LDHA in neuroblastoma is associated with poor outcome and its … · 2018-06-20 · LDHA in neuroblastoma is associated with poor outcome ... Christian Beltinger, Section of Experimental

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LDHA in neuroblastoma is associated with poor outcome

and its depletion decreases neuroblastoma growth

independent of aerobic glycolysis

Carmen Dorneburg1, Matthias Fischer2, Thomas F.E. Barth3, Wolfgang Mueller-

Klieser4, Barbara Hero2, Judith Gecht2, Daniel R. Carter5, Katleen de Preter6,

Benjamin Mayer7, Lisa Christner1, Frank Speleman6, Glenn M. Marshall5,8, Klaus-

Michael Debatin1, and Christian Beltinger1

1Dept. of Pediatrics and Adolescent Medicine, University Medical Center Ulm, Ulm,

Germany; 2Children's Hospital, Department of Pediatric Oncology and Hematology,

University of Cologne, Germany; 3Institute of Pathology, University Medical Center

Ulm, Ulm, Germany; 4Institute of Pathophysiology, University Mainz, Germany;

5Children’s Cancer Institute Australia, Lowy Cancer Centre, University of New South

Wales, Sydney, Australia; 6Center for Medical Genetics (CMGG), Ghent University,

Ghent, Belgium; 7Institute of Epidemiology and Medical Biometry, Ulm University,

Ulm, Germany; 8Kids Cancer Centre, Sydney Children's Hospital, Sydney, Australia.

Running title: LDHA and aerobic glycolysis in neuroblastoma

Keywords: LDHA, aerobic glycolysis, neuroblastoma, MYCN, survival

Financial support: This work was supported in part by a grant of the Deutsche

Krebshilfe to CB (grant number 70112002)

Corresponding author: Christian Beltinger, Section of Experimental Pediatric

Oncology, Dept. of Pediatrics and Adolescent Medicine, University Medical Center

Research. on October 4, 2020. © 2018 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 20, 2018; DOI: 10.1158/1078-0432.CCR-17-2578

http://clincancerres.aacrjournals.org/

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Ulm, Eythstr. 24, 89075 Ulm, Germany. Phone: 49-731-500-57032; Fax: 49-731-500-

57042; E-mail: [email protected]

Conflict of interest: none.

Notes about the manuscript: 5312 words and 6 figures




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Translational relevance:

Being independently associated with poor outcome, LDHA expression could be

incorporated into signatures to assess if LDHA expression improves risk prediction in

NB. LDHA may also be considered as a therapeutic target in NB if the growth-

controlling effects of LDHA inhibition can be improved by combining it with additional

therapeutic approaches. The data suggest that for this purpose targeting

mechanisms other than aerobic glycolysis should be considered.




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Abstract

Purpose: To investigate whether lactate dehydrogenase A (LDHA), an important

component of the LDH tetramer crucial for aerobic glycolysis, is associated with

patient outcome and constitutes a therapeutic target in neuroblastoma (NB).

Experimental Design: Expression of LDHA mRNA and protein were determined in

709 and 110 NB patient samples, respectively, and correlated to survival and risk

factors. LDHA and LDHB were depleted in human NB cell lines by CRISPR/Cas9

and shRNA, respectively, and aerobic glycolysis, clonogenicity and tumorigenicity

were determined. Expression of LDHA in relation to MYCN was measured in NB cell

lines and in the TH-MYCN NB mouse model.

Results: Expression of LDHA, both on the mRNA and the protein level, was

significantly and independently associated with decreased patient survival.

Predominant cytoplasmic localization of LDHA protein was associated with poor

outcome. Amplification and expression of MYCN did not correlate with expression of

LDHA in NB cell lines or TH-MYCN mice, respectively. Knockout of LDHA inhibited

clonogenicity, tumorigenicity and tumor growth without abolishing LDH activity or

significantly decreasing aerobic glycolysis. Concomitant depletion of LDHA and the

isoform LDHB ablated clonogenicity while not abrogating LDH activity or decreasing

aerobic glycolysis. The isoform LDHC was not expressed.

Conclusions: High expression of LDHA is independently associated with outcome of

NB and NB cells can be inhibited by depletion of LDHA or LDHB. This inhibition

appears to be unrelated to LDH activity and aerobic glycolysis. Thus, investigations

of inhibitory mechanisms beyond attenuation of aerobic glycolysis are warranted,

both in NB and normal cells.




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Introduction

Neuroblastoma (NB) is the most common extracranial solid tumor of childhood. The

transcription factor MYCN, whose gene is often amplified in poor prognosis NB (1, 2),

has long been known to convey a poor prognosis by inducing diverse target genes

(3). While the poor prognosis of high-risk patients has improved in the last decades,

many patients still die from their disease (4, 5). Therefore, novel prognostic markers

and therapeutic targets are needed.

Aerobic glycolysis, also known as the Warburg effect, is a hallmark of cancers,

including NB (6-8). Aerobic glycolysis increases the provision of metabolic building

blocks and renders the tumor microenvironment permissive for tumor growth, both of

which endows cancer cells with a growth advantage despite the energetic inefficiency

of aerobic compared to anaerobic glycolysis (9). As aerobic glycolysis occurs in

cancer but not in normal cells, it constitutes a promising therapeutic target. Along this

line, inhibition of aerobic glycolysis has been shown to decrease growth of cancer

cells, including NB (10-12).

Lactate dehydrogenase (LDH) is a tetrameric enzyme composed of either

lactate dehydrogenase A (LDHA) or B (LDHB) subunits, or combinations thereof, or

LDHC (13). LDHA is utilized by cancer cells to bypass oxidative phosphorylation by

reducing pyruvate to lactate (9). This diverts metabolic precursors of pyruvate into

the pentose phosphate pathway, which supplies metabolic building blocks for cancer

cell growth (9). Elevated extracellular lactate levels enhance tumor angiogenesis,

immune escape and additional parameters of the tumor microenvironment conducive

for tumor growth (14-17). LDHA is known to be a target gene of c-MYC (18) and of

hypoxia-inducible factor 1 alpha (HIF-1α) (19), and may be a target of MYCN (20),

consistent with a role of LDHA in tumor maintenance. Along this line, overexpression




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of LDHA is associated with unfavorable prognosis of several cancers (21, 22) and

with resistance to radiotherapy (23). Conversely, inhibition of LDHA can reduce

cancer progression (24-29). Similar to LDHA, LDHB is also associated with

aggressive cancer phenotypes (30, 31). Targeting LDHB decreases cancer cell

proliferation (30, 31) and autophagy (32).

As little is known about aerobic glycolysis, LDHA and LDHB in NB, we

investigated these aspects in detail. In a large patient cohort we show that LDHA

mRNA and protein expression and predominant cytoplasmic localization of LDHA

protein are associated with increased tumor aggressiveness and decreased patient

survival. Knocking out LDHA and knocking down LDHB decreased malignant

characteristics of NB cells. Surprisingly, aerobic glycolysis remained unaffected.




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Materials and Methods

Analysis of LDHA mRNA expression, patient outcome and risk factors

Clinically annotated gene expression profiles previously generated from NB patients

using a 44k oligonucleotide array (33) were analyzed for LDHA mRNA expression, as

described in Supplementary Information.

Analysis of LDHA protein expression, patient outcome and risk factors

The Neuroblastoma Tumor Bank in Cologne, Germany and the Pediatric Tumor

Registry in Kiel, Germany provided a panel of tumors from initial diagnosis that are

described in Supplementary Information.

The formalin-fixed and paraffin-embedded NB specimens were subjected to

H&E and immunohistochemical staining for LDHA according to standard methods. To

quantify LDHA protein, slides were analyzed using a Keyence microscope BZ-9000

(Neu-Isenburg, Germany) and Keyence image analysis software. The LDHA-positive

tumor area was determined and expressed as the percentage of total tumor area on

the slide. For subcellular investigation, the percentages of cells with LDHA-positive

cytoplasm and LDHA-positive nuclei in each sample were determined by a

pathologist blinded to patient data. Tumors with ≥5% or <5% positive cells in the

respective cellular compartment were categorized as positive or negative,

respectively. Statistical analysis was performed as detailed in Supplementary

Information.

LDHA in the TH-MYCN NB progression model

The TH-MYCN NB progression model has been described elsewhere (34). Briefly,

TH-MYCN+/+ mice (35) were sacrificed at weeks 1 and 2 postnatally to isolate




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sympathetic ganglia containing foci of neuroblast hyperplasia, and at week 6 to

harvest advanced NB tumors. Sympathetic ganglia from TH-MYCN-/- (wild-type) mice

at week 1, 2 and 6 were used as control for expression changes during normal

development. Murine total RNA was isolated using the RNeasy Mini Kit (Qiagen).

The samples were profiled on Agilent SurePrint G3 Gene Expression Microarrays

according to the manufacturer’s protocol. Data were summarized and normalized

with the vsn method (36) in the R statistical programming language using the limma

package. Linear regression analysis was performed to evaluate the differential

temporal expression pattern in ganglia from wild-type mice and ganglia and tumors

from transgenic mice.

Generation of LDHA knockout NB cells

Three different sgRNAs (Supplementary Information) were cloned into GeneArt®

CRISPR Nuclease Vector (life technologies). NB cells were transiently transfected

with sgRNA plasmids, sorted and seeded as single cells. Single cell clones were

expanded and DNA was isolated using DirectPCR® Lysis Reagent Cell (Peqlab).

Exon 2 of LDHA was PCR-amplified (Supplementary Tab. S1) and Sanger-

sequenced. Sequences were analyzed for indels in the target region and for absence

of off-target mutations in the 5 genes most likely to harbor such mutations. The

clones were classified as having LDHA wild-type sequence or homozygous knockout

sequences, i.e. indels leading to premature stop codons. Knockout of LDHA was

verified by Western blot analysis.

Knockdown of LDHB by shRNA

NB cells were stable transduced with three LDHB shRNA lentiviruses and one non-

silencing control virus at a MOI of 20 (TRIPZ, Dharmacon, #RHS4740-EG3945, and




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#RHS4743) according to the manufacturer’s protocol. After stable selection with

puromycin cells were treated with 1 µg/ml doxycycline (Sigma) to induce shRNA

expression. To determine knockdown efficiency Western blots for LDHB protein were

performed.

Glucose and lactate determination in vitro

Glucose and lactate levels in cell culture medium were determined using colorimetric

glucose and lactate assays (BioVision kits K606-100 and K627-100), according to the

manufacturer’s instructions. Briefly, NB cells were seeded in 6-well plates and after

indicated time points medium was collected and deproteinized. Subsequently, 50 µl

of diluted samples were used to measure lactate and glucose concentrations using a

BioTek ELISA reader.

LDH activity assay

The LDH activity of NB cells was determined according to the manufacturer’s

protocol (Sigma, MAK066). 1-2x106 cells were suspended with 500 µl LDH assay

buffer and centrifuged with 10000 x g at 4°C for 15 min. Lactate dehydrogenase

activity was measured in supernatants and cell lysates at 450 nm using a BioTek

ELISA reader.

Animal experiments

6-8 weeks old male and female immunodeficient RAG2-/- common gamma chain-/-

(RAG2-/- / cγc-/-) mice bred in the Animal Research Center of Ulm University and

housed in sterile isolators were used. NB cell line clones KELLY, LAN-5, SK-N-AS

and SK-N-BE(2)C were chosen for injection. 5x105 viable cells (1x106 of SK-N-AS

cells) in 25% high concentration matrigelTM (BD Biosciences) in DMEM/F12 (Gibco)




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were subcutaneously injected. Mice were monitored regularly and tumor size was

measured using a caliper (v= ½(W*W*H)). Doubling time was calculated from tumor

volume over time. Mice were sacrificed when tumors reached 1.5 cm in diameter or

when tumors penetrated the skin. All experiments were done according to state and

institutional guidelines for the care and protection of research animals.

Glucose, lactate and ATP determinations in vivo

Glucose, lactate and ATP content in tumor sections were measured by induced

metabolic bioluminescence imaging, as described previously (37). In brief, cryostat

sections of shock-frozen tissue specimens were immersed into an enzyme solution.

Defined increase of temperature made the tissue sections melt and allowed for

enzymatic reactions to take place, eventually leading to emission of light. The light

was registered with a precision microscope (Axiophot, Zeiss, Germany) and an

ultrasensitive video system (Ancor, UK) to calculate local metabolite content in

micromoles per gram (μmol/g) of tissue.

Additional information is provided in Supplementary Material and Methods.




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Results

High expression of LDHA mRNA is independently associated with poor outcome

We assessed association of LDHA transcripts with survival and with established risk

factors of NB. To this end, we performed in silico analysis in a large number of

clinically annotated NB. High LDHA expression was significantly associated with

markedly lower overall and event-free survival (Figure 1A, Supplementary Fig. S1),

independent of MYCN, age and stage (Figure 1B, Supplementary Tab. S2).

Increased levels of LDHA mRNA were significantly associated with MYCN

amplification, increased age, advanced stage and undifferentiated histology (Figure

1C). Taken together, in this study, increased expression of LDHA mRNA in NB was

independently associated with poor outcome and correlated with established risk

factors.

High LDHA protein levels, and increased cytoplasmic and decreased nuclear LDHA

protein are significantly associated with poor outcome

To investigate whether protein expression of LDHA in NB is associated with outcome

and established risk factors of NB, we performed immunohistochemistry of LDHA in

110 clinically annotated patient samples. For LDHA protein quantification, the fraction

of LDHA-positive tumor area of total tumor area was determined on the slides (Figure

2A). Kaplan-Meier analysis showed a marked association of LDHA protein

expression with decreased overall and event-free patient survival (Figure 2A), which

was independent of MYCN and age for overall but not for event-free survival (Figure

2B, Supplementary Tab. S3). Validation of the results in additional cohorts is




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required. Increased expression of LDHA protein was significantly associated with

amplification of MYCN, increased age and advanced stage (Figure 2C).

Given its known function, we hypothesized that LDH compartmentalized into

the cytoplasm but not the nucleus would be negatively associated with survival.

Indeed, this compartmentalization (Figure 2D) appeared to be associated with

decreased overall survival (Figure 2E).

More MYCN-amplified, advanced stage and undifferentiated NB contained

cells with cytoplasmic LDHA (Figure 2F). The increase of cytoplasmic LDHA in NB of

older patients was small.

Thus, in this study, high LDHA protein levels were associated with poor

outcome of NB and correlated with risk factors of NB.

Expression of LDHA in NB of TH-MYCN mice is not induced by MYCN

While LDHA is a known target gene of c-MYC (18), it is unknown whether it is also a

target gene of MYCN. To start to address this question, we first investigated LDHA

mRNA expression during the development of NB from tumor-prone ganglia at 2

weeks of age to tumors at 6 weeks of age in TH-MYCN transgenic mice by in silico

analysis (35). While expression of ODC1, a bona fide target gene of MYCN,

markedly increased during neuroblastomagenesis compared to developing ganglia of

wild-type mice, expression of LDHA did not (Figure 3A). This shows that LDHA in NB

of TH-MYCN mice is not induced by expression of MYCN.




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Expression of LDHA and LDHB in human NB cell lines is not increased when MYCN

is amplified

To address the question whether MYCN amplification is associated with enhanced

LDHA expression, we determined LDHA transcript and protein levels in 10 NB cell

lines and 2 derivatives with single and increased copy number of MYCN (Figure 3B).

No difference was detected. Interestingly, SK-N-BE(2)C cells were completely devoid

of LDHA. As LDHA, LDHB was not differentially expressed in the amplified and non-

amplified NB cell lines (Figure 3B).

Forced overexpression of MYCN does not increase expression of LDHA and the

regulator HIF-1α in human SH-EP NB cells

Next, we investigated whether acute exposure of NB cells to MYCN would induce

LDHA. To this end, we triggered nuclear translocation of MYCN-ER in SH-EP MYCN-

ER cell line by adding tamoxifen (38). While the MYCN target gene ODC1 was

robustly induced LDHA expression was not (Figure 3C). MYCN is known to induce

key Warburg effect enzymes, either directly or via HIF-1α. We therefore analyzed

whether MYCN and HIF-1α cooperate in inducing key Warburg effect enzymes. As

HIF-1α protein is only present when stabilized by hypoxic conditions, we simulated

the effect of hypoxia on HIF-1α by stabilizing HIF-1α with deferoxamine. MYCN did

not significantly increase HIF-1α protein (Supplementary Fig. S2A) nor mRNA levels

of LDHA or of other Warburg enzymes examined in SH-EP MYCN-ER cells

(Supplementary Fig. S2B). Deferoxamine significantly increased mRNA of Glut1,

PDK1 and LDHA mRNA (Supplementary Fig. S2B). Of note, MYCN and HIF-1α did

not cooperate in the transcription of the Warburg enzymes investigated.




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Taken together, the data do not support the notion that increased MYCN, per

se or together with HIF-1α, enhances expression of LDHA and other key enzymes of

aerobic glycolysis in the NB cell lines investigated.

LDHA depletion decreases aggressiveness of NB cells

To investigate the relevance of LDHA for aggressiveness of NB cells, LDHA was

knocked out in SK-N-AS and KELLY NB cell lines using CRISPR/Cas9. SK-N-AS

and KELLY cell lines were chosen because they are representative of MYCN non-

amplified and MYCN-amplified NB cell lines, respectively. Clones expanded from

single cells after transfection of CRISPR/Cas9 and LDHA sgRNAs were Sanger-

sequenced and probed for LDHA protein by Western blotting. In all clones without

LDHA protein (Figure 4A) and with unambiguous sequence analysis LDHA was

homozygously knocked out (data not shown), while all clones expressing LDHA were

wild-type. Homozygous knockout clones and wild-type clones were randomly chosen

for further experiments.

Knockout of LDHA significantly decreased clonogenicity of both SK-N-AS and

KELLY NB cells (Figure 4B).

To determine the influence of LDHA on tumorigenicity and in vivo growth of

NB cells, LDHA-knockout and wild-type clones of SK-N-AS and KELLY cells were

transplanted subcutaneously into Rag2-/- cγc-/- mice. LDHA depletion decreased

tumor incidence in SK-N-AS cells and prolonged tumor latency (Figure 4C) and

tumor-doubling time (Figure 4D) of both SK-N-AS and KELLY NB cells.

Together, these results show that knockout of LDHA decreases

aggressiveness of NB cells.




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LDHA is dispensable for the Warburg effect in NB cells

To assess the metabolic effects of LDHA depletion in NB cell lines, we determined

LDH activity of LDHA-depleted and -replete NB cells. While LDH activity was reduced

in cells with knockout of LDHA residual LDH activity was maintained (Figure 5A). Of

note, glucose consumption and lactate production were not significantly altered in

LDHA-depleted cells under aerobic culture conditions (Figure 5A).

To determine the situation in vivo, SK-N-AS, LAN-5 and SK-N-BE(2)C cells

were subcutaneously transplanted into immunodeficient mice and tumor tissues were

stained for LDHA. While LDHA was highly expressed in SK-N-AS and LAN-5 tumors,

SK-N-BE(2)C cell tumors were completely devoid of LDHA (Figure 5B). This was not

due to mutations in the coding sequence of LDHA (data not shown). Tumors derived

from SK-N-AS, LAN-5 and SK-N-BE(2)C cells were analyzed for the metabolites

glucose, lactate and ATP (Figure 5C). Despite the complete absence of LDHA, SK-

N-BE(2)C tumors consumed glucose and generated lactate similar to the LDHA-

replete NB cell lines SK-N-AS and LAN-5. SK-N-BE(2)C tumors also generated

similar amounts of ATP as the LDHA-replete LAN-5 tumors. These data show that

NB cells completely lacking LDHA can still maintain aerobic glycolysis.

Concomitant depletion of LDHA and LDHB in NB cells does not abrogate LDH

activity and the Warburg effect while abolishing clonogenic growth

LDH activity is supposed to be essential for aerobic glycolysis in tumor cells. We

reasoned that LDHB could rescue LDH activity when LDHA is depleted. Seemingly

supporting this notion, we found that LDHB was clearly and invariably expressed in

NB cell clones both replete with and depleted of LDHA (Figure 6A). To directly




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assess the relevance of LDHB in NB cells, doxycycline-dependent inducible short-

hairpin RNAs (shRNA) were used to knockdown LDHB. LDHB was strongly reduced

in both wild-type and LDHA-depleted clones while LDHC was not expressed (Figure

6B). Expression of LDHC mRNA was very low in NB cell lines and patient NB

(Supplementary Fig. S3). Knockdown of LDHB did not decrease LDH activity in SK-

N-AS cells and only marginally in KELLY cells (Figure 6C). Of note, combined

depletion of LDHA and LDHB did not abrogate LDH activity (Figure 6C). Neither

depletion of LDHB alone nor in combination with LDHA depletion affected glucose

consumption and lactate production (Figure 6C).

To assess the effect of combined depletion of LDHB and LDHA on malignant

behavior in vitro, we investigated clonogenic growth. Depletion of LDHB alone

decreased clonogenic growth of SK-N-AS cells significantly and of KELLY cells

profoundly (Figure 6D). Combined depletion of LDHA and LDHB abrogated

clonogenic growth in both cell lines (Figure 6D).

Taken together, simultaneous depletion of LDHB and LDHA in SK-N-AS and

KELLY cells does not decrease aerobic glycolysis while abolishing clonogenicity.




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Discussion

This paper shows that increased expression of LDHA is associated with decreased

survival in NB. The data support the notion that LDHA and its isoform LDHB

contribute to aggressiveness of NB cells while being dispensable for aerobic

glycolysis. This is compatible with non-metabolic pro-tumor functions of LDHA and

LDHB in NB. Additional studies are necessary to validate these novel results.

We have shown in this large cohort of NB patients that increased LDHA

mRNA was associated with markedly decreased overall survival while correlating

with parameters of aggressive disease, i.e. amplification of MYCN, older age, stage 4

and undifferentiated histology. Similarly, an increased fraction of tumor cells

expressing LDHA protein was associated with poor outcome in NB. The latter

supports the notion that enhanced expression of LDHA is not an epiphenomenon and

may facilitate clinical application, as immunohistochemical analysis is a standard

method in the diagnostic work-up of NB. Along this line, we found that subcellular

detection of LDHA protein might provide additional prognostic information, as

cytoplasmic but not nuclear expression of LDHA appeared to be associated with

poorer survival. Future prospective validation of this conclusion is warranted.

At first glance, the association of increased overall and cytoplasmic expression

of LDHA with prognosis of NB may be readily explained by the role of LDHA in the

Warburg effect. Along this line of argument, nuclear LDHA would represent enzyme

sequestered from its cytoplasmic compartment, where aerobic glycolysis functions of

LDHA are located. However, we provide evidence that LDHA is not necessary for

aerobic glycolysis in the NB cell lines analyzed. This raises the question whether

LDHA has functions unrelated to aerobic glycolysis. Indeed, LDHA has been reported

to regulate mRNA stability of non-glycolytic genes (39) and may impact on replication




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independent of aerobic glycolysis (40). It is tempting to speculate that non-glycolytic

targets of LDHA, both in the cytoplasm and the nucleus, include some that bestow an

aggressive phenotype upon NB cells.

The data do not provide evidence that increased expression of MYCN

enhances expression of LDHA. LDHA mRNA was not increased in the NB of TH-

MYCN mice compared to non-tumorous sympathetic ganglia in wild-type mice, in line

with other data (41, 42). In our limited number of NB cell lines no difference in LDHA

transcript and protein levels was found between MYCN-amplified and non-amplified

cell lines. Acute activation of MYCN in SH-EP MYCN-ER NB cells did not induce

LDHA and MYCN did not cooperate with HIF-1to induce LDHAThese data do not

contradict the finding of us and others (20) that LDHA mRNA and protein are

increased in patient NB with amplification of MYCN, given that chromosomal

amplification of 2p, where MYCN is located, also amplifies additional genes which

may induce LDHA. Our data apparently contrast with reports showing that

knockdown of MYCN in LAN-5 NB cells decreases LDHA mRNA and that MYCN

cooperates with HIF-1to induce LDHA (20). This may be explained by species- and

cell type-specific differences, and by MYCN and HIF-1being knocked down rather

than activated. Other events yet to be elucidated may drive LDHA expression while

impacting on aggressiveness of NB, either via LDHA or independent thereof.

We show that LDHA contributes to aggressiveness of NB cells in vitro and in

vivo while being dispensable for the Warburg effect. By employing CRISPR/Cas9-

mediated knockout of LDHA and by analyzing several cellular clones verified at the

genomic and protein level, complete absence of LDHA was assured. Given the

complete lack of LDHA, the decrease of aggressiveness was surprisingly moderate,

if one assumes that LDHA plays a pivotal and non-redundant role in the Warburg




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effect and that aerobic glycolysis is crucial for NB cells. It is possible that during the

selection of LDHA knockout clones cells may have grown out that depend less on

LDHA. In addition, the oncogenic context of LDHA may determine its role in NB.

Along this line, lack of LDHA did not decrease development of lymphoma driven by c-

MYC in transgenic mice (43), in contrast to RAS-driven tumors that were susceptible

to LDHA ablation (43, 44). In NB cells, the balance between MYCN, which belongs to

the MYC family of transcription factors, and activated RAS, present in some NB

including the SK-N-AS cell line, may influence their response to LDHA depletion.

Further supporting the notion that oncogenic context influences the role of LDHA,

outcome after depletion of LDHA in other cancers has ranged from severely

diminished tumorigenicity (45, 46) to no effect (47).

The surprisingly moderate decrease of aggressiveness by LDHA depletion

may also be explained by the LDHB isoform, that we have shown to be amply

expressed in NB, substituting for LDHA. By employing inducible shRNA we achieved

near-complete depletion of LDHB. Depletion of LDHB decreased growth of NB cells

while not impacting on the Warburg effect. The former may have been caused by

loss of LDHB-mediated control of lysosomal function and thus decreased autophagy,

as described to occur specifically in cancer cells (32). Combined depletion of LDHB

and LDHA ablated clonogenicity.

Of note, LDH activity was not ablated despite homozygous knockout or

constitutive lack of LDHA, and glucose consumption, lactate production and

generation of ATP were maintained in vitro and in vivo. This shows that LDHA can be

dispensable for the Warburg effect in NB cells, a possibility entertained previously

(41, 42, 48).




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Intriguingly, combined depletion of LDHA and LDHB did not abrogate LDH

activity. This may be explained by the residual expression of LDHB observed

because of incomplete knockdown. LDHC protein, which might have substituted for

LDHA and LDHB, was not expressed in the cells, in line with near-absent mRNA

expression of LDHC in NB cell lines and patient NB.

Strikingly, concomitant depletion of LDHA and LDHB did not decrease aerobic

glycolysis, possibly because of the residual LDH activity. An alternative explanation

could be that LDH may be dispensable for aerobic glycolysis in NB cells.

Irrespectively, it can be concluded that therapeutic inhibition of aerobic glycolysis in

neuroblastoma by targeting LDH will be challenging. However, inhibition of LDHA

and LDHB decreased growth of NB cells independent of the Warburg effect.

Elucidation of the mechanisms involved and of the impact on non-malignant cells is

warranted.

In summary, high expression of LDHA in NB is independently associated with

poor patient survival and inhibiting LDHA and LDHB decreases NB growth

independent of aerobic glycolysis. This may have implications for future risk

assessment and therapy of NB patients.




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Acknowledgements

We thank Anneleen Beckers for help with the TH-MYCN mouse data, Ali

Gawanbacht for FACS sorting and Nicole Heymann and Helgard Knauß for technical

assistance.

We are grateful to the Deutsche Krebshilfe for financial support (grant

70112002 to CB).




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28

Figure legends

Figure 1. Increased LDHA mRNA expression in patient NB is independently

associated with poor outcome

(A) High transcript levels of LDHA in NB are significantly associated with

decreased survival of patients. Overall and event-free survival of 481 NB

patients according to LDHA transcript levels. The prognostic LDHA cut-off

had been determined in a training set by maximally selected log-rank statistic.

(B) The association of LDHA mRNA with survival is independent of known

risk factors of NB. Cox proportional hazard regression analyses using the

optimal prognostic cut-off expression for LDHA were performed. LDHA

expression and the risk factors MYCN status (na, non-amplified; a, amplified),

age and tumor stage were analyzed.

(C) Increased LDHA transcript levels are associated with risk factors of NB.

LDHA mRNA levels of the 709 patients were analyzed depending on risk

factors. Data are presented as box plots; na, non-amplified MYCN; a, MYCN-

amplified; n, number of samples. p values were calculated using the Mann-

Whitney test, except for the INPC where the Kruskal-Wallis test was used; ***,

p<0.001; **, p<0.01.

Figure 2. Increased expression of LDHA protein in patient NB is associated

with poor outcome

(A) High LDHA protein expression in NB is significantly associated with

decreased survival of patients. Overall and event-free survival of 110 NB

patients depending on LDHA protein expression, as determined by

immunohistochemistry (left panels). Statistical analysis by log-rank test. The




29

panels on the right show a representative LDHA-stained tumor slide and its

image analysis. Bars equal 50 µm. The LDHA-positive tumor area was

expressed as the percentage of total tumor area and used as a surrogate for

LDHA protein expression.

(B) The association of LDHA protein expression with survival is

independent of known risk factors of NB. Cox proportional hazard

regression analyses for LDHA protein expression, MYCN status and age are

shown.

(C) Increased LDHA protein levels are associated with risk factors of NB.

The NB patient samples stained for LDHA were analyzed in relation to risk

factors. Data are presented as box plots. na, non-amplified MYCN; a, MYCN-

amplified; n, number of samples. p values were calculated using the Mann-

Whitney test, except for INPC where the Kruskal-Wallis test was used. **,

p<0.01; *, p<0.05; n.s., not significant.

(D) LDHA in human NB is located in cytoplasm and nuclei. The NB stained

for LDHA were analyzed using higher magnification. Representative images

of tumor sections with LDHA-positive cytoplasm (top) and LDHA-positive

nuclei (bottom) are shown. Bars equal 50 µm.

(E) Increased and decreased numbers of cells with cytoplasmic and nuclear

LDHA, respectively, are associated with decreased overall survival of

NB patients. In the NB stained for LDHA the percentages of cytoplasmic-

positive and nuclear-positive cells within each tumor were determined.

Tumors with ≥5% or <5% positive cells in the respective cellular compartment

were categorized as positive or negative, respectively. Kaplan-Meier survival

analysis of the NB patients according to the subcellular distribution of LDHA

is shown. Statistical analysis by log-rank test.




30

(F) Increased number of cells with cytoplasmic LDHA in NB is associated

with risk factors. The fraction of tumors with LDHA in the cytoplasm was

analyzed for association with MYCN status (na, non-amplified; a, amplified),

age, stage and differentiation (diff., differentiated; p., poorly differentiated;

undiff., undifferentiated); CI, 95% confidence interval.

Figure 3. LDHA is expressed in NB of TH-MYCN mice and in human NB cell

lines, independent of MYCN

(A) LDHA mRNA does not increase during MYCN-induced transformation of

superior cervical ganglion cells to NB. mRNA expression of ODC1 and

LDHA during NB progression from tumor-prone ganglia at 2 weeks to tumors

at 6 weeks in TH-MYCN+/+ mice (red) is shown and compared to ganglia in

wild-type mice (blue). Linear regression analysis was performed and p values

were determined. ***, p<0.001; n.s., not significant.

(B) LDHA and LDHB are expressed in human NB cell lines independent of

MYCN amplification. MYCN-amplified and non-amplified human NB cell lines

and two primary NB cultures (U-NB1 and U-NB2) were analyzed by qRT-

PCR. LDHA mRNA expression is shown relative to HPRT expression. The

means of replicates of individual cell lines are shown in the upper left panel,

the means of all MYCN non-amplified vs. all amplified cell lines in the upper

right panel. Protein levels of LDHA and LDHB were determined by

immunoblotting, with TUBULIN as control (lower panel). Statistical analysis

was performed using Student’s t-test. ns; not significant.

(C) Forced overexpression of MYCN does not significantly increase

expression of LDHA and HIF-1α in SH-EP NB cells. SH-EP-MYCN-ER

cells were treated either with vehicle (-) or with tamoxifen (+) for 48h. Using




31

qRT-PCR, the amount of ODC1, LDHA and HIF-1α mRNA in relation to

ACTIN was determined in duplicates. Results represent three independent

experiments, calculated by the 2-ΔΔCt method. Statistical analysis was

performed with the unpaired, two-tailed Student’s t-test. **p<0.01.

Figure 4. LDHA depletion in SK-N-AS and KELLY NB cells decreases

clonogenicity, tumorigenicity and tumor growth

(A) LDHA protein is depleted in CRISPR/Cas9 knockout clones. SK-N-AS

and KELLY cell clones expanded from single cells after transfection with

CRISPR/Cas9 and sgRNA1 or sgRNA3 were probed for LDHA by Western

blot. ACTIN was used as loading control.

(B) LDHA depletion decreases clonogenicity. Wild-type (wt) and homozygous

LDHA knockout (ko) clones of SK-N-AS cells (wt clones sgRNA1 11 and 12,

and sgRNA3 1 and 12; ko clones sgRNA1 4 and 8, and sgRNA3 3, 4 and 6)

and of KELLY cells (wt clones sgRNA1 1 and 3, and sgRNA3 8; ko clones

sgRNA1 4 and 7, and sgRNA3 2, 5 and 6) were used. Clones were seeded

at low density into soft agar (KELLY) or onto plastic (SK-N-AS). Colonies

were stained and counted. Shown are the means and SD of three

independent experiments with the sample size (n) indicated. Statistical

analysis was performed using Student’s t-test. **, p<0.01; *, p<0.05.

(C) LDHA depletion decreases tumor incidence and increases tumor

latency. 1x106 cells of SK-N-AS clones (wt clones sgRNA1 11 and sgRNA3

1; ko clones sgRNA3 3 and 4) and 5x105 cells of KELLY clones (wt clones

sgRNA1 3 and sgRNA3 8; ko clones sgRNA1 7 and sgRNA3 2) were

injected subcutaneously into Rag2-/- cγc-/- mice (n=10-13 per group). The

development of tumors was determined regularly. Shown is the percentage




32

of tumor-free mice at times post transplantation. Statistical analysis was

performed using the log-rank test.

(D) LDHA depletion decreases tumor growth. Size of the tumors generated in

(C) was measured sequentially using a caliper. Tumor volumes and tumor-

doubling times of individual tumors were calculated. Statistical analysis was

performed using Student’s t-test; *, p<0.05; n.s., not significant.

Figure 5. LDHA is dispensable for the Warburg effect in SK-N-AS, KELLY and

SK-N-BE(2)C cells

(A) LDHA depletion in SK-N-AS and KELLY cells does not abrogate LDH

activity and does not significantly inhibit aerobic glycolysis. Wild-type

(wt) and homozygous LDHA knockout (ko) clones of SK-N-AS cells (wt clones

sgRNA1 11 and 12, and sgRNA3 12; ko clones sgRNA1 4, and sgRNA3 3 and

6) and of KELLY cells (wt clones sgRNA1 1 and 3, and sgRNA3 8; ko clones

sgRNA1 4 and 7, and sgRNA3 2) were cultured for the times indicated. LDH

activity, glucose and lactate were determined in the medium normalized to cell

numbers. Shown are the means and SD of three independent experiments

with the sample size (n) indicated. p values were determined using the

Student’s t-test. ***, p<0.001; *, p<0.05; n.s., not significant.

(B) SK-N-BE(2)C NB tumors constitutively lack LDHA. SK-N-AS, LAN5 and

SK-N-BE(2)C cell lines were injected subcutaneously into RAG2-/-/ cγc-/- mice.

Formalin-fixed and paraffin-embedded tumors were stained for human LDHA.

Bars equal 100 µm, in the insets 50 µm.

(C) Intact Warburg effect in SK-N-BE(2)C NB cells despite complete

constitutive lack of LDHA. Glucose, lactate and ATP per g tissue mass were

determined by induced metabolic bioluminescence imaging in cryostat




33

sections of subcutaneous tumors derived from SK-N-AS (n=8), LAN-5 (n=10)

and SK-N-BE(2)C (n=6). p values were calculated using the Mann-Whitney

test. ***, p<0.001; *, p<0.05; n.s., not significant.

Figure 6. Combined depletion of LDHA and LDHB in SK-N-AS and KELLY cells

does not abrogate LDH activity and aerobic glycolysis while ablating

clonogenicity

(A) LDHB is expressed in NB clones depleted of LDHA. Cell lysates from SK-

N-AS and KELLY clones were used for Western blot analysis of LDHA and

LDHB. ACTIN was used to control for equal loading. Shown is one

representative result of three independent Western blots.

(B) LDHB is strongly decreased after shRNA induction in wild-type and

LDHA knockout cells while LDHC remains not expressed. Wild-type (wt)

and LDHA knockout (ko) clones of SK-N-AS cells and of KELLY cells were

stably transduced with inducible non-silencing shRNA (ns) or silencing shRNA

against LDHB (sh2 and sh3). shRNA expression was induced by doxycycline

treatment for 72 h. LDHA, LDHB and LDHC proteins were detected by

Western blot and ACTIN was used as loading control.

(C) Combined depletion of LDHA and LDHB does not abrogate LDH activity

and does not reduce aerobic glycolysis. Wild-type (wt) and LDHA knockout

(ko) clones of SK-N-AS cells and of KELLY cells expressing ns, sh2 or sh3

were cultured. LDH activity, determined in cell lysates, and glucose and

lactate in culture supernatants were normalized to cell numbers. Shown are

the results of three independent experiments. p values were determined using

2-way ANOVA. ***, p<0.001; **, p<0.01; *, p<0.05; n.s., not significant.




34

(D) Combined depletion of LDHA and LDHB ablates clonogenicity. LDHA wt

and ko clones of SK-N-AS and KELLY cells expressing ns, sh2 and sh3 were

seeded at low density onto plastic. Colonies were stained and counted. Shown

are the results of three independent experiments. Statistical analysis was

performed using 2-way ANOVA. ***, p<0.001; *, p<0.05.




Fig. 1

0 5 10 15 20 25

A

Time after diagnosis (y)

Ove

rall

surv

ival

LDHA high (n=169)LDHA low (n=312)

<18 ≥18na 1-3, 4s 4

LDH

A m

RN

A ex

pres

sion

(x10

8 )

B

Age (months) Stage

*********

a

C17

16

14

13

12

15

11

17

16

14

13

12

15

11

17

16

14

13

12

15

11

MYCN

17

16

14

13

12

15

11

INPC

undiff. poorly diff. diff.

**

cut-off = 14.28p < 0.001

cut-off = 14.15p < 0.001

1.0

0.8

0.6

0.4

0.2

0.0

Even

t-fre

e su

rviv

al

1.0

0.8

0.6

0.4

0.2

0.00 5 10 15 20 25

Time after diagnosis (y)


Stage

Age

MYCN

LDHA

Hazard ratio (95% CI)

low high

na a

<18m ≥18m

1-3,4s 4

1 2 3 4 5 6 7

Stage

Age

MYCN

LDHA low

na

<18m

1-3,4s

high

a

≥18m

4

Hazard ratio (95% CI)1 2 3 4 5 6 7

Overall survival Event-free survivalp=0.002

p<0.001

p<0.001

p<0.001

p<0.001

p<0.001

p<0.001

p=0.009

(n=581) (n=122) (n=259)(n=334)(n=278)(n=431) (n=43)(n=333)(n=22)




Fig. 2

1-3, 4s 4 undiff.p.diff.MYCN

a

A

B

0 5 10 15Time after diagnosis (y)

p = 0.395

LDHA−neg. nuclei (n=77) LDHA−pos. nuclei (n=33)

0 5 10 15

Ove

rall

surv

ival

p = 0.040

LDHA−neg. cytoplasm (n=45) LDHA−pos. cytoplasm (n=65)

C

LDHA−pos. cytoplasm + LDHA−neg. nuclei (n=48)

LDHA−neg. cytoplasm + LDHA−pos. nuclei (n=16)

p = 0.028

020406080

100

020406080

100

020406080

100

020406080

100

≥18<18

0.0

0.2

0.4

0.6

0.8

1.0

naAge (months) Stage INPC

0 5 10 15

na a <18 ≥18

Age (months)

1-3, 4s 4

Stage

LDH

A-p

ositi

ve tu

mor

are

a (%

of t

otal

tum

or a

rea)

0

20

40

60

80

100

0.0

0.2

0.4

0.6

0.8

1.0

Ove

rall

surv

ival

0 5 10 15Time after diagnosis (y)

p<0.001


***

LDHA-neg. tumor areaLDHA-pos. tumor area

0.0

0.2

0.4

0.8

1.0

Even

t-fre

e su

rviv

al

0Time after diagnosis (y)

p=0.002


E

0.6

MYCN

undiff. p.

INPC

n.s.

diff.

F

20 5 10 15 20

Hazard ratio (95% CI)4 5 6321 7 8 9 10111213

Age

MYCN

LDHA low high

na a

<18m ≥18m

p=0.008

p=0.001

p=0.002

D

(n=75) (n=32) (n=57)(n=53)(n=63)(n=47) (n=7)(n=82)(n=7)

(CI: 3

2-55%

)

(CI: 7

9-99%

)

(CI: 4

2-72%

)

(CI: 4

7-72%

)

(CI: 3

4-64%

)

(CI: 4

9-74%

)

(CI: 4

-71%)

(CI: 4

9-69%

)

(CI: 4

2-99%

)

cut-off = 61 cut-off = 61

Overall survival

Tum

ors

with

cyt

opla

smic

LD

HA

(% o

f all

tum

ors)

**

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100




Fig. 3

A

B

amplifiedMYCN-MYCN

non-amplified

LD

HA

mR

NA

ex

pre

ss

ion

(re

lati

ve

to

HP

RT

)

n.s.

LDHA

TUBULIN

10

15

5

0

n.s.

C

0

2

4

6

8**

0

2

4

6

8

10

12

OD

C1

mR

NA

ex

pre

ss

ion

(re

lati

ve

to

AC

TIN

)

LD

HA

mR

NA

ex

pre

ss

ion

(re

lati

ve

to

AC

TIN

)

4-OHT+ +- -

4-OHT+-

4-OHTHIF

-1α

mR

NA

ex

pre

ss

ion

(re

lati

ve

to

AC

TIN

) n.s.n.s.

0

0.5

1.0

1.5

LDHB

TUBULIN

0

5

10

15 n.s.

LD

HA

mR

NA

ex

pre

ss

ion

(re

lati

ve

to

HP

RT

)

MYCN non-amplified amplified

MYCN-

Postnatal age (w)

TH−MYCN+/+ miceWild−type mice

1 2 6 1 2 6

12

9.7

10

5.8

ODC1 LDHA

***

SH-E

P

SH-S

Y5Y

SK-N

-SH

SK-N

-AS

NB69

GI-M

E-N

U-N

B1

KELLY

LAN-5

IMR32

SK-N

-BE(2

)C

U-N

B2




A

C

D

Co

lon

ies

pe

rw

ell

0 50 100 1500

20

40

60

80

100

p=0.016

Time post transplantation (d)

Tu

mo

r-fr

ee

mic

e(%

)

0

5

10

15

20 *

Do

ub

lin

gti

me

(d)

0

100

200

300

400

Co

lon

ies

pe

rw

ell

Time post transplantation (d)

p=0.009

0

20

40

60

80

100

Tu

mo

r-fr

ee

mic

e(%

)

0 50 150100

Do

ub

lin

gtim

e(d

)

0

5

10

15

20n.s.

SK-N-AS KELLY

**

0

100

200

300

400* LDHAwt clones

LDHAko clones

LDHAwt clones

LDHAko clones

LDHAwt clones

LDHAko clones

LDHA

sgRNA1

ACTIN

1 2 3 4 5 6 7 8 9 10 11 12

2 9 121 3 4 5 6 7 8 10 11

Clone #

sgRNA3

LDHA

ACTIN

B

1 2 3 4 5 6 7 8 9

1 2 3 4 5 6 7 8 9

LDHA

sgRNA1

ACTIN

Clone #

sgRNA3

LDHA

ACTIN

Clone # Clone #

(n=4)(n=5) (n=3)(n=3)

Fig. 4




Fig. 5A

SK-N-AS LAN-5 SK-N-BE(2)C

SK-N-A

SLAN5

SK-N-B

E(2)C

0

2

4

6

8

10

Glu

cose

(µm

ol/g

)

SK-N-A

SLAN5

SK-N-B

E(2)C

0

10

20

30

40

Lact

ate

(µm

ol/g

)

SK-N-A

SLAN5

SK-N-B

E(2)C

0

1

2

3

4

ATP

(µm

ol/g

) ****

n.s.n.s.

n.s.

n.s.

n.s.n.s.

n.s.

0.01.02.03.04.0

x10-6

5.0

Glu

cose

(nm

ol/µ

l/cel

l)

0 24 48 960.00.51.01.52.02.5

Time (h)

Lact

ate

(nm

ol/µ

l/cel

l)

n.s.

n.s.

*

24 48 9602468

1012

Time (h)

B

C

x10-6

24 48 72Time (h)

05

10152025

***

Lact

ate

(nm

ol/µ

l/cel

l)

0 24 48 72Time (h)

x10-4

0

2

4

6

8

n.s.

x10-4

Glu

cose

(nm

ol/µ

l/cel

l)

02468

10

n.s.

SK-N-AS KELLYLDHA wt clones (n=3)LDHA ko clones (n=3)

LDH

act

ivity

(m

U/m

l/106

cells

)

LDH

act

ivity

(m

U/m

l/106

cells

)




LDHA wt LDHA ko

11 312 412 6

LDHB

ACTIN

LDHA wt LDHA ko

3 21 48 7

LDHA

A

B

C

SK-N-AS KELLY

LDHB

ACTIN

LDHA

#3 (wt) #2 (ko)

ns nssh2 sh2sh3 sh3

#11 (wt) #4 (ko)

ns nssh2 sh2sh3 sh3

LDHA clone #

LDHB shRNA #

LDHA clone #

D

0.0

1.0

2.0

3.0

4.0x10-5

0 24 48 72 96

0 24 48 72

Time (h)

960.0

x10-5

#3 (wt)

#3 (wt)

#2 (ko)#2 (ko)

x10

0

10

20

30

40

24 48 72 96

0

50

100

150

200

#3 (wt) #2 (ko)

ns nssh2 sh2sh3 sh3

LDHA clone #

LDHB shRNA #

x10

0

20

40

60

#11 (wt)

#11 (wt)

#4 (ko)#4 (ko)

24 48 72 96

******

******

0.0

2.0

4.0

6.0

8.0x10-5

Lacta

te(n

mo

l/µl/

cell

)

0 24 48 72

Time (h)

96

0

50

100

150

200

Co

lon

ies p

er

well

#11 (wt) #4 (ko)

sh2ns nssh2 sh3 sh3

n.s.******

n.s.

1.0

2.0

3.0

4.0

0.0

1.0

2.0

3.0

4.0x10-5

Glu

co

se

(nm

ol/µ

l/cell

)

0 24 48 72 96

*

n.s.

n.s.

LDHA LDHB LDHA LDHB

LDHC

LD

H a

cti

vit

y

(mU

/10

3cell

s)

n.s.

n.s.

n.s.

n.s.

n.s.

sh2,3

ns

ns

sh2,3

sh2,3

ns

ns

sh2,3

Fig. 6




Published OnlineFirst June 20, 2018.Clin Cancer Res Carmen Dorneburg, Matthias Fischer, Thomas F.E. Barth, et al. aerobic glycolysis

ofits depletion decreases neuroblastoma growth independent LDHA in neuroblastoma is associated with poor outcome and

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LDHA in neuroblastoma is associated with poor outcome and its … · 2018-06-20 · LDHA in neuroblastoma is associated with poor outcome ... Christian Beltinger, Section of Experimental