DTIC ADA423213: CHK2, A Candidate Prostate Cancer Susceptibility Gene

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Rochester, ‘isseaota 55805

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7 ABSTAAET Mans 205 Woe]
40 idensify prostate canter susceptibility genus, we apglied a mitation screening of
eandigate gone sppronch. Ia’our last yosr's reporter, se identified a total of 22 14.98)
Sermline CHER? mutations in 176 cline Laora, 149 fani‘1al proscace cancer (HPC)
fomilics, and 409 eporacie cazea, Sixtaed of tho 19 unique CAB? mtations identified in
tbls study were not ceversce sprig <2) unaffected men, suggastior pultolayieal. elfect of
Chere mitations te prostate cancer cevclogrent. In thie year's reporter, we identified tee
poratic CEK2 mutations it 24 clirde prestate tane= sampls ludiceLicg thal COKD mutation
iu prostate canesr ola 30 pot germline oF gomatic. To invastigave tke fusctuon of those
tuatations su prustate tomorigenseis, we generstn stable ce:l tines ané analyzed the Ca
Kinaes sotivities in 5 of the eatante befave ané after irradiation. Waile sost of the
puvarccne have modest reduced C4K0 kicsse activity in comparison with wilé-eype CHR2, ove
somatic rulalion {Glusaitys) cotsLly abolishes sinage ectivicy. our data provide first
Svedence that the CHK? putations Identified i prostate cancee, both germine ane somatic,
Shiwie CHR? kinase activity auggesting that vatationa in CHK@ may contriscto to the
Gevelopnont of peetate cancer through altering 0&2 kinsse activity.

TESORO Tama 5 RONDE OF FOES
GRAD, peogtate cancer, and suacepsibitity a
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Mumber: DAMD17~02-1-0093

TITLE: CHK2, A Candidate Prostate Cancer Susceptibility ceue

PRINCIPAL TWESTIGATOR: anguo Liu, Ph.D.

CONTRACTING ORGANTZATION: Yayo Clinic, Rochester

Rochester, Minnesota 55905

RSpeaT DATE: January 2004

(yes OF REFORT: Annual

PREFARED FOR: U.S. Army Medical Research and vateriel comand
Foet Detrick, Maryland 21702-5012

DISTRIBUTION STATEMENT: Approved [or Public Release;
Pistcibutioa Unlimited

‘The views, opinions and/or findings contained in this repor= are
those of the author{s) ané should net be construed as an officia’
Departne:t of the arny position, policy or devisica unless so
designated by other decunentation.
Cover

SF 298

Table of Contents...

Introduction...

Bouy..

Key Research Accomplishments.

Reportable Outcomes...

Table of Contents

Appendices,
Amuual report
DOD grant DAMD17-02-0093
CHK, A Candidate Prostate Cancer Susceptibility Gene
‘Wanguo Li, PD.
Mayo Clinic

Untroduefion:
‘Prostate cancer isthe seccnd ment coraraon cancer aad the second leading sure of euncar raovtality it

-Amesican mon. Previeus sles ef ferly history and twins with prostate cancots have shosvm thal genetics
plays entical role in the development ofthis cances, However, penetic components coutt ating t prostate
‘cancer {SLM 200200) have boon diffieul Io identity, largely due to the complenily of this deus andthe
Jresence of phienocapics in high-risk files. Revaviny Io Ue diicaltics to idensty high penetrt genes
Fused on Wakage ana‘ysis and positional cloniny, i hax been suggested thatthe pathogenesis of the dineare ik
retuted ut Teun, th par, to acne mutadens in mulljpls low-penetrant genes. Although lese penctrant, such
igenes might play an importaar ole aca population level, Que lahoraturies have recently applied a now approxch
tt iderify pronidle cancer absceptibility geno(a) based on Tanllion screening of candiGate genes involved in the
DNA damage-signaling palhway and identifice m.vations ir the CHK2 ge, CHK? is ¢ key regulator in tals
pathway, It regulates umber a devirstream effector proteins such ns p53 sm phiys esenal niles in
Soordinaing DNA repair. voll eyele prognéssion, tanstipiousl regulation und spaplosis in response to various
[DNA-damaging events, Because mufalions in p33 is infeeguent in prostate cancer, while s¢mmion [more than
'507%) in all ether caacers, we hypotheslze that CHIK2, the upsttoam regulator of 33, eculd he w candidate
prostate cancer susceptbility gone. Therefore, we propose tb: |) screat for CHK. mutations in 163 fanilial
prostate cancer Familes, collected atthe Muy Clinic, and dete-mine whether the matations segregate with
postace cancer in Families: 2) perfarra functional analyses Io letorrine the impact of mutant CHK? in te DNA

umage-sigcaliny pathway using a kinase activity asenyand 3) perform Jot al helevaaygosity (LOH) studies to
Aelermine if CHIC furtinns a a luck suppressor in prastase cancer, These resulls will vance au
“understanding ef hs stinlogy of prostate eanvar cad may also enabtc us to develop diagnostic tools for che eaty
detoetion and prevention of prostale cancer.

Body:

In yen one, we uecomplished lasks 1-4 of che proposal. Ths task S, which we propesed to fulfil in year
‘000, andl the zeearnplishonents aapciated with tis task ats surtmarized below:

Task 5, Kinase activity analynen will he performed on all of the mutations identified inciod’ne thase identitied
in the fein! premiie cancer Tanides, According to the frequency of C#HK2 motation Wend in
primary prostate cance tines, we many identify moe then 30 different mutations in Camiial cases. Sa
Ye neod to analyze about 20-20 mutations. This ork will be done in Moachs 17-28.

A) Cregie CHR? muhim.s using misiagenesis Kit and cloae thorn into PDEST20 sector
1b) ransfozt the mains into an insect cal line (S19) to generat: rocombinam baculbvirus
encoding OST used wild-type and ayatants of CHIC
Jutatnome ulfinity chaomaiograpby:
4) Kira actisity will he measured using GST-Cae25C as substrate.

As we showed fn the ropnet las! yeu, we have identified a total of 18 unique gemmline CHK2 mutations
in she analyaes of the sporadic nnd forihal prostous cancer samples (1). In addition, we identifica wo navel
sometic CHK mutations in owe posite Lumar somples this year (A349G, Argl17Gly and GO6LS,

Glu32iL ys). Ths, CHI mutations in prostale cancer could he eller germlice mutations or somatic mutations
ulthough the enajoricy of chern are gerimines,

Since some of the mutaitons have been analyzce andl repuried previnusly by us or others, four of dem
have the sume changes in the neighbouring Arg amino acids (Arg L#0C¥ys wd Argl8ICys), and thee truncating
rmutalicns ctivinusly abs kinase acticity due tothe deletion af the kinase domein (245de11Shp, £VS241G-
SA,und Glu249Stop, we Facused on eftoxt ou the focctional amelysis ofthe remaining (2 mutations (Ife 1},

4
‘able 1
CHK? inuttons densified in prostate canov for kinase aetvity ws

amram Rulon ‘Ani ack change Donate
n cpa Gliese ‘wroeien germline
2 Ansl20)y FHA muatic
3 ‘arg145P0 FHA senting
a tet s7Tr FHA gerne
5 Gy 1STANg FHA fermline
6 aaulsieys Uskossn

7 Guasetys Kinase

8 (e381 Phe Kinase gennline
5 Angi Kinase ‘geomline
0 Ghegattys Kinase somatic
u Tase3Pia ease

n TuaTéLys Kinase

1 We firs! iatmdaced above point mutations iu the CHK? corti sequence imta the Matomalisn
expression plasmid encriding FTA-tagaed CHIC2 nsing the QuickChange Site Direct Mutagenesis Kit
Cineatrogen) for Ihe generation of expiession constracts encoding cach ruuants. The mutant coustnets were
confirmed by sequeace malysin, GST-Cadc25¢. containing the C-terminal (ragenent (residues 200-256) af
Cae25C, was used asa CITCD subserate

b ¢) To detecmine CIIK? kinane activity mare ace-rate and ro make the wusny possible befose and attr
ivadiation, we performed Kinase sssiy in momimnian system instead inthe insoct cell line (S19), We, therefore,
‘stall shed stable call Lines expressing HA tagged wild-type ir he matant CHK2 constrvets in EICT(S veil,
‘which ave CTIK2 deficient, Gal 8-resistant clones were isblated ond exagencus CHIC2 exptession was
‘confirmed by Western blotcng using anti-HA er ani-CFI2 uncihnuy. Only stable cell Lines expressing mutant
‘CHR? ul the levels sinila to that of wild fype CHK? wore uses for CTIK2 kinase activity assay, We
succesfully generated 9 aut ot 12 stable coll lines sve for CITK2 Kinase anaysis.

ds To exumine how these mutations affect CHK? activation, we exymined CTTK2 kinase activation
before and alter DNA, chime, Tiefly, neve cells wero radiated, CHIK2 wes iram ynorrecipitated trom wtoole
cell extract ath ank-HTA antibody and CTIIC2 Kinase assays were performed asing CST-Céc2SC fragment as
Substrate, Westen blots with unticphospho-ClUK2 Tod and ant] CHA? amtibexlies were alsa performed to
examine CHK? protein level ans] phenphiorylucion of CHIC2 at Uhr 6 site following DNA damage. Compared
ta wild-type CHK2, the Glu371 Ts mucant did noc have any detectable CHK kinins acivity Figure 4)
Incerestingty, Yas mutant still underseent the ATM-deyendent phosphorylation of Thr-68 site following DNA.

“eat » fee

enn nee etree FTE
damage. In rltion, mutans of AspEITGHy, ArgISLCys, Thed 76 ys, UlusALys. Glu239L yg, Thr323Pe0
partially coduced CHK? Kkinmve uelivity (Figure 1), Execpt for the GlubsLys miccant, non of those CHE 2
Inutuais had a rednction of Thr-68 preephorylation of CHK2 following DNA damag re 1). Those
absecvations are quite differemt fram: dhnse seen in the Arg] 4SPro mutsml (2). The Azgl43Pt0 mutant abolishes
bherh C1IK2 kinase zeeiviey and Thr-68 phosphorylation oF CHK? fellovving DNA darbage. Thus. its tikely thet
These raulations oF CHIC? identified in prostate cancer affect some steps in CHK? tivation afer the initial
phospherslavion af CHK2 at Thr-68 site by ATM,

key Research Accomplishraent

| We genecsred acotant expcessiom suystrut:s and established corresponding slaN cell lines for 9 CHK2
mutations identified in prostato cancer palients or tumors. Kinase activity amalysex demonstrated that those
CII? anuteions eiice covupletey abolishet or modest eeduced CHK2 Kinase aetivity in comparison with
swild-iype CFTK?2 indicating that chose CHK?. mutatis may indce prostate tumorigenesis due t lacks or
Tess kinase activity in the veeponse to DNA damage

2, We hase “denlified two navel somatic CHK? mutatfans ia 2/84 prostate mumor samples indicating that
CHIC2 snvteHiom in arasiate cancer cond be clther germline or scmavic. In addition, kinase activity axsuy
demenstrated Una ore snrnacie mutation (RTF) hid modest reduced CHIK2 kines activity whils the other
‘ce (H321K) totaly uholished CHK? kinase

Reportable outcomes:

THE mutations, both gerraline ant somatic. identified in prostate camer patients of tusnors, impair
Kinase activity which is seeessary or preper mesponding wo DNA damage and for prevention of neoplast.c
tmansfecmatio,

Cemetusions:

‘Our reacts pcvide first evidenoo thal CHK2 mutations identified in prostax cancer patients ot issues
{germltce or somite) may coatiibute ro the development of prostale cancer by altering khase activity of CTTK2
‘Which is entical in wesaoeding to the DNA damage aed inthe mainloin geno at

References:

J Dung X, Wane ., Taniguchi k. Wang X, Cunsing’nun JM, MeDuraell SK, Qian C, Marks AP, Slayer Sl,
Peienon BI, Smith DI, ChevilloJC, BhutsML, Jschsen J, Schaid DJ, TindsifDY, Thibodkena SN Ke Tin
(2003) Mucations in CHEK Associated with Prostate Cuncer Risk. Am J Hum Genet 72:270-280.

2. Wu X. Websler SR. und Chen J. 2001) Characterization. of iomorasociated CHIR mutations. J Bial
hem. 276:2971-4,

Appendices:

|. “The draft of our mamuseript ended "Characterizacion of CHAK? mutations (a prostate cancer" hy
2anelin Wr, Ktamgyamg Dong, Wanguo Liu, sud Junjie Chon, which seas submitted te Cancer
Roscarch (CHR? has heen named as CHEK2 by HUGO)
Characterization of CHK2 Mutations in Prostute Canece

Ninnglin Wu.! Xangyang Dong,’ Wang Lit? and Junie Chen!

"Depurimenx of Oncology. Mayo Clinie und Foundation Rachoster, MN 35905

vision of Experiment Pathology, Departecent of Laboralory Medicine ad

Pathology, Mayo Clinie*btayo Medical Scbocl, Rochester, MIN 5S905

Word cont
Absunct: 158

Intraéuction, Results and Discussion: 2530

5a whe vorsespondence should be addressed

hens: [phone]

Fax: [phone]

E-mail en junitttemyo.c

Running tile:
(Cha nctams in proto cancer

kes words: Chk2, prostate cauccr, mblilion, Zinase
ARSTRACT.

“The checkpoint kinase 2 (Chk2) is » wma aupptessor that participates in the INA

demage-signaling parhwuy. Tis hosphorylatod snd activated following DNA damage,
resulting coll eyele arrest an apoptosis. brcviously, ws idemtilill yermline mutations of

nal wo

Chk in paces with prostate cancer. ln this study, we ave idee al
somat ialems oF CK? in peste cancer patients and investigated the fictions uf
hos: mutans da ot, While most of he genie tations of Chk? an one somatic
smut (1176) have moo eve Ce? kisase ati in comparison wh wid
ype ChKd, one sometic mutalor, (721K) totaly abolishes Cok? Kinase activity. Given
chat several clini! Chk2 mutations residing inthe FHA domain of CH42, ve frther

analyved the role of Chk? FHA domain and demonstrated the requirement of an irl

PHA domain for fully nlivation of Chk2. These cosults provide evidence thal Ch
_mntetions identified in prostate cancer may contribute (athe development of prostate

cancer in sone pationts.
the abbreviations esl ure: ChK2, checkpoint kinase 2: 8Q, SQ-sichs IR, oniving

radintions PHA Furkhead-cssocinted: HA, hemagylutinia; DOD, deparuaent of velénse;

SPORR, specialized program of zescarch excellence.
INTRODUCTION

‘Maintenance e€ goncunic.inteprty depends on the coordinatiun of ecll eycle cheek swints, repair

systems and apoptosis (1), DNA damage checkpoints play important roles in maintaining geromic

Jnugrty following stesses, MuLariens in genes involved in these checkpoint pathways, ach as p33 aud
ATM, cesule in gorumve iestabilicy and cancer predisposition (2).
Checkpoint kinase 2 (ChE2) is 4 major downstream effector of ATM. As a protein kinase

involved in the DNA damage response, CHK? i rapidly phosphoryhted al Tu66 and activated in er.

ATM dependent manner folowing 1R (4) Ths it tum monies p58 sesponses (5-7). Chia diecty
phosphorylates p53 i rn, al psibly mediates p53 sabiliation folowing DNA damage 6,83
C2 also phoophorylutenbreasecencer tumor suppressor: (BRCAD, The phosphorylation of BRCAL
by C142 is mpontant forthe ails of BRCAL to eexore cel survival aller INA dontage (9) Resent

111). These phosphorylation events may

Fincings suggest that Ch sl regulates PML (Hn E
contibuts fo a p53-independent apoptosis pathwny following DNA domage

Several sunfes suggest shat Chk? ise wrner suppressor. Horerozygous geal mutations in
CK? were dette in a sbsct of -Franmeni syndrome pacts with wild-type p33 (12), suagesting
thal rotation of either p53 ot Chk? in sulcient for devclopraen wf L-trmumeai syndrome, Siri
vie p53 mutations are race in myelodysplastic systomes (MDS) and acu myloid Ikea (AML
(U3). nrecent sty identified a CHK2 moalon in PTA down and anathsr tation in C2 Klaas
cio same pation (14, ln bone aro samples fom individ with myelodysplastic
syroms (9DS), Mofhrann etal (13) found ome patient with a CHK2 mutation a eon $07 a! CIN. A
teton which lends to impired Ohk2 kinase setivty was als found in a tang cancer pti! (16)

“Three aldvianal Chis? mutations iden!ifed from patients with careinoue he hreast, colon, lung,