Abstract
Neuroblastoma (NB) is a childhood cancer arising from sympatho-adrenal
neural crest cells. MYCN amplification is found in half of high-risk NB
patients; however, no available therapies directly target MYCN. Using
multi-dimensional metabolic profiling in MYCN expression systems and
primary patient tumors, we comprehensively characterized the metabolic
landscape driven by MYCN in NB. MYCN amplification leads to
glycerolipid accumulation by promoting fatty acid (FA) uptake and
biosynthesis. We found that cells expressing amplified MYCN depend
highly on FA uptake for survival. Mechanistically, MYCN directly
upregulates FA transport protein 2 (FATP2), encoded by SLC27A2. Genetic
depletion of SLC27A2 impairs NB survival, and pharmacological SLC27A2
inhibition selectively suppresses tumor growth, prolongs animal
survival, and exerts synergistic anti-tumor effects when combined with
conventional chemotherapies in multiple preclinical NB models. This
study identifies FA uptake as a critical metabolic dependency for
MYCN-amplified tumors. Inhibiting FA uptake is an effective approach
for improving current treatment regimens.
Subject terms: Cancer metabolism, Targeted therapies, Paediatric cancer
__________________________________________________________________
Half of high-risk neuroblastoma patients have MYCN amplification. Here,
the authors show that MYCN induces fatty acid uptake and synthesis to
support neuroblastoma and inhibition of a fatty acid transporter
impairs tumor progression in preclinical models.
Introduction
Neuroblastoma (NB), a childhood solid tumor of the sympathetic nervous
system, accounts for 15% of total childhood cancer mortality^[62]1.
Despite multimodal therapies, the 5-year survival rate for high-risk
patients with NB remains below 50%^[63]1. Almost half of high-risk NB
patients harbor the amplified MYCN oncogene, the primary oncogenic
driver of NB, leading to resistance to therapy and poor clinical
outcomes^[64]2,[65]3. Pleiotropic effects and the lack of enzymatic
activity^[66]4 make directly targeting the MYCN oncoprotein
challenging. Therefore, new strategies for disrupting MYCN oncogenic
programming are critical for developing effective NB therapies. MYCN
facilitates NB growth in complex microenvironments with limited
nutrient availability by overriding hypoxia-inducible factor
(HIF1α)-mediated cell cycle arrest and promoting intratumoral vascular
development to facilitate nutrient access by interior tumor
regions^[67]5,[68]6. However, the mechanisms underlying the MYCN
reprogramming of NB tumors that permit nutrient recruitment from the
microenvironment remain unknown.
Compared with normal cells, tumor cells are metabolically reprogrammed
to promote biomass and energy production to support rapid cell
growth^[69]7. These metabolic changes are driven by oncogenic stimuli
(e.g. phosphatidyl inositol 3-kinase [PI3K]–AKT–mechanistic target of
rapamycin complex 1 [mTORC1]^[70]8,[71]9, MYC^[72]10, and RAS^[73]11),
loss of tumor suppressors (p53)^[74]12, and metabolic enzyme
dysregulation (e.g. isocitrate dehydrogenase 1, IDH1)^[75]13. In tumors
with activated MYC, increased growth demand is sustained through
metabolic reprogramming^[76]14, including stimulation of glucose and
glutamine consumption^[77]15,[78]16, mitochondrial biogenesis^[79]17,
and biosynthesis of lipids, proteins, and nucleotides^[80]18–[81]20.
MYCN appears to exert similar metabolic functions, promoting
glycolysis^[82]21, lipogenesis^[83]22,[84]23, and metabolism of
glutamine^[85]21,[86]24, serine^[87]25, and polyamine^[88]26 to enhance
macromolecular biosynthesis and energy production. However, we lack a
comprehensive understanding of how MYCN-amplified tumors rewire their
metabolism and how to incorporate these findings into novel treatment
modalities. To address these knowledge gaps, we comprehensively
characterized the metabolic landscape driven by MYCN in primary patient
samples and multiple in vitro inducible systems.
Lipids play essential roles in supporting tumor survival as membrane
components, energy reservoirs, and signaling mediators^[89]27.
Glycerolipids, such as diacylglycerols (DGs), function as secondary
messengers that directly bind protein kinase C, RAS guanyl
nucleotide-releasing protein, and chimaerins to modulate downstream
oncogenic signaling^[90]28. DGs and fatty acids (FAs) assemble into
triacylglycerols (TGs), which are stored in lipid droplets (LDs) as a
FA reservoir. Under nutrient deprivation conditions, TGs are
hydrolyzed, releasing FAs to maintain lipid homeostasis and meet energy
requirements (via β-oxidation)^[91]29. Cancer cells actively obtain FAs
through endogenous biosynthesis and uptake from the
microenvironment^[92]27. Endogenous lipogenic mechanisms include de
novo synthesis from acetyl-CoA, FA desaturation, and elongation.
Numerous studies support a role for de novo lipogenesis in
oncogenesis^[93]27,[94]30. We and others have shown that MYC or MYCN
[MYC(N)] promotes FA synthesis by cooperating with Mondo A–sterol
regulatory element-binding protein 1 (SREBP1) or directly activating
transcription of key lipogenic enzymes, including acetyl-CoA
carboxylase (ACC), fatty acid synthase (FASN), and stearoyl-CoA
desaturase (SCD1)^[95]18,[96]23,[97]31. Recently developed FA synthesis
inhibitors (e.g., ACC inhibitor ND-646^[98]32; FASN inhibitors TVB-2640
and orlistat^[99]22,[100]33; and SCD1 inhibitor A939572^[101]34) show
varying degrees of anti-tumor activity in preclinical models. However,
few have progressed to clinical trials, including TVB-2640
([102]NCT03808558, [103]NCT03179904, and [104]NCT03032484) and orlistat
(non-cancer trials). The limited clinical efficacy of these inhibitors
may be due to limited compound specificity or the activation of
compensatory FA uptake mechanisms mediated by oncogenic signaling, such
as HIF1α^[105]35, mTOR^[106]36, and RAS^[107]11. FAs are imported by
the membrane CD36 FA translocase and FA transport proteins (FATP1–6,
encoded by SLC27A1–6) and trafficked intracellularly via FA binding
proteins (FABP1–12)^[108]37. Emerging evidence supports a role for FA
transporters in oncogenesis and chemoresistance. CD36 promotes prostate
cancer growth and oral carcinoma metastases^[109]38,[110]39, and drives
acquired resistance to lapatinib in HER2-postive breast cancer^[111]40.
FATP1 (SLC27A1) accelerates melanoma initiation, and FATP2 (SLC27A2)
confers melanoma resistance to BRAF and MEK inhibition by promoting FA
uptake from the microenvironment^[112]41,[113]42. However, the
mechanisms underlying MYC(N) regulation of FA transport in
proliferation, disease progression, and resistance to therapy remain
unclear.
Using untargeted metabolomics in both in vitro models and patient
samples, we characterized the metabolic landscape driven by MYCN in NB
and identified key metabolic nodes relevant to NB patients and suitable
for therapeutic intervention. We found that MYCN promotes glycerolipid
accumulation, a finding validated across multiple systems and supported
by targeted lipidomic studies. FAs are required for glycerolipid
synthesis. We found that MYCN enhances FA uptake to support cell
survival. Mechanistically, we identified the FATP2-encoding SLC27A2 as
a novel MYCN transcriptional target required for NB growth. Inhibiting
FA uptake by targeting SLC27A2 blocked tumor growth, prolonged animal
survival, and enhanced the efficacy of conventional chemotherapy in
multiple preclinical NB models. This metabolic dependency identified in
MYCN-driven NBs suggests that targeting FA uptake represents a
promising strategy for improving current therapies in high-risk
disease.
Results
MYCN reprograms NB metabolism and promotes glycerolipid accumulation
MYCN promotes various metabolic adaptations to support tumor
growth^[114]21–[115]26,[116]43,[117]44. However, whether these
adaptations are directly relevant to NB patients or can be targeted for
therapeutic interventions remains incompletely understood. To address
this knowledge gap, we performed untargeted metabolomics (Metabolon
Inc., Discovery HD4^™ platform) in two NB cell lines with perturbed
MYCN expression (LAN5 shMYCN and MYCN3 Tet-On) and NB primary tumors
(MYCN-amplified [MNA] n = 18; non MYCN-amplified [non MNA] n = 18,
Supplementary Data [118]1). In the shMYCN model, MYCN was conditionally
knocked down (KD) using small hairpin RNA (shRNA)-mediated gene
silencing (1 µg/mL doxycycline [DOX] 0–96 h) in MNA LAN5 cells. In the
MYCN3 Tet-On model (MYCN-ON), MYCN was conditionally turned on (1 µg/mL
DOX, 0–72 h) in non-MNA SHEP sub-cloned cells (Fig. [119]1a, top, and
Supplementary Fig. [120]1a, left). In total, 580 and 789 metabolites
were analyzed in cells and primary tumors, respectively (Supplementary
Data [121]2). MYCN-induced metabolic alterations across different
models are presented in pathway classification networks (Fig. [122]1b).
Blue and red circles indicate significantly downregulated and
upregulated metabolites (p ≤ 0.05), respectively. Circle sizes
represent the absolute log[2] fold change (FC). Notably, MYCN
differentially altered metabolite levels in lipid, amino acid,
carbohydrate, and nucleotide super pathways (p ≤ 0.05), with lipid
metabolism as the most represented category (>35% differential
metabolites, median log[2]FC > 0.56, Supplementary Fig. [123]1a, right
and bottom). These findings suggest that MYCN globally reprograms NB
metabolism, particularly lipid metabolism.
Fig. 1. MYCN reprograms NB metabolism.
[124]Fig. 1
[125]Open in a new tab
a Untargeted metabolomics profiling workflow using UHPLC-MS/MS and
GC-MS (Discovery HD4^™ platform, Metabolon Inc.) in LAN5 cells (MYCN KD
for 0, 72, and 96 h, n = 4 each), MYCN3 cells (MYCN-ON for 0, 48, and
72 h, n = 4 each), and primary tumors (MNA, n = 18; non-MNA, n = 18). b
Metabolite classification network. Each circle represents a metabolite.
Circle size indicates absolute log[2]FC of metabolite level in
comparisons of MYCN KD 72 h vs. CTRL, MYCN-ON 72 h vs. MYCN-OFF and MNA
vs. non-MNA. Red=upregulated metabolite; blue=downregulated metabolite
(p ≤ 0.05). One-way ANOVA or Welch’s two-sample t-test was used to
compare metabolite levels between groups. c Metabolic changes within
subpathways. Comparison groups are the same as in (b). Differential
abundance scores were calculated from subpathways containing at least
three measured metabolites. 100 or −100 = all metabolites in the
subpathway are upregulated or downregulated (p ≤ 0.05). Circle
size=number of differentially altered metabolites (p ≤ 0.05).
*indicates FDR < 0.25, hypergeometric test with p-value adjusted by
Benjamini–Hochberg procedure. d Pathway enrichment analysis using
GSEA-based algorithm. Subpathways with FDR < 0.25 were selected and
ranked by –log[10](FDR). Red=upregulated subpathway; blue=downregulated
subpathway. e Lipidomics profiling in LAN5 shMYCN (CTRL and MYCN KD for
72 h), MYCN3 [MYCN-OFF (-DOX) and MYCN-ON (+DOX) for 72 h] and SK-N-AS
MYCN-ER^™ [MYCN-OFF (−4-OHT) and MYCN-ON (+4-OHT) for 48 h] cells
(n = 4 each). Two-sided unpaired t-test; p-value adjusted by
Benjamini–Hochberg procedure to obtain FDR. Significantly altered
lipids (FDR < 0.25) were selected for heatmap (color is scaled by
Z-score: red=upregulated; blue=downregulated). Percentages of
upregulated and downregulated lipid classes are shown in stacked bar
graphs. DG diacylglycerol, TG triacylglycerol, PC phosphatidylcholine,
PE phosphatidylethanolamine, PG phosphatidylglycerol, PI
phosphatidylinositol, PS phosphatidylserine, CE cholesteryl ester,
plasmenyl. PE plasmenyl phosphatidylethanolamine. KD knockdown, MNA
MYCN-amplified, non-MNA non MYCN-amplified. Source data are provided in
the Source Data file.
To identify metabolic changes in each subpathway, we used differential
abundance analysis^[126]45 for three comparisons (MYCN KD vs. CTRL,
MYCN-ON vs. MYCN-OFF, and MNA vs. non-MNA). An abundance score of 100
indicates that 100% of the subpathway metabolites are significantly
upregulated (p ≤ 0.05), whereas a score of −100 indicates that 100% of
the subpathway metabolites are significantly downregulated (p ≤ 0.05).
Significantly enriched subpathways (FDR < 0.25, Benjamini–Hochberg
adjusted p-value) are marked with asterisks “*”. Subpathways with the
same number of “*” designations were ranked by the average absolute
differential abundance score. Notably, the DG group was the most
differentially abundant pathway, consistently upregulated by MYCN in
all three systems (differential abundance score: MYCN KD = − 25; MYCN
ON = 70; MNA = 56; Fig. [127]1c). Other MYCN-altered subpathways
included: phospholipid (PL), glutamate, endocannabinoid, methionine,
cysteine, S-adenosylmethionine (SAM) and taurine metabolism, and
phosphatidylcholine (PC) (FDR < 0.25, Fig. [128]1c and Supplementary
Fig. [129]1b). To further support our findings, we performed gene set
enrichment analysis (GSEA; Fig. [130]1d and Supplementary Data [131]2).
DG was the most enriched subpathway in MNA patients compared with
non-MNA patients and was consistently induced by MYCN in both in vitro
systems (FDR < 0.25, Fig. [132]1d). Polyamine metabolism was also
upregulated in MNA patients, consistent with previous studies showing
that MYCN activates polyamine metabolism^[133]26. The fatty acyl
carnitine group was highly enriched upon MYCN activation, suggesting
the upregulation of FA oxidation, but no enrichment was observed in
other systems. Collectively, these results suggest that MYCN alters
lipid metabolism and promotes DGs accumulation in NB tumors.
To specifically evaluate the lipid metabolic changes induced by MYCN,
we performed targeted lipidomics in LAN5 shMYCN, MYCN3 Tet-On cells,
and SK-N-AS MYCN-ER^™ cells, in which MYCN-mediated transcription is
conditionally activated by 4-hydroxytamoxifen (4-OHT)^[134]23.
Significantly altered lipids are represented in the heatmap
(FDR < 0.25, Fig. [135]1e and Supplementary Data [136]3). All TGs
(downstream of DGs) were significantly downregulated upon genetic
depletion of MYCN (FDR < 0.25, Fig. [137]1e, left), whereas most DGs
and TGs were upregulated upon MYCN induction (FDR < 0.25, Fig. [138]1e,
middle and right). Because FAs are required for glycerolipid synthesis,
we analyzed the FA chain compositions of MYCN-upregulated glycerolipids
across the three systems. The FA chains 14:0, 16:0, 16:1, and 18:1 were
the most represented (>30%, Supplementary Fig. [139]1c), suggesting
that these FAs contribute to glycerolipid synthesis. To examine how
MYCN alters FA compositions, we applied mass spectrometry to profile
FAs in MYCN3 Tet-On cells and TH-MYCN^+/+ transgenic mice, in which
neural crest-specific MYCN expression drives spontaneous NB formation
recapitulating human disease^[140]3. NB cells and tumors expressing
high MYCN levels also contained high levels of 14:0, 16:1, or 18:1
(FDR < 0.25, Supplementary Fig. [141]1d–e), suggesting that MYCN
upregulates these FAs for glycerolipid synthesis. In addition, MYCN
increased the ratios of 14:1 to 14:0 and 16:1 to 16:0 (Supplementary
Fig. [142]1d–e), thus MYCN also promotes FA desaturation. Collectively,
these results indicate that MYCN changes the abundance of FAs required
for glycerolipid accumulation.
MNA cell survival relies highly on FA uptake
FAs can be synthesized in cells or imported from the microenvironment.
To dynamically examine how MYCN regulates FA synthesis, we added
deuterated water (D[2]O) and [U-^13C]16:0 to SK-N-AS MYCN-ER^™ cell
culture medium to trace de novo FA synthesis and desaturation,
respectively. MYCN activation (4-OHT for 24 h) promoted de novo FA
synthesis by increasing deuterium-labeled 16:0, and enhanced FA
desaturation by increasing the ratios of [^13C[16]]16:1 to
[^13C[16]]16:0 and [^13C[16]]18:1 to [^13C[16]]18:0 (p < 0.05,
Fig. [143]2a). To determine the effects of MYCN on FA uptake, we traced
changes in cell media supplemented with [^13C[16]]16:0, the most
abundant FA in mammalian cells, as an indicator of uptake. MYCN
activation significantly enhanced FA uptake (p < 0.05, Fig. [144]2a),
suggesting that MYCN promotes lipid accumulation by enhancing both FA
synthesis and uptake. These findings were validated in a second MYCN
system (MYCN3 Tet-On cells, p < 0.05, Supplementary Fig. [145]2a),
indicating that MYCN enhances intracellular FA availability for lipid
synthesis.
Fig. 2. MNA cell survival relies on FA uptake.
[146]Fig. 2
[147]Open in a new tab
a Stable isotope tracing of FA synthesis and FA uptake in SK-N-AS
MYCN-ER^™ cells with or without 4-OHT (500 nM) for 24, 48, and 72 h.
Mean ± SD (n = 3); two-sided unpaired t-test per time point. b
Viability of NB and normal cells upon 72 h treatment with FA synthesis
inhibitors (A939572, orlistat) and FA uptake inhibitors (CB16.2 and
CB5). IC[50] calculated in GraphPad Prism (7.01). Mean ± SD (n = 3). c
Top, cell viability in complete media, delipidized media, and
delipidized media supplemented with 0.025% FAs. MNA: LAN5 (0–6 day),
IMR32 (0–4 day) and SK-N-BE(2c) (0–6 day); non-MNA: SHEP (0–6 day) and
SK-N-AS (0–6 day). Mean ± SD (n = 3); two-way ANOVA with Dunnett’s
multiple comparisons test. Bottom, Caspase 3/7 activity in complete
media, delipidized media, and delipidized media supplemented with
0.025% FAs for 4 days. Mean ± SD (n = 3); one-way ANOVA with Dunnett’s
multiple comparisons test. FC fold change, CM complete media, DLM
delipidized media. Source data are provided in the Source Data file.
To assess the impact of FA synthesis and uptake on cell survival, we
evaluated NB cell viability [MNA: LAN5, IMR32, SK-N-BE(2c); non-MNA:
SH-SY5Y, SHEP, SK-N-AS] and normal cells (ARPE-19, C2C12, and HS-5)
after treatment with two FA synthesis inhibitors (A939572, an SCD1
inhibitor and orlistat, an FASN inhibitor)^[148]22,[149]34 or two FA
uptake inhibitors (CB16.2 and CB5, which target FATP2)^[150]46
(Fig. [151]2b). NB cells were more sensitive to FA uptake inhibition
(MNA IC[50]: 0.5–7.9 µM; non-MNA IC[50]: 1.4–10.4 µM) than to FA
synthesis inhibition (MNA IC[50]: 7.6–60.7 µM; non-MNA IC[50]:
9.2–77.5 µM), suggesting that NB cells actively use exogenous FA for
survival. CB16.2 was toxic to all tested normal cells, despite having
the highest efficacy in NB cells. Conversely, CB5 was highly effective
in NB cells and did not elicit cytotoxicity against normal cells. MYCN
enhances SCD1 activity (Supplementary Figs. [152]1d–e and [153]2a),
whereas A939572 suppresses SCD1 activity (p < 0.0001) and de novo FA
synthesis (p < 0.05) in MNA cells (Supplementary Fig. [154]2b, left).
However, SCD1 inhibition did not effectively inhibit cell growth and
stimulated compensatory dose-dependent FA import from the media
(p < 0.05; Supplementary Fig. [155]2b, right), suggesting that
exogenous FA uptake may reduce cell sensitivity to FA synthesis
inhibition. To test this hypothesis, we evaluated the viability of MNA
cells in complete and delipidized media with and without A939572. The
removal of exogenous lipids significantly enhanced the cytotoxicity of
A939572 (IC[50] from 41.0 µM to 2.2 µM), which was partially rescued by
FAs supplementation (Supplementary Fig. [156]2c). Pharmacological
inhibition of FA uptake via CB5 also enhanced the cytotoxic effects of
A939572 (IC[50] from 39.3 µM to 0.4 µM) and increased cell apoptosis
(p < 0.05, Supplementary Fig. [157]2d). These results suggest that NB
cells import exogenous FAs as a compensatory mechanism to evade FA
synthesis inhibition.
To determine whether MYCN-driven FA uptake supports cell survival, we
examined the viability of three MNA (LAN5, IMR32, and SK-N-BE(2c)) and
two non-MNA (SHEP and SK-N-AS) cell lines and SK-N-AS MYCN-ER^™ cells
with and without MYCN activation under complete and delipidized media
conditions. Deprivation of exogenous lipids reduced the viability of
MNA and MYCN-ER activated cells (+4-OHT) to a greater extent than
non-MNA and MYCN-ER CTRL cells (−4-OHT) (Fig. [158]2c and Supplementary
Fig. [159]2e). Moreover, FA supplementation partially restored
viability of MNA and MYCN-ER activated cells (p < 0.05; Fig. [160]2c
and Supplementary Fig. [161]2e), suggesting that exogenous FAs support
MYCN-driven cell survival. Similarly, lipid deprivation induced cell
apoptosis in MNA and MYCN-ER-activated cells (Fig. [162]2c and
Supplementary Fig. [163]2e), which was significantly alleviated by FA
supplementation (p < 0.05), suggesting that exogenous FAs selectively
promote cell survival in MYCN-driven cells. Collectively, our data
indicate that MYCN-driven cell growth depends on exogenous FAs.
The FA transporter SLC27A2 is a direct target of MYCN
To further elucidate how MYCN regulates FA import, we assessed the
expression of membrane FA transporter genes (SLC27A1–6 and CD36) in
multiple MYCN models. SLC27A2 was remarkably upregulated in SK-N-AS
MYCN-ER^™ cells upon MYCN activation (10 fold at 48 h, p = 0.0001,
Fig. [164]3a) and in MYCN3 cells upon MYCN induction (p = 0.006,
Supplementary Fig. [165]3a). Moreover, SLC27A2 was the only
significantly downregulated transporter when MYCN was turned off in
Tet-21/N cells (p = 0.01, Fig. [166]3b), suggesting that SLC27A2 is
selectively regulated by MYCN.
Fig. 3. MYCN directly upregulates FA transporter SLC27A2.
[167]Fig. 3
[168]Open in a new tab
a FA transporters (SLC27A1–6, CD36) and ODC1 mRNA expression in SK-N-AS
MYCN-ER^™ cells (1 µM 4-OHT for 0, 24, 48 h). Mean±SEM (n = 3); two-way
ANOVA with Dunnett’s multiple comparisons test. b FA transporters
(SLC27A1–6, CD36) mRNA expression in Tet21/N cells (±2 µg/mL DOX for
24 h). Mean±SEM (n = 3); two-sided unpaired t-test. MYCN protein
expression in Tet21/N cells (−DOX, MYCN-ON and +DOX, MYCN-OFF). c MYCN
ChIP-qPCR assays in TET21/N cells (±2 µg/mL DOX for 48 h). Input and
MYCN ChIP samples were analyzed by qPCR using specific primers for
SLC27A1–6 and CD36. Mean±SD (n = 3); two-way ANOVA with Dunnett’s
multiple comparisons test. d Gene expression analysis in Cohort 1
([169]GSE45547). Left, correlation matrix of transporter gene
expression, MYCN expression/activity, and c-MYC expression.
Correlations with p-values < 0.05 are represented in the heatmap.
Red = positive; blue = negative correlation. Middle, SLC27A2 expression
in MNA (n = 93) and non-MNA patients (n = 550). Two-sided unpaired
Welch’s t-test. Right, SLC27A2 expression in stage 1–4 S patients
(stage 1: n = 153; stage 2: n = 113; stage 3: n = 91; stage 4: n = 214;
stage 4 s: n = 78). One-way ANOVA with Tukey’s multiple comparisons
test. Box plots indicate median (middle line), 25th and 75th
percentiles (box), as well as min and max (whisker). e, f Survival
analyses in Cohort 1 ([170]GSE45547). e OS and EFS rates for stage
1–4 S and stage 3–4 patients with high (top third) or low (bottom
third) SLC27A2 expression. f OS and EFS predictions for genes in the
long-chain FA transport geneset (GO: 0015909); –log[10](p-value).
Kaplan–Meier method was used to plot survival curves, and log-rank test
was used for statistical analysis. Red=high expression has poor
prognosis (p < 0.05); blue = low expression has poor prognosis
(p < 0.05); gray = no significance. FC fold change, OS overall
survival, EFS event-free survival. Source data are provided in the
Source Data file.
To assess MYCN binding to promoter regions of membrane FA transporters
(SLC27A1–6 and CD36), we performed MYCN ChIP-qPCR analysis in both MNA
cells and MYCN Tet-Off cells (Tet-21/N). We found significant
enrichment (>15 fold) of MYCN binding to the promoter region of SLC27A2
(chr15 [hg38]: 50,182,222-50,182,302), which contains a non-canonical
E-box (CACCTG) in MNA cells (LAN5 and IMR32, Supplementary
Fig. [171]3b–c and Supplementary Data [172]4). Moreover, turning off
MYCN almost completely abrogated MYCN binding (Fig. [173]3c). We then
asked whether c-MYC could also bind to SLC27A2. c-MYC ChIP-qPCR
analysis in SH-SY5Y NB cells with high c-MYC expression showed that
c-MYC binds to the same promoter region but with lower affinity (4 fold
enrichment; Supplementary Fig. [174]3b–c). To determine whether MYCN
promoter binding results in gene transcription, we fused a downstream
luciferase gene to the promoters of SLC27A1, wild-type and mutant
SLC27A2, and ODC1 (a known MYCN target)^[175]47 and evaluated
luciferase activity. Turning off MYCN significantly reduced wild-type
SLC27A2-fused luciferase activity (p = 0.01, Supplementary
Fig. [176]3d). However, mutation of the non-canonical E-box (CACCTG to
GAATTC) in the SLC27A2 promoter abrogated this effect (Supplementary
Fig. [177]3d), suggesting that MYCN activates SLC27A2 transcription via
binding to this region. Although MYCN also binds to the SLC27A1
promoter (Fig. [178]3c and Supplementary Fig. [179]3b), no changes in
SLC27A1 transcription activity were detected when MYCN was turned off
(Supplementary Fig. [180]3d). Altogether, our study identified SLC27A2
as a direct transcriptional target of MYCN.
Because MYCN selectively upregulates SLC27A2 expression, we next asked
whether SLC27A2 expression correlates with MYCN expression, activity,
or amplification status in NB patients. SLC27A2 expression positively
correlated with MYCN expression and activity, as indicated by the
summed expression score for 157 MYCN target genes^[181]48 (p < 0.05,
Fig. [182]3d) in a large patient cohort (cohort 1: [183]GSE45547,
n = 649). By contrast, mRNA levels of other FA transporters did not
correlate with MYCN expression or activity, although CD36 expression
positively correlated with c-MYC expression (p < 0.05, Fig. [184]3d,
left). High expression of SLC27A2 also correlated with MYCN
amplification (p = 8.1e^−45) and stage 4 disease (p < 0.01)
(Fig. [185]3d), and strongly predicted poor overall (OS) and event-free
survival (EFS) in patients of all stages (OS: p = 1.0e^−8; EFS:
p = 4.7e^−6) and stage 3–4 high-risk patients (OS: p = 2.7e^−4; EFS:
p = 4.2e^−2, Fig. [186]3e). Compared with other long-chain FA
transport-associated genes ([187]GO: 0015909, n = 75), SLC27A2 ranked
top in predicting poor clinical outcomes (R2, Fig. [188]3f, red dots).
These findings were validated in a second patient cohort (cohort 2:
[189]GSE85047, n = 283, Supplementary Fig. [190]3e–g), suggesting that
SLC27A2 is a critical transporter for NB survival. To explore potential
for targeting SLC27A2, we compared SLC27A2 mRNA expression in three NB
cohorts (Lastowska, [191]GSE13136, n = 30; Hiyama, [192]GSE16237,
n = 51; Versteeg, [193]GSE16476, n = 88) with normal tissue cohort
(Adrenal Gland, [194]SN_ADGL, n = 13; Neural Crest, [195]GSE14340,
n = 5; Normal Various, [196]GSE7307, n = 504 including 108 types of
normal tissues; datasets generated from the same u133p2 platform and
normalized by MAS5.0). Median SLC27A2 expression was higher in MNA
patients than in normal tissues (Supplementary Fig. [197]3h),
suggesting that SLC27A2 could function as a therapeutic target in NB.
Genetic and pharmacological inhibition of SLC27A2 impairs NB survival
To determine the impact of SLC27A2 expression on cell survival, we
analyzed survival outcomes in 789 cell lines after SLC27A2 knockout
using the CRISPR (Avana) Public 20Q4V2 dataset (Broad Institute,
USA)^[198]49. The results were grouped according to primary disease and
ranked using a dependency score. NB ranked tenth among 30 primary
diseases (dependency score = −0.23), suggesting that NB cells depend on
SLC27A2 for survival (Supplementary Fig. [199]4a).
To confirm whether SLC27A2 is required for NB survival, we genetically
depleted SLC27A2 via shRNA-mediated gene silencing (two sequences) in
MNA LAN5 cells (Fig. [200]4a), which express high basal SLC27A2 levels
(Supplementary Fig. [201]4b). SLC27A2 KD in LAN5 cells effectively
reduced FA uptake (p < 0.01, Fig. [202]4b) and impaired cell growth
(p < 0.0001, Fig. [203]4c) and colony forming capacity (p < 0.05,
Fig. [204]4d), suggesting that SLC27A2 is required for cell growth.
This phenotypic outcome was confirmed in a second MNA cell line (IMR32)
(Supplementary Fig. [205]4c) and was selective to MNA cells, as SLC27A2
depletion in non-MNA cells (SK-N-AS) did not affect FA uptake or cell
growth (Supplementary Fig. [206]4c). To further examine whether SLC27A2
is required for in vivo tumor growth, we orthotopically implanted MNA
LAN5 shCTRL and shSLC27A2 cells into NCr nude mice. Silencing SLC27A2
significantly reduced tumor growth (assessed by MRI imaging, p = 0.048)
and tumor weights (p = 0.01, Fig. [207]4e–f). These effects were
associated to a significant reduction of intratumoral neutral lipids
(Oil Red O staining, p = 0.003, Fig. [208]4g). Furthermore, depletion
of SLC27A2 reduced tumor proliferation (Ki67 staining, p = 0.004,
Supplementary Fig. [209]4d) and increased tumor apoptosis (cleaved
Caspase-3 staining, p < 0.0001, Supplementary Fig. [210]4e) without
altering MYCN expression (p = 0.4, Supplementary Fig. [211]4f).
Lipidomic profiling of shCTRL (n = 8) and shSLC27A2 (n = 8) tumors
demonstrated downregulation of the majority of DGs and TGs upon SLC27A2
KD (FDR < 0.25, absolute FC > 2, Fig. [212]4h and Supplementary
Data [213]3), confirming that inhibition of SLC27A2 effectively reverts
MYCN-induced glycerolipid accumulation. Collectively, these data
indicate that SLC27A2 is required for NB cell survival and tumor
growth.
Fig. 4. Suppressing FA uptake impairs NB cell survival.
[214]Fig. 4
[215]Open in a new tab
a Silencing SLC27A2 in LAN5 cells. Two shSLC27A2 GIPZ vectors tested,
with empty GIPZ vector as control. Mean ± SEM (n = 3). b FA uptake in
LAN5 shCTRL and shSLC27A2 cells. Cells stained with the FA analog
BODIPY^™ 558/568 C12 and quantified as CTCF by ImageJ2. Mean±SD
(n = 3). c Cell growth in LAN5 shCTRL and shSLC27A2 cells. Mean ± SD
(n = 3). d Clonogenic assay in LAN5 shCTRL and shSLC27A2 cells.
Mean ± SD (n = 3); e–h LAN5 shCTRL and shSLC27A2 orthotopic xenograft
model. e Tumor volumes at weeks 3 and 4 post-implantation. CTRL = 15;
shSLC27A2 = 14; two-way ANOVA with Sidak’s multiple comparisons test. f
Tumor weights at week 5 post-implantation. Mean ± SEM (CTRL = 13,
shSLC27A2 = 12); two-sided unpaired Mann–Whitney test. g Oil Red O
staining of intratumoral lipids. Mean ± SEM (CTRL = 6, shSLC27A2 = 8);
two-sided unpaired Mann–Whitney test. h Lipidomics profiling of shCTRL
and shSLC27A2 tumors (n = 8 each). Lipids (FDR < 0.25, absolute FC > 2)
are shown in the heatmap (color scaled by Z-score: red = upregulated;
blue = downregulated). MGDG monogalactosyldiacylglycerol, DGDG
digalactosyldiacylglycerol, dhCER dihydroceramides, CL cardiolipin, SM
sphingomyelin, PA phosphatidic acid, lyso.PC lysophosphatidylcholine,
lyso.PE lysophosphatidylethanolamine, plasmenyl.PC
plasmenylphosphatidylcholine, all other abbreviations consistent with
Fig. [216]1e. i FA uptake following CB5 treatment in LAN5 and SHEP
cells (0–15 µM, 5 min). Cells stained with BODIPY^™ 500/510 C1, C12 and
quantified as CTCF by ImageJ2. Mean ± SD (n = 3). j Caspase 3/7
activity of NB and normal cells after CB5 treatment (0–10 µM, 24 h).
Mean ± SD (n = 3). k Apoptosis, p53/p21, and MYCN/c-MYC protein
expression in LAN5, IMR32, and SHEP with CB5 (0–20 µM, 16–24 h). VP16
(10 µM, 24 h) = positive CTRL. Representative blots from three
independent experiments are shown. FC fold change, Arb. Unit arbitrary
unit. a, b, d, and i, one-way ANOVA with Dunnett’s multiple comparisons
test; c and j, two-way ANOVA with Dunnett’s multiple comparisons test.
Source data are provided in the Source Data file.
To pharmacologically target FATP2 (encoded by SLC27A2), we used the
small-molecule FATP2 inhibitor CB5^[217]46, which elicits selective
cytotoxicity against NB cells but spares normal cells (Fig. [218]2b).
We first validated the ability of CB5 to block FA uptake in NB cells by
both stable isotope tracing (Supplementary Fig. [219]2d) and staining
with a fluorescent-labeled FA analog BODIPY^™ 500/510 C1, C12. CB5
preferentially blocked FA uptake in MNA LAN5 cells (p < 0.05) compared
with non-MNA SHEP cells (Fig. [220]4i). Because the FATP2 inhibitor
CB16.2 also suppresses FATP1 activity^[221]41, we verified the
specificity of CB5 for FATP2. Ectopic overexpression of both FATP1 and
FATP2 increased FA uptake in MNA cells. However, CB5 only reduced FA
uptake in FATP2-overexpressing cells (Supplementary Fig. [222]4g),
suggesting that CB5 specifically targets FATP2 in NB. CB5 more
effectively inhibited cell viability and induced apoptosis (determined
by Caspase 3/7 activity) in MNA cells than in non-MNA cells, without
affecting normal cells (Figs. [223]2b and [224]4j), suggesting that
targeting FATP2 selectively impairs MNA cell survival. The selectivity
of CB5 was further supported by the preferential induction of cleaved
PARP and cleaved Caspase-3 expression (markers of apoptosis) in MNA
LAN5 and IMR32 cells compared with non-MNA SHEP cells (p < 0.05,
Fig. [225]4k and Supplementary Fig. [226]5a). CB5 also inhibited MYCN
but not c-MYC protein expression, supporting the selective targeting of
MNA cells. p53 and its downstream target p21(Waf1/Cip1) play critical
roles in cell cycle, proliferation, and apoptosis in the setting of
MYCN amplification. Although p53 can be directly upregulated by MYCN as
a mechanism for MYCN-induced apoptosis^[227]50, we found that CB5
increased both p53 and p21(Waf1/Cip1) protein expression in MNA cells
(p < 0.05, Fig. [228]4k and Supplementary Fig. [229]5a). Thus, CB5
inhibits MYCN and activates p53 signaling to suppress cell growth and
promote apoptosis.
Targeting FA transport effectively suppresses NB tumor growth
To determine the contribution of FA transport to tumor growth, we
evaluated the anti-tumor activity of CB5 in multiple preclinical NB
models. We orthotopically implanted MNA LAN5 luciferase-expressing
cells and non-MNA SK-N-AS cells into the renal capsule of NCr nude mice
(Fig. [230]5a). After tumor engraftment, mice were randomly assigned to
CTRL (vehicle) or CB5 (25 mg/kg, twice a day [b.i.d.], intraperitoneal
injection (i.p.) for 2 weeks) treatment groups. CB5 significantly
inhibited the growth of LAN5 xenografts as measured by luciferase
activity (p = 0.002) and tumor weights (p = 0.03, Fig. [231]5b and
Supplementary Fig. [232]5b). However, CB5 did not reduce tumor volumes
and weights of SK-N-AS xenografts (Fig. [233]5c and Supplementary
Fig. [234]5c), suggesting that CB5 preferentially inhibits MNA tumors.
Notably, CB5 treatment did not cause toxicity assessed by mouse general
clinical conditions and weight changes during treatment (Supplementary
Fig. [235]5b–c).
Fig. 5. Suppressing FA uptake exerts anti-tumor effects in multiple
preclinical models.
[236]Fig. 5
[237]Open in a new tab
a–c NB cell line-derived orthotopic xenograft model. a LAN5 or SK-N-AS
cells were orthotopically implanted in NCr nude mice. Two weeks later
mice were treated with vehicle or CB5 (25 mg/kg, b.i.d., 6 days/week)
for 2 weeks. b LAN5 tumor sizes (IVIS) and weights after treatment.
Mean±SEM (CTRL = 6, CB5 = 6); two-way ANOVA with Sidak’s multiple
comparisons test (left); two-sided unpaired Mann–Whitney test (right).
c SK-N-AS tumor volumes (MRI) and weights after treatment. Mean ± SEM
(CTRL = 8, CB5 = 8); two-way ANOVA with Sidak’s multiple comparisons
test (left); two-sided unpaired Mann–Whitney test (right). d–g
TH-MYCN^+/+-derived orthotopic allograft model. d Cells from one
TH-MYCN^+/+ tumor were orthotopically implanted in NCr nude mice. Two
weeks later mice were treated with vehicle or CB5 (25 mg/kg, b.i.d.,
6 days/week) for 2 weeks. e Tumor volumes (MRI) on treatment days 1 and
14. Tumors were framed and quantified; representative images and
mean ± SEM are shown (CTRL = 10, CB5 = 9). f Tumor weights at treatment
day 14. Mean ± SEM (CTRL = 10, CB5 = 9); two-sided unpaired
Mann–Whitney test. g Oil Red O staining of intratumoral lipids.
Mean±SEM (CTRL = 6, CB5 = 5 responsive tumors); two-sided unpaired
Mann–Whitney test. h Cells from one TH-MYCN^+/+ tumor were
orthotopically implanted in syngeneic 129 × 1/svj wild-type mice. Two
weeks later mice were treated with vehicle or CB5 (30 mg/kg, b.i.d.,
6 days/week) for 2 weeks. Tumors were weighed on treatment day 14.
Mean ± SEM (CTRL = 14, CB5 = 13); two-sided unpaired Mann–Whitney test.
i Cells from one MNA patient tumor (P6) were orthotopically implanted
in NCr nude mice, and 2 weeks later mice were treated with vehicle or
CB5 (25 mg/kg, b.i.d., 6 days/week) for 6 weeks. Tumor incidence
analyzed by Fisher’s exact test (CTRL = 8, CB5 = 9). Kaplan–Meier
survival analyzed by log-rank test. Arb. Unit arbitrary unit, Px
treatment period. Source data are provided in the Source Data file.
The TH-MYCN transgenic model is an aggressive MYCN-induced de novo NB
model^[238]3. To assess the anti-cancer activity of CB5, we generated
an orthotopic allograft model of NB by implanting a TH-MYCN^+/+ tumor
into NCr nude mice (Fig. [239]5d). Tumors developed after 2 weeks, at
which time mice were randomly assigned to CTRL (vehicle) or CB5
(25 mg/kg, b.i.d.) treatment groups. MRI was performed on treatment
days 1 and 14 to monitor tumor growth. CB5 significantly reduced tumor
volumes (p = 0.006, Fig. [240]5e and Supplementary Fig. [241]5d) and
weights (p = 0.01, Fig. [242]5f) in this model, and no signs of
toxicity were observed during the study (Supplementary Fig. [243]5d).
Moreover, mice responsive to CB5 treatment showed lower neutral lipid
levels than CTRL mice (p = 0.004, Fig. [244]5g), suggesting that CB5
blocks lipid accumulation in vivo. This is likely due to the
CB5-mediated inhibition of MYCN and MYCN-targeted FA synthesis and
transport protein expression (SCD1, ACC, and FATP2; Supplementary
Fig. [245]5d). Two tumors escaped CB5 treatment (Fig. [246]5f).
Supporting our findings, these tumors did not exhibit lipid inhibition
and showed signs of MYCN activation and FA synthesis/transport activity
(SCD1 and FATP2, Supplementary Fig. [247]5d).
One caveat to using nude mice as preclinical models is that they lack
an immune microenvironment. To evaluate the efficacy of blocking FA
transport in the presence of an intact immune microenvironment, we
generated a TH-MYCN^+/+-derived orthotopic syngeneic mouse model
(Fig. [248]5h) by implanting a TH-MYCN^+/+ tumor into the renal capsule
of wild-type immunocompetent 129×1/svj mice. After 2 weeks, mice were
treated with either CTRL (vehicle) or CB5 (30 mg/kg, b.i.d. i.p.) for 2
weeks. CB5 treatment blocked tumor growth (Fig. [249]5h, p < 0.0001)
without apparent toxicity (Supplementary Fig. [250]5e) in this model.
To evaluate the long-term effects of blocking FA uptake in MNA NBs, we
used a patient-derived orthotopic xenograft model (Fig. [251]5i). Cells
prepared from a primary MNA stage 4 NB tumor (P0) were implanted into
the renal capsule of NOG mice (P1). The tumor was then passaged in NOG
mice to P4 and in NCr nude mice to P6. In this model, tumors initiate
approximately 5 weeks after implantation and mice succumb to disease
burden 8–10 weeks after implantation. We asked whether blocking FA
uptake through long-term CB5 treatment could prevent MNA tumor
development and prolong animal survival. CB5 treatment was applied 2
weeks after implantation when tumors had not initiated. Mice received
CTRL (vehicle) or CB5 (25 mg/kg, b.i.d., i.p.) for 6 weeks and tumor
growth was monitored by MRI. CB5 did not prevent tumor initiation
(Fig. [252]5i). However, chronic CB5 treatment significantly prolonged
animal survival (p = 0.004, Fig. [253]5i) without notable toxicity
(Supplementary Fig. [254]5f), suggesting that blocking FA uptake can
suppress primary MNA tumor growth and extend survival.
Targeting FA transport sensitizes NB to conventional chemotherapies
Elevated lipid metabolism promotes acquired resistance to chemotherapy.
Targeting FA synthesis (FASN), oxidation (CPT1), or uptake (CD36)
sensitizes cancer cells to chemotherapy in adult
models^[255]40,[256]51,[257]52. Thus, we asked whether FATP2 inhibition
enhanced the anti-tumor activity of conventional chemotherapies, such
as etoposide (VP16, a topoisomerase II inhibitor) or temozolomide (TMZ,
a DNA alkylating agent), which are used as induction or relapse
therapies. We first evaluated the synergy between CB5 and either VP16
or TMZ by assessing cell viability under single and combination
treatment conditions, assigning a synergy score using the Bliss model
(>10 indicates synergy)^[258]53. CB5 synergizes with both VP16 and TMZ
in MNA cells (Fig. [259]6a and Supplementary Fig. [260]6a–b). In
addition, both CB5 + VP16 and CB5 + TMZ combinations increased
Caspase-mediated apoptosis compared with single-drug treatments in MNA
cells (p < 0.05, Fig. [261]6b and Supplementary Fig. [262]6c),
suggesting that these combination therapies are highly effective in the
setting of MYCN amplification.
Fig. 6. Suppressing FA uptake promotes the efficacy of conventional
chemotherapies.
[263]Fig. 6
[264]Open in a new tab
a Synergy analyses in MNA (LAN5 and IMR32) and non-MNA (SHEP) cells
treated with CB5 (0.4–2.5 µM), VP16 (5–50 nM) and their combination for
72 h. Heatmap represents mean Bliss scores from three independent
experiments. Bliss score > 10 indicates synergy. b Cell viability and
Caspase 3/7 activity after single and combination treatments. Cells
were treated with CTRL, CB5 (3 µM), VP16 (80 nM), or their combination
for 72 h. Mean ± SD (n = 3); one-way ANOVA with Tukey’s multiple
comparisons test. c Anti-tumor activity of CB5 + VP16 combination
therapy in LAN5-derived orthotopic xenografts. LAN5 cells were
implanted in the renal capsule of NCr nude mice. Two weeks after
implantation, mice were treated with CTRL (vehicle), CB5 (15 mg/kg,
b.i.d. 6 days/week), VP16 (8 mg/kg daily, 3 days/week) or their
combination for 2 weeks. Tumor weights after 2 weeks of treatment are
shown. Mean ± SEM (CTRL = 11, CB5 = 11, VP16 = 10, CB5 + VP16 = 12);
two-sided unpaired Mann–Whitney test. d Survival analysis in
patient-derived orthotopic xenografts. Cells prepared from one MNA
patient tumor (P8) were implanted in the renal capsule of NCr nude
mice. Five and half weeks after implantation, mice were treated with
CTRL (vehicle), CB5 (15 mg/kg, b.i.d. 6 days/week), VP16 (6 mg/kg
daily, 3 days/week), or their combination for 5 weeks. Survival was
plotted as a Kaplan–Meier curve and analyzed by the log-rank test
(CTRL = 12, CB5 = 10, VP16 = 10, CB5 + VP16 = 11). Px treatment period;
FC fold change. Source data are provided in the Source Data file.
Because the CB5 + VP16 combination showed promising in vitro
synergistic effects, we assessed the anti-tumor activity of this
combination in MNA LAN5-derived orthotopic xenografts (Fig. [265]6c).
Combination therapy (CB5: 15 mg/kg, b.i.d., i.p. 6 days/week and VP16:
8 mg/kg, i.p. 3 days/week) markedly reduced final tumor weights
compared with monotherapies (p < 0.0005, Fig. [266]6c). Moreover,
CB5 + VP16 demonstrated enhanced effects on tumor cell proliferation
and apoptosis (p ≤ 0.05, Supplementary Fig. [267]6d–e) without further
reducing MYCN expression compared with CB5 alone (Supplementary
Fig. [268]6f) and with no evidence of body weight loss or normal organ
toxicity (Supplementary Fig. [269]6g). These data suggest that blocking
FA transport effectively enhances tumor responses to VP16. To then
determine the long-term effects of combination therapy on animal
survival, we used our MNA patient-derived orthotopic xenograft model
(Fig. [270]6d). Mice were subjected to single or combination therapy
(CB5: 15 mg/kg, b.i.d., i.p. 6 days/week and VP16: 6 mg/kg, i.p. 3
days/week) for 5 weeks. Animals treated with CB5 + VP16 combination
therapy survived significantly longer than those receiving single-drug
therapies (p < 0.05, Fig. [271]6d). No treatments caused significant
body weight loss or clinical signs of toxicity (Supplementary
Fig. [272]6h). Collectively, our data suggest that blocking
MYCN-induced metabolic reprogramming effectively enhances the
anti-tumor effects of conventional chemotherapy.
Discussion
Using a variety of biochemical and analytical approaches, we
comprehensively characterized the metabolic landscape of NB tumors and
defined MYCN amplification as a major driver of distinct metabolic
adaptations. Our metabolic screening and analyses in human tumors and
cell lines across various models revealed that MYCN induces a
consistent accumulation of glycerolipids. Glycerolipids function as
secondary messengers that activate downstream oncogenic signaling and
serve as FA reservoir for energy storage and the prevention of toxic
FAs accumulation to support tumor growth^[273]29. MYCN induction or
amplification resulted in a distinct glycerolipid signature mainly
characterized by a robust increase in DGs. FAs can be funneled into
various metabolic pathways to synthesize more complex lipid species,
including DGs (which are precursors of TGs), contributing to the
structural diversity of the cellular lipids. MYCN may directly regulate
glycerolipid synthesis and degradation, for example, by upregulating
diacylglycerol-acyltransferase 2 (DGAT2) to store excess FAs in TGs and
LDs (Supplementary Fig. [274]7). DGAT1 and DGAT2 catalyze the
esterification of acyl-CoA with DGs to form TGs^[275]54. Although their
roles in oncogenesis remain largely unexplored, targeting DGAT1 to
block FA storage induces severe oxidative stress in
glioblastoma^[276]55. Future investigations remain necessary to
elucidate how MYCN maintains lipid homeostasis and protects NB cells
from oxidative damage. Here we show that targeting FA uptake
effectively inhibits MYCN-induced glycerolipid accumulation and tumor
growth, suggesting that FA transport is critical for these functions.
Cancer tissues show aberrant activation of de novo lipogenesis, and
inhibition of enzymes within the FA biosynthesis pathway can block
tumor growth^[277]30. FA biosynthesis contributes to cancer by
providing the building blocks for biological membranes and ATP
synthesis during nutrient depletion, in addition to regulating membrane
trafficking and signaling pathways critical for cell survival, such as
phosphatidylinositol-3,4,5-trisphosphate and
sphingolipids^[278]56,[279]57. MYC(N) dynamically alters de novo
lipogenesis, lipid storage, and β-oxidation to generate ATP. Inhibition
of MYC(N) induces LDs formation as a consequence of impaired FA
oxidation^[280]58. We and others have also shown that MYC(N) promotes
FA biosynthesis and aberrant activation of SREBP1^[281]18 in cancer,
including NB^[282]22,[283]23, and genetic and pharmacological
interference with FA biosynthesis blocks MYC-driven tumor
growth^[284]18. Moreover, ACC or FASN inhibition partially blocks
cancer growth in a subcutaneous NB model^[285]22. However, the
long-term efficacy and normal tissue toxicity associated with this
approach remain largely unknown. We found that inhibiting FA
biosynthesis via the SCD1 inhibitor A939572 triggered compensatory FA
uptake, suggesting that NB cells can utilize exogenous lipids when
precursors are limited or FA synthesis is impaired. We demonstrated
that blocking FA uptake either through exogenous lipid deprivation or
pharmacological FATP2 inhibition remarkably reduced the viability of
MYCN-induced cells, promoted apoptosis, and enhanced sensitivity to FA
biosynthesis inhibition. These effects could be partially rescued by FA
supplementation, indicating that MYCN-driven cells depend on FA uptake
for survival. The dependency on exogenous FAs is emerging in other
cancers. In prostate cancer and melanoma, the membrane transporters
CD36 and FATP1 respectively promote lipid accumulation and tumor
progression^[286]38,[287]41. Moreover, melanoma cells use FATP2 to
acquire lipids from aged fibroblasts, conferring resistance to targeted
therapy^[288]42. In NB, we found that oncogenic MYCN drives FA uptake
to maintain tumor growth. By screening mRNA expression and MYCN binding
to membrane FA transporters, we identified the FA transporter gene
SLC27A2 (encoding FATP2) as a direct MYCN target required for NB
survival. SLC27A2 expression in NB patients uniquely correlates with
MYCN expression and activity. Moreover, high SLC27A2 expression
strongly predicts poor clinical outcomes when compared to other FA
transporters. Collectively, these data suggest that MNA NB depends on
SLC27A2-mediated FA uptake, making SLC27A2 an attractive therapeutic
target for high-risk disease.
SLC27A2 is necessary for NB survival, as both genetic interference and
pharmacological inhibition via the small-molecule inhibitor CB5
impaired MYCN-induced tumor growth. Supporting the selectivity of
targeting SLC27A2 in NB, normal cells were not affected by CB5
treatment, suggesting the possibility of new therapeutic opportunities.
We demonstrated the anti-tumor activity of CB5 in multiple MYCN-induced
preclinical NB models, including cell line-derived orthotopic models,
both allograft and syngeneic models, and patient-derived models,
suggesting that FA uptake via SLC27A2 represents an intrinsic
vulnerability of MYCN-driven tumors that can be targeted
therapeutically. SLC27A2 is also expressed in immune cells, such as
oncogenic polymorphonuclear myeloid-derived suppressor cells, and
SLC27A2 inhibition blocks immune-suppressive activity in these cells,
delaying tumor progression^[289]59. Future studies remain necessary to
determine whether SLC27A2 inhibition also interferes with immune cell
functions and their metabolic dependencies to prohibit tumor growth.
MYCN-induced drug resistance limits the anti-tumor effects of
conventional chemotherapy, and thus remains a major clinical
obstacle^[290]60. Moreover, metabolic reprogramming and altered lipid
metabolism also promote acquired drug resistance^[291]27. Increased
FASN, CPT1B, and CD36 expression correlate with poor drug response in
cancer patients^[292]40,[293]51,[294]52. We found that targeting a key
MYCN-dependent metabolic vulnerability, the FA uptake pathway, strongly
enhanced the clinical activity of cytotoxic agents that inhibit DNA
synthesis, such as VP16 and TMZ. Because FA-derived acetyl-CoA can fuel
the TCA cycle to generate aspartate, the nucleotide synthesis
precursor^[295]61, we speculate that inhibiting FA uptake may limit
nucleotide reservoirs for DNA synthesis. Future studies are needed to
test whether FA uptake contributes to FA oxidation and nucleotide
synthesis and investigate the molecular mechanisms underlying the
observed synergy between CB5 and conventional chemotherapy.
In conclusion, we uncovered that MYCN promotes glycerolipid
accumulation in NB. MYCN drives FA uptake by directly upregulating
SLC27A2, which is required for glycerolipid synthesis and MYCN-induced
cell survival. Genetic and pharmacological interference (via CB5) of
SLC27A2 blocks NB tumor growth. Moreover, CB5 prolongs animal survival,
and synergizes with conventional chemotherapy in multiple preclinical
NB models. Our study shows that MYCN-driven tumors rely heavily on FA
uptake for survival, suggesting that FA uptake may represent a
promising therapeutic target in high-risk MNA patients.
Methods
Patient sample analyses
Frozen primary tumors (MNA, n = 18; non-MNA, n = 18) were provided by
the Research Tissue Support Service (RTSS) at Texas Childrens’ Hospital
(TCH). Patient clinical information is summarized in Supplementary
Data [296]1. This study was approved by the Institutional Review Board
for Human Subject Research at Baylor College of Medicine and Affiliated
Hospitals (BCM IRB, H-42596, H-6650). Informed consent was obtained
from all participants. Compensation was not provided.
Publicly available patient data were retrieved from R2: Genomics
Analysis and Visualization Platform ([297]http://r2.amc.nl). Cohort 1
(Kocak, [298]GSE45547, n = 649): data included single-color gene
expression profiles from 649 NB tumors based on 44 K oligonucleotide
microarrays^[299]62. Survival annotations are available for 479
patients. Cohort 2 (NRC, [300]GSE85047, n = 283): data were profiled
using the Affymetrix Human Exon 1.0 ST Array^[301]63. The MYCN activity
signature was derived from a signature composed of 157 differentially
expressed genes identified upon genetic depletion of MYCN from MNA cell
lines^[302]48. Gene expression was then converted to a Z-score, and the
MYCN activity score of each patient was computed by adding the Z-scores
of upregulated genes and subtracting the Z-scores of downregulated
genes. Pearson’s correlation coefficient and p-value (Python scientific
library) were used to analyze correlations between the expression
levels of two genes or between gene expression and MYCN activity.
Correlations with p-values < 0.05 are shown in the heatmap. To compare
gene expression between NB and normal tissues, SLC27A2 mRNA expression
was extracted from three NB datasets (Lastowska, [303]GSE13136, n = 30;
Hiyama, [304]GSE16237, n = 51; and Versteeg, [305]GSE16476, n = 88) and
three normal tissue datasets (Adrenal Gland, [306]SN_ADGL, n = 13;
Neural Crest, [307]GSE14340, n = 5; and Normal Various, [308]GSE7307,
n = 504 including 108 types of normal tissues). NB and normal tissue
datasets were acquired with the u133p2 platform and normalized by
MAS5.0 (R2). Kaplan–Meier analysis was used to assess how FA
transporter gene expression predicts patient survival by computing OS
and EFS or PFS rates of the top and bottom tertiles, stratified by
expression of each gene in the long-chain FA transport gene set (GO:
0015909). P-values were computed by the log-rank test. OS and EFS
significance [-log[10](p-value)] of all transporters were ranked and
are shown in the scatter plot. Red dots indicate that high expression
predicts poor outcome (p < 0.05); blue dots indicate that low
expression predicts poor outcome (p < 0.05); gray dots indicate no
significance.
Cell culture and drugs
The human NB cell lines IMR32 (male, RRID: CVCL_0346, CCL-127), SK-N-AS
(female, RRID: CVCL_1700, CRL-2137), SH-SY5Y (female, RRID: CVCL_0019,
CRL-2266) (from the American Type Culture Collection, ATCC), SHEP
(female, RRID: CVCL_0524, Shohet lab, University of Massachusetts),
Kelly (female, RRID: CVCL_2092, Sigma 92110411) and SK-N-BE(2c) (male,
RRID: CVCL_0529, ATCC CRL-2268) (Bernardi lab, BCM), LAN5 (male, RRID:
CVCL_0389, Metelitsa lab, BCM), LAN5 shMYCN (Bernardi lab), and MYCN3
(Shohet lab) were maintained in RPMI 1640 media (Lonza, NJ, USA)
containing 10% FBS (Germini Bio-Products, CA, USA), 4 mM L-glutamine
(Thermo Fisher Scientific, MA, USA), and 1% streptomycin-penicillin
(Thermo Fisher Scientific). SK-N-AS MYCN-ER^™ cells (Altman lab,
University of Rochester, NY), as well as HS-5 bone marrow stroma cells
(male, RRID: CVCL_3720, Redell lab, TCH, ATCC CRL-11882) and C2C12
mouse myoblast cells (female, RRID: CVCL_0188, Neilson lab, BCM, ATCC
CRL-1772) were maintained in DMEM media (Thermo Fisher Scientific)
containing 4.5 g/L glucose, 110 mg/L sodium pyruvate, 10% FBS, 4 mM
L-glutamine, and 1% streptomycin-penicillin. ARPE-19 retinal pigmented
epithelial cells (male, RRID: CVCL_0145, Zoghbi lab, BCM, ATCC
CRL-2302) were maintained in DMEM/F12 medium (Thermo Fisher Scientific)
containing 3.2 g/L glucose, 10% FBS, 2.5 mM L-glutamine, and 1%
streptomycin-penicillin. Tet21/N cells (female, RRID: CVCL_9812, Perini
lab, University of Bologna, Italy) were maintained in DMEM media
(Fisher Scientific) containing 4.5 g/L glucose, 10% FBS, 4 mM
L-glutamine, and 1% streptomycin-penicillin under geneticin selection
(0.2 mg/ml; G418, neomycin, Santa Cruz, CA, USA) and hygromycin
(0.15 mg/ml; Sigma, MO, USA). All cell lines were validated by short
tandem repeat analysis within the past 12 months and regularly tested
for mycoplasma.
To knock down MYCN in LAN5 shMYCN cells, DOX (Santa Cruz) was added at
a final concentration of 1 µg/mL for 72 or 96 h. To induce MYCN in
MYCN3 cells, DOX was added at a final concentration of 1 µg/mL for 48
or 72 h. To turn off MYCN in Tet21/N cells, DOX was added at a final
concentration of 2 µg/mL for 24 h. To activate MYCN-mediated
transcription, SK-N-AS MYCN-ER^™ cells were treated with 5 nM–1 µM
4-OHT (Sigma) for 1–6 days. Expression of MYCN and MYCN targets was
verified before using the cell lines for further experiments.
To determine the role of exogenous FAs on cell growth, LAN5, IMR32,
SK-N-BE(2c), SHEP, SK-N-AS and SK-N-AS MYCN-ER^™ cells were cultured in
complete media containing 10% FBS (Capricorn Scientific, Germany,
FBS-12A) or in delipidized media containing 10% delipidized FBS
(Capricorn Scientific, FBS-DL-12A) for up to 6 days. An FA mixture
(Sigma, F7050) was added to delipidized media to rescue the effects of
lipid deprivation. Drugs: A939572 (APExBIO, TX, USA, B3607), orlistat
(Sigma, O4139), CB16.2 (ChemBridge, CA, USA, 5830995), CB5 (ChemBridge,
5674122), VP16 (Selleck Chemicals, TX, USA, S1225) and TMZ (Selleck
Chemicals, S1237).
Plasmids
MYCN3 (MYCN Tet-On): To generate MYCN3 cells, MYCN cDNA was cloned into
a pTR2-Hygro vector (BD Biosciences, NJ, USA) containing a
tetracycline-responsive promoter. The construct was then transfected
into a SHEP subclone stably expressing the tetracycline response
element and selected with hygromycin. LAN5 shMYCN: GIPZ human MYCN
lentiviral vectors (V2LHS_36755 and V3LHS_322662) were obtained from
the Cell-Based Screening Service (C-BASS) core at BCM. V3LHS_322662
(with better KD efficiency) was inserted into pINDUCER11 (C-BASS),
containing a constitutive cassette (rtTA3 and eGFP) to detect infection
efficiency and a turboRFP (tRFP)-shRNA cassette activated upon DOX
treatment. shSLC27A2: To knock down SLC27A2 expression, two GIPZ
lentiviral shRNA vectors with GFP reporters (V2LHS_27492 and
V3LHS_398610, GE Healthcare Dharmacon, CO, USA) were used in LAN5
cells. V3LHS_398610 (with better KD efficiency) was used in IMR32 and
SK-N-AS cells. FATP1 or FATP2 overexpression: FATP1 (C-BASS) or FATP2
(OriGene) cDNAs were cloned into pINDUCER20, a Tet-inducible lentiviral
vector for ORF expression. Second generation lentiviruses were prepared
as previously described^[309]64.
Mouse models
In vivo studies were approved by the BCM Institutional Animal Care and
Use Committee (AN7089 and AN6190). Mice were housed at the TCH Animal
Facility, a temperature (21 ± 1^oC) and humidity (60%)-controlled and
specific pathogen-free environment under a 14 h:10 h light/dark cycle.
Mice were fed standard chow diet (LabDiet, 3002906-704) ad libitum. The
maximal tumor size permitted was 1.5 cm by diameter, and this size was
not exceeded in this study. TH-MYCN transgenic model: TH-MYCN^+/− mice
(129×1/svj), kindly provided by the Weiss lab (University of California
San Francisco, CA, USA), were bred and pups were genotyped to identify
TH-MYCN^-/-, TH-MYCN^+/-, and TH-MYCN^+/+ mice. TH-MYCN^+/+ 129×1/svj
mice develop tumors (almost 100% incidence) at week 3–4 and succumb to
disease by week 7–8^[310]3. NB cell-derived orthotopic xenograft
model^[311]65: An inoculum of 10^6 NB cells was surgically injected
under the renal capsule of 6-week-old female NCr nude mice
(CrTac:NCr-Foxn1^nu, Taconic, NY, USA). TH-MYCN^+/+-derived orthotopic
allograft model: An inoculum of 10^6 cells prepared from one
TH-MYCN^+/+ tumor were injected under the renal capsule of 6-week-old
female NCr nude mice (CrTac:NCr-Foxn1^nu). TH-MYCN^+/+-derived
orthotopic syngeneic model: An inoculum of 10^6 TH-MYCN^+/+ tumor cells
from a TH-MYCN^+/+ tumor (different tumor from allograft model) was
injected under the renal capsule of 6-week-old female TH-MYCN^−/− mice
(129×1/svj). Patient-derived orthotopic xenograft model: a primary MNA
NB tumor (male, stage 4, P0) was implanted into the renal capsule of
6–8-week-old female NOG mice (P1, NOD.Cg-Prkdc^scid
Il2rg^tm1Sug/JicTac, Taconic). The NB tumor was verified
histologically, then passaged in 6 to 8-week-old female NOG mice
(NOD.Cg-Prkdc^scid Il2rg^tm1Sug/JicTac) to P4 (Vasudevan lab, BCM), and
finally passaged in 6 to 8-week-old female NCr nude mice
(CrTac:NCr-Foxn1^nu, 2 × 10^6 cells) to P6 − 8 before initiating drug
studies. Tumor engraftment and growth were monitored by bioluminescent
imaging (Xenogen IVIS 100 System, Caliper Life Sciences, MA, USA) or
MRI (echo time = 80 ms, repetition time = 3030 ms, slice
thickness = 1.2 mm, field of view = 80 mm, number of slices = 18,
matrix = 256 × 250, number of signal averages = 2, dwell time = 25 µs,
scan time = 3.5 min, 1.0 T permanent MRI scanner, M2 system, Aspect
Technologies, Israel). Bioluminescent images were analyzed by Living
Image 4.7.3, and data are expressed as total flux. MRI images were
analyzed and processed in Osirix (5.8.5, Pixmeo, Bernex, Switzerland),
and data are expressed as tumor volume in cm^3. Power Analysis: based
on our previous studies^[312]23, at least six mice per group are
required to reach 80% statistical power at p < 0.05
([313]https://www.stat.ubc.ca/~rollin/stats/ssize/n2). Mice were
randomized and evenly allocated into treatment groups. Non-engrafted
mice were excluded from data collection. Vehicle composition: 10%
polyethylene glycol 400 (Sigma, 202398), 10% Tween 20 (Fisher
Scientific, BP337-500), and 80% PBS (Lonza, 17-516 F). CB5 (15–30 mg/kg
b.i.d.) was administered i.p. for 2–6 weeks. VP16 (6–8 mg/kg) was
administered i.p. three times per week for 2–5 weeks. At the end point
(tumor diameter > 1.5 cm), mice were euthanized to determine tumor
weight or survival. Intratumoral lipids were assessed by Oil Red O
staining (Pathology Core, TCH). Briefly, frozen tumor sections were
fixed in 60% isopropanol for 15 min before staining with 0.003% Oil Red
O solution (Sigma, O0625) for 30 min. Nuclei were counterstained with
hematoxylin (Richard-Allen Scientific, MI, USA, 7211) for 30 sec. The
total intensity of LDs was quantified by ImageJ2
([314]https://imagej.net/ImageJ2) from two representative images per
tumor (blinded pathology review) and normalized by cell number. Samples
with poor staining quality or without tumor areas were excluded from
the ImageJ2 analysis. To assess tumor proliferation, apoptosis and MYCN
expression, paraffin-embedded tumor sections were blocked with horse
serum (10%) and incubated with Ki67 antibody (1:50, Biocare Medical,
CRM325A, clone SP6, RRID: AB_2721189; Agilent, M7240, clone MIB1, RRID:
AB_2142367), cleaved Caspase-3 antibody (1:400, Cell Signaling, 9661 L,
RRID: AB_2341188) and MYCN antibody (1:100, Sigma, OP13-100UG, clone
NCM II 100, RRID: AB_213284) at 4 °C overnight. Sections were washed
with PBS and incubated with biotinylated anti-rabbit IgG (1:200, Vector
Laboratories, BA1000, RRID: AB_2313606) and anti-mouse IgG (1:200,
Vector Laboratories, BA9200, RRID: AB_2336171) antibodies at room
temperature for 30 min, followed by incubation with
3,3'-diaminobenzidine and counterstaining with hematoxylin (Fisher
Scientific, 7211). Five representative fields were captured by Nikon
ECLIPSE 90i (x40, blinded pathology review). Ki67 positive cells,
cleaved Caspase-3 positive cells, MYCN expression intensity, and the
total number of cells were analyzed by ImageJ2. Data are presented as
the percentage of Ki67 or cleaved Caspase-3 positive cells and MYCN
intensity per cell. Samples with poor staining quality or without tumor
regions were excluded from the ImageJ2 analysis.
In vitro functional assays
Cell viability
Cell viability was determined using a Cell Counting Kit-8 (CCK, Dojindo
Molecular Technologies, MD, USA, CK04-05) according to the
manufacturer’s instructions. For synergy studies, cells were treated
with different doses of two drugs either alone or in combination. Cell
viability was converted to inhibition% = 100%−viability% and synergy
scores were calculated using the Bliss model (>10 indicates synergy;
<−10 indicates antagonism)^[315]53. Synergy heatmaps were prepared in
GraphPad Prism (7.01). Caspase 3/7 activity: Caspase 3/7-mediated
apoptosis was measured using a Caspase-Glo assay kit (Promega, WI, USA,
G8091) as previously described^[316]23. FA uptake assay: Real-time FA
uptake was determined using a QBT Fatty Acid Uptake Assay Kit
(Molecular Devices, CA, USA, R6132) according to the manufacturer’s
instructions. To determine compensatory FA uptake induced by FA
synthesis inhibition, cells were serum-starved for 48 h, treated with
0–15 µM A939572 for 15 min, and a proprietary assay reagent was added.
Plates were read at λ[ex] = 485 nm and λ[em] = 515 nm every 30 s for
30 min. To determine drug specificity, IMR32 FATP1 and
FATP2-overexpressing cells were serum-starved and induced (DOX 20 ng/mL
for 48 h or 72 h) before adding the assay reagent containing CB5
(0–6 µM). Plates were read at λ[ex] = 485 nm and λ[em] = 515 nm every
30 sec for 100 min. FA uptake imaging was performed by fluorescence
staining of an FA analog (BODIPY^™ 558/568 C12, D3835 and BODIPY^™
500/510 C1, C12, D3823, Thermo Fisher) using an Olympus IX71 (Olympus,
Japan). The staining solution contained 10 µM fluorescence dye, 5 µM
BSA and 0.2% trypan blue in phenol red-free delipidized RPMI media,
modified from Li et al.^[317]66. Images were quantified by ImageJ2:
corrected total cell fluorescence (CTCF) = integrated density–(area of
selected cell×mean fluorescence of background readings)^[318]67.
Clonogenic assay: LAN5 shCTRL and shSLC27A2 cells were seeded at low
density on a 6-well plate. After 12 days of culture, with media
replaced every 3 days, the plate was stained with 0.25% Coomassie
Brilliant Blue G (Sigma, 27815) for 20 min at room temperature and
rinsed with distilled water to remove excess dye. The plate was then
imaged using a FluorChem R System (ProteinSimple, CA, USA) and the
number of colonies was counted using alpha View-FluorChem Q software
(3.4.0.0).
Real-time qPCR and western blotting
qPCR analysis
Total RNA isolated using an RNeasy Mini Kit (Qiagen, MD, USA, 74104)
was directly mixed with the reagents supplied in the QuantiTect SYBR®
Green RT-PCR Kit (Qiagen, 204243) and subjected to one-step RT-PCR
performed on StepOnePlus^™ Real-Time PCR System (Thermo Fisher
Scientific). Primer sequences are listed in Supplementary Data [319]4.
Western blotting: Cells and tumors were lysed with RIPA buffer (Thermo
Fisher, 89900) containing protease inhibitors (Roche, 04693116001).
Protein concentrations were measured by Bradford assay and 50–75 μg
protein was electrophoresed and transferred. Primary antibodies: MYCN
(1:500, Cell Signaling, 9405 S, RRID: AB_10692664), FATP1 (1:500,
Abcam, ab69458, RRID: AB 1270734), FATP2 (1:500, Abcam, ab83763, RRID:
AB 1859828; Thermo Fisher Scientific, PA5-30420, RRID: AB 2547894;
Thermo Fisher Scientific, PA5-42429, RRID: AB 2610399), total and
cleaved PARP (1:500, BD Biosciences, 551024, clone 7D3-6, RRID:
AB_394008), total Caspase-3 (1:500, Santa Cruz, sc-65497, clone 4.1.18,
RRID: AB 1120001), total cleaved Caspase-3 (1:300, Cell Signaling,
9661 S, RRID: AB 2341188), p21 (Waf1/Cip1) (1:300, Cell Signaling,
2947 S, clone 12D1, RRID: AB 823586), p53 (1:500, Santa Cruz, sc-126,
clone DO-1, RRID: AB 628082), SCD1 (1:1000, Cell Signaling, 2438 S,
RRID: AB 823634), ACC (1:1000, Cell Signaling, 3662 S, RRID: AB
2219400), CypB (1:500, Santa Cruz, sc-130626, clone k2E2, RRID: AB
2169421), and ACTB (1:5000, Sigma, A2228, clone AC-74, RRID: AB
476697). Secondary antibodies (LI-COR Biosciences, NE, USA): IRDye®
680RD Goat anti-mouse IgG (1:10000, 925-68070, RRID: AB 2651128),
IRDye® 800CW Goat anti-mouse IgG (1:10000, 926-32210, RRID: AB 621842),
and IRDye® 800CW Goat anti-rabbit IgG (1: 10000, 926-32211, RRID: AB
621843). Membranes were scanned using an Odyssey Infrared Imaging
System (Odyssey® Application Software 3.0, LI-COR Biosciences).
Metabolomics, lipidomics, and FA analyses
Global metabolomics
Cell samples (MYCN KD 0, 72, 96 h; MYCN-ON 0, 48, 72 h; n = 4 per
condition) and primary tumor samples (MNA, n = 18; non-MNA, n = 18)
were submitted to Metabolon Inc. for untargeted metabolomic profiling
(DiscoveryHD4 Metabolic Platform, n = 545 compound library, NC, USA).
One-way ANOVA was used to compare metabolite levels between groups.
Welch’s two-sample t-test was used to compare metabolite levels between
MNA and non-MNA primary tumors (p-value ≤ 0.05). The metabolite
classification network is presented by Cytoscape 3.8.2 using
subpathways as the source nodes and metabolites as the target nodes.
Target node sizes indicate the absolute log[2]FC of metabolite levels
between MYCN KD 72 h vs. CTRL, MYCN-ON 72 h vs. MYCN-OFF and MNA vs.
non-MNA. Red: upregulated metabolites; blue: downregulated metabolites
(p ≤ 0.05); gray: no significant change (p > 0.05). To determine
metabolic changes in each subpathway, a differential abundance score
was calculated for each condition (adapted from Hakimi et al.^[320]45).
Differential abundance score = (# of significantly increased
metabolites
[MATH: − :MATH]
# of significantly decreased metabolites) / # of metabolites measured
in subpathway
[MATH: × :MATH]
100%. Differential abundance scores were calculated in subpathways
containing at least three differentially altered metabolites in at
least one condition (p ≤ 0.05). A score of 100 indicates that all
metabolites in a subpathway are upregulated; a score of −100 indicates
that all metabolites in a subpathway are downregulated (p ≤ 0.05). To
identify subpathway enrichment, two enrichment algorithms were used.
Algorithm 1: Over-representation analysis using the hypergeometric
distribution with p-values adjusted by the Benjamini–Hochberg
procedure. “*” Indicates that a subpathway is significantly enriched in
one comparison group (adjusted p-value < 0.25), and subpathways were
ranked by the number of “*” designations. Subpathways with the same
number of “*” designations were ranked by the average absolute
differential abundance score from three comparison groups. Algorithm 2:
GSEA (4.0.3)^[321]68 used subpathways as the ‘gene’ set reference and
included subpathways containing at least three metabolites. GSEA was
conducted with 10,000 randomized metabolite sets to estimate
statistical significance, and the signal-to-noise metric (Z-score)
between the two phenotypes was used to rank all metabolites.
Subpathways with FDR < 0.25 were selected for presentation.
Lipidomics
Briefly, lipid extracts (n ≥ 4 per group) were analyzed by liquid
chromatography (LC)/triple time-of-flight (TOF) (Shimadzu CTO-20A
Nexera X2 41 UHPLC system, Kyoto, Japan)^[322]23. Mass spectrometry
(MS) analysis was conducted on a Triple TOF 5600 equipped with a Turbo
V^™ ion source (AB Sciex, Concord, Canada). Data were acquired with
Analyst TF software 1.8 (AB Sciex). Data were normalized by median
interquantile range normalization and were log[2] transformed.
Student’s t-test was used to compare differences between two groups.
P-values were adjusted by the Benjamini–Hochberg procedure to obtain
FDR. Changes with FDR < 0.25 (LAN5 shMYCN and MYCN3 cells) and with
absolute log[2]FC > 1 and FDR < 0.25 (SK-N-AS MYCN-ER^™ cells; LAN5
shCTRL and shSLC27A2 tumors) are presented in heat maps (R software
4.0.4).
FA profiling
Intracellular total FAs were analyzed by either LC-MS (n = 4 per group)
or gas chromatography (GC)-MS (n = 6 per group). For LC-MS analysis,
extracted FAs were analyzed by high-performance LC coupled with Agilent
6495 QQQ MS (Agilent Technologies, CA, USA) using ESI negative
ionization via single-reaction monitoring. Data analyzed with Agilent
mass hunter quantitative software (10.0) were normalized with an
internal standard and log[2] transformed per sample. For GC-MS, FA
concentration and tracer enrichment were measured using
pentaflurobenzylbromide derivatization and GC-MS negative chemical
ionization as previously reported^[323]69, with a slight modification:
an SP-2380 column (Supelco, PA, USA) was used to improve the separation
of palmitoleic acid (16:1n7) and sapienic acid (16:1n10) peaks. Data
were acquired in selective ion-monitoring mode. Analyte and standard
peak areas were determined. The ratio of the area from all (M0-M2)
analyte-derived ions relative to that of the internal standard
tridecanoic acid (m/z, 213–215) was calculated. Ratios were compared
with the calibration curves of serial FA concentrations to determine
each FA concentration. Student’s t-test was used to compare FA levels
between groups. P-values were adjusted using the Benjamini–Hochberg
procedure to obtain FDR. Changes with FDR < 0.25 were selected for data
presentation.
FA tracing
Briefly, deuterated water (D[2]O) (99 atom%, Cambridge Isotope
Laboratory, MA, USA) was used to determine de novo lipogenesis^[324]23.
[U-^13C]palmitic acid (16:0) potassium salt (99 atom%^13C, Cambridge
Isotope Laboratories) was used to determine FA desaturase activity
(ratio of [^13C[16]]monounsaturated FA to [^13C[16]]saturated FA) and
FA uptake (original [^13C[16]]FAs in medium—remaining [^13C[16]]FAs in
medium[^13C[16]]). FAs were analyzed by GC-MS and normalized by the
total protein contents of each sample.
Chromatin immunoprecipitation (ChIP) and luciferase activity assay
MYC(N) ChIP
Briefly, 1x10^7 cells were cross-linked with 1% formaldehyde, and the
reaction was stopped with 0.125 M glycine before harvest. DNA was
sheared by sonicating using Bioruptor PLUS (Diagenode, NJ, USA). A
small aliquot of sonicated material was retained as input and the
remaining sample was immunoprecipitated using 5 µg ChIP-grade
antibodies (MYCN, Santa Cruz, sc-53993, clone B8.4.B, RRID: AB 831602;
c-MYC, Santa Cruz, sc-764, clone N-262, RRID: AB 631276). Rec-sepharose
protein A beads (Invitrogen, CA, USA, 101141) were used to immobilize
immuno-complexes. Crosslinking was reversed using RNase-A treatment
(37 °C, 1 h) and proteinase K (Roche) for 6 h at 65 °C.
Immunoprecipitated DNA was purified by phenol/chloroform and ethanol
precipitation. MYCN and c-Myc binding to the SLC27A promoter was
analyzed by qPCR (SSOADVANCE-BIORAD) using primers listed in
Supplementary Data [325]4 (amplicon regions included), and normalized
by the fold enrichment (2^-ΔΔCT) method using ABCA10 TSS as a negative
control. Dual luciferase reporter assay: The effects of MYCN on SLC27A1
and A2 promoter activities were analyzed using a dual luciferase gene
reporter assay as previously described^[326]70. Luciferase reporter
activity was measured using the Dual Luciferase Assay System (Promega,
E1980). Chemiluminescence values for firefly and renilla luciferases
were measured using a GloMax 20/20 instrument (Promega), and data are
reported as percentage of the MYCN-OFF/MYCN-ON ratio in Tet21/N cells.
MYCN expression was turned off (DOX 2 µg/mL for 24 h) 6 h after
transfection (Effectene, Qiagen), and activities were compared with
those in untreated cells. Empty vector (pGL3b) and pGL3-ODC1 promoter
were used as negative and positive controls, respectively. pGL3
constructs carrying SLC27A1, SLC27A2 and ODC1 promoters were obtained
by directional cloning using primers listed in Supplementary
Data [327]4 (promoter regions included). An E-BOX SLC27A2 mutant (from
CACCTG to GAATTC) promoter construct was obtained by whole-around PCR
technology using primers listed in Supplementary Data [328]4.
Data and statistical analysis
SLC27A2 dependency scores were extracted from the CRISPR (Avana) Public
20Q4V2 dataset^[329]49, which is part of the DepMap project (The Broad
Institute, USA). This dataset was generated from a CRISPR knockout
screen of 18,119 genes in 789 cell lines across 30 primary diseases. A
low dependency score indicates a high dependency on SLC27A2 for
survival.
Microsoft Excel (2013) was primarily used for data collection, and
GraphPad Prism (7.01) and R (4.0.4) were used for data analyses, unless
otherwise specified. BioRender.com was used to generate diagrams in
Figs. [330]1a, [331]5a, d, h, i, [332]6c and d. Investigators were
blinded to cell identity during metabolomics, lipidomics, FA profiling,
and pathological analyses. For in vitro experiments, we assumed a
normal distribution and performed two-sided unpaired parametric tests
(Student’s t-test) for two-group comparisons. Multiple-group
comparisons used one-way or two-way ANOVA with Dunnett’s or Tukey’s
multiple comparisons test. For animal studies, differences in tumor
incidence between groups were computed by Fisher’s exact test. Tumor
growth and mouse weights were analyzed by two-way ANOVA followed by
Sidak’s multiple comparisons test. Tumor weights and protein markers
were compared using two-sided unpaired Mann–Whitney tests. Survival was
plotted using Kaplan–Meier curves and analyzed using the log-rank test.
Data are shown as the mean, mean ± SD or mean ± SEM (n indicates number
of biologically independent samples and is provided in the figure
legend). P-values < 0.05 were considered significant.
Reporting summary
Further information on research design is available in the [333]Nature
Research Reporting Summary linked to this article.
Supplementary information
[334]Supplementary Information^ (71.8MB, pdf)
[335]Peer Review File^ (1.6MB, pdf)
[336]41467_2022_31331_MOESM3_ESM.pdf^ (516.5KB, pdf)
Description of Additional Supplementary Files
[337]Supplementary Data 1^ (13.4KB, xlsx)
[338]Supplementary Data 2^ (194.2KB, xlsx)
[339]Supplementary Data 3^ (394KB, xlsx)
[340]Supplementary Data 4^ (13.6KB, xlsx)
[341]Reporting Summary^ (6MB, pdf)
Acknowledgements