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https://doi.org/10.1038/s41467-020-15289-7

OPEN

CDK1 dependent phosphorylation of hTERT
contributes to cancer progression

1234567890():,;

Mami Yasukawa 1,13, Yoshinari Ando 1,13, Taro Yamashita 2,13, Yoko Matsuda3,12, Shisako Shoji 4,
Masaki Suimye Morioka5, Hideya Kawaji 5,6, Kumiko Shiozawa 7, Mitsuhiro Machitani1, Takaya Abe8,9,Shinji Yamada 10, Mika K. Kaneko 10, Yukinari Kato 10,11, Yasuhide Furuta8,9, Tadashi Kondo7,
Mikako Shirouzu 4, Yoshihide Hayashizaki 6, Shuichi Kaneko 2 & Kenkichi Masutomi 1 ✉The telomerase reverse transcriptase is upregulated in the majority of human cancers andcontributes directly to cell transformation. Here we report that hTERT is phosphorylated at
threonine 249 during mitosis by the serine/threonine kinase CDK1. Clinicopathologicalanalyses reveal that phosphorylation of hTERT at threonine 249 occurs more frequently inaggressive cancers. Using CRISPR/Cas9 genome editing, we introduce substitution mutations
at threonine 249 in the endogenous hTERT locus and ﬁnd that phosphorylation of threonine
249 is necessary for hTERT-mediated RNA dependent RNA polymerase (RdRP) activity but
dispensable for reverse transcriptase and terminal transferase activities. Cap Analysis ofGene Expression (CAGE) demonstrates that hTERT phosphorylation at 249 regulates theexpression of speciﬁc genes that are necessary for cancer cell proliferation and tumor formation. These observations indicate that phosphorylation at threonine 249 regulates hTERTRdRP and contributes to cancer progression in a telomere independent manner.

1 Division of Cancer Stem Cell, National Cancer Center Research Institute, Tokyo 104-0045, Japan. 2 Department of Gastroenterology, Kanazawa University
Graduate School of Medical Science, Kanazawa 920-8641, Japan. 3 Department of Pathology, Tokyo Metropolitan Geriatric Hospital and Institute ofGerontology, Tokyo 173-0015, Japan. 4 Laboratory for Protein Functional and Structural Biology, RIKEN Center for Biosystems Dynamics Research, Yokohama
230-0045, Japan. 5 Preventive Medicine and Applied Genomics Unit, RIKEN Center for Integrative Medical Sciences, Yokohama 230-0045, Japan. 6 RIKEN
Preventive Medicine and Diagnosis Innovation Program, Wako 351-0198, Japan. 7 Division of Rare Cancer Research, National Cancer Center ResearchInstitute, Tokyo 104-0045, Japan. 8 Animal Resource Development Unit, RIKEN Center for Life Science Technologies, Kobe 650-0047, Japan. 9 Genetic
Engineering Team, RIKEN Center for Life Science Technologies, Kobe 650-0047, Japan. 10 Department of Antibody Drug Development, Tohoku University
Graduate School of Medicine, Sendai 980-8575, Japan. 11 New Industry Creation Hatchery Center, Tohoku University, Sendai 980-8579, Japan. 12Present
address: Oncology Pathology, Department of Pathology and Host-Defense, Kagawa University, Kagawa 761-0793, Japan. 13These authors contributed
equally: Mami Yasukawa, Yoshinari Ando, Taro Yamashita. ✉email: kmasutom@ncc.go.jp

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R

ecent analyses of human cancer genomes revealed thatsomatic mutations within the promoter of human telomerase reverse transcriptase (hTERT) occur at high frequency inover 50 distinct cancer types1–6. These common promoter mutations are associated with higher expression levels of hTERT and apoor prognosis5,7–9. In humans, experiments involving live-cell
imaging techniques combined with CRISPR-Cas9 genome editing
demonstrated that recruitment of hTERT and hTERC to telomeres
occurs through dynamic interactions between telomerases and thechromosome end during S-phase10. Although these observations
indicate that recruitment of telomerase holoenzyme to the telomere
is regulated in cell cycle-dependent manner, only a small subset ofhTERT forms interactions with telomeres and Cajal bodies even in
S-phase10 and the regulation and function of the majority ofhTERT outside S-phase is poorly understood. In addition, cell
cycle-dependent regulation of hTERT messenger RNA (mRNA) isobserved by several groups and the highest level of hTERT mRNA
are detected in mitotic phase11,12.
We have previously reported that hTERT has an RNAdependent RNA polymerase (RdRP) activity, which generates
double-stranded RNAs (dsRNAs) from a single-stranded RNA
not only in a primer-dependent manner, but also in a primerindependent manner12,13. We also detected upregulation ofhTERT protein, as well as RdRP activity, in mitotic phase using
several cell lines12,14.
Here, we report that hTERT is phosphorylated in a cell cycledependent manner and that this phosphorylation is essential for
the RdRP activity and tumor formation via regulation of target
gene expression independent of hTERT-mediated elongation of
telomeres.
Results
Mitotic-speciﬁc accumulation of hTERT. Since it has beenchallenging to detect endogenous hTERT11,15, we extensively
validated available hTERT-speciﬁc antibodies against hTERT,
including the mouse monoclonal antibody (mAb) (clone 10E9-2),
the mouse mAb (clone 2E4-2), the sheep polyclonal Abs (pAbs)
abx120550, and the rabbit mAb ab3202. Speciﬁcally, we performed validation experiments by (i) immunoprecipitation (IP)with anti-hTERT antibodies followed by immunoblotting (IB)(Fig. 1a), (ii) suppression of hTERT by small interfering RNAs(siRNAs) speciﬁc for hTERT12,16 followed by IP-IB (Supplementary Fig. 1a), and (iii) recovery of telomerase activity withthese antibodies as gauged by quantitative direct telomerase
assay11,17,18 (Supplementary Fig. 1b, c). In each case, we foundthat the 10E9-2, the 2E4-2, the abx120550 and the ab32020
identiﬁed hTERT (Fig. 1a and Supplementary Fig. 1), conﬁrming
the sensitivity and speciﬁcity of these antibodies.
We previously reported that hTERT protein localizes to mitotic
spindles14 and hTERT protein expression is enriched in mitosis12(Fig. 1a). We reconﬁrmed the enrichment of hTERT protein inmitosis using the antibodies described above11 (Fig. 1a, two rightpanels). In addition, we conﬁrmed mitotic-speciﬁc accumulationof hTERT at the mRNA level and RNA-dependent RNA
polymerase (RdRP) activities of hTERT in a cell cycledependent manner by IP-RdRP assay19 using hTERT immune
complexes immunoprecipitated from cell lysates with antihTERT mAb (10E9-2) (Supplementary Fig. 2a, b). Furthermore,
we manipulated cells in mitotic phase with nocodazole treatment
or double thymidine block treatment and observed the increase ofhTERT expression in both cases (Supplementary Fig. 2c).
Consistent with our data, Xi et al.11 also reported that hTERT
expression is enriched in mitotic phase by double thymidine
block treatment. These observations suggest that expression ofhTERT protein is regulated in a cell cycle-dependent manner and2

is not due to nocodazole treatment (through stress kinases such as
p3820) but due to mitotic entry.
Phosphorylation of hTERT in mitosis. To investigate hTERT
regulation in mitosis, we ﬁrst treated HeLa cells with nocodazole.
We conﬁrmed that cells accumulated in mitotic phase by assessing phospho-histone H3 (Ser10) levels (Fig. 1b, lower panel).
When we examined the migration of endogenous hTERT in the
mitotic phase by sodium dodecyl sulfate–polyacrylamide gelelectrophoresis (SDS-PAGE), we found that endogenous hTERT
isolated by immunoprecipitation with anti-hTERT mAb (clone
10E9-2)14 migrated slower than ectopically expressed FLAGtagged hTERT (Fig. 1b, upper panel). We thus speculated that
endogenous hTERT in mitotic phase is post-translationally
modiﬁed. We treated hTERT immunoprecipitated with antihTERT mAb (clone 10E9-2) from mitotic cells with λ phosphatase and found that phosphatase treatment diminished themobility shift of hTERT protein (Fig. 1c). This observation suggested that hTERT is phosphorylated in mitosis.
To identify the mitotic phosphorylation sites in hTERT, weectopically expressed hTERT in HEK-293T (293T) or HeLa cells
followed by treatment with nocodazole to arrest cells in mitosis. We
isolated hTERT by immunoprecipitation and performed mass
spectrometry (MS) analysis using liquid chromatography-tandem
mass spectrometry (LC-MS/MS). We used the Mascot software
package (version 2.5.1; Matrix Science) to search for the mass ofeach peptide ion peak against the SWISS-PROT database (Homosapiens, 20,205 sequences in the Swiss prot_2015_09.fasta ﬁle) andto identify the phosphorylation sites. We identiﬁed four phosphopeptides in 293T cells and one phosphopeptide in HeLa cells.
The phosphopeptide 241GAAPEPERpTPVGQGSWAHPGR261 wasfound in both 293T and HeLa cells (Fig. 1d). Moreover,phosphorylation sites analysis by the Mascot software revealed that
only the residue at threonine 249 (T249) was phosphorylated in thephosphopeptide (amino acids 241-261) (Supplementary Data 1).
We found that T249 is conserved only in primates (Fig. 1e).
To interrogate the consequences of hTERT phosphorylation at
T249, we generated rabbit polyclonal antibodies that speciﬁcallyrecognize phosphothreonine 249 (anti-249T-P). We conﬁrmedthat treatment of hTERT isolated from mitotic HeLa cells with λphosphatase or suppression of hTERT by hTERT-speciﬁc siRNAs
ablated the signal detected by this antibody (Fig. 1f, g). Taken
together, these observations indicate that hTERT at T249 isphosphorylated in mitosis.
We noted that the sequence surrounding T249 contains [S/T]P
motif that is implicated as a target of the serine-threonine kinasecyclin-dependent kinase 1 (CDK1)21. To investigate whether
CDK1 phosphorylates hTERT, we performed in vitro kinase assay
using recombinant hTERT fragment proteins (amino acids
191–306) and CDK1-cyclinB proteins followed by MS analysis.
We used phosphate-afﬁnity gel electrophoresis (Phos-tag SDSPAGE) to detect the phosphorylation status of hTERT fragment
incubated with puriﬁed CDK1-cyclinB or IKK2_2-664 used as acontrol of serine-threonine kinase. Phos-Tag SDS-PAGE allows
the migration delay of phosphorylated proteins relative totheir unphosphorylated counterparts in gels containing aphosphate-binding tag22. The Phos-tag SDS-PAGE revealed thathTERT_191-306 proteins were phosphorylated by CDK1-cyclinB
and IKK2_2-664 in vitro (Fig. 1h, upper panel). The hTERT_191306 protein phosphorylated by CDK1-cyclinB was detected withthe anti-249T-P antibodies, but we were unable to detect theprotein phosphorylated by IKK2_2-664 with the anti-249T-P
antibodies (Fig. 1h, lanes 6 and 7 on lower panel), suggesting that
IKK2 does not phosphorylate hTERT at T249 in vitro. To further
conﬁrm that hTERT T249 is phosphorylated by CDK1-cyclinB

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non M

non M

+
(kDa)
225
150

150
IB: hTERT(2E4-2)

-150

150 -

–

IP: hTERT(10E9-2 )

225

-225

225 -

λ phosphatase(kDa)

IP: hTERT(10E9-2)(kDa)c

FLAG-hTERT
(O/E)

non M

Endogenous
hTERT
non M

293T

M

HeLa

M

293T

M

HeLa
non M

(kDa)

b

IP: hTERT (abx120550)
IB: hTERT (ab32020)

M

IP: hTERT (10E9-2)
IB: hTERT (2E4-2)

Ma

IB: hTERT(2E4-2)

102

102

-102

102 -

76

76
-76

76 -

Cell lysate
IB: phosphoHH3 (Ser10)dPhosphopeptides

HeLa

e

Phospho/(phospho+nonphospho)

M

non M

M

non M

M

2

9

0

0

100%

100%

AWNHSVR

1

3

22

26

4%

10%

EKPQGSVAAPEEEDTDPR

1

0

27

40

4%

0%

VTYVPLLGSLR

0

1

36

49

0%

2%

GAAPEPERTPVGQGSWAHPGR

0

1

0

0

–

100%

1-230:
Telomerase essential
N

Nonphosphopeptides

non M
GAAPEPERTPVGQGSWAHPGR
293T

Cell lysate
IB: phosphoHH3 (Ser10)

17

100

f

191–306TEN

17

TERC binding

NLS

DNA polymerization

TRBD
300

200

400

Telomeric DNA positioning

RT
500

600

700

C

CTE
800

900

1000

λ phosphatase

1100

–

+
(kDa)

IP: hTERT(10E9-2 )TERT [Homo sapiens]
TERT [Macaca mulatta]
TERT [Gorilla gorilla gorilla]
TERT [Pan troglodytes]
TERT [Propithecus coquereli]Tert [Mus musculus]
TERT [Rattus norvegicus]
TERT [Canis lupus familiaris]
TERT [Bos taurus]
Est2p [Saccharomyces cerevisiae S288C]

225
150

IB: hTERT(249T-P)

TERT [Homo sapiens]
TERT [Macaca mulatta]
TERT [Gorilla gorilla gorilla]
TERT [Pan troglodytes]
TERT [Propithecus coquereli]Tert [Mus musculus]
TERT [Rattus norvegicus]
TERT [Canis lupus familiaris]
TERT [Bos taurus]
Est2p [Saccharomyces cerevisiae S288C]

102
76

NLS
TERT [Homo sapiens]
TERT [Macaca mulatta]
TERT [Gorilla gorilla gorilla]
TERT [Pan troglodytes]
TERT [Propithecus coquereli]Tert [Mus musculus]
TERT [Rattus norvegicus]
TERT [Canis lupus familiaris]
TERT [Bos taurus]
Est2p [Saccharomyces cerevisiae S288C]

g

h
hTERT
siRNA
NC

IP: hTERT(10E9-2)#1#2+
CDK1-cyclinB–
IKK2_2-664
hTERT_191–306 –
–
ATP

–
–
+
–

+
–
–
+

–
–
+
+

+
–
+
–

+
–
+
+

–
+
+
+

102

+

+

+

CDK1-cyclinB

+

–

+

PhosphorylatedhTERT fragments

(kDa)
-52
-38
-31

-17
hTERT fragment
(kDa)
17

102

–

-24

Phosphate affinity
SDS-PAGE76

IB: hTERT(2E4-2)

IB: hTERT
249T-P TpMab-1

hTERT_191–306 +

(kDa)
225
150

IB: hTERT(249T-P)

i

– – –
+ + +
+ – –
– + –

IB: hTERT(249T-P)

-12

12
-3.5
1 2 3 4 5

in vitro, we performed MS analysis using LC-MS/MS and identiﬁedthat threonine 249 was phosphorylated by CDK1-cyclinB, not by
IKK2_2-664 (Supplementary Fig. 3 and Supplementary Table 1).
These observations conﬁrmed that CDK1-cyclinB phosphorylates
hTERT T249.

6

7 8

9 10

To further study hTERT phosphorylation at T249, we
generated a mouse monoclonal antibody that speciﬁcallyrecognize phosphothreonine 249 (TpMab-1). We conﬁrmed thatthe antibody speciﬁcally detected phosphorylation signals of

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Fig. 1 Identiﬁcation of phosphorylation site of hTERT in mitosis. a Detection of endogenous hTERT. The cells were treated with DMSO (denoted as “nonM”) or nocodazole to manipulate cells in mitosis (denoted as “M”), and immunoprecipitated with anti-hTERT mouse mAb (10E9-2) or anti-hTERT sheeppAbs (abx120550). The hTERT proteins were detected by anti-hTERT mouse mAb (2E4-2) (two left panels) or anti-hTERT rabbit mAb (ab32020) (tworight panels). b hTERT proteins were isolated by immunoprecipitation with 10E9-2 and detected by 2E4-2 (upper panel). Cells arrested in mitosis with
nocodazole were conﬁrmed by anti-phospho-histone H3 (Ser10) antibodies (lower panel). c Endogenous hTERT immunoprecipitated from 293T cells weretreated with λ phosphatase to remove the phosphate groups and detected by 2E4-2 (upper panel). d Summary of MS analysis to identify phosphorylationsites in hTERT. The numbers of phosphopeptides and nonphosphopeptides are based on pep_expect (Expectation value for PSM) < 0.05 used as a cutoff
value. Bold letters indicate amino-acid residues identiﬁed as phosphorylated residues. See the details in Supplementary Data 1. e Multiple sequencealignment of telomerases using COBALT85. Red letters denote conserved residues and blue letters denote non-conserved residues with no gaps. Yellow is
the phosphorylated threonine residue detected in this study. f The immune complexes were treated with λ phosphatase. Phosphorylation of hTERT at T249
was detected by anti-249T-P rabbit pAbs. g Endogenous hTERT proteins from cells transfected with two different siRNAs speciﬁc for hTERT.
Phosphorylation of hTERT at T249 was detected by anti-249T-P pAbs (upper panel). Whole-hTERT proteins were detected by 2E4-2 (lower panel). h The
recombinant hTERT fragment proteins (191–306 a.a) were phosphorylated by CDK1-cyclinB or IKK2_2-664 in vitro and phosphorylated proteins weredetected in Phos-Tag SDS-PAGE (upper panel). Phosphorylation of hTERT at threonine 249 by CDK1-cyclinB was conﬁrmed by anti-249T-P pAbs (lower
panel). The protein bands enclosed with square were analyzed by MS to conﬁrm the phosphorylation sites. i Phosphorylation of threonine 249 by CDK1cyclinB was conﬁrmed by anti-249T-P pAbs and TpMab-1 mouse mAb. Experiments were repeated three times (for a, g, i), ﬁve times (for b, c, f) and twice(for h) with similar results. Source data are provided in the Source Data ﬁle.

hTERT T249 phosphorylated by CDK1-cyclinB in vitro similar toanti-249T-P polyclonal antibodies (Fig. 1i).
Phosphorylation of hTERT at T249 occurs in cancers. To assess
whether T249 was phosphorylated in human cancers, we ﬁrst
validated the speciﬁcity of the anti-249T-P pAbs for immunohistochemical (IHC) staining in Huh7 xenografts. The observed
signals were fully abolished by the absorption treatment withphosphopeptide (Supplementary Fig. 4a, b), whereas treatment
with nonphosphopeptide did not alter the signals (Supplementary
Fig. 4c), indicating that the antibodies are speciﬁc and applicablefor immunohistochemical analyses.
To investigate the relationship between a progression of
pancreatic cancer and hTERT phosphorylation, we stained eachpancreatic precancerous lesions with anti-249T-P antibodies. We
found no evidence for anti-249T-P staining in normal ducts,
PanIN-1 and -2 but observed robust staining in PanIN-3(Fig. 2a–e). In pancreatic adenocarcinomas, we found stainingof hTERT phosphorylation with anti-249T-P pAbs (Fig. 2f). Bothnormal and atypical multipolar mitotic images exhibited stronghTERT phosphorylation (Fig. 2g, h). We also found strong
staining with anti-CDK1 pAbs in pancreatic cancer cells (Fig. 2i).
Furthermore, we examined the number of cells stained with anti249T-P antibodies, referred as 249T-P-positive cells, in eachpancreatic ductal lesion (Fig. 2j). The number of 249T-P-positive
cells was the highest in carcinomas, followed by PanIN-3, PanIN2, PanIN-1, and duct epithelium (p < 0.05). To consolidate thespeciﬁcity of the data for IHC with anti-249T-P pAbs, we stained
the pancreatic cancer samples with TpMab-1 monoclonal antibody, that also speciﬁcally recognize phosphothreonine 249(Fig. 1i), in serial sections prepared from the identical pancreaticcancer lesions (Supplementary Fig. 5a–f). We observed theidentical staining pattern with these antibodies in the same cells
and conﬁrmed that phosphorylation signals of hTERT T249 weredetected in pancreatic carcinoma. Further, we found that 249T-Ppositive cases showed higher incidence of lymph node metastasis
than 249T-P-negative cases (Supplementary Table 2 andSupplementary Fig. 6a). Surprisingly, clinicopathological analysis
indicated the strong correlation between CDK1 expression andhTERT T249 phosphorylation in pancreatic cancer (p = 0.0289,
Supplementary Table 2). We conﬁrmed that the same pancreaticcancer cells were co-stained with anti-CDK1 and TpMab-1 byimmunoﬂuorescence images (Supplementary Fig. 7a–d). The
signal intensities between these antibodies demonstrated positive
correlation at single-cell level (p = 0.0350, Supplementary
Fig. 7e).
4

In addition, we conducted IHC with anti-249T-P pAbs andanti-CDK1 pAbs on HCC samples. We failed to detect the
speciﬁc signals in non-cancerous lesions obtained from two
individual patients (Fig. 2k, l) but found strong staining with anti249T-P (Fig. 2m, n) and anti-CDK1 (Fig. 2o, p) in HCC lesions
obtained from the identical patients, respectively. We foundstrong hTERT phosphorylation signals mostly in mitotic cells of
more advanced cancers, suggesting that hTERT phosphorylation
at T249 is associated with HCC grade (Supplementary Table 3
and Supplementary Fig. 6b). 249T-P-positive HCC showed high
CDK1 expression with statistical signiﬁcance (p < 0.0001, Supplementary Table 3). In addition, CDK1 expression correlated withpoorly differentiated morphology (Supplementary Table 4).
In consonance with this observation, we found that patients
whose tumors lacked anti-249T-P or anti-CDK1 staining had
longer overall survival (OS; p = 0.048 and 0.036, respectively)
than patients whose cancers showed with anti-249T-P staining(Fig. 2q, r). These observations suggest that both hTERT
phosphorylation at T249 and high expression of CDK1 occurs
in cancers, more frequently in aggressive cancers.
Consequences of hTERT phosphorylation. To study the consequence of hTERT phosphorylation, we used λ phosphatase toremove all phosphate groups from hTERT immunoprecipitatedwith the anti-hTERT mAb (clone 10E9-2) and then performed aquantitative direct telomerase assay and an RdRP assay (Fig. 3a).
We found that phosphatase treatment of hTERT reduced hTERTdependent RdRP activity but had no measurable effect on telomerase activity.
To investigate whether endogenous hTERT protein is phosphorylated by CDK1 in vitro and the phosphorylation event
regulates RdRP activity, we treated the hTERT immune complex
with phosphatase to remove phosphate groups from hTERT
protein and then added CDK1 and ATP to phosphorylate T249 in
endogenous full-length hTERT. We conﬁrmed that T249 residueof hTERT was phosphorylated by CDK1 using in vitro kinase
assay (Fig. 3b, left panel). In addition, we noted that CDK1
treatment after phosphatase treatment did not completely
relocate the hTERT signal to the original position. This indicatesthat there may exist other phosphorylation site(s) besides T249 by
CDK1. We further conﬁrmed that this phosphorylation event by
CDK1 regulates RdRP activity of hTERT (Fig. 3b, right panel).
To conﬁrm that CDK1 phosphorylates hTERT at T249, we
suppressed CDK1 with CDK1-speciﬁc siRNAs (Fig. 3c). Suppression of CDK1 decreased cell proliferation by 70%, and we
observed that introduction of CDK1-speciﬁc siRNAs #1, 2, andNATURE COMMUNICATIONS | (2020)11:1557 | https://doi.org/10.1038/s41467-020-15289-7 | www.nature.com/naturecommunications

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NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15289-7aN-smallbN-largecdPanIN-2e

PanIN-3

f

g

50
45
40
35
30
25
20
15
10
5
0

l

Survival (%)

Cancer(n = 47)

PanIN-3(n = 12)PanIN-2(n = 18)

PanIN-1(n = 28)

N-large(n = 38)

q 100
N-small(n = 41)hTERT phosphorylation (%)

k

i

h

j

PanIN-1

90

*
80
70
60
0

o

n

p

r
Survival (%)

m

200

400
Days

600

800

100
90

*

80
70
60
0

3 suppressed hTERT T249 phosphorylation by 98%. Altogether,
these observations strongly suggest that CDK1 is required for thephosphorylation of hTERT at T249.
Moreover, we found that the RdRP activity of hTERT was
substantially diminished by CDK1 knockdown while suppression of
CDK1 had no effect on telomerase activity (Fig. 3d), indicating that

200

400
Days

600

800

phosphorylation by CDK1 is required for the RdRP activity ofhTERT. These results suggest that hTERT proteins are phosphorylated by CDK1 in mitosis and the phosphorylation is not required
for the telomerase activity but for the RdRP activity of hTERT.
To further conﬁrm that CDK1 speciﬁcally phosphorylates
hTERT T249, we treated HeLa cells with CDK1 inhibitor, RO-

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Fig. 2 Phosphorylation of hTERT T249 in pancreatic cancer and HCC. a–e Representative images of precancerous lesions stained with anti-249T-P pAbs.
N-small, intercalated duct to intralobular duct; N-large, interlobular duct to main pancreatic duct; PanIN, pancreatic intraepithelial neoplasia; Cancer,
pancreatic invasive ductal adenocarcinoma. Original magniﬁcation x400, scale bar: 50 µm. f Phosphorylation of hTERT T249 was detected in the nucleus ofpancreatic adenocarcinomas. Original magniﬁcation x400, scale bar: 50 µm. g, h Both normal mitotic (pointed by arrows, g) and atypical multipolar mitotic
images (pointed by arrow, h) exhibited phosphorylation of hTERT T249 (inserts). Original magniﬁcation x400. i Expression of CDK1 was detected in the
nucleus of pancreatic adenocarcinomas. Original magniﬁcation x400, scale bar: 50 µm. j Relationship between pancreatic ductal lesions and hTERT
phosphorylation at T249. Box-and-whisker plot of hTERT phosphorylation for each of the pancreatic ductal lesions surgically resected from patients withpancreatic cancer (n = 47). The box portion of the box plot is deﬁned by two lines at the 25th percentile and 75th percentile. A line is drawn inside the box
at the median. The two whisker boundaries are the 5th percentile and 95th percentile. p < 0.05 by Speaman’s rank correlation test. k–p IHC staining with
anti-249T-P pAbs in non-cancerous lesions (k, l) and HCC lesions (m, n) obtained from two individual patients. IHC staining with anti-CDK1 pAbs in HCC
lesions (o, p) obtained from two identical patients. Original magniﬁcation x200, scale bar: 100 µm. q, r Kaplan–Meier survival analysis of primary patientswith HCC grouped for 249T-P-positive (red) or negative (blue). Phosphorylation status of hTERT T249 (q) and CDK1 expression (r) showed statisticallysigniﬁcant correlations with overall survival (OS, p = 0.048 and 0.036, respectively, log rank test, two-sided) in patients.

330623. Inhibition of CDK1 activity diminished the phosphorylation of hTERT T249 (Fig. 3e) and reduced the RdRP activity ofhTERT without affecting telomerase activity (Fig. 3f). Thus, bothsuppressing CDK1 expression or inhibiting CDK1 kinase activity
decreased the phosphorylation of hTERT at T249 and hTERTmediated RdRP activity.

Consequences of phosphorylation of hTERT on T249. To further assess the consequences of phosphorylation of hTERT T249
in the human cell lines, we created hTERT mutants where we
substituted the threonine 249 of hTERT with alanine (T249A) or
glutamic acid (T249E). We introduced these mutants intelomerase-negative human ﬁbroblast BJ cells and analyzed thetelomerase activity of the mutant proteins by the telomeric repeat
ampliﬁcation protocol (TRAP) assay (Fig. 4a, left panel andSupplementary Fig. 8a) and direct telomerase assay (Supplementary Fig. 8b). We found that both of these mutant hTERT
alleles exhibited telomerase activity equivalent to that observed
for wild-type hTERT (WT-hTERT). In contrast, when we
examined RdRP activity using BJ cells stably overexpressing thesehTERT mutants, BJ cells with WT-hTERT and T249A exhibited
little RdRP activity while T249E cells demonstrated steady statelevel of RdRP activity. These data indicate that normal cells show
little RdRP activity due to the lack of phosphorylation at T249 ofhTERT (Supplementary Fig. 8c).
We conﬁrmed the ﬁndings by introducing these hTERT
mutants in 293T cells and measured the telomerase activity by aquantitative assay as measured by the direct telomerase assay(Fig. 4b). We veriﬁed that these mutants retained telomerase
activity comparable to WT-hTERT and did not interfere withendogenous hTERT. Consistent with a previous report11, ectopic
expression of WT-hTERT protein slightly increased measured
telomerase activity. We also assessed whether these two mutants
elongated telomeres when expressed in cells by performingSouthern blotting for telomere restriction fragments. We analyzed
the telomere length in the BJ cells at 8 population doubling (PD)after infection of the two mutants and found that T249A and
T249E mutants elongated telomeres (Fig. 4a, right panel). These
data demonstrated that telomerase activity of the mutants was
intact.
We next examined whether introduction of hTERT mutants in
293T cells altered hTERT-dependent RdRP activity13. We foundthat expression of the phosphorylation-defective mutant hTERT
T249A decreased RdRP activity (Fig. 4c), while ectopic expressionof hTERT T249E increased RdRP activity, suggesting that thismutant acts as a phosphomimetic mutant. These observations
conﬁrmed that the phosphorylation of hTERT T249 is required
for the RdRP activity but not for reverse transcriptase activityof hTERT.
6

To further assess the biological effects of phosphorylation ofhTERT T249 in cancer cell lines, we ectopically expressed hTERT
T249A, hTERT T249E, or a control vector expressing only a drug
resistance marker into several well-characterized cancer cell lines;
two telomerase/RdRP-positive ovarian cancer cell lines12, PEO1
and PEO14, and a telomerase/RdRP-positive patient-derived
hepatocellular carcinoma (HCC) MT cells (Fig. 4d). In PEO1,
PEO14, and MT cells, the expression of hTERT T249A
dramatically decreased the cell proliferation (0.73-, 0.18- and0.50-fold, respectively) while the hTERT T249E showed a minor
effect on cell proliferation (1.03-, 1.01- and 1.19-fold, respectively) (Fig. 4e). These observations suggest that phosphorylationdefective hTERT T249A interferes with hTERT-mediated RdRP
activity and the inhibition of RdRP activity causes inhibitory
effects on the cell growth. In contrast, hTERT T249E failed toalter the proliferation of these cell lines.
To verify the effects of hTERT mutants on the cell proliferationin an hTERT null cell line, we introduced WT-hTERT, hTERT
T249A, hTERT T249E, or a control vector in Saos2 cells that lacktelomerase activity. We observed that T249E mutant mosteffectively accelerates the cell proliferation, WT-hTERT is thesecond and that T249A has no effects on cell proliferation(Supplementary Fig. 9).
To examine whether suppression of hTERT affects the cell
viability, we introduced hTERT-speciﬁc siRNAs to several
different cancer cell lines known to be hTERT positive (A549,
293T, and HeLa cells) and an hTERT null cell line (VA13). We
conﬁrmed that cancer cells addicted to TERT rapidly die by
suppression of hTERT (Supplementary Fig. 10).
To further evaluate the inﬂuence of hTERT phosphorylation at
T249 using patient-derived xenograft (PDX) models in vivo, we
introduced hTERT T249A (MT+T249A) or a control retroviral
vector (MT-Control) into MT (patient-derived HCC) cells andimplanted these cells subcutaneously into non-obese diabetic/severe combined immunodeﬁciency (NOD/SCID) mice. Six ofeight mice harboring MT+T249A failed to form tumors (Fig. 4f,
left panel) and the volume of tumors from mice harboring MT+
T249A was signiﬁcantly smaller than those from mice with MTControl (Fig. 4f, right panel; p = 0.0028). Taken together, theseobservations indicate that phosphorylation of hTERT at T249 isrequired for cancer cell proliferation through activation of RdRP
activity of hTERT. Although the expression of these hTERT
alleles clearly separated hTERT-mediated telomerase and RdRP
activity, endogenous hTERT is expressed at low levels even incancer cells.
CRISPR-Cas9 genome editing to introduce mutations at T249.
To assess whether the phosphorylation of T249 affected endogenous hTERT function, we used CRISPR-Cas9 genome editing
to introduce alanine or glutamic acid substitutions at T249 in the

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ab

Directtelomerase
RdRP assay
assay

λ phosphatase

–

+

–

RdRP assay

+

(nt)
80
70

(nt)
- 40

60

λ phosphatase

–

+

+

λ phosphatase

–

+

+

CDK1-cyclinB

–

–

+

CDK1-cyclinB

–

–

+

IP: hTERT(10E9-2)

(kDa)
225
150

IB: hTERT(249T-P)

102

- 30

50

40
30

76
- 20

40

30

1

20
1

0.24

20

150

IB: hTERT(2E4-2)

Relative
RdRP
activity1

0.18 0.60

102

Relative
RdRP
activity

Relative
1.14 telomerase
activitycdCDK1 siRNA
IP: hTERT(10E9-2)

(nt)

NC

#1

#2

#3

Direct telomerase assay

(kDa)

#4

RdRP assay

CDK1 siRNA

150

NC #1

CDK1 siRNA#2 #3 #4

NC #1

#2 #3 #4(nt)
IB: hTERT(249T-P)

102
150

IB: hTERT(2E4-2)

80
70

(nt)
- 40

60
- 30

50
102

- 20

40
Cell lysate
31

IB: CDK1

1

0.11 0.05 0.09 0.75

30

Relative
RdRP
activity

20

e

f

CDK1
inhibitor
IP: hTERT(10E9-2)

–

+
(kDa)
- 225
- 150

Direct telomerase
assay
CDK1
–
+
inhibitor
(nt)
80
70

RdRP assay
CDK1
inhibitor

–

+

(nt)
- 40

60
IB: hTERT(249T-P)

- 102

50

- 75

40

- 30

- 20
30
IB: hTERT(2E4-2)

1

- 102

0.35

Relative
RdRP
activity20

endogenous hTERT locus in 293T cells (Fig. 5a). After introduction of Cas9 and guide RNA with donor DNAs into
293T cells, we selected clones and conﬁrmed that the mutations
were introduced by PCR ampliﬁcation, restriction enzyme
digestion, and Sanger sequencing (Fig. 5b, c). Control cells,
Control-CRISPR, were selected from clonal selection of cells
expressing Cas9 and no guide RNAs. We examined potential offtarget (OT) mutations in six candidate loci corresponding to
guide RNA sequence and no OT mutations were detected in thecells (Supplementary Fig. 11). We conﬁrmed that T249A-CRISPR

and T249E-CRISPR were expressed at a level similar to that ofendogenous hTERT in the Control-CRISPR cells (Fig. 5d, lower
panel). We further ascertained that introduction of these mutations into hTERT had no effect on the protein stability, reported
protein-protein interactions with hTERT, such as nucleostemin(NS)16 and SMG624–26, cellular localization, and a templateindependent terminal transferase activity known as a noncanonical activity of hTERT27 (Supplementary Fig. 12a–d).
Using these genome-edited 293T cells, we conﬁrmed that
substitution of T249 to alanine ablated hTERT-dependent RdRP

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Fig. 3 CDK1 phosphorylates hTERT. a Direct telomerase assay (left panel) and IP-RdRP assay (right panel) using HeLa cells. Immune complexes weretreated with λ phosphatase to remove the phosphate groups from hTERT proteins. The relative activities of telomerase and RdRP are noted below thepanel, respectively. b In vitro CDK1 kinase assay of hTERT immune complexes treated with λ phosphatase, followed by IP-IB assay (left panel) and IP-RdRP
assay (right panel). The relative RdRP activities are noted below the panel. c Detection of endogenous hTERT proteins using HeLa cells transfected withfour different siRNAs speciﬁc for CDK1 followed by nocodazole treatment. Phosphorylation of hTERT proteins were detected by anti-249T-P pAbs (upper
panel). Suppression of CDK1 proteins was conﬁrmed by anti-CDK1 mAb (lower panel). d Direct telomerase assay (left panel) and IP-RdRP assay (rightpanel) using HeLa cells transfected with four different siRNAs speciﬁc for CDK1. e Detection of phosphorylated hTERT proteins using HeLa cells treatedwith 5 µM of CDK1 inhibitor, RO-3306. The cells were then treated with nocodazole. Phosphorylation of hTERT proteins was detected by anti-249T-P pAbs
(upper panel) and hTERT proteins isolated by immunoprecipitation were detected by anti-hTERT mAb (clone 2E4-2) (lower panel). f Direct telomerase
assay (left panel) and IP-RdRP assay (right panel) using HeLa cells treated with 5 µM of CDK1 inhibitor, RO-3306. The relative RdRP activities are noted
below the panel. Experiments were repeated three times (for a (right panel), c, e, f (right panel)) and twice (for a (left panel), b, d, f (left panel)) withsimilar results. Source data are provided in the Source Data ﬁle.

activity (Fig. 5d, upper panel). Similar to ectopic expression of themutant proteins (Fig. 4c), hTERT with T249E mutation increased
the RdRP activity while hTERT with T249A mutation decreased
the activity. These results conﬁrmed that phosphorylation of
T249 is essential for RdRP activity of hTERT.
To evaluate the direct inﬂuence of hTERT phosphorylation at
T249 using xenograft models in vivo, we inoculated ControlCRISPR or T249A-CRISPR subcutaneously into NOD/SCID
mice. Four of eight mice harboring T249A-CRISPR failed to form
tumors (Fig. 5e, upper panel) and the volume of tumors frommice with T249A-CRISPR was signiﬁcantly smaller than thosefrom mice with Control-CRISPR (Fig. 5e, lower panel; p =0.0469). We conﬁrmed that RdRP-deﬁcient hTERT proteinsisolated from T249A-CRISPR retained telomerase activity andbound to hTERC RNA comparable to the hTERT from ControlCRISPR (Fig. 5f). Taken together, these observations favor that
phosphothreonine 249 in hTERT is important for cancer cellproliferation through activation of RdRP activity of hTERT.
Effects of hTERT phosphorylation on gene expression. In prior
work, we showed that hTERT-mediated RdRP activity affects
gene expression by affecting RNA levels14. Global, low-leveltranscription are retained during mitosis28. We hypothesized thatalteration of hTERT RdRP activity would change transcription
regulatory network to trigger proliferation of cancer cells. To
assess the impact on transcriptome by hTERT phosphorylation at
T249, we examined gene expression at promoter level using Cap
Analysis of Gene Expression (CAGE) method29 for T249ACRISPR phosphorylation deﬁcient cells, by contrasting toControl-CRISPR cells in triplicate. We found 3,482 CAGE peaks,
indicating each transcriptional start site (TSS) and the expression
level as a promoter activity, that exhibited altered the activity inthese cells (FDR < 1e-4), where 1,926 and 1,556 were up- anddown-regulated in T249A-CRISPR. In particular, we found thatthree independent CAGE peaks of the transcription factor,
forkhead box O4 (FOXO4), were upregulated in T249A-CRISPR
while the most upstream TSS, p1, showed no change (Fig. 6a). We
conﬁrmed the increase in expression of FOXO4 proteins in
T249A-CRISPR by immunoblotting (Fig. 6b). We further monitored FOXO4 mRNA level by manipulating cells in mitosis andconﬁrmed that FOXO4 expression level is lower in mitosis than inother cell cycle phases in Control-CRISPR cells, while we failed toobserve the differences in T249A-CRISPR, which defects in RdRP
activity. This ﬁnding favors that CDK1 activity and upregulatedhTERT-RdRP activity may downregulates FOXO4 expression inmitosis (Supplementary Fig. 13). To further conﬁrm that thephosphorylation of hTERT T249 by CDK1 speciﬁcally affects theexpression of FOXO4, we treated HeLa cells with CDK1 inhibitor,
RO-3306. Inhibition of CDK1 activity increased the expression of
FOXO4 mRNA in a dose-dependent manner (Supplementary
Fig. 14).
8

Gene ontology (GO) term analysis showed enrichment of genes
associated with “cell cycle phase transition” (GO:0044770,
Supplementary Table 5) within the upregulated genes in the
T249A-CRISPR. Notably, FOXO4 is annotated in this GO term
and functions as tumor suppressor proteins in various cancers by
preventing proper cell cycle regulation30–35. These ﬁndings
indicate that phosphorylation of T249 on hTERT regulates theexpression of FOXO4 negatively in cancer cell lines and increaseof FOXO4 expression in T249A-CRISPR is correlated with adecrease of tumor formation.
To determine whether FOXO4 is a direct target of hTERT
phosphorylated at T249, we performed IP-RT-PCR experimentswith RNAs isolated from hTERT-IP samples of Control-CRISPR
and T249A-CRISPR. We isolated hTERT-RNA complexes byimmunoprecipitation with an anti-hTERT mAb (clone 10E9-2)
from Control-CRISPR and T249A-CRISPR cells, and puriﬁed
RNAs from the immune complexes with DNase treatment.
cDNAs were synthesized with strand-speciﬁc primers, and we
performed PCR using primers to amplify the ﬁrst exon of FOXO4
mRNAs. We conﬁrmed that endogenous hTERT immunoprecipitated from Control-CRISPR interacts with the ﬁrst exon of
FOXO4 mRNAs and that there was a 50% reduction of hTERT
associated with FOXO4 mRNAs isolated from T249A-CRISPR
cells (Fig. 6c). We also found that hTERT from Control-CRISPR
interacts with antisense RNAs (asRNAs) of FOXO4 mRNA undercondition in which we failed to recover RNase P RNA13. To ruleout the non-speciﬁc association between hTERT and FOXO4
RNAs, we performed UV crosslinking followed by conventional
immunoprecipitation with anti-hTERT antibody. Moreover, we
washed the immune complex under stringent conditions such as
high-salt condition. We conﬁrmed that the interaction inControl-CRISPR is maintained even in such a stringent condition(Supplementary Fig. 15).
To further assess the function of asRNAs in regulation of
FOXO4 mRNA, we synthesized asRNA complementary to theﬁrst exon of FOXO4 mRNA and transfected it in T249A-CRISPR
and Control-CRISPR. We conﬁrmed that introduction of FOXO4
asRNA downregulates FOXO4 mRNA in T249A-CRISPR(Fig. 6d).
To examine whether CDK1 directly regulates FOXO4 protein,
we treated T249A-CRISPR phosphorylation deﬁcient cells with
CDK1 inhibitor (RO-3306). We found that there are nodifferences of FOXO4 protein expression level and the FOXO4
protein migrates at the similar molecular weight (Supplementary
Fig. 16). These data suggest that CDK1 might not involve in
FOXO4 protein regulation by a direct phosphorylation of FOXO4
protein.
Taken together, we propose a model for regulation of FOXO4
expression via phosphorylation of hTERT at T249 (Supplementary Fig. 17). Speciﬁcally, in Control-CRISPR, hTERT proteins
phosphorylated at T249 bind to the ﬁrst exon of FOXO4 mRNAs.

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(nt)

c

(kb)

160

T249E

T249A

Control

Wild type

RdRP assay

T249E

T249A

Wild type

Control

Wild type

T249E

Control

(nt)

(nt)

80

40

70

120
100
80
60

30

60

10

50

5
4
3

20
40

40
2

IC

1

2.15 0.86 2.84

T249E

Wild type

T249E

T249A

Controlb Direct telomerase assay

Telomere length

TRAP assay

T249A

a

(kDa)

30

Relative
RdRP
activity1

Control

MT
T249E

(kDa)

Control

PEO14
T249E

T249A

PEO1
Control

20
Relative
1 1.27 1.21 1.23 telomerase
activity

TRF
averagelength (kb)

T249A

d

7.3

(kDa)

T249A

6.2 7.6 7.2

IB: hTERT(2E4-2)
102

102

102

38

38

38

IB: β-actin

PEO1
10
9
8
7
6
5

PEO14

MT (patient-derived HCC)
1.6

6

1.4

4
3
2
1
0

5

*

Cell number (×10 )5
Cell number (×105)

Cell number (×105)

e

4
3

*

2
1

1.2
1
0.8

*

0.6
0.4
0.2
0

0
1 2 3 4 5 6 7 8 9 10 11

1 2 3 4 5 6 7 8 9 10 11

1 2 3 4 5 6 7 8 9 10 11

Days

Days

Days

f
MT-Control

MT +T249A

Tumor volume (mm3)

300

200

*

100

0
0

These RNAs serve as templates for dsRNA synthesis by hTERTRdRP. These dsRNA complexes are degraded and the expressionof FOXO4 is reduced. In T249A-CRISPR, RdRP-deﬁcienthTERT-T249A proteins interact with less FOXO4 mRNAs thanhTERT proteins in Control-CRISPR. Without producing asRNA,
more FOXO4 mRNAs are retained in the T249A-CRISPR and

10

20
Days

30

increase of FOXO4 expression causes tumor suppression via
cancer cell growth arrest.
To corroborate the effects of FOXO4 expression in cancer cells,
we established stable cell lines introduced with pBABE-hygro(pBh) control vector or FOXO4-expressing retroviral vector (pBhFOXO4) in HeLa cells and 293T cells. Increase of FOXO4 protein

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Fig. 4 Consequences of hTERT phosphorylation on T249. a TRAP assay using BJ cells stably introduced with control retrovirus, hTERT mutants or wildtype hTERT (left panel). hTERT mutants were created by substitution of the threonine 249 with alanine (T249A) or glutamic acid (T249E). Telomere
length in the BJ cells was determined by Southern blot analysis for telomere restriction fragments (right panel). White bars indicate the position of mean
radioactivity and the TRF average length were noted below the panel. Telomere structure in BJ cells was analyzed at 8 PD after infection. b Direct
telomerase assay using hTERT proteins immunoprecipitated from 293T cells transfected with control vector, wild-type hTERT, T249A, or T249E mutants.
The relative telomerase activities are noted below the panel. c IP-RdRP assay using the cells prepared with identical manipulations as used in the b. The
relative RdRP activities are noted below the panel. d Immunoblotting to conﬁrm ectopically overexpressed hTERT mutant proteins in PEO1, PEO14 andMT cells infected with hTERT-T249A, hTERT-T249E, or control retroviruses. hTERT proteins were detected by anti-hTERT mAb (clone 2E4-2) (upper
panel). β-actin was used as an internal control (lower panel). e Cell proliferation assay using PEO1, PEO14 and MT cells infected with hTERT-T249A (redline), hTERT-T249E (blue line) or control retrovirus (black line). This assay was done in triplicate and data are shown as the mean ± SD. Asterisk:
statistically signiﬁcant values (p < 0.05 by Student’s t-test, two-sided). f MT cells introduced a control retroviral vector (MT-Control) or hTERT-T249A(MT+T249A) were implanted into eight NOD/SCID mice for each and tumors generated from the cells are presented (left panel). The volume of tumors
from mice harboring MT-Control (black line) or MT+T249A (red line) is shown as the mean ± SEM (n = 8, p = 0.0028 by unpaired t-test, two-sided)(right panel). Experiments were repeated three times (for c, d) and twice (for a, b) with similar results. Source data are provided in the Source Data ﬁle.

expression introduced with pBh-FOXO4 (HeLa+pBh-FOXO4)
was conﬁrmed by immunoblotting whereas expression level of
FOXO4 introduced with pBh (HeLa+pBh) was below thedetection limit of anti-FOXO4 antibody (Fig. 6e, left panel).
Upregulation of FOXO4 signiﬁcantly inhibited the cell proliferation in vitro by 77.0% (Fig. 6e, right panel). To evaluate the effects
of FOXO4 overexpression in HeLa using xenograft models, we
inoculated HeLa+pBh or HeLa+pBh-FOXO4 subcutaneously into
NOD/SCID mice. Retroviral expression of FOXO4 resulted in
reduction of tumor formation in ﬁve of eight mice (Fig. 6f, upper
panel). The volume of tumors from mice harboring HeLa+pBhFOXO4 was signiﬁcantly smaller than those from mice with HeLa
+pBh (Fig. 6f, lower panel, p = 0.0092). Ectopic expression of
FOXO4 in Control-CRISPR 293T cells also showed the same trend
to delay cell proliferation and reduce tumor formation (Supplementary Fig. 18a, b). Taken together, these observations conﬁrmedthat increase of FOXO4 expression in T249A-CRISPR brings about
a decrease of tumor formation in vivo.
To further assess the biological inﬂuences of increase of FOXO4
expression in T249A-CRISPR, we established stable cell lines
expressing short-hairpin RNAs (shRNAs) speciﬁc for FOXO4(shFOXO4-1, 2) or shGFP control in T249A-CRISPR. Knockdown of FOXO4 protein expression in the cell lines was conﬁrmed
by immunoblotting (Fig. 6g, upper panel). Down-regulation of
FOXO4 in T249A-CRISPR signiﬁcantly increased the cellproliferation by 1.31- and 1.26-fold, respectively (Fig. 6g, lower
panel). To evaluate the effects of FOXO4 suppression in T249ACRISPR on tumor formation, we used xenograft models by the
subcutaneous inoculation of T249A-CRISPR expressing shFOXO4(T249A-CRISPR+shFOXO4-1, 2) or shGFP (T249A-CRISPR+shGFP) into NOD/SCID mice. While ﬁve of seven mice with
T249A-CRISPR+shGFP formed xenograft tumors, T249ACRISPR+shFOXO4-1 and -2 developed tumors in all seven mice
(Fig. 6h, upper panel). Compared to T249A-CRISPR+shGFP andControl-CRISPR, T249A-CRISPR+shFOXO4 exhibited a higher
tumor growth rate and the volume of tumors from mice with
T249A-CRISPR+shFOXO4-1 and -2 was signiﬁcantly larger thanthose from mice with T249A-CRISPR + shGFP (Fig. 6h, lower
panel; p = 0.0008 and p = 0.0136, respectively). In ControlCRISPR cells, stable knockdown of FOXO4 by expressing shorthairpin RNAs (shRNAs) speciﬁc for FOXO4 (shFOXO4-1, 2) did
not show any statistically signiﬁcant differences on cell proliferation and tumor formation (Supplementary Fig. 19a, b). Theseobservations veriﬁed that perturbation of FOXO4 expression by
speciﬁc shRNAs rescued the defects in tumor formation ability in
T249A-CRISPR.
We previously reported that hTERT proteins interact with the
RNA component of mitochondrial RNA processing endoribonuclease (RMRP) and produce dsRNA by RdRP activity13. RMRP
10

RNA is a non-coding RNA and mainly transcribed by RNA
polymerase III36. We conﬁrmed that the interaction between
hTERT and RMRP was increased in mitotic cells in 293T and
HeLa cells (Supplementary Fig. 20a). We also found that hTERT
complexes from T249E-CRISPR bind to both sense and antisense
RMRP RNAs while hTERT complexes from T249A-CRISPR failto interact with the RNAs (Supplementary Fig. 20b). While wefailed to observe the phosphorylation of hTERT causes any effects
on the protein stability, protein-protein interactions, cellular
localization and the known enzymatic activities (Supplementary
Fig. 12a–d and Fig. 5f), we conﬁrmed that the regulation of target
RNAs expression occurs due to the alteration of the binding
afﬁnity between hTERT and endogenous template RNAs, such as
FOXO4 and RMRP, and the production of antisense RNAs. Theseﬁndings indicate that phosphorylation affects the binding afﬁnity
between hTERT and template RNAs for RdRP activity.
Discussion
Telomerase plays an essential role in maintaining telomeres. The
expression of the catalytic subunit of telomerase hTERT is
upregulated in the majority of human cancers, most often due tomutations in its promoter. Here, we demonstrate that hTERT is
phosphorylated by CDK1 in mitosis and that this phosphorylation event affects a second hTERT function, RNA-dependent
RNA polymerase activity without affecting hTERT function at
telomeres. This mitotic function of hTERT correlates with tumor
aggressiveness and is necessary for tumor formation. Altogether
these observations separate the telomere and non-telomere
directed functions of hTERT and identify a mechanism that
regulates hTERT RdRP activity.
In this study, we focused on the characterization of T249
phosphorylation of hTERT in pancreatic cancer and HCC, sincethese cancers are most deadly cancers with poor survival outcome37. In addition, both cancers probably occur in correlation
with functional role of hTERT protein38,39.
It is well accepted that telomerase recruitment to telomeres
occurs in S-phase in many organisms40,41. While these observations indicate that telomerase recruitment to telomeres and telomerase activation are regulated in a cell cycle-dependent
manner, there have been two reports that studied telomerase
activity outside the S-phase42,43. Speciﬁcally, these reports showedthat Xenopus telomerase activity was active in mitotic phase andmodulated chromatin conﬁguration. In consonance with theseﬁndings, we previously reported that hTERT localizes to both
mitotic spindles and centromeres, that hTERT protein expression
level is enriched in mitosis, and that hTERT-RdRP activity ispresent in nuclear extracts derived from several cancer cell linesarrested in mitosis12,14,19. Here, we have conﬁrmed and extended

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aWild type
E

R

T249A

P

T

V

E

G

R

PAM

BsaHI

T249E
P

A

V

G

E

R

E

PAM

ApaI

b

P

V

G
PAM

AgeI

c
Control-CRISPR(bp)

T249E-CRISPR

T249E-CRISPR

Plasmid control

Control-CRISPR

T249E-CRISPR
Plasmid control

Plasmid control

Control-CRISPRNo enzyme BsaHI cut AgeI cut

T249A-CRISPR

Control-CRISPR

Plasmid control

T249A-CRISPR

Control-CRISPR

T249A-CRISPR
Plasmid control

Control-CRISPR

Plasmid control

No enzyme BsaHI cut ApaI cut

Control-CRISPR

T249A-CRISPR(bp)
1000 800 600 500 400 -1000 700 500 400 300 -

300 -

200 -

T249E-CRISPR

200 100 100 -efControl-CRISPR

Directtelomerase
assay

(nt)

T249A-CRISPR

ControlCRISPR
T249ACRISPR

T249E-CRISPR

T249A-CRISPR

Control-CRISPR

d

40

3000

(nt)

30

RdRP assay

20

1
IP: hTERT (10E9-2)
IB: hTERT(2E4-2)0.21 2.43

Relative
RdRP
activity(kDa)

Tumor volume (mm3)

80
70
60
2000

50
40

1000

*

30

0
1

3

5

7

9 11 13 15 17 19 21
Days

- 102
1

0.99

IP: hTERT(10E9-2 )

(bp)
200
100

hTERC
1

these ﬁndings by identifying the mitotic kinase CDK1 as a key
regulator of hTERT RdRP but not telomerase activity.
Several laboratories have reported that hTERT is phosphorylated44–47. Speciﬁcally, four putative phosphorylation sites havebeen reported, serine 22746, serine 45747, tyrosine 70745, andserine 82444. Serine 227 is located in a canonical nuclear localization signal, might be phosphorylated by Akt kinase, and thephosphorylation of S227 may promote nuclear localization of20
Relative
telomerase
activity

1.07

Relative
expression

hTERT indicated by the experiments using phosphorylation
mutant proteins46. Serine 824 also might be phosphorylated andthe phosphorylation of S824 is suggested to enhance hTERTmediated telomerase activity via Akt activation44. This previous
report did not provide any direct evidence that hTERT S824
phosphorylation itself is really essential and prerequisite for the
maintenance of basal telomerase activity. We speculated that
although hTERT S824 phosphorylation may enhance the

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Fig. 5 Threonine 249 in the endogenous hTERT locus is edited by CRISPR-Cas9 in 293T cells. a Genome editing strategy to introduce alanine or glutamic
acid substitution in the endogenous hTERT locus. Green letters denote the PAM (protospacer adjacent motif) sequence for genome editing by CRISPRCas9. b PCR fragments surrounding target regions (1,184 bp for T249A and 456 bp for T249E) were ampliﬁed from genomic DNA of genome-edited 293T
clones. PCR fragments from pBABE-puro-hTERT (plasmid control), 293T control cells (Control-CRISPR) and mutant cells (T249A-CRISPR, T249E-CRISPR)
were digested with the indicated restriction enzymes and electrophoresed in 5% polyacrylamide gels. The bands enclosed with square and indicated byarrows show that the cleavage patterns of PCR fragment were changed in CRISPR-mutant clones. c Sanger sequencing traces of a control and two mutant
clones generated from gel-puriﬁed PCR fragments of the genomic DNAs of the respective clones. The sequences boxed in red and blue are the base triplet
coding for alanine and glutamic acid in mutant clones, respectively. d IP-RdRP assay using hTERT proteins immunoprecipitated from 293T CRISPR clones(upper panel). The relative RdRP activities are noted below the panel. hTERT proteins isolated by immunoprecipitation from the cells were detected by antihTERT mAb (clone 2E4-2) (lower panel). e Tumors generated from 1 × 105 cells of 293T Control-CRISPR or 293T-T249A-CRISPR in eight NOD/SCID micefor each are presented (upper panel). The volume of tumors from mice with Control-CRISPR (black line) or T249A-CRISPR (red line) is shown as the mean
± SEM (n = 8, p = 0.0469 by unpaired t-test, two-sided) (lower panel). f Direct telomerase assay using hTERT proteins immunoprecipitated from ControlCRISPR or T249A-CRISPR. The relative telomerase activities are noted below the panel (upper panel). Immune complexes were isolated from ControlCRISPR and T249A-CRISPR with anti-hTERT mAb (clone 10E9-2) and associated hTERC RNA were subjected to RT-PCR (lower panel). Experiments wererepeated ﬁve times (for d, f) with similar results. Source data are provided in the Source Data ﬁle.

telomerase activity, phosphatase treatment to remove phosphategroup(s) in hTERT did not affect the minimal basal activity oftelomerase. Serine 457 is phosphorylated by DYRK2 during G2/
M and promotes ubiquitination of hTERT proteins47. Tyrosine
707 might be phosphorylated by C-Src under oxidative stress andthe phosphorylation of Y707 is suggested to alter subcellular
localization of hTERT, shown by phosphorylation mutant protein
replacing tyrosine 707 with nonphosphorylatable phenylalanine45. We were unable to detect these previously reported
phosphorylation sites by MS analysis in this study with mitotic
cells. These putative phosphorylation sites were predicted fromconsensus sequences for speciﬁc kinases, and MS analysis was notconducted in these previous reports (Supplementary Table 6). We
speculate that these sites may be difﬁcult to detect using MS
analysis since the phosphorylation occurs only under speciﬁc
conditions such as Akt overexpression or under oxidative stress.
In addition, previous reports demonstrated that phosphorylation
affect either telomerase activity or subcellular localization ofhTERT using phosphorylation mutant proteins whereas ourstudy identiﬁes another phosphorylated site that does not affecttelomerase activity (Figs. 4b and 5f).
We have identiﬁed CDK1 as a kinase that phosphorylates hTERT
at T249 in mitosis. CDK1 is essential for cell division in the embryoand deﬁciency in CDK1 in mouse model results in embryoniclethality in the ﬁrst cell divisions48. Moreover, CDK1 is sufﬁcient todrive the cell cycle in all cell types48. Previous reports demonstrated
that CDK1 expression is upregulated in many human malignant
tumor tissues and that CDK1 activity correlates with the prognosisof patients with tumors49–57. In addition, loss of CDK1 in the liverin CDK1 conditional knockout mice showed complete resistanceagainst tumorigenesis58, indicating that CDK1 is required fortumorigenesis in liver cancer. Here, we report a link between CDK1
and phosphorylation of hTERT at T249, and the phosphorylation
occurs more frequently in aggressive and advanced cancers inclinical samples, suggesting an additional role for CDK1 in cancer
progression. These observations implicate CDK1 and phosphorylation of hTERT T249 as an approach to inhibit a key cancerassociated function of hTERT.
CDK1 is essential for progression into mitotic phase and isoften overexpressed in human cancers59. Moreover, a previous
report indicated that CDK1 overexpression correlated with poor
clinicopathological features and survival in HCC56, consistent
with our current study. Therefore, CDK1 is considered as apivotal molecular target. Indeed, following the success of a panCDK inhibitor, palbociclib, in advanced breast cancer, two phase
II clinical trials (milciclib and palbociclib) are currently ongoingto evaluate their efﬁciency on advanced HCC. Unfortunately,
milciclib targets CDK2/4/5/7 and palbociclib targets CDK4/6, and12

no pan-CDK inhibitors that also targets CDK1 is currently
evaluated in clinical trials60. Since our data demonstrated thealternative role of CDK1 to phosphorylate hTERT T249 fortumorigenesis, selective CDK1 inhibitors may have the profound
impact on the treatment of aggressive cancers. Pan-CDK inhibitor(s) that also targets CDK1 would be a better candidate fortreating these cancers.
Threonine 249 in hTERT is conserved in human and primates
but not in the other mammals and yeast (Fig. 1e), suggesting that
phosphorylation-dependent RdRP activity of TERT is restricted
to primates. When we introduced T249A substitution into
endogenous hTERT, we found that the mutant did not affecttelomerase activity at telomeres but instead affected RdRP activity
and inhibited the tumorigenic potential of human cancer celllines. These observations suggest that phosphorylation of hTERT
at T249 contributes to tumor formation independent of its reverse
transcriptase activity to elongate telomeres. Furthermore, we
found that phosphorylation of hTERT at T249 regulates theexpression of the tumor suppressor gene, FOXO4, in cancer celllines (Supplementary Fig. 17).
Expression levels of FOXO4 inversely correlate with tumor formation and incidence of clinical metastasis30–35. Consistent with
FOXO4 providing a role of tumor suppressor, T249A-CRISPR
expressing higher levels of FOXO4 formed less and smaller tumors inmouse xenograft model (Figs. 5e and 6h). Our ﬁndings indicate that
phosphorylation of hTERT T249 regulates FOXO4 expression
negatively by the RdRP activity in cancer cells and is implicated inpivotal event for carcinogenesis. We note that FOXO4 expression isvery low and difﬁcult to detect in endogenous protein level. In thecurrent study, while we monitored FOXO4 expression level as mainly
gauged by mRNA, the mRNA level does not necessarily reﬂect the
FOXO4 protein expression level nor FOXO4 functional aspect. In
case of FOXO1 known as a putative tumor suppressor, phosphorylation of FOXO1 by CDK1 inhibits the transcriptional activity of
FOXO1 and promotes cell proliferation61 while CDK1 phosphorylation of FOXO1 stimulates FOXO1-dependent transcription andleads to cell death in neurons62. Along the same line, FOXOs, such as
FOXO1 and FOXO3, both support and suppress metastatic breast
cancer progression63. While our data indicate that expression of
FOXO4 is regulated at least in part via RdRP activity of hTERT
phosphorylated by CDK1, regulation of FOXO4 might be complicated and yet to be elucidated. Further studies are required for a
detailed understanding of gene expression regulation by hTERT in
mitosis.
Methods
Antibodies. Anti-hTERT mouse monoclonal antibodies (mAbs, clones 10E9-2
and 2E4-2) were generated and described the speciﬁcity as reported previously14.

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p4@FOXO4
p5@FOXO4

5

p1

p2@FOXO4
p1@FOXO4

0

T249A-CRISPRbControl-CRISPR

Log2 fold change([log2 CPM in T249A] - [log2 CPM in Control])

a

76

p5

p2

(kDa)

p4

IB: FOXO4

–5

–10
0

5

IB: β-actin

10

Average expression (log2 CPM)

38

d

Input

ControlCRISPR
T249ACRISPR
ControlCRISPR
T249ACRISPR
ControlCRISPR
T249ACRISPR
ControlCRISPR
T249ACRISPR

Control- T249A- Control- T249ACRISPR CRISPR CRISPR CRISPR

Relative expression

1 2.67

0.52

1

400
300
200

1

–

1

4.30 4.51 1.24

10.98 0.95 1.01––

RNaseP RNA

1 0.07

RNaseP RNA
Relative expression

Relative expression

800
600

1 0.67–

FOXO4 mRNA

FOXO4 asRNA
Relative expression

(bp)
500
400
300

FOXO4 mRNA

–RT

+RT

hTERT-IP IgG-IP

LucasRNA
FOXO4asRNA

–RT

+RT

LucasRNA
FOXO4asRNAc

Relative expression

f

0.91

HeLa +pBh

9
8

76

*

7

1500

6

Tumor volume (mm3)(kDa)

IB: FOXO4

5
4
3
2

38

IB: GAPDHHeLa+pBh-FOXO4

10

Cell number (×105)

HeLa +pBh
HeLa+pBh-FOXO4

e

1
0
1

2

3

4

5

6

7

1000

*

500

8
0

Days

1 13 15 17 19 21 23 25 27 29 31 33 35

T249A-CRISPR+shFOXO4-2

T249A-CRISPR+shGFP

T249A-CRISPR+shFOXO4-1

Days

g

h
Control-CRISPR(kDa)
T249A-CRISPR+shGFP- 76
IB: FOXO4

T249A-CRISPR+shFOXO4-1- 38
IB: GAPDH

T249A-CRISPR+shFOXO4-2

18
16

5000

**

12
10
8
6
4
2
0

1

2

3

4

5

6

7

Tumor volume (mm3)

Cell number (×105)

14
4000
3000

* *
*
*

2000
1000

8

Days

Phosphospeciﬁc rabbit polyclonal antibodies (pAbs) and mouse mAb against
phosphorylated threonine 249 of hTERT (anti-249T-P and TpMab-1, respectively)
were generated in this study (see below). Anti-hTERT mouse mAb (1:100, clone
2E4-2), anti-hTERT rabbit mAb (1:1000, Abcam, ab32020), anti-249T-P pAbs
(1:1000), TpMab-1 mAb (1:50), anti-phospho-histone H3 (Ser10) rabbit pAbs
(1:2000, Merck, 06-570), anti-cdc2 [CDK1] (POH1) mouse mAb (1:1000, Cell
Signaling Technology, 9116), anti-β-actin (AC-15) mouse mAb (1:20000, Sigma-

0
1

3

5

7

9 11 13 15 17 19 21 23 25
Days

Aldrich, A5441), anti-FOXO4 rabbit mAb (1:4000, Abcam, ab128908), antiGAPDH mouse mAb (1:1000, MBL Co., Ltd., M171-3), and anti-SMG6 rabbit
pAbs64 (1:1000) were used for immunoblotting (IB). Anti-hTERT mAb (clone
10E9-2: MBL Co., Ltd., M216-3) and anti-hTERT sheep pAbs (Abbexa Ltd.,
abx120550) were used for immunoprecipitation (IP). Anti-CDK1 rabbit pAbs
(1:250, Merck, HPA003387), anti-249T-P pAbs (1:250) and TpMab-1 mAb (1:250)
were used for immunohistochemistry.

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Fig. 6 Expression of FOXO4 is regulated by hTERT phosphorylated on T249. a MA plot of CAGE peaks differentially expressed between the T249ACRISPR and Control-CRISPR (left panel). x- and y-axes indicate average expression levels and fold changes with triplicates, respectively. Individual dots
represent CAGE peaks corresponding to promoters deﬁned by the FANTOM5 project77. CAGE peaks with statistical signiﬁcance are indicated by pink andlight blue for higher and lower expression in the T249A-CRISPR, respectively. FOXO4 are indicated with red on MA plot and the positions of each FOXO4
CAGE peak are visualized by IGV (right panel). b The endogenous proteins were detected by anti-FOXO4 mAb in T249A-CRISPR while not in ControlCRISPR. β-actin: an internal control. c Immune complexes were isolated with 10E9-2 and associated RNAs were subjected to RT-PCR. d Transfection of
asRNA complementary to the ﬁrst exon of FOXO4 or ﬁreﬂy luciferase mRNA. Cellular RNAs were subjected to RT-PCR. “RT” indicates reverse transcriptase
reaction. The relative expressions are noted below the panel (for c, d). e Immunoblotting of ectopically overexpressed FOXO4 (left panel) and cellproliferation (right panel); infected with pBABE-hygro (pBh) (black) and pBh-FOXO4 (red) retroviruses. The cell proliferation assay was done in triplicate
and data are shown as the mean ± SD. Asterisk: statistically signiﬁcant values (p < 0.05 by Student’s t-test, two-sided). f Tumor appearances generated
from subcutaneous injection in eight mice (upper panel). The volume curves of tumors are demonstrated as the mean ± SEM (n = 8) (lower panel).
Asterisk: statistically signiﬁcant values (p = 0.0092 by unpaired t-test, two-sided). g Immunoblotting of FOXO4 (upper panel). Cell proliferation assay(lower panel) was done in triplicate and data are shown as the mean ± SD. Asterisk: statistically signiﬁcant values (p < 0.05 by Student’s t-test, two-sided).
h Tumors were established from subcutaneous injection in seven mice (upper panel). Tumor-volume curves of xenografts are shown as the mean ± SEM(n = 7) (lower panel). Asterisk: statistically signiﬁcant values (p < 0.05 by unpaired t-test). Control-CRISPR (black), T249A-CRISPR+shGFP (red), T249ACRISPR+shFOXO4-1 (blue), and T249A-CRISPR + shFOXO4-2 (green) (for g, h). Experiments were repeated three times (for b) and twice (forc–e (left panel), g (upper panel)) with similar results. Source data are provided in the Source Data ﬁle.

Generation of phosphospeciﬁc polyclonal antibodies. To generate phosphospeciﬁc antibodies against phosphorylated threonine 249 of hTERT (anti-249T-P),
hTERT phosphopeptide 244CEPERpTPVGQG254 was used to immunize rabbits.
The antibodies were puriﬁed from the antisera by using hTERT phosphopeptideconjugated resin and then further puriﬁed by passing it through hTERT
nonphosphopeptide-conjugated resin.
Generation of hybridoma producing TpMab-1 antibody. Female 4-week-old
BALB/c mice were purchased from CLEA Japan and kept under speciﬁc pathogenfree (SPF) conditions. The Animal Care and Use Committee of Tohoku University
approved animal experiments to produce antibody in this study. BALB/c mice were
immunized by intraperitoneal (i.p.) injection of 100 μg of hTERT phosphopeptide
244CEPERpTPVGQG254 together with Imject Alum (Thermo Fisher Scientiﬁc).
After two additional immunizations of 100 μg, a booster injection of 100 μg was
given i.p. 2 days before spleen cells were harvested. The spleen cells were fused with
P3U1 cells (ATCC) using PEG1500 (Roche Diagnostics,). The hybridomas weregrown in RPMI 1640 medium (Nacalai Tesque) at 37 °C in a humidiﬁed atmosphere containing 5% CO2 and 95% air, supplemented with 10% heat-inactivatedfetal bovine serum (Thermo Fisher Scientiﬁc), hypoxanthine, aminopterin, andthymidine (HAT) selection medium supplement (Thermo Fisher Scientiﬁc), and
5% BriClone Hybridoma Cloning Medium (QED Bioscience). One hundred units/
ml penicillin, 100 μg/ml streptomycin, and 25 μg/ml amphotericin B (Nacalai
Tesque) were added to the culture medium. Plasmocin (5 μg/ml; InvivoGen) was
also used to prevent Mycoplasma contamination. The culture supernatants werescreened using enzyme-linked immunosorbent assay (ELISA) for binding tohTERT phosphopeptide and nonphosphopeptide.
Enzyme-linked immunosorbent assay (ELISA). Peptides were immobilized on
Nunc Maxisorp 96-well immunoplates (Thermo Fisher Scientiﬁc) at 1 μg/ml. After
blocking with SuperBlock T20 (PBS) Blocking Buffer (Thermo Fisher Scientiﬁc),the plates were incubated with culture supernatant with subsequent 1:2000 diluted
peroxidase-conjugated anti-mouse IgG (Dako). The enzymatic reaction was conducted with 1-Step Ultra TMB-ELISA (Thermo Fisher Scientiﬁc). The opticaldensity was measured at 655 nm using an iMark microplate reader (Bio-Rad
Laboratories). These reactions were performed with a volume of 50–100 μl at 37 °C.
Cell culture and mitotic cell synchronization. The human cervical carcinoma cellline HeLa, the SV40-transformed human embryonic kidney cell line HEK-293T(293T) and human patient-derived hepatocellular carcinoma cell line MT were
cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum(IFS). The human ovarian carcinoma cell lines PEO1 and PEO14 were cultured in
RPMI-1640 medium supplemented with 10% IFS and 2 mM sodium pyruvate(Gibco). The human foreskin ﬁbroblast cell line BJ was cultured in K/O DMEM/
Medium 199 (4:1) supplemented with 15% IFS and L-Gultamine (Nacalai Tesque).
Mitotic cell synchronization was performed by the following method. Cells were
switched to medium containing 2.5 mM thymidine (Nacalai Tesque) and incubatedfor 24 h. Six hours after release, cells were incubated in medium containing 0.1 µg/
ml nocodazole (Sigma-Aldrich) for 16 h. After shake-off, mitotic cells wereretrieved14. Cells arrested in mitosis with nocodazole were conﬁrmed by IB using
anti-phospho-histone H3 (Ser10) antibodies.
Transfection of siRNAs. For suppression of hTERT or CDK1 expression, HeLa
cells were transfected with siRNAs using Lipofectamine 2000 (Thermo Fisher
Scientiﬁc). After 48 h of incubation, cells were treated with 0.1 µg/ml nocodazole
14

for 16 h. The sequences of siRNAs against hTERT16 are listed in Supplementary
Table 7. MISSION siRNAs Hs_CDC2_4049_s and Hs_CDC2_4049_as,
Hs_CDC2_4053_s and Hs_CDC2_4053_as, Hs_CDC2_6210_s and
Hs_CDC2_6210_as, and Hs_CDC2_4050_s and Hs_CDC2_4050_as were used for
CDK1 siRNA #1, CDK1 siRNA #2, CDK1 siRNA #3 and CDK1 siRNA #4,
respectively. MISSION siRNA Universal Negative Control #1 (Sigma-Aldrich) wasused as a negative control.
Statistics and reproducibility. Error bars in graphs represent SD or SEM as
indicated in the ﬁgure legends. The “n” numbers in the legends indicate biologicallyindependent samples (cells or animals) and statistics such as error bars are derived
from only the case of n ≥ 3. Statistically signiﬁcant differences between conditions
were determined using two-sided unpaired t-tests or as speciﬁed in ﬁgure legends.
Statistical signiﬁcance is represented as p-values.
IP-IB of hTERT. In all, 1 × 107 cells were lysed in 1 ml of Lysis buffer A (0.5% NP40, 20 mM Tris-HCl (pH 7.4), and 150 mM NaCl). After sonication, lysates werecleared of insoluble material by centrifugation at 21,000 × g at 4 °C for 15 min. One
milliliter of lysate was pre-absorbed with 40 μl of Pierce Protein A Plus Agarose(Thermo Fisher Scientiﬁc) for 30 min at 4 °C. Pre-absorbed lysate was mixed with
10 μg of anti-hTERT mAb (clone 10E9-2)14 or 30 μg of anti-hTERT pAbs
(abx120550) and 40 μl of Pierce Protein A Plus Agarose, and incubated overnightat 4 °C. Immune complexes were washed three times with Lysis buffer A and eluted
in 2× SDS loading buffer (2% β-mercaptoethanol, 20% glycerol, 4% SDS, and 100
mM Tris-HCl (pH 6.8)), and then subjected to SDS-PAGE in 8% polyacrylamidegels. Anti-hTERT mouse mAb (clone 2E4-2) and Mouse TrueBlot ULTRA AntiMouse Ig HRP (Rockland) or anti-hTERT rabbit mAb (ab32020) and anti-rabbit
IgG HRP (GE Healthcare) were used for IB to detect whole-hTERT proteins. Antiphospho hTERT rabbit pAbs (anti-249T-P) and anti-rabbit IgG HRP (GEHealthcare) or anti-phospho hTERT mouse mAb (TpMab-1) and anti-mouse IgG
HRP (GE Healthcare) were used for IB to detect phosphorylated hTERT proteins.
For the λ phosphatase treatment, the beads suspension with immune complexes
was treated with 2000 U of λ protein phosphatase (Bio Academia) and 2 mM
MnCl2 in λ-PPase reaction buffer (50 mM Tris-HCl (pH 7.6), 100 mM NaCl, 2 mM
DTT, 100 µM EDTA and 0.01% Brij 35) and incubated at 30 °C for 30 min.
Identiﬁcation of phosphopeptides by MS analysis. The protein samples
immunoprecipitated with anti-hTERT mAb (clone 10E9-2) were separated by
12.5% SDS-PAGE and subjected to in-gel digestion using trypsin65. The tryptic
digests were subjected to liquid chromatography coupled with nanoelectrospraytandem mass spectrometry (Finnigan LTQ Orbitrap XL mass spectrometer;
Thermo Fisher Scientiﬁc). The Mascot software package (version 2.5.1; Matrix
Science) was used to search for the mass of each peptide ion peak against the
SWISS-PROT database (Homo sapiens, 20,205 sequences in the Swiss
prot_2015_09.fasta ﬁle) using the following parameters: 1 missed cleavage; ﬁxed
modiﬁcation: carboxymethylation (C); variable modiﬁcation: oxidation (M),phosphorylation (ST), phosphorylation (Y); search mode: MS/MS ion search withdecoy database search included; peptide mass tolerance ± 10 ppm; MS/MS masstolerance ± 0.8 Da; peptide charge: 2+ and 3 + .
In vitro kinase assay using recombinant hTERT proteins. For recombinant
protein expression using cell-free synthesis-coupled transcription–translation66,
the target cDNA fragment, corresponding to 191–306 residues of hTERT was subcloned into the pCR2.1-TOPO vector (Thermo Fisher Scientiﬁc). Proteins, subjected to afﬁnity puriﬁcation, were N-terminally fused with a modiﬁed natural

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polyhistidine N11-tag (amino-acid sequence: MKDHLIHNHHKHEHAHAEH)
with a TEV (Tobacco Etch virus) protease recognition site and a GSSGSSG linker
sequence. These sequences were introduced using TOPO cloning. The cell-freesynthesized N11-tagged hTERT_191-306 protein was puriﬁed using an AKTA 10 S
system (GE Healthcare) with a HisTrap columns (GE Healthcare); the AKTA 10 S
system was washed with a concentration gradient buffer (50 mM Tris-HCl buffer at
pH 8.0, containing 1 M NaCl and 10 mM imidazole). The N11-tagged recombinant
proteins were eluted with a concentration gradient of imidazole (from 10 to 500mM) in elution buffer (50 mM Tris-HCl buffer at pH 8.0, containing 0.5 M NaCl).
Imidazole was removed by overnight dialysis at 4 °C in wash buffer. The afﬁnitytags were removed by incubation at 4 °C for 20 h with TEV protease. The resulting
tag-cleaved proteins were puriﬁed by ion exchange chromatography with a HiTrap
SP column (GE Healthcare) and gel ﬁltration with the ﬁnal buffer (25 mM TrisHCl buffer at pH 7.0, containing 450 mM NaCl, 0.25 mM TCEP) using HiLoad 16/
600 Superdex columns (GE Healthcare).
For baculovirus–insect cell expression of active form of human IKK2 (IKK2_2664), we used the Bac-to-Bac Baculovirus Expression System (Thermo Fisher
Scientiﬁc). The target cDNA fragment, corresponding to 2-664 residues of human
IKK2, was sub-cloned into the pDEST vector (Thermo Fisher Scientiﬁc).
Polyhistidine afﬁnity-tagged IKK2_2-664 was expressed in insect Sf9 cells at a
multiplicity of infection (MOI) of 1.0 with a recombinant baculovirus that
expresses IKK2_2-664. The cells from each culture were harvested 48 h postinfection, and the cell pellets were washed once with phosphate-buffered saline(PBS) and were immediately frozen in liquid N2. The recombinant IKK2_2-664
protein was puriﬁed to use as a negative control in vitro kinase assay67.
Puriﬁed hTERT_191-306 proteins were incubated with CDK1-cyclinB (NewEngland Biolabs) or puriﬁed IKK2_2-664 proteins. In vitro kinase assays werecarried out in 30 µl of kinase reaction buffer (50 mM Tris-HCl pH7.5, 10 mM
MgCl2, 0.1 mM EDTA, 2 mM DTT, 0.01% Brij 35) containing 4 mM ATP, 10 µM
of hTERT_191-306 proteins, and 30 units of CDK1-cyclinB or 1 µM of IKK2_2664. Reactions were incubated at 37 °C for 2 h. Reaction samples were terminatedby adding 2.5x SDS sample loading buffer (5% (w/v) SDS, 250 mM DTT, 15% (v/v)
glycerol, 140 mM Tris-HCl pH 6.8, and 0.01% (w/v) bromophenol blue), boiled
and subjected to Phos-tag SDS-PAGE22. There are no speciﬁc molecular weightmarkers that is available for this assay.
For mass spectrometry analyses, gel regions, containing proteins, were excisedand digested with trypsin (Promega) for 20 h at 37 °C. The resulting peptides wereanalyzed by LC-ESI-MS/MS (liquid chromatography-electrospray ionization
tandem mass spectrometry) at the Support Unit for Bio-Material Analysis in
RIKEN CBS Research Resources Center.
In vitro kinase assay using endogenous hTERT proteins. TERT protein was
immunoprecipitated from human cell lines as described for the IP-IB assay with
anti-hTERT mAb (clone 10E9-2). The beads suspension with immune complexes
was treated with 2000 U of λ protein phosphatase (Bio Academia) and 2 mM
MnCl2 in λ-PPase reaction buffer (50 mM Tris-HCl (pH 7.6), 100 mM NaCl, 2 mM
DTT, 100 µM EDTA and 0.01% Brij 35), and incubated at 30 °C for 30 min.
Immune complexes were washed three times with Lysis buffer A and twice withkinase reaction buffer (50 mM Tris-HCl pH7.5, 10 mM MgCl2, 0.1 mM EDTA, 2
mM DTT, 0.01% Brij 35) containing 1x PhosStop phosphatase inhibitors (Sigma).
In vitro kinase assays were carried out in 20 µl of kinase reaction buffer containing
1x PhosStop, 6.5 mM ATP, and 0.2 µg of CDK1-cyclinB (Carna Biosciences).
Reactions were incubated at 37 °C for 2 h. Reaction samples were washed once withkinase reaction buffer containing 1x PhosStop, added 2x SDS sample loading
buffer, boiled and subjected to SDS-PAGE in 8% polyacrylamide gels.
IP-RdRP assay. TERT protein was immunoprecipitated from human cell lines as
described for the IP-IB assay with anti-hTERT mAb (clone 10E9-2)14. The beadsuspension with immune complexes was washed four times with 1× acetate buffer(10 mM HEPES-KOH (pH 7.8), 100 mM potassium acetate, and 4 mM MgCl2)
containing 10% glycerol, 0.1% Triton-X, and 0.06× cOmplete EDTA-free (Roche),and once with AGC solution (1× acetate buffer containing 10% glycerol and 0.02%
CHAPS) containing 2 mM CaCl2. The bead suspension was treated with 0.25 unit/
μl Micrococcal Nuclease (Takara Bio) at 25 °C for 15 min. Immunoprecipitates
were subsequently washed twice with AGC solution containing 3 mM EGTA andonce with 1× acetate buffer containing 0.02% CHAPS. Forty microliter of reaction
mixture was prepared by combining 20 μl of the bead suspension with 6 μl of [α32P] UTP (3,000 Ci/mmol) and 25 ng/μl (ﬁnal concentration) of RNA template,and incubated at 32 °C for 2 h. The sequence of RNA template is as follows: 5ʹ-GG
GAUCAUGUGGGUCCUAUUACAUUUUAAACCCA-3ʹ68. This RNA has
hydroxyl groups at both the 5ʹ and 3ʹ ends. The ﬁnal concentrations of ribonucleotides were 1 mM ATP, 0.2 mM GTP, 10.5 μM UTP, and 0.2 mM CTP. The
resulting products were treated with Proteinase K to stop the reaction, puriﬁed
several times with phenol/chloroform until the white interface disappeared, andprecipitated using ethanol. The RdRP products were treated with RNase I (2 U,
Promega) at 37 °C for 2 h to digest single-stranded RNAs completely, followed byProteinase K treatment, phenol/chloroform puriﬁcation, and ethanol precipitation.
The products were electrophoresed in a 10% polyacrylamide gel containing 7 M
urea, and detected by autoradiography. The signal intensities were quantiﬁed using
Fiji/ImageJ software.

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Generation of phosphorylation mutant plasmids. For site-directed mutagenesis,
QuikChange II XL site-directed mutagenesis Kit (Agilent Technologies) was used.
In brief, site-directed mutagenesis PCRs were performed using PfuUltra HighFidelity DNA polymerase following the manufacturer’s protocol with either thepBABE-puro-hTERT retroviral vector or the pNK-FLAG-Z-hTERT expression
vector as template plasmids. Sequences of mutagenic primers are listed in Supplementary Table 7. PCR products were digested with DpnI restriction enzyme for
1 h at 37 °C and then transformed into XL-10-Gold ultracompetent cells. Mutations were conﬁrmed by Sanger sequencing.
Telomere analysis. For the telomeric repeat ampliﬁcation protocol (TRAP) assay,
1 × 105 cells were suspended in 200 μl of TRAP lysis buffer (10 mM Tris-HCl (pH7.5), 1 mM MgCl2, 1 mM EGTA, 0.5% CHAPS, 10% glycerol, 100 μM Pefabloc SC,
and 0.035% 2-mercaptoethanol). The TRAP assay was performed with 5 μl of thesuspension by a conventional method16.
To measure telomere length by Southern blotting, genomic DNAs were isolated,digested with HinfI and AfaI, electrophoresed in a 0.8% agarose gel and hybridized
with a 32P-labeled telomeric (CCCTAA)369.
Direct telomerase assay. For the direct telomerase assay, we modiﬁed the original
methods17,18. Brieﬂy, TERT protein was immunoprecipitated from human celllines as described for the IP-IB assay without sonication. Immune complexes were
washed three times with Lysis buffer A, and then suspended in 30 μl of TRAP lysis
buffer. The direct telomerase assay was carried out with 10 μl of the suspension and40 μl of reaction mixture (2.5 μl of [α-32P] dGTP (6000 Ci/mmol), 50 mM Tris-HCl(pH 8.0), 50 mM KCl, 1 mM MgCl2, 1.25 mM Spermidine, 5 mM 2-mercaptoethanol, 2.5 mM dTTP, 2.5 mM dATP, 25 μM dGTP and 2.5 μM a5 primer (5ʹTTAGGGTTAGGGTTAGCGTTA-3ʹ)) by incubation at 37 °C for 2 h. 5ʹ-32Plabeled synthetic 18-mer DNA was added as an internal recovery/loading standard17. The products were puriﬁed with phenol/chloroform, and precipitated using
ethanol. The products were electrophoresed in a 10% polyacrylamide gel containing 7M urea, and detected by autoradiography. The signal intensities were
quantiﬁed using Fiji/ImageJ software.
Generation of stable cell lines and proliferation assay. Amphotropic retroviruses were created using the retroviral vectors pBABE-puro, pBABE-purohTERT T249A or pBABE-puro-hTERT T249E for making stable cell lines
expressing hTERT mutants. After infection, polyclonal cell populations were
puriﬁed by selection with puromycin (2 µg/ml) for 3 days70.
We used the following short-hairpin RNA (shRNA) vectors, shFOXO4-1(TRCN0000010291) and shFOXO4-2 (TRCN0000039720), constructed by The
RNAi Consortium and the sequences are listed in Supplementary Table 7. shGFP
was used as the control. These vectors were used to make amphotropic lentiviruses,
and the cell populations were selected with puromycin (2 µg/ml) for 3 days.
For generating stable cell lines expressing FOXO4, we constructed the retroviral
vector pBABE-hygro (pBh)-FOXO4. After infection of pBABE-hygro or pBhFOXO4, the cells were selected with hygromycin B (200 µg/ml) for 7 days.
To generate proliferation curves, cells were plated in triplicate and counted in a
Z2 Particle Count and Size Analyzer (Beckman-Coulter).
RT-PCR and quantitative reverse transcription PCR (qRT-PCR). Total cellular
RNA was isolated using TRIzol (Thermo Fisher Scientiﬁc), treated with RQ1
DNase (Promega), and subjected to RT-PCR and qPCR. For IP-RT-PCR, RNAsamples were extracted from the immune complexes as described for the IP-IB
assay without sonication. Immune complexes were washed three times with Lysis
buffer A containing 300 mM NaCl and associated RNAs were isolated with TRIzol.
The RT reaction was performed with oligo(dT)12-18 (Thermo Fisher Scientiﬁc)primer or target- and strand-speciﬁc primers using PrimeScript Reverse Transcriptase (Takara Bio) for 60 min at 42 °C, followed immediately by PCR. qPCR
was performed with a LightCycler 480 II (Roche) using LightCycler 480 SYBRGreen I Master (Roche) according to the manufacture’s protocols. Sequences of
PCR primers are listed in Supplementary Table 7.
Genome editing using CRISPR-Cas9. The PuroCas9 plasmid, which consists ofthree cassettes for Cas9 cDNA, chimeric guide RNA, and puromycin cDNA, wasgenerated by modifying the px330 plasmid (addgene #42230)71. Cas9 and puromycin resistance genes are expressed under the control of the CAG promoter72
and the PGK promoter73, respectively. The guide RNA sequence targeted against
hTERT was cloned into the BbsI site of the PuroCas9 plasmid (PuroCas9-hTERT).
For generating T249A-CRISPR and T249E-CRISPR, the T249A donor plasmid orT249E single-stranded oligo included the point mutation, leading to amino-acidchange were transfected with PuroCas9-hTERT using FuGeneHD (Promega) into
293T cells, respectively. For generating Control-CRISPR, PuroCas9 plasmid wastransfected using FuGeneHD into 293T cells. Following selection with puromycin,
single-cell was cloned. Genomic DNA was extracted with the GenElute Mammalian genomic DNA miniprep kit (Sigma) according to the manufacturer’s
instructions. Presence of mutation in single-cell clones was conﬁrmed by PCR
ampliﬁcation, restriction enzyme digestion and Sanger sequencing. Potential offtarget (OT) mutation sites of the guide RNA were predicted74 and veriﬁed the

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sequences by Sanger sequencing following PCR ampliﬁcation using OT site-speciﬁcprimers. Sequences of PCR primers, donor plasmid/oligo and guide RNA are listed
in Supplementary Table 7.
Xenotransplantation. Six-week-old male Non-obese diabetic/severe combined
immunodeﬁciency (NOD/SCID) mice (NOD/NCrCRL-Prkdcscid) were purchasedfrom Charles River Laboratories, Inc. and used as recipients for xenotransplantation. They were maintained in SPF rooms at 20 °C, 50% humidity, and 12-h light/
12-h dark cycle condition. 1 × 105 cells were suspended in a mixture of serum-free
medium and Matrigel (BD Biosciences; 1:1 volume). The mixture was injectedsubcutaneously through a 26-gauge needle into the right dorsal areas of anesthetized NOD/SCID mice. We monitored tumor formation and tumor size every twoor three days, and dissected out the tumors within a month after engraftment.
CAGE sequencing and data analysis. Of each of the total RNAs extracted fromtwo triplicates (Control-CRISPR and T249A-CRISPR), 3 µg was used to prepare asequencing library according to the non-Ampliﬁed non-Tagging Illumina Cap
Analysis of Gene Expression (nAnT-iCAGE)29 by using CAGE library preparation
kit (DNAFORM), and sequenced by NextSeq500 platform (Illumina). After discarding sequences with ambiguous (N) and low-quality (Phred < 30), theremaining reads were aligned with the human reference genome (GRCh37) bySTAR v2.6.0a75 with a guide of known junctions of RefSeq transcripts76. The
alignments with mapping quality more than 20 were selected, and their 5ʹ-ends
were counted based on the robust set of CAGE peaks identiﬁed in a previous
study77 and provided at the FANTOM5 web resource78. The read counts werenormalized as counts per million (CPM) with relative log expression (RLE)
method79. The sequencing results were visualized using Integrative Genomics
Viewer (IGV)80. This normalization and subsequent differential analysis wereconducted with edgeR81, where FDR < 1e-4 were used as a criteria for statisticalsigniﬁcance. p-value of GO term enrichment was assessed with DAVID v6.882
using all human genes as a background set, and only the terms that have less than
100 corresponding genes were chosen to exclude very general GO terms such as“biological process”.
Clinical pancreatic tissue samples. The pancreatic tissues used in this study wereobtained from patients who underwent surgical treatment at Tokyo Metropolitan
Geriatric Hospital (n = 47; female, n = 25; male, n = 22; 62–91 years old; mean age,
74.8 ± 7.0 years old) and all patients provided written informed consent. The
present study was conducted in accordance with the principles embodied in the
Declaration of Helsinki, 2013, and all experiments were approved by the ethics
committees of Tokyo Metropolitan Geriatric Hospital and Institute of Gerontology(permit-#260219).
Tissues were ﬁxed in 10% buffered formalin and then subjected to standard
tissue processing and parafﬁn embedding. The tissues were sliced serially into
sections 3 μm thick for hematoxylin and eosin (H&E) and immunohistochemical
staining. Pathological specimens were diagnosed by our pathologists based on theWorld Health Organization Classiﬁcation of Tumors of the Digestive System83.
The most common precursor lesions of pancreatic ductal adenocarcinoma are
PanIN lesions. PanINs are microscopic papillary or ﬂat, noninvasive epithelial
neoplasms that are usually <5 mm in diameter and conﬁned to pancreatic ducts.
PanINs are divided into three grades according to the degree of cytological andarchitectural atypia. Lesions with minimal, moderate or marked atypia aredesignated PanIN-1, PanIN-2, and PanIN-3, respectively. PanIN-1A is ﬂatepithelial lesion composed of tall columnar cells with basally located nuclei andabundant supranuclear mucin. PanIN-1B has a papillary, micropapillary, or basally
pseudostratiﬁed architecture but are otherwise identical to PanIN-1A. PanIN-2(pancreatic intraepithelial neoplasia 2) may be ﬂat but are mostly papillary.
Cytologically, by deﬁnition, PanIN-2 must have some nuclear abnormalities.
PanIN-3 is usually papillary or micropapillary; however, they may rarely be ﬂat.
True cribriforming, the appearance of “budding off” of small clusters of epithelial
cells into the lumen, and luminal necrosis should all suggest the diagnosis of
PanIN-3. PanIN-3 resembles carcinoma at the cytonuclear level, but invasion
through the basement membrane is absent84.
Immunohistochemistry (IHC) of pancreatic tissue samples. Parafﬁn-embedded
sections (3 μm) were subjected to immunostaining. After deparafﬁnization, thetissue sections were preheated in HEAT PROCESSOR Solution pH 6 (Nichirei) for
20 min at 100 °C. Then, sections were incubated for 5 min at room temperature
with Protein Block Serum-Free (Dako). The tissue sections were then incubatedwith the anti-249T-P pAbs, TpMab-1 mAb (1: 250 in dilution) or anti-CDK1 pAbs
(1: 250 in dilution) for 1 h at room temperature. Endogenous peroxidase activity
was blocked by 3% H2O2 for 5 min at room temperature. Sections were incubatedwith Second antibody (REAL EnVision, Dako). Bound antibodies were detectedusing diaminobenzidine tetrahydrochloride as the substrate. The sections were thencounterstained with Mayer’s hematoxylin. Negative control tissue sections wereprepared by omitting the primary antibody. As for the evaluation of immunostaining, proportion of positively stained nucleus of cancer cells were analyzed at
magniﬁcation x200. For statistical analysis, the patient groups with 249T-P and
CDK1 positive/negative were divided at 10% cutoff value.
16

Fluorescent staining of pancreatic tissue samples. Parafﬁn-embedded sections(3 μm) were subjected to ﬂuorescent staining. Fluorescent labeling of primary
antibodies were performed using conjugation kit (abcam). After deparafﬁnization,
the tissue sections were incubated overnight at 4 °C with anti-CDK1 pAbs (1:100 in
dilution) and TpMab-1 mAb (1:100 in dilution). After incubation, the sections
were mounted with Vectashield H-1200 containing DAPI. Fluorescent images were
captured by a CCD camera (ORCA-ER-1394, Hamamatsu Photonics KK) mounted
on a microscope (80i, Nikon). Intensity of ﬂuorescence was analyzed using WinROOF2015 (Mitani Corporation).
Statistical analysis of pancreatic tissue sample. The level of signiﬁcance was set
at p < 0.05 for all analyses. Statistical analyses were performed using the StatView J
version 5.0 software package (SAS Institute) and SPSS version 22 (IBM Corp.).
Clinical HCC samples. A total of 100 HCC patients who received surgery at
Kanazawa University Hospital from 2008 to 2013 were enrolled in the study. This
study was approved by the Institutional Review Board at Kanazawa University (IRB
# 1065) and all patients provided written informed consent.
IHC of HCC samples. IHC of HCC tissues and adjacent non-cancerous liver tissues
was performed using DAKO Envision+ kits (Agilent Technologies) according to the
manufacturer’s instruction. Brieﬂy, formalin-ﬁxed parafﬁn-embedded tissue slides
were deparafﬁnized, rehydrated, and immediately proceeded for antigen retrieval(120 °C for 5 min) using autoclaves and target retrieval solution, citrate pH 6 (Agilent
Technologies). Slides were immersed with blocking solution (Agilent Technologies) for
15 min and subsequently replaced with anti-249T-P pAbs or anti-CDK1 pAbs diluted
at 1:250 with antibody diluent solution (Agilent Technologies). Slides were incubatedat 4 °C overnight and then washed and visualized with DAB + substrate chromogen(Agilent Technologies). The patient groups with 249T-P and CDK1 positive/negative
were divided at 10% cutoff value.
Statistical analysis of HCC samples. Kaplan–Meier survival analysis was performed in GraphPad Prism software 6.0 (GraphPad Software). The association ofphosphorylation of hTERT threonine 249 and clinicopathologic characteristics was
examined with either Student’s tests or χ2 tests.
Transfection of FOXO4-asRNA. An asRNA complementary to the ﬁrst exon of
FOXO4 mRNA was synthesized using T7 RNA polymerase. A part of the ﬁrst exonof FOXO4 mRNA was ampliﬁed by PCR and the PCR amplicon was used as the
template for in vitro transcription by T7 RNA polymerase (Promega). After thein vitro transcription reaction, the transcribed RNA was treated by DNase (RQ1
DNase; Promega) and puriﬁed with MicroSpin G-25 column (GE Healthcare) andphenol/chloroform. An asRNA complementary to ﬁreﬂy luciferase mRNA was
similarly synthesized using a ﬁreﬂy luciferase-expressing plasmid (pGL4.10; Promega). The sequences of the primers used are shown in Supplementary Table 7.
The synthesized asRNA were transfected in T249A-CRISPR cells using Lipofectamine 2000 (Thermo Fisher Scientiﬁc).
Reporting summary. A reporting summary for this article is available as a Supplementary Information ﬁleData availability
The CAGE sequencing data reported in this study are available in the DDBJ Sequence
Read Archive (DRA) with accession number (DRA007587). All peptide information of
TERT proteins detected by mass spectrometry analysis in this study is shown in
Supplementary Data 1 and Supplementary Table 1. The source data underlying Figs. 1a,b, c, f, g, h, i, 3a, b, c, d, e, f, 4a, b, c, d, 5b, d, f, 6b, c, d, e, g, and Supplementary Figs. 1a, b,
c, d, 2a, b, c, 8a, b, c, 12a, b, c, d, 13, 14, 15, 16, 18a, 19a 20a, 20b are provided as the
Source Data ﬁle. All the other data supporting the ﬁndings of this study are available
within the article and its supplementary information ﬁles and from the corresponding
author upon reasonable request. A reporting summary for this article is available as a
Supplementary Information ﬁle.

Received: 12 September 2019; Accepted: 3 March 2020;

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was supported in part by the Grant-in-Aid for challenging Exploratory Research underGrant Number 15K14482, AMED under Grant Number JP18fk0210005, The National
Cancer Center Research and Development Fund (30-A-4), Daiichi Sankyo Foundation ofLife Science to K.M. and the Platform Project for Supporting Drug Discovery and Life
Science Research (Basis for Supporting Innovative Drug Discovery and Life Science
Research [BINDS]) from AMED under Grant Number JP17am0101078 to Y.K.

Author contributions
M.Y., Y.A., and M.M. performed biochemical and cellular experiments. T.Y., Y.M., and
S.K. performed immunohistological and statistical analysis of clinical samples. S.S. and
M.S. designed and performed in vitro kinase assay. M.S.M., H.K., and Y.H. performed
CAGE sequencing and the bioinformatics analyses. K.S. and T.K. carried out mass
spectrometric protein proﬁling and the bioinformatics analyses. T.A. and Y.F. designed
the experiments for CRISPR-Cas9 gene editing of hTERT. S.Y., M.K.K., and Y.K. generated a phosphospeciﬁc monoclonal antibody against hTERT. M.Y., Y.A., T.Y., andK.M. designed the experiments, discussed the interpretation of the results and wrote
the manuscript. All authors read and approved the ﬁnal manuscript.

Competing interests
The authors declare no competing interests.

Additional information
Supplementary information is available for this paper at https://doi.org/10.1038/s41467020-15289-7.
Correspondence and requests for materials should be addressed to K.M.
Peer review information Nature Communications thanks Boudewijn Burgering, and theother, anonymous, reviewer(s) for their contribution to the peer review of this work. Peerreviewer reports are available.
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Acknowledgements

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We would like to thank Yasuhiro Murakawa for useful discussions and Saori Uekifor technical supports. We are grateful to the Support Unit for Bio-Material Analysis,
RIKEN CBS RRD, for technical help with the mass spectrometry analysis. This work

© The Author(s) 2020

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