1

PML-RARa interaction with TRIB3 impedes PPARg/RXR function and 1

triggers dyslipidemia in acute promyelocytic leukemia 2

3

Ke Li1,4#, Feng Wang3#, Zhao-Na Yang3#, Bing Cui3, Ping-Ping Li3, Zhen-Yu Li5, 4

Zhuo-Wei Hu1* and Hong-Hu Zhu2* 5 6 1National Clinical Research Center for Metabolic Disease, Department of 7

Metabolism and Endocrinology, the Second Xiangya Hospital, Central South 8

University, Changsha, Hunan, 410011, China 9 10 2Department of Hematology & Institute of Hematology, Zhejiang Province Key 11

Laboratory of Hematology Oncology Diagnosis and Treatment, The First Affiliated 12

Hospital, Zhejiang University, Hangzhou, Zhejiang, 310058, China 13

14 3Immunology and Cancer Pharmacology Group, State Key Laboratory of Bioactive 15

Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese 16

Academy of Medical Sciences & Peking Union Medical College, Beijing, 100050, 17

China 18

19 4NHC Key Laboratory of Biotechnology of Antibiotics, Institute of Medicinal 20

Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical 21

College, Beijing, 100050, China 22

23 5Department of Hematology, Affiliated Hospital of Xuzhou Medical University, 24

Xuzhou, Jiangsu, 230031 China 25

26

Running title: TRIB3 promotes dyslipidemia development in APL 27

28

*Co-correspondence: zhuhhdoc@163.com; huzhuowei@csu.edu.cn 29

30 #Contributing equally 31

32

Figure/table count: 8 main figures, 2 supplementary figure and 2 supplementary 33

tables 34

2

Abstract 35

Although dyslipidemia commonly occurs in patients with acute promyelocytic leukemia 36

(APL) in response to anti-APL therapy, the underlying mechanism and the lipid statuses 37

of patients with newly diagnosed APL remain to be addressed. 38

Methods: We conducted a retrospective study to investigate the lipid profiles of APL 39

patients. PML-RARa transgenic mice and APL cells-transplanted mice were used to 40

assess the effects of APL cells on the blood/liver lipid levels. Subsequently, gene set 41

enrichment analysis, western blot and dual luciferase reporter assay were performed to 42

examine the role and mechanism of PML-RARa and TRIB3 in lipid metabolism 43

regulation in APL patients at pretreatment and after induction therapy. 44

Results: APL patients exhibited a higher prevalence of dyslipidemia before anti-APL 45

therapy based on a retrospective study. Furthermore, APL cells caused secretion of 46

triglycerides, cholesterol, and PCSK9 from hepatocytes and degradation of low-density 47

lipoprotein receptors in hepatocytes, which elevated the lipid levels in APL cell-48

transplanted mice and Pml-Rara transgenic mice. Mechanistically, pseudokinase TRIB3 49

interacted with PML-RARa to inhibit PPARg activity by interfering with the interaction 50

of PPARg and RXR and promoting PPARg degradation. Thus, reduced PPARg activity in 51

APL cells decreased leptin but increased resistin expression, causing lipid metabolism 52

disorder in hepatocytes and subsequent dyslipidemia in mice. Although arsenic/ATRA 53

therapy degraded PML-RARa and restored PPARg expression, it exacerbated 54

dyslipidemia in APL patients. The elevated TRIB3 expression in response to 55

3

arsenic/ATRA therapy suppressed PPARg activity by disrupting the PPARg/RXR dimer, 56

which resulted in dyslipidemia in APL patients undergoing therapy. Indeed, the PPAR 57

activator not only enhanced the anti-APL effects of arsenic/ATRA by suppressing TRIB3 58

expression but also reduced therapy-induced dyslipidemia in APL patients. 59

Conclusion: Our work reveals the critical role of the PML-RARa/PPARg/TRIB3 axis in 60

the development of dyslipidemia in APL patients, potentially conferring a rationale for 61

combining ATRA/arsenic with the PPAR activator for APL treatment. 62

63

Key words: AML, Cancer, leukemia, lipid metabolism, tribbles, 64

65

4

66

67

Graphical Abstract: The crosstalk of PML-RARa/PPARg/TRIB3 contributes to the 68

abnormal lipid metabolism associated with APL 69

70

5

Introduction 71

Acute promyelocytic leukemia (APL) is the M3 subtype of acute myelogenous leukemia 72

(AML), which is driven by a chimeric PML-RARa oncoprotein [1]. Although increased 73

body mass index (BMI) and a high prevalence of obesity were reported in patients with 74

APL [2-6], the status of the serum lipid profile in newly diagnosed APL patients remains 75

unclear. All-trans retinoic acid (ATRA) and arsenic trioxide (As2O3) have long been used 76

successfully against APL [7-9]. However, in recent years, more attention has been paid to 77

ATRA-induced hypertriglyceridemia in APL patients undergoing ATRA therapy [10-13]. 78

Two mechanisms are assumed to account for the hypertriglyceridemia induced by ATRA. 79

First, ATRA stimulation increases the synthesis of cholesterol and triglycerides in the liver 80

to elevate the blood lipid levels of APL patients [10]. Second, metabolites, including 81

cytokines and adipokines, produced by APL cells may contribute to ATRA-induced 82

hypertriglyceridemia [14, 15]. However, the molecular mechanism of anti-APL therapy-83

mediated dyslipidemia remains elusive and needs to be further clarified. 84

85

As a nuclear receptor with transcription factor functions, peroxisome proliferator-activated 86

receptor-g (PPARg) controls lipid and glucose metabolism by forming PPARg-retinoid X 87

receptor (RXR) heterodimers to bind a PPAR-response element (PPRE) [16]. Retinoic acid 88

receptors (RARs) are ligand-controlled transcription factors that act as heterodimers with 89

RXRs to regulate cell growth and survival and are also implicated in the pathogenesis of 90

metabolic diseases [17]. In APL, the oncoprotein PML-RARa can heterodimerize with 91

6

RXRs, which bind strongly to retinoic acid response elements and represses the 92

transcription of RAR targets [18, 19]. Given that crosstalk exists between the nuclear 93

receptors PPARg, RARs and RXRs and that PML-RARa and PPARg share the same 94

partner, RXRs, we presumed that PML-RARa contributes to abnormal lipid metabolism 95

by competing with PPARg for RXR partners to inhibit PPARg target genes. 96

97

Tribbles homologue 3 (TRIB3), a member of the pseudokinase family, acts as a stress 98

sensor that responds to a diverse range of stressors, including inflammatory, metabolic and 99

endoplasmic reticulum (ER) stress [20-22]. Our recent study reported that TRIB3 100

promotes APL progression by interacting with the oncoprotein PML-RARa and inhibiting 101

p53-mediated senescence [23, 24]. Increases in TRIB3 expression induced by ATRA or 102

arsenic treatment decreased the therapeutic efficacy of treatment [23]. TRIB3 functions as 103

a metabolic stress factor to participate in the regulation of lipid and glucose metabolism 104

by interacting with the E3 ubiquitin ligase COP1, decreasing phospho-AKT and negatively 105

regulating PPARg transcriptional activity [25-27]. Therefore, we herein hypothesize that 106

the increased TRIB3 expression functions together with PML-RARa to participate in the 107

regulation of dyslipidemia in patients with APL. We first conducted a retrospective study 108

to investigate the lipid profiles of an adequate sample of APL patients. Furthermore, we 109

examined the roles and mechanisms of PML-RARa and TRIB3 in lipid metabolism in 110

APL patients before treatment and after induction therapy. Overall, our study not only 111

defines a mechanism by which the crosstalk of PML-RARa/PPARg/TRIB3 contributes to 112

7

the abnormal lipid metabolism associated with APL but also provides a rationale for the 113

combination of ATRA/arsenic with PPAR activator for APL therapy. 114

115

8

Materials and Methods 116

Patients and samples 117

We conducted a retrospective study of 120 patients with AML (APL and non-APL) at our 118

center from January 2014 through June 2016. The eligibility criteria included patients with 119

an age ranging from 15 to 65 years old and newly diagnosed AML. APL patients received 120

ATRA and arsenic, and non-APL patients received idarubicin (10 mg/m2/d × 3 days) or 121

daunorubicin (45 mg/m2/d × 3 days) and cytarabine (100 mg/m2/d × 7 days) as induction 122

therapy. In addition to age, gender, height, weight and hematological parameters, the total 123

cholesterol (TC), triglyceride (TG), high-density lipoprotein (HDL) cholesterol, and low-124

density lipoprotein (LDL) cholesterol concentrations were measured before and after 125

induction therapy in all patients. 126

127

Intravenous blood was collected from all subjects after 10 ± 2 h of fasting to measure 128

serum lipids. Blood samples were collected in vials containing an EDTA anticoagulant 129

agent. The plasma was promptly separated (< 4 h after collection of whole blood). We 130

used an Abbott ARCHITECT c 16000 instrument and TG, TC, HDL, and LDL test kits 131

(Merit Choice Bioengineering (Beijing) Co., Ltd.), which used the GPO-PAP, CHOP-PAP, 132

catalase clearance and surfactant clearance methods, respectively. Abnormal lipid status 133

was determined by utilizing criteria established by the expert panel of the National 134

Cholesterol Education Program (NCEP), Adult Treatment Panel III (ATP III). The cut-off 135

values, including the upper limits of normal, for TGs, TC, and LDL were 1.7 mmol/L (150 136

9

mg/dL), 5.2 mmol/L (200 mg/dL), and 3.4 mmol/L (130 mg/dL), respectively, and the 137

lower limit of normal for HDL was 1.04 mmol/L (60 mg/dL). Informed consent was 138

obtained from all participants in accordance with the Declaration of Helsinki. The 139

procedure was approved by the Ethics Committee of the Institute of Hematology and 140

Blood Diseases Hospital of PUMC (KT2019055-EC-1) and the institutional review board 141

at Affiliated Hospital of Xuzhou Medical University. Patient-related information is shown 142

in Supplementary Table 1. 143

144

Definitions of variables 145

According to the European Society of Cardiology (ESC)/European Atherosclerosis 146

Society (EAS) Guidelines for the Management of Dyslipidemias, hypertriglyceridemia is 147

defined as TGs >1.7 mmol/L (150 mg/dL). For TG-based analysis, the study groups were 148

categorized into two major TG groups: TG group-1 (TG ≤ 1.7 mmol/L) and TG group-2 149

(TG > 1.7 mmol/L). BMI was calculated as weight in kilograms/(height in meters)2, and 150

the current WHO criteria were used to categorize patients as underweight/normal (BMI < 151

25 kg/m2) or overweight/obese (BMI ≥ 25 kg/m2). The initial white blood cell (WBC) 152

count was evaluated and adjusted for the APL patients as follows: WBC counts ≤ 10 × 153

109/L and > 10 × 109/L for the low- and high-risk categories, respectively [28]. 154

155

Animal Studies 156

10

The myeloid cell-specific Pml-Rara knockin (Pml-RaraKI), Pml-RaraKI Trib3 knockin 157

(PR-T3KI), and Pml-RaraKI Trib3 knockout (PR-T3KO) transgenic mice (C57BL/6, male) 158

were constructed as described previously [23]. hMRP8-Pml-Rara transgenic mice were 159

obtained from Kan-kan Wang’s laboratory [29, 30]. These mice were maintained in the 160

animal facility at the Institute of Materia Medica under specific-pathogen-free (SPF) 161

conditions. For the animal studies, the mice were earmarked before grouping and were 162

then randomly separated into groups by one person; however, no particular method of 163

randomization was used. The sample size was predetermined empirically according to 164

previous experience using the same strains and treatments. No animals were excluded from 165

the analysis. Generally, the investigator was not blinded to the group allocation when 166

assessing the outcome. We ensured that the experimental groups were balanced in terms 167

of animal age and weight. All animal studies were approved by the Animal 168

Experimentation Ethics Committee of the Chinese Academy of Medical Sciences (permit 169

no. 002802), and all procedures were conducted in accordance with the guidelines of the 170

Institutional Animal Care and Use Committees of the Chinese Academy of Medical 171

Sciences. The animal study was also conducted in accordance with the Animal Research: 172

Reporting of In Vivo Experiments (ARRIVE) guidelines. 173

174

Statistical analysis 175

The Wilcoxon Mann-Whitney test was used to compare the distributions of numerical 176

variables between patients with APL and patients with other types of AML. The 177

11

associations between qualitative variables were assessed by the χ2 test. All statistics were 178

computed using SPSS software, version 22.0. P values < 0.05 were considered statistically 179

significant. 180

12

Results 181

APL patients have a higher prevalence of dyslipidemia than non-APL AML patients 182

A retrospective study (Supplementary Table 1) was conducted to investigate lipid profiles 183

and other major clinical parameters in 120 newly diagnosed AML patients (60 APL 184

patients versus 60 non-APL AML patients). More patients were overweight (BMI > 25) in 185

the APL group (52%, 31/60) than in the non-APL AML group (32%, 19/60) (p = 0.02). 186

However, the obesity rate (BMI > 30) was not different between the APL and non-APL 187

AML patients (13% vs. 5%, p = 0.11). Hyperlipidemia was found in 65% (39/60) of APL 188

patients and in 36% (22/60) of non-APL patients (p = 0.0019). The initial levels of TGs 189

before treatment were higher in the APL patients than in the non-APL patients (Figure 1A). 190

Moreover, the TC, HDL and LDL levels in the APL patients were higher than those in the 191

non-APL patients (Figure 1B-D), indicating that a higher proportion of APL patients had 192

dyslipidemia. 193

194

To examine the effect of APL cells on dyslipidemia in vivo, normal FVB mice were 195

transplanted with APL cells from hMRP8-Pml-Rara mice, and the body/liver weight, liver 196

lipid levels, and blood lipid levels of recipient mice were assessed over time (Figure 1E). 197

The body and liver weights of mice transplanted with APL cells were higher than those of 198

mice transplanted with normal spleen cells (Figure 1F-H). Additionally, the APL cell-199

transplanted mice showed elevated TG and cholesterol levels in the liver (Figure 1I). 200

Moreover, the serum TC and TG levels in APL cell-transplanted mice were higher than 201

13

those in normal spleen cell-transplanted mice (Figure 1J). To explore whether APL cells 202

are the main inducers of dyslipidemia in vivo, we assessed the liver and serum TC levels 203

in NOD scid gamma (NSG) mice transplanted with APL or non-APL C1498 AML cells 204

(Figure 1K). Consistent with the clinical data, the liver and serum TC levels in NSG mice 205

transplanted with APL cells were higher than those of C1498 cell-transplanted NSG mice 206

over time after inoculation (Figure 1L-M). Overall, these data indicate that APL cells play 207

a critical role in the enhancement of liver and serum lipid levels in both APL patients and 208

mice. 209

210

Given that the lipid production capacity of leukemia cells is less than that of metabolic 211

organs, such as the liver, we next examined the effects of APL cells on the lipid metabolism 212

of hepatocytes (Figure 2A). We found that the triglyceride and cholesterol production in 213

mouse primary hepatocytes was increased by APL cells derived from hMRP3-Pml-Rara 214

mice but not by murine non-APL AML cells (Figure 2B). Simultaneously, APL cells 215

promoted the secretion of the proprotein convertase subtilisin/kexin type 9 (PCSK9) in 216

hepatocytes as indicated by ELISA (Figure 2C) and subsequently reduced low-density 217

lipoprotein receptor (LDLR) expression in hepatocytes (Figure 2D). PML-RARa acts as 218

the driver of genetic alteration and the most critical factor responsible for the pathogenesis 219

of > 95% of APL cases. To investigate whether PML-RARa contributed to the 220

dyslipidemia associated with APL, we examined the serum TG, HDL and LDL levels in 221

myeloid cell-specific Pml-Rara knockin (Pml-RaraKI) mice (Figure 2E). Serum TG, HDL, 222

14

and LDL levels were higher in 3- to 4-month-old Pml-RaraKI mice (no APL symptoms) 223

than in wild-type (WT) mice of the same age (Figure 2F-H), indicating that PML-RARa 224

plays a critical role in the lipid metabolism disorder associated with APL. 225

226

PML-RARa inhibits PPARg transcriptional activity to induce dyslipidemia in APL 227

patients 228

To investigate the mechanisms underlying the high prevalence of dyslipidemia in APL 229

patients, we performed gene set enrichment analysis (GSEA) on differentially expressed 230

genes (DEGs) enriched in APL and non-APL AML subtypes. Notably, the lipid 231

metabolism process was identified as one of the most significantly enriched Kyoto 232

Encyclopedia of Genes and Genomes (KEGG) gene sets associated with APL (Figure 3A). 233

Moreover, several lipid metabolism-related genes (RETN, GPHN, ME1, LEP, LTC4S, 234

DHCR7, TRIB3, MOSC2, ABCA1, and PPARG) ranked among the top dysregulated genes 235

in APL patients versus non-APL AML patients. We further analyzed the individual 236

expression of the lipid metabolism genes in these AML subtypes and found that the 237

expression levels of only RETN, LEP, PPARG and TRIB3 were significantly dysregulated 238

in APL cells compared with non-APL AML cells (Figure 3B, Figure S1A and Table S2). 239

Leukemia cells from most APL patients showed reduced PPARg protein expression and 240

enhanced resistin and TRIB3 protein levels compared with those in leukemia cells from 241

non-APL patients (Figure 3C). The adipokines leptin and resistin, transcriptionally 242

regulated by PPARg, are involved in the regulation of glucose and lipid metabolism. Serum 243

15

resistin, but not leptin, was also dysregulated in APL patients (Figure 3D and Figure S1B). 244

Furthermore, overexpression of PML-RARa decreased the protein level of PPARg in non-245

APL U937 cells, accompanied by increased resistin and reduced leptin expression (Figure 246

S1C-D). Thus, the serum resistin and PCSK9 levels in APL cell-transplanted FVB mice 247

were higher than those in the control mice over time (Figure 3E-F). To verify whether the 248

adipokine resistin secreted from APL cells could enhance lipid levels in hepatocytes, we 249

treated normal liver cells with resistin (Figure 3G) and found that resistin stimulation 250

lowered LDLR expression in hepatocytes and enhanced the levels of TG, TC and PCSK9 251

in the culture supernatants of hepatocytes (Figure 3H-I). Consistently, resistin-knockdown 252

APL cells almost lost the ability to enhance the levels of TG, TC and PCSK9 in the culture 253

supernatants of hepatocytes (Figure 3J-L). Collectively, these data indicate that APL cells 254

elevate lipid levels in APL mice and that PML-RARa acts as the core factor causing the 255

dyslipidemia associated with APL. 256

257

To determine how PML-RARa is involved in the metabolic disorders associated with APL, 258

we examined the expression of PM-RARa and PPARg in leukemia cells from APL 259

patients. The expression of PML-RARa was negatively correlated with that of PPARg in 260

APL cells (Figure 4A). The transcriptional activity of PPARg, but not PPARa, was 261

inhibited by overexpression of PML-RARa in APL cells (Figure 4B). Indeed, adipose 262

PPARg is a well-known mediator of organism-wide metabolism [16]; however, it is still 263

unclear whether myeloid PPARg has similar effects. Given that APL cells have a low level 264

16

of endogenous PPARg protein, PPARg expression was stably depleted in non-APL 265

myeloid leukemia cells, and the effects of these cells on the liver and serum lipid levels 266

were examined in the mice (Figure 4C). Non-APL myeloid leukemia cells induced no liver 267

TG or TC enhancement over time in vivo, but PPARg knockdown cells increased both the 268

liver TG and TC levels of the transplanted mice (Figure 4D-E). Similarly, increases in 269

serum TG and TC levels were observed in mice transplanted with PPARg-depleted 270

leukemia cells but not in mice transplanted with control leukemia cells (Figure 4D-E). 271

Moreover, the lipid levels of mice transplanted with PPARg-depleted leukemia cells were 272

much higher than those of control mice (Figure 4D-G). These data indicate that altered 273

PPARg activity in APL cells is responsible for the dyslipidemia observed in APL mice. 274

275

PPARg is a ligand-activated transcription factor and functions as a heterodimer with an 276

RXR [31]. Mechanistically, this finding was verified by the interaction of PML-RARa 277

and PPARg in human APL cells (Figure 5A). PML-RARa overexpression decreased the 278

interaction of PPARg and RXR in APL cells (Figure 5B). Elevated TRIB3 expression 279

promotes lipid metabolism and sustains the oncogenic function of PML-RARa in APL 280

patients via protein-protein interactions [23]. Indeed, TRIB3 coimmunoprecipitated with 281

PPARg in APL cells (Figure 5C). Furthermore, TRIB3, PPARg and PML-RARa formed 282

a heterotrimer (Figure 5D), and elevated TRIB3 promoted the binding of PML-RARa and 283

PPARg in APL cells (Figure 5E-F). Moreover, TRIB3 depletion rescued the interaction 284

between PPARg and RXR by decreasing PML-RARa expression (Figure 5G). These data 285

17

indicate that the collaboration of PML-RARa and TRIB3 inhibits PPARg activity by 286

disrupting the PPARg/RXR heterodimer. 287

288

Additionally, overexpression of PML-RARa decreased PPARg protein expression (Figure 289

5H). We assessed the role of PML-RARa in the regulation of PPARg ubiquitination and 290

degradation mediated by ubiquitin ligases [32, 33]. In the presence of the protein synthesis 291

inhibitor cycloheximide (CHX), the PPARg protein exhibited a reduced half-life in PML-292

RARa-overexpressing cells compared with that in the control group. TRIB3 293

overexpression further accelerated the degradation of PPARg mediated by PML-294

RARa overexpression (Figure 5I). PML-RARa overexpression increased the 295

ubiquitination of PPARg and TRIB3 further increased the ubiquitination of PPARg 296

mediated by PML-RARa overexpression (Figure 5J). Furthermore, Trib3 knockin Pml-297

Rara (PR-T3KI) mice [23] showed elevated serum resistin levels compared with those in 298

the Pml-RaraKI mice (Figure 5K). Given the interconnectivity between PML-RARA 299

expression and PPARG/TRIB3, we investigated whether these genes are direct targets 300

(transcriptionally) of PML-RARa by transfecting Myc-tagged PML-RARA plasmids into 301

NB4 cells and used an anti-Myc antibody to capture protein-DNA complexes. The ChIP-302

qPCR results verified that both PPARG and TRIB3 are direct target genes of PML-RARA 303

(Figure S2). Thus, these gain-of-function studies indicate crucial roles of PML-RARa and 304

TRIB3 in the inhibition of PPARg activity by interrupting the PPARg/RXR heterodimer 305

and promoting ubiquitination-dependent PPARg degradation. 306

18

ATRA/arsenic rescues PPARg expression in APL cells but does not ameliorate 307

dyslipidemia in APL patients 308

ATRA plus arsenic trioxide (As2O3) with or without chemotherapy induces high remission 309

rates in APL patients by degrading the oncoprotein PML-RARα [34, 35]. Given that PML-310

RARa inhibited PPARg activity in APL cells, we next examined whether ATRA/As2O3 311

rescued PPARg activity and improved dyslipidemia in APL patients by decreasing PML-312

RARa expression. The combination of ATRA and As2O3 enhanced PPARg expression in 313

leukemia cells from APL patients after 1 week of induction therapy (Figure 6A). 314

Furthermore, As2O3 treatment rescued the colocalization of PPARg and RXR by degrading 315

the PML-RARa protein (Figure 6B). However, the mRNA and protein levels of resistin 316

were enhanced, whereas the protein level of leptin was decreased in APL cells after As2O3 317

or ATRA treatment (Figure 6C-D), which was accompanied by elevated resistin secretion 318

and downregulated leptin secretion (Figure 6E) in the culture supernatant of APL cells 319

after As2O3 treatment. 320

321

We next assessed the effects of leptin on the secreted TC and TG levels of the supernatant 322

of hepatocytes cells cocultured with APL cells treated with ATRA (Figure 6F). Increased 323

TC and TG levels were observed in the ATRA-treated coculture system of primary mouse 324

APL cells and hepatocytes. Leptin partially protected against the ATRA-induced TC and 325

TG enhancement of hepatocytes cocultured with APL cells (Figure 6G-H). These data 326

indicated that decreased leptin contributed to the dyslipidemia phenotypes caused by 327

19

ATRA treatment. We further analyzed the lipid profiles of APL patients during first-line 328

therapy. The TG concentrations of APL patients increased after 3 weeks of induction 329

therapy (Figure 6I). Consistently, the serum resistin levels of most APL patients were also 330

elevated after 1 week of induction therapy (Figure 6J and Table S2). The observation that 331

anti-APL therapy reduced PML-RARa and enhanced PPARg but could not normalize 332

dyslipidemia in APL patients indicates that other important regulator(s) may participate in 333

the regulation of lipid metabolism disorder in APL patients during induction therapy. 334

335

High TRIB3 mediates ATRA/arsenic therapy-induced dyslipidemia in APL patients 336

We found that combined ATRA and As2O3 therapy increased TRIB3 abundance in APL 337

cells from most patients after 1 week of therapy (Figure 7A and Table S2). Furthermore, 338

ATRA or As2O3 enhanced the transcription of TRIB3 in APL cells (Figure 7B). TRIB3 339

was reported to suppress adipocyte differentiation by negatively regulating PPARg 340

transcriptional activity [26]. Indeed, TRIB3 overexpression inhibited the transcriptional 341

activity of PPARg (Figure 7C) by interrupting the interaction between RXR and PPARg 342

(Figure 7D) in APL cells. TRIB3 deletion not only increased the transcriptional activity of 343

PPARg but also rescued the reduced PPARg activity induced by As2O3 treatment (Figure 344

7E). Although TRIB3 depletion showed no effects on leptin expression in APL cells, 345

silencing TRIB3 impeded the enhancement of resistin expression induced by ATRA 346

treatment in APL cells (Figure 7F). Moreover, TRIB3 depletion ameliorated the elevated 347

resistin production in the culture supernatants of APL cells treated with ATRA (Figure 348

20

7G). Additionally, we examined the effects of ATRA on the levels of TC, TG and PCSK9 349

in the supernatant of primary hepatocytes cocultured with mouse APL cells with or without 350

TRIB3 depletion (Figure 7H). Loss of TRIB3 moderately counteracted the increased levels 351

of TC, TG and PCSK9 secretion from hepatocytes cocultured with APL cells treated with 352

ATRA (Figure 7I-K). Importantly, ATRA treatment induced increased serum TG levels in 353

Pml-Rara transgenic mice but did not elevate TG levels in Trib3-knockout Pml-Rara 354

transgenic mice (PR-T3KO) (Figure 7L-M), indicating that TRIB3 is involved in the 355

regulation of anti-APL-therapy-induced dyslipidemia in individuals with APL. Overall, 356

arsenic/ATRA treatment enhances TRIB3 abundance in APL cells, and TRIB3 interacts 357

with PPARg to impede the heterodimer formation of PPARg and RXR, inhibiting PPARg 358

transcriptional activity and abnormal lipid metabolism in APL cells (Figure 7N). 359

360

Collaboration of PPAR agonists and ATRA/arsenic improves dyslipidemia in APL 361

patients 362

Given that APL patients had a high prevalence of dyslipidemia before and during induction 363

therapy, the use of a lipid-lowering drug combined with ATRA and As2O3 therapy may 364

further benefit APL patients and improve their clinical outcomes. First, we examined the 365

effects of pharmacological PPAR activation in APL cells. A PPARg activator (pioglitazone, 366

PZD) synergized with ATRA-mediated differentiation in primary APL cells (Figure 8A). 367

Similarly, the PPARa agonist fenofibrate (FN) potently increased the differentiation and 368

apoptosis percentages of APL cells induced by ATRA or an arsenic agent (Figure 8B-C). 369

21

Furthermore, the PPAR activator decreased PML-RARa expression and impeded the 370

increased resistin induced by As2O3 treatment (Figure 8D). Interestingly, the arsenic-371

induced increase in TRIB3 expression was hindered by FN treatment (Figure 8D), and the 372

PPAR activators reduced the increase in TRIB3 transcription induced by arsenic treatment, 373

indicating that PPAR activators nonspecifically inhibit TRIB3 (Figure 8E-F). Accordingly, 374

FN treatment protected against elevated resistin secretion and reduced leptin secretion in 375

APL cells treated with As2O3 (Figure 8G-H). 376

377

We next evaluated the therapeutic effect of ATRA/arsenic and FN in combination in the 378

PML-RARa mouse model. Treatment of PML-RARa APL mice with the combination for 379

3 weeks improved lipid metabolism disorder associated with APL, as indicated by 380

reductions in the elevated serum TG and TC levels in ATRA/arsenic-treated APL mice 381

(Figure 8I). Although the combination did not significantly improve the survival rate of 382

APL mice compared with those treated with ATRA/arsenic alone (Figure 8J), the PPAR 383

agonist FN synergized with ATRA/arsenic therapy to decrease spleen weights in APL 384

mice (Figure 8K). Similarly, treatment of APL patients (n = 17) with the combination for 385

3 weeks improved the dyslipidemia associated with APL, as indicated by reductions in the 386

elevated TG and TC levels of APL patients (Figure 8L). Overall, these data showed that 387

combined therapy with ATRA/arsenic and a PPAR agonist is suggested for APL patients. 388

389

22

In summary, our study suggests that the collaboration of PML-RARa with elevated TRIB3 390

expression inhibits PPARg activity and causes lipid metabolism abnormalities in newly 391

diagnosed APL patients. The TRIB3 expression that was increased by ATRA/As2O3 392

treatment further impeded PPARg activity by forming the heterotrimer of TRIB3, PML-393

RARa and PPARg, contributing to dyslipidemia in APL patients undergoing anti-APL 394

therapy. 395

23

Discussion 396

Previous studies demonstrated that TRIB3 interaction with PPARg negatively regulates 397

PPARg activity in adipose tissue [26]. Our recent work indicates that elevated TRIB3 398

expression stabilizes the oncoproteins PML-RARa and PML in APL [23, 24] and that 399

PML exerts its essential role in breast cancer cell and hepatic stellate cell (HSC) 400

maintenance through regulation of PPAR signaling and fatty acid oxidation (FAO) [36, 401

37]. However, we do not know whether or how TRIB3, PPARg and PML-RARa 402

contribute collaboratively to the regulation of lipid metabolism in APL cells. In this study, 403

we verified the hypothesis that the metabolic stress sensor TRIB3 collaborates with the 404

oncoprotein PML-RARa to inhibit PPARg activity and subsequently cause dyslipidemia 405

in newly diagnosed APL patients. Moreover, elevated TRIB3 expression in response to 406

arsenic/ATRA therapy further suppressed PPARg activity by disrupting the PPARg/RXR 407

dimer, which contributes to abnormal lipid metabolism in arsenic/ATRA-treated APL 408

patients. Accordingly, the PPAR activator not only enhanced the anti-APL effects of 409

arsenic/ATRA in vitro but also reduced arsenic/ATRA-induced dyslipidemia in APL 410

patients. Thus, our study may provide a rationale for the combination of ATRA/arsenic 411

therapy with the PPAR activator for the treatment of patients with APL. 412

413

Several studies have shown that APL patients have a higher percentage of obesity [38], 414

which is considered an adverse prognostic indicator for clinical outcome in APL. However, 415

the mechanism of the association between APL and obesity or overweight remains unclear. 416

24

Less is known regarding the lipid profile statuses of newly diagnosed APL patients, 417

although dyslipidemia has been associated with several types of cancer [39-41]. In this 418

study, we showed that newly diagnosed APL patients indeed had a higher prevalence of 419

dyslipidemia than non-APL AML patients, indicated by elevated serum levels of TC, TG, 420

HDL and LDL. PPARg is a transcription factor that plays a key role in adipogenesis and 421

insulin sensitization, and polymorphisms in PPARg have been associated with obesity and 422

diabetes-related phenotypes, such as hyperinsulinemia and dyslipidemia [42, 43]. Here, 423

we identified that APL cells with defective PPARg function is likely the essential factor 424

triggering the dyslipidemia of APL patients by secreting resistin and subsequently 425

disrupting the lipid metabolism of hepatocytes. Although Jansen et al provided a 426

preliminary clue that PML-RARa interferes with PPAR signaling pathways [19], little is 427

known regarding the role and mechanism of PML-RARa in the lipid metabolism 428

modulated by PPARg. Based on these observations, our study revealed that PML-RARa 429

interacts with PPARg to disrupt the PPARg/RXR heterodimer and promote PPARg 430

ubiquitination and degradation, which may partially explain the obesity and dyslipidemia 431

in newly diagnosed patients with APL that is driven by PML-RARa. A recent study 432

reported that the cytokine galectin-12, a negative regulator of lipolysis, is selectively 433

overexpressed in APL cells [44] and may participate in lipid metabolism regulation in APL 434

patients via lipid droplet accumulation. Our study showed that the production and secretion 435

of the adipokines resistin and leptin were dysregulated in APL cells. Thus, galectin-12, 436

resistin, leptin, and other adipokines/cytokines regulated by PPARg may act 437

25

synergistically to affect lipid metabolism in metabolic tissues, contributing to the 438

dyslipidemia associated with APL. 439

440

The application of ATRA/arsenic treatment in APL makes APL a curable chronic disease, 441

a great victory in the war against cancer in humans. However, although ATRA/arsenic 442

treatment degraded PML-RARa and restored PPARg expression, it did not improve but 443

rather exacerbated dyslipidemia in these APL patients. One major reason is that retinoids 444

induce hypertriglyceridemia in APL patients treated with ATRA. Several studies have 445

indicated that the increased synthesis of cholesterol and TGs, the disproportion of 446

apoprotein constituents in the liver, and defects in very-low-density lipoprotein (VLDL) 447

clearance in skeletal muscle contribute to ATRA-induced hypertriglyceridemia in APL 448

[45-47]. However, the detailed molecular mechanism of therapy-related dyslipidemia in 449

APL has not been clear until now. Pseudokinase TRIB3, a critical modulator of 450

glucose/lipid metabolism and cancer progression [48-50], has been reported to inhibit 451

PPARg activity in adipocytes via its interaction with PPARg [26]. Although we previously 452

found that TRIB3 expression is enhanced in APL cells [23] and arsenic enhances the 453

mRNA and protein expression of TRIB3 [51], it is still unclear whether TRIB3 plays a 454

role in the abnormal lipid level of treated APL patients. Our study revealed that TRIB3 455

induced by ATRA/arsenic therapy further inhibits PPARg activity to dysregulate TG and 456

adipocytokine secretion in APL cells, which subsequently contributes to the disordered 457

lipid metabolism associated with APL. Thus, the inhibition of TRIB3 combined with 458

26

ATRA/arsenic treatment may improve the lipid metabolism of APL patients. Interestingly, 459

we found that PPAR agonists can reduce arsenic/ATRA-induced dyslipidemia in APL 460

patients and decrease TRIB3 expression in APL cells. This effect may derive from the fact 461

that PPAR agonists inhibit TGFβ1/Smad3 signaling [52] and that TRIB3 is a target gene 462

of TGFβ1/Smad3 [53]. Thus, the beneficial effects of PPAR agonists in APL may be 463

derived from not only the direct lowering of blood lipids but also a potential inhibition of 464

TRIB3 expression; this concept needs further clarification. 465

466

In summary, our study not only reveals a critical role of the PML-RARa/PPARg/TRIB3 467

axis in dyslipidemia for patients with APL but also confers a rationale for the combination 468

of ATRA/arsenic with the PPAR activator for the treatment of patients with APL. 469

470

Abbreviations 471

APL: acute promyelocytic leukemia; ATRA: all-trans retinoic acid; AML: acute 472

myelogenous leukemia; BMI: body mass index; DEGs: differentially expressed genes; ER: 473

endoplasmic reticulum; FN: fenofibrate; GSEA: gene set enrichment analysis; HDL: high-474

density lipoprotein; KEGG: kyoto encyclopedia of genes and genomes; LDL: low-density 475

lipoprotein; PPARg: peroxisome proliferator-activated receptor-g; PPRE: PPAR-response 476

element; PZD: pioglitazone; RARs: Retinoic acid receptors; TRIB3: tribbles homologue 477

3; TC: total cholesterol; TG: triglyceride. 478

479

27

Acknowledgments 480

This work was supported by grants from the National Key R&D Program of China 481

(2017YFA0205400), the National Natural Science Foundation of China (81530093, 482

81773781 to ZWH; 81570128 and 81970133 to HHZ; and 81872904 to KL), Beijing 483

Outstanding Young Scientist Program (BJJWZYJH01201910023028), the CAMS 484

Innovation Fund for Medical Sciences (2016-I2M-1-007 to ZHW; 2016-I2M-1-011 to KL; 485

and 2016-I2M-3-008 to FW), and the “Ten thousand plan" - National high-level talents 486

special support plan to KL. 487

488

Conflicts of interest 489

The authors have no conflicts of interest in relation to this submission.490

28

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Figures and Legends

Figure 1. Lipid profiles of patients with APL and mice transplanted with APL cells from hMRP8-Pml-Rara

APL mice. (A-D) Serum triglyceride (TG), total cholesterol (TC), high-density lipoprotein (HDL) and low-density

lipoprotein (LDL) levels in APL patients (n = 60) and non-APL AML patients (n = 60) were evaluated before induction

therapy. (E) Approaches to define the effects of murine APL cells or normal spleen cells on the body/liver weights,

liver lipid levels, and blood lipid levels of FVB recipient mice. (F and G) Body weights (F) and liver weights (G) of

the FVB recipient mice transplanted with normal spleen cells (CTRL) and Pml-Rara APL cells (Pml-Rara) at the

34

indicated times after inoculation. (H) Representative liver morphologies of CTRL and Pml-Rara recipient mice at 3

weeks after inoculation. (I) Total triglyceride (TG) and cholesterol levels of the liver lipids extracted using the

chloroform/methanol method. The data were normalized to the liver weights and are represented as the mean ± the

standard error (SEM). N = 3 mice per group. (J) Serum TG and total cholesterol (TC) levels in the CTRL and Pml-

Rara recipient FVB mice at the indicated times after inoculation. (K) Approaches to define the effects of murine APL

cells or non-APL AML cells (C1498) on liver and blood TC levels in NSG recipient mice. (L and M) Liver and serum

TC levels in recipient NSG mice transplanted with normal spleen cells (CTRL), Pml-Rara APL cells, or C1498 AML

cells at the indicated times after inoculation. For panels F-M, n = 4 mice per group. APL: acute promyelocytic leukemia;

AML: acute myeloid leukemia; ULN: upper limits of normal.

35

Figure 2. PML-RARa-positive APL cells induced lipid production in hepatocytes. (A) Approaches to define the

effects of murine APL cells or C1498 AML cells on lipid production and LDLR expression in hepatocytes. (B) Total

triglyceride (TG) and cholesterol levels in culture supernatants from mouse hepatocytes were determined by enzymatic

assay. (C) The PCSK9 levels in the culture supernatants of mouse hepatocytes cocultured with the indicated cells were

detected by ELISA. (D) Confocal assay of LDLR expression in mouse hepatocytes cocultured with C1498 AML or

APL cells. (E) Approaches to define murine serum TG/HDL/LDL levels in myeloid cell-specific Pml-Rara knockin

(Pml-RaraKI) mice (3-4 months old). (F-H) Serum TG, HDL, and LDL levels in WT mice and Pml-RaraKI mice at the

age of 3-4 months.

36

Figure 3. PPARg signaling is dysregulated in PML-RARa-positive cells. (A) Gene set enrichment analysis (GSEA)

shows the enrichment of lipid metabolism process-related genes (GSE13204) in APL cells (n = 50) versus non-APL

AML cells (n = 50). (B) qRT-PCR was performed to analyze the mRNA levels of RETN, LEP, PPARG, and TRIB3

(normalized to GAPDH) in primary APL cells and non-APL AML cells. (C left) The expression of PML-RARa,

PPARg, resistin, leptin, and TRIB3 was detected by Western blotting in primary APL cells and non-APL AML cells.

37

(C right) Statistical analyses of PPARg, resistin, leptin, and TRIB3 expression in primary APL cells (n = 16) and non-

APL AML cells (n = 8). (D) Serum resistin levels in newly diagnosed APL patients (n = 34) and non-APL AML

patients (n = 13). (E) Approaches to define the effects of murine APL cells or normal spleen cells on serum resistin

and PCSK9 levels in FVB recipient mice. (F) Serum resistin (left) and PCSK9 (right) levels in FVB recipient mice

transplanted with normal spleen cells (CTRL) and APL cells (Pml-Rara) at the indicated times after inoculation (n =

4 per group). (G) Approaches to define the effects of resistin on the LDLR expression and lipid secretion of hepatocytes.

(H) The LDLR expression in normal mouse hepatocytes was detected by confocal assay after resistin (50 ng/mL)

stimulation for 24 h. (I) The levels of TG, cholesterol and PCSK9 in the supernatants of normal mouse hepatocytes

after resistin (50 ng/mL) stimulation for 24 h. (J) Approaches to define the effects of resistin-depleted murine APL

cells on the TG, cholesterol and PCSK9 secretion of hepatocytes. (K and L) The levels of TG, cholesterol and PCSK9

in the supernatants of normal mouse hepatocytes after coculture with APL cells with or without resistin depletion for

24 h.

38

Figure 4. Reduced PPARg activity in APL cells causes lipid metabolism disorder in APL patients. (A) Correlation

between PML-RARa and PPARg expression (protein level) in human primary APL cells (n = 16). Each data point

represents the value from an individual patient. Statistical significance was measured by Pearson’s correlation test. (B)

PML-RARa decreased the transcriptional activity of PPARg. HEK 293T cells were transiently transfected with the

indicated plasmids. After 24 h of transfection, luciferase activities were measured. (C) Approaches to define the effects

of C1498 AML cells with or without PPARg depletion on the liver and blood lipid levels of C57 BL/6 recipient mice.

(D-G) Liver and serum TG and TC levels in mice transplanted with CTRL shRNA C1498 cells or PPARg shRNA

C1498 cells at the indicated times after inoculation. For panel D-G, n = 4 mice per group.

39

Figure 5. The combination of PML-RARa and TRIB3 inhibits PPARg activity by disrupting the PPARg/RXR

heterodimer and promoting PPARg degradation. (A) The interaction between endogenous PML-RARa and PPARg

was detected by a coimmunoprecipitation (co-IP) assay in human primary APL cells. (B) PML-RARa reduced the

interaction between PPARg and RXR. NB4 cells were transfected with or without a PML-RARa-expressing plasmid.

After 24 h of transfection, co-IP analysis was performed to detect the PPARg/RXR interaction. (C) The interaction

between TRIB3 and PPARg was detected by a co-IP assay in HEK 293T cells transfected with TRIB3- and PPARg-

expressing plasmids. (D) TRIB3, PML-RARa and PPARg trimers were detected by a co-IP assay in HEK 293T cells.

40

HEK 293T cells were transiently transfected with PPARg-, TRIB3-, and PML-RARa-expressing plasmids. After 24 h

of transfection, a co-IP assay was performed to detect the interactions between TRIB3, PML-RARa and PPARg. (E)

TRIB3 increased the interaction between PPARg and PML-RARa. NB4 cells were infected with an adenovirus

expressing TRIB3 or an HA tag (CTRL). After 24 h of transfection, a co-IP analysis was performed to detect the

PPARg/PML-RARa interaction. (F) TRIB3 increased the colocalization of PPARg and PML-RARa. Human primary

APL cells were infected with adenovirus expressing TRIB3 or CTRL. After 24 h of transfection, the colocalization of

PPARg/PML-RARa was detected by an IF staining assay. (G) TRIB3 depletion increased the interaction between

PPARg and RXR. A co-IP analysis was performed to detect the PPARg/RXR interaction in NB4 cells with or without

TRIB3 depletion. (H) The effects of PML-RARa on the protein level of PPARg in U937 cells. (I) The effect of PML-

RARa or PML-RARa and TRIB3 overexpression on PPARg degradation. HEK 293T cells were transfected with the

indicated plasmids, and 12 h later, the cells were incubated with CHX (10 µg/mL) for the indicated times. (J) The

effect of PML-RARa overexpression or PML-RARa and TRIB3 overexpression on PPARg ubiquitination. HEK 293T

cells were transfected with the indicated plasmids, and 12 h later, cell extracts were IP with anti-Myc Ab. Ubiquitinated

PPARg was detected by immunoblotting. (K) Serum resistin levels in Pml-Rara mice (nonleukemic) with or without

Trib3 knockin (3 months old).

41

Figure 6. ATRA/As2O3 treatment increases PPARg expression but does not improve dyslipidemia. (A)

ATRA/As2O3 treatment increased PPARg expression in APL cells. The PPARg expression in primary APL cells

isolated from the bone marrow of APL patients was detected by Western blotting before treatment and 1 week after

42

combined ATRA and arsenic treatment. (B) Colocalization of PPARg (red) and RXR (green) in primary human APL

cells (APL-1#) was detected by immunostaining. Scale bar, 10 µm. (C) qRT-PCR was performed to analyze the RETN

mRNA levels in primary APL cells treated as indicated for 24 h. (D) The expression of leptin and resistin in primary

APL cells was detected by Western blotting after administration of the indicated treatment for 24 h. (E) Effects of

arsenic treatment on resistin and leptin secretion in primary APL cells after 2 days of treatment. (F) Approaches to

define the effects of ATRA or ATRA and leptin on the lipid production of murine hepatocytes cocultured with murine

APL cells. (G and H) The levels of TG (G) and cholesterol (H) in the supernatants of normal mouse hepatocytes after

coculture with primary APL cells treated with ATRA or ATRA and leptin for 12 h. (I) ATRA and arsenic treatment

increased serum TG levels in APL patients. The serum TG levels of APL patients were measured by Abbott

ARCHITECT c16000 at the indicated treatment times (n = 60), ULN: upper limits of normal. (J) ATRA and arsenic

treatment increased serum resistin levels in APL patients (n = 12).

43

Figure 7. ATRA/As2O3-enhanced TRIB3 inhibits PPARg activity and increases dyslipidemia in APL. (A)

ATRA/As2O3 treatment induced TRIB3 abundance in APL cells. TRIB3 expression in primary APL cells isolated from

the bone marrow of APL patients (n = 8) was detected by Western blotting before treatment and 1 week after combined

ATRA and arsenic treatment. (B) ATRA/arsenic treatment inhibited TRIB3 transcription. HEK 293T cells were

transiently transfected with the pTRIB3-luc and TK plasmids. After 24 h of transfection, cells were treated with ATRA

or arsenic for 24 h, and luciferase activities were measured. (C) TRIB3 decreased the transcriptional activity of PPARg.

HEK 293T cells were transiently transfected with PPRE-Luc, TK, and CTRL or TRIB3 overexpression plasmids. After

24 h of transfection, the cells were treated with PZD (5 µM), and luciferase activities were measured. (D) TRIB3

reduced the interaction between PPARg and RXR. NB4 cells were transfected with or without a TRIB3-expressing

plasmid. After 24 h of transfection, co-IP analysis was performed to detect the PPARg/RXR interaction. (E) The effect

of As2O3 treatment on the transcriptional activity of PPARg in NB4 cells with or without TRIB3 deletion. CTRLCas9

44

and TRIB3cas9 NB4 cells were transfected with PPARg reporter genes. After 24 h of transfection, the cells were treated

with vehicle or As2O3 for 24 h, and luciferase activities were measured. (F) The effect of ATRA treatment on the

protein levels of leptin and resistin in primary APL cells with or without TRIB3 depletion. (G) The effect of ATRA

treatment on the resistin level of culture supernatants of primary APL cells with or without TRIB3 depletion. (H)

Approaches to define the effects of TRIB3-depleted murine APL cells on the TG, cholesterol and PCSK9 secretion of

hepatocytes. (I-K) The levels of TG (I), cholesterol (J) and PCSK9 (K) in the supernatants of normal mouse

hepatocytes after coculture with TRIB3-depleted APL cells treated with ATRA for 12 h. (L) Strategy for studying the

effects of ATRA treatment on the serum TG levels in Pml-Rara (Pml-RaraKI) and Trib3 knockout Pml-Rara (PR-

T3KO) mice. (M) Serum TG levels in the indicated mice treated with or without ATRA (8 months old). (N) Schematic

diagram illustrating the role of TRIB3 in the regulation of PPARg activity in APL cells treated with ATRA/arsenic.

45

Figure 8. PPAR agonists induce the synergism of anti-APL with ATRA/As2O3 by improving dyslipidemia. (A)

CD11b expression was evaluated by flow cytometry in primary APL cells that were subjected to the indicated treatment.

The percentage of CD11b-positive cells was calculated with FCS Express software. The data are presented as the mean

± SEM of 3 assays. (B and C) The PPARa agonist (fenofibrate, FN) increased the percentage of differentiated and

apoptotic APL cells induced by ATRA or As2O3 treatment. Primary APL cells were treated with the indicated treatment

for 24 h and then stained with a CD11b antibody or Annexin-V-PI and evaluated by flow cytometry. The data are

presented as the mean ± SEM of 3 assays. (D) The expression of PML-RARa, PPARg, TRIB3, and resistin in primary

APL cells treated as indicated was detected by Western blotting. (E and F) PPAR agonists suppressed the elevated

TRIB3 transcription induced by As2O3. HEK 293T cells were transiently transfected with the TRIB3 reporter plasmids

(p-TRIB3&TK). After 24 h of transfection, the cells were treated with the indicated treatment for 24 h, and luciferase

46

activities were then measured. (G and H) The PPARa agonist (FN) enhanced the secreted leptin level and reduced the

resistin level in primary APL cells that were subjected to As2O3 treatment. The data are presented as the mean ± SEM

of 3 assays. (I) Serum TG and TC levels in Pml-Rara recipient FVB mice treated as indicated. (J) Kaplan-Meier

survival curves for the Pml-Rara recipient FVB mice treated as indicated (n = 8). (K) The data indicate the spleen

weights of the Pml-Rara recipient FVB mice treated as indicated. (L) Serum TG and TC levels in APL patients (n =

17) were evaluated after combined treatment with ATRA/arsenic plus FN.

1

Supplemental Methods

Cell lines and culture

NB4, U937 and C1498 AML cells (mouse non-APL acute myeloid leukemia cells) were

purchased from Shanghai Bioleaf Biotech Co., Ltd, where they were recently authenticated by

STR profiling and characterized by mycoplasma and cell vitality detection. These cells were

cultured and maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum

(FBS, Invitrogen, CA, USA) under 5% carbon dioxide.

Leukemia cell transplantation

Mouse APL-like spleen cells from hMRP8-Pml-Rara transgenic mice or normal spleen cells

were intravenously injected into nonirradiated FVB mice (2 ´ 106 cells per mouse) aged

between 6 and 10 weeks. To compare the effects of the mouse APL cells and non-APL leukemia

cells on the lipid metabolism of the recipient mice, APL cells from hMRP8-Pml-Rara mice or

C1498 (non-APL) leukemia cells were intravenously injected into NSG mice (2 ´ 106 cells per

mouse) aged between 4 and 6 weeks. One week later, transplanted mice were sacrificed and

analyzed for lipid levels once a week for 4 weeks.

ATRA, arsenic and FN treatment in vivo

APL cells from hMRP8-Pml-Rara transgenic mice were intravenously injected into

nonirradiated FVB mice (2 ´ 106 cells per mouse) aged between 6 and 10 weeks. Mice

implanted with leukemic cells were randomly assigned to either type of treatment. One day

later, when transplanted cells had established, 400 mg/kg FN (100 µL, p.o., once a day), 2

2

mg/kg ATRA (100 µL, p.o., once a day) and 2 mg/kg As2O3 (100 µL, i.p., once a day), or 400

mg/kg FN (100 µL, p.o., once a day), 2 mg/kg ATRA (100 µL, p.o., once a day) and 2 mg/kg

As2O3 (100 µL, i.p., once a day) were administered for 3 consecutive weeks. To evaluate the

survival rate, these mice were monitored for 28 days.

Chromatin immunoprecipitation (ChIP)-qPCR assay

ChIP assays were performed according to the manufacturer’s protocol using a SimpleChIP®

Plus Sonication Chromatin IP Kit (Cell Signaling Technology, Danvers, MA, USA, #56383).

Briefly, NB4 cells transfected with Myc-tagged PML-RARa plasmids were fixated with 1%

formaldehyde for 8 min, incubated with glycine (50 mM final) for 10 min and washed three

times with PBS. After cell lysis and chromatin extraction, chromatin was sonicated to 100-500

bp using a BioRuptor sonicator (Diagenode), followed by centrifugation at 16,000 g for 10 min

at 4 °C. The lysates were incubated overnight at 4 °C with ChIP-grade antibodies specific for

Myc (Abcam, #ab9132), which were coupled to magnetic beads. Precipitated material was

eluted (input chromatin was used as a control), the crosslink was reverted, and DNA was

purified by chloroform/phenol extraction and resuspended in DNA elution buffer. qPCR

analysis was performed using specific primers corresponding to the TRIB3 and PPARG

promoters. The primers used for the ChIP-qPCR assays are shown below: TRIB3-1-F: 5'-

CCCCACAACTTATATCTAGTGCAGG-3', TRIB3-1-R: 5'-CAGCTGGCATTTAG

GGAGCATGTCT-3'; TRIB3-2-F: 5'-TCCCTAAATGCCAGCTGTTATTATG-3', TRIB3-2-

R: 5'-CATGGACAAGCGCTTGTCTCTCACT-3'; TRIB3-3-F: 5'-TGAGAGACAAGCGC

3

TTGTCCATGCC-3', TRIB3-3-R: 5'-AGAATCTGTGTGGTCATTTCTG CAT-3'; TRIB3-4-

F: 5'-AATGACCACACAGATTCTACCAATG-3', TRIB3-4-R: 5'-TGTC TGTGAGGTCTT

CAGAGCTGAT-3'; TRIB3-5-F: 5'-AGCTCTGAAGACCTCACAGACA CAT-3', TRIB3-5-

R: 5'-GCTGAGATTACAGGCGTGAGCCACA-3'; TRIB3-6-F: 5'-TGTAATCTCAG

CGCTTTGGGAGGCC-3', TRIB3-6-R: 5'-GCTGGAGTGCAATGGCGTGATCTTG-3';

TRIB3-7-F: 5'-TTGCACTCCAGCCTGGGTGACAGAA-3'; TRIB3-7-R: 5'-GGCCATGC

CATGTCCAAGGTCACAG-3'; TRIB3-8-F: 5'-ATTCCCTGTGAC CTTGGACATGGCA-3',

TRIB3-8-R: 5'-AGAGCTTGGTTTTGAGCCATGTGCT-3'; TRIB3-9-F: 5'-ACATGGCTCA

AAACCAAGCTCTGGG-3', TRIB3-9-R: 5'-TGCCACACCTGGTCCATGGACCCTG-3';

TRIB3-10-F: 5'-AGGGTCCATGGACCAGGTGTGGCAG-3', TRIB3-10-R: 5'-AGGATGG

AAGCAAAGCTGCAGCCCT-3'; PPARG-1-F: 5'-ACATTGCTGGTGGGATTGTAAA

ATG-3', PPARG-1-R: 5'-TCCATTATACACATATACCACATTT-3'; PPARG-2-F: 5'-

ATGTGGTATATGTGTATAATGGAAT-3', PPARG-2-R: 5'-ACCCCCTCTCCATTA

ACAGTCATTC-3'; PPARG-3-F: 5'-ATGACTGTTAATGGAGAGGGGGTTC-3', PPARG-3-

R: 5'-ATGACTGTTAATGGAGAGGGGGTTC-3'; PPARG-4-F: 5'-AGGGTCAAGCG

ATTCTACTGCCTCA-3', PPARG-4-R: 5'-TACAATTCAGGCCGGGTATGGCAGC-3';

PPARG-5-F: 5'-ACCCGGCCTGAATTGTACATTT TAC-3', PPARG-5-R: 5'-TAATTTTAA

TTGTTTAGTAGAGACT-3'; PPARG-6-F: 5'-AACATGTCAAGACACAGTCTCTACT-3',

PPAR G-6-R: 5'-TCTTTTCTTTCTTTCTTCCATGAGA-3'; PPARG-7-F: 5'-

AAAGAAAGAAAAGAAAGGAAGAAAG-3', PPARG-7-R: 5'-GTCCTTCCTCCAC

AGCCCCTAAGAT-3'; PPARG-8-F: 5'-CTGTGGAGGAAGGACATGATTATGT-3',

4

PPARG-8-R: 5'-TTCTGGGCCTGATCCTCTTTGGGGA-3'; PPARG-9-F: 5'-AGGATCAGG

CCCAGAACAGTATGCT-3', PPARG-9-R: 5'-AGGCA CGAGAAACAGTTTCTCATGT-3';

PPARG-10-F: 5'-AAACTGTTTCTCGTGCCTCACGTCC-3', PPARG-10-R: 5'-ACATGG

TTATTCACAAGTCA CTGAC-3'.

In vivo study of PPARg function

To generate cells stably expressing PPARg-shRNA, PPARg-shRNA viral particles were

purchased from Genecopoeia, and the target sequence of the shRNA was 5’-

CAACAGGCCTCATGAAGAA-3’. After 24 h of infection, PPARg-shRNA stable C1498 cells

were selected in medium containing 1 µg/mL puromycin for 14 days. After 2-3 passages in the

presence of puromycin, the cultured cells were used for experiments without cloning. Control-

shRNA or PPARg-shRNA C1498 cells (2 x 106) were injected intravenously into C57BL/6

mice aged between 6 and 8 weeks. One week later, transplanted mice were sacrificed and

analyzed for lipid levels once a week for 4 weeks.

Measurement of liver lipids

The fresh mouse liver was isolated and ground uniformly, and 100 mg of liver tissue was

accurately weighed into the EP tube. Then, 1 mL of a chloroform/methanol solution

(chloroform: methanol = 2:1, V/V) was added, followed by vigorous vortexing of the mixture

for 1 min and letting it stand for 2 h on ice. Then, the mixture was centrifuged at 1650 g for 10

minutes at 4 °C. The mixture was separated into 3 layers: the upper methanol layer, the middle

5

protein disc, and the bottom chloroform and lipids. The bottom phase was transferred to a new

EP tube and stored at -80 °C before use. The total TG and TC levels were measured by using

commercial kits according to the manufacturers’ protocols.

Isolation of primary mouse hepatocytes

Primary hepatocytes were isolated from normal mouse livers as described in JoVE 1. Briefly,

mouse livers were perfused in situ with EGTA solution followed by pronase (Sigma, P5147)

solution and collagenase D (Roche, 11088882001) solution at 37 °C for primary hepatic cell

isolation. After perfusion, the liver was transferred into a sterile Petri dish containing protease

and collagenase D solution. After being gently minced, the liver and cell suspension were

filtered through a 70-µm cell strainer and centrifuged at 300 g for 3 min at 4 °C to isolate

hepatocytes. Primary hepatocytes were cultured in DMEM (Gibco, 10564029) supplemented

with 10% FBS, insulin (Sigma, I9278), dexamethasone (Sigma, D4902) and penicillin-

streptomycin (Gibco, 15070063) on collagen-coated dishes (Corning, 354236).

Coculture of APL cells with hepatocytes

For coculture, primary mouse hepatocytes were preseeded in 24-well plates and cultured with

DMEM supplemented with 10% FBS, insulin, dexamethasone and penicillin-streptomycin on

collagen-coated dishes overnight. Primary isolated APL or AML cells (5 ́ 104 cells/well in 10%

FBS in RIPM 1640) were added to the transwell (STEMCELL, #38024) upper chamber, and

hepatocytes and leukemia/normal spleen cells were added at a ratio of 10:1. The

6

leukemia/normal cells and hepatocytes were cocultured together at 37 °C for 48 h and then

separated for further analysis.

ELISA and TC/TG measurement

Human resistin and leptin levels in plasma or culture supernatant were determined by enzyme-

linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (Beijing 4A

Biotech Co., Ltd, China). Mouse PCSK9 levels in plasma or culture supernatant were

determined by ELISA according to the manufacturer’s instructions (Sino Biological, Beijing,

China). Cholesterol and triglycerides (TGs) in the cell culture were assessed by an enzymatic

assay according to the supplier’s protocols (Applygen Technologies Inc., Beijing, China).

Stable cell lines

To generate cells stably expressing TRIB3-shRNA1/2 (T3 sh) or control-shRNA (CTRL),

plasmids were transfected into NB4 cells with Lipofectamine LTX with Plustransfection

reagent (Invitrogen, CA, USA) according to the manufacturer’s instructions. After 24 h of

transfection, stable transfectants were selected in medium containing 1 µg/mL puromycin

(Gibco, CA, USA) for 14 days. After 2-3 passages in the presence of puromycin, the cultured

cells were used for experiments without cloning. To generate cells stably expressing TRIB3Cas9,

TRIB3Cas9 viral particles were purchased from TransOMIC Technologies Inc., and the gRNA

target sequence was 5’-GTGCTGGTGACAGTGCGCCA-3’. Cas9 then cut at this sequence,

and nonhomologous end-joining (NHEJ) took place. TRIB3-C (CRISPR Clone ID) was used

7

to construct stable TRIB3Cas9 NB4 cells. After 24 h of infection, stable cells were selected in

medium containing 1 µg/mL puromycin for 14 days. After 2-3 passages in the presence of

puromycin, the cultured cells were used for experiments without cloning.

Flow cytometry

Fluorescently labeled antibodies against the following surface proteins were used for human

cell staining: CD11b (human), annexin-V FITC, and proprium iodide (PI). Data were acquired

using a FACSCanto II flow cytometer (BD, CA, USA). FCS Express software was used for data

analysis.

Immunoprecipitation, immunoblotting, and immunostaining

Co-IP experiments were performed as described previously23. Briefly, cells were collected and

lysed for 30 min on ice. Soluble lysates were incubated with the indicated antibodies at 4 °C

overnight, followed by incubation with Protein A/G Plus-Agarose (Santa Cruz Biotechnology,

TX, USA) at 4 °C for 2 h. Immunocomplexes were separated from the beads and then boiled

for 10 min. The precipitated proteins were subjected to SDS-PAGE and blotted with specific

antibodies. For immunoblotting assays, proteins were extracted from cells using RIPA buffer

(Cell Signaling Technology, MA, USA). A BCA Protein Assay Kit was used to determine

protein concentrations. Protein extracts were separated by SDS-PAGE, transferred onto PVDF

membranes, and subjected to immunoblot analysis. Western blot images were captured by a

Tanon 5200 chemiluminescent imaging system (Tanon, Shanghai, Beijing).

8

Real-time PCR and RNA interference

Total RNA was extracted using TRIzol (Invitrogen, CA, USA) according to the manufacturer’s

instructions. Reverse transcription of the total cellular RNA was carried out using oligo (dT)

primers and M-MLV reverse transcriptase (Transgen Biotech, Beijing, China). PCR was

performed using a Mycycler thermal cycler and analyzed using agarose gels. The sequences of

the PCR primers were as follows: PPARG forward, 5′-TCTCTCCGTAATGGAAGACC-3′;

PPARG reverse, 5′-CCCCTACAGAGTATTACG-3′; RETN forward, 5′-

AGCCATCAATGAGAGGATCCAG-3′; RETN reverse, 5′-TCCAGGCCAATGCTGCTT-3′;

GPHN forward, 5′-TCGCCTCTCTACAGCTTCCT-3′; GPHN reverse, 5′-

CTGCACCTGGACTGGACATT-3′; ME1 forward, 5′-CGGAACCCTCACCTCAACAA-3′;

ME1 reverse, 5′-GTTGAAGGAAGGTGGC AACA-3′; LEP forward, 5′-ATGCATTGG

GGAACCCTGTGCGG-3′; ME1 reverse, 5′-TGAGGTCCAGCTGCCACAGCATG-3′;

LTC4S forward, 5′-ACGAGGTAGCTCTACTGGCTG-3′; LTC4S reverse, 5′-

ACCTGCAGGGAGAAGTAGGC-3′; DHCR7 forward, 5′-GCCATGGTCAAGGGCTACTT-

3′; DHCR7 reverse, 5′-ACTTCCCGA TCCGAGGGTTA-3′; NKIRAS1 forward, 5′-

GCTGCAAGGTTGTGGTTTGT-3′; NKIRAS1 reverse, 5′-CACGCCTTCCTGTAGACCTC-

3′; MOSC2 forward, 5′-GCAAGCAGCCTTCCTCAAAC-3′; MOSC2 reverse, 5′-

GCCTCATTGCCACAGTCTCT-3′; ABCA1 forward, 5′-ATGGCTTGTTGGCCTCAGC-3′;

ABCA1 reverse, 5′-GCAGCAGCTGACATGTTTGT-3′; GAPDH forward, 5′-

GTGGACATCCGCAAAGACC-3′; and GAPDH reverse, 5′-CCTAGAAGCATTTGCGGTG-

9

3′. TRIB3 siRNAs were produced by RiboBio (Guangzhou, China) and transfected using

Lipofectamine RNA interference MAX Transfection Reagent (Life Technologies, CA, USA)

according to the manufacturer’s instructions.

Gene set enrichment analysis (GSEA)

We ranked the 11,378 genes by their association with the APL (n = 50) and non-APL (n = 50)

groups (GSE13204) using the signal-to-noise measure in the GSEA program according to log2-

fold changes. Lipid metabolic gene sets were collected from the database

(http://software.broadinstitute.org/gsea/msigdb/index.jsp).

Antibody table

ANTIBODY SOURCE IDENTIFIER Anti-TRIB3 antibody Abcam ab75846

Anti-TRIB3 antibody Abcam ab137526

PPARγ antibody CST C26H12

Anti-leptin antibody Abcam Ab3583

Anti-resistin antibody Abcam Ab124681

Gapdh Zsjqbio Ta-08

Anti-PML protein antibody Abcam Ab96051

PML protein antibody Novus NB100-59787

RXRα/β/γ antibody Santa Cruz sc-46659

Anti-Myc-tag mAb MBL BIOTECH M047-3

Anti-Flag-tag mAb MBL BIOTECH PM020

Ubiquitin antibody CST 3933S

Anti-retinoic acid receptor alpha antibody Abcam ab28767

β-Actin (D6A8) rabbit mAb CST 8457S

Anti-Myc magnetic beads Biotool B26302

Anti-Flag magnetic beads Biotool B26102

ALEXA FLUOR(R) 488 RABBIT Invitrogen A21210

Mounting medium with DAPI Zsbio ZLI-9557

10

Donkey anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor 647

Invitrogen A31573

Donkey anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor 555

Invitrogen A31570

Alexa Fluor® 647 anti-mouse/human CD11b Biolegend 101220

11

Supplementary Figures and Legends

Supplementary Figure S1. (A) qRT-PCR was performed to analyze the mRNA levels of GPHN, DHCR7, ABCA1, LTC4S,

MOSC2, and ME1 in primary APL cells and non-APL AML cells. (B) Serum leptin levels in newly diagnosed APL patients

(n = 34) and non-APL AML patients (n = 13). (C) The effects of PML-RARa overexpression on the protein levels of

PPARg, resistin, leptin, and TRIB3 in U937 cells. (D) The effects of PML-RARa overexpression on the mRNA levels of

RETN and LEP in U937 cells. The indicated mRNA levels in U937 cells with or without PML-RARa overexpression were

detected by qRT-PCR.

12

Supplementary Figure S2. TRIB3 and PPARG genes are direct targets of PML-RARa. (A) The TRIB3 promoter was

analyzed. Fragments located upstream of ATG were used as the promoter region. (B) ChIP-qPCR assays were performed

using different specific primers corresponding to the TRIB3 promoter region. (C) The PPARG promoter was analyzed.

Fragments located upstream of ATG were used as the promoter region. (D) ChIP-qPCR assays were performed using

different specific primers corresponding to the PPARG promoter region.

13

References

1. Au - Cui A, Au - Hu Z, Au - Han Y, Au - Yang Y, Au - Li Y. Optimized Analysis of In Vivo and In VitroHepatic Steatosis. JoVE. 2017(121):e55178.

PatientNo

APL =1; non-APLAML = 2 Gender Age

Height(cm)

Body weight(kg)

BMI>25=1; <25=0 BMI

1 1 Male 45 168 77 1 27.2817462 1 Female 51 162 53 0 20.19509223 1 Female 37 163 50 0 18.81892434 1 Male 19 176 70 0 22.59814055 1 Female 30 163 47 0 17.68978896 1 Female 29 156 65 1 26.70940177 1 Female 25 160 54 0 21.093758 1 Male 38 172 69 0 23.32341819 1 Female 61 162 55 0 20.957171210 1 Female 52 161 65 1 25.07619311 1 Female 34 160 60 0 23.437512 1 Female 15 160 50 0 19.5312513 1 Male 52 179 90 1 28.08901114 1 Female 61 157 60 0 24.341758315 1 Female 44 162 60 0 22.862368516 1 Male 34 180 85 1 26.234567917 1 Male 27 178 107 1 33.770988518 1 Male 66 173 70 0 23.388686619 1 Female 26 160 55 0 21.48437520 1 Female 34 160 62 0 24.2187521 1 Female 13 156 45 0 18.491124322 1 Male 29 170 65 0 22.491349523 1 Female 24 153 72 1 30.75740124 1 Female 21 165 60 0 22.038567525 1 Female 29 170 73 1 25.259515626 1 Male 33 168 72 1 25.510204127 1 Male 41 175 75.5 0 24.653061228 1 Male 39 168 69 0 24.447278929 1 Male 57 170 74 1 25.605536330 1 Male 34 170 81 1 28.027681731 1 Female 44 162 54 0 20.576131732 1 Female 30 160 54 0 21.0937533 1 Female 61 154 60 1 25.299375934 1 Male 49 165 71 1 26.078971535 1 Female 62 156 76 1 31.229454336 1 Female 63 172 70 0 23.661438637 1 Male 43 180 78 0 24.0740741

Table S1. Clinical information of patients samples used in Figure 1

38 1 Male 47 170 77 1 26.643598639 1 Female 45 162 75 1 28.577960740 1 Male 45 170 80 1 27.681660941 1 Female 28 160 75 1 29.29687542 1 Female 29 165 57.5 0 21.120293843 1 Male 44 162 67.5 1 25.720164644 1 Male 54 167 65 0 23.306680145 1 Female 16 152 65 1 28.133656546 1 Male 41 168 74 1 26.218820947 1 Male 33 173 59 0 19.713321548 1 Female 19 163 68 1 25.593737149 1 Male 40 165 62 0 22.773186450 1 Male 30 174 94 1 31.047694551 1 Male 47 160 61 0 23.82812552 1 Male 27 177 95 1 30.323342653 1 Male 46 160 72 1 28.12554 1 Female 17 165 62 0 22.773186455 1 Male 50 170 78 1 26.989619456 1 Male 32 174 78 1 25.762980657 1 Male 59 174 81 1 26.753864458 1 Male 26 179 95 1 29.649511659 1 Male 17 171 80 1 27.358845560 1 Male 28 175 80 1 26.12244961 2 Male 50 168 77 1 27.28174662 2 Male 37 178 77 0 24.302487163 2 Female 29 166 49 0 17.781971364 2 Male 39 178 79 0 24.933720565 2 Female 48 165 58 0 21.303948666 2 Male 48 176 91 1 29.377582667 2 Male 55 168 70 0 24.801587368 2 Female 57 160 48 0 18.7569 2 Female 53 165 49 0 17.998163570 2 Male 43 170 70 0 24.221453371 2 Male 59 169 75.5 1 26.434648672 2 Male 26 181 65 0 19.840664273 2 Male 43 172 81 1 27.379664774 2 Male 17 183 100 1 29.860551275 2 Male 24 165 61 0 22.40587776 2 Male 45 174 73.5 0 24.276654877 2 Male 24 180 95 1 29.320987778 2 Male 42 172 90 1 30.421849679 2 Female 28 150 53 0 23.5555556

80 2 Male 54 170 77 1 26.643598681 2 Male 38 176 95 1 30.66890582 2 Female 17 150 61 1 27.111111183 2 Female 49 158 55 0 22.031725784 2 Female 26 162 55 0 20.957171285 2 Male 54 183 97 1 28.964734786 2 Female 39 160 66 1 25.7812587 2 Male 41 165 65 0 23.875114888 2 Male 39 174 72 0 23.781212889 2 Male 57 155 54 0 22.476586990 2 Male 51 165 65 0 23.875114891 2 Male 45 170 85 1 29.411764792 2 Female 45 166.5 63 0 22.725428193 2 Male 21 167 56 0 20.079601394 2 Female 43 159 59 0 23.337684495 2 Male 42 159 48.5 0 19.184367796 2 Female 25 165 70 1 25.711662197 2 Female 22 160 54 0 21.0937598 2 Male 52 176 95 1 30.66890599 2 Male 49 165 58.5 0 21.4876033100 2 Female 33 165 66 0 24.2424242101 2 Male 24 172 57 0 19.2671714102 2 Female 59 168 55 0 19.4869615103 2 Male 25 188 95 1 26.8786781104 2 Male 29 175 80 1 26.122449105 2 Female 23 165 60.5 0 22.2222222106 2 Female 55 168 55 0 19.4869615107 2 Male 31 181 81 0 24.72452108 2 Female 26 164 60 0 22.3081499109 2 Male 37 168 62.5 0 22.1442744110 2 Female 43 159 70 1 27.6887781111 2 Male 43 165 55 0 20.2020202112 2 Female 31 167 50.5 0 18.1074976113 2 Female 61 158 52 0 20.8299952114 2 Female 28 170 56 0 19.3771626115 2 Male 45 170 75 1 25.9515571116 2 Male 44 172 62 0 20.9572742117 2 Female 38 172 64 0 21.6333153118 2 Female 25 170 59 0 20.4152249119 2 Female 62 159 52 0 20.5688066120 2 Female 19 165 62 0 22.7731864

Name AML types Sample type Gender Age Treatment history1 APL PB+BM M 27 ATRA + Arsenic2 APL PB+BM F 24 Ara-C + ATRA3 APL PB+BM M 62 ATRA + Arsenic4 APL PB+BM M 41 ATRA + Arsenic5 APL PB+BM M 20 ATRA + Arsenic6 APL PB+BM F 49 ATRA + Arsenic7 APL PB+BM M 40 ATRA + Arsenic8 APL PB+BM M 34 ATRA + Arsenic9 APL PB+BM M 34 ATRA + Arsenic10 APL PB+BM F 49 ATRA + Arsenic11 APL PB+BM M 34 ATRA + Arsenic12 APL PB+BM M 44 ATRA + Arsenic13 APL PB+BM M 33 ATRA + Arsenic14 APL PB+BM M 38 ATRA + Arsenic15 APL PB+BM F 41 IA, CAG16 APL PB+BM M 31 CAG17 APL PB+BM M 20 CAG18 APL PB+BM M 38 ATRA19 APL PB+BM F 33 IA20 APL PB+BM M 18 IA21 APL PB+BM M 38 ATRA22 APL PB+BM F 61 HAA23 APL PB+BM M 27 ATRA24 APL BM F 54 HAA25 APL PB M 53 ATRA26 APL PB F 61 HAA27 APL BM+PB M 27 ATRA28 APL BM F 48 ATRA29 APL BM F 16 ATRA30 APL BM F 50 ATRA31 APL BM F 35 ATRA32 APL BM M 58 ATRA33 APL BM F 17 ATRA34 APL BM M 23 Dasatinib35 APL BM F 30 ATRA36 APL BM F 48 ATRA37 APL BM M 58 ATRA38 APL BM M 55 ATRA + Sorafenib39 APL BM F 17 ATRA40 APL BM F 35 ATRA41 APL BM F 16 ATRA42 AML-M2 ETO+ KITPB M 48 HAA43 AML-ETO BM M 20 IA, HAA44 AML PB F 1945 AML-ETO BM M 2146 AML-ETO BM M 48 HAA47 B-ALL PB M 46 Dasatinib48 AML (FLT3-ITD) PB M 50 Sorafenib49 AML (FLT3-ITD) PB F 68 Sorafenib

Table S2. APL patient treatment history and sample details, related to Figure 2, 3, 5,6, and 7.

50 AML PB F 55 HAA51 non-APL PB M 18 CAG52 non-APL PB F 54 HAA53 T-ALL PB M 29 CAG54 non-APL (FLT3-ITD)BM F 15 Dasatinib

APL, Acute Promyelocytic Leukemia; AML, Acute Myelogenous Leukemia; PB, Peripheral Blood; BM, Bone Marrow; F, Female; M, Male; ATRA, All-trans retinoic acid; HAA, Homoharringtonine combined aclarubicin and cytarabine; CAG, Cytarabine, Aclarubicin and G-CSF; IA, Idarubicin.