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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: [email protected]; [email protected] 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.