rna helicase a is a downstream mediator of kif1bb tumor ... · research article rna helicase a is a...

RESEARCH ARTICLE

RNA Helicase A Is a Downstream Mediator of KIF1Bb Tumor-Suppressor Function in Neuroblastoma Zhi Xiong Chen 1 , Karin Wallis 1 , Stuart M. Fell 1 , 2 , Veronica R. Sobrado 1 , Marie C. Hemmer 1 , Daniel Ramsköld 1 , 2 , Ulf Hellman 4 , Rickard Sandberg 1 , 2 , Rajappa S. Kenchappa 6 , Tommy Martinson 5 , John I. Johnsen 3 , Per Kogner 3 , and Susanne Schlisio 1,2

on July 11, 2020. © 2014 American Association for Cancer Research. cancerdiscovery.aacrjournals.org Downloaded from

Published OnlineFirst January 27, 2014; DOI: 10.1158/2159-8290.CD-13-0362

http://cancerdiscovery.aacrjournals.org/

APRIL 2014�CANCER DISCOVERY | 435

ABSTRACT Inherited KIF1B loss-of-function mutations in neuroblastomas and pheochromo-

cytomas implicate the kinesin KIF1B as a 1p36.2 tumor suppressor. However, the

mechanism of tumor suppression is unknown. We found that KIF1B isoform β (KIF1Bβ) interacts with RNA

helicase A (DHX9), causing nuclear accumulation of DHX9, followed by subsequent induction of the proa-

poptotic XIAP-associated factor 1 (XAF1) and, consequently, apoptosis. Pheochromocytoma and neuro-

blastoma arise from neural crest progenitors that compete for growth factors such as nerve growth factor

(NGF) during development. KIF1Bβ is required for developmental apoptosis induced by competition for

NGF. We show that DHX9 is induced by and required for apoptosis stimulated by NGF deprivation. More-

over, neuroblastomas with chromosomal deletion of 1p36 exhibit loss of KIF1Bβ expression and impaired

DHX9 nuclear localization, implicating the loss of DHX9 nuclear activity in neuroblastoma pathogenesis.

SIGNIFICANCE: KIF1Bβ has neuroblastoma tumor-suppressor properties and promotes and requires

nuclear-localized DHX9 for its apoptotic function by activating XAF1 expression. Loss of KIF1Bβ alters

subcellular localization of DHX9 and diminishes NGF dependence of sympathetic neurons, leading to

reduced culling of neural progenitors, and, therefore, might predispose to tumor formation. Cancer

Discov; 4(4); 434–51. ©2014 AACR.

See related commentary by Bernards, p. 392.

Authors’ Affi liations: 1 Ludwig Institute for Cancer Research Ltd.; 2 Depart-ment of Cell and Molecular Biology, Karolinska Institutet; 3 Department of Women’s and Children’s Health, Karolinska University Hospital, Stockholm; 4 Ludwig Institute for Cancer Research Ltd., Biomedical Center, Uppsala; 5 Department of Clinical Genetics, Institute of Biomedicine, University of Gothenburg, Sahlgrenska University Hospital, Göteborg, Sweden; and 6 Moffi tt Cancer Center, Neuro-Oncology Program, Tampa, Florida

Note: Supplementary data for this article are available at Cancer Discovery Online (http://cancerdiscovery.aacrjournals.org/).

Current address for Z.X. Chen: Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore.

Corresponding Author: Susanne Schlisio, Ludwig Institute for Cancer Research Ltd., Karolinska Institutet, Nobels väg 3, SE-17177, Stockholm, Sweden. Phone: 46-8-52487117; Fax: 46-8-332812; E-mail: [email protected]

doi: 10.1158/2159-8290.CD-13-0362

©2014 American Association for Cancer Research.

INTRODUCTION During development of the peripheral nervous system,

neural progenitor cells depend on and compete for growth

factors, such as nerve growth factor (NGF). Mutations affect-

ing NGF-dependent neuronal survival have been associated

with sympathetic nervous system tumors such as neuro-

blastoma and later-developing malignancies of neural crest

origin, such as paraganglioma and pheochromocytoma ( 1–5 ).

Germline mutations associated with paragangliomas and

pheochromocytomas ( VHL , RET , NF1 , and SDHB/C/D ) are

thought to defi ne a pathway that is activated when NGF is

limiting, leading to apoptosis mediated by the EGLN3 prolyl

hydroxylase ( 4 ). Failure to properly cull the neuronal progeni-

tor cells during development might predispose to neoplastic

transformation ( 2 , 6 ). Recently, we identifi ed the KIF1B gene

as a downstream mediator of the proapoptotic effects of the

prolyl hydroxylase EGLN3 and demonstrated that KIF1B

isoform β (KIF1Bβ) is necessary and suffi cient for apoptosis

when NGF is limiting. KIF1B is a member of the kinesin

3 family and encodes two alternatively spliced isoforms,

KIF1Bα and KIF1Bβ ( 7–9 ). Both share an N-terminal motor

domain but contain different C-terminal cargo domains.

KIF1Bα and KIF1Bβ are motor proteins implicated in antero-

grade transport of mitochondria and synaptic vesicle precur-

sors, respectively ( 10 ). However, the recently identifi ed role

of KIF1Bβ in NGF-mediated neuronal apoptosis implicates

this kinesin as an important player during sympathetic neu-

ron development. Moreover, KIF1B maps to chromosome

1p36.2, a chromosomal region that is frequently deleted in

neural crest–derived tumors, including neuroblastomas ( 5 ).

The identifi cation in neuroblastomas and pheochromocy-

tomas of inherited KIF1B missense mutations that remove

KIF1Bβ’s ability to induce neuronal apoptosis ( 5 , 11 ) suggests

that KIF1Bβ is a pathogenic target of 1p36 deletion in these

diseases. Therefore, we investigated the tumor-suppressive

mechanism by which KIF1Bβ regulates apoptosis.

RESULTS DHX9 Is a Binding Partner of the KIF1Bb Apoptotic Domain

To investigate how EGLN3 regulates KIF1Bβ and how

this promotes cell death, we mapped the domain of KIF1Bβthat is necessary and suffi cient to induce apoptosis ( Fig. 1A ).

We tested a series of N- and C-terminal truncated KIF1Bβvariants for apoptotic function by electroporating them

into primary rat sympathetic neurons ( Fig. 1B ). Neurons

were 4′,6-diamidino-2-phenylindole (DAPI) stained, and the

nuclei of FLAG-positive neurons were visualized for apoptotic

changes. In addition, we transfected NB1 neuroblastoma cells

with the KIF1Bβ variants and stained cells with crystal violet

to determine cell viability ( Fig. 1C ). These results confi rm

earlier observations that both full-length and motor-defi cient




436 | CANCER DISCOVERY�APRIL 2014 www.aacrjournals.org

Chen et al.RESEARCH ARTICLE

Figure 1. DHX9 is a binding partner of KIF1Bβ apoptotic domain. A, schematic representation of KIF1Bβ deletion mutants and their ability to induce cell death, DHX9 binding, and DHX9 nuclear localization. FL, full-length. B, percentage of apoptosis in FLAG-positive, rat primary sympathetic neurons after electroporation with plasmids encoding FLAG-KIF1Bβ mutants as indicated. DAPI-stained nuclei were evaluated for apoptotic changes such as nuclear condensation and fragmentation. C and D, crystal violet staining to measure cell viability in NB1 and CHP212 cells after transfection with FLAG-KIF1Bβ plasmids as indicated. Transfected cells were selected with G418 (500 μg/mL) for several weeks. Empty vector (empty) served as negative control. E, immunoblot analysis of NB1 cells that were coinfected with lentivirus encoding Flag-KIF1Bβ mutants together with adenovirus encoding EGLN3 or coinfected with lentivirus encoding short hairpin targeting EGLN3 (shE3) or nontargeting control (SCR) as indicated. F, large-scale immunoprecipitation in NB1 cells that were infected with lentivirus encoding Stag-FLAG-KIF1Bβ600–1400. Silver-stained gel indicates (arrow) identifi ed coimmunoprecipitated proteins and table listing peptides identifi ed by mass spectrometry. G, immunoblot showing coimmunoprecipitation of endogenous DHX9 and KIF1Bβ in SK-N-SH cells that were immunoprecipitated with KIF1Bβ antibody. H, anti-DHX9 immunoblot analysis of NB1 cells transfected to produce Flag-KIF1Bβ protein and immunoprecipitated with anti-Flag antibody as indicated (arrow).

A

FL

60

0

80

0

10

00

12

00

14

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death:

DHX9

binding:

Yes Yes YesFlag-

600–1400

Ad-EgIN3

EGIN3

Tubulin

Flag

– + – + –

–+

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Bait

ID In silica Protein ID

RNA helicase A

(DHX9)

Exportin-2

(XPO2)

Acc #

NP_001348 23 22/77

NP_001307

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7 6/50

Seq.

cov.

# pep

pl MW

6.4 142

5.5 111

1a

1b

40

30

20

10

0

GFP

KIF-W

T

KIF-6

00–1

000

KIF-6

00–1

200

KIF-6

00–1

400

KIF-6

00–1

600

KIF-6

00–1

770

KIF-8

00–1

600

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000–

1600

KIF-1

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DHX9 binding

DHX9 localization

Apoptotic activity

600–1600 600–1400 600–1200 600–1000

600–1770

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Empty WT 1000–1400 600–1400

800–1600 1000–1600 1200–1600

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ells

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21

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localization:

Motor

600–1770

600–1600

600–1400

600–1200

600–1000

800–1600

1000–1600

1200–1600

1000–1400

600–1400Δ1100–1300

B

C G

D H

F

E

KIF1Bβ





RNA Helicase A Is Vital for KIF1Bβ Tumor Suppression RESEARCH ARTICLE

KIF1Bβ induce apoptosis when ectopically expressed ( 5 , 12 ).

The apoptosis-inducing region of KIF1Bβ was previously

mapped to amino acids 637 to 1576 ( 12 ). Moreover , we identi-

fi ed the domain that retains apoptotic activity residing within

the 600–1400 amino acid region, whereas the 600–1200 and

the 1200–1600 domains failed to induce apoptosis ( Fig. 1B

and C ). Therefore, we tested additional KIF1Bβ deletions

and identifi ed the domain-spanning amino acids 1000–1400

(KIF1Bβ1000–1400) as suffi cient to induce apoptosis similar

to full-length KIF1Bβ in NB1 and CHP-212 neuroblastoma

cells ( Fig. 1D ). In contrast, internal deletion of amino acids

spanning 1100–1300 abrogated the ability of KIF1Bβ600–

1400Δ1100–1300 to induce apoptosis. Comparable levels of

KIF1Bβ protein production were confi rmed by immunoblot

analysis (Supplementary Fig. S1A).

Because KIF1Bβ was previously identifi ed downstream of

the prolyl hydroxylase EGLN3 during apoptosis, we ques-

tioned whether the truncated KIF1Bβ variants are respon-

sive to EGLN3. When ectopically expressed, the protein

level of KIF1Bβ600–1400 domain that retains apoptotic

activity was induced by EGLN3 ( Fig. 1E , left). Conversely,

knockdown of EGLN3 expression by lentivirus encoding

short hairpin RNA (shRNA) markedly decreased ectopic

KIF1Bβ600–1400 abundance ( Fig. 1E , right), similar to

what we observed with endogenous KIF1Bβ protein (Sup-

plementary Fig. S1B). However, the proapoptotic variant

KIF1Bβ1000–1400 was not regulated by EGLN3, in contrast

with the nonapoptotic variant KIF1Bβ600–1200 ( Fig. 1E ).

Therefore, regulation by EglN3 and induction of apop-

tosis are mediated through distinct domains within the

KIF1Bβ600–1400 region.

Next, we searched for proteins that interact specifi cally with

the proapoptotic and EGLN3-responsive KIF1Bβ600–1400

domain. NB1 cells were transiently transduced with Stag-

FLAG-KIF1Bβ600–1400 lentivirus and bound proteins were

resolved by SDS-PAGE and analyzed by mass spectrome-

try ( Fig. 1F ). Among the peptides identifi ed, 22 peptides

from RNA helicase A (DHX9) were recovered ( Fig. 1F ). The

association between KIF1Bβ and DHX9 was confi rmed in

SK-N-SH neuroblastoma cells by coimmunoprecipitation of

endogenous KIF1Bβ with endogenous DHX9 ( Fig. 1G ). Fur-

thermore, exogenously expressed FLAG-KIF1Bβ600–1400 in

NB1 cells also confi rmed the interaction with endogenous

DHX9 ( Fig. 1H ). This interaction was specifi c, because DHX9

did not coprecipitate with nonapoptotic variant FLAG-

KIF1Bβ600–1200 ( Fig. 1H ).

KIF1Bb Requires DHX9 to Induce Apoptosis Next, we asked whether DHX9 is necessary for KIF1Bβ

apoptotic function. Knockdown of DHX9 in NB1 ( Fig. 2A )

and SK-N-SH cells ( Fig. 2B ) using lentiviral shRNA resulted

in protection from apoptosis induced by ectopic expression

of KIF1Bβ600–1400 as measured by crystal violet staining

for viability. Cells transduced with nontargeting shRNA

(shSCR) served as control. Protection from apoptosis was

observed with multiple independent shRNAs targeting

DHX9, indicating that the knockdown was “on-target”

(Supplementary Fig. S2A). The same result was obtained

using SK-N-SH cells that were engineered to induce KIF1Bβ

upon tetracycline treatment ( Fig. 2C ). Cells treated with

tetracycline died upon KIF1Bβ induction; however, trans-

duction with shRNA against DHX9 resulted in resistance to

KIF1Bβ-induced cell death ( Fig. 2C ). Conversely, coexpres-

sion of DHX9 together with KIF1Bβ600–1400 in CHP-212

cells had a synergistic effect on cell death, as measured by

crystal violet staining for viability and cleavage of caspase-3

( Fig. 2D ). This synergy was specifi c to the apoptotic domain

of KIF1Bβ (KIF1Bβ600–1400), as coexpression of DHX9

with the nonapoptotic mutant KIF1Bβ600–1200 did not

have the same effect ( Fig. 2E ). In addition, we quantifi ed

apoptosis by scoring the nuclei of cells expressing GFP–

histone fusion protein ( Fig. 2F and Supplementary Fig.

S2B). The nuclei of NB1 cells transfected to produce GFP–

histone alone or together with WT-DHX9 were healthy

and uniform. In contrast, cells transfected with full-length

RFP-KIF1Bβ ( Fig. 2F ) or RFP-KIF1Bβ600–1400 (Supple-

mentary Fig. S2B) displayed signs of apoptosis (nuclear

condensation and fragmentation) in 22% and 30% of cells,

respectively. However, this proportion increased synergisti-

cally to 68% and 72% when RFP-KIF1Bβ was coexpressed

together with DHX9 ( Fig. 2F and Supplementary Fig.

S2B). Previous studies demonstrated that DHX9 functions

in the nucleus as a transcriptional activator ( 13–18 ). To

determine whether this function of DHX9 is required for

KIF1Bβ-induced apoptosis, we used previously character-

ized DHX9 mutants ( 19, 20 ) that are defective in either

nuclear transport (ΔNTD-DHX9) or transcriptional activa-

tion (TD-DHX9; Supplementary Fig. S2C). When tested,

both DHX9 mutants, ΔNTD-DHX9 and TD-DHX9, failed

to synergize with KIF1Bβ in apoptosis induction, implying

DHX9 nuclear activity in the induction of apoptosis by

KIF1Bβ ( Fig. 2F and Supplementary Fig. S2B). Expression

and localization of RFP-KIF1Bβ and eCFP-DHX9 mutants

was confi rmed by confocal microscopy ( Fig. 2G and Sup-

plementary Fig. S2D).

KIF1Bb Promotes DHX9 Nuclear Localization Motivated by our fi ndings that nuclear localization of

DHX9 is needed to synergize with KIF1Bβ in apoptosis, we

investigated DHX9 cellular localization upon KIF1Bβ expres-

sion. Endogenous DHX9 and exogenous eCFP-DHX9 were

predominantly nuclear in 1p36-intact SK-N-SH neuroblas-

toma cells (KIF1Bβ +/+ ), in line with earlier reports describ-

ing nuclear DHX9 localization ( Fig. 3A and B ; refs. 13,

14 , 16 ). However, in 1p36.2 homozygous-deleted NB1 cells

(KIF1Bβ −/− ), DHX9 localized mainly in the cytoplasm ( Fig.

3A and B ). To understand whether cytoplasmic DHX9 in NB1

cells is a consequence of KIF1Bβ loss, we restored KIF1Bβ

by ectopically expressing RFP-KIF1Bβ and visualized endog-

enous DHX9 and exogenous eCFP-DHX9 (Supplementary

Fig. S3A, S3C, and S3D). Nuclear localization of endog-

enous DHX9 in NB1 cells was restored in 89% of cells upon

expression of RFP-KIF1Bβ or 80% of cells upon expression

of RFP-KIF1Bβ600–1400 (Supplementary Fig. S3A). Simi-

larly, ectopically expressed eCFP-DHX9 together with RFP-

KIF1Bβ or RFP-KIF1Bβ600–1400 in NB1 cells resulted in

nuclear localization of DHX9 in 92% and 80% of cells, respec-

tively ( Fig. 3C and D ). In contrast, the nonapoptotic KIF1Bβ

mutant (KIF1Bβ600–1200) that does not interact with DHX9

displayed signifi cantly less nuclear localization of DHX9






Figure 2. DHX9 is required for KIF1Bβ-induced apoptosis. A, crystal violet staining of NB1 cells and (B) SK-N-SH cells after infection with lentivirus encoding shRNA targeting DHX9 (sh DHX9 ) or control virus (shSCR) and transfected with Flag-KIF1Bβ600–1400. Cells were selected with G418 (500 μg/mL) for several weeks. Bottom, corresponding immunoblot analysis. C, crystal violet staining of tetracycline-inducible SK-N-SH cells that were infected with lentivirus encoding shRNA targeting DHX9 (sh DHX9 ) or control virus (shSCR) and treated with tetracycline (0.5 μg/mL) to induce Flag-KIF1Bβ. Bottom, corresponding immunoblot analysis. D, crystal violet staining of CHP-212 cells that were transfected with His-DHX9 plasmid as indicated and cotransfected with increasing amounts of Flag-KIF1Bβ600–1400 plasmid and selected with G418 (500 μg/mL) for several weeks. Bottom, correspond-ing immunoblot analysis. E, performed as D with addition of Flag-KIF1Bβ600–1200) plasmid as indicated (arrow). F, NB1 cells transfected with plasmid encoding GFP-histone along with plasmid encoding RFP-KIF1Bβ or WT-DHX9 (wild-type) alone, or RFP-KIF1Bβ in combination with WT-DHX9 or mutant DHX9 (ΔNTD-DHX9 or TD-DHX9) as indicated. Shown is the percentage of GFP-positive nuclei exhibiting apoptotic changes 48 hours after transfection (mean ± SD; n = 3; *, P < 0.05). G, corresponding immunofl uorescence studies of F 24 hours after transfection with plasmids encoding RFP-KIF1Bβ (red) and eCFP-DHX9 (green).

A B

D E

F G

CshSCR shDHX9

shSCR

Empty

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KIF1Bβ600–1400

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– + – +

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shSCR shDHX9

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– + – +

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–

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RFP-KIF1BβBars –10 μm

eCFP-DHX9 Hoechst Merge

TD-DHX9

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DHX9

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–

+

–

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Figure 3. KIF1Bβ promotes DHX9 nuclear localization. A, left, anti-KIF1Bβ immunoblot analysis of NB1 and SK-N-SH cell lines. Right, immunofl uores-cence images of NB1 and SK-N-SH cells stained for DHX9 (green) and counterstained with Hoechst to visualize nuclei (blue). B, fl uorescence studies of NB1 and SK-N-SH cells 24 hours after transfection with plasmid encoding eCFP-DHX9 (green) as indicated. C, fl uorescence studies in NB1 cells 24 hours after transfection with plasmid encoding eCFP-DHX9 together with either RFP-KIF1Bβ-WT or RFP-KIF1Bβ mutants as indicated (arrow). D, graphical representation showing percentage of transfected cells with nuclear or cytoplasmic eCFP-DHX9 localization (mean ± SD; n = 3; **, P < 0.01; ***, P < 0.001). E, graphical representation of immunofl uorescence images of NB1 cells (Supplementary Fig. S3D) transfected with Flag-KIF1Bβ mutants as indicated. Shown is the percentage of nuclear or cytoplasmic eCFP-DHX9 or both 24 hours after transfection (mean ± SD; n = 3; *, P < 0.05; **, P < 0.01; ***, P < 0.001). F, anti-DHX9 (green) immunofl uorescence staining of NB1 cells 24 hours after transfection with plasmids encoding Flag-KIF1Bβ wild-type (WT) or disease-causing variants as indicated (red). The percentage of transfected cells displaying nuclear DHX9 is shown in G (mean ± SD; n = 3; *, P < 0.05; **, P < 0.01; ***, P < 0.001).

A endg. DHX9 Hoechst Merge

endg. DHX9 Hoechst Merge

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CRFP-KIF1Bβ FLAG-KIF1Bβ

Bars –10 μm

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Bars –20 μm

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Bars –20 μm

eCFP-DHX9 Hoechst Merge






as compared with RFP-KIF1Bβ and RFP-KIF1Bβ600–1400

( Fig. 3C and D ).

In addition to NB1 cells, KIF1Bβ-inducible SK-N-SH

cells (Tet-SK-N-SH) showed an enhanced nuclear accumu-

lation of endogenous DHX9 24 hours after FLAG-KIF1Bβ

induction by tetracycline (Supplementary Fig. S3B). Nuclear

DHX9 in tetracycline-treated cells was concentrated in spe-

cifi c nuclear regions and colocalized with the nucleoli, visu-

alized by anti-Fibrillarin counterstaining (Supplementary

Fig. S3B).

Because DHX9 nuclear localization depends upon KIF1Bβ

expression, we reasoned that silencing of KIF1Bβ in SK-N-SH

cells (KIF1Bβ +/+ ) should result in the reverse. Knockdown of

KIF1Bβ in SK-N-SH cells using lentiviral shRNA resulted

in predominantly cytoplasmic DHX9 (Supplementary Fig.

S3C). This was also achieved using shRNA targeting EGLN3,

resulting in downregulation of KIF1Bβ protein and subse-

quent cytoplasmic localization of DHX9 (Supplementary

Fig. S3C).

We next examined the ability of additional KIF1Bβ vari-

ants to infl uence eCFP-DHX9 localization. The proapoptotic

domain 1000–1400, as well as the proapoptotic domain

1000–1600, signifi cantly stimulated DHX9 nuclear localiza-

tion in 40% and 52% of the cells, respectively, with 27% of

the cells displaying partial DHX9 nuclear localization ( Fig.

3E and Supplementary Fig. S3D). In contrast, the apoptotic-

defective mutants 1200–1600 and 600–1400Δ1100–1300

were impaired in stimulating eCFP-DHX9 nuclear localiza-

tion ( Fig. 3E and Supplementary Fig. S3D), similar to what

we observed for the apoptotic-defective mutant 600–1200

( Fig. 3D ). However, although mutant 600–1200 is defi cient

in DHX9 binding, DHX9 nuclear localization, and apoptosis,

the mutants 1200–1600 and 600–1400Δ1100–1300 retained

the ability to bind to DHX9 despite being defective in apopto-

sis and DHX9 nuclear localization ( Fig. 1A and Supplemen-

tary Fig. S3E). On the basis of the DHX9-binding data, we

conclude that the region required for DHX9 binding resides

on amino acids 1300–1400, as 600–1400Δ1100–1300 retained

the ability to bind DHX9, whereas 600–1200 did not ( Fig. 1A

and H and Supplementary Fig. S3E). In contrast, the bind-

ing site does not overlap with the DHX9 nuclear localization

site. We conclude that the DHX9 nuclear localization site

resides on amino acids 1100–1200, as 1000–1400 localizes

DHX9 to the nucleus, whereas 1200–1600 and Δ1100–1300

do not ( Figs. 1A and 3E ). This indicates that DHX9 binding

and DHX9 localization are dictated by two distinct adja-

cent sites. It further implies that both sites are required for

KIF1Bβ-induced apoptosis, because mutants that either lack

the DHX9 binding site (600–1200) or lack the DHX9 localiza-

tion site (1200–1600 and Δ1100–1300) failed to induce apop-

tosis ( Fig. 1A, C, and D ). Collectively, these results suggest

that additional KIF1Bβ-interacting partners/modifi ers at the

amino acid region 1100–1200 might be required to mediate

DHX9 nuclear localization.

Next, we tested putative disease-causing KIF1Bβ variants

that were identifi ed in neuroblastomas and pheochromocy-

tomas and were defective in apoptosis ( 5 ). The variants T827I

and P1217S failed to relocate DHX9 to the nucleus ( Fig. 3F

and G ). Moreover, ectopic expression of the variant E1628K

displayed a signifi cant reduction of nuclear DHX9 compared

with wild-type FLAG-KIF1Bβ. However, the variants E646V

and S1481N stimulated DHX9 nuclear localization similar

to wild-type KIF1Bβ despite their impairment in apoptosis

( Fig. 3F and G ), indicating that DHX9 nuclear localization is

necessary but not suffi cient for KIF1Bβ to induce apoptosis,

and additional mechanisms might be required for KIF1Bβ

apoptotic function.

Exportin-2 Is Necessary for DHX9 Nuclear Localization and KIF1Bb Apoptotic Function

To understand how KIF1Bβ regulates DHX9 nuclear

localization, we investigated other KIF1Bβ-associated pro-

teins that were identifi ed by large-scale immunoprecipita-

tion ( Fig. 1F ). In addition to DHX9, we identifi ed Exportin-2

(XPO2) as a specifi c binding partner of the KIF1Bβ600–1400

apoptotic domain ( Fig. 1F ). XPO2 regulates nuclear import

and export of cellular proteins via its ability to reexport

Importin from the nucleus to the cytoplasm after imported

substrates have been released into the nucleoplasm ( 21 ).

Knockdown of XPO2 in SK-N-SH cells using two independ-

ent lentiviral shRNAs caused robust reduction of nuclear

DHX9 (5%) compared with shSCR control (71%; Fig. 4A ).

XPO2 knockdown effi ciency in these cells was verifi ed by

Western blot analysis ( Fig. 4B and Supplementary Fig. S4).

Because nuclear localization-defi cient DHX9 (ΔNTD-DHX9;

Fig. 2F and Supplementary Fig. S2B) failed to cooperate

with KIF1Bβ in apoptosis, we tested whether silencing of

XPO2 protects from KIF1Bβ-induced apoptosis. Coexpres-

sion of GFP-KIF1Bβ, together with plasmids encoding shR-

NAs targeting XPO2, demonstrated signifi cant protection

from KIF1Bβ-mediated apoptosis in NB1 cells ( Fig. 4C ).

Together, these results demonstrate that XPO2 is required

for nuclear localization of DHX9 and that activity is essential

for KIF1Bβ-induced apoptosis.

DHX9 Nuclear Localization Induced by KIF1Bb Stimulates Proapoptotic XAF1

Because transcription-dead mutant DHX9 (TD-DHX9)

failed to cooperate with KIF1Bβ in apoptosis, we asked

whether KIF1Bβ-mediated DHX9 nuclear localization

results in activation of specific target genes. We performed

RNA-seq to analyze gene expression in NB1 cells that were

transduced with either shRNA targeting DHX9 (sh DHX9 )

or control virus (shSCR) and subsequently transfected

with plasmid encoding KIF1Bβ600–1400. Principal com-

ponent analysis (PCA) of overall gene expression profiles

revealed transcriptional changes in shSCR cells express-

ing KIF1Bβ600–1400 compared with pcDNA3 control

( Fig. 5A ). Although DHX9 knockdown in cells alone

generally resulted in differences in gene expression com-

pared with the shSCR control, there was minimal differ-

ence in gene expression upon expressing pcDNA3 and

KIF1Bβ600–1400 in the context of DHX9 knockdown

( Fig. 5A ). Differential expression analysis using DESeq

revealed 58 genes significantly upregulated (false dis-

covery rate, FDR, < 0.05) by KIF1Bβ600–1400, and their

expression is depicted as a heatmap ( Fig. 5B ). Twenty-

three genes were upregulated at least 2-fold and, among

those, 18 genes were dependent on DHX9 expression






Figure 4. Exportin-2 is necessary for nuclear localization of DHX9 and KIF1Bβ to induce apoptosis. A, anti-DHX9 (green) immunofl uorescence staining of SK-N-SH cells stably expressing short hairpins targeting Exportin-2 (sh XPO2 ) or nontargeting control (shSCR) as indicated. Right, percentage of cor-responding cells with nuclear or cytoplasmic DHX9 or both: nuclear, percentage of cells with higher nuclear fl uorescence intensity (green) compared with the cytoplasm; cytoplasmic, percentage of cells displaying higher cytoplasmic fl uorescence intensity (green) compared with the nuclear region. Nuclear and cytoplasmic, percentage of cells displaying similar fl uorescence intensity in the nucleus and cytoplasm. B, corresponding anti-XPO2 immunoblot analysis of stably infected cells with shRNA lentivirus targeting XPO2 that were displayed in A. C, NB1 cells were cotransfected with either empty pcDNA3 plasmid or GFP-KIF1Bβ600–1400 together with plasmids encoding short hairpin targeting XPO2 (pLKO-sh XPO2 ) or nontargeting control (pLKO-shSCR) as indicated. Shown is the percentage of apoptotic cells at 96 hours after transfection determined by fl uorescence-activated cell sorting (FACS) analysis using TMRE staining (mean ± SD; n = 3; ****, P < 0.0001). MOI, multiplicity of infection.

Bars –20 μm

shSC

R

A

B C

KIF1Bβ

pcDNA3

DHX9

sh

SC

R

shSC

R

sh

XP

O2

(90

)

shXPO

2(90)

shXpo

2(90)

shXpo

2(92)

sh

XP

O2

(92

)

shXPO

2(92)

pLKO

-shX

PO(9

0)

pLKO

-shX

PO(9

2)

DAPI Merge Nuclear

Cytoplasmic

Nuclear and

cytoplasmic

100

80

60

40

20

0

Ce

ll nu

mb

er

(%)

100 ********

80

XPO2

MOI 2 20 2 220

Tubulin

60

40

20

0

pLKO

-shS

CR

Ap

op

tosis

(%

)






Figure 5. KIF1Bβ-driven nuclear DHX9 induces the expression of proapoptotic XAF1. A, PCA of overall gene expression profi les identifi ed by RNA-seq analysis. RNA was obtained from NB1 cells stably expressing short hairpins targeting DHX9 (sh DHX9 ) or nontargeting control (shSCR) that were trans-fected with plasmids encoding FLAG-KIF1Bβ600–1400 or pcDNA3 as indicated. PCA plot displaying three independent experiments. B, heatmap depict-ing expression of genes in A that were signifi cantly upregulated (FDR < 0.05): green, lower expression; red, higher expression. C, list of genes identifi ed by differential expression analysis using DESeq that were upregulated greater than 2-fold (FDR < 0.05) by KIF1Bβ600–1400 and dependent on DHX9expression as displayed in A. D, relative mRNA levels of XAF1 determined by qRT-PCR ( XAF1.1 corresponding to transcript variant 1; mean ± SD; n = 3; *, P < 0.05). E, anti-XAF1 immunoblot analysis of CHP-212 and NB1 cells transfected with plasmids encoding FLAG-KIF1Bβ600–1400 and FLAG-KIF1Bβ, respectively. F, anti-DHX9 (green) and anti-XAF1 (red) immunofl uorescence analysis in SK-N-SH cells with tetracycline-inducible KIF1Bβ (−Tet, nonin-duced; +Tet, induced).

Bars –20 μm

A

C D

F

E

B

shSCR + pcDNA3

shSCR + KIF1BβshDHX9 + pcDNA3

7.0

6.0

5.0

4.0

3.0

2.0

Re

lative

exp

ressio

n

1.0

0.0

shDHX9 + KIF1Bβ

Gene

4.92

4.02

2.88

2.84

2.80

2.79

2.35

2.35

2.34

2.27

2.16

2.15

2.14

2.13

2.12

2.11

2.03

2.01

CXCL10

DDX60L

DDX60

TRIM22

SAMD9

SAMD9L

XAF1

MX 2

IFIT1B

IFIT1

IFIT3

IFITM1

IFI44L

OAS2

OAS1

OAS3

OASL

SELE

FDRFold

change

IFIT

M1

IFIT

1

IFIT

2

IFIT

3

IFIT

M3

2.0

1.8

1.6

1.4

1.2

1.0

0.8

0.6

0.4

0.2

0.0

–0.2

–0.4

–0.6

–0.8

–1.0

–1.2

–1.4

–1.6

–1.8

–2.0

log2 RPKM

shSCR + pcDNA3

shDHX9 + KIF1BβshSCR + KIF1BβshDHX9 + pcDNA3

AP

OL6

SE

LE

IFI3

5IF

I44

NM

IR

SA

D2

ICA

M1

PN

PT

1X

AF

1

SP

100

PA

RP

9

PA

RP

10

CM

PK

2D

DX

60L

IL32

US

P18

ISG

15

BAT

F2

PLE

KH

A4

SA

MH

D1

PA

RP

14

UB

E2L6

DH

X58

C19orf

66

MX

2

PLS

CR

1D

DX

58

HE

RC

6

VC

AM

1

RN

F213

IFIT

5D

TX

3L

PR

IC285

OA

SL

OA

S2

OA

S1

OA

S3

UB

A7

BS

T2

EIF

2A

K2

STAT

2

STAT

1

IFIT

1B

IFI2

7D

DX

60

SA

MD

9L

CX

CL10

IFI6

TR

IM22

IFI4

4L

SP

110

SA

MD

9

pcDNA3

shSCR

pcDNA3

shDHX9

KIF1BβshDHX9

KIF1BβshSCR

–Tet

+Tet

XAF1

FLAG

Tubulin

KIF1BβpcDNA3

CHP212

KIF1BβpcDNA3

NB1

XAF1

XAF1.1

4.56 × 10–3

2.96 × 10–19

2.96 × 10–19

2.96 × 10–19

2.96 × 10–19

6.29 × 10–17

9.34 × 10–11

5.80 × 10–3

4.37 × 10–18

6.47 × 10–15

4.82 × 10–13

6.02 × 10–17

1.53 × 10–4

1.01 × 10–11

5.75 × 10–10

5.80 × 10–3

6.87 × 10–3

1.32 × 10–2

XAF1DHX9 Hoechst Merge

*

*

*

*






(Supplementary Tables S1 and S2 and Fig. 5C ). Most

of the KIF1Bβ-induced, DHX9-dependent targets were

IFN-induced or IFN-related, consistent with earlier

reports implicating DHX9 in transcriptional regulation

of IFN-α–inducible genes ( 22 ). Some of these genes are

known NF-κB downstream targets ( 23–25 ), in line with

earlier observations demonstrating a DHX9–NF-κB inter-

action resulting in the transactivation of specific promot-

ers ( 18 , 26 ). In addition, the proapoptotic XIAP-associated

factor 1 ( XAF1 ) was identified ( Fig. 5C ). XAF1 functions

as a negative regulator of members from the inhibitors

of the apoptosis (IAP) family ( 27 ). Quantitative real-time

PCR (qRT-PCR ) confirmed that XAF1 mRNA is upregu-

lated by KIF1Bβ in a DHX9-dependent manner ( Fig. 5D ).

Moreover, exogenous expression of KIF1Bβ600–1400 or

full-length KIF1Bβ in CHP-212 and NB1 cells resulted in

XAF1 protein induction ( Fig. 5E ). In addition, inducible

KIF1Bβ neuroblastoma cells (Tet-SK-N-SH) resulted in

enhanced DHX9 nuclear localization and XAF1 induction

( Fig. 5F ).

Loss of DHX9 Promotes Neuronal Survival in the NGF Signaling Pathway

We used differentiated PC12 cells to study the regulation

of DHX9 during neuronal survival by NGF. Consistent with

previous reports ( 5 ), NGF withdrawal from PC12 cells caused

the induction of KIF1Bβ protein, followed by the induction of

apoptosis ( Fig. 6A and Supplementary Fig. S5A). We observed

that, like KIF1Bβ, XAF1 protein was also induced with similar

kinetics in PC12 cells after NGF deprivation and associated

with increased XAF1 mRNA ( Fig. 6A and Supplementary

Fig. S5A and S5B). To determine whether induction of XAF1

depends on KIF1Bβ-mediated DHX9 nuclear accumulation,

we fi rst assayed endogenous DHX9 localization in PC12 cells

followed by NGF withdrawal. NGF-deprived PC12 cells dis-

playing apoptotic characteristics showed enhanced nuclear

DHX9 (67% of cells), in contrast with cells maintained in

NGF, which showed only 1% of cells with nuclear DHX9

( Fig. 6B ). However, PC12 cells transduced with shRNA target-

ing KIF1Bβ prevented nuclear accumulation of DHX9 ( Fig.

6C ). Likewise, knockdown of EGLN3 reduced nuclear DHX9

and caspase-3 cleavage compared with shSCR control ( Fig.

6D ). Moreover, PC12 cells transduced with shRNA target-

ing KIF1Bβ abolished the induction of XAF1 upon NGF

withdrawal ( Fig. 6E ). Furthermore, we observed induction of

DHX9 protein upon NGF withdrawal in shSCR PC12 cells

and in primary mouse sympathetic neurons ( Fig. 6E, F, and

G ). Next, we asked whether loss of DHX9 blocks apopto-

sis in NGF-deprived PC12 cells. PC12 cells transduced with

shRNAs targeting DHX9 were protected from apoptosis

upon NGF withdrawal compared with control cells, as deter-

mined by cleaved caspase-3 quantifi cation ( Fig. 6H ). Fur-

thermore, silencing of DHX9 in NGF-deprived PC12 cells

showed ablated XAF1 induction similar to that observed fol-

lowing KIF1Bβ knockdown ( Fig. 6F ). Silencing of DHX9 also

abolished the induction of endogenous KIF1Bβ protein in

NGF-deprived PC12 cells and SK-N-SH neuroblastoma cells,

possibly by regulating the translation of KIF1Bβ mRNA ( Fig.

6F and I and Supplementary Fig. S5C).

DHX9 Nuclear Localization Is Impaired in KIF1Bb-Defi cient Neuroblastoma Tumors

To investigate whether nuclear localization of DHX9

depends on KIF1Bβ in vivo , we studied the expression of

DHX9 and KIF1Bβ in the mouse sympathetic nervous system

when developmental apoptosis peaks, for example, around

birth ( 28 ). In situ hybridization revealed that KIF1Bβ was

highly and exclusively expressed in the sympathetic ganglia

but not in non-neuronal tissue surrounding the ganglia,

highlighting its specifi c role in the sympathetic nervous

system ( Fig. 7A and B ). The identity of the superior cervical

ganglia (SCG) was confi rmed with immunohistochemistry

for tyrosine hydroxylase, a marker for noradrenergic and

adrenergic neurons ( Fig. 7C ). To investigate whether nuclear

DHX9 localization coincides with KIF1Bβ expression in the

SCG, DHX9 immunofl uorescence studies were performed.

We observed DHX9 in the nuclei of cells within the SCG, but

in the cytoplasm of cells in the surrounding, non-neuronal

tissue that lack KIF1Bβ expression ( Fig. 7D and Supplemen-

tary Fig. S6A). This is in accordance with our in vitro obser-

vation that DHX9 nuclear localization is dependent upon

KIF1Bβ expression.

We next analyzed 13 primary neuroblastoma tumors for

KIF1Bβ and DHX9 protein expression, determined DHX9

cellular localization, and sequenced the KIF1B exome. Only

two of 13 neuroblastomas, K14 and K33, were 1p36-intact

(1p36 +/+ ), whereas the remaining 11 samples harbored

hemizygous 1p36 deletions (Supplementary Table S3). We

observed a complete lack of KIF1Bβ protein expression in

most of the 1p36-deleted tumors, in contrast with tumor K14

(1p36 +/+ ) and the SK-N-SH cell line (1p36 +/+ ; Fig. 7E ). Also,

tumor K33 (1p36 +/+ ) did not express KIF1Bβ, and tumors

K36 and K56 expressed faster migrating forms of KIF1Bβ

protein, likely due to splicing alterations ( Fig. 7E ). Exome

sequencing did not reveal any missense mutations in KIF1B b

alleles, although polymorphic variants were identifi ed in two

tumors, K14 (V1554M) and K10 (M807I; Supplementary

Table S4). The lack of KIF1Bβ protein expression in KIF1Bβ-

hemizygous tumors might result from epigenetic silencing,

translation, or splicing alterations. Notably, DHX9 protein

expression varied across the different neuroblastoma tumors

( Fig. 7E ).

Next, we investigated DHX9 localization in these tumors

in paraffi n-embedded sections ( Fig. 7F and Supplemen-

tary Fig. S6B and S6C). Specifi city of the DHX9 staining

in these paraffi n-embedded sections was confi rmed using

the K11 tumor as negative control, because it lacks DHX9

protein expression (Supplementary Fig. S6C and Fig. 7E ).

In tumors that lacked KIF1Bβ protein expression (K10,

K12, K33, K7, and K9), DHX9 was observed in both the

cytoplasm and nucleus, with the majority in the cytoplasm

( Fig. 7F and Supplementary Fig. S6B). However, the K14

tumor expressing KIF1Bβ protein displayed predominantly

nuclear DHX9. These results accord with our observations

in the mouse sympathetic nervous system and in vitro stud-

ies that nuclear localization of DHX9 depends on KIF1Bβ

protein.

Collectively, our results demonstrate that DHX9 nuclear

localization is impaired in KIF1Bβ-defi cient neuroblastoma






Figure 6. Induction of KIF1Bβ increases nuclear DHX9 and XAF1 expression during NGF withdrawal. A, anti-KIF1Bβ and anti-XAF1 immunoblot analy-sis of differentiated PC12 cells subjected to NGF withdrawal. B–D, immunofl uorescence images of differentiated PC12 cells that were subjected to NGF withdrawal (24 hours; −NGF). Antibodies against DHX9 (green) and cleaved caspase-3 (red) were used in addition to Hoechst staining to visualize nuclei (blue). B, right, percentage of PC12 cells exhibiting nuclear DHX9 and cleaved caspase-3 before (+) and after (−) NGF withdrawal at indicated times (mean ± SD; n = 3; *, P < 0.05). C, before NGF withdrawal, cells were infected with lentivirus encoding short hairpins targeting KIF1Bβ (sh KIF1Bβ ) or nontarget-ing control virus (shSCR). Right, corresponding percentage of cells displaying DHX9 nuclear accumulation (mean ± SD; n = 3; *, P < 0.05). D, before NGF withdrawal, cells were infected with lentivirus encoding short hairpins targeting rat-EGLN3 (sh EGLN3 ). E and F, immunoblot analysis of differentiated PC12 cells before (+) and after (−) NGF withdrawal as indicated. Before NGF withdrawal, differentiated cells were infected with lentivirus encoding short hairpins targeting KIF1Bβ (sh KIF1Bβ ), DHX9 (sh DHX9 ), or nontargeting control virus (shSCR). G, immunoblot analysis of primary mouse sympathetic neurons subjected to NGF withdrawal at indicated times. H, percentage of apoptosis in differentiated PC12 cells before (+) and after (−) NGF withdrawal that were transduced with lentivirus encoding short hairpins targeting DHX9 (sh DHX9 ) or nontargeting control virus (shSCR). Apoptosis was scored by cleaved caspase-3 quantifi cation (mean ± SD; n = 5; *, P < 0.05). I, immunoblot analysis of SK-N-SH cells infected with lentivirus encoding short hairpins targeting DHX9 (sh DHX9 ) or control virus (shSCR). GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

*

Bars –20 μm

Bars –20 μm Bars –20 μm

A

endg. DHX9

KIF1Bβ

Tubulin

XAF1

16 24 40 48 h

8 16 24 48 8 16 24 48 h%

of cells

shS

CR

shSC

R

shKIF

1Bβ

shE

gIN

3

Nuclear DHX9

Cl. casp-3100

80

60

40

20

0

Nucle

ar

DH

X9 in %

100

80

60

40

20

0

+ N

GF

+N

GF

+ NGF

(+)

NGF

(+)

NGF

(–)

NGF

(+)

NGF

(–)

NGF

18 h 24 h

(–) NGF

–N

GF

– NGF

shSC

R

shDHX9

Apopto

sis

(%

)

0

10

20

30

40

50+ NGF– NGF

– NGF – NGF

*

NGF withdrawal

KIF1Bβ

KIF1Bβ

KIF1Bβ DHX9

DHX9

DHX9

GAPDH

GAPDH

GAPDH

XAF1 Cl. casp-3

6 12 18 24 h+ N

GF

+ N

GF

+ N

GF

NGF withdrawal

B

D

E

F

G H

I

C cl. casp-3 Hoechst Mergeendg. DHX9

shS

CR

shSCR

shSCR

shDHX9

shK

IF1B

β

shKIF1Bβ

8 16 24 8 16 24 h

– NGF – NGF

KIF1Bβ

DHX9

GAPDH

XAF1

+ N

GF

+ N

GF

shSCR shDHX9

(+)

NGF

(–)

NGF

(+)

NGF

(–)

NGF

Hoechst Merge

endg. DHX9 cl. casp-3 Hoechst Merge*

*






Figure 7. DHX9 is localized to the nuclei of developing mouse sympathetic neurons but not in human neuroblastoma tumors defi cient in KIF1Bβ. A, in situ hybridization using a probe for KIF1Bβ on sagittal sections of wild-type mouse at embryonic day E17.5 showing KIF1Bβ expression in the SCG adjacent to the cochlea as indicated. B, in situ hybridization for KIF1B b at P2 using both antisense and sense probes in adjacent sections. C, in situ hybridization for KIF1B b in mouse SCG at P2 and immunohistochemistry for tyrosine hydroxylase showing expression in the same area. D, anti-DHX9 (red) immunofl uorescence images in the SCG (1), non-neuronal surrounding tissue (2), and the cochlea (3) of mouse sagittal sections at postnatal day 1. E, immunoblot analysis of primary neuroblastoma tumors. 1p36 status has been characterized and is outlined in Supplementary Table S3 (1p36 +/+ , wild-type; 1p36 +/− , 1p36 deletion). SK-N-SH cells (1p36-intact) served as positive control. F, immunohistochemistry for DHX9 on paraffi n-embedded tissue sections of human neuroblastoma tumors counterstained with hematoxylin. K10, K12, K33, and K7 do not express KIF1Bβ but are positive for DHX9 based on the immunoblot analysis in E. K14 is positive for KIF1Bβ and DHX9 based on immunoblot analysis in E. The image in A was acquired using a ×4 objective; images in F were acquired using a ×20 objective, whereas higher magnifi cation images of selected regions were acquired using a ×100 objec-tive. G, signaling model linking familial genetic lesions associated with sympathetic nervous system cancer (pheochromocytoma or neuroblastoma) to apoptosis when NGF becomes limiting during sympathetic neuronal development.

Bar –40 μm

AKIF1Bβ KIF1Bβ KIF1Bβ

KIF1Bβsense

Tyrosine

hydroxylase

NGF

DHX9

Apoptosis

KIF1Bββ

KIF1Bβ positive KIF1Bβ negative

KIF1Bβ

KIF1Bβ

DHX9

DH

X9

SKNSHShort

exp.

Long

exp.

K61K56K36K43K34K9K11K55K33

K33

K14

K14×20 ×20

×100 ×100 ×100 ×100 ×100

×20 ×20 ×20

K12

K12

K10

K10

K7

K7

GAPDH

1p36

+/+

1p36

+/+

1p36

+/–

1p36

+/–

B

D

E

F

G

C

NF1

VHL

TrkA

c-JUN

EGLN3

XAF1

? ?

SuccinateSDH

Jun-B

c-RET

DAPI MergeDHX9 DHX9






tumors. This suggests that loss of KIF1Bβ during normal

development of the sympathetic nervous system may impair

NGF-deprived apoptosis due to mislocalization of DHX9.

Indeed, low expression of KIF1Bβ is correlated with poor

prognosis and reduced survival of patients with neuro-

blastoma, providing further evidence to suggest that

KIF1Bβ is a neuroblastoma tumor suppressor (Supplemen-

tary Fig. S6D).

DISCUSSION KIF1Bβ was previously characterized as a potential 1p36

tumor-suppressor gene that mediates neuronal apoptosis

when NGF is limiting in the developing nervous system

( 5 , 11 , 12 ). Here, we provide a mechanistic understanding

of KIF1Bβ-mediated tumor suppression. We identifi ed the

RNA helicase DHX9 as an interacting partner of KIF1Bβ

and found that DHX9 is necessary for KIF1Bβ to induce

apoptosis and is required for apoptosis when NGF is

limiting.

DHX9 is a member of the DEAH-box DNA/RNA helicase

family that catalyzes the ATP-dependent unwinding of

double-stranded RNA and DNA–RNA complexes ( 14 ).

Recently, DHX9 has been characterized in multiple cellular

functions, including translation, RNA splicing, and miRNA

processing. In addition, DHX9 localizes to both the nucleus

and the cytoplasm and functions as a transcriptional regu-

lator ( 13 , 15 – 17 ). Here, we report that KIF1Bβ induces

neuronal apoptosis by directing DHX9 nuclear accumula-

tion, leading to induction of proapoptotic XAF1 . XAF1 has

been reported as an antagonist of antiapoptotic XIAP and

has been shown to convert XIAP into a proapoptotic pro-

tein to degrade survivin ( 27 , 29 ). Inhibition of XIAP is nec-

essary and suffi cient for sympathetic neurons to acquire

apoptotic competence during NGF withdrawal-induced

apoptosis ( 30 ). Similarly, expression of survivin is strongly

correlated with advanced stages of disease and unfavorable

neuroblastoma outcomes ( 31 ).

Abnormal NGF signaling has been linked to nervous

system tumors such as neuroblastoma, medulloblastoma,

and pheochromocytoma ( 1–5 , 32, 33 ). Our fi ndings imply

that alterations in NGF-mediated developmental apoptosis

may play a role in these types of cancers. We found that

DHX9 is induced when NGF is limiting, localizes and accu-

mulates in the nucleus, and that nuclear accumulation is

dependent upon KIF1Bβ expression. Furthermore, down-

regulation of DHX9 diminished the expression of proapop-

totic XAF1 and caused escape from NGF withdrawal-

dependent apoptosis. However, silencing of DHX9 also

abolished KIF1Bβ induction and, therefore, loss of DHX9

could also cause escape from apoptosis due to its effect on

KIF1Bβ. Therefore, KIF1Bβ’s function in apoptosis might

involve additional mechanisms that account for NGF-

dependent apoptosis. In this regard, we tested recently

identifi ed putative disease-causing KIF1Bβ mutants for

their ability to regulate DHX9 localization. Indeed, variants

T827I, P1217S, and E1628K—all of which are defective in

apoptosis—failed to stimulate nuclear localization of

DHX9, supporting our fi ndings that nuclear DHX9 medi-

ates KIF1Bβ apoptotic function. However, variants E646V

and S1481N stimulated DHX9 localization to a compara-

ble degree as wild-type KIF1Bβ despite their impaired apop-

totic abilities. This indicates that DHX9 nuclear localization

is necessary but not suffi cient for KIF1Bβ to induce apop-

tosis and that additional events are needed for KIF1Bβ to

induce apoptosis.

In an attempt to mechanistically understand how

KIF1Bβ regulates DHX9 nuclear localization, we exam-

ined other KIF1Bβ binding partners. In addition to DHX9,

we found that XPO2 binds to the KIF1Bβ proapoptotic

600–1400 domain. XPO2 has been reported to regulate

nuclear import and export of cellular proteins. Here, we

show that loss of XPO2 impairs DHX9 nuclear localiza-

tion and, consequently, impedes KIF1Bβ-induced apop-

tosis, further implicating DHX9 nuclear localization as

a requirement for KIF1Bβ apoptotic function. XPO2 was

previously implicated in regulating apoptosis induced by

Pseudomonas exotoxin ( 34 ), and resistance to Pseudomonas

exotoxin is phenocopied in Egl-9 −/− worms ( 35 ), suggesting

that XPO2 acts in the same apoptotic program mediated

by EGLN3 and KIF1Bβ. Moreover, our results suggest that

DHX9 binding and DHX9 nuclear localization are medi-

ated by two distinct and adjacent sites, KIF1Bβ1300–1400

and KIF1Bβ1100–1200, respectively. Because both sites

are required for KIF1Bβ apoptosis function, we concluded

that additional KIF1Bβ binding partners on amino acid

region 1100–1200 might participate in DHX9 localiza-

tion. It was previously demonstrated that arginine meth-

ylation of DHX9 determines its subcellular localization

( 36 ). Indeed, we identified arginine methyltransferase

PRMT5 in the large-scale KIF1Bβ600–1400 immunopre-

cipitation (data not shown). Therefore, PRMT5 might be

such a modifier.

Finally, we demonstrate that the regulation of DHX9 by

KIF1Bβ is relevant in neuroblastoma. Our in vivo studies

in the mouse sympathetic nervous system demonstrate

that DHX9 is specifically found in the nuclei of cells that

express KIF1Bβ and that nuclear DHX9 is present in 1p36-

intact tumors that express wild-type KIF1Bβ. In contrast,

analysis of 1p36-deleted neuroblastomas with complete

loss of KIF1Bβ protein expression showed impaired DHX9

nuclear localization. In addition to our findings that

implicate impairment of nuclear DHX9 in neuroblastoma

pathogenesis, other evidence points to dysfunction of RNA

helicases in pediatric nervous system cancers. Medullob-

lastoma exome sequencing uncovered recurrent somatic

missense mutations within RNA helicases and showed that

15% of medulloblastomas seemed to have some disruption

in RNA helicase activity ( 37 ). Given the apparent impor-

tance of RNA helicase function in medulloblastoma, we

searched the Catalogue of Somatic Mutations in Cancer

(COSMIC) database ( 38 ) for DHX9 mutations in cancer

(Supplementary Table S5). We found numerous tumors

with DHX9 missense mutations within the CBP-binding

domain, nuclear transport domain (NTD), minimal trans-

activation domain (MTAD), and helicase ATP-binding

domain, all of which were predicted to impair DHX9’s abil-

ity to mediate transcription. Moreover, 44 of 144 unique

samples were found to contain mutations.






In summary, we propose that loss of nuclear DHX9 due

to impaired EGLN3 activity or loss of KIF1Bβ promotes

neuronal survival during the NGF-dependent developmen-

tal culling of sympathetic neurons. On the basis of our

studies, we propose that failure to properly cull neuronal

progenitors during development predisposes to sympathetic

nervous system tumors such as neuroblastoma. Alterations

in developmental apoptosis due to dysfunction of EGLN3,

KIF1Bβ, and DHX9 might play a role in the pathogenesis

of these tumors by allowing neuronal progenitors to escape

from developmental culling and thereby predisposing them

to neoplastic transformation ( Fig. 7G and Supplementary

Fig. S7).

METHODS Cell Culture

Human neuroblastoma cell lines (NB1, CHP-212, and SK-N-

SH) and PC12 cells were maintained as previously described ( 5 ).

CHP-212, SK-N-SH, and PC12 cell lines were obtained from the

American Type Culture Collection, and NB1 cells were obtained

from the Japanese Collection of Research Bioresources. The cell

lines used were not further authenticated. Sympathetic neurons

from P1 mice were isolated from the SCG and cultured as described

previously ( 39 ).

Plasmids FLAG-tagged KIF1Bβ plasmids and corresponding mutants

were generated as described previously ( 5 ). RFP-KIF1Bβ, RFP-

KIF1Bβ(600–1400), and RFP-KIF1Bβ(600–1200) were generated by

cloning TagRFP into FLAG-KIF1Bβ plasmids. RFP was cloned into

the Kpn I-digested 5′ region of FLAG-KIF1Bβ pcDNA3.1 expression

vectors. Lentivirus expressing RFP-KIF1Bβ(600–1400) was gener-

ated as previously described using pLenti-Flag-KIF1Bβ(600–1400).

Stag was cloned into FLAG-KIF1Bβ(600–1400) by annealing 5′ phosphorylated oligonucleotides containing S-tag sequence and

Xba I restriction site, followed by ligation with the Xba I-digested 5′ region of pLenti-FLAG-KIF1Bβ(600–1400). His-DHX9 and eCFP-

DHX9 were purchased from Genocopoeia. eCFP-ΔNTD-DHX9, and

eCFP-TD-DHX9 were generated from eCFP-DHX9 (Genocopoeia).

eCFP-ΔNTD-DHX9 was created by introducing a premature STOP

codon at residue position 1146 of DHX9 to result in a truncated

DHX9 lacking in NTD. eCFP-TD-DHX9 was created by introducing

W332A, W339A, and W342A mutations at the MTAD domain of

DHX9. eCFP-ΔNTD-DHX9, eCFP-TD-DHX9, and eCFP-R1166L-

DHX9 were all generated using the QuikChange Site-Directed

Mutagenesis Kit (Stratagene).

Primers RFP- XbaI forward primer: 5′-TGCTCTAGAATGGTGTCTAAGGG

CGAAGA-3′; RFP- XbaI reverse primer: 5′-TGCTCTAGAATTAAG

TTTGTGCCCCAGTTTG-3′; S-tag- XbaI sense oligo: 5′-CTAGAA

TGAAGGAGACCGCCGCCGCCAAGTTCGAGAGACAGCACAT

GGACAGCT-3′; S-tag- XbaI antisense oligo: 5′-CTAGAGCTGTCCAT-

GTGCTGTCTCTCGAACTTGGCGGCGGCGGTCTCCTTCATT-3′. Site-directed mutagenesis primers: XbaI -pLenti-KIF1Bβ(600–1400)

forward: 5′-CTCAGCTTATAATCGAGAGGGCCCGCGGT-3′; XbaI-

pLenti-KIF1Bβ(600–1400) reverse: 5′-ACCGCGGGCCCTCTCGATT

ATAAGCTGAG-3′; eCFP-ΔNTD-DHX9_STOP forward: 5′-CTCAG

CTGCTGGTATCTAACCTTATGATTGGC-3′; eCFP-ΔNTD-DHX9_

STOP reverse: 5′-GCCAATCATAAGGTTAGATACCAGCAGCTGAG-3′; eCFP-TD-DHX9_MTAD forward: 5′-GGTTCCTGCGTCACCTCCAC

AATCCAACGCGAATCCTGCGACTAGT-3′; eCFP-TD-DHX9_MTAD

reverse: 5′-ACTAGTCGCAGGATTCGCGTTGGATTGTGGAGGTG

ACGCAGGAACC-3′.

shRNA and siRNA shRNA-expressing lentiviral plasmids targeting DHX9 were

obtained from Mission shRNA (Sigma). shRNA sequence target-

ing human DHX9 was (#1): 5′-TCGAGGAATCAGTCATGTAAT-3′, (#2): 5′-CCAGAAGAATCAGTGCGGTTT-3′, (#3): 5′-GGGCTATATC

CATCGAAATTT-3′. shRNA (#1) was also used to target DHX9

in rat PC12 cells. shRNA sequences targeting rat KIF1Bβ and

rat EGLN3 in PC12 cells as well as human KIF1Bβ and human

EGLN3 have been previously described ( 5 ). siRNAs targeting rat

DHX9 were purchased from Eurofi ns MWG Operon; siDHX9(1)

5′-GCAUGGACCUUAAGAAUGA-3′ and siDHX9(3) 5′-CCACG

CAAGUUCCACAAUA-3′. siRNA targeting human XAF1 was pur-

chased from Dharmacon under ON-TARGETplus SMARTpool

siRNA. Cotransfections of siRNA with pcDNA3 plasmids were per-

formed using DharmaFECT Duo Transfection Reagent according to

the manufacturer’s recommendations.

Viral Expression and Infection Adenovirus encoding rat EGLN3 (SM-20) was a gift from Robert

Freeman (University of Rochester, Rochester, NY). Virus amplifi ca-

tion and infection has been previously described ( 5 ). Lentiviral infec-

tion for gene silencing using shRNA was performed according to the

manufacturer’s instructions (MISSION shRNA; Sigma).

Immunoprecipitation and Mass Spectrometry A total of 160 × 10 6 NB1 cells were transduced with lentivirus

expressing S-tag-KIF1Bβ(600–1400). Two days later, cells were har-

vested with lysis buffer (150 mmol/L NaCl, 50 mmol/L Tris–HCl,

5 mmol/L EDTA, 0.1% CHAPS, and pH 7.4) and incubated for

2 hours at 4°C with rotation. The lysate was centrifuged at 20,000

× g for 20 minutes. The resulting supernatant was precleared for

2 hours and, subsequently, S-protein agarose beads (Novagen) were

added to lysates and incubated for 3 hours at 4°C with rotation.

Samples were centrifuged and beads were washed fi ve times in wash

buffer (500 mmol/L NaCl, 50 mmol/L Tris–HCl, 5 mmol/L EDTA,

pH 7.4). Bound protein complexes were eluted in Laemmli buffer

containing 10 mmol/L Dithiothreitol and heated for 10 minutes

at 95°C. Eluates were analyzed by SDS-PAGE, gels were subjected

to silver stain, and bands of interest were excised for mass spec-

trometry identifi cation. Mass spectrometry protocol was previously

described ( 40 ).

Coimmunoprecipitation of Endogenous Proteins One confl uent p150 plate of SK-N-SH neuroblastoma cells was

harvested with 1-mL immunoprecipitation (IP) buffer (20 mmol/L

Tris, 150 mmol/L NaCl, 2 mmol/L EDTA, 10% glycerol, 0.1%

CHAPS, and protease inhibitors, pH 7.4), incubated for 2 hours,

and then centrifuged at 14,000 × g for 30 minutes. The resulting

supernatant was precleared with 40-μL Protein A agarose slurry for

1 hour and then centrifuged at 2,500 × g for 3 minutes. Either 5-μg

rabbit polyclonal KIF1Bβ antibody or rabbit immunoglobulin G

(IgG) isotype control antibody (Cell Signaling Technology; DA1E,

#3900) was added to 1-mg lysate overnight at 4°C. Subsequently,

40-μL Protein A agarose slurry was added and incubated on a rota-

tor for 3 hours and then centrifuged at 2,500 × g for 30 seconds,

and the resulting resin was washed with 1-mL IP buffer. The resin

was washed three times in total, and reducing 2× sample buffer

was added to the resin and boiled for 5 minutes and analyzed by

immunoblotting. Coimmunoprecipitation of exogenous FLAG-

KIF1Bβ with Anti-FLAG M2 Affi nity Gel was prepared according to

the manufacturer’s instructions (Sigma).






Apoptosis Assays Apoptosis was assessed using GFP-histone to quantify apoptotic

nuclei as previously described ( 5 ). Alternatively, immunofl uorescence

staining for cleaved caspase-3 allowed for visualization and quantifi -

cation of apoptotic cells via microscopy. Apoptosis in primary sym-

pathetic neurons was scored by DAPI staining, to visualize apoptotic

changes as previously described ( 5 , 41 ). KIF1Bβ-induced apoptosis

assays in XPO2-knockdown cells were performed by fl uorescence-

activated cell sorting (FACS) analysis using tetramethylrhodamine

ethyl esters (TMRE; Invitrogen Corporation) 92 hours after trans-

fection. Statistical analysis was performed by one-way ANOVA, fol-

lowed by the Bonferroni posttest using the GraphPad Prism software

(GraphPad Software Inc., version 6.00).

Tetracycline-Regulated Expression System Tetracycline-responsive inducible KIF1Bβ expression in SK-N-

SH cells was constructed using the T-REx System (Invitrogen).

Tetracycline-inducible cells were plated in a 6-well plate and trans-

duced with either shSCR or sh DHX9 lentivirus. After 24 hours,

cells were grown in selection medium (1 μg/mL puromycin) for

3 days before being replenished with fresh medium containing

0.5 to 1 μg/mL tetracycline. Fresh medium containing tetracy-

cline and puromycin was replenished every 2 days. After 4 days

of induction or upon reaching confl uence, cells were transferred

into p100 plates to undergo further selection and induction

until resistant colonies were identifi ed. Cells were maintained in

blasticidin and zeocin at all times at concentrations previously

mentioned.

Immunofl uorescence and Immunohistochemistry Cells were fi xed with 4% paraformaldehyde (PFA) and stained with

1 μg/mL Hoechst for 10 minutes at room temperature. Immunofl uo-

rescence was performed by fi xation with 4% PFA for 15 minutes, fol-

lowed by quenching with 10 mmol/L glycine for 20 minutes. Cells

were then permeabilized with 0.1% Triton X-100, blocked with 5%

goat serum, and incubated with primary antibodies in PBS contain-

ing 0.1% bovine serum albumin (BSA) overnight at 4°C. Secondary

antibodies conjugated to fl uorophores were incubated in PBS with

0.1% BSA at 1:1,000 for 1 hour, followed by 1 μg/mL Hoechst for 10

minutes (Invitrogen). All steps were interspersed with two to three

washes with PBS or PBS with 0.1% BSA and performed at room tem-

perature unless otherwise stated.

C57BL/6 mice were decapitated at postnatal day 1 (P1) and frozen

on dry ice. Immunohistochemistry was performed on 12-μm cryostat

sagittal sections. The sections were fi xed with PFA and blocked with

Mouse on Mouse blocking reagent, followed by incubation with 5%

normal goat serum (NGS) in 0.3% Triton X-100 in PBS for 1 hour

(Vector Laboratories). After blocking, the sections were incubated

with mouse anti-DHX9 at 1:250 dilution (Novus Biologicals) and

rabbit anti-tyrosine hydroxylase at 1:1000 dilution (Pel-Freeze) in

5% NGS and Triton X-100 overnight at 4°C. The sections were sub-

sequently washed in 0.1% Triton X-100 in PBS and incubated with

anti-rabbit Alexa Fluor 488 and anti-mouse Alexa Fluor 594 second-

ary antibodies at 1:1,000 dilution (Invitrogen) for 1 hour at room

temperature, washed, and coverslipped with Vectashield mounting

medium containing DAPI (Vector Laboratories). Sections were ana-

lyzed using an LSM 5 Exciter confocal laser-scanning microscope

(Zeiss). Alternatively, sections were incubated with a biotinylated

goat anti-rabbit antibody at 1:500 dilution for 1 hour at room tem-

perature, followed by the Vectastain ABC Kit and visualization with

3,3′-diaminobenzidine (DAB kit; Vector Laboratories).

Human primary neuroblastoma paraffi n-embedded tissue sections

were deparaffi nized in xylene and rehydrated. The sections were

treated with 10 mmol/L sodium citrate for 10 minutes at 98°C.

Blocking was performed with 1% BSA followed by 2% NGS in 1%

BSA/PBS, after which the sections were incubated with mouse anti-

DHX9 at 1:1,000 dilution in NGS/BSA overnight at 4°C (Novus Bio-

logicals). The sections were subsequently washed and incubated in

biotinylated goat anti-mouse antibody at 1:250 dilution, followed by

the Vectastain ABC Kit and DAB visualization as above. The sections

were counterstained with Mayer’s hematoxylin, dehydrated, cleared

in xylene, coverslipped (Histolab), and viewed with a Nikon Eclipse

E1000 microscope.

Microscopy Immunofl uorescence and fl uorescent protein-tagged images were

acquired and analyzed using Zeiss LSM 5 EXCITER or Zeiss LSM

510 META laser-scanning confocal microscopes together with Zeiss

LSM 5 EXCITER or Zeiss LSM 510 software, respectively. Images

were acquired using 63X Plan-Apochromat/1.4 NA Oil with DIC

capability objective. The excitation wavelengths for TagRFP/Alexa

Fluor 555, eCFP, GFP/Alexa Fluor 488, and DAPI/Hoechst were

543, 458, 488, and 405 nm, respectively. Images were captured at

frame size: 1024, scan speed: 7, and 12-bit acquisition and line aver-

aging mode: 8. Pinholes were adjusted so that each channel had the

same optical slice of 1 to 1.2 μm. Image scaling was performed using

the Photoshop CS6 “Place Scale Marker” tool, whereby the number

of pixels was divided by the fi eld size and multiplied by the desired

distance to indicate the respective scales. Approximately 50 to 100

cells per sample were counted for quantifi cation analysis. Images

of neuroblastoma paraffi n-embedded tissue sections were acquired

using ×20 and ×100 objectives mounted on a Nikon Eclipse E1000

microscope.

Antibodies Rabbit polyclonal anti-Flag (F7425) and mouse monoclonal anti-

Flag (F3165 and F1804) were purchased from Sigma. Mouse mono-

clonal anti-EGLN3 was generously provided by Dr. Peter Ratcliffe

(Oxford University, Oxford, UK). Mouse monoclonal anti-DHX9 was

purchased from Novus Biologicals (3G7; H00001660-M01). XPO2

mouse monoclonal antibody (#610482; BD Transduction Labora-

tories) was used at a dilution of 1:1,000 for immunoblot analysis.

Polyclonal KIF1Bβ-antibody was raised in rabbits against a syn-

thetic peptide (GHYQQHPLHLQGQELNSPPQPC) by Peptide Spe-

cialty Laboratories GmbH. Rabbit monoclonal anti-cleaved caspase-3

(D175; 5A1E; #9664), rabbit monoclonal anti-Fibrillarin (C13C3;

#2639), and rabbit poly clonal anti-His (#2365) were purchased from

Cell Signaling Technology. Rabbit polyclonal anti-XAF1 (ab17204)

was purchased from Abcam. Rabbit anti-tyrosine hydroxylase was

purchased from Pel-Freeze.

NGF Withdrawal NGF withdrawal in primary sympathetic neurons and PC12 cell

has been recently described ( 39 ).

RNA-seq Analysis NB1 cells stably transduced with either shSCR or sh DHX9 were trans-

fected with either empty pcDNA3.1 plasmid or KIF1Bβ(600–1400)

expression plasmid, in conjunction with pMACS K k .II plasmid in a

1.5:1 ratio (Miltenyi Biotec). We made three biologic replicates for

each condition. After 48 hours, cells expressing both the plasmid of

interest and selection plasmid were isolated using the MACSelect

Transfected Cell Selection System and performed as recommended

by the vendor (Miltenyi Biotec). RNA purifi cation of isolated cells was

carried out with the RNeasy Mini Kit according to the manufacturer’s

instructions (Qiagen). Purifi ed total RNA was sent for mRNA-seq






analysis (Fasteris) using TruSeq library preparation (polyA + , not

strand-specifi c) and sequencing on Illumina HiSeq 2000, with a 50-bp

single-end read length. Between 6,972,104 and 13,963,048 reads were

generated per sample. Reads were aligned to genome assembly hg19

and exon–exon junctions using bowtie with setting best , and fi ltered

for unique hits ( 42, 43 ). We generated gene expression values and

read counts with the rpkmforgenes.py May 3, 2011, version with set-

tings fulltranscript – mRNAnorm ( 44 ). Genes were tested for differential

expression using DESeq 1.0.6 with default settings ( 45 ). DESeq uses

the Benjamini–Hochberg method to calculate FDRs from its P values.

PCA plot and heatmap of signifi cantly regulated genes were generated

using Qlucore Omics Explorer 2.2.

qRT-PCR Analysis qRT-PCR was performed on cDNA libraries previously created

for RNA-seq using KAPA SYBR FAST Universal 2× qPCR Mas-

ter Mix according to the manufacturer’s instructions (Kapa Bio-

systems) in an Applied Biosystems VIIA7 machine in 384-well

format. Reactions (10 μL) were prepared in technical triplicate

and total XAF1 and XAF1.1 (NM_017523) mRNAs were quanti-

fi ed relative to RPL13A mRNA by relative standard curve quan-

tifi cation using the Applied Biosystems VIIA7 software bundle.

qRT-PCR primers were as follows: RPL13A forward primer: 5′-TCC

AAGCGGCTGCCGAAGATG-3′; RPL13A reverse primer: 5′-ACCTT

CCGGCCCAGCAGTACC-3′; XAF1 forward primer: 5′-AAGCCCAGG

ACCAGCTCCCCTA-3′; XAF1 reverse primer: 5′-AGACCACCACAGC

AAGTAGGCAGG-3′; XAF1.1 forward primer: 5′-ACCAGCAGGTTG

GGTGTACGATGT-3′; XAF1.1 reverse primer: 5′-CGCTCCTGGCA

CTCATTGGCCTT-3′.

In Situ Hybridization C57BL/6 mice were decapitated at embryonic day 17.5

(E17.5) or P1 and were processed as for immunohistochemistry.

Digoxigenin-labeled RNA probes were generated from a cDNA sub-

clone in the pGEM-T easy plasmid (Promega). In vitro transcription

was carried out using the DIG RNA Labeling Kit (Roche), according

to the manufacturer’s instructions. Sense and antisense probes were

generated from a cDNA fragment corresponding to nucleotides

3709-4292 of the KIF1Bβ tail domain (accession no. NM_207682.2)

and designed not to cross-react with the KIF1Bα isoform. Probes

were diluted in hybridization buffer [50% formamide, 5% saline-

sodium citrate buffer , 5× Denhardt’s solution, 250 μg/mL tRNA,

500 μg/mL sonicated salmon sperm DNA, 20 mg/mL blocking rea-

gent for nucleic acid hybridization and detection (Roche)] to a fi nal

concentration of 10 ng/μL. Hybridization was performed at 70°C

overnight and detected using anti-digoxigenin (DIG) antibody,

followed by visualization with nitroblue tetrazolium (NBT) and

5-bromo-4-chloro-3-indolyl phosphate (BCIP; Roche). The SCG was

identifi ed on the basis of its proximity to the cochlea and through

immunohistochemical staining in adjacent sections, using an anti-

body against tyrosine hydroxylase, which is a marker for adrenergic

and noradrenergic sympathetic neurons.

Mice C57BL/6 mice were kept at 21°C on a 12-hour light and 12-hour

dark cycle. Animal care procedures were in accordance with the

guidelines set by the European Community Council Directives

(86/609/EEC). Required animal permissions were obtained from the

local ethical committee.

KIF1B Exome Sequencing Genomic DNA was isolated from primary neuroblastoma tumors

and submitted for exome sequencing performed by Otogenetics.

KIF1B exome mutation reports of primary neuroblastomas were

based on custom target sequencing of human chromosome 1 from

10,270,764 to 10,441,661 using HiSeq2000 PE100 with minimum

80× coverage.

Tumor Material All available neuroblastoma tumors were from the Swedish NB

Registry. Tumors were staged according to the International Neu-

roblastoma Staging System (INSS; ref. 46 ) and INRG criteria ( 47 ).

Ethical permission was granted by the local ethics committee. Tumor

analysis has been previously described ( 48 ).

Disclosure of Potential Confl icts of Interest No potential confl icts of interest were disclosed.

Authors’ Contributions Conception and design: Z.X. Chen, K. Wallis, P. Kogner, S. Schlisio

Development of methodology: Z.X. Chen, K. Wallis, V.R. Sobrado,

S. Schlisio

Acquisition of data (provided animals, acquired and managed

patients, provided facilities, etc.): Z.X. Chen, K. Wallis, S.M. Fell,

V.R. Sobrado, M.C. Hemmer, U. Hellman, T. Martinson, J.I. Johnsen,

P. Kogner, S. Schlisio

Analysis and interpretation of data (e.g., statistical analysis,

biostatistics, computational analysis): Z.X. Chen, K. Wallis, V.R.

Sobrado, D. Ramsköld, U. Hellman, T. Martinson, J.I. Johnsen, P.

Kogner, S. Schlisio

Writing, review, and/or revision of the manuscript: Z.X. Chen,

K. Wallis, S.M. Fell, V.R. Sobrado, T. Martinson, J.I. Johnsen, P. Kogner,

S. Schlisio

Administrative, technical, or material support (i.e., reporting or

organizing data, constructing databases): Z.X. Chen, T. Martin-

son, J.I. Johnsen, P. Kogner, S. Schlisio

Study supervision: S. Schlisio

Supervision of RNA-seq analyses: R. Sandberg

Performed one experiment for this article: R.S. Kenchappa

Acknowledgments The authors thank Robert Freeman, Peter Ratcliffe, and Shazib

Pervaizfor for valuable reagents, and Anita Bergstrom for technical

assistance.

Grant Support Z.X. Chen is supported by the Swedish Children Cancer Founda-

tion and the National University of Singapore. K. Wallis is supported

by the Swedish Brain Foundation and the Swedish Children Cancer

Foundation. S. Schlisio is supported by grants from the Swedish

Children Cancer Foundation, the Swedish Research Council, the

Swedish Cancer Society, and the Åke Wiberg Foundation, and is

an Assistant Member of Ludwig Institute for Cancer Research Ltd

(LICR).

Received July 11, 2013; revised January 7, 2014; accepted January

22, 2014; published OnlineFirst January 27, 2014.

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2014;4:434-451. Published OnlineFirst January 27, 2014.Cancer Discovery Zhi Xiong Chen, Karin Wallis, Stuart M. Fell, et al. Tumor-Suppressor Function in Neuroblastoma

βRNA Helicase A Is a Downstream Mediator of KIF1B

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