University of Groningen

University of Groningen

Identifying aneuploidy-tolerating genes Simon, Judith Elisabeth

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CHAPTER 2

Chromosome instability induced by Mps1 and p53 mutation generates aggressive lymphomas exhibiting

aneuploidy-induced stress

Floris Foijer

, Stephanie Z. Xie

, Judith E. Simon

, Petra L. Bakker

, Nathalie Conte

, Stephanie Davis

, Eva Kregel

, Jos Jonkers

, Allan Bradley

and Peter K. Sorger

1

European Research Institute for the Biology of Ageing, University of Groningen, University Medical Center Groningen, 9713 AV Groningen, the Netherlands

2

Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115, USA

3

Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, CB10 1SA, UK

4

The Netherlands Cancer Institute, Division of Molecular Pathology, 1066 CX, Amsterdam, the Netherlands

†

Present address: Princess Margaret and Toronto General Hospitals, University Health Network, Toronto, M5G2C1, Canada

&

These authors contributed equally

This chapter is based on:

Proc Natl Acad Sci U S A. 2014 Sep 16;111

ABSTRACT

Aneuploidy is a hallmark of human solid cancers that arises from errors in mitosis and results in gain and loss of oncogenes and tumour suppressors. Aneuploidy poses a growth disadvantage for cells grown in vitro, suggesting that cancer cells adapt to this burden. To better understand the consequences of aneuploidy in a rapidly proliferating adult tissue we engineered a mouse in which chromosome instability (CIN) was selectively induced in T cells. A flanked-by LOX (FLOX) mutation was introduced into the Mps1 spindle assembly checkpoint (SAC) gene so that Cre-mediated recombination would create a truncated protein (Mps1

) that retained the kinase domain, but lacked the kinetochore- binding domain and thereby weakened the checkpoint. In a sensitized p53

background we observed that Mps1

mice suffered from rapid-onset acute lymphoblastic lymphoma (T-ALL). The tumours were highly aneuploid and exhibited a metabolic burden similar to that previously characterized in aneuploid yeast and cultured cells. The tumours nonetheless grew rapidly and were lethal within 3-4 months of birth.

SIGNIFICANCE

Normal cells mis-segregate chromosomes only very rarely but the majority of cancer cells have a chromosome instability (CIN) phenotype that makes errors more common and results in abnormal chromosomal content (aneuploidy). Although aneuploidy promotes transformation via gain of oncogenes and loss of tumour suppressors, it also slows cell proliferation and disrupts metabolic homeostasis. Aneuploidy therefore represents a liability as well as a source of selective advantage for cancer cells. In this paper, we provoke CIN in murine T cells by weakening the spindle assembly checkpoint and then study the consequences. We find that CIN dramatically accelerates cancer in a genetically predisposed background and that the resulting aneuploid cancers are metabolically deranged, a vulnerability that may open new avenues to treating aneuploid cancers.

A B S T RAC T

Aneuploidy is a hallmark of human solid cancers that arises from errors in mitosis and results in gain and loss of oncogenes and tumour suppressors. Aneuploidy poses a growth disadvantage for cells grown in vitro, suggesting that cancer cells adapt to this burden. To better understand the consequences of aneuploidy in a rapidly pro- liferating adult tissue we engineered a mouse in which chromosome instability (CIN) was selectively induced in T cells. A flanked-by LOX (FLOX) mutation was introduced into the Mps1 spindle assembly checkpoint (SAC) gene so that Cre-mediated recom- bination would create a truncated protein (Mps1 ^DK ) that retained the kinase domain, but lacked the kinetochore-binding domain and thereby weakened the checkpoint.

In a sensitized p53 ^+/- background we observed that Mps1 ^DK/DK mice suffered from rapid-onset acute lymphoblastic lymphoma (T-ALL). The tumours were highly aneu- ploid and exhibited a metabolic burden similar to that previously characterized in aneuploid yeast and cultured cells. The tumours nonetheless grew rapidly and were lethal within 3-4 months of birth.

SIGNIFICANCE

Normal cells mis-segregate chromosomes only very rarely but the majority of cancer

cells have a chromosome instability (CIN) phenotype that makes errors more com-

mon and results in abnormal chromosomal content (aneuploidy). Although aneu-

ploidy promotes transformation via gain of oncogenes and loss of tumour suppres-

sors, it also slows cell proliferation and disrupts metabolic homeostasis. Aneuploidy

therefore represents a liability as well as a source of selective advantage for cancer

cells. In this paper, we provoke CIN in murine T cells by weakening the spindle as-

sembly checkpoint and then study the consequences. We find that CIN dramatically

accelerates cancer in a genetically predisposed background and that the resulting

aneuploid cancers are metabolically deranged, a vulnerability that may open new

avenues to treating aneuploid cancers.

INTRODUCTION

Aneuploidy is a hallmark of oncogenesis, affecting two out of three cancers

. Aneuploidy arises during mitosis as a result of chromosome instability (CIN)

. The frequent occurrence of CIN in solid human tumours suggests a fundamental link between aneuploidy and cancer

. However, primary mouse embryonic fibroblasts (MEFs) carrying a supernumerary chromosome have decreased proliferative potential, as do cells isolated from Down syndrome patients

implying that chromosome imbalance imposes a physiological burden that lowers fitness, at least in untransformed cells

. In some mouse models, CIN appears to have a significant impact on lifespan at the organismal level, with increased aneuploidy decreasing life expectancy and vice versa

. However, it remains poorly understood how the fitness cost imposed by CIN is balanced by its potential to promote oncogenic transformation.

Mouse models of CIN involving conditional or hypomorphic mutations in SAC genes provide a means to study aneuploidy and assess its impact on cell fitness and oncogenesis.

The SAC detects the presence of maloriented or detached kinetochores during mitosis and arrests cells in metaphase until all pairs of sister chromatids achieve the bioriented geometry that is uniquely compatible with normal disjunction

. The SAC constitutes a signaling cascade (comprised of Mps1, Bub, Mad, CenpE and RZZ proteins) that blocks activation of the anaphase promoting complex (APC/C), and thus mitotic progression, until all chromosomes are properly aligned

. In the mouse, germline deletion of SAC genes results in early embryonic lethality whereas heterozygous knockout of Mad2 and other SAC genes generates relatively weak tumour phenotypes late in life

. Paradoxically, some SAC mutations appear to be both tumour predisposing and tumour suppressing depending on the context (e.g. CenpE heterozygosity)

. Hypomorphic BubR1 mutations also have the unexpected property of promoting progeria

.

Conditional mutations typically yield tumour phenotypes more representative of human disease than germline mutations

, but conditional alleles have been little studied in the case of the spindle checkpoint. We therefore engineered a conditional flanked by Lox (FLOX) mutation into Mps1, a gene thought to function upstream in the SAC pathway

and then selectivity truncated the protein by expressing Cre recombinase in T cells.

The Mps1 truncation (Deletion in Kinetochore domain; Mps1

) removes the kinase-

targeting domain but leaves the rest of the protein intact. We show that expression of this

truncated protein causes chromosome instability in MEFs and aneuploidy in either of two

different Cre-expressing mouse lines. Mps1 truncation in combination with heterozygous p53 deletion leads to early-onset lymphoblastic lymphoma and consequent death. In lymphoma cells, changes in the expression of metabolic, splicing, and DNA synthesis genes are very similar to changes previously identified in aneuploid yeast and cultured murine cells

and appear to constitute a hallmark of chromosomal imbalance.

RESULTS

To provoke CIN in a tissue-restricted fashion, we engineered a conditional Mps1

truncation allele by flanking exons 3-4 of the Mps1 locus with lox-sites; correct targeting of Mps1 in mouse ES cells was confirmed by Southern blotting and RT-PCR (Sup. Fig.1A-C; Sup.

methods). Upon expression of Cre-recombinase

the Mps1

allele generates a truncated Mps1 kinase lacking residues 47-154, a domain involved in kinetochore binding

(Fig.

1A). When expressed in MEFs as a GFP fusion, the Mps1

protein had the anticipated molecular weight, but unlike wild type Mps1-GFP, did not accumulate to the same levels on prometaphase kinetochores (Fig. 1B, Sup. Fig. 1D compare upper and lower panels, Sup. Movies 1, 2, Sup. Fig. 1E). We conclude that the Mps1

mutation impairs but does not prevent kinetochore binding, a conclusion supported by over-expression studies in human MCF10A cells (Sup. Fig. 1F, G, Sup. Movies 3, 4).

The Mps1

truncation weakens the SAC and causes CIN

To determine the consequences of Mps1 mutation for chromosome segregation at a

cellular level, we isolated Mps1

embryos, generated MEFs and transduced them with

retroviruses expressing doxycycline-inducible Cre (GFP-T2A-Cre). We found that

exposure of these cells to doxycycline (Dox) resulted in highly efficient switching,

yielding Mps1

MEFs within 24-48 hours (compare Fig. 1C lanes 1-2 to lane 4; some

recombination was also observed in the absence of Dox; lane 3). When treated with the

spindle poison nocodazole both control Mps1

and Mps1

MEFs arrested in mitosis,

showing that both cell types can respond to spindle disassembly (Fig. 1D). However,

when time-lapse imaging was used to assay the duration of arrest, Mps1

cells were

observed to exit mitosis 150 ± 16 min after DNA condensation in contrast to 264 ± 15

min in control cells (congenic Mps1

cells not exposed to Cre) a significant difference

(p<0.0001; Fig. 1E). The observation that cells expressing Mps1

are unable to sustain

mitotic arrest in the presence of spindle damage suggests that the SAC is impaired but not

inactivated by the Mps1

mutation

which was our goal in creating the allele.

Time-lapse imaging in the absence of nocodazole showed that H2B-Cherry-transduced Mps1

MEFs spent ~40% longer in mitosis than control cells (Fig. 1F, Sup. Fig. 1H). In addition, they frequently contained lagging chromosomes (Fig. 1F, G Sup. Movies 5-7;

control cells are shown in Sup. Movies 8, 9) and half of all cells failed to form a proper metaphase plate (Sup. Movie 10) resulting in polyploidy (Fig. 1G, Sup. Movie 11). While this might appear to be a paradoxical phenotype for a SAC hypomorph, it has been shown that other SAC proteins both promote and sense chromosome-microtubule attachment and that partial inactivation of these proteins actually lengthens mitosis because residual SAC function is able to sense incomplete attachment

. We conclude that the Mps1

mutation causes a partial loss of checkpoint function and also impairs kinetochore- microtubule attachment preventing Mps1

cells from stably arresting in the presence of spindle poisons and mis-segregating chromosomes under normal growth conditions

.

Mps1

provokes aggressive T-ALL in a p53 heterozygous background

The Mps1

truncation was introduced into a highly mitotic, non-essential adult tissue by crossing Mps1

mice with animals bearing a T cell specific Lck-Cre transgene

. PCR revealed efficient switching of Mps1

to Mps1

in T cells from 8-10 weeks old animals (Sup. Fig. 2A) concomitant with changes in the DNA content of G1 thymocytes (compare peak width and coefficient of variation for G1 peaks in Mps1

and wild type animals;

Fig. 2A-B) but life span was unaffected (Fig 2C, red line). When Mps1

Lck-Cre

and wild type control mice were injected with Paclitaxel, a microtubule-stabilizing drug that interferes with spindle assembly, elevated levels of mitotic cells were observed 5 hr. later in both genotypes (Fig. 2D), consistent with data from MEFs showing that Mps1

– expressing cells arrest in the presence of spindle damage.

To assay tumour formation in a sensitized environment we generated Mps1

Lck-Cre

animals heterozygous for a FLOX-p53 allele

. Loss of p53 suppresses aneuploidy- associated apoptosis in multiple cell types and is oncogenic in thymocytes

. Mps1

p53

Lck-Cre

mice rapidly developed acute T cell acute lymphoblastic lymphomas (T-ALL) and ~50% of animals were dead of the disease by 3.5 months and 100% by 5 months (Fig.

2D, dark green line). In contrast, heterozygosity at Mps1 had little effect on survival of

p53-null mice: Mps1

p53

Lck-Cre

animals had survival curves indistinguishable from

p53

Lck-Cre

mice (Sup. Fig. 2B). In addition, Mps1 wild-type p53

Lck-Cre

mice

rarely developed disease and tumour-free survival was indistinguishable from that of Lck-

Cre

control animals (Fig. 2D, compare dark blue and black lines)

. Thus, the Mps1

mutation is strongly oncogenic in T cells on a p53

background.

A

50 aa

1 200 520 830

Kinetochore-

binding domain Kinase domain

Deleted in Mps1^DK

C

E D

Cre^Dox-ind +

- + + -

+ - - Dox

Mps1^f Mps1^DK

B

+

+ + +

-

Cre^Dox-ind -

+

- - +

- Dox -

+

- + -

Noco + Number of mitotic cells (% pH3pos)

- 0 2 4 6 8 10

Mps1^WT-GFP Mps1^WT-GFP

Mps1^DK-GFP Mps1^DK-GFP 6:00

6:00

12:00

12:00

p<0.0001

***

0 20 40 60 80 100

Time from metaphase

Time in mitosis

Mps1^f/f + Cre^Dox-ind Mps1^f/f

***

p<0.0001

N=36 N=90

N=82 N=99

Time (min)

F

0 50 100 150 200 250 300

N=34 N=64

Time before exiting from mitosis in nocodazole (min)

***p<0.0001

G

Mps1^f/f MEFs Cre^Dox-ind

+ -

Normal mitosis Lagging chr.

Tetraploidyzation Binucleation Polyploidyzation Failed mitosis Cell death Unclear 0

20 40 60 80 100

Percentage of cells

Cre^Dox-ind - +

Mps1^f/f MEFs

Figure 1 Figure 1. Mps1 truncation leads to mitotic delay, severe abnormalities, and a weakened SAC.

(A) Schematic representation of Mps1 truncation allele. (B) Time-lapse image stills showing clear kinetochore localization of retrovirally-transduced wild type GFP-Mps1 in pro-metaphase (upper panels), and less binding of GFP-Mps1

(bottom panels) to kinetochores in MEFs. DNA was labelled with retroviral H2B-Cherry. (C) PCR detecting the truncation/deletion alleles for Mps1 in genomic DNA isolated from control- or Cre-infected Mps1

MEFs. (D) Average mitotic index of Dox-inducible Cre-transduced MEFs following 6 hours of nocodazole treatment. Mitotic index refers to the percentage of mitotic cells as measured by phospho-Histone H3 staining. Error bars show the SEM of at least four biological replicates.

(E) Average time of mitotic exit for nocodazole-arrested control- Mps1

(blue) or Cre-infected Mps1

(red) MEFs. (F) Average duration of mitosis (upper bars), and time from metaphase to cytokinesis (lower

bars) of Mps1

control- (blue) and Cre-infected MEFs (red) as determined by time-lapse microcopy.

Error bars show the SEM of within bar depicted number of cells. (G) Distribution of mitotic phenotypes for control- and Cre-infected Mps1

MEFs as observed by time-lapse microscopy. Explanation of used terms:

Tetraploidization: a seemingly normal cell failed cytokinesis resulting in one large tetraploid cell. Binucleation:

a seemingly normal cell failed cytokinesis resulting in one cell with two nuclei. Polyploidization: a seemingly tetraploid or polyploid cell failed cytokinesis resulting in a polyploid cell. Other failed mitosis: a combination of mitotic errors.

Mps1

promoted loss of heterozygosity (LOH) at the p53 locus: the PCR product corresponding to p53

was substantially more abundant than the product corresponding to wild-type p53 in DNA from Mps1

p53

Lck-Cre

tumours (Sup. Fig. 2C, compare tumours 9-17 with 18-21). qPCR data were consistent with this finding: p53 mRNA was virtually undetectable in tumours recovered from Mps1

p53

animals (Fig. 2E). To characterize the LOH event, we extracted probe values from array-based comparative genomic hybridization (aCGH) for 17 tumours arising in Mps1

p53

animals (Sup. Fig. 2D). In all but two animals (tumours 12 and 39) hybridization to p53 probes was low, similar to that of p53-null tumours from Mps1

p53

animals (compare Sup. Figs. 2D, E). Hybridization to neighbouring probes was unaffected, suggesting that the wild type copy of p53 had been replaced by p53

, either through CIN or mitotic recombination. We conclude that Mps1 truncation facilitates p53 LOH, a highly oncogenic event in thymocytes

.

The pro-tumourigenic effects of Mps1 truncation do not appear to involve p53 LOH alone. Tumour induction was significantly faster in Mps1

p53

Lck-Cre

(and Mps1

p53

Lck-Cre

) mice than in p53

Lck-Cre

mice: death of 50% of the former animals by 3.5 months as opposed to 5 months for the latter (Fig. 2D, p<0.0001). Analysis of mRNA and genomic DNA confirmed efficient Cre-mediated deletion of p53 in tumours having either genotype (Fig. 2E and Sup. Figs. 2C-E). Moreover, accelerated tumourigenesis in double Mps1

p53

mutant animals relative to p53

animals was confirmed with a second Cre driver, MMTV-Cre, which is transcribed in both T cells and the mammary gland

(Sup. Fig. 2F). No mammary tumours were observed in these animals, however, presumably because T-ALL developed before breast cancers could emerge.

To show that tumours were comprised of cells in which the Mps1

loci had been excised

and thus, that lymphomagenesis was not driven by p53 loss alone (a concern because p53

is such a strong tumour suppressor in T cells), we measured the efficiency of Cre-mediated

recombination at the Mps1 genomic locus using PCR and aCGH, we assayed Mps1

mRNA levels and we performed Western blotting. In tumours isolated from Mps1

p53

and Mps1

p53

mice, bands corresponding to Mps1

were the predominant amplified

products (Sup. Fig. 2G, Sup. info S1) both in Lck-Cre

and MMTV-Cre

backgrounds. In aCGH data, near-complete loss of hybridization to sequences excised by Cre-mediated recombination of the Mps1

locus was observed (Fig. 2F). qPCR of tumour RNA also confirmed loss of Mps1 expression: probes selective for the non-mutated 3’domain (Mps1 probe set B, Fig. 2G, Sup. Fig. 2H) yielded a strong qPCR product, whereas probes corresponding the 5’ region of Mps1 deleted in Mps1

(Mps1 probe set A) were ~20-fold less abundant in tumours than in wild-type thymus DNA. Moreover, RT-PCR followed by Sanger sequencing confirmed the presence of correctly recombined Mps1

transcript (in tumours 13 and 23) and full length Mps1 in p53

tumours (tumours 36 and 63; Sup. Fig.

2I, Sup. info S2). qPCR also showed that Mps1

is overexpressed in tumours ~4-fold relative to wild-type Mps1 in parental cells, presumably because elevated expression of the hypomorphic allele confers a selective advantage on cells. Finally, by Western blotting we could detect a protein band corresponding to the expected length of Mps1

protein in Mps1

p53

Lck-Cre

tumour samples (tumours 18, 19, Fig. 2H). We conclude that the Mps1

allele was maintained in the vast majority of T cells throughout the development of a tumour and thus, the acceleration in tumourigenesis observed for double mutant animals reflects ongoing synergy between Mps1 and p53 mutations.

Mps1

-driven tumours exhibit recurring chromosomal abnormalities

To determine the extent of aneuploidy in Mps1

T-ALL we used array CGH to quantify

chromosome copy number across the genome and interphase Fluorescence in Situ

Hybridization (FISH) to quantify chromosome number in single cells. Array CGH

revealed frequent loss and gain events for multiple chromosomes (4 representative plots

are shown in Fig. 3A with normalized aCGH data summarized in Sup. info S3). As a

simple measure of CIN we summed the total number of chromosome gain and loss events

in each tumour to create an “aneuploidy index”. The aneuploidy index ranged from

3-19 in 30 tumour samples examined, and was significantly higher in tumours arising in

Mps1

p53

or Mps1

p53

animals (average indices of 8.2 and 7.6 respectively) than in

p53

animals (average index 2.4, p=0.047 and p=0.028 respectively; Fig. 3B). Similarly,

unsupervised single linkage hierarchical clustering of cumulative aCGH data showed that

tumours from Mps1

mice heterozygous or homozygous for p53 deletion (Mps1

p53

and Mps1

p53

animals) clustered together and had more chromosomal abnormalities

(Fig. 3C; green numbers) than tumours from p53

animals (blue numbers), which had

less severe aneuploidy. Probes lying on the same chromosomes co-clustered across all

tumour samples, demonstrating that a significant fraction of the aneuploidy in these

tumours involved gain and loss of whole chromosomes.

A B

0 2 4 6 8

Coefficient of variation G1

Control Mps1^f/f Lck-Cre⁺

D

Mitotic cells (%pH3pos)

- +

Paclitaxel + -

- - + +

Lck-Cre 0.0 0.5 1.0 1.5 2.0 2.5

C

Age (months) 0

20 40 60 80 100

Percent surviving

12

0 4 8

Mps1^f/f p53^f/f Lck-Cre⁺ (n=11)

Lck-Cre⁺ (n=12) Mps1^f/f Lck-Cre⁺ (n=29) Mps1^f/f p53^f/+ Lck-Cre⁺ (n=27)

p53^f/f Lck-Cre⁺ (n=20) p53^f/+ Lck-Cre⁺ (n=23)

E

Wild type 0 1 2 p53 Set A

p53 Set B

Mps1^f/f p53^f/+

Cre⁺

Relative fold expression

Mps1^f/f p53^f/f Cre⁺

F

G

H

Mps1^WT Mps1^DK Actin

*

38 63 18 19 TID

ControlMps1f/f Lck-Cre+

DNA-content

2n 4n

Cell number

6

Mps1^f/f p53^f/f Cre⁺ Wildtype

0 1 2 3 4 5

Mps1 Set A Mps1 Set B

Mps1^f/f p53^f/+

Cre⁺

Relative fold expression

-5.0 1:1 5.0 Log2 ratio

Mps1f/f p53f/f Cre+ p53f/f Cre+

TID 1819 2021 2223 2425 4849 5052

3637 3863 64 Mps1 locus Mps1 Cref/f+

2653 55 107

1211 1314 1516 1739 4243 4445 4647 54

Mps1 locus TID

Mps1f/fp53f/+ Cre+

Mps1 locus TID

Figure 2

Figure 2. Mps1 truncation provokes aneuploidy in vivo and decreases T-ALL latency in a p53-

compromised background. (A) DNA content distribution in control and Mps1

T cells. At least 10,000

were counted. (B) Coefficient of variation (CV) values for the DNA content within G1 peaks in cell cycle

profiles of Mps1

and Cre-negative T cells. Error bars show SEM of > 5 biological replicates (experiment

animals) or > 2 replicates for control animals. (C) Kaplan Meier curves showing overall survival of indicated

genotypes. (D) Average mitotic index (% phosphorylated Histone H3) of thymocytes isolated from Paclitaxel-

or control-injected mice 4-6 hours post-treatment. Error bars show SEM of > 5 biological replicates

(experiment animals) or > 2 replicates for control animals. (E) Quantitative PCRs showing complete loss

of expression of p53 (p53 probes A and B) in lymphomas of indicated genotypes. Error bars show SEM for

at least three tumours per genotype. (F) Array CGH data showing loss of the kinetochore binding sequence

in Mps1 for tumours with indicated genotypes. Each rectangle represents a single aCGH probe value: three

probes values are shown: one probe recognizing the kinetochore binding domain (middle) and two probes

flanking the 5’and 3’ sides of the deleted region. Log2 ratios < -5 indicate complete loss of the indicated

probe. Numbers refer to tumour IDs (TID, Sup. info 1). (G) Quantitative PCRs showing full conversion of

Mps1

to Mps1

in tumours with indicated genotypes. Primer set A recognizes the sequence deleted in

Mps1

, set B detects a fragment in the kinase domain. (H) Mps1 protein levels in p53

(TIDs 38, 63, full

length Mps1) and Mps1

p53

tumours (TIDs 18, 19) showing conversion of full length to truncated Mps1

in the latter genotype. The asterisk refers to a background band recognized in all lysates, and runs just below

Mps1

that is only detected in TIDs 18 and 19.

Amplification of Chr15 was particularly frequent in aCGH data, regardless of genotype, and is known to be common feature of mouse T-ALLs

. In addition, amplification of Chr4 and 14 and to a lesser extent Chr9 was observed in many tumours and Chr13 and 19 were commonly deleted, suggesting that those chromosomes carry genes important for transformation or aneuploid tumour progression. Interphase FISH confirmed aneuploidy in non-transformed Mps1 mutant thymocytes and heterogeneity in chromosome number within a single tumour. For example, Chr15 and 17 were aneuploid in a greater number of cells in Mps1

Lck-Cre

thymocytes than in wild-type thymocytes (an average of 5%

vs. 11% of cells for Chr15 and 6% vs. 12% of cells for Chr17, Sup. Fig. 2J). In Mps1

p53

Lck-Cre

T-ALL tumours we observed Chr15 trisomy in up to 80% cells but with differences in the fraction of cells involved from one animal to the next (Fig. 3D, Sup.

Fig. 2J).

To identify common focal loss and gain events we performed genome-wide cumulative segmental gain or loss analysis (SGOL) comparing Mps1

-driven and p53

Lck-Cre

tumours. For both tumour classes, SGOL revealed strong deletion peaks on Chr6 and 14, consistent with a unique pattern of recombined T cell receptor alpha/beta loci. These results strongly suggest that tumours arose from a single parental T cell (Sup. Fig. 3A, B). We can reconcile the SGOL and FISH data by hypothesizing that T-ALLs are clonal early in their development (at the time of TCR rearrangement) but that ongoing CIN results in subsequent chromosome loss and gain. Selection is expected to maintain some aneuploidies, for example Chr15 amplification, whereas other chromosomes (e.g. Chr17) might be subjected to ongoing loss and gain.

Recent studies on budding yeast and MEFs carrying supernumerary chromosomes have shown proportional increases in gene copy number and transcription

. To determine if this is also true for tumours driven by Mps1 truncation we used Illumina expression arrays to analyse the transcriptomes of 22 tumour samples that had previously been studied by aCGH as well as thymus DNA isolated from 6 week-old wild type mice. A strong correlation between mRNA and gene copy number was observed when aCGH values and expression levels were sorted based on chromosomal position (Sup. Fig.

3C and D respectively). When we calculated the average expression changes per

individual chromosome for each tumour (using healthy thymic samples as a control)

and then compared the value to aCGH intensity (Fig 4A-B, Sup. info S4) the correlation

between expression and copy number was R

= 0.44 – 0.76 for the most commonly

aneuploid chromosomes (Chr4, 14, and 15; Fig. 4C; Sup. Fig. 4 shows correlation for

all chromosomes). We conclude that in tumours, as in cultured cells, chromosomal imbalances are on average translated into increases and decreases in transcription and thus, that there is little or no dosage compensation.

0 5

-5

Tumor 15

Mps1^f/f p53^f/+ Lck-Cre⁺ 1

2 3

4 5

6 7

8 9

10 11

12 13

14 15

16 17

18 19

X Y 0

5

-5

Tumor 20

Mps1^f/f p53^f/f Lck-Cre⁺ 1

2 3

4 5

6 7

8 9

10 11

12 13

14 15

16 17

18 19

X Y

0 5

-5

Tumor 38

p53^f/f Lck-Cre⁺ 1

2 3

4 5

6 7

8 9

10 11

12 13

14 15

16 17

18 19

X Y 0

5

-5

Tumor 36

p53^f/f Lck-Cre⁺ 1

2 3

4 5

6 7

8 9

10 11

12 13

14 15

16 17

18 19

X Y

A

1314 12

1920 1017

24 21

1849

16 23 4852 50 47

46 7 4443

1525 37 3663 38

1 5 2, var. 313 7 17619810

4 14 915 11 12 16

X 18

Mps1 p53 tumors p53 tumors

Hierarchically clustered CGH probes

Hierachically clustered tumors

39 22 11 24 3545

-1.0 1:1 1.0

log2 CGH ratio of tumor DNA over reference liver DNA

C

B

Aneuploidy index

p53^f/f Mps1^f/f p53^f/f Mps1^f/f

p53^f/+

0 5 10 15 20

p=0.0047 p=0.0278

Mps1^f/f Lck-Cre⁺ Mps1^f/f Lck-Cre^-

Mps1^f/f p53^f/+ Lck-Cre⁺ Chr

D

Figure 3

Figure 3. Mps1 truncation leads to CIN and clonally stable karyotypes in tumours. (A) Representative

aCGH profiles for four tumours showing whole chromosome instability. (B) Aneuploidy index (total number

of gained and lost whole chromosomes as assessed by aCGH) for tumours with indicated genotypes. (C)

Single linkage hierarchical cluster analysis for individual tumours (top to bottom) and CGH probes (left to

right). Clustering was separated in 20 clear groups by eye. (D) Representative interphase FISH images of

control (upper panel), Mps1

T cells (middle panel) and Mps1

T-ALL cells (lower panel) showing copy

numbers for Chr15 (green) and 17 (red).

Average aCGH signal per chromosome in individual tumors

A

B

0 1.0

0.5

-0.5

-1.0

p53^f/f tumors

Average log2 signal

0

123 45

67 89

1011 1213

1415 1617

1819 1.0

0.5

-0.5

-1.0

Mps1^f/f p53^f/f / Mps1^f/f p53^f/+ tumors

Average log2 signal

Average RNA expression array signal per chromosome in individual tumors

0 1.0

0.5

-0.5

-1.0

Mps1^f/f p53^f/f / Mps1^f/f p53^f/+ tumors

Average log2 signal

0 1.0

0.5

-0.5

-1.0

Average log2 signal

Chr. 15 R²= 0.44 Chr. 4

R²= 0.77 Chr. 14

R²= 0.63

Average log2 signal expression array

Average log2 signal aCGH array Correlation copy number alterations and expression changes

C

-0.2 0.2 0.4 0.6 0.8

-0.2 0 0.2 0.4 0.6 0.8 -0.2 0.2 0.4 0.6 0.8

-0.2 0 0.2 0.4 0.6 0.8 -0.2 0.0 0.2 0.4 0.6 0.8

0 0.2 0.4 0.6 0.8 p53^f/f tumors

123 4 5

67 89

1011 1213

1415 1617

1819

123 45

67 89

1011 1213

1415 1617

1819 1

23 4 5

67 89

1011 1213

1415 1617

1819

Figure 4 Figure 4. Gene copy number results in proportional transcription changes in aneuploid tumours.

Average aCGH (A) or RNA expression values (B) were calculated per chromosome for each tumour and

plotted for each tumour group. Each individual symbol represents the average value of that chromosome in

one tumour; black crosses show the mean and SEM across all tumours per chromosome. (C) Linear regression

plots showing the correlation strength (coefficient of correlation, R

) between copy number changes (aCGH)

and expression changes (expression arrays) for frequently gained Chr4, 14, and 15.

Mps1

tumours show evidence of aneuploidy-induced stress

To identify genes significantly over and under-expressed in T-ALLs, we sorted genes based on their cumulative expression changes across all samples (annotated in Sup. Fig.

5A, Sup. info S4). For Chr4, 14 and 15, the majority of genes had a positive cumulative score and the reverse was true for Chr19, reflecting the correlation between changes in transcription and gene copy number. Among the outliers, we found several that exhibited an inverse correlation between copy number and expression including the Keratins Krt, 5, 7, 8 and 18, Epsi1 and Chdr1. These genes are expressed in the thymic cortex and not in tumour cells, and lower expression in mutant animals is likely to reflect T-ALL-mediated depletion of cortical tissue. A second set of outlier genes was significantly over-expressed relative to other genes on the same chromosome. This set include genes involved in cell metabolism (Srm, Gln3, Cox6a, Drospha, Adk), cellular stress (Serp2, Hsf1), cell cycle (Recql4, Cdkn2a, Skp2, Tnc), and epigenetic regulation (Prmt5, Cbx5). Surprisingly, Myc, a known oncogene in T-ALL

was not among the strongest positive outliers on Chr15. In future work it should be possible to use Mps1

-driven aneuploidy to identify new oncogenes or tumour suppressors involved in T-ALL as well as genes involved in cell survival in the presence of CIN.

To begin to identify biological pathways altered by aneuploidy, we compared probes that were up- or downregulated at least 1.5-fold in > 15% of tumours analysed (4 out of 22); ~3300 genes were identified by this analysis. Webgestalt

was then used to find GO categories that were significantly enriched (using a Bonferroni corrected p value

< 0.05). The most commonly deregulated pathways were T cell receptor signaling, mRNA processing, cell cycle, and pathways involved in cellular metabolism (Sup. Fig.

3E). When we performed hierarchical clustering of deregulated genes (Fig. 5), dividing clusters into five groups based on whether genes were strongly or weakly up- or down regulated, T cell differentiation and signaling factors were down-regulated, consistent with histological data showing that tumours are poorly differentiated. In contrast, pathways involved in cellular metabolisms (GO terms for metabolic pathways, RNA metabolic pathways, spliceosome, translation factors, nucleotide synthesis, etc.) were upregulated.

Misregulation of these processes has previously been associated with aneuploid stress

in cultured mammalian cells and budding yeast

. We conclude that dysregulation of

metabolic pathways is a common feature of CIN in multiple organisms and cell types,

including rapidly growing tumours.

45 44 42 13 11 10 7 38 63 46 47 18 19 20 21 22 23 48 49 50 52 36

I:Macrophage markers, antigen processing and presentation,T-cell proliferation, T-cell differentiation

II: T-cell receptor signaling, chemokine signaling, interferon signaling, MAPK signaling, T-cell differentiation

III: mRNA processing, Amico acid metabolism,Metabolic pathways, Purine/ pyrimidine biosynthesis, Ribosome biogenesis, RNA transport

IV: Spliceosome, Proteasome,mRNA processing, Metabolic pathways

V: RNA transport, Translation factors, Metabolic processes

Tumor ID

-1.0 1:1 1.0 log2 ratio of tumor RNA expression over RNA

expression from non-transformed proliferating T-cells

Figure 5

Figure 5. Mps1

driven CIN activates genes regulating various aspects of cellular metabolism. Single linkage hierarchical clustering of transcriptome data shows the most significant deregulated pathways per group. Groups I-V were grouped by visual inspection. Full gene lists and enrichment analyses are in Sup.

info S4.

DISCUSSION

In this paper we report the development and analysis of mice in which CIN and consequent aneuploidy are induced in a rapidly proliferating but non-essential adult tissue by conditionally truncating the SAC kinase Mps1. We show that the Mps1 truncation, which deletes a kinetochore-targeting domain but leaves kinase activity and other functions intact, can provoke but not sustain a checkpoint arrest in the presence of spindle poison;

it also causes chromosome misalignment and generates lagging chromosomes, consistent

with the dual role of SAC proteins in promoting and sensing kinetochore attachment. When

mutated in murine T cells, Mps1 truncation causes aneuploidy but this is insufficient for

efficient oncogenic transformation. However, in a predisposed p53 heterozygous deletion

background, Mps1 truncation results in rapidly growing acute lymphoblastic lymphoma

(T-ALL). We observe frequent p53 LOH in Mps1

p53

Lck-Cre

animals, consistent

with the know role of p53 as a potent tumour suppressor in T cells. However, p53 LOH

cannot be the only oncogenic event in T-ALLs because tumour latency is significantly

shorter in Mps1

p53

Lck-Cre

animals than in p53

Lck-Cre

animals. aCGH also reveals higher levels of aneuploidy in compound mutant animals than in single mutants.

Taken together these data suggest that Mps1 mutation causes p53 LOH and the combined Mps1-p53 mutant genotype results in more efficient gain and loss of cancer genes than p53 mutation alone.

We interpret aCGH and interphase FISH data to show that T-ALLs are initially clonal (based on TCR rearrangement) but that ongoing CIN results in tumours with recurrent aneuploidies in Chr4, 14, 15 and 19 as well as sporadic changes in other chromosomes.

The net result is changes in the expression of oncogenes and tumour suppressors driving T-ALL as well as changes in as-yet unidentified pathways involved in tolerizing cells to aneuploidy. A significant body of literature has emerged over the past few years examining the consequences of aneuploidy for the physiology of yeast and cultured murine fibroblasts. These studies have revealed recurrent up-regulation of pathways involved in mRNA processing and other metabolic processes

. Our data show that T-ALLs generated by Mps1 truncation and p53 loss also exhibit similar transcriptional signatures as aneuploid MEFs

and untransformed tissues

. Thus T-ALLs cells can grow rapidly in the face of frequent chromosome loss and gain, but that CIN nonetheless imposes a burden on tumour cells similar to that observed in MEFs and budding yeast cells. This is a potentially significant finding, since “proteotoxic stress” and metabolic dysregulation have become important targets for cancer therapy and may represent a means to selectively kill highly aneuploid cancers.

ACKNOWLEDGEMENTS

We thank members of the Sorger lab, S.W.M. Bruggeman, L. Albacker and L. Kleiman,

for critically reading the manuscript and fruitful discussions, B. Bakker for assistance

with cloning, Roderick Bronson for help with pathology, and A. Burds for sharing Lck-

Cre and MMTV-Cre mice. This work was supported by Dutch Cancer Society (RUG

2012-5549), Stichting Kinder Oncologie Groningen (SKOG) and EMBO grants to FF,

by NIH grants CA084179 and CA139980 to PKS, by P30-CA14051 to the MIT mouse

transgenic facility and Wellcome Trust funding (FF and AB).

AUTHOR CONTRIBUTIONS

F.F., S.Z.X. and P.K.S. designed the project; F.F., S.Z.X., J.E.S., P.L.B., performed experiments; S.D. assisted with mouse experiments; E.K. and J.J. provided reagents; F.F., S.Z.X. and N.C. analysed data; F.F., A.B. and P.K.S. provided funding; F.F. and P.K.S.

wrote the paper.

MATERIALS & METHODS Analysis of mice

Mice used in this study had a mixed C57BL/6 and 129/Sv D3 genetic background. Mps1 conditional mice harbouring a deletion of residues 47 to 154 were generated as described in supplemental methods. p53 conditional knockout mice were obtained from Anton Berns (25), Lck-Cre transgenic mice from Taconic

, and MMTV-Cre mice

from the MIT mouse repository. Mice were intercrossed to obtain the described strains and genotyped as described previously

(genotyping PCR primers in Sup. info S5). For survival studies, mice were monitored for tumour development weekly starting at 2.5 months of age by looking for difficulty in breathing (a consequence of thymic hypertrophy). Tissues were fixed in 10% formalin and then paraffin-embedded for histology. Animal protocols were approved by the MIT, HMS and UMCG Committees on Animal Care and the UK Home Office.

MEF isolation, transduction, flow cytometry and time-lapse analysis.

MEFs were isolated as described before

at E13.5 from Mps1

embryos and cultured

under low oxygen (3%) conditions. Retroviral particles (pRetrox system, Clontech) were

produced in Lipofectamine 2000-transfected (Life) 293T cells. MEFs were first transduced

with pRetrox-rtTA virus and next with pRetrox-GFP-T2A-Cre avirus, allowing us to

assess Cre expression without linking Cre to GFP directly

or pRetrox GFP-Mps1

or

GFP-Mps1

virus (constructs described in detail in Supplementary Methods). For time-

lapse imaging, cells were subsequently transduced with pRetrox-H2B-Cherry or pRetrox-

H2B-GFP to visualize the DNA and treated with 250 ng/ml nocodazole (Sigma) where

indicated. For flow cytometry transduced cells were treated with 1 μg/ml doxycycline

(Sigma) for 48-72 hours to induce the retroviral inserts, treated with 250 ng/ml nocodazole

for 6 hours and then fixed in 70% ethanol. Cells were then stained with PE-labelled

pHistoneH3 antibody (Cell Signaling) and FxCycle Brilliant blue (Life) and analysed on

a LSRII analyzer (BD Biosciences). Data was analysed using FlowJo software. For time- lapse analysis, cells were seeded on LabTek imaging chambers (Thermo Fisher), induced with 1 μg/ml Dox for 48-72 hours and imaged for up to 36 hours on a DeltaVision Elite imaging station (Applied Precision, GE Healthcare) in a low-oxygen imaging chamber (Oko-labs). Movies were deconvolved and analysed using SoftWorx suite.

DNA/RNA/protein isolation, array and blotting experiments

Genomic DNA from tumour samples and control liver was isolated using genomic DNA tissue kits (Qiagen). Genotyping primers can be found in Sup. info S5. Protein was isolated using a total protein extraction kit (Millipore). For protein detection, proteins were blotted using standard Western blotting protocols. Used antibodies were TTK (Santa Cruz, C19) and HRP-conjugated goat ant rabbit (Cell Signaling). For aCGH experiments, labelled DNA (see Sup. Methods) was hybridized to 244K mouse genome CGH arrays (Agilent) according to manufacturer’s protocol and analysed as described in supplemental methods. RNA was isolated from tumour samples and thymuses from 6 weeks old control mice using the RNeasy kit (Qiagen). Labelled RNA (see Sup. methods) was hybridized to Illumina v6.2 beadchip arrays, scanned and analysed as described in the supplemental methods. qPCR primers can be found in Sup. info S5. All array data has been deposited at NCBI GEO under accession number GSE57334.

SUPPLEMENTAL MATERIALS & METHODS Generation of Mps1

conditional and Mps1

mutant mice

A 129/Sv mouse genomic lambda-phage library was screened for the genomic locus of

Mps1 as described

. A 14 kb portion of the Mps1 genomic locus encoding exons 1-9

was isolated and cloned into pBluescript (Life) via NotI sites. DNA encoding Diphtheria

toxin A (DT-A) was cloned into the NotI site as a negative selection marker. A loxP site

was cloned into an NcoI site between exons 2 and 3. A 4 kb selection cassette containing

thymidine-kinase (TK) and the neomycin-resistance gene flanked by two loxP and FRT

recombination sites (LFNT-cassette) was cloned into a SphI site between exons 4 and

5. The resulting 22.6 kb targeting vector (Sup. Fig. 1A, line 2) was introduced into

embryonic stem (ES) cells and clones isolated as previously described

. Incorporation of

the gene targeting vector by homologous recombination into ES clones was determined

by Southern blotting using a 664 bp 5’ probe and a 665’ 3’ probe after digestion with NheI

or BglI as indicated in Sup. Fig. 1A, line 3 as described

. The 5’ probe was generated

by digesting a lambda phage clone with SacI and XmaI which contained sequences 5’

of the targeting vector and then subcloned into pBluescript (Invitrogen). The 5’ probe yields an 11.7 kb wild type band and a 9 kb targeted band. The 3’ probe is homologous to sequences containing exon 9 of Mps1 and was generated by PCR yielding a 13.4 kb wild type band and a 9.4 kb targeted band. Two correctly targeted ES clones were isolated.

The LFNT cassette was removed in these ES clones by transfecting cells with vectors expressing Cre recombinase (pCrePac) or Flp recombinase (pFlpe), which resulted in the DK (Sup. Fig 1A line 5) or conditional (f) (Sup. Fig. 1A line 4) allele, respectively.

Southern blotting after restriction digestion with NcoI and probing with the 5’ 664 bp probe yielded 8 kb for wild type, 12 kb for the targeted locus (containing LFNT), 15.2 kb for the DK allele and 18.6 kb for the f-allele. Three DK/+ and two f/+ subclones from the two original clones were injected into blastocyts and successfully generated founder lines for the conventional DK and conditional alleles.

To assess the consequences of Mps1 truncation in developing embryos, we generated Mps1

ES cells by Cre electroporation, established the Mps1

allele in the germ line and intercrossed Mps1

mice. No Mps1

mice were recovered in 33 litters comprising 237 pups showing that the Mps1 truncation allele is embryonic lethal (Mps1

mice comprised 38% of viable pups and Mps1

62% of pups, the expected Mendelian ratio; Sup. Fig. 5B). Timed matings (E10.5 embryos were isolated and the yolk sac taken to determine genotype by PCR as described

revealed that Mps1

embryos died at or before E10 (Sup. Fig. 5C), as previously described for germline deletion of other checkpoint genes

. We conclude that aneuploidy generated by the Mps1

allele is lethal to developing embryos.

Generation of pRetrox GFP-Mps1 full length, pRetrox GFP-Mps1DK and Cre-T2A-GFP.

For pRetrox GFP-Mps1 vectors, we first PCR amplified eGFP (Phusion polymerase,

Thermo Scientific) with BglII and BamHI+NotI Sites 5’ and 3’ respectively. This PCR

fragment was cloned into BamH1, NotI-digested (New England Biolabs) a pRetrox

backbone (Clontech) thus destroying the BamH1 site 5’ of the GFP. In case of full length

Mps1, we then PCR-amplified Mps1 from a human Mps1 cDNA clone (Harvard clone

library) flanked by BamH1 and NotI sites 5’ and 3’ respectively, which was cloned into

pRetrox-eGFP digested with BamH1 and NotI. For Mps1

, we first PCR-amplified a

fragment encompassing the 5’ ATG to an endogenous HindII site in exon 1 of human

Mps1, which was ligated into a pMSCV backbone (Clontech) digested with BamH1 and

HindIII. We next PCR-amplified Mps1 from the start of exon 4 fused to remained sequence

of exon 1 downstream of the endogenous HindIII site to its stop codon, which was ligated into pMSCV containing exon 1 of Mps1 from the previous step, thus Screating Mps1

. We then PCR-amplified Mps1

from pMSCV Mps1

using BglII and NotI sites and cloned it into pRetrox eGFP similar to full length Mps1. For Cre-T2A-GFP, we first PCR amplified GFP while adding a T2A sequence to its 3’ end

flanked by BglII and BamH1- NotI sites, which was cloned into pRetrox using BamH1 and NotI sites. We next cloned Cre downstream of GFP-T2A using BamH1 and NotI sites 5’ and 3’ respectively. All used primers can be found in Sup. info S5.

Quantification of kinetochore-bound Mps1

To quantify the levels of GFP-Mps1 bound to kinetochores, fluorescence arbitrary units on a horizontal line through a kinetochore were analysed in SoftWorx software (GE Healthcare) using the line-profiling tool. To correct for GFP-tagged protein expression levels, we normalized kinetochore-bound values by subtracting the average GFP expression counts in the cell from the counts at the kinetochore (line-profiling tool). We then calculated the fluorescence ratios between GFP-Mps1 and GFP-Mps1

for both cell types, as presented in Sup. Figs. 1E and G.

Comparative Genome Hybridization and analysis

For aCGH experiments DNA was labelled with Cy3 or Cy5 fluorescent nucleotides

according to the BioPrime array CGH genomic labelling protocol (Life) and cleaned

using purelink PCR purification kit (Life). Many liver samples showed evidence of T

cell infiltration, disqualifying them as control samples. Therefore we used pooled sex-

matched reference samples from un-infiltrated livers from littermates. Labelled DNA

was hybridized to 244K mouse genome CGH microarrays (Agilent) according to the

manufacturer’s protocol, which were subsequently scanned, background corrected and

normalized using the loess algorithm with Bioconductor limma package

. Data was

then segmented and split into regions of estimated equal copy number using a CBS

(Circular Binary Segmentation) algorithm (Bioconductor) with the DNAcopy package

.

To further reduce noise, we used the “undo” method to remove all splits that are not

at least 3 standard deviations apart. Log2 ratios (tumour/reference) were plotted along

the chromosome coordinates using the same software. To create a multi-sample matrix,

we assigned segment means to mouse genes (NCBIM37, 63) on all segments for each

sample using the Bioconductor CNTools package. Data was further processed and sorted

on chromosomal position in Microsoft Excel. The aneuploidy score was calculated by

assessing whole chromosome losses and gains in Agilent Genomic Workbench software

and statistical analyses were performed using Graphpad Prism software. In order to find chromosome regions showing common gains/losses, we computed SGOL (Segment Gain or loss) scores by calculating all positives and negative log2 ratio values set over or under a threshold (threshold used was log2 ratio = |0.3| using the Bioconductor cghMCR package

.

RT-PCR, qPCR, and expression arrays

Synthesis of biotin-labelled cRNA was performed using a Illumina totalprep RNA

amplification kit (Ambion). 1.5µg of amplified biotinylated cRNA was hybridized to

llumina Sentrix BeadChips (v6.2) and subsequently labelled with streptavidin-conjugated

Cy3 (Amersham) according to manufacturer’s protocol. Following scanning, data were

quantile normalized

and analysed using Bioconductor lumi

and limma packages

. Data

were p-value adjusted to yield a sorted list of differentially expression genes

and sorted in

chromosomal position or frequency of deregulation in Microsoft Excel. For GO analysis,

all genes with 2log values between -0.6 and 0.6 (less than 1.5 fold deregulated) and genes

deregulated in fewer than 4 tumours were excluded. For RT-PCR, reverse transcription

was performed using a OligoDT, random hexamer mixture and MMLV-RT (New England

Biolabs) according to the manufacturer’s instructions. Primer sequences can be found in

Sup. info S5. For qPCR reactions, 1 mg of total RNA was used for a reverse transcriptase

reaction (Superscript II, Life). The resulting cDNA was used as a template for qPCR

(ABI PRISM 7700 Sequence Detector) in the presence of SYBR-green (Life) to label the

product. The relative amounts of cDNA were compared to Actin to correct for the amount

of total cDNA. Average values and standard deviations were calculated as indicated in

figure legends and compared to the expression values in control mice (normalized to the

value of 1). Primer sequences can be found in Sup. info S5.

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