding highlights the

32 potential of LAMP5 as a target in the treatment of this disease.

41 2

42 Abstract

43 Purpose: Despite many attempts to understand mixed-lineage leukemia (MLL

44 leukemia), effective therapies for this disease remain limited. We identified a

45 lysosome-associated membrane protein (LAMP) family member, LAMP5, that is

46 specifically and highly expressed in MLL leukemia patients. The purpose of the study

47 was to demonstrate the functional relevance and clinical value of LAMP5 in the

48 disease.

49 Experimental Design: We first recruited a large cohort of leukemia patients to

50 validate LAMP5 expression and evaluate its clinical value. We then performed in vitro

51 and in vivo experiments to investigate the functional relevance of LAMP5 in MLL

52 leukemia progression or maintenance.

53 Results: LAMP5 was validated as being specifically and highly expressed in MLL

54 leukemia patients and was associated with a poor outcome. Functional studies showed

55 that LAMP5 is a novel autophagic suppressor and protects MLL fusion proteins from

56 autophagic degradation. Specifically targeting LAMP5 significantly promoted

57 degradation of MLL fusion proteins and inhibited MLL leukemia progression in both

58 an animal model and primary cells. We further revealed that LAMP5 is a direct target

59 of the H3K79 histone methyltransferase DOT1L. Downregulating LAMP5 with a

60 DOT1L inhibitor enhanced the selective autophagic degradation of MLL oncoproteins

61 and extended survival in vivo; this observation was especially significant when

62 combining DOT1L inhibitors with LAMP5 knockdown.

63 Conclusion: This study demonstrates that LAMP5 serves as a “bodyguard” for MLL

64 fusions to evade degradation and is the first to link H3K79 methylation to autophagy

65 regulation, highlighting the potential of LAMP5 as a therapeutic target for MLL

66 leukemia.

80 Introduction

81 Mixed-lineage leukemia (MLL leukemia) is an extremely aggressive blood

82 malignancy with unique biological and clinical characteristics. The primary genetic

83 lesions in MLL leukemia are chromosomal translocations of MLL that fuse in-frame

84 with more than 70 different partners, accounting for up to 80% of infant leukemia

85 cases and approximately 5%-10% of adult leukemia cases (1, 2). Owing to the

86 development of resistance and high relapse rates with currently available therapies,

87 MLL leukemia is frequently associated with a poor prognosis; therefore, novel

88 therapeutic targets are urgently needed (3, 4). Recently, targeting the MLL degradation

89 pathway has been shown to be a promising approach to treat MLL leukemia.

90 Stabilization of wild-type MLL by the E2/E3 ligase UBE2O was reported to

91 outcompete MLL chimeras and deregulate MLL chimera target gene expression,

92 indicating that MLL fusion proteins are genuine therapeutic targets (5). However, in

93 contrast to its wild-type counterpart, MLL fusion proteins appear to be resistant to

94 ubiquitin-proteasome system (UPS) degradation because of their domain deficiency

95 or possibly because of the degradation resistance conferred by the fusion partner (5-7).

96 As the resistance of MLL fusions to degradation likely contributes to the first

97 universal insult by all MLL chimeras (7), a better understanding of the stabilization of

98 mutant MLLs is an important endeavor. Although the genetic depletion of MLL fusion

99 proteins (MLL-AF4 and MLL-AF9) has been shown to exert tumor-suppressive

100 effects (8), methods of targeting the degradation pathway of MLL chimeras remain

101 unknown.

102 In addition to ubiquitin-proteasome system (UPS)-mediating degradation of

103 proteins, autophagy is widely regarded as another major degradation pathway that

104 participates in the degradation of PML-RARA and BCR-ABL fusion proteins (9, 10).

105 However, no report to date has linked autophagy to the degradation of MLL chimeras.

106 Recently, Watson and colleagues proposed that reduced autophagy with ATG5

107 depletion accelerates the development of AML with MLL-ENL in a mouse model (11),

108 supporting an anti-leukemic effect of autophagy for MLL leukemia. We previously

109 investigated epigenetic targets of noncoding RNAs in MLL leukemia (12) and found a

110 lysosome-associated membrane protein (LAMP) family member, LAMP5, to be

111 specifically and highly expressed in MLL leukemia patients. As two LAMP family

112 members, LAMP1 and LAMP2, have been reported to act as enhancers in autophagy

113 pathways (13, 14), we sought to determine whether LAMP5 is also involved in

114 autophagy and whether LAMP5 links autophagy to the degradation of MLL chimeras.

115 More importantly, LAMP5 expression is restricted to non-activated plasmacytoid

116 dendritic cells (pDCs) (15), and we speculated whether the exclusive expression

117 pattern of LAMP5 in MLL leukemia might serve as a target in treatment of the

118 disease.

119 Here, we show that LAMP5 is a direct target of the H3K79 methyltransferase

120 DOT1L and that its protein product acts as a novel autophagy suppressor associated

121 with MLL leukemia progression. We reveal that LAMP5 is specifically activated by

122 DOT1L to protect MLL fusion oncoproteins from selective autophagic degradation.

123

124 Materials and Methods

125 Patient samples

126 Bone marrow samples were obtained from 216 patients at the time of initial

127 diagnosis, including 53 MLL leukemia and 163 MLL-wt leukemia patients. All

128 samples were obtained with informed consent from the first Affiliated Hospital of Sun

129 Yat-sen University. Sample collection was approved by the Hospital’s Protection of

130 Human Subjects Committee. The detailed clinicopathological characteristics of the

131 patients are summarized in Supplementary Table S1.

132

133 Statistical analysis

134 Fisher’s exact test was used to determine the significance of differentially

135 expressed mRNA levels between the two groups. Data are expressed as the mean ±

136 SEM of three independent experiments. One-way ANOVA was performed to compare

137 multiple groups, and the Tukey's Multiple Comparison Test was used to analyze

138 multiple comparisons. ROC curves were used to determine the diagnostic utility of

139 LAMP5 mRNA. The sensitivity and specificity were identified at the optimal cutoff

140 point that was chosen at which the youden’s index was maximal. The probability of

141 leukemia-free survival at 5 years was the study end-point. Leukemia-free survival was

142 calculated from the date of CR until either relapse or death in remission.

143 Leukemia-free survival was analyzed using the Kaplan-Meier method with a log-rank

144 test. Two-tailed tests were used for univariate comparisons. For univariate and

145 multivariate analysis of prognostic factors, a Cox proportional hazard regression

146 model was used. p<0.05 was considered statistically significant.

147

148 Cell culture

149 Human THP1 and MOLM13 cells (ATCC, USA) were cultured in RPMI-1640

150 medium (HyClone, USA), MV4-11 cells (ATCC, USA) were cultured in IMDM

151 (HyClone, USA), and 293T (ATCC) were cultured in DMEM (Gibco, USA)

152 supplemented with 10% fetal bovine serum (HyClone, USA) at 37°C in a 5% CO2

153 atmosphere. The primary cells were from the patients with MLL fusion leukemia and

154 cultured in IMDM (HyClone, USA) supplemented with 10% FBS.

155

156 RNA isolation and quantitative real-time PCR (RT-PCR)

157 Total RNA was extracted from bone marrow and cell samples using an

158 Invitrogen™ TRIzol™ Kit (Thermo Fisher, USA) according to the manufacturer’s

159 instructions. All RNA samples were stored at -80°C before reverse transcription and

160 quantitative RT-PCR. RNA was reverse-transcribed into cDNA with the PrimeScript®

161 RT reagent Kit with gDNA Eraser (Takara, Japan). Quantitative RT-PCR for mRNA

162 was performed using the SYBR Premix ExTaq real-time PCR Kit (Takara, Japan)

163 according to the manufacturer’s instructions. All of the data were normalized to

164 GAPDH expression as a control. The expression level for each mRNA was

165 determined using the 2-△△Ct method. All primers were confirmed by sequencing the

166 PCR product fragments, as shown in Supplementary Table S2.

167

168 Protein extraction

169 Total protein was extracted from cells using RIPA lysis buffer (Beyotime

170 Biotechnology, China) with 1× complete ULTRA protease inhibitor (Roche, USA);

171 protein was extracted from bone marrow samples using an Invitrogen™ TRIzol™ Kit

172 (Thermo Fisher, USA) with the Thermo Scientific™ Halt™ Protease Inhibitor

173 Cocktail (Thermo Fisher, USA) according to the manufacturer’s instructions.

174

175 Flow cytometric analysis and cell proliferation CCK-8 assays

176 Cells were stained with Annexin V/FITC and propidium iodide (PI) (Lianke,

177 China) and then analyzed by flow cytometry (BD, USA) according to the

178 manufacturer’s guidelines. Cell proliferation was measured using the Cell Counting

179 Kit-8 (CCK-8, Dojindo Molecular Technologies, China). For the CCK-8 assay, cells

180 transfected with siRNAs were seeded at a density of 20,000 cells per well in 100 ml

181 of complete medium in 96-well plates. Absorbance was measured on a VICTOR™

182 X5 Multilabel Plate Reader (PerkinElmer, USA) at wavelengths of 480 and 630 nm at

183 0, 24, 48, 72 and 96 h time points.

184

185 RNA interference and plasmid construction

186 RNA interference using siRNA (Ribobio, China) was performed to knockdown

187 LAMP5, DOT1L, etc. Lentiviral shRNA templates were cloned into the

188 pGreenPuro™ shRNA Cloning and Expression Vector (System Biosciences,

189 Germany), and pSIH1-H1-siLuc-copGFP was used as negative control. Recombinant

190 vectors encoding human DOT1L (NM_032482.2), and P62/ SQSTM1

191 (NM_001142298) were constructed by PCR-based amplification from the cDNA of

192 human THP1 cells and then subcloned into the pCDH-CMV-MCS-EF1-Puro-copGFP

193 eukaryotic expression vector (Addgene, USA). PMIG- FLAG-MLL-AF9 (N- terminal)

194 was purchased from Addgene. All constructs were confirmed by DNA sequencing.

195 The primers and siRNA/shRNA sequences are listed in Supplementary Table S3.

196

197 Animal model

198 Five-week-old male NOD-SCID mice were maintained under specific

199 pathogen-free conditions in the Laboratory Animal Center of Sun Yat-sen University.

200 All experimental procedures were performed according to the institutional ethical

201 guidelines for animal experiments. Mice were randomly assigned to five groups of ten

202 mice each. In each group, lentiviral stably transduced sh-NC, sh-LAMP5-1,

203 sh-LAMP5-2 cells (4×106) were subcutaneously injected into the dorsal right flanks

204 of the mice, and the mice were monitored for 2 days each for tumor growth(16-18).

205

206 The NOD-scid-IL2Rg-/- (NSI) mice were intravenously (tail vein) implanted by

207 sh-RNA established MOLM13 cells. NSI mice are TALEN-mediated gene targeting in

208 the NOD background. Male NSI mice age 6 weeks and sub-lethally (1Gy) irradiated

209 were used. Tail vein injection of 5×106 shRNA lentivirus infected MOLM13 cells in

210 150 μL PBS was performed to generate xenograft leukemia. The xenografted mice

211 were randomized into different groups, and 8 mice in each. For the control, 150 μL of

212 PBS without cells was injected. We killed three mice in each group after two weeks.

213 Subsequently, the peripheral blood mononuclear cell (PBMC), spleen and BM from

214 xenograft mice were treated with a red blood cell lysis buffer (Biolegend, USA). Flow

215 cytometry for the GFP+ MOLM13 cells was performed on a C6 cytometer (BD, USA)

216 and analyzed using FlowJo software. The remaining 5 mice were performed the

217 survival assay.

218 Animal Treatment

219 5×106 sh-NC and sh-LAMP5 MV4-11 cells were firstly injected into the mice.

220 Ten days after transplantation, mice were randomized and treated with 40 mg/kg

221 EPZ5676, or vehicle (2% DMSO + 30% PEG 300 + 5% Tween 80 + 63% PBS)

222 every other day for 10 days(19, 20). At day 35, three mice in each group were

223 randomly killed and checked the infiltration degree of MV4-11 by Wright-Giemsa

224 staining and flow cytometry.

225

226 Cell transfection and lentivector expression systems

227 Transient transfections of recombinant vectors were performed using the

228 Lipofectamine 2000/3000 (Invitrogen, USA) system, and transient transfections of

229 siRNAs was performed using the Neon Transfection System (Invitrogen, USA) with

230 10 μl reactions according to the manufacturer’s guidelines. For stable expression

231 assays, lentiviral expression vectors, including pGreenPuro™ shRNA and

232 recombinant pCDH-CMV-MCS-EF1-Puro-copGFP eukaryotic expression vector,

233 were packaged into lentiviruses using Lentivector Expression Systems (System

234 Biosciences, Germany) consisting of pPACKH1-GAG, pPACKH1-REV, and pVSV-G.

235 Finally, the lentiviruses were transformed into THP1, MV4-11 and MOLM13 cells,

236 and the transformed cells were then selected with puromycin.

237

238 Immunoblotting and immunoprecipitation

239 For IP assays, FLAG- or HA-tagged fusion proteins were used. In the Co-IP

240 experiment with MLL-AF9-FLAG (N- terminal) and P62-HA (C- terminal) in 293T

241 cells, we used the FLAG® Immunoprecipitation Kit and EZview™ Red Anti-HA

242 Affinity Gel (Sigma, USA) and then used the FLAG and Influenza Hemagglutinin

243 (HA) peptides (Sigma, USA) to elute the proteins of interest. In the Co-IP experiment

244 with MLL-AF9 and P62-HA in P62-HA-expressing THP1 cells, anti-HA (Sigma,

245 USA), anti-mouse IgG (Thermo, USA), and MagnaBind Protein G Beads (Thermo,

246 USA) were used, and the samples were boiled after vigorous washing for three times.

247 All proteins for IP were lysed with cell lysis buffer supplemented with Thermo

248 Scientific™ Halt™ Protease Inhibitor Cocktail (Thermo Fisher, USA). Finally, all

249 samples were suspended in 5x loading buffer and then denatured for 5 min at 100°C,

250 separated via SDS-PAGE, transferred to PVDF membranes, and blotted. All

251 antibodies used in this work are listed in Supplementary Table S4.

252

253 Chromatin immunoprecipitation

254 ChIP analyses were performed on chromatin extracts from THP1 and MOLM13

255 cells using a Magna ChIP™ G - Chromatin Immunoprecipitation Kit (17-611) (Merck

256 Millipore, Germany) with trimethyl-Histone H3 (Lys79), dimethyl-Histone H3

257 (Lys79), and FLAG-MLL-AF9 according to the manufacturer’s standard protocol. In

258 this assay, samples incubated with Rabbit IgG served as the negative control. The

259 fold-enrichment of H3K79me2/me3 was quantified by quantitative RT-PCR and

260 calculated relative to the input chromatin.

261 Immunofluorescence and transmission electron microscopy

262 TEM images of autophagosomes were obtained from thin sections using a

263 JEM1400 electron microscope (JEOL, Japan). For Immunofluorescence studies, after

264 washing 2 times with PBS, cells were fixed for 10 min in 4% formaldehyde on the

265 slide and then permeably treated by 1% Triton X-100 for 10 min and followed by 1%

266 BSA, PBS pH 7.5 blocking for 30 min at room temperature. Subsequently, the cells

267 were incubated with primary and second antibodies successively. The autophagy

268 fluorescence signals were obtained using an anti-LC3B antibody (Sigma, USA)

269 analyzed by Zeiss7 DUO NLO confocal laser microscope (Carl Zeiss, Germany).

270

271

272 Results

273 LAMP5 is highly expressed in MLL leukemia patients and is associated with a

274 poor outcome

275 To validate whether LAMP5 is specifically and highly expressed in MLL

276 leukemia and to evaluate its clinical relevance, we recruited a cohort of 216 leukemia

277 patients, including 53 patients with different MLL fusion proteins (including

278 MLL-AF9, MLL-AF4 and MLL-ENL) and 163 patients without fusion proteins to

279 detect LAMP5 expression (the detailed clinical parameters are presented in

280 Supplementary Table S1). As shown in Fig. 1A, LAMP5 displayed a significantly

281 higher expression level in the MLL leukemia patient group than that in the MLL

282 wild-type (MLL-wt) group (p<0.001) and in all leukemic cell lines with different

283 MLL translocations (Supplementary Fig. S1A). We also reanalyzed microarray

284 data for 2,096 patient samples classified into 18 subtypes (GSE13204) (21), including

285 AML with t(11q23)/MLL, AML1-ETO, PML-RAR, and CBFβ-MYH11A, and ALL

286 with t(11q23)/MLL, IGH-MYC, and E2A-PBX. The results clearly showed that

287 LAMP5 is differentially expressed among these groups, with the highest expression

288 levels in MLL leukemia patients (Supplementary Fig. S1B, C). Next, we evaluated

289 the clinical value of the aberrantly expressed LAMP5. Receiver operating

290 characteristic (ROC) curve analysis was performed to distinguish patients with MLL

291 leukemia from those without, and LAMP5 achieved a high AUC value in both the

292 GES13204 data set (n=108 for MLL and n=1988 for MLL-wt) and our leukemia

293 sample validation set (n=53 or MLL and n=163 for MLL-wt), with considerably

294 significant sensitivity and specificity at the optimal cutoff point, as calculated by

295 Youden’s index (Fig. 1B, and Supplementary Fig. S1D). We also used a cohort of

296 patients (n=200) with complete clinical information for prognosis analyses. As shown

297 in Fig. 1C, patients with a higher level of LAMP5 exhibited reduced 5-year

298 leukemia-free survival (p=0.0118). Notably, in MLL leukemia, the 5-year

299 leukemia-free survival of patients with a high LAMP5 level was less than that of

300 patients with a low expression level of LAMP5, although the p value was not

301 significant (Fig. 1D, n=45, p=0.0831). We further examined LAMP5 expression in an

302 independent data set from the BloodSpot database (22-24), specifically LAMP5

303 expression relative to the patient outcome as well as LAMP5 expression in 11q23

304 compared with other AML sub-types and healthy cells. The results showed that

305 LAMP5 was more highly expressed in AML with t(11q23)/MLL compared with other

306 AML sub-types and healthy cells (Supplementary Fig. S1E) and that a higher level

307 of LAMP5 may lead to reduced 5-year leukemia-free survival (Supplementary Fig.

308 S1F). These data indicated that LAMP5 may play a role in aggressive MLL leukemia.

309

310 LAMP5 is positively correlated with the MLL fusion protein level and promotes

311 MLL leukemia maintenance in vitro

312 The expression patterns in clinical samples suggested that LAMP5 might be

313 associated with MLL translocations. Thus, we evaluated the correlation between

314 LAMP5 and MLL fusion proteins in paired patients at diagnosis and after a complete

315 response (CR). A positive correlation between the expression of LAMP5 and MLL

316 fusion proteins was found: both proteins showed higher expression levels in patients

317 at diagnosis, and their expression levels were markedly decreased in CR patients (Fig.

318 1E-G). We also applied RNAi approaches to knock down LAMP5 expression in MLL

319 leukemia cell lines. Supplementary Fig. S2A shows the efficiency of siRNAs against

320 LAMP5. We found that MLL fusion proteins were significantly downregulated, but

321 the wild-type MLL level was only slightly changed (Fig. 1H and Supplementary Fig.

322 S2B). However, LAMP5 knockdown had only a minimal effect on the mRNA level of

323 MLL fusion proteins (Supplementary Fig. S2C). These results indicated the

324 preferential involvement of LAMP5 in regulating the MLL fusion protein levels.

325 We next performed in vitro and in vivo experiments to investigate the effects of

326 LAMP5 on MLL leukemia progression and MLL fusion protein maintenance.

327 Knocking down LAMP5 in both MV4-11 and THP1 cells led to a considerable

328 decline in cell proliferation and an increase in apoptosis (Fig. 2A and

329 Supplementary Fig. S3A, B). Arsenic trioxide (ATO) is a clinical drug in leukemia

330 treatment and generally induces apoptosis in many leukemia cells (25, 26). As shown

331 in Supplementary Fig. S3C, the apoptosis rate was significantly higher in

332 LAMP5-knockdown MLL leukemia cells treated with ATO. We also used primary

333 cells from two distinct MLL leukemia patients (one was confirmed to harbor

334 MLL-AF4, and the other harbored MLL-ENL) for functional validations (27). As

335 shown in Fig. 2B, upon LAMP5 knockdown, ATO-induced apoptosis of both samples

336 was significantly increased, and MLL fusion protein levels were decreased (Fig. 2C),

337 showing the potential of LAMP5 as an important therapeutic target.

338

339 LAMP5 promotes MLL leukemia maintenance and progression in vivo

340 We further applied a NOD-scid-IL2Rg-/- mouse model (16, 28) and

341 intravenously (tail vein) implanted the mice with MOLM13 cells carrying LAMP5

342 short hairpin RNA (named as sh-LAMP5) and control (named as sh-NC)

343 (Supplementary Fig. S2D, E). We sacrificed the mice after two weeks and found that

344 the percentages of GFP+ cells were decreased in the peripheral blood, bone marrow,

345 spleen, liver and kidney of the mice treated with the sh-LAMP5 MOLM13 cells

346 compared with those in the animals treated with sh-NC-transfected cells (Fig. 2D and

347 Supplementary Fig. S4A). We also found that the mice treated with sh-LAMP5 cells

348 had significantly smaller and lighter spleens than mice from the sh-NC groups at two

349 weeks after implantation (Fig. 2E). Consistently, hematoxylin and eosin (H&E)

350 staining results showed that the amounts of leukemia cells in the bone marrow and

351 spleen from sh-LAMP5-transfected mice were reduced compared with those from

352 sh-NC-transfected mice (Fig. 2F, G). Notably, the sh-LAMP5 groups survived longer

353 than did the control groups (Fig. 2H, p=0.0033), suggesting that LAMP5 knockdown

354 could inhibit MLL leukemia progression. Additionally, we generated a NOD/SCID

355 xenograft mouse model (19) by subcutaneously injecting sh-LAMP5 Molm13 cells.

356 As shown in Supplementary Fig. S4B, shRNA-mediated knockdown of LAMP5

357 inhibited the malignant proliferation of MLL-positive tumors. Moreover, tumor

358 growth and weight were dramatically decreased (Supplementary Fig. S4C, D).

359 MLL-AF9 protein levels in the sh-LAMP5 NOD/SCID mice were also lower than

360 those in the control mice (Fig. 2I), a finding that was consistent with that from MLL

361 leukemia cell lines and patient primary cells (Fig. 1H and Fig. 2C). These results

362 suggested that MLL leukemia may depend on LAMP5 for its maintenance and

363 progression.

364

365 LAMP5 acts as an autophagic suppressor in MLL leukemia

366 We then determined how LAMP5 maintains the level of MLL fusion proteins

367 and promotes leukemogenesis. We first performed a cycloheximide (CHX) assay to

368 investigate whether LAMP5 influences protein synthesis. CHX is a specific inhibitor

369 of translation elongation to suppress protein synthesis. (29, 30), and we used 50 µg/ml

370 of cycloheximide (CHX) to block MLL protein synthesis in sh-NC and sh-LAMP5

371 cells and measured the decay rate of MLL fusion proteins. In the control group, only

372 slight reduction of MLL-AF4 protein was found under treatment of CHX for 1.5 h;

373 however, knockdown of LAMP5 led to a significant decrease in the MLL-AF4 fusion

374 protein level, indicating that LAMP5 regulates the degradation of MLL fusion protein

375 (Supplementary Fig. S4E). Previous studies have suggested that two LAMP family

376 members, LAMP1 and LAMP2, play important roles in autophagy (13, 14). More

377 importantly, a very recent study showed that LAMP5 is localized to endo-lysosomes

378 in pDC (15). These previous findings suggest that LMAP5 might be associated with

379 autophagy processes. Because MLL chimeras were reported to have innate resistance

380 to proteasome degradation, we addressed whether autophagy causes the degradation

381 of MLL fusion proteins following LAMP5 depletion. To this end, we performed three

382 assays (31, 32) to investigate whether LAMP5 is physiologically relevant in

383 autophagy. First, immunofluorescence (IF) labeling of the autophagy-related marker

384 LC3B was evaluated by laser scanning confocal microscopy (LSCM). The results

385 showed significant LC3B puncta accumulation in LAMP5-downregulated MLL cells

386 (Fig. 3A, and Supplementary Fig. S5A). Next, we observed autolysosome formation

387 by ultrastructural analysis. As shown in Fig. 3B (arrows indicated the autolysosome),

388 autolysosome formation was enhanced in LAMP5-knockdown cells. Third, we

389 measured LC3B-II protein levels using Western blotting. Fig. 3C and Fig. S5B

390 showed that LC3B-II was enriched following the knockdown of LAMP5 in the MLL

391 cell lines THP1 and MV4-11, as well as in the primary MLL leukemia cells (Fig. 3D).

392 Together, these results clearly showed that autophagy is enhanced when LAMP5 is

393 knocked down, suggesting that LAMP5 is an autophagy suppressor.

394 LAMP5 protects MLL oncogenic fusion proteins from autophagic degradation

395 To explore the possible mechanism by which LAMP5 regulates the autophagy

396 phenomenon in MLL leukemia. Co-immunoprecipitation (Co-IP) assay was

397 performed to verify the interaction between LAMP5 and several major

398 autophagy-associated proteins, including ATG5, Becline-1 and LC3B. However, only

399 ATG5 was found to directly interact with LAMP5 (Fig. 3E, F and Supplementary

400 Fig. S5C-F). Co-localization analysis followed by immunofluorescence further

401 confirmed this interaction (Fig. 3G). Knockdown of LAMP5 expression dramatically

402 enhanced the protein level of ATG5 but not the mRNA level (Fig. 3H, and

403 Supplementary Fig. S5G), further showing that LAMP5 acts as a suppressor to

404 regulate autophagy.

405 The observations above showed that LAMP5 is a novel autophagic suppressor

406 and is associated with MLL fusion protein level. Because we speculated that LAMP5

407 might protect MLL oncogenic fusion proteins from autophagic degradation, we first

408 treated cells with rapamycin, an autophagy inducer, and two autophagy inhibitors,

409 bafilomycin A1 and chloroquine (31). We found that the protein levels of both MLL

410 chimeras (MLL-AF9 and MLL-AF4), but not wild-type MLL, were dramatically

411 decreased upon rapamycin treatment and were increased upon bafilomycin A1 or

412 chloroquine treatment (Fig. 4A, B, and Supplementary Fig. S5H), indicating that

413 MLL fusion proteins were degraded via the autophagy pathway. Furthermore, we

414 detected the MLL fusion protein levels upon knocking down LAMP5 and treating the

415 cells with or without the autophagy inducer rapamycin (31). As shown in Fig. 4C, the

416 MLL-AF9 protein level declined dramatically with LAMP5 knockdown, especially

417 when cells were simultaneously exposed to rapamycin. Additionally, after knocking

418 down LAMP5, a decrease in the MLL-AF9 protein levels was reversed upon

419 treatment with the autophagy inhibitor bafilomycin A1 (Fig. 4D). Given that the ATO

420 could enhance the apoptosis rate when knocking down LAMP5 in MLL leukemia

421 (Supplementary Fig.S3C), we assessed whether the combination of ATO and

422 LAMP5 inhibition can impact autophagy and MLL-translocation. As shown in

423 Supplementary Fig. S5I and J, ATO indeed induced autophagy in MLL leukemia

424 cells, and the combination of ATO and LAMP5 inhibition accelerated the autophagy

425 process and degradation of MLL fusion proteins (Supplementary Fig. S5K). We also

426 found that ATG5 knockdown considerably restored the apoptosis rate and cell

427 proliferation induced by knocking down LAMP5 in MLL cells (Fig. 4E, F, and

428 Supplementary Fig. S5L). Notably, both MLL-AF9 and MLL-AF4 protein levels

429 were partially recovered when knocking down ATG5 concomitantly (Fig. 4G, H).

430 These data suggested that suppressing LAMP5 expression could release MLL fusions

431 from its defense system, leading to their degradation by selective autophagy.

432 To further demonstrate whether MLL fusion proteins are involved in the

433 autophagic removal process, we next investigated the interaction between MLL fusion

434 protein and p62/SQSTM1, a critical “cargo receptor” (9, 10) for proteins targeted to

435 be degraded by autophagy. We co-transfected both p62 and MLL-AF9 plasmids into

436 293T cells to explore whether the degradation of MLL fusion proteins is dependent on

437 p62. As shown in Fig. 4I, a p62-plasmid-dependent decrease in the MLL-AF9 levels

438 was revealed. Co-IP experiments also showed that MLL-AF9 directly bound to p62

439 (Fig. 4J, K, and Supplementary Fig. S5M); the interaction between MLL-AF9 and

440 p62 was strengthened when knocking down LAMP5 (Fig. 4L). These data indicate

441 that MLL-AF9 aggregates were shuttled to the autolysosome by p62, and LAMP5

442 regulates the p62-mediated selective autophagic degradation of MLL fusion proteins

443 in leukemia.

444

445 LAMP5 is activated by the H3K79 histone methyltransferase DOT1L

446 We finally sought to determine the mechanism of LAMP5 expression activation

447 in MLL leukemia. Interestingly, similar to the HOX gene cluster, we found that

448 H3K79me2 was located at the genetic locus of LAMP5 in the GSE38338 data set (33,

449 34), suggesting that LAMP5 might be targeted by DOT1L. DOT1L has been reported

450 to be indispensable for MLL leukemia maintenance and progression through

451 enhancing H3K79 methylation at its target gene loci, such as Meis1 and the HOX

452 gene cluster (19, 33, 34). Accordingly, we performed chromatin immunoprecipitation

453 (ChIP) for H3K79me2 and H3K79me3, both of which are important modifications

454 triggered by DOT1L(33-36). The results showed a considerable occupancy level of

455 H3K79 methylation at the LAMP5 gene loci (Fig. 5A). Notably, the enrichment

456 degree of H3K79me3 located at the LAMP5 gene loci was much greater than that of

457 H3K79me2, suggesting that H3K79me3 is the major modification for LAMP5

458 regulation. ChIP assays further showed the MLL-fusion protein to be located at the

459 LAMP5 locus, suggesting that expression of LAMP5 may be regulated by the

460 MLL-fusion-proteins/DOT1L complex (36-38) (Fig. 5B and Supplementary Fig.

461 S6A). We next investigated the effect of the DOT1L/MLL-fusion protein complex on

462 LAMP5 expression. Similar to the pattern of H3K79 methylation, knocking down

463 DOT1L dramatically decreased LAMP5 both at the mRNA and protein levels (Fig.

464 5C, D), whereas overexpressing DOT1L significantly increased LAMP5 levels (Fig.

465 5E, and Supplementary Fig. S6B). We also enhanced MLL-AF9 expression in THP1

466 cells and found that LAMP5 was upregulated when MLL-AF9 overexpressed (Fig.

467 5F). These results demonstrated that LAMP5 is the direct downstream target of the

468 DOT1L complex

469 Because DOT1L appears to be the critical regulatory factor at the LAMP5 gene

470 locus and directly modulates LAMP5 expression, we further evaluated whether

471 LAMP5 responds to a DOT1L inhibitor (19, 39). We treated both THP1 and

472 MOLM13 cells with the DOT1L inhibitors SGC0946 and EPZ5676 (19, 39) and

473 detected H3K79 methylation levels of LAMP5 (Supplementary Fig. S6C, D). The

474 ChIP experiment with an H3K79me3 antibody showed decreased H3K79me3

475 occupancy at the LAMP5 locus (Fig. 5G, H, and Supplementary Fig. S6E,F).

476 Notably, the levels of LAMP5 and MLL fusion proteins were also significantly

477 decreased after treatment with DOT1L inhibitors (Fig. 5I, J, and Supplementary Fig.

478 S6G-I), further showing that LAMP5 is a direct target of DOT1L. Importantly, the

479 autophagy-related marker LC3B dramatically accumulated in MLL leukemia cells

480 exposed to DOT1L inhibitors, and LC3B-II was significantly increased (Fig. 5I, J,

481 and Supplementary Fig. S6 G-I), indicating that LAMP5 is sensitive to a DOT1L

482 inhibitor and, for the first time, linking H3K79 methylation to autophagy regulation.

483 Moreover, we found that MLL fusion protein levels were partially restored under

484 LAMP5 overexpression with DOT1L inhibitor treatment (Fig. 5K, and

485 Supplementary Fig. S6J), suggesting that LAMP5 serves as a bodyguard for MLL

486 fusion proteins to evade degradation.

487

488 Knockdown of LAMP5 combined with the DOT1L inhibitor significantly delays

489 the progression and improve survival of the MLL leukemia in vivo

490 We finally assessed the effects of the combination of suppressing LAMP5 and the

491 DOT1L inhibitors using the MV4-11 transplantation NOD-SCID mice model. Sh-NC

492 /Sh-LAMP5 MV4-11 cells were first injected into the mice, and then we initiated the

493 intraperitoneal injection of the animals with the DOT1L inhibitor on day 10 after

494 transplantation, before they succumbed to leukemia (5, 19, 39). EPZ5676 is a DOT1L

495 inhibitor that has been under evaluation as a targeted therapy for MLL leukemia (19,

496 40). Intraperitoneal injection with 40 mg/kg of EPZ5676 or vehicle was performed

497 every other day for 10 days (5, 19, 39). Four treatments by injection were

498 administered— sh-NC cells, sh-LAMP5 cells, sh-NC + EPZ5676, and sh-LAMP5 +

499 EPZ5676. At day 35, we randomly killed three mice in each group and investigated

500 the infiltration degree of MV4-11 cells. Wright-Giemsa staining of the blood and

501 bone marrow was applied to show the blasts of MLL leukemia in the mice. The

502 results showed that the cellular morphology was partially transformed in those

503 inoculations, essentially significant in the group treated with sh-LAMP5 + EPZ5676

504 (Supplementary Fig. S7A). The percentages of GFP+ cells were also dramatically

505 decreased in the group of sh-LAMP5 + EPZ5676 (Fig. 6A, and Supplementary Fig.

506 S7B). Notably, suppressing LAMP5 (Sh-LAMP5) and EPZ5676 (Sh-NC + EPZ5676)

507 could extend the survival of the recipients beyond 48 days when all the

508 vehicle-treated mice succumbed to the disease, and the combination group

509 (sh-LAMP5 + EPZ5676) strikingly extended the life of the MLL leukemia mice to

510 more than 80 days (Fig. 6B). Together, these data indicated that the combination with

511 the DOT1L inhibitor and LAMP5 suppression can delay the progression and improve

512 the survival of aggressive MLL leukemia in vivo.

513 In conclusion, the observations above revealed that LAMP5 serves as a

514 “bodyguard” for MLL fusions to evade degradation. In contrast to the intrinsic

515 resistance of MLL chimeras to UPS degradation, the sustained active transcription of

516 LAMP5 in MLL leukemia favors the suppression of the cellular autophagy process to

517 maintain the level of MLL fusion proteins. We showed that genetic depletion of

518 LAMP5 effectively releases MLL fusions from its defense system, resulting in its

519 degradation. A model for specific activation of LAMP5 by DOT1L serving as a

520 “bodyguard” for MLL fusions to evade degradation is shown in Fig. 6C.

521 Discussion

522 Targeting the protein degradation pathway is an efficient paradigm in malignant

523 cancers (9, 10, 41, 42). In MLL leukemia, wild-type MLL has previously been shown

524 to be a valuable target (5), and its function depends on its accumulation and E3 ligase

525 SCFskp2- and APCcdc20-mediated degradation by the ubiquitin-proteasome system to

526 coincide with the cell cycle. However, in MLL leukemia, recurrent MLL fusions such

527 as MLL-AF4, MLL-AF9, MLL-ENL and MLL-ELL show diminished interactions

528 with these two E3 ligases, leading to the stabilization of MLL chimeras and onset of

529 MLL leukemia(7). Moreover, due to the loss of the PHD/Bromo domain in MLL

530 fusions, the other two UPS system ligases, ECSASB2 and UBE2O, cannot bind to MLL

531 chimeras; thus, MLL fusion proteins are resistant to UPS degradation (5, 6). Therefore,

532 it is necessary to identify an alternative approach to degrade MLL fusion proteins. In

533 this study, we revealed that LAMP5, a member of the lysosome-associated membrane

534 protein family, serves as a “bodyguard” for MLL fusions to evade degradation.

535 Suppressing LAMP5 expression effectively released MLL fusion from its defense

536 system, resulting in degradation. In contrast to the intrinsic resistance of MLL

537 chimeras to UPS degradation, the sustained activation of LAMP5 in MLL leukemia

538 suppresses the cellular autophagy process to maintain the level of MLL fusion

539 proteins. Notably, LAMP5 expression is restricted only to a few tissue types, including

540 non-activated pDCs in normal hematopoiesis (15, 43) and MLL fusion leukemia. This

541 exclusive expression pattern in MLL leukemia suggests that LAMP5 is a valuable

542 target in these diseases, and targeting LAMP5 may be effective without major

543 unwanted effects.

544 LAMP5 belongs to the LAMP family (44). Unlike other family members, such

545 as LAMP1 and LAMP2, LAMP5 shows robust suppression of autophagy flux in MLL

546 leukemic cells, therefore resulting in the stabilization of MLL chimeras. Although

547 autophagy has been reported to affect the degradation of oncoproteins PML-RARA

548 and BCR-ABL (9, 10), the role of autophagy in MLL leukemia is complicated. Chen

549 et al. reported that disruption of Atg5 or Rb1cc1 does not affect MLL-AF9 leukemia

550 maintenance (45). Another report also mentioned that autophagy did not contribute to

551 chemoresistance but to the initiation of leukemia in an MLL-AF9 mouse model (46).

552 By contrast, Watson and colleagues proposed that diminished autophagy with ATG5

553 depletion accelerates the development of AML with MLL-ENL in a mouse model (11).

554 Although the effect of autophagy in the initiation of the leukemic transformation stage

555 is inconclusive, the anti-cancer effect of autophagy in MLL leukemia maintenance

556 and progression is consistent. Our study showed that the genetic depletion of LAMP5

557 effectively releases MLL fusions from its defense system and results in degradation,

558 suggesting that a higher level of LAMP5 supports MLL leukemia maintenance. Based

559 on the universally high expression of LAMP5 in MLL leukemia, the anti-leukemic

560 effect of autophagy might be an intrinsic property at the maintenance stage. Thus,

561 enhancing autophagy may be beneficial for treatment.

562 It is also noteworthy that LAMP5 has been suggested to function as a

563 co-chaperone with UNC93B1, a known Toll-like Receptor (TLR) chaperone, to

564 shuttle TLRs to their respective locations in the endosomes (43) and indirectly

565 responses to immunomodulation (15). Thus, further investigation into whether

566 LAMP5 plays any other autophagy-independent roles in MLL leukemia will assist in

567 fully understanding its specific function for the disease. The combination of knocking

568 down LAMP5 together with the DOT1L inhibitor may be an effective regimen for

569 MLL leukemia therapy. Finally, how LAMP5 might be suppressed in a therapeutic

570 setting should also be considered for further translational application. Based on recent

571 progress in oncolytic viruses (47-50), using an adenovirus-mediated short hairpin

572 RNA delivery system containing shRNA targeting LAMP5 or CRISPR-Cas9

573 containing sgRNA targeting LAMP5 may be an alternative method for suppressing

574 LAMP5 in future clinical application.

575

576 Acknowledgments

577 We thank Prof. Pan jingxuan at State Key Laboratory of Ophthalmology, Zhongshan

578 Ophthalmic Center, Sun Yat-sen University, Guangzhou, China, for providing the

579 protocols for primary cell culture. We also thank Dr. Li-Bing Huang, Ms Cong Liang

580 at The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China, for

581 clinical sample collection.

582 This research was supported by National Key R&D Program of China (No.

583 2017YFA0504400 to Y-Q Chen) and National Natural Science Foundation of China

584 (No. 81770174 to Y-Q Chen and 31870818 to W-T Wang)，and grants from

585 Guangdong Province (No. 2014T70833 to Y-Q Chen). 29

586 Author contributions

587 W.T.W., C.H., and Y.M.S., designed and performed the research, analyzed data and

588 wrote the manuscript. Z.H.C., K.F., W.H., L.Y.S. and Z.C.Z performed the research

589 and analyzed data. X.Q.L. collected and analyzed clinical data. Y.Q.C. designed the

590 research, analyzed data and wrote the manuscript.

591

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753 Figure legends

754 Fig. 1. LAMP5 is highly expressed in MLL patient samples and is a predictor of a

755 poor outcome. (A) LAMP5 expression is significantly upregulated in MLL leukemia

756 (n=53) compared with its expression in MLL-wt (n=163) patients (t-test, p<0.001).

757 Relative expression (2-ΔCT) was used to quantify the LAMP5 mRNA expression

758 relative to that of a housekeeping gene (GAPDH). (B) ROC curve analysis showed

759 that LAMP5 had high AUC values of 0.8988 (95% confidence interval (CI):

760 0.8549-0.9447, p<0.001) and 0.9174 (95% CI: 0.8739-0.9608, p<0.001) in the

761 GES13204 data set (n=108 for MLL and n=1988 for MLL-wt) and validation set

762 (n=53 for MLL and n=163 for MLL-wt), respectively, with considerably significant

763 sensitivity (sen.) and specificity (spe.) at the optimal cutoff point calculated by

764 Youden’s index. (C) The 5-year leukemia-free survival indicated that LAMP5 may

765 serve as a useful biomarker for the prognosis of leukemia (n=200, p=0.0078). (D) The

766 5-year leukemia-free survival of patients with a high expression level of LAMP5 is

767 less than that of patients with a low LAMP5 level in MLL leukemia although the p

768 value was not significant (n=45, p=0.0831). (E, F) LAMP5 and MLL fusion protein

769 levels in 7 paired MLL leukemia patients (initial diagnosis versus complete response,

770 CR), and (G) the MLL-fusion protein levels were positively correlated with those of

771 LAMP5 mRNA (-△CT) at preliminary diagnosis (Pearson r=0.9033, p=0.0053).

772 Relative expression (2-ΔCT) was used to quantify the LAMP5 mRNA expression

773 relative to a housekeeping gene (GAPDH). (H) Knockdown of LAMP5 reduced the

774 MLL-AF4 and MLL-AF9 protein levels. The MLL-AF9 or -AF4/beta-tubulin

775 densitometric ratio was recorded by ImageJ.

776 Fig. 2. LAMP5 regulates MLL leukemia progression in primary cells and in vivo.

777 (A) Cell proliferation was measured using the CCK-8 assay at 0, 24, 48, and 96 h.

778 Error bars reflect ±SEM (**, p<0.01). (B) Apoptosis was measured by flow cytometry

779 in two primary cell lines from MLL leukemia patients transfected with LAMP5

780 siRNA and the control under 5 μM ATO treatment for 24 h. Error bars reflect ±SEM

781 (***, p<0.001, right) in three independent experiments. (C) Western blotting was

782 performed to detect MLL-AF4 and MLL-ENL protein levels when LAMP5 was

783 knocked down in primary cells. The MLL-AF4 or -ENL/beta-tubulin densitometric

784 ratio was recorded by ImageJ. (D) The NOD-scid-IL2Rg-/- mouse model was

785 intravenously (tail vein) injected with sh-RNA-established MOLM13 cells, PBS

786 groups are mice not injected with MOLM13 cells. The percentages of GFP+

787 MOLM13 cells were checked in the peripheral blood, bone marrow, spleen, liver and

788 kidney 14 days after implantation. Error bars reflect ±SEM (*, p<0.05). (E) Size and

789 weight of the spleen in the xenografts. PBS group is mice not injected with MOLM13

790 cells. Error bars reflect ±SEM (*, p<0.05). H&E staining in bone marrow (F) and

791 spleen (G) sections from mice. (H) Kaplan–Meier curves show the survival of mice

792 intravenously injected with sh-NC and sh-LAMP5 MOLM13 cells (n=10 mice per

793 group). p values were calculated using the log-rank (Mantel-Cox) test (**, p=0.0033).

794 (I) NOD-SCID mice with subcutaneous inoculation of sh-RNA-transformed

795 MOLM13 cells into the flanks. MLL-AF9 protein levels in LAMP5-knockdown

796 MOLM13 cell-inoculated NOD/SCID mice. The MLL-AF9 or -AF4/beta-tubulin

797 densitometric ratio was recorded by ImageJ.

798

799 Fig. 3. LAMP5 regulates autophagy in . (A) Immunofluorescence experiments

800 showed LC3B puncta accumulating in LAMP5-downregulated THP1 cells. The

801 number of LC3B puncta per cell was calculated by Image-Pro Plus (right, n=100 cells)

802 (***P<0.001). Scale bar, 10 μm. A representative image from three independent

803 experiments is shown. (B) Autolysosomes (indicated by arrows), as detected by

804 transmission electron microscopy (TEM), are accumulated in LAMP5-downregulated

805 THP1 cells. Error bars reflect ±SEM (ten cells per group, ***, p<0.001). Western

806 blotting showed LC3B-II enrichment following the knockdown of LAMP5 in MLL

807 leukemia cells (C) and primary cells (D). The LC3B-II/beta-tubulin densitometric

808 ratio was recorded by ImageJ. (E, F) The Co-IP outcomes revealed that LAMP5

809 interacted with ATG5 in THP1. (G) Immunofluorescence showed LAMP5 interacted

810 with ATG5 in MLL leukemia cells. (H) Knockdown of LAMP5 expression

811 dramatically enhanced the protein level of ATG5. The ATG5/beta-tubulin

812 densitometric ratio was recorded by ImageJ.

813

814 Fig. 4. LAMP5 regulates the selective autophagic degradation of MLL fusion

815 proteins. MLL-AF9 (A, THP1) and MLL-AF4 (B, MV4-11) protein levels were

816 dramatically decreased in response to rapamycin but were increased upon bafilomycin

817 A1 or chloroquine treatment. The densitometric ratios of MLL-AF9/beta-tubulin and

818 MLL-AF4/beta-tubulin were calculated by ImageJ. (C) MLL-AF9 protein level

819 detection under the knockdown of LAMP5 in THP1 cells exposed to rapamycin. The

820 densitometric ratios of MLL-AF9/beta-tubulin and LAMP5/beta-tubulin were

821 calculated by ImageJ. (D) The resulting decline in MLL-AF9 levels by knocking

822 down LAMP5 was reversed upon treatment with the autophagy inhibitor bafilomycin

823 A1 (25 nM, 12 h). The densitometric ratio of MLL-AF9/beta-tubulin was calculated

824 by ImageJ. (E) Apoptosis was measured by flow cytometry in THP1 and MV4-11

825 cells transfected with LAMP5 and/or ATG5 siRNA and the control. Error bars reflect

826 ±SEM (***, p<0.001, right) in three independent experiments. (F) Cell proliferation

827 was measured using the CCK-8 assay at 0, 24, 48, and 96 h. Error bars reflect ±SEM

828 (**, p<0.01). MLL-AF9 (G) and MLL-AF4 (H) protein levels under the knockdown

829 of LAMP5 and/or ATG5 in MLL leukemia cells. The densitometric ratios of

830 MLL-AF9/beta-tubulin and MLL-AF4/beta-tubulin were calculated by ImageJ. (I)

831 Decline in the MLL-AF9 levels with the concentration gradient of p62 plasmids. The

832 FLAG-MLL-AF9/beta-tubulin densitometric ratio was recorded by ImageJ.(J, K)

833 Co-transfection of FLAG-MLL-AF9 and p62-HA in 293T cells and co-IP experiments

834 showed MLL-AF9 binding to p62. (L) Co-IP experiments indicated that the

835 MLL-AF9-p62 interaction increased after LAMP5 knockdown.

836

837 Fig. 5. DOT1L targets LAMP5 through enhancing H3K79 methylation at the

838 LAMP5 gene locus. (A) The anti-H3K79 methylation ChIP assay showed the

839 enrichment of H3K79me2/3 located at the LAMP5 gene locus in MLL leukemia cells.

840 Western blotting for the H3K79 methylation of ChIP/IP; GAPDH served as the

841 negative control. (B) The anti-FLAG methylation ChIP assay showed the enrichment

842 of FLAG-MLL-AF9 located at the LAMP5 gene locus in THP1 and 293T. The

843 LAMP5 and H3K79me2/me3 levels were decreased dramatically following DOT1L

844 knockdown in THP1(C) and MV4-11(D). The densitometric ratios of DOT1L and

845 LAMP5/beta-tubulin, H3K79me2/H3, H3K79me3/H3 were recorded by ImageJ.

846 LAMP5 was increased significantly when DOT1L (E) and MLL-AF9 (F) were

847 overexpressed. The densitometric ratios of DOT1L, MLL-AF9 and

848 LAMP5/beta-tubulin were recorded by ImageJ. (G, H) The ChIP experiment

849 showed decreased H3K79me3 occupancy on the LAMP5 gene in MLL leukemia cells

850 treated for 2 days with 1 μM SGC0946, a DOT1L inhibitor. Upon exposure to 1 μM

851 SGC0946 for 2 days, the levels of LAMP5 and MLL fusion proteins were decreased

852 in THP1(I) and MV4-11(J) cells. Immunofluorescence assay demonstrated LC3B

853 accumulation with or without SGC0946. The densitometric ratios of MLL-AF9,

854 LAMP5,LC3B-II /beta-tubulin were recorded by ImageJ. (K) Western blotting

855 showed the MLL-AF9 protein levels under LAMP5 overexpression and SGC0946

856 treatment. The densitometric ratios of MLL-AF9 and LAMP5/beta-tubulin were

857 recorded by ImageJ.

858

859 Fig. 6. Combination of LAMP5 knockdown and EPZ5676 significantly improves

860 survival in vivo. (A) The percentages of GFP+ MV4-11 cells were detected in the

861 peripheral blood, bone marrow, spleen and liver 35 days after implantation. Error bars

862 reflect ±SEM (*, p<0.05). PBS groups are mice not injected with MOLM13 cells. (B)

863 Kaplan-Meier survival curves of mice injected with sh-NC or sh-LAMP5 MV4-11

864 cells after vehicle and EPZ5676 treatment at day 10. Vehicle or 40 mg/kg of EPZ5676

865 was administered every other day by intraperitoneal injection for a total of five

866 treatments. The number (n) indicates the number of mice in each group. The p values

867 were calculated using the log-rank test. (C) A working model proposed for the

868 specific activation of LAMP5 by H3K79 methyltransferase to serve as a “bodyguard”

869 for MLL fusions to evade degradation.

870

871

Activation of the lysosome-associated membrane protein LAMP5 by DOT1L serves as a bodyguard for MLL fusion oncoproteins to evade degradation in leukemia

Wen-Tao Wang, Cai Han, Yu-Meng Sun, et al.

Clin Cancer Res Published OnlineFirst January 16, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-18-1474

Supplementary Access the most recent supplemental material at: Material http://clincancerres.aacrjournals.org/content/suppl/2019/01/16/1078-0432.CCR-18-1474.DC1

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