US 2013 O14932OA1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2013/0149320 A1 Almouzni et al. (43) Pub. Date: Jun. 13, 2013
(54) ASF1B ASA PROGNOSS MARKER AND Publication Classification THERAPEUTIC TARGET INHUMAN CANCER (51) Int. C. GOIN33/68 (2006.01) A6II 45/06 (2006.01) A613 L/7088 (2006.01) (75) Inventors: Genevieve Almouzni, Neuilly Sur Seine A638/17 (2006.01) (FR); Armelle Corpet, Paris (FR) CI2O I/68 (2006.01) (73) Assignees: CENTRE NATIONAL DE LA A 6LX39/395 (2006.01) RECHERCHE SCIENTIFIOUE, (52) U.S. C. PARIS (FR), INSTITUTCURIE, CPC ...... G0IN33/6875 (2013.01); C12O 1/6886 PARIS (FR) (2013.01); A61K.39/39558 (2013.01); A61 K 31/7088 (2013.01); A61K 38/17 (2013.01); (21) Appl. No.: 13/700,792 A61K 45/06 (2013.01) USPC ...... 424/174.1; 435/7.92; 435/6.12:506/9; (22) PCT Filed: May 31, 2011 514/44 A: 514/44 R: 514/19.3: 436/501 (86) PCT NO.: PCT/EP2011/058939 (57) ABSTRACT S371 (c)(1), The present invention provides a prognostic marker in human (2), (4) Date: Feb. 5, 2013 cancer, ASflb, a high expression thereof being associated with a poor prognosis. The present invention also provides a (30) Foreign Application Priority Data method for selecting a subject affected with a cancer for an adjuvant therapy. Finally, the present invention provides a May 31, 2010 (EP) ...... 1O164424.3 new therapeutic target for treating cancer.
Patent Application Publication Jun. 13, 2013 Sheet 2 of 26 US 2013/014932.0 A1
FIG.2A
O K4. AS 200 400 600 800 1000 Pl intensity
AS G1 G1/S S S/G2 M
... m.a...wu wa-wrix wa-ana. "" " " -26
Cyclin A - - - - - usuruoup-assists --55 p60 - un pure us uruses --72
FIG.2C
O,03 O,02 0.01 O Patent Application Publication Jun. 13, 2013 Sheet 3 of 26 US 2013/O14932O A1
Fig. 3A Celineis a : X "an PrimaryYoung fibroblastOld Senescent IMR90 or --- PO27 PD72 PD8O 1x2x 1X 2x 1x2x 1x2x -2BM (kDa) 1X 2X 4X X 2X 4X 1X 2X 4X MD Asf1 (a+b) is see s Asf1 (a+b) - sesse-res a -23 Cyclin A it is w -55 Cyclin A - m as - E. -35 CAF-1 p60 - as - - -72 CAF-1 p60 ------2 o-Tubulin up u us up us w ur-ss O-Tubulin to purveyenous uvue -ss Fig. 3B Fig. 3C
P intensity
Patent Application Publication Jun. 13, 2013 Sheet 4 of 26 US 2013/014932.0 A1
Fig. 4A Fig. 4B MCF7 120 MCF7 AS GO 2h 4h 8h 24h c X2X4XX2X4X1X2X4X 1X2X4X 1x2x4x 1x2x4x Moog As?1 (a+b)-is- a - - - - - a Cyclin A ------F - s - O
CAF-1 peo - so ------2 PCNA woo as a s - m or sor or - - - we as - o-Tubulin ------two we ------ss Fig. 4C MCF7
Fig. 4D
PI intensity Patent Application Publication Jun. 13, 2013 Sheet 5 of 26 US 2013/O14932O A1
Fig. 5A Fig. 5B
ASf1 2 3 CAF-1 s p150 200 40 PI intensity Merge
DAP Patent Application Publication Jun. 13, 2013 Sheet 6 of 26 US 2013/014932.0 A1
Fig. 6A Fig. 6B
Mammary cells 3. - f T BSt E 1X 2X 4X 1X 2X 4X MoDa & . -, -26
Asfi (a+b) - : R. '' CAF-1 peo - or . -72 O-Tubulin - - us - or up - % S phase 25% 13% Fig. 6C
ASf1
CAF-1 p150
DAP
Fig. 6D Asfa ASffb T Bst Bst
Asf1 a-p150+ Asfib+ p150+ Asfia p150- E. Asf1 b-p150 Asfa-4-p150- Asf1 b+ p150 Patent Application Publication Jun. 13, 2013 Sheet 7 of 26 US 2013/O14932O A1
Fig. 7A
siCon siAsfia siASf 1b siAsf1(a+b) g siaSf (a+b) 1x2x4x 1x2x 4x 1x2x4x 1x2x4x MoD 2's siAs?ib Asf1 (ab) - e - - - -2s (-, siAsfia. a --- mm YM - siCon. O-Tubulin - oup - upu - us use -55 Pinto: SO
Fig. 7B Fig. 7C siAsfia siASfb siGFP siAsfia siAst 1b siAsf1(a+b)
8 y
- 8 f
siAsf1(a-b)
Fig. 7D Jun. 13, 2013 Sheet 8 of 26 US 2013/014932.0 A1
Fig. 7F DAP LaminA DAP LaminA
Fig. 7G
SiCOn SiASfa SASfo SiAsf1(a+b)
siCon. siAsfah2 siAsfib+2 siAsf1(a+b)+2 Patent Application Publication Jun. 13, 2013 Sheet 9 of 26 US 2013/O14932O A1
Fig. 8A Asfa Asfb
- - 0
6- . g 2-2 2-3
-s -
s 2S N Fig. 8B
es - g g-2 is 54 s 3.g- Seasas m *::::::: 12 - - - S Fig. 8C
EGFR Neurofigina
s -- siAsfia siAst1b - - siAsfia--b - G-15 s e - -2 a - FC O-RT-PCR 03 -19 -1.6 '" -0.1 -13 -0.9 -2.2 -0.8 - 17 FC Affymetrix 0.2 -1.6 -2.2 -0.1 -0.5 -0.2 -0.6 -0.2 -O.7 Patent Application Publication Jun. 13, 2013 Sheet 10 of 26 US 2013/O14932O A1
Figure 9 siasfa GO term -log(p-value) 2.5 3. 3,5 4. 45 5 55 cytokinesis (6) organelle organization (26) Mphase of mitotic cell cycle (8) response to stress (34) anatorica structure in morphogeneSISIR YESS (9) protein complex localization (2) regulation of blood vessel size (5) regulation of systemic arterial blood pressure (4) Smooth muscle hyperplasia (2) GIS transition of mitotic cell cycle (5) endothelial cell migration (5) siasfib GO term -log(p-value) 2.5 3. 3,5 4. 4.5 5 55 DNA-dependent DNA replication (10) iron-sulfur cluster assembly (3) Y. G1 Stransition of mitotic cell cycle (8)
siasfahb GO terra -log(p-value) 2.5 3. 3.5 4. 4.5 5. 5.5
regulation of cell shape (9) cellular macromolecule catabolic process (36) intracellular transport (58 cardiac muscle cell development (4) biopolymer catabolic process (31) negative regulation of DNA metabolic process (10) aminosugar metabolic process (8) coenzyme metabolic process (4) Ci Patent Application Publication Jun. 13, 2013 Sheet 11 of 26 US 2013/O14932O A1
Fig. 10A p60/p150 Asfa/ASfb
-15 - -0.5 O 0.5 log(p60) log(Asfia)
Asfia/p60 Asf1b/p60
r O - 9 sgo Sun g", cy
t -1 O 1. 2 -2 - O 2 log(p60) log(p60) Asfa/Ki67 Asfb/Ki67
s s ge Se.g
-1.5 - -0.5 log(Asfia)O O5 -2 log(Asf1b)- Fig. 10B OVeral Survival p=0.01
c
ce s 2 cc s ce wit ed
c cs o Aso as 0.7 . . , AS O.7
O 24 48 72 96 20 144 Time (months) Nunbeat risk As K-7 57 53 49 46 44 42 3. Asby O.7 28 18 15 Patent Application Publication Jun. 13, 2013 Sheet 12 of 26 US 2013/0149320 A1
Fig. 11A Tumor size Asfia
s r go ge g 3: g". cy
t - TO/T T2 p=0.06 Mitotic index
p w g se g 3. 33 g T cy t
Grade
goun a o 3. 3. g r y te p=0.06 Fig. 11B Disease free interval p=0.017 Metastasis free interval p=0.0002
2 s 8 O i.
O 24 48 72 S6 120 44 0 24 48 72 96. 120 144 Time (months) - Time (months) Nui Title at risk Nutbeat risk Asif K07 57 53 46 40 37 33 19 Asfb as 0.7. 57 53 49 46 44 41 29 Asix O.7 28 23 19 15 4. 13 10 AS-07 28 24 22 17 16 15 12 Patent Application Publication Jun. 13, 2013 Sheet 13 of 26 US 2013/0149320 A1
Fig. 12A Tumor grade Distant metastasis at 5 years Disease Free survival 5 years p=3x10-5 p=1.7x10-5 p=5.9x10-5 1. 1.5 20 ge 1. se g o 51. 1.0 s S 30.5 2 a 0. Ot g 0. o 300 iosas s CA. 0.5 e 31. c 0. e -1. 1.5 -2. 3-0. s:-20 -2. -3.0 -3.0 -1.0. -3.5 no distant distant no disease relapse metastasis metastasis (n=9) (n=33) (n=33) (n=154) (n=35) (n=196) (n=79) Fig. 12B Skin Breast Liver p=1.5x10-14 p=2.6x10-5 1.0 p=1.1x10-8 1.5 0.5 2 2 s g s g 0. 51. 3 5-05 e e C 1. 0. So. 5-1.5 d e s c 3-2
2. 2 2-3. -3.5 -0.5 -0.5 -4. normal benign cutanenous normal breast non-tumor hepatocellular Skin nevus melanoma breast carcinoma liver carcinoma (n=7) (n=18) (n=45) (n=7) (n=40) (n=76) (n=104) Ovarian Lung p=6.7x10-5 p=1x10-7
1.
25" e.g.05 < 0. 3. Soo g s 0. 2-0.
-0.5 -10 normal ovarian normal lung ovary SeOUS lung adenocarcinoma adenocarcinoma (n=4) (n=41) (n=31) (n=31) Patent Application Publication Jun. 13, 2013 Sheet 14 of 26 US 2013/0149320 A1
Fig. 13A Cox MODEL FOR METASTASIS FREE INTERVAL (n = 73) Variables included in the model: mitotic index (qualitative), tumor size (qualitative and quantitative), tumor grade, Asf1b, CAF-1 peo, CAF-1 p150, HP1, Ki67 Wariables RR 95% CI p-value Asf1b. ASF 1b a 0.70 1 ASF 1b > 0.70 71 2.0 - 26.0 8.66x10-4
cox MoDEL FOR DISEASE FREE INTERVAL (n = 73) Variables included in the model: mitotic index (qualitative), tumor size (qualitative and quantitative), tumor grade, Asf1b, CAF-1 peso, CAF-1 p150, HP1, Ki67 Variables RR 95% C. p-value CAF-1 p50 CAF-1 p60s 0.60 1 - - CAF-1 péO > 0.60 5.5 2.5 - 11.9 6.58x10-5
COX MODEL FOR OVERALL SURVIVAL (n = 73) Variables included in the model age, mitotic index (qualitative), tumor size (quantitative), hormonal receptor status (ERPR), Asf1b, CAF-1 peso, CAF-1 p150, HP1, Ki67 Watiables RR 95% Ci p-value CAF-1 peo CAF-1 p50s 0.60 1 CAF-1 péO > 0.60 12.9 2.6 - 64.2 5.95x10-4 Fig. 13B Asfb 1.8x10-5 19x10-4 57.11 7x10-6
BC LUMNAL MEC MICRO NORMA BLC UMINAL MBC MICRO NORMAL Group Group Patent Application Publication Jun. 13, 2013 Sheet 15 of 26 US 2013/014932.0 A1
FIGURE 14 Age p = 0.034 Mitotic index p = 3e-04
as s o
as 10 mitosis >10 mitosis
Elston-Ellis grade (mitosis) p = 2e-04 Elston-Ellis grade p = 0.002
e a g 3. 3.
t EE- EE- EE-i
Hormonal receptors p = 0.01 Triple negative p = 0.059
2S s g g g 3.
ER+ and of PR- ER- and PR Patent Application Publication Jun. 13, 2013 Sheet 16 of 26 US 2013/O14932O A1
FIGURE 15 Metastasis free survival (p=0.018) Disease free interval (p=0.045) 100 100
80 - 80
e 60 - 60
fd 9. 40 - 40
20 - 20
O O O 12 24 36 48 6O 72 84 96 08 32 156 0 2 24 36 48 60 72 84 96 108 132 15S Time (Months) Time (Months) number at risk number at risk - 34 33 32 3, 28 25 24 23 22 21 is 17 15 5 - 34 33 32 3i 28 22 21 20 19 9 6 4 12 3 35 34 33 30 29 28 28 26 24 22 18 15 12 4 - 35 34 32 27 24 23 22 19 7 6 13 11 9 3 Disease free survival (p=0.018) 100
80
60
40
20
O 12 24 36 48 so 72 84 96 08 132 156 Tire (Months) number at risk - 34 33 32 3 28 22 2 20 S 19 16 14 2 3 - 35 34 32 27 24, 23 22 19 17 16 13 11 9 3 Patent Application Publication Jun. 13, 2013 Sheet 17 of 26 US 2013/O14932O A1
FIGURE 16
Cut-Off Wale 0.7
O TurnOurS TurnOurS MCF-7 BU "low Asf1b" "high Asf1b" Breast tumour samples from 1995 Patent Application Publication Jun. 13, 2013 Sheet 18 of 26 US 2013/014932.0 A1
Fig. 17A so HS578T 48h Of SiRNA sSN {, -Ss is es 2 s 2S. \ 9 S & S S S S. 1X 2X 1X2X 1x2x 1X 2x 1x2x 1x2x 1X2X MoDa Asf1(a+b)a=sese so - - -o o-tubulin
Fig. 17B
CN O CN 1A. N. cus CN C. CN 1S v. S $ is SS 2 32is S Sis its 5 to S to3. it S.is t 5 g g c) Patent Application Publication Jun. 13, 2013 Sheet 19 of 26 US 2013/0149320 A1
SiASfail2
Fig. 18C
DAP LaminA
SiCOn g 3. ts o siasfah? 25ac
s as
siAsf1b siAsf1(a+b). se:
Patent Application Publication Jun. 13, 2013 Sheet 21 of 26 US 2013/O14932O A1
COX MODEL FOR METASTASIS FREE INTERVAL (n = 62) Variables included in the model: mitotic index (qualitative), menopause status, ablation surgery quality, progesteron receptor status, Asf1b, Ki67 Variables RR 95% CI p-value ASf 1b ASf1b < 0.3 1 ASf1 b > 0.3 5.7 1.3 - 25.7 O.024 Menopaused 1 D Non menopaused 4.2 14 - 12.8 O.O13
COX MODEL FOR DISEASE FREE INTERVAL (n = 62) Variables included in the model: mitotic index (qualitative), tumor size (qualitative), menopause status, Asf1b, CAF-1 p60, Ki67 Variables RR 95% C p-value Asfb ASf 1b < O-3 1 ae r ASf b > 0.3 3.24 1.2 - 8.5 O.O163 Menopaused 1 a Non menopaused 2.59 1.1 - 6.2 O.O322
COX MODEL FOR OVERALL SURVIVAL (n = 62) Variables included in the model: mitotic index (dualitative), ablation surgery quality, Asf1b, CAF-1 p60, Ki67 Variables RR 95% C. p-value K67 Ki67 340 1 Ki67 > 40 6.18 1.6 - 24.0 0.008 Complete ablation surgery 1 re Incomplete ablation surgery 4.O 10 - 15.4 O.O46
Figure 20 Patent Application Publication Jun. 13, 2013 Sheet 22 of 26 US 2013/0149320 A1
Fig. 21A O 20 30 40 50 60 70 80 90 100 Consensus Asfia AKVOYNNWLINPSPFYPFGFEITFEEDLSEDLEkIINGSAEEEYDQVLDSLVGPvPAGR4 VFQADAPPGLIPDADANGVIWLITc Consensus Asfib AKVSVNVAVENPSPFHSPFRFEISFECSEALADDFWKTTYWGSAESEEFDQIDSWVGPWPAGRHMFVFQADAPNPSLIPETDAVGWTWITCT - 10 120 130 140 150 10 170 130 190 290 Consensus Asfia YRGQEFIRVGYYVNNEYTETELRENPPVKPDFSKLQRNILASNPRVTRFHINEDNTEKLEDAESSNPNLQSLSTDALPSASKGISTSENSLNVLESHDCM Consensus Asfib YHGQEFIRVGYYVNNEYPNPELRENPPKPOFSQLQRNILASNPRVTRFHINADNNDRLEAIENQDPSLGCGLPLSCTP--IKGLGLPGCIPGLLPENSIDCI s: it is a 4 ex: sk...... highly divergentamino acids 190 200 : mildly divergent amino acids
Mildly divergent a amino acids between Asfa and Asfib
Histone interacting region hAsfa (1-156) Structure
D 180° Highly divergent A. . . .Y. amino acids s between Asfa and Asf1b HIRA/CAF-1 --Y interacting region
Fig. 21C & Sëys M a-la P ASf b-u M - 17kDa HRA - 130kDa CAF-1 peo ---up -72kDa Patent Application Publication Jun. 13, 2013 Sheet 23 of 26 US 2013/O14932O A1
Fig. 22A Flowthrought Bound (10%) (100%)
ers m m9 Š 5 : 33 is 3S N- it E is E is 8 as to z8 & a 3 Ö 33 3 £5 ög
Fig. 22B GST HRAB GST HRA B domain domain 461D
–2
1.
EtBr Patent Application Publication Jun. 13, 2013 Sheet 24 of 26 US 2013/O14932O A1
Asfa/As Asfb/CAF-1 peso
s e t g
s cor=0.52 ps1.19e-09 f - -4 -3 2 - 2 togasfit log.caf As HURP As/cm2
e e 3. 3.
cor=0.83 p=6.45e-32
Figure 23 Patent Application Publication Jun. 13, 2013 Sheet 25 of 26 US 2013/O14932O A1
Histological grade Histological stage HEHy
p = 2e-04
grade 1 grade 2 grade 3 stage stage i Stage ilia-lib Stage lic-IV Timor resection Family antecedents of breast cancer
complete incomplete ou
Figure 24 Patent Application Publication Jun. 13, 2013 Sheet 26 of 26 US 2013/O14932O A1
Pleomorphic liposarcoma p=9.6e-10
2. O
1.5
1.O
0.5. Gastric adenocarcinoma 0.O. p=2.1e-9
-0.5 Pancreatic carcinoma p=3.2e-7
10 ; -5 Gastric mucosa (n-31) Gastricadenocarcinoma (R-26) infiltrating bladder urothelial carcinoma
p=1.6e-14 1 5. 3.5 -2.0- Pancreas ?n=6 Pancreatic carcinoma (n-36) 3.0 2.5 Vulvar intraepithelial neoplasia 2.0 e3.2e-6 15 10 0.5
Colon adenoma
Wulva (n=10) Vulwar neoplasia (n=9)
Gioblastona =4.3e-1 3
Fiore a US 2013/O 14932O A1 Jun. 13, 2013
ASF1B ASA PROGNOSS MARKER AND 0006. Histone proteins form the core of the repeated unit THERAPEUTIC TARGET INHUMAN of chromatin, the nucleosome, in which 146bp of DNA is CANCER wrapped aroundan octamer comprising (H3-H4-H2A-H2B) histones. They are handled by histone chaperones (De Koning FIELD OF THE INVENTION et al., 2007), which are critical for histone dynamics during DNA replication (Corpet and Almouzni, 2009; Ransometal. 0001. The present invention relates to the field of medi 2010). Chromatin Assembly Factor 1 (CAF-1), a complex of cine, in particular of oncology. It provides a new prognostic three polypeptides RbAp48, p.60 and p150 in mammals, is the marker in human cancer. primary histone chaperone involved in the deposition of H3-H4 histones coupled to DNA replication or repair (De BACKGROUND OF THE INVENTION Koning et al., 2007). Interestingly, CAF-1 pé0 is a prolifera 0002 Cancer occurs when cell division gets out of control tion marker with diagnostic and prognostic value in breast and results from impairment of a DNA repair pathway, the cancer (Polo et al., 2004; Polo et al., 2010; Mascolo et al., transformation of a normal gene into an oncogene or the 2010; Staibano et al., 2011). CAF-1 subunits as well as malfunction of a tumor Supressor gene. Many different forms CAF-1 partners; ASfla (Anti-silencing function 1), Asflb, of cancer exist. The incidence of these cancers varies but it PCNA (Proliferating Cell Nuclear Antigen), and HP la (Het represents the second highest cause of mortality, after heart erochromatin protein 1) (see below) are more abundantly disease, in most developed countries. While different forms expressed in tumoral versus normal cells (WO 2005/085860; of cancer have different properties, one factor which many Polo et al., 2004). cancers share is the ability to metastasize. Distant metastasis 0007. In the present invention, the inventors focused on the of all malignant tumors remains the primary cause of death in histone H3-H4 chaperone Asfl. First identified by its ability patients with the disease. to de-repress transcriptional silencing when overexpressed in 0003. The therapeutic care of the patients having cancer is yeast, ASfl has been implicated in transcriptional regulation primarily based on Surgery, radiotherapy and chemotherapy in yeast and Drosophila. Current data Suggest a conserved and the practitioner has to choose the most adapted therapeu role of Asfl during DNA replication as a histone donor for tic strategy for the patient. In the majority of the cases, the CAF-1 which in turn deposits histones onto DNA. However, choice of the therapeutic protocol is based on the anatomo while a single isoform of ASfl is present in yeast, higher order pathological and clinical data. Currently, the methods to organisms such as plants, worms or mammals possess more determine prognosis and select patients for adjuvant therapy than one gene encoding Asfl. Phylogenetic studies indicate rely mainly on pathological and clinical staging. However, it that the distinction of two Asfl iso forms, called Asfla and is very difficult to predict which localized tumor will eventu Asflb, is specific to mammals. Their highly conserved N-ter ate in distant metastasis. Indeed, due to insufficiently accurate minus provides a binding interface with the H3-H4 histones prognosis predictions, a Substantial proportion of cancer Sub (De Koning et al., 2007), and their most divergent C-terminal jects with inherently good outcome receive adjuvant systemic part is less characterized (FIG. 1A). Both Asfl isoforms can therapy without gaining any benefit. interact with p60, the mid-subunit of CAF-1. Collectively, the 0004. Therefore, there is a great need for the identification two human Asf1 isoforms have both been implicated in buff of prognostic markers that can accurately distinguish tumors ering the transient overload of replicative histone H3.1 that associated with poor prognosis including high probability of accumulates during replication stress, as well as in the control metastasis, early disease progression, increased disease of S phase progression (Grothet al., 2005; Groth et al., 2007). recurrence or decreased patient Survival, from the others. Found in the nucleus, in complex with the MCM2-7 proteins, Using Such markers, the practitioner can predict the patients the putative helicase that unwinds DNA ahead of the replica prognosis and can effectively target the individuals who tion fork, human ASfla and ASf1b participate in regulating would most likely benefit from adjuvant therapy. replication fork progression (Groth et al., 2007; Jasencakova 0005. The understanding of the molecular basis of cancer et al., 2010). This is likely caused by defects in the transfer of has advanced tremendously with the identification of muta parental histones. Notably, defects in S phase progression tions in the genome of tumor cells. Yet, while numerous occurred only upon combined depletion of both Asf1 iso studies Support a major role for genetic events in cancer forms, indicating that ASfla and ASflb can Substitute/com Susceptibility, in particular for breast cancers, this genetic pensate for one another in this function. However, the pos contribution alone does not explain the clinical complexity sible compensation of one iso form by the other does not and heterogeneity of cancers, therefore suggesting that other exclude a regulation of task distribution under normal situa mechanims may contribute to the process of tumorigenesis tions. The higher expression of ASflb in human tissues such and aggressiveness. A current challenge is to find how, as thymus or testis does provide a first hint in this direction beyond genetic alterations, changes in the higher order (Umehara and Horikoshi, 2003). Furthermore, human Asfla nuclear organization of DNA into a complex chromatin struc proves most efficient to rescue defects in the DNA damage ture participate in tumorigenesis. Abnormal gene expression response in yeast depleted of endogenous ASfl, while human (mostly gene silencing) in cancer cells associates with Asflb compensates for the growth defects and the sensitivity changes in DNA methylation and aberrant histone post-trans to replicational stress (Tamburini et al., 2005). In mammalian lational modifications in corresponding promoter regions. In cells, a specific interaction of Asfla with HIRA is critical for addition, genome-wide changes of specific histone modifica senescence-associated cell cycle exit (Daganzo et al., 2003; tions are predictive of clinical outcome in specific cancers Tang et al., 2006: Zhang et al., 2005). In addition, Asfla has (Kurdistani, 2007). Thus, to consider how particular alter been involved in the regulation of H3K56 acetylation in ations in chromatin organization and histone dynamics occur human cells (Das et al., 2009;Yuan et al., 2009) which is a key in cancer and how they could promote tumorigenesis is of histone mark during processes such as replication or repair. major interest. Taken together, while the distinct Asfl iso forms share the US 2013/O 14932O A1 Jun. 13, 2013 common molecular properties enabling them to handle 0012. Accordingly, in a first aspect, the present invention H3-H4 histone pools, how they exploit these properties in concerns a method for predicting or monitoring clinical out different context by involving a regulated distribution of tasks come of a subject affected with a cancer, wherein the method between the two Asfl iso forms remained still poorly char comprises the step of determining the expression level of acterized. ASflb in a cancer sample from said subject, a high expression 0008 DNA microarrays have compared normal cells to level of Asflb being indicative of a poor prognosis. Prefer breast cancer cells and found differences in hundreds of ably, a poor prognosis is a decreased patient Survival and/or genes, but the significance of most of those differences is an early disease progression and/or an increased disease unknown. Several Screening tests are commercially mar recurrence and/or an increased metastasis formation. keted, but the evidence for their value is limited. The only test 0013. In a second aspect, the present invention concerns a supported by Level II evidence is Oncotype DX, which is not method for selecting a subject affected with a cancer for a approved by the U.S. Food and Drug Administration (FDA) therapy, preferably an adjuvant therapy, or determining but is endorsed by the American Society of Clinical Oncol whether a subject affected with a cancer is susceptible to ogy. MammaPrint (vant Veer et al., 2002) is approved by the benefit from a therapy, preferably an adjuvant therapy, FDA but is only supported by Level III evidence. Two other wherein the method comprises the step of determining the tests have Level III evidence: Theros and MapQuant DX. No expression level of ASflb in a cancer Sample from said Sub tests have been verified by Level I evidence (in a prospective, ject, a high expression level of ASflbindicating that atherapy, randomized controlled trial, patients who used the test had a preferably an adjuvant therapy, is required. better outcome than those who did not). In a review, Sotirou 0014. In a last aspect, the present invention concerns a concluded, "The genetic tests add modest prognostic infor method for monitoring the response to a treatment of a subject mation for patients with HER2-positive and triple-negative affected with a cancer, wherein the method comprises the step tumors, but when measures of clinical risk are equivocal (e.g., of determining the expression level of Asf1b in a cancer intermediate expression of ER and intermediate histologic sample from said subject before the administration of the grade), these assays could guide clinical decisions'. Various treatment, and in a cancer sample from said Subject after the gene expression signatures including ASfla or ASflb for can administration of the treatment, a decreased expression level cer prognosis have been disclosed. For instance in WO07/ of Asflb in the sample obtained after the administration of the 070621, a signature comprising at least 25 genes to be treatment indicating that the Subject is responsive to the treat selected among a list of 100 genes (including Asflb) for ment. predicting solid tumor outcome has been disclosed, without 0015. In one embodiment, the expression level of Asf1b is any supporting data. Rosty et al., 2005 disclosed a cervical determined by measuring the quantity of ASflb protein or cancer proliferation gene cluster composed of 163 highly ASfb mRNA. correlated transcripts among which Asf1b. In addition, Wira 0016. In a further embodiment, the quantity of Asf1b pro pati et al (2008) disclose a prognosis signature for breast tein is measured by immuno-histochemistry, semi-quantita cancer of 355 genes including Asflb. tive Western-blot or by protein or antibody arrays. 0009. A comprehensive analysis of gene-expression 0017. In another further embodiment, the quantity of based classifiers recently compared nine gene expression sig ASflb mRNA is measured by quantitative or semi-quantita natures thought to be associated with breast cancer outcome tive RT-PCR, or by real time quantitative or semi-quantitative (Regal et al., 2008). Overall, the nine signatures had similar RT-PCR or by transcriptome approaches. performance in terms of assigning a sample to either a poor 0018. In another embodiment, the methods of the inven outcome group or a good outcome group. However, Such tion comprise the step of comparing the expression level of signatures exhibited a large degree of discordance with 50% Asflb to a reference expression level. Additionally, the meth of the samples receiving at least one outcome assignment that ods of the invention further comprise the step of determining is discordant with the assignments of the other signatures. In whether the expression level of Asf1b is high compared to addition, no signature showed strong association with Sur said reference expression level. vival when applied to subgroups of tumor with aggressive 0019. In a further embodiment, the reference expression feature i.e. lymph node positive, ER negative or high grade. level is the expression level of Asf1b in a normal sample. 0010. Therefore, due to the adverse effects of chemo 0020. Alternatively, the reference expression level is the therapy and radiotherapy, there is still astrong need to provide expression level of a gene having a stable expression in dif the most accurate prognosis as possible to avoid the unnec ferent cancer samples, such as the RPLPO (ribosomal protein essary treatment of patients. Po-like protein) gene. 0021. In still another embodiment, the method according SUMMARY OF THE INVENTION to the invention further comprises assessing at least another 0011. The inventors demonstrated a clear association of cancer or prognosis marker Such as tumor grade, hormone human Asflb, but not Asfla, with proliferation capacity and receptor status, mitotic index, tumor size, HJURP (Holliday the prognostic value of ASflb in early stage cancer. Remark junction recognition protein, also called DKFZp762E1312, ably, high ASflb expression levels significantly correlates URLC9, hFLEG1, FAKTS) expression level or expression of with the tumor proliferation status, and with a poor prognosis proliferation markers such as Ki67 (antigen identified by outcome including the appearance of metastasis, increased monoclonal antibody Ki-67), MCM2 (minichromosome disease recurrence, and the overall Survival of the patients. maintenance 2, also called D3S3194, KIAA0030, BM28, Asflb thus represents a new proliferation marker, which is cdcl.9), CAF-1 pé0 (also called CAF1B or CHAF1B for Chro relevant for the prognosis in cancer. In addition, inhibition of matin assembly factor 1, subunit B) and CAF-1 p 150 Asflb expression was shown to decrease cell proliferation, (CAF1A or CHAF1A for Chromatin assembly factor 1, sub highlighting the role of ASflb as a new target for drug dis unit A) or prognosis marker Such as HP1C. (heterochromatin covery in cancer or for treating cancer. protein 1-alpha, CBXS). US 2013/O 14932O A1 Jun. 13, 2013
0022. In addition, the present invention concerns the use of (gi74735206) located on chromosome 6q22, and Asf1b ASflb as a prognosis marker in cancer, as a marker for select (gi74734533) located on chromosome 19p 13, was performed ing a subject affected with a cancer for a therapy, preferably using ClustalW software (www.ebi.ac.uk/Tools/clustalw2/). an adjuvant therapy, or determining whether a subject Secondary structures in the conserved N-terminal region are affected with a cancer is susceptible to benefit from a therapy, indicated above the sequences. The C-terminus of Asfla and preferably an adjuvant therapy, or as a marker for monitoring Asflb is more variable, harboring potential phosphorylation the response to a treatment of a subject affected with a cancer. sites. Amino-acids that differ between Asfla and Asflbare in 0023. In still another embodiment, the cancer is a solid grey. Asterisks: identical residues, double dots: conserved cancer or a hematopoietic cancer, preferably a solid cancer residues; single dots: semi-conserved substitutions. (lower and more preferably, an early stage solid cancer without local panel) Scheme depicting the percentage of homology or systemic invasion. Preferably, the cancer is selected from between the different parts of Asfla and Asf1b. Specific anti the group consisting of breast cancer, osteosarcoma, skin bodies against Asfla were raised against the full GST-Asfla cancer, ovarian cancer, lung cancer, liver cancer, cervix can protein, whereas specific Asf1b antibodies were raised cer, liposarcoma, gastric cancer, pancreatic cancer, bladder against a GST-C-Term-Asf1b (amino-acids 156 to 202). FIG. cancer, Vulvar cancer, colon cancer and brain cancer. More 1B: Western blot analysis of the two Asf1 antibodies on preferably, the cancer is breast cancer. Even more preferably, recombinant His-C-Terminal Asfla and His-C-Terminal the cancer is an early stage breast cancer without local or Asflb showing a high specificity of these two antibodies on systemic invasion. recombinant proteins. M: molecular weight marker. FIG. 1C: 0024. The present invention also concerns a kit (a) for Western blot analysis of Asflantibodies on total extracts from predicting or monitoring clinical outcome of a subject human U-2-OS cells, depleted of Asfla, Asflb, or Asfla+b by affected with a cancer; and/or (b) for selecting a subject siRNA for 48 h. Increasing amounts (x) of total cell extracts affected with a cancer for a therapy, preferably an adjuvant are loaded and C-tubulin serves as a loading control. While therapy, or determining whether a subject affected with a Asfla antibody #28134 highly recognizes Asfla and detects cancer is susceptible to benefit from a therapy, preferably an a faint band of Asf1b, Asf1b antibody #18143 is highly spe adjuvant therapy; cific. M: molecular weight marker. FIG. 1D: IF analysis of 0025 and/or (c) for monitoring the response to a treatment U-2-OS cells treated as in (C) underscores the high specificity of a subject affected with a cancer, wherein the kit comprises of the two Asf1 antibodies. Scale bar is 20 Lum. (i) at least one antibody specific to Asf1b; and/or (ii) at least (0029 FIG. 2. Asfla and Asf1b levels across the cell cycle one probe specific to the Asflb mRNA or cDNA and/or (iii) at in HeLa cells. FIG. 2A: Flow cytometry analysis of HeLa least one nucleic acid primer pair specific to Asf1b mRNA or cells asynchronously growing (AS) or released from a cDNA and optionally, a leaflet providing guidelines to use double-thymidine block at the following times: Oh (G1/S), 4 such a kit. Preferably, the kit further comprises means for h (S), 8 h (S/G2), and 14 h (G1). Mitotic cells (M) were detecting the formation of the complex between Asflb and collected after a 20h nocodazole block. FIG.2B: Westernblot said at least one antibody specific to ASflb; and/or means for analysis of Asfla and Asf1b levels in synchronized HeLa detecting the hybridization of said at least one probe specific cells treated as in (A). Increasing amounts (X) of total cell to the Asf1b mRNA or cDNA on Asf1b mRNA or cDNA; extracts are loaded. Cyclin A, CAF-1 p60, and PCNA are and/or means for amplifying and/or detecting said ASflb shown for comparison. M: molecular weight marker. FIG. mRNA or cDNA. 2C: Quantitative RT-PCR analysis of Asfla and Asf1b mRNA 0026. The invention further concerns methods for select levels across the cell cycle in HeLa cells. Levels are normal ing or identifying a molecule of interest capable of inhibiting ized to the reference gene ribosomal protein Po-like protein Asflb, preferably to selectively inhibit it. Therefore, the (RPLPO) (de Cremoux et al., 2004). (a.u.): arbitrary units. present invention concerns a method for selecting or identi Error bars represent data from two independent experiments. fying a molecule useful for the treatment of cancer, in par ticular for improving the clinical outcome of a patient having 0030 FIG. 3. Expression of Asf1b depends on the cycling cancer, comprising testing a molecule for its ability to inhibit status of cells. FIG. 3A (left panel) Western blot analysis of Asflb and selecting the molecule capable of inhibiting Asflb. total cell extracts from non treated asynchronous (AS) and Preferably, the ability of the test molecule to inhibit Asflb is quiescent (GO) MCF7 breast cancer cells and BJ primary measured by a binding assay to ASflb protein, by an assay foreskin fibroblasts. (right panel) Comparison of the behav measuring the ASflb expression in a cell, by a nuclear mor iour of Asfl isoforms in primary IMR90 human diploid fibro phology assay, by a Colony Formation Assay, by an in vitro blasts young (PD27), old (PD72) and senescent (PD80). Increasing amounts (X) of total cell extracts are loaded. C-tu nucleosome assembly assay or by a combination thereof. bulin is a loading control. Asfla and Asflb have been revealed 0027 Finally, the present invention concerns a molecule with a mix of the specific Asf1 antibodies (FIG. 1 and Table inhibiting Asf1b, preferably through direct interaction with 3). CAF-1 pé0 and Cyclin A have been used as markers for Asflb, preferably selectively in respect to Asfla, for use in cell proliferation. M: molecular weight marker. (See also the treatment of cancer, in particular a cancer with a high FIG. 2). FIG. 3B: Specific expression of Asfla, Asflb (FIG. expression level of Asflb. Preferably, the molecule inhibiting 1) and the largest subunit of CAF-1 (p150) revealed by immu ASflb is selected from the group consisting of a small mol nofluorescence in MCF7 cells asynchronous (AS) or quies ecule, anaptamer against ASflb, an antibody against ASflb, a cent (GO). DAPI stains nuclei. Scale bar is 20 um. FIG. 3C: nucleic acid against ASflb, a molecule preventing the inter Flow cytometry analysis of the cell cycle distribution of action between Asf1b and one of its binding partners. MCF7 and BJ cells asynchronous (AS) or quiescent (GO). FIG. 3D: (left panel) Asfla and Asf1b mRNA levels in pro BRIEF DESCRIPTION OF THE DRAWINGS liferating (AS), quiescent (GO) and MCF7 cells and BJ pri 0028 FIG.1. Specificity of Asflantibodies. FIG. 1A: (up mary fibroblasts as determined by Quantitative RT-PCR. per panel) Alignment of the two ASfl isoforms, ASfla (right panel) Asfla and Asf1b mRNA levels in young, old or US 2013/O 14932O A1 Jun. 13, 2013
senescent IMR90 human diploid primary fibroblasts as deter staining for the given protein. Black arc lines indicate an mined by Quantitative RT-PCR. Levels have been normalized absence of correlation between Asfla/b presence and CAF-1 to the reference gene ribosomal protein Po-like protein p150 presence. Numbers represent the mean of 3 independent (RPLPO) (de Cremoux et al., 2004) and levels in proliferating experiments (n=100 nuclei counted by experiment). cells are set to 100%. The error bars represents.d. from 2-3 independent experiments. 0037 FIG. 7. Distinct effects of Asfla and Asf1b deple tions. FIG. 7A Specific depletion of Asfl isoforms. (Left 0031 FIG. 4. Asflb levels increase upon re-entry into the panel) Western blot analysis of total extracts from human cell cycle. FIG. 4A Westernblotanalysis oftotal MCF7 breast U-2-OS cells showing the specific depletion of Asfla, Asf1b cancer cell extracts from asynchronous (AS), quiescent (GO) or Asfl(a+b) for 48 h by siRNA treatment. Increasing or cells released from quiescence for the indicated times (2, 4, amounts (X) of total cell extracts are loaded and C-tubulin 8 and 24 hours). For increasing amounts (X) of total cell serves as a loading control. A mix of the specific ASfl anti extracts, we revealed Asfla and Asflb with a mix of the bodies (FIG. 1) reveals Asfla and Asf1b. M: molecular specific ASfl antibodies. C-tubulin was used as a loading weight marker. (Right panel) Flow cytometry analysis of the control, CAF-1 pé0, PCNA and Cyclin as markers for cell cell cycle distribution of the cells shown in the left panel. FIG. proliferation. M: molecular weight marker. FIG. 4B Asfla 7B Venn diagram showing the overlap between the signifi and cantly (p<0.05) differentially expressed genes determined in 0032. Asflb mRNA levels in proliferating (AS), quiescent each siRNA condition indicated (siAsfla, siAsflb, and (GO) and MCF7 cells released from GO are determined by siAsf1(a+b)) versus the control siRNA. Numbers indicate the Quantitative RT-PCR. Levels are normalized as in FIG.3. The quantity of genes overlapping between 2 conditions. (See also error bars represent S.d. from 3 independent experiments. FIGS. 8 and 9). FIG.7C Colony Formation Assay for HeLa FIG. 4C Specific expression of Asfla, Asflb and the largest cells treated with the indicated siRNA. The mean surviving subunit of CAF-1 (p150) revealed by immunofluorescence in fraction (%) is indicated in the histograms below. Error bars MCF7 cells asynchronous (AS), quiescent (GO), or released represent data from 3 independent experiments. FIG. 7D from GO for the indicated times. DAPI stains nuclei. Scale Cellular defects upon specific depletion of Asf1 isoforms. bar is 20 Lum. FIG. 4D Flow cytometry analysis of the cell Immunofluorescence analysis of U-2-OS cells treated as in cycle distribution of the cells shown in FIG. 4A. (A). DAPI stains nuclei. Scale bar is 20 Lum. FIG. 7E Histo 0033 FIG. 5. Asfl expression levels in mammary cell grams show quantitative analysis of the proportion of aber lines. FIG. 5A Flow cytometry analysis of the breast cancer rant nuclear structures in U-2-OS cells treated as in (A). The cell line HSS78T(T) and the non-tumoral mammary cell line mean percentage of altered nuclei (lobulated) and the percent 0034). Hs578Bst (Bst), which are derived from the same age of micronucleated cells after 48 h of siRNA treatment are patient, in order to assess polyploidy. Tumoral (T) and normal plotted. Evalues indicate the standard deviation from counts (Bst) cells contain 25% and 13% of cells in S phase respec of three and two independent experiments, respectively. FIG. tively (De Koning et al., 2009). FIG.5B Expression of Asfla, 7F Immunofluorescence analysis of LaminA staining in U-2- Asflb and CAF-1 p 150 is revealed by immunofluorescence OS cells treated as in (A). DAPI stains nuclei. Scale bar is 10 analysis of tumoral (T) and normal (Bst) mammary cell lines. m. FIG. 7G Colony Formation Assay for U-2-OS cells Total levels of each protein are visualized as cells were not treated with two independent sets of siRNAs. The mean Sur pre-extracted before fixation. Arrowheads indicate nuclei viving fraction (%) is indicated in the histogramms. Error negative for CAF-1 p 150 staining but positive for ASfla stain bars represent data from 2 independent experiments. ing Normal arrows point out nuclei double negative for CAF-1 p 150 and Asf1b staining, underscoring that Asf1b 0038 FIG. 8. Validation of transcriptomic data. FIG. 8A levels parallels the proliferative status of the cells. Nuclei Quantitative RT-PCR analysis of Asfla and Asf1b mRNA were stained with DAPI. Scale bar is 20 m. levels in U-2-OS cells depleted for Asfla, Asflb or Asf1(a+b) 0035 FIG. 6. Asf1b levels reflect the proliferating status of for 48 h by siRNA treatment. mRNA levels are normalized to breast cell lines. FIG. 6A the reference gene Glyceraldehyde 3-phosphate dehydroge 0036) Asfla and Asf1b levels analyzed by Western blot nase (GAPDH) and expressed as the log2(fold change) rela analysis of total extracts from the tumoral (T) and the normal tive to the control siRNA. The error bar represents data from (Bst) mammary cell lines. Asfl(a+b) were revealed with a three independent experiments. FIG. 8B mRNA extracted mix of the specific Asf1 antibodies and CAF-1 p60 is shown from human U-2-OS cells treated as in(A) were hybridized to for comparison. Increasing amounts of cell extracts (X) are Affymetrix HG-U133-Plus2 oligonucleotide microarrays. loaded. C-tubulin is a loading control. M: molecular weight mRNA expression levels of Asfla and Asflb obtained from marker. The percentage of cells in S phase (FIG. 5A) is the Affymetrix hybridization are expressed as a log2(fold indicated below the Western Blot. FIG. 6B Quantitative RT change) relative to the control siRNA depletion. Error bars PCR analysis of Asfla and Asflb mRNA levels in tumoral (T) represents data from two independent experiments. FIG. 8C and normal (Bst) mammary cell lines. Levels were normal Quantitative RT-PCR analysis of mRNA levels for the indi ized to the reference gene RPLPO and set to 100% in normal cated genes in U-2-OS cells treated as in (A). mRNA levels cells. Error bars represents.d. from 3 independent experi are normalized to the reference gene GAPDH and expressed ments. FIG. 6C Specific expression of Asfla, Asf1b and as the log2 fold change relative to the control siRNA. The CAF-1 p150 revealed by immunofluorescence analysis of error bar represents data from three independent experiments tumoral (T) and normal (Bst) mammary cell lines. Total levels respectively. Below each graph is indicated the numerical of each protein are visualized as cells were not pre-extracted value for the mean log2 fold change (FC) obtained by Q-RT before fixation. Dapi stains nuclei. Scale bar is 10 um. (See PCR or on the Affymetrix microarray. also FIG. 5B). FIG. 6D Pie charts show quantitative analysis 0039 FIG. 9. Gene Ontology (GO) analysis of differen of the proportion of cells in each of the categories indicated. tially expressed genes after the specific knock-down of ASfla + indicates a positive staining while - indicates a negative or Asflb or Asf1(a+b). US 2013/O 14932O A1 Jun. 13, 2013
0040 Gene Ontology (GO) annotation of genes were different types of cancer compared to normal tissue. Results obtained on the microarray from the Ensemble database in from transcriptome studies on different tumor types are ana March 2009. For each GO term, a Hypergeometric test was lyzed and plotted using ONCOMINE database (Rhodes et al., performed to determine whether genes of the differentially 2004). p-values, based on Student's T-test, were considered expressed list showed a more frequent association with a as significant when ps0.05. certain term than could be expected by chance given the GO 0044 FIG. 13. Asf1b levels predict the occurrence of annotation of all genes represented on the microarray. Histo metastasis and distinguishes specific breast tumor Subtypes. gram bars represent the -log(p-value) for each significant GO FIG. 13A Multivariate analysis adjusted for known prognos term. Terms for which the test resulted in a p-value of less or tic factors (such as mitotic index (qualitative and quantita equal to 0.001 (-log(p-value) equal to 3 as shown by the tive), tumor size (qualitative and quantitative), tumor grade dashed line) were considered to be significantly over-repre and Ki67 levels) and for our genes of interest (Asflb, CAF-1 sented for the given list. Numbers in brackets indicate the p60, CAF-1 p150 and HPlcc) in n=73 samples. Only CAF-1 number of significant genes found in the list of differentially p60 expression is an independent prognostic factor for the expressed genes for the given GO category. Arrows indicate if disease free interval and overall survival, while only Asf1b the genes are up- or down-regulated in the indicated siRNA stands out as an independent prognostic factor for the occur relative to the control siRNA. rence of metastasis. In each case, the significant p-value (p<0. 0041 FIG. 10. Asflb, a proliferation marker with prog 05), the Relative Risk (RR) and the 95% Confidence Interval nostic value in small breast cancers. FIG. 10A Correlations (CI) is indicated. FIG. 13B Asfla and Asflb mRNA expres between the logarithmic mRNA expression levels of the indi sion levels in different subtypes of breast cancer were ana cated genes are depicted. The Pearson coefficient of correla lyzed from available transcriptomic data selected from the tion (r) and its associated p-value are indicated. Red numbers Institut Curie together with an asterisk * indicates a significant p-value 0045 Human Tumor database. BLC (Basal-Like Cancers) (p<0.05). FIG. 10B Prognostic value of Asf1b in (TO/T1/T2 (n=17) and MBC (Medullary Basal-like Cancers) (n=19) are NO-MO) breast cancers. Univariate Kaplan-Meier curves of basal-like subtypes of breast tumors with MBC tumors char the survival in patients expressing low (Asflbs0.7) or high acterized by an inflammatory stroma. LUMINAL (n=23) (Asf1b-0.7) levels of Asflb. The number of patients at risk at groups breast Subtypes from the Luminal A and Luminal B each time point is indicated below each graphic. Significant category. MICRO (Micropapillary cancers) (n=22) indicates p-values are sO.05. breast subtypes belonging to the Luminal B subtype. NOR 0042 FIG. 11. Asflb levels correlate with proliferation MAL (n=6) corresponds to normal breast tissue. Compari and have a prognostic value in breast cancer patients. FIG. sons of the expression levels of Asfla and Asflb between 11A Relative mRNA levels of Asfla?b, p60 and Ki67 in sample groups were performed using two-sample Wilcoxon (TO/T1/T2-NO-MO) breast cancers. Box plots representing rank-Sum tests. Boxes represent the 25th-75th percentile, logarithmic expression levels of Asfla, Asf1b, CAF-1 pé0 brackets: range; black line: median; black dots: outliers. Sig and Ki67 mRNAs, according to the indicated clinico-patho nificant p-values of these tests (<0.05) were corrected for logical factors in breast cancer samples with a >10 years multiple testing using the Bonferroni method. NS: Non-sig patient follow-up. Boxes represent the 25th-75th percentile, nificant. brackets: range; black line: median; black dots: outliers. 0046 FIG. 14. Correlations of Asf1b with clinical data: set Below each graph the p-values determined by a Kruskal of patients from 1996. Box plots representing logarithmic Wallis’ test are indicated. Anasterisk* indicates a significant expression levels of Asflb mRNA according to the indicated p-value (p<0.05). FIG. 11B Prognostic value of Asflb in clinicopathological factors in breast cancer Samples with a (TO/T1/T2-NO-MO) breast cancers. Univariate Kaplan-Meier >10 years follow-up. Boxes represent the 25th-75th percen curves of the disease free interval (interval before the occur tile, brackets: range; black line: median; black dots: outliers. rence of local recurrence, regional lymph node recurrence, P-values determined by a Kruskal-Wallis' test are indicated. contro-lateral breast cancer or metastasis), and the occur A significant p-value is <0.05. rence of metastasis (metastasis free interval) in patients 0047 FIG. 15. Prognostic value of Asf1b: set of patients expressing low (Asflbs0.7) or high (Asf1b-0.7) levels of from 1996. Univariate Kaplan-Meier curves of the metastasis Asflb. An asterisk* indicates a significant p-value (p<0.05). free interval (interval before the occurrence of metastasis), The number of patients at risk at each time point is indicated the disease free interval (interval before the occurrence of below each graphic. (See also FIGS. 10 and 12). local recurrence, regional lymph node recurrence, contro 0043 FIG. 12. Asflb correlates with prognosis in breast lateral breast cancer or metastasis), and the disease free Sur cancer and is overexpressed in multiple types of cancer. FIG. vival in patients expressing low (Asflb <0.27) or high 12A Prognosis value in breast cancer. Boxplot representation (Asfle=0.27) levels of Asf1b. A significant p-value is p-0.05. of microarray expression levels of ASflb in relation to prog The number of patients at risk at each time point is indicated nosis in different breast cancer transcriptomes. ASflb expres below each graphic. sion levels significantly correlate with the grade of the tumor 0048 FIG. 16. Asf1b mRNA levels in different cell types (left), with the occurrence of metastasis at 5 years (middle) compared to its levels in breast tumor samples from 1995. and with the disease free survival at 5 years (right). Results 0049. The Asflb mRNA levels were determined by quan are analyzed and plotted using ONCOMINE database titative RT-PCR in proliferating HeLa cells, in proliferating (Rhodes et al., 2004). Boxes represent the 25th-75th percen (AS) or quiescent (GO) MCF7 breast adenocarcinoma cells, tile, brackets: range; black line: median; black dots: outliers; in proliferating (AS) or quiescent (GO) BJ primary fibro n: sample number. p-values, based on Student's T-test, were blasts, in tumoral (Hst) and normal (Bst) mammary cell lines, considered as significant when ps0.05. FIG. 12B Tumoral and in breast tumor samples extracted in 1995 at Institut Versus normal microarray expression data. Boxplot represen Curie. The same primers and amplification kit were used to tation as in (A) of microarray expression levels of Asf1b in detect Asf1b mRNA levels in cell lines or in breast tumor US 2013/O 14932O A1 Jun. 13, 2013
samples. Levels were normalized to the reference gene ribo the proportion of aberrant nuclear structures in MDA-MB somal protein Po-like protein (RPLPO) (de Cremoux et al., 231 cells treated as in (A). The mean percentage of altered 2004). The cut-off value indicated corresponds to the cut-off nuclei (lobulated) and the percentage of DNA bridges after 48 defining two populations of patients: one with “low” Asf1b h of siRNA treatment are plotted. Evalues indicate the stan values which have a good prognosis, and another population dard deviation from counts of two independent experiments. of patients with “high’ Asf1b values and which have a bad FIG. 19D Colony Formation Assay for MDA-MB-231 cells disease outcome. This cut-off value is consistent with Asflb treated with two independent sets of siRNAs against Asf1 mRNA levels obtained in reference cell lines and may there isoforms. The mean surviving fraction (%) is indicated in the fore be determined as a value which is above the levels of histogramms. Error bars represent data from 2 independent Asfb mRNA in MCF-7GO, BJ GO and/or Bst cell lines. experiments. 0050 FIG. 17. Specific depletion of Asfl isoforms in 0054 FIG. 20. Multivariate analysis in patients of 1996. Hs578T cells. FIG. 17A. A. Western blot analysis of total Multivariate analysis adjusted for known prognostic factors extracts from human Hss78T cells showing the specific (such as mitotic index, tumor size, tumor grade and Ki67 depletion of Asfla, Asf1b or Asfl(a+b) for 48 h by siRNA levels) and for our genes of interest (Asflb, CAF-1 p60, treatment with two independent sets of oligonucleotides. CAF-1 p 150 and HP1C. when significant in univariate analy Increasing amounts (X) of total cell extracts are loaded. C-tu sis) in n-62 samples. This analysis performed on an indepen bulin serve as a loading control. Asfla and Asf1b were dent set of patients confirms the prognostic value of ASflb in revealed with a mix of the specific Asf1 antibodies. M: predicting metastasis occurence. In each case, the significant molecular weight marker. FIG. 17B Flow cytometry analysis p-value (p<0.05), the Relative Risk (RR) and the 95% Con of the cell cycle distribution of the cells shown in (A). Asf1b fidence Interval (CI) are indicated. depletion slightly increases the number of cells in S/G2 0055 FIG. 21: Structural divergences between Asfla and phases. ASfl(a+b) depletion impairs S phase progression as Asflbisoforms in Amniotes. FIG. 21A Consensus sequences demonstrated previously in U-2-OS cells (Grothet al., 2007). of Amniota Asfla and Asflb were determined based on their FIG. 17C Quantitative RT-PCR analysis of Asfla and Asf1b alignment in JalView. Asterisks (*) indicate amino acids with mRNA levels in Hss78T cells treated as in (A). mRNA levels a strong divergence while double dots (:) indicate a substitu are normalized to the reference gene GAPDH and expressed tion of amino acids with a mild effect (conservation of the as the log2(fold change) relative to the control siRNA. The property of the amino acids). Divergentamino acids from the error bar represents data from two independent experiments. N-terminal region were reported on the structure of human 0051 FIG. 18. Asf1b depletion impairs proliferation in Asfla. FIG. 21B Structural surface view (left) and ribbon Hs578T cells. FIG. 18A Immunofluorescence analysis of view (right) of human Asfla N-terminus (aa 1-155) deter human Hss78T cells showing the specific depletion of Asfla, mined by NMR (PDB accession number 1TEY.pdb.) (Mous Asf1b or Asf1 (a+b) for 48 h by RNA interference. DAPI son et al., 2005) and visualized using PYMOL software. stains nuclei. Scale bar is 10 um. FIG. 18B Histograms show 0056 White surfaces indicate the histone interacting quantitative analysis of the proportion of aberrant nuclear region determined as the amino-acids that are less than 4A in structures in Hss78T cells treated as in (A). The mean per distance from H3-H4 in the crystal structure of Asfl-H3-H4 centage of altered nuclei (lobulated) and the percentage of (PDB accession number 2IO5.pdb.) (Natsume et al., 2007). micronucleated cells after 48 h of siRNA treatment are plot Lightgrey surfaces indicate the HIRA/CAF-1 p60 interacting ted. Evalues indicate the standard deviation from counts of region determined as the amino acids that are less than 4A in four independent experiments. FIG. 18C (left panel) Immu distance from the B-domain in the crystal structure of Asfl nofluorescence analysis of LaminA staining in Hss78T cells B-domain of HIRA (PDB accession number 2I32.pdb.) (Tang treated as in (A). Arrowheads marks DNA bridges. DAPI et al., 2006). Dark grey and black surfaces indicate the diver stains nuclei. Scale bar is 10 Lum. (right panel) Histograms gent amino-acids between ASfla and ASflb as determined by show quantitative analysis of the proportion of DNA bridges a comparison of the consensus sequence obtained for each in Hss78T cells treated as in (A). Evalues indicate the stan Asfl protein through the multiple alignment of vertebrate dard deviation from counts of two independent experiments. ASfla and ASflb protein sequences (see A). Dark grey Sur FIG. 18D Colony Formation Assay for Hss78T cells treated faces (marked with an arrowhead) depict relatively mild with two independent sets of siRNAs against Asfl isoforms. amino-acids changes while black Surfaces (marked with an The mean surviving fraction (%) is indicated in the histo arrow) show highly divergent amino-acids. FIG. 21C Immu gramms. Error bars represent data from 3 independent experi noprecipitation performed on total cell extracts from human mentS. Hela S3 cells with either a control rabbit IgG antibody or the 0052 FIG. 19. Asf1b depletion impairs proliferation in purified specific antibodies against ASfl isoforms. Input is MDA-MB-231 cells. FIG. 19A 5% of the immunoprecipitated material. Asfla and Asf1b 0053 Western blot analysis of total extracts from human were revealed with a mix of the specific Asfl antibodies, MDA-MB-231 cells showing the specific depletion of Asfla, CAF-1 pé0 and HIRA by western blotting. M: molecular Asflb or Asfl (a+b) for 48 h by siRNA treatment. Increasing weight marker. The inventors hypothesize that the divergent amounts (x) of total cell extracts are loaded. C-tubulin serve regions between Asfla and Asf1b uncovered in (B) could as a loading control. Asfla and Asflb were revealed with a mediate the preferential interaction of Asfla with HIRA and mix of the specific Asfl antibodies. M: molecular weight Asf1b with CAF-1 pé0. marker. FIG. 19B Flow cytometry analysis of the cell cycle 0057 FIG. 22. Inhibition of chromatin assembly with the distribution of the cells shown in (A). FIG. 19C (left panel) B-domain of HIRA. FIG. 22A Binding of the wild-type but Immunofluorescence analysis of human MDA-MB-231 cells not the mutated form (I461D) of the B-domain of HIRA to treated as in (A). Arrowheads marks DNA bridges with Asfl. GST pull-down was performed on HSE extracts with 3 micronuclei between cells. DAPI stains nuclei. Scalebar is 10 mg of recombinant GST B-domain of HIRA proteins on um. (right panel) Histograms show quantitative analysis of beads. Input and flow through are 10% of the immunoprecipi US 2013/O 14932O A1 Jun. 13, 2013
tated material. Asfl and HIRA were revealed by western highlight distinct functions for ASfla and Asflb and identify blotting. FIG.22B The B-domain of HIRA inhibits nucleo a novel molecular target for the development of new thera Some assembly independent of replication. In vitro nucleo peutic strategies. some assembly reactions independent of DNA synthesis were performed with increasing amounts (2, 4 or 8 mg) of the Definitions recombinant GST HIRA B-domain or its mutated form 0064. As used herein, the term 'Asflb’ refers to the Anti (I461D) added directly into the nucleosome assembly reac Silencing Function 1 homolog B or CIA-II. Unigene Cluster tion mix. The purified plasmid DNA was analyzed by agarose for Asf1b is Hs.26516. Representative mRNA and protein elecrophoresis and was visualized by staining with ethidium sequences are NM 018154.2 or AF279307, and bromide (EtBr). The migration positions of DNA plasmid NP 060624.1, respectively. form 1 (Supercoiled), form 11 (nicked circular) and lir (closed 0065. The term “cancer or “tumor, as used herein, refers circular) are indicated. Below the EtBr gel, GST proteins to the presence of cells possessing characteristics typical of were revealed by Western Blotting. cancer-causing cells, such as uncontrolled proliferation, and/ 0058 FIG. 23. Asflb levels strongly correlate with CAF or immortality, and/or metastatic potential, and/or rapid 1p60, HJURP and Mcm2 levels. Correlations between the growth and/or proliferation rate, and/or certain characteristic logarithmic mRNA expression levels of the indicated genes morphological features. This term refers to any type of malig are depicted. The Pearson coefficient of correlation (r) and its nancy (primary or metastases) in any type of subject, male of associated p-value are indicated. Significant p-values (p<0. female. In particular, the term encompasses breast cancer at 05) are indicated. any stage of progression, such as “T” (primary), "N’ (cancer 0059 FIG. 24. Asflb correlates with histological grade has spread to lymph nodes) or “M” (cancer has metastasized). and stage in ovarian cancer. Box plots representing logarith Typical cancers are solid or hematopoietic cancers such as mic expression levels of Asf1b mRNA according to the indi breast, stomach, oesophageal, sarcoma, ovarian, cated clinico-pathological factors in ovarian cancer samples. endometrium, bladder, cervix uteri, rectum, colon, lung or Boxes represent the 25th-75th percentile, brackets: range; ORL cancers, paediatric tumors (neuroblastoma, glyoblas black line: median; black dots: outliers. Below each graph the toma multiforme), lymphoma, leukaemia, myeloma, semi p-values determined by a Kruskal-Wallis' test are indicated. noma, Hodgkin and malignant hemopathies. Preferably, the Significant p-values (p<0.05) are indicated. cancer is selected from the group consisting of breast cancer, 0060 FIG. 25: Asf1b is overexpressed in a variety of can osteosarcoma, skin cancer, ovarian cancer, lung cancer, liver cers. Boxplot representation of microarray expression levels cancer, cervix cancer, liposarcoma, gastric cancer, pancreatic of Asf1b in different types of cancer compared to normal cancer, bladder cancer, Vulvar cancer, colon cancer and brain tissue. Results from transcriptome studies on different tumor cancer. Preferably the cancer is a solid cancer, more prefer types (Barretina et al., 2010; D’Errico et al., 2009; Pei et al., ably an early stage Solid cancer without local or systemic 2009; Sabates-Bellver et al., 2007: Sanchez-Carbayo et al., invasion. Preferably, the solid cancer is breast cancer, more 2006; Santegoets et al., 2007; Sun et al., 2006) are analyzed preferably an early stage breast cancer without local or sys and plotted using ONCOMINE database (Rhodes et al., temic invasion. 2004). p-values, based on Student's T-test, were considered 0066. As used herein, the term “treatment”, “treat' or as significant when ps0.05. Boxes represent the 25th-75th “treating refers to any act intended to ameliorate the health percentile, brackets: range; black line: median; black dots: status of patients such as therapy, prevention, prophylaxis and outliers; n: Sample number. retardation of the disease. In certain embodiments, such term refers to the amelioration or eradication of a disease or symp DETAILED DESCRIPTION OF THE INVENTION toms associated with a disease. In other embodiments, this term refers to minimizing the spread or worsening of the 0061 Recent data have uncovered a number of alterations disease resulting from the administration of one or more in chromatin organization in the context of cancer. How they therapeutic agents to a Subject with Such a disease. occur and connect with other genetic alterations during the 0067. The term “therapy”, as used herein, refers to any development of this disease is currently a major issue. In this type of treatment of cancer (i.e., antitumoral therapy), includ context, understanding the role of histone dynamics repre ing an adjuvant therapy and a neoadjuvant therapy. Therapy sents a significant step forward which can help to classify comprises radiotherapy and therapies, preferably systemic cancer types. This is particularly important when considering therapies such as hormone therapy, chemotherapy, immuno the complexity and heterogeneity encountered in breast can therapy and monoclonal antibody therapy. CS. 0068. The term “adjuvant therapy”, as used herein, refers 0062 Mammalian cells possess two closely related iso to any type of treatment of cancer given as additional treat forms of the histone H3-H4 chaperone Anti-silencing func ment, usually after Surgical resection of the primary tumor, in tion 1 (Asf1), Asfla and Asfb. Asfla and Asf1b are two a patient affected with a cancer that is at risk of metastasizing isoforms encoded by two distinct genes. Up to date, their and/or likely to recur. The aim of such an adjuvant treatment specific or redundant functions have remained elusive. is to improve the prognosis. Adjuvant therapies comprise 0063. In the present study, the inventors aimed at unravel radiotherapy and therapy, preferably systemic therapy, Such ling the respective importance of the two isoforms of the as hormone therapy, chemotherapy, immunotherapy and histone chaperone Asfl, Asfla and Asflb, in relation to pro monoclonal antibody therapy. liferation and tumorigenesis. Indeed, they reveal a clear asso 0069. The term “neoadjuvant therapy”, as used herein, ciation of human Asf1b, but not Asfla, with proliferation refers to any type of treatment of cancer given prior to Surgical capacity in cultured cells which proves relevant in breast resection of the primary tumor, in a patient affected with a tumors. Furthermore, they demonstrate the prognostic value cancer. The most common reason for neoadjuvant therapy is of ASflb in early stage breast cancer. Together, their findings to reduce the size of the tumor so as to facilitate a more US 2013/O 14932O A1 Jun. 13, 2013 effective Surgery. Neoadjuvant therapies comprise radio or cell samples. The sample may be treated prior to its use. therapy and therapy, preferably systemic therapy. Such as The term "cancer Sample” refers to any sample containing hormone therapy, chemotherapy, immunotherapy and mono tumoral cells derived from a patient, preferably a sample clonal antibody therapy. which contains nucleic acids. Preferably, the sample contains 0070. As used herein, the term “chemotherapeutic treat only tumoral cells. The term “normal sample” refers to any ment” or “chemotherapy” refers to a cancer therapeutic treat sample which does not contain any tumoral cells. ment using chemical or biochemical Substances, in particular 0078. The methods of the invention as disclosed below, using one or several antineoplastic agents. may be in vivo, ex vivo or in vitro methods, preferably invitro (0071. The term “radiotherapeutic treatment” or “radio methods. therapy” is a term commonly used in the art to refer to mul 007.9 The present invention relates to a method for pre tiple types of radiation therapy including internal and external dicting or monitoring clinical outcome of a Subject affected radiation therapies or radioimmunotherapy, and the use of with a cancer, wherein the method comprises the step of various types of radiations including X-rays, gamma rays, determining the expression level of ASflb in a cancer sample alpha particles, beta particles, photons, electrons, neutrons, from said subject, a high expression level of Asf1b being radioisotopes, and other forms of ionizing radiations. indicative of a poor prognosis. It is important to note that the 0072 The term “immunotherapy” refers to a cancer thera expression level of ASflb is a significant prognosis marker for peutic treatment using the immune system to reject cancer. clinical outcome taken alone. In addition, ASflb has proven to The therapeutic treatment stimulates the patient’s immune be a significant prognostic marker that can be used for all system to attack the malignant tumor cells. It includes immu types of breast cancer tumors tested including aggressive nization of the patient with tumoral antigens (eg. by admin breast cancer tumors such as the basal-like Subtype and the istering a cancer vaccine), in which case the patients own Erbb2 positive subtype. immune system is trained to recognize tumor cells as targets 0080. In a further aspect, the present invention concerns a to be destroyed, or administration of molecules stimulating method for selecting a subject affected with a cancer for a the immune system such as cytokines, or administration of therapy, preferably an adjuvant therapy, or determining therapeutic antibodies as drugs, in which case the patients whether a subject affected with a cancer is susceptible to immune system is recruited to destroy tumor cells by the benefit from a therapy, preferably an adjuvant therapy, therapeutic antibodies. In particular, antibodies are directed wherein the method comprises the step of determining the against specific antigens Such as the unusual antigens that are expression level of ASflb in a cancer Sample from said Sub presented on the surfaces of tumors. As illustrating example, ject, a high expression level of Asflbindicating that atherapy, one can cite Trastuzumab or Herceptin antibody which is preferably an adjuvant therapy, is required. A high expression directed against HER2 and approved by FDA for treating level of Asflb indicates a decreased patient survival and/oran breast cancer. early disease progression and/or an increased disease recur 0073. The term “monoclonal antibody therapy” refers to rence and/or an increased metastasis formation. Accordingly, any antibody that functions to deplete tumor cells in a patient. this type of cancer associated with poor prognosis has to be In particular, therapeutic antibodies specifically bind to anti treated with a therapy, preferably an adjuvant therapy in order gens present on the Surface of the tumor cells, e.g. tumor to improve the patient’s chance for survival. The type of specific antigens present predominantly or exclusively on therapy is chosen by the practitioner. It includes radiotherapy, tumor cells. Alternatively, therapeutic antibodies may also chemotherapy, hormonal therapy, immunotherapy and mono prevent tumor growth by blocking specific cell receptors. clonal antibody therapy. However, these therapies are usually 0074 The term “hormonetherapy” or “hormonal therapy” aggressive and cause several side effects. By using the refers to a cancer treatment having for purpose to block, add method according to the invention it is therefore possible to or remove hormones. For instance, in breast cancer, the limit adjuvant therapy to Subjects who really need them and female hormones estrogen and progesterone can promote the spare a large Subgroup of subjects (those identified as having growth of Some breast cancer cells. So in these patients, a good prognosis) of a harmful, useless and expensive treat hormonetherapy is given to blockestrogen and a non-exhaus ment. tive list commonly used drugs includes: Tamoxifen, Fareston, I0081. In a last aspect, the present invention further con Arimidex, Aromasin, Femara, ZoladeX/Lupron, Megace, and cerns a method for monitoring the response to a treatment of Halotestin. a subject affected with a cancer, wherein the method com 0075. As used herein, the term “poor prognosis” refers to prises the step of determining the expression level of Asflb in a decreased patient Survival and/or an early disease progres a cancer sample from said subject before the administration sion and/or an increased disease recurrence and/or an of the treatment, and in a cancer Sample from said subject increased metastasis formation. after the administration of the treatment, a decreased expres 0076. As used herein, the term “subject” or “patient” sion level of Asflb in the sample obtained after the adminis refers to an animal, preferably to a mammal, even more pref tration of the treatment indicating that the Subject is respon erably to a human, including adult, child and human at the sive to the treatment. The treatment may be any type of prenatal stage. However, the term “subject' can also refer to treatment Such as chemotherapy, hormonetherapy, immuno non-human animals, in particular mammals such as dogs, therapy, monoclonal antibody, radiotherapy or any other type cats, horses, cows, pigs, sheep and non-human primates, of cancer treatment. A first cancer sample is obtained from the among others, that are in need of treatment. subject before the administration of the treatment. A second 0077. The term “sample', as used herein, means any sample is obtained from the same Subject after the adminis sample containing cells derived from a Subject, preferably a tration of the treatment. Preferably, the second sample is sample which contains nucleic acids. Examples of Such collected when a significant effect on cell proliferation can be samples include fluids such as blood, plasma, Saliva, urine expected, dependent on the chosen treatment. In an embodi and seminal fluid samples as well as biopsies, organs, tissues ment, the second sample is collected at least 2 days after US 2013/O 14932O A1 Jun. 13, 2013
administration of the treatment, preferably one week after distinct migration on SDS-PAGE, ASfla and Asflb can be said administration. The responsiveness of the patient to the distinguished even with a cross-reacting antibody. The quan treatment is evaluated by determining the expression level of tity of Asf1b protein may be measured by semi-quantitative Asflb in tumoral cells before and after the treatment. The Western blots, enzyme-labeled and mediated immunoassays, expression level of Asflb in the cancer sample is determined Such as ELISAS, biotin/avidin type assays, radioimmunoas as described above, preferably by measuring the quantity of say, immunoelectrophoresis or immunoprecipitation or by Asflb protein or Asf1b mRNA. A lower expression level of protein or antibody arrays. The protein expression level may Asflb in tumoral cells contained in the sample collected after be assessed by immunohistochemistry on a tissue section of the treatment in comparison with the expression level of the cancer sample (e.g. frozen or formalin-fixed paraffin Asflb in tumoral cells contained in the sample collected embedded material). The reactions generally include reveal before the treatment indicates that the subject is responsive to ing labels such as fluorescent, chemiluminescent, radioactive, the treatment. This method may also provide an indication of enzymatic labels or dye molecules, or other methods for the required duration and/or intensity of the treatment. In this detecting the formation of a complex between the antigen and case, a follow-up after each cycle of treatment permits to the antibody or antibodies reacted therewith. Preferably, the determine if the expression level of Asf1b is lower than before quantity of ASflb protein is measured by immunohistochem the treatment, and thus to adjust the treatment duration and/or istry or semi-quantitative western-blot. Asf1b detection by intensity accordingly. In particular, this method could be immunohistochemistry does not require an antigen-unmask indicative of the efficacy of the treatment for increasing the ing step, unlike the routinely used marker Ki-67, and thus disease free interval and/or, the overall survival, and/or for speeds up the staining process. decreasing the metastasis occurrence. 0085. In another embodiment of these above mentioned 0082 In an embodiment of these above mentioned meth methods, the expression level of Asf1b is determined by ods, the method further comprises the step of providing a measuring the quantity of Asflb mRNA. Methods for deter cancer sample from the Subject. mining the quantity of mRNA are well known in the art. For 0083. The expression level of Asf1b can be determined example, the nucleic acid contained in the sample (e.g., cellor from a cancer sample by a variety of techniques. In an tissue prepared from the patient) is first extracted according to embodiment, the expression level of Asf1b is determined by standard methods, for example using lytic enzymes or chemi measuring the quantity of Asf1b protein or Asf1b mRNA. cal Solutions or extracted by nucleic-acid-binding resins fol 0084. In a particular embodiment of these above men lowing the manufacturer's instructions. The extracted mRNA tioned methods, the expression level of Asflb is determined is then detected by hybridization (e.g., Northern blot analy by measuring the quantity of ASflb protein. The quantity of sis) and/or amplification (e.g., RT-PCR). Preferably quanti Asflb protein may be measured by any methods known by tative or semi-quantitative RT-PCR is preferred. Real-time the skilled person. Usually, these methods comprise contact quantitative or semi-quantitative RT-PCR is particularly ing the sample with a binding partner capable of selectively advantageous. Preferably, primer pairs were designed in interacting with the Asf1b protein present in the sample. The order to overlap an intron, so as to distinguish cDNA ampli binding partner is generally a polyclonal or monoclonal anti fication from putative genomic contamination. An example of body, preferably monoclonal. Polyclonal and monoclonal primer pair which may be used in this method is presented in antibodies anti-Asflbare commercially available. Examples the experimental section and is constituted by the primers of of marketed antibodies are the rabbit monoclonal anti-Asflb SEQID Nos 3 and 4. Other primers may be easily designed by antibodies from Cell Signaling Technology (#2902, #2769, the skilled person. Other methods of Amplification include produced by immunizing animals with a synthetic peptide ligase chain reaction (LCR), transcription-mediated amplifi corresponding to the carboxy-terminus of the human ASflb cation protein) and the mouse monoclonal anti-Asflb from Abnova I0086 (TMA), strand displacement amplification (SDA) (#H00055723-M01 and # H00055723-M03). In some and nucleic acid sequence based amplification (NASBA). embodiments. Such as for immunofluorescence and immuno Preferably, the quantity of Asflb mRNA is measured by histochemistry applications, the antibody is specific to ASflb quantitative or semi-quantitative RT-PCR or by real-time compared to Asfla, i.e. the antibody specific to Asf1b does quantitative or semi-quantitative RT-PCR or by transcriptome not cross-react with Asf1b. For example, the rabbit mono approaches. clonal anti-Asf1b antibodies from Cell Signaling Technology 0087. In an embodiment of these above mentioned meth (#2902, #2769) have been proved to be specific by the inven ods, the method further comprises the step of comparing the tors. However, other antibodies such as Abnova expression level of Asf1b to a reference expression level. HHOOO55723-MO1 and HHOOO55723-MO3 could also be I0088. In particular, the reference expression level can be tested for their specificity to Asf1b and their cross-reactivity the expression level of Asf1b in a normal sample. The normal with Asfla. Other antibodies which can be used in the differ sample is a non-tumoral sample, preferably from the same ent methods to quantify the Asflb protein are well known by tissue than the cancer Sample. The normal sample may be the skilled person and are commercially available. In addi obtained from the subject affected with the cancer or from tion, the methods for producing anti-Asf1b antibodies are another Subject, preferably a normal or healthy Subject, i.e. a well-known in the art. In a preferred embodiment, the anti subject who does not suffer from a cancer. Preferably, the body is specific to Asflb in comparison to Asfla, i.e. the normal sample is obtained from the same Subject than the antibody specific to Asf1b does not cross-react with Asfla cancer sample. Expression levels obtained from cancer and (e.g., #2902, #2769 of Cell Signaling Technology). An anti normal samples may be normalized by using expression lev body specific for Asflb compared to Asfla may be prepared els of proteins which are known to have stable expression by using either the C-terminal part of the protein where the such as RPLPO (acidic ribosomal phosphoprotein PO), TBP sequence identity is lower and/or a segment including several (TATA box binding protein), GAPDH (glyceraldehyde amino acid substitutions (see FIG. 1A). However, due to their 3-phosphate dehydrogenase) or 3-actin. In particular, expres US 2013/O 14932O A1 Jun. 13, 2013 sion of RPLPO may be assayed by Q-PCR with the following study of a reference population as used in the Example section primers set: SEQID Nos 5 and 6. with the breast cancer samples collected in 1995 or 1996. The 0089 Alternatively, the reference expression level may be cut-off value will be chosen so as to obtain a significant the expression level of a gene having a stable expression in p-value. different cancer samples. Such genes include for example, 0094. In a particular embodiment of these above men RPLPO, TBP, GAPDH or B-actin. Preferably, the reference tioned methods, the present method further comprises assess expression level is the expression level of the RPLPO gene. ing at least another cancer or prognosis marker Such as tumor The use of the human acidic ribosomal phosphoprotein PO grade, hormone receptor status, mitotic index, tumor size, (RPLPO) gene as reference was described in the article of de HJURP expression level or expression of proliferation mark Cremoux et al. (de Cremoux et al., 2004). In a preferred ers such as Ki67, MCM2, CAF-1 pé0 and CAF-1 p 150 or embodiment, the quantity of Asf1b mRNA is normalized prognosis marker such as HP1C, CAF-1 pé0 and CAF-1 p 150 according to the quantity of RPLPO mRNA. The quantity of (Polo et al., 2010; Mascolo et al., 2010; Staibano et al., 2011), RPLPO mRNA is used as reference quantity (i.e. 100%). The preferably expression of CAF-1 proliferation markers and quantity of Asf1b mRNA is expressed as a relative quantity HPla prognosis marker. These markers are commonly used with respect to the quantity of RPLPO mRNA. and the results obtained with these markers may be combined 0090 Expression of RPLPO may be assayed by Q-PCR with the results obtained with the present method in order to with the following primers set: SEQ ID Nos 5 and 6 and provide a better prognostic value than the individual factor expression of GAPDH may be assayed by Q-PCR with the alone. This combination of chromatin-related factors to better following primers set: SEQ ID Nos 21 and 22. Of course, predict prognosis is reminiscent of the combination of differ alternative primers set may be designed by the skilled in the ent chemotherapeutic agents which are used together to target art a cancer with a better efficiency. The use of the proliferation 0091. In a further embodiment of these above mentioned and prognosis markers is well-known by the skilled person. methods, the method further comprises the step of determin HPla as a prognosis marker has been described in PCT appli ing whether the expression level of Asf1b is high compared to cation PCT/EP2010/055423 and in De Koninget al., 2009. As the reference expression level. example, the tumor grade may be determined according the 0092. The expression level of Asf1b in the cancer sample Elston & Ellis method (Elston & Ellis, 2002), the hormone is considered as significantly different (i.e. high) compared to receptor status (estrogen and progesterone) may be deter the reference expression level in a normal sample, if, after mined at the protein or at the mRNA level, the mitotic index normalization, difference is in the order of 2.5-fold higher may be determined by counting mitotic cells in ten micro than the expression level in the normal sample or more. Pref scopic fields of a representative tissue section and the tumor erably, the expression level of Asf1b in the cancer sample is size can be detected by imaging techniques (e.g. mammog considered as high if the level is at least 3-fold higher, or 4, 5 raphy), by palpation or after Surgery in the excised tissue. or 6-fold higher, than the expression level in the normal Expression of Ki67 and CAF-1 can be assessed at the protein sample. level or at the mRNA level. High grade, high mitotic index, 0093. If the reference expression level is the expression and/or large size are indicative for a worse prognosis. The level of a gene having a stable expression in different cancer HJURP expression level may be assessed as described in Hu samples, in particular the expression level of the RPLPO et al., 2010. High HJURP expression is associated with poor gene, the expression level of Asf1b is considered as high if the clinical outcomes. Preferably, in order to preserve the effi level or quantity of Asf1b mRNA is of at least or about 0.2, ciency of the method, the number of assayed markers will be 0.25, 0.3, 0.35, 0.4, 0.45, 0.5,0.55, 0.6, 0.65, 0.7, 0.75, 0.8, as low as possible. Indeed, the efficiency of a test is the 0.85, 0.9, 0.95 or 1% of the RPLPO mRNA level or quantity combination of its predictability and the number of assayed reference or in the range of 0.2-1% of the RPLPO mRNA markers. Accordingly, the ASflb marker is combined with a level or quantity reference. The level or quantity of Asf1b limited number of selected markers, more preferably below relative to the quantity of RPLPO mRNA may be calculated 5-10, still more particularly with 5, 4, 3, 2 or 1 marker(s). by applying X=100/(EA(Cp Gene Cp RPLPO)), where E is 0.095 For instance, expression of Ki67 may be assayed by the mean efficiency of the primers. For instance, with the Q-PCR with the following primers: Forward primer: primers set of SEQ ID Nos 5 and 6, E is 2. By “about” is ATTGAACCTGCGGAAGAGCTG (SEQ ID No 9) and intended +/-5% of the value. The exact value should be Reverse primer: GGAGCGCAGGGATATTCCCTT (SEQID determined using a reference kit. Specifically, Asf1b mRNA No 10). For instance, expression of HP1C. may be assayed by levels should be determined in a number of reference cell Q-PCR with the following primerset: SEQID Nos 11 and 12. lines with the same primers, reference gene and QPCR ampli For instance, expression of CAF-1 pé0 may be assayed by fication kit that are intended to be used with the tumoral Q-PCR with the following primerset: SEQID Nos 17 and 18. samples. For instance, see FIG. 16 clearly showing that the For instance, expression of CAF-1 p 150 may be assayed by cut-off value may be defined with reference cell lines as a Q-PCR with the following primerset: SEQID Nos 19 and 20. value which is above the levels of ASfb mRNA in MCF-7 Of course, alternative primers set may be designed by the GO, BJ GO and/or Bst cell lines. A cut-off value may then be skilled in the art. determined dependent on expression levels of Asf1b in the 0096. Using multivariate statistical analyses, the inventors various cell types containing both normal and tumoral cells. have demonstrated, in the experimental section below, that This cut-off value may be easily adjusted by the skilled person ASflb expression levels predict disease outcome (metastasis using another reference gene. This cut-off value is in particu occurrence) better than these standard prognostic markers. lar applicable for breast cancer. This value may vary depend 0097. The present invention further concerns the use of ing on the type of cancer and will be easily adapted by the one ASflb as a prognosis marker in cancer, preferably in human skilled in the art. In particular, the skilled in the art can define cancer and more preferably in human breast cancer. In a the cut-off value for the expression level of Asf1b based on the preferred embodiment, ASflb is used as a prognosis marker in US 2013/O 14932O A1 Jun. 13, 2013 an early stage breast cancer without local or systemic inva a cancer comprising testing a molecule for its ability to inhibit Sion. As used herein, the term “prognosis marker” refers to a Asflband selecting the molecule capable of inhibiting Asf1b. compound, i.e. ASflb, used to predict or monitor clinical 0112. In particular, the present invention further concerns outcome of a Subject affected with a cancer. methods for screening or identifying a molecule Suitable for 0098. The present invention also concerns the use of Asf1b treating a cancer, in particular for improving the clinical as a marker for selecting a subject affected with a cancer for outcome of a patient having cancer, comprising 1) providing a therapy, preferably an adjuvant therapy, or determining a cell expressing the ASflb gene; 2) contacting said cell with whether a subject affected with a cancer is susceptible to a test molecule; 3) determining the expression level of said benefit from a therapy, preferably an adjuvant therapy. Pref Asflb gene or the activity of the Asf1b protein in said cell; erably, the cancer is a human cancer and more preferably for and, 4) selecting the molecule which decreases the expression human breast cancer. level or activity of said Asf1b gene or protein, respectively. In 0099. The present invention further concerns the use of a particular embodiment, the cell-expression the ASflb gene ASflb as a marker for monitoring the response to a treatment is obtained from a patient sample. Preferably, the cell is a of a subject affected with a cancer. Preferably, the cancer is a mammalian cell expressing the ASflb gene. More preferably, human cancer and more preferably for human breast cancer. the cell has a high expression level of Asflb. The expression 0100. In another aspect, the present invention further con level of said Asflb gene may be determined by measuring the cerns a kit, and its use, quantity of Asf1b protein or Asf1b mRNA, in particular as 0101 (a) for predicting or monitoring clinical outcome detailed above. The “high expression level is determined of a subject affected with a cancer; and/or according to the criteria detailed above. Alternatively, the 0102 (b) for selecting a subject affected with a cancer activity of Asf1b protein or the expression level of Asflb gene for a therapy, preferably an adjuvant therapy, or deter can be measured directly or indirectly. For instance, a mining whether a Subject affected with a cancer is sus decreased expression of Asflb leads to an altered nuclear ceptible to benefit from atherapy, preferably an adjuvant morphology and micronuclei formation (see Example section therapy; and/or and FIGS. 7D-E). For instance, nuclei can be observed by 0103 (c) for monitoring the response to a treatment of a microscopy with DAPI or other revelation systems. There subject affected with a cancer, fore, the Asf1b activity may be measured by observing 0104 wherein the kit comprises nuclear morphology and an altered nuclear morphology is 0105 (i) at least one antibody specific to Asf1b and, indicative of a decreased Asf1b activity or Asf1b inhibition. optionally, means for detecting the formation of the In addition oralternatively, the activity of Asf1b protein or the complex between Asf1b and said at least one antibody; expression level of Asflb gene can be measured by a Colony and/or Formation Assay. Indeed, a decreased expression of ASflb 0106 (ii) at least one probe specific to the Asf1b mRNA leads to a decreased number of colonies (see Example section or cDNA and, optionally, means for detecting the and FIG. 7C). A Colony Formation Assay can be carried out hybridization of said at least one probe on Asf1b mRNA as described in Franken et al., 2006. In a preferred embodi or cDNA; and/or ment, the activity of Asflb protein or the expression level of 0107 (iii) at least one nucleic acid primer pair specific ASflb gene is measured both by the nuclear morphology and to Asf1b mRNA or cDNA and, optionally, means for by the Colony Formation Assay. amplifying and/or detecting said mRNA or cDNA; and, 0113 Alternatively, the present invention further concerns 0.108 (iv) optionally, a leaflet providing guidelines to in vitro methods for Screening or identifying a molecule Suit use Such a kit. able for treating a cancer, in particular for improving the 0109. In addition, the kit may further comprise the detec clinical outcome of a patient having cancer, comprising 1) tion means specific of one or several markers selected from contacting a test molecule with ASflb protein, 2) determining the group consisting of HJURP, Ki67, MCM2, HP 1a, CAF-1 the ability of the test molecule to bind said Asf1b protein, and p60 and CAF-1 p 150. The detection means may be an anti 3) selecting the test molecule capable of binding said Asf1b body specific of the protein, or a probe or a primer pair protein. Binding to said ASflb protein provides an indication specific of the mRNA or cDNA. Preferably, the Asf1b marker as to the ability of the molecule to inhibit or decrease the is combined with less than 20 other markers, preferably less activity of said Asf1b protein. The determination of binding than 10,9,8,7,6, 5 or 4 other markers, still more preferably may be performed by various techniques, such as by labeling less than 7, 6, 5 or 4 other markers. of the test molecule, by competition with a labeled reference 0110. The inventors have also shown that inhibition of ligand, etc. The binding capacity or affinity to ASflb can be Asflb expression using a RNA interference approach cells measured by well-known technologies, such as Biacore. In prevented continued cell proliferation, thereby showing that particular, the binding assay can be combined with functional ASflb is a therapeutic target against which new anti-cancer assay Such as the nuclear morphology determination or the drugs could be developed. Colony Formation Assay. 0111. The present invention also relates to methods for 0114. The present invention also concerns methods for selecting or identifying a molecule of interest for treating a screening or identifying a molecule Suitable for treating a cancer. In particular, the molecule is useful for improving the cancer, in particular for improving the clinical outcome of a clinical outcome of a patient having a cancer. Indeed, the patient having cancer, based on an in vitro nucleosome present invention provides a new therapeutic target, ASflb. assembly assay. This assay is based on the measurement of Therefore, any molecule able to inhibit Asf1b is of interest for the ability of the test molecule to interfere with nucleosome treating cancer. By inhibiting Asflb is intended that the mol assembly, in the replication-coupled or the replication-inde ecule is able to inhibit the histone H3-H4 chaperone activity pendent pathway or both pathways. Details on in vitro of Asflb. Therefore, the present invention relates to a method nucleosome assembly assays can be found for instance in for selecting or identifying a molecule of interest for treating Ray-Gallet et al., 2004 (in particular pages 123-127). US 2013/O 14932O A1 Jun. 13, 2013
0115 Accordingly, in the screening methods of the inven ment of tumors, reduce tumor burden, produce tumor regres tion, the ability of the test molecule to inhibit Asf1b is mea sion in a mammalian host and/or prevent metastasis Sured by a binding assay to ASflb protein, by an assay mea occurrence and cancer relapse. In treating the cancer, the Suring the ASflb expression in a cell, by a nuclear pharmaceutical composition of the invention is administered morphology assay, by a Colony Formation Assay, by an in in a therapeutically effective amount. vitro nucleosome assembly assay or by a combination thereof I0127. By “effective amount' it is meant the quantity of the (combination of two, three or four assays). pharmaceutical composition of the invention which prevents, 0116. The test molecule can be any organic or inorganic removes or reduces the deleterious effects of cancer in mam molecule and in particular, without being limiting thereto, a mals, including humans. It is understood that the adminis Small molecule, an aptamer, a lipid, an antibody, a nucleic tered dose may be adapted by those skilled in the art accord acid or a peptide, polypeptide or protein. The molecule may ing to the patient, the pathology, the mode of administration, be all or part of a combinatorial library of products, for etc. instance. I0128. The cancer may be a solid cancer or a hematopoietic 0117 Finally, the invention relates to a method for treating cancer, preferably a solid cancer and more preferably, an a cancer by administering a therapeutic effective amount of a early stage Solid cancer without local or systemic invasion. molecule inhibiting the Asflb or the use of a molecule inhib Preferably, the cancer is selected from the group consisting of iting the ASflb for treating a cancer. In particular, the treat breast cancer, osteosarcoma, skin cancer, ovarian cancer, lung ment allows the improvement of the clinical outcome of a cancer, liver cancer, cervix cancer, liposarcoma, gastric can patient having cancer. cer, pancreatic cancer, bladder cancer, Vulvar cancer, colon 0118. Accordingly, the present invention relates to cancer and brain cancer. More preferably, the cancer is breast 0119 a pharmaceutical composition comprising a mol cancer. Even more preferably, the cancer is an early stage ecule inhibiting ASflb, and optionally a pharmaceuti breast cancer without local or systemic invasion. cally acceptable carrier, in particular for use in the treat I0129. In a particular embodiment, the cancer to be treated ment of cancer, optionally in combination with is a cancer with a high expression level of ASflb. A high radiotherapy or an anti-tumoral agent; expression of ASflb can be assayed as above-detailed. I0120 a molecule inhibiting Asf1b, and optionally a 0.130. In a preferred embodiment, the molecule inhibiting pharmaceutically acceptable carrier, for use in the treat Asflb is selective in respect to Asfla. That means that the ment of cancer, optionally in combination with radio molecule inhibits Asflb and not Asfla, or with a greater therapy or an anti-tumoral agent; efficacy for Asflb in comparison to ASfla (for instance by a I0121 the use of a molecule inhibiting Asf1b for the factor of at least 10, 100 or 1000). However, due to the manufacture of a medicament for the treatment of can differential expression/activity of the iso forms Asflb and cer, optionally in combination with radiotherapy or an Asfla, a non-selective inhibitor of Asf1 can also be useful anti-tumoral agent; especially in cases where such inhibitor could be selectively 0.122 a method for treating a cancer in a Subject in need targeted to proliferating cancer cells. thereof, comprising administering an effective amount I0131 The Asf1b inhibitor can be, without being limiting of a pharmaceutical composition comprising a molecule thereto, a small molecule, an aptamer, an antibody, a nucleic inhibiting ASflb and optionally a pharmaceutically acid or a molecule preventing the interaction ASflb with an acceptable carrier; Asflb interacting partner. Preferably, the molecule inhibiting 0123 a combined preparation, productor kit containing ASflb is selected from the group consisting of an antibody (a) a molecule inhibiting ASflb and (b) an anti-tumoral against ASflb, an antisense or siRNA against ASflb, prefer agent as a combined preparation for simultaneous, sepa ably the antibody, antisense or siRNA being specific of Asf1b rate or sequential use, in particular in the treatment of in comparison to ASfla. Cancer, (0132. The term “small molecule' refers to a molecule of 0.124 a method for treating a cancer in a Subject in need less than 1,000 daltons, in particular organic or inorganic thereof, comprising administering an effective amount compounds. Structural design in chemistry should help to of a pharmaceutical composition comprising a molecule find such a molecule. The molecule may have been identified inhibiting ASflb, and an effective amount of a pharma by a screening method disclosed in the present invention. ceutical composition comprising an anti-tumoral agent; I0133. In a preferred embodiment of the invention, the and, Asflb inhibitor is a nucleic acid molecule interfering specifi 0.125 a method for treating a cancer in a subject in need cally with ASflb expression, thereby decreasing or Suppress thereof, comprising administering an effective amount ing the expression of this protein. Such nucleic acids are more of a pharmaceutical composition comprising a molecule amply detailed below. Preferably, this nucleic acid is selected inhibiting Asf1b in combination with radiotherapy: from the group consisting of a RNAi, an antisense nucleic 0126 Within the context of the invention, the term treat acid or a ribozyme. Said nucleic acid can have a sequence ment denotes curative, symptomatic, and preventive treat from 15 to 50 nucleotides, preferably from 15 to 30 nucle ment. Pharmaceutical compositions and preparations of the otides. In a preferred embodiment, said nucleic acid com invention can be used in humans with existing cancer or prises a sequence of SEQ ID No. 14. By a “decrease' in tumor, including at early or late stages of progression of the expression is meant, for example, a decrease of 30%, 50%, cancer. The pharmaceutical compositions and preparations of 70%, 80%, 90% or 95% of the gene expression product. the invention will not necessarily cure the patient who has the I0134) The term “RNAi or “interfering RNA” means any cancer but will delay or slow the progression or prevent RNA which is capable of down-regulating the expression of further progression of the disease, ameliorating thereby the the targeted protein. It encompasses Small interfering RNA patients’ condition. In particular, the pharmaceutical compo (siRNA), double-stranded RNA (dsRNA), single-stranded sitions and preparations of the invention reduce the develop RNA (ssERNA), micro-RNA (miRNA), and short hairpin US 2013/O 14932O A1 Jun. 13, 2013
RNA (shRNA) molecules. RNA interference, designate a viral or non-viral vectors), or produced by in vivo transcrip phenomenon by which dsRNA specifically suppresses tion from viral or non-viral vectors. expression of a target gene at post-translational level. In nor 0141 Antisense nucleic acid may be modified to have mal conditions, RNA interference is initiated by double enhanced Stability, nuclease resistance, target specificity and stranded RNA molecules (dsRNA) of several thousands of improved pharmacological properties. For example, anti base pair length. In vivo, dsRNA introduced into a cell is sense nucleic acid may include modified nucleotides cleaved into a mixture of short dsRNA molecules called designed to increase the physical stability of the duplex siRNA. The enzyme that catalyzes the cleavage, Dicer, is an formed between the antisense and sense nucleic acids, e.g., endo-RNase that contains RNase III domains (Bernstein et phosphorothioate derivatives and acridine substituted nucle al., 2001). In mammalian cells, the siRNAs produced by otides. Dicer are 21-23 by in length, with a 19 or 20 nucleotides 0.142 Ribozyme molecules can also be used to block the duplex sequence, two-nucleotide 3' overhangs and 5'-triphos expression of Asflb. Ribozymes are catalytic RNA molecules phate extremities (Elbashir et al., 2001a; Elbashir et al., with ribonuclease activity which are capable of cleaving a 2001b; Zamore et al., 2000). A number of patents and patent single-stranded nucleic acid, such as an mRNA, to which they applications have described, in general terms, the use of have a complementary region. Thus, ribozymes can be used to siRNA molecules to inhibit gene expression, for example, catalytically cleave mRNA transcripts to thereby inhibit WO99/32619, US 2004.0053876, US 2004.0102408 and WO translation of the protein encoded by the mRNA. Ribozyme 2004/OO7718. molecules specific for ASflb can be designed, produced, and 0135 siRNA are usually designed against a region 50-100 administered by methods commonly known to the art (see nucleotides downstream the translation initiator codon, e.g., Fanning and Symonds, 2006, reviewing therapeutic use whereas 5'UTR (untranslated region) and 3'UTR are usually of hammerhead ribozymes and small hairpin RNA). avoided. The chosen siRNA target sequence should be sub jected to a BLAST search against EST database to ensure that 0143. In a particular embodiment, the interfering nucleic the only desired gene is targeted. Various products are com acid molecule is expressed by a vector, preferably a viral mercially available to aid in the preparation and use of siRNA. vector comprising a contruct allowing the expression of inter In a preferred embodiment, the RNAi molecule is a siRNA of fering nucleic acid molecule. For instance, the viral vector at least about 15-50 nucleotides in length, preferably about can be an adenovirus, an adeno-associated virus, a lentivirus 20-30 base nucleotides. or a herpes simplex virus. 0.136 RNAi can comprise naturally occurring RNA, syn 0144. In another preferred embodiment of the invention, thetic RNA, or recombinantly produced RNA, as well as the Asf1b inhibitor is an antibody specific of Asflb and able altered RNA that differs from naturally-occurring RNA by to decrease its activity. Preferably, the antibody is specific of the addition, deletion, substitution and/or alteration of one or Asflb, and capable of discriminating binding to Asf1b in more nucleotides. Such alterations can include addition of comparison with ASfla. non-nucleotide material. Such as to the end of the molecule or 0145 As used in the present invention, the term “anti to one or more internal nucleotides of the RNAi, including body' includes monoclonal antibodies, chimeric antibodies, modifications that make the RNAi resistant to nuclease diges humanized antibodies, recombinant antibodies and frag tion. ments thereof. Antibody fragment means, for example, F(ab) 0.137 RNAi may be administered in free (naked) form or 2, Fab, Fab' or sP v fragments. According to a particular by the use of delivery systems that enhance stability and/or embodiment, the antibody can be IgG, IgM, IgA, Ig) or IgE, targeting, e.g., liposomes, or incorporated into other vehicles, preferably IgG or IgM. Methods for producing antibodies are Such as hydrogels, cyclodextrins, biodegradable nanocap well known to those persons skilled in the art. Sules, bioadhesive microspheres, or proteinaceous vectors 014.6 Antibodies specific of Asf1b have been disclosed in (WO 00/53722), or in combination with a cationic peptide details above. However, in order to be effective, such anti (US 2007275923). They may also be administered in the form bodies might have to be engineered to be able to penetrate the of their precursors or encoding DNAs. cellular membrane into the nucleus. 0138 Antisense nucleic acid can also be used to down 0147 As used here, the term “aptamer” means a molecule regulate the expression of ASflb. The antisense nucleic acid of nucleic acid or a peptide able to bind specifically to Asf1b can be complementary to all or part of a sense nucleic acid protein or to a binding partner of Asflb. In a preferred encoding ASflb e.g., complementary to the coding strand of a embodiment, the aptamers are nucleic acids, preferably RNA, double-stranded cDNA molecule or complementary to an generally comprising between 5 and 120 nucleotides (Os mRNA sequence, and is thought to interfere with the transla borne et al., 1997). They can be selected in vitro according to tion of the target mRNA. Preferably, the antisense nucleic a process known as SELEX (Systematic Evolution of Ligands acid is an RNA molecule complementary to a target mRNA by Exponential Enrichment). encoding ASflb. 0.148. The molecule preventing interaction of Asf1b with 0.139. An antisense nucleic acid can be, for example, about one or several of its binding partners can be an aptamer, an 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. antibody, a peptide, a polypeptide or a protein. Preferably, the Particularly, antisense RNA molecules are usually 18-50 binding partner for which preventing the interaction with nucleotides in length. ASflb is sought is selected from the group consisting of 0140. An antisense nucleic acid can be constructed using Histone H3, Histone H4, CAF-1, or HIRA. chemical synthesis and enzymatic ligation reactions using 0149. In particular, the structure of Asf1 complexed with procedures known in the art. Particularly, antisense RNA can histones H3 and H4 is known (English et al., 2006; Natsume et be chemically synthesized, produced by in vitro transcription al, 2007). Accordingly, based on this knowledge, the skilled from linear (e.g. PCR products) or circular templates (e.g., in the art can design molecules preventing the binding of US 2013/O 14932O A1 Jun. 13, 2013
Asflb to histone H3 and/or H4. For instance, a segment of relevant both for the diagnosis and the prognosis in breast ASflb or of its binding partner can be prepared and used to cancer and potentially a new target for drug discovery in prevent the binding. breast cancer. 0150. In addition, the structure of Asf1 complexed with a specific domain of HIRA or CAF-1 pé0 called the B-domain Results is known (Tang et al., 2006; Malay et al., 2008). Accordingly, based on this knowledge, the skilled in the art can design Asfla and Asflb Levels in Proliferating and Non molecules preventing the binding of Asf1b to p60 or HIRA. Proliferating Cells For instance, a segment of ASflb or of its binding partner can 0157 To examine the expression of Asf1 isoforms in dis be prepared and used to prevent the binding. In particular, a tinct proliferating states, the inventors used specific antibod fraction of the B-domain of HIRA represents an attractive ies that they developed against full-length Asfla or the C-ter candidate for an inhibitory molecule and can act as a domi minal region of ASflb, and which recognize only one or the nant negative as illustrated in the experimental section. For other isoform (FIG. 1A-D). Importantly, while their distinct example, the B-domain may comprise or consist of the migration on SDS-PAGE enables to distinguish Asfla and sequence of SEQID No. 28. Interestingly, as interaction with Asflb, for immunofluorescence studies the use of these anti the histones H3-H4 occurs on the opposite site, an inhibiting bodies was essential to specifically assess the presence of molecule could be designed covering both H3-H4 and HIRA/ Asfla or Asflb in single cells. The inventors first analyzed the CAF-1 pé0 interaction sites. expression of ASfla and ASflb during the cell cycle in Syn 0151. Furthermore, the inventors have herein identified chronized HeLa cells (FIG. 2A). Western blot analysis novel structural differences between Asfla and Asflb. They revealed that Asfla and b are expressed at comparable levels have defined two regions mediating the specificity of the during all stages of the cell cycle as previously reported (FIG. ASfla and ASflb isoforms, these regions being distinct from 2B). In addition, the inventors only detected minor variations the previously characterized interaction domains of Asfl with for Asfla and Asf1b at the RNA level as analyzed by quanti histones and with HIRA or CAF-1 pé0 (FIG.21B). They have tative RT-PCR during the cell cycle (FIG. 2C). This showed also demonstrated the preferential interaction of Asflb with that Asfla and Asf1lb expression are not restricted to S phase CAF-1 pé0 (FIG. 21C). Accordingly, based on this knowl but may have other functions besides their known activity edge, the skilled person can easily design molecules, in par during DNA replication. ticular peptides, that specifically prevent the binding of Asf1b 0158 Since, the inventors did not observe significant with HIRA or CAF-1 p60, more particularly with CAF-1 p60. variation during the cell cycle and given the differential 0152 The molecule inhibiting Asflb may be administered expression of ASfl isoforms in human tissues, they investi by topical, transdermal, oral, rectal, Sublingual, intranasal or gated the levels of ASfl isoforms in non-cycling cells. To parenteral route. address this issue, they used various cell lines in which a transient cell cycle exit can be induced in a controlled manner. 0153. Further aspects and advantages of the present inven An anti-estrogen treatment puts MCF7 breast carcinoma cells tion will be described in the following examples, which in quiescence (Carroll et al., 2000), and under these condi should be regarded as illustrative and not limiting. tions the inventors observed a dramatic decrease in the levels of Asflb protein (FIG.3A). Quantification by chemilumines EXAMPLES cence revealed a major downregulation of ASflb of at least 5-fold in quiescent cells. In contrast, the inventors only Example 1 observed a minor decrease of Asfla. Cyclin A and the histone 0154 Here, the inventors investigated the specific expres chaperone CAF-1 p60 served as cell cycle controls (Polo et sion of the two human Asfl isoforms, Asfla and Asflb, in al., 2004). The inventors further confirmed this distinct relation to cell proliferation and tumorigenesis. In model cell expression of Asf1 iso forms in relation to proliferation by lines, they showed that Asf1b displays a specific prolifera immunofluorescence microscopy using our specific antibod tion-dependent expression pattern not shared by ASfla. The ies (FIG.3B). specific depletion of each isoform by siRNA enabled them to 0159. The inventors then also examined the levels of Asfla evaluate their respective functional importance. They and Asf1b mRNA in asynchronous or quiescent cells by revealed a distinct genome-wide specificity for the two Asf1 quantitative RT-PCR. While Asfla mRNA levels remained iso forms by a transcriptional signature in human cells stable upon quiescent induction, they observed a significant reduction of Asflb mRNA levels (about 7-fold) in quies depleted of Asfla, Asf1b or both isoforms. In addition, deple cence. These findings indicate that the most important regu tion of Asflb led to an increased number of aberrant nuclear latory impact on Asf1lb expression occurs at the RNA level structures and micronuclei and severely impaired prolifera (transcription activity and/or RNA stability) (FIG. 3D). tion, defects that they did not observe upon Asfla depletion. Serum starvation induced quiescence in a human primary 0155 As a further means to explore the physiological fibroblast cell line (BJ), as attested by flow cytometry (FIG. relevance of these differences, they pursued an analysis on a 3C), provided similar results (FIGS. 3A-D). Further, as a selection of breast tumor samples for which a long-term means to monitor the expression of ASfl isoforms upon cell patient follow-up was available. Remarkably, high Asf1b cycle exit, the inventors choose replicative senescence, a per expression significantly correlates with the tumor prolifera manent cell cycle arrest, which occurs through replication tion status, the appearance of metastasis and a decreased dependent telomere shortening in human primary cells after overall survival of the patients. prolonged division in culture. This was important given the 0156 The inventors thus reveal here distinct functions for role of Senescence in tumor Suppression and its connection each Asf1 isoform with a key role for Asflb in proliferation. with the ageing process. They found that Asflb isoform, both Asflb thus represents a new proliferation marker, which is at the protein and mRNA levels in young (PD30), old (PD72) US 2013/O 14932O A1 Jun. 13, 2013
and senescent (PD80) IMR90 human diploid primary fibro examine whether there could be different requirements for blasts, follows the replication capacity of the cells showing a each Asfl isoform. For this, they knocked-down Asfla and major downregulation in senescent cells (about 5-fold) while Asflb, individually or together, in human U-2-OS cells by Asfla expression was only mildly affected (FIGS.3A and D). RNA interference. They first verified the specific depletion of 0160 They conclude that Asflb, in contrast to Asfla, is a one or the other isoform by Western blot (FIG. 7A). While a specific marker for discriminating between cycling and non single band corresponding to Asf1b or Asflaremained visible cycling cells, whether transiently or permanently arrested. after the single depletion of Asfla or Asf1b respectively, the two bands corresponding to ASfla and ASflbisoforms disap Expression Pattern of Asfl Isoforms Upon Cell Cycle Entry peared in the double knockdown as seen by Western blot analysis (FIG. 7A). Importantly, while knockdown of both iso 0161 The inventors’ observation of a major downregula forms produced a strong accumulation of cells in S phase as tion of Asflb in quiescence (GO), prompted them to follow shown previously, cell cycle profiles by FACS analysis did how and when Asf1b is re-expressed upon release from G0 in not change significantly after depletion of ASfla or ASflb MCF7 cells. They monitored cell cycle progression both by flow cytometry and by the expression of the known cell cycle alone (FIG. 7A). regulator CyclinA, or the proliferation markers CAF-1 p60 or 0164. Since Asfl has been implicated in the regulation of p150 (FIGS. 4A, C and D) (Polo et al., 2004). Western blot transcription in yeast and Drosophila, the inventors examined ting revealed that, during release from GO, re-expression of the impact of each ASfl iso form on transcription genome Asflb correlated with the reappearance of Cyclin A (FIG. wide. Using RNA extracted from cells depleted of the differ 4A). Remarkably, the changes observed at the protein level ent ASfl isoforms, the inventors performed a transcriptome paralleled those observed at the mRNA level as measured by analysis on a GeneChip Human Genome U133 Plus 2.0 Array quantitative RT-PCR (FIG. 4B). The inventors obtained simi (Affymetrix). Depletion of Asf1a, Asflb or Asf1(a+b) was lar results in BJ primary foreskin cells released from G0. By effectively detected under our hybridization conditions (FIG. microscopy, they also found that the number of cells positive 8B) and by Q-RT-PCR (FIG.8A). The inventors observed that for ASflb increased after release from quiescence and corre Asflb depletion led to a slight but reproducible upregulation sponded to cells expressing CAF-1 p150 (FIG. 4C). The rapid of ASfla relative to the mock depletion, Suggesting that a upregulation of Asf1b following exit from G0 and entry into compensating mechanism was at play (FIG. 8B). In contrast, the cell cycle is consistent with important cellular demands at Asfla depletion did not significantly alter Asf1b levels. The early steps prior to S phase. Together, these data demonstrate changes observed by the Affymetrix microarray analysis that ASflb is expressed in a manner dependent on the cycling were recapitulated by quantitative-RT-PCR in 3 independent experiments for a set of selected genes (FIGS. 8A and 8C). Status. Remarkably, the inventors did not find any obvious conser Asflb Correlates with the Proliferation Status of Breast Can Vation of transcriptomic data with the one obtained in yeast cer Cell Lines Suggesting that, beside sharing common molecular proper 0162 Since tumoral cells often show a high proliferation ties, human ASfl isoforms and yeast ASfl might have distinct rate, the inventors wondered whether Asfl isoforms are dif functions. Interestingly, the Venn diagram showed that while ferentially expressed in human tumoral mammary cell lines, the highest proportion of affected genes (2151) is shared relative to normal cells. To examine this issue, they used between the three different siRNA conditions, Asflb has the mammary cells derived from the same patient: Hs578T highest contribution to the overall changes observed in Asf1 tumoral and Hss78Bst normal cells which provide a direct (a+b) depletion (FIG.7B). This is consistent with the heatmap comparison between cells of similar origin (Hackett et al., representation of the differentially expressed genes. Aware 1977). Hs578T tumoral and Hsis78Bst normal cells contain that the effects detected are modest compared to those 25 and 13% of cells in S phase respectively (FIG.5A). West observed for known transcription factors, the inventors per ern blot analysis of total cell extracts revealed a marked formed a Gene Ontology (GO) analysis on the differentially increase in the levels of Asf1b protein in tumoral versus expressed genes. Each of their siRNA conditions affected normal cells (FIG. 6A). Quantitative RT-PCR analysis various functional classes of genes further emphasizing a revealed a 2.8-fold increase in the levels of Asf1b mRNA in clear distinct transcriptional signatures for ASfla and ASflb. tumoral mammary cells indicating that ASflb protein expres The class of genes linked to cell proliferation significantly sion is regulated in part at the level of transcription (FIG. 6B). stood out in ASflb depletion, Supporting the importance of In contrast, the inventors did not observe significant changes in Asfla protein or mRNA levels. Interestingly, expression of Asflb in cell proliferation (FIG.9). Asflb paralleled the proliferative status of tumoral cells as 0.165. The inventors then investigated in closer details the assessed by CAF-1 p60 in Western blot (FIG. 6A) or CAF-1 effects of Asfla?b depletion at the cellular level by immunof p150 staining in immunofluorescence (FIGS. 6C and 5B). A luorescence microscopy, in the U-2-OS model cell line (FIG. closer analysis by immunofluorescence microscopy revealed 7), and in two breast cancer cell lines: Hss78T cells (FIGS. 17 that, in individual cells, Asf1b staining perfectly matched and 18) and MDA-MB-231 cells (FIG. 19) in which they CAF-1 p 150 staining, while in contrast, Asfla did not (FIGS. could obtain a significant depletion of Asfl isoforms (FIGS. 6D and 5B). The inventors thus conclude that Asf1lb expres 17 and 19). Intriguingly, Asf1b depletion alone led to a sion directly correlates with the proliferative status of mam remarkable and reproducible increase in the number of aber mary cells. rant nuclear structures including altered nuclear morphology (3 to 10 times more than in the control siRNA) and micronu Asfla Versus Asf1b Depletion: Distinct Effects on Cellular clei formation (FIGS. 7D-E, 18A-B and 19C). In addition, Fate Asflb depletion also increased the number of internuclear DNA bridges (FIGS. 18C and 19C). Quantification of the 0163 The differential expression pattern of Asfla and number of altered nuclei as well as the number of micronuclei Asflb in relation to proliferation prompted inventors to in U-2-OS cells depleted with two independent sets of siRNA US 2013/O 14932O A1 Jun. 13, 2013 confirmed the specificity of the phenotype observed upon breast carcinoma of a size that permitted primary conserva Asflb depletion only (FIG. 7E). The expression pattern of tive tumorectomy (median 18 mm; range 6-50 mm). The lamin A, a marker of the nuclear periphery, was specifically standard treatment received by the patients at the Institut altered in ASflb-depleted cells Suggesting the presence of an Curie for Such localized breast cancers was tumor excision abnormal nuclear lamina in the lobulated nuclei (FIGS. 7F with radiotherapy. However, adjuvant systemic therapy can and 18C), not observed upon Asfla depletion. The inventors increase the chance of long-term Survival and determining then assayed the ability of cells depleted of the different Asf1 which patients with localized breast cancers would benefit iso forms to undergo “unlimited' division. For this, they from these treatments is a current challenge. Thus, new clas performed a Colony Formation Assay (CFA) ((Franken et al., sifiers could provide better guidelines for the administration 2006) on HeLa cells and mammary tumoral Hs578T cells of adjuvant chemotherapy. The inventors therefore measured transfected with control, Asfla, Asf1b or Asfl(a+b) siRNAs. Asfla, Asflb, CAF-1 p60 and CAF-1 p150 mRNA expres In this assay, a decrease in the number of colonies reflects sion levels by quantitative RT-PCR in 86 breast tumor either impaired proliferation, or increase in cell death or both. samples and normalized the expression levels to the known They observed a striking difference in the number of colonies reference gene RPLPO (de Cremoux et al., 2004). For statis obtained after the single Asfl isoform depletions underscor tical analysis they retained data that fulfilled their amplifica ing a distinct impact of ASfla and ASflb on proliferation tion quality criteria (reproducible duplicates, consistent (FIG.7C and 18D). Asflb-depleted cells formed fewer colo primer efficiency between samples). TABLE 1A Description of the samples from patients of 1995 with small breast tumors. Age Median: 53 (range: 26-70) Size classification TO/T1 62% ER (+) 86%f(-) 14% Menopaused Yes 54% T2 38% PR (+) 69%/(-) 31% No 46% Tumor size (mm) Median: 18 (range: 6-50) Ki67 <=15 52% Histological ductal 88% Mitotic index Median: 8 (range: 0-105) 15-40 24% type lobular 9% Grade EE I 33% >40 24% papiliary 190 II 42% Adjuvant No 93% tubular 190 III 25% chemotherapy Yes 79% nies than the control cells Suggesting that the absence of 0.167 First, the inventors studied the correlation between Asflb prevented continued proliferation. These results were the levels of each Asfl isoform and that of another prolifera confirmed in U-2-OS cells (FIG. 7G) and MDA-MB-231 tion marker such as CAF-1 subunits (Polo et al., 2004) or cells (FIG. 19D). Importantly, given the decrease in a number Ki67 (Schonk et al., 1989). Asfla levels only weakly corre of genes required for proliferation in Asflb depleted cells, but lated with that of Asflb, CAF-1 pé0 or CAF-1 p150 and did not ASfla, in transcriptomic data (data not shown), the effects not correlate with Ki67. In contrast, Asf1b levels significantly observed upon Asflb depletion most likely reflect an acute correlated with p60 (r–0.7: p.<10), p150 (r–0.6:)p-10' effect on proliferation leading to cell death as a consequence. and Ki67 (r-0.5; p<10), which again demonstrated its impor This is further supported by transcriptomic data in which the tant link with cell proliferation (Table 1B and FIG.10A). The inventors could not find any bias towards genes involved in inventors then investigated the correlation of Asfla, Asflb, cell death in Asflb depleted cells. Collectively, the depletion CAF-1 or Ki67 levels with clinical parameters to evaluate a analysis underscored distinct functions of Asfl isoforms with potential diagnostic value. They found a high significant posi an importance of Asflb for proliferation. tive correlation of Asf1b levels, but not Asfla, with the tumor size (p=0.0063), the number of mitotic cells (p<10), and the Asflb Correlates with Proliferation in Breast Tumor Samples grade of the tumor (p<10) (FIG. 11A). Notably, Asf1b 0166 To assess the relevance of the inventors’ findings proved even more significant than the other proliferative connecting ASflb with proliferation in a physiological con markers p60 and Ki67 (FIG. 11A). Table 1B (here under) text, they analyzed a selection of cryopreserved breast carci Summarizes all correlations. Taken together, the present noma samples collected in 1995 at the Institut Curie. Table 1A observations put forward Asf1b as a new proliferation marker (here under), provides the patients and tumor characteristics. of clinical interest and prompted the inventors to examine its They focused on node-negative and metastasis-free invasive prognostic value in the context of breast cancer. TABLE 1B Comparison of Asfla, Asf1b, CAF-1 pé0, CAF-1 p150 and Ki67 between multiple groups of prognostic factors. p60 p150 Ki67 p-value N p-value N p-value N p-value Clinicopathological factors
Age O.70 O.36 1.O 0.79 4.7 x 10-3 <=50 34 53 44 53 53 >50 21 32 31 33 33 US 2013/O 14932O A1 Jun. 13, 2013
TABLE 1 B-continued Tumor size O.O6 6.3 x 10-3 O.O28 O.34 6.6 x 10-3 no tumor T1a 33 53 46 53 53 T2a 22 32 29 33 33 Pathological Tumor O.18 2.1 x 10-4 O.O7 O.17 O.O13 <=20 mm 38 59 51 59 59 >20 mm 17 26 24 27 27 Number of mitosis O.O7 6.3 x 10-6 1.4 x 10-3 1.2 x 10-3 2.3 x 10-4 <=10 31 53 44 53 53 >10 24 32 31 33 33 Grade EE O.O6 1.2 x 10-6 9.5 x 10-3 O.04 5.4 x 10-5 I 18 29 25 29 29 II 24 34 29 35 35 III 13 22 21 22 22 Asf1a ASflb p60 p150 Ki67
r p-value r p-value r p-value r p-value r p-value
Corrections with other markers
CAF-1 pé0 0.42 2.5 x 10-3 0.66 2.4 x 10-10 — 0.84 p < 2.2 x 10–16 0.26 O.O26 CAF-1 p150 0.53 2.6 x 10-5 0.64 3.6 x 10–11 0.84 p < 2.2 x 10-16 O.21 O.OS4 Ki67 O.O2 O.86 O52 3.6 x 10-7 O.26 O.O26 O.21 O.OS4 Asf1a O.32 O.O17 O.42 2.5 x 10-3 O.S3 2.6 x 10-5 O.O2 O.86 ASf1b O.32 O.O17 O.66 2.4 x 10-10 O.64 3.6 x 10-11 O52 3.6 x 10-7
0168 (Upper part) Correlations between the indicated the prognostic value of Asf1b. At 10 years, 98% 93-100 of genes and clinicopathological factors. N: number of samples the patients with low Asflb expression had not developed included in the statistical analysis for each gene. Significant metastasis compared to 66% 50-87 of the patients with high p-values (sO.05) are noted in bold. (Lower part) Correlations ASflb levels. between the genes. r: Pearson coefficient of correlation. Sig nificant p-values (sO.05) are noted in bold. 0170 In a second independent set of 71 breast cancer samples collected in 1996 at the Asflb has a Prognostic Value in Breast Cancer (0171 Institut Curie, the inventors confirmed the obtained 0169. The inventors first investigated the relationships results. Table 2 provides the patients and tumor characteris between Asflb levels and disease outcome, as determined by tics for this set. TABLE 2 Description of the samples from patients of 1996 with small breast tumors. Age Median: 55 (Range: 30-69) Size classification TOT1 66% ER (+) 80%/(-) 20% Menopaused Yes 37% T2 34% PR (+) 77%/(-) 23% No 63% Tumor size (mm) Median: 20 (Range: 8-35) Ki67 <=15 44% Histological ductal 74% Mitotic index Median: 5 (Range: 0-120) 15-40 25% type lobular 16% Grade EE I 30% >40 31% papiliary 190 II 48% Adjuvant No 93% tubular 9% III 22% chemotherapy Yes 79% the disease free interval, the overall survival and the occur 0172 Asflb expression correlates with the tumor charac rence of metastasis. Since ASfla did not show significant teristics, in particular the mitotic index (p<10), the tumor correlation with any of the clinical markers studied, they did grade (p=0.002) and the hormone receptor status (p=0.01) not consider it in this analysis. At 10 years, the overall sur (FIG. 14). In addition, as shown in FIG. 15, they confirmed vival, the distant recurrence and the disease progression rates that ASflb expression is a prognosis marker. They determined were 90 83-97), 87 80-95 and 70% 61-81, respectively. a cut-off value of 0.27 for ASflb mRNA levels which divided patients into two groups: one with low Asflb levels (41% of They determined a cut-off value of 0.7 for Asf1b mRNA patients with Asf1bs0. 27), and the second with high Asf1b levels which divided patients into two groups: one with low levels (>0. 27) which was significantly associated with dis Asflb levels (67% of patients with Asf1bs0.7), and the sec ease progression (p=0.045, Relative Risk (RR)-2.71.0–7.4) ond with high Asflb levels (>0.7) which was significantly in univariate analysis (FIG. 15). Moreover, higher Asf1b lev associated with disease progression (p=0.017, Relative Risk els significantly associated with shorter overall survival (p=0. (RR)=2.31.1-4.8) in univariate analysis (FIG. 11B). More 018, RR=2.91.2-7.2) and with an increased occurrence of over, higher Asf1b levels significantly associated with shorter distant metastasis (p=0.007, RR-9.91.3-76.7) (FIG. 15) overall survival (p=0.01, RR=6.3 1.3-31.3) and an further underlining the prognostic value of Asflb. increased occurrence of distant metastasis (p=0.0002, 0173 Next, the inventors compared the prognostic value RR-7.8.2.1-28.3) (FIGS. 11B and 10B) further underlining of Asf1b with CAF-1 p60, CAF-1 p150 and HP1c. (De Kon US 2013/O 14932O A1 Jun. 13, 2013
ing et al., 2009) in multivariate analysis adjusted for known Asfl in yeast is best rescued by human Asfla for defects in the prognostic factors and for the genes of interest. They found DNA-damage response, while Asf1b best compensates for that only high CAF-1 pé0 expression was an independent the growth defects and the sensitivity to replication stress prognostic factor for disease progression (p<10, RR=5.5 (Tamburini et al., 2005). In mammalian cells, Asfla specifi 2.5-11.9) and decreased overall survival (p<10, RR=12.9 cally interacts with HIRA and is required together with HIRA 2.6-64.2) (FIG. 13A). Their observations thus confirm that for senescence-associated cell-cycle exit (Zhanget al., 2005). CAF-1 is not only of interest for diagnosis (Polo et al., 2004) Recent evidence also suggests that ASfla could play an but also show its relevance for the prognosis of breast cancer. important role in the regulation of H3K56 acetylation levels Notably, Asflb stood out as the only independent prognostic in human cells and in transcriptional activation mediated by factor for the metastasis free interval (p<10). High Asf1b H3.3 incorporation together with HIRA (Das et al., 2009: levels are associated with a higher risk of developing distant Yang et al., 2010; Yuan et al., 2009). In contrast, as shown metastasis (RR=7.1 2.0-26.0) (FIG. 13A). In the set of herein, Asflb is the Asfl isoform essential for proliferation. tumor samples from 1996, they found menopausal status 0176) However to date, the structural basis for such a pos (p=0.013, RR=4.21.4-12.8) and Asf1b mRNA levels (p=0. sible divergence in function is poorly understood. The inter 024, RR=5.7 1.3-25.7) as independent prognostic markers action domain of Asfl with histones H3-H4 is highly con for metastasis free interval (FIG. 20), therefore confirming served between Asfla and Asf1b isoforms (English et al., data obtained in the first series of tumor samples. Interest 2006; Natsume et al., 2007), as well as its hydrophobic core ingly, together with menopausal status, ASflb expression lev interacting with the B-domain of HIRA or CAF-1 pé0 (Malay els also significantly predicted disease progression (FIG. 20). et al., 2008; Tang et al., 2006). In addition, both Asfla and Thus, they demonstrate for the first time that Asflb is a new Asflb are known to interact with both H3.1 and H3.3 histone proliferation marker of prognostic value in breast cancer that variants (English et al., 2006; Natsume et al., 2007; Tagami et is highly predictive for the occurrence of metastasis. al., 2004). Thus, Asf1 is not the primary factor that discrimi nates between the histone variants, and the distinct functions Asfl Levels in Breast Tumor Subtypes of ASfl isoforms are most probably not a consequence of a 0.174. In breast cancer, expression-profiling studies have preferential interaction with a given histone variant. This is helped to distinguish different Subtypes of tumors according consistent with the fact that among the four residues that to a specific expression profile (Sotiriou and Piccart, 2007) differ between the two histone variants, three are clustered on defining the following molecular classes: luminal-A cancers the opposite site of the Asf1 binding site and the fourth is and luminal-B cancers, which are predominantly Estrogen located in the N-terminal tail which most likely does not Receptor (ER)-positive: basal-like cancers, which mostly interact with Asf1 (English et al., 2006; Natsume et al., 2007). correspond to ER-negative, Progesterone Receptor (PR)- It is therefore intriguing to realize that despite the high degree negative and HER2-negative tumors; and HER2-overex of similarity between the two human Asfl isoforms (71% of pressing cancers corresponding to tumors with amplification identity), the small divergence observed in the first 30 amino of the ERBB2 gene. Importantly, these molecular subgroups acids of the sequence, and the different C-terminal domains have distinct clinical outcomes and responses to therapy, with (aa 156-202) may be sufficient to elicit specific interactions/ the basal-like tumors and HER2 positive tumors having a functions with other histone chaperones, as Suggested by more aggressive clinical picture (Sotiriou and Piccart, 2007). swapping experiments of the Asfla and Asf1b N- and C-ter Taking advantage of an available transcriptome database minal domains (Tang et al., 2006). derived from breast tumor samples of cryopreserved tissues 0177. Here, the inventors determined the divergentamino selected from the Institut Curie, the inventors examined Asf1 acids between the consensus sequences of ASfla and ASflb levels in specific subtypes of breast tumors. Asfla mRNA isoforms in Amiotes (FIG. 21). Since the distinctamino-acids levels were similar in normal breast samples, luminal tumors are dispersed along the Asfl sequence (FIG. 21A), they then (luminal and micropapillary) and basal-like (BLC) subtypes, visualized the localization of these amino-acids on the known and only significantly increased in the medullary basal-like structure of the human Asfla N-terminus (aa 1-155) (Mous (MBC) subtype which has an inflammatory stroma (FIG. son et al., 2005). They found a striking clustering of these 13B). In contrast, Asf1b mRNA levels were low in normal amino-acids in 3D at the top and bottom of Asf1, defining two breast tissue and significantly increased in all breast tumor potential regions mediating the specificity of the ASfla and subtypes (FIGS. 13B). Interestingly, the inventors found the Asflb isoforms (FIG. 21B). Importantly, these two regions highest expression levels for Asflb in the BLC and MBC are distinct from the previously characterized interaction Subgroups, corresponding to the basal-like highly prolifera domains of Asf1 with histones (FIG.21B) and with HIRA or tive tumors (FIG. 13B). Thus, Asflb expression levels CAF-1 pé0 (FIG. 21B) which are highly conserved between showed a clear association with the proliferation rate and Asfla and Asf1b. They hypothesize that these two regions aggressiveness of distinct breast cancer Subtypes. could reveal additional specific interaction of Asfla?b with Identification of Novel Structural Differences between Asfla the known histone chaperones HIRA or CAF-1 p60, or with and Asflb Isoforms and their Functional Implications other partners yet to be identified. Interestingly, using specific 0175 While Asfl exists as a single isoform in Fungi (eg antibodies against Asfla and Asflb as previously described Saccharamyces cerevisiae), the inventors uncovered a major herein, they performed an immunoprecipitation of ASfla or duplication event leading to the clear distinction of ASfla and Asflb from Hela S3 total cell extracts. While they confirmed Asflbisoforms in Amniotes (data not shown). They therefore the preferential interaction of Asfla with HIRA, they uncov investigated the structural divergence between these two iso ered for the first time a preferential interaction of Asf1b with forms to try and assign distinct functions to each individual CAF-1 pé0 (FIG. 21C). ASfl isoform. Evidence suggesting a potential difference in 0.178 Since the C-terminal extention of Asfl is unfolded the functions of the two mammalian Asfl isoforms came both in the isolated proteins, they restricted their analysis to the from studies in yeast and mammalian cells. The depletion of N-terminal domain of human Asfl. Nevertheless, it is pos US 2013/O 14932O A1 Jun. 13, 2013
sible that the C-terminus adopts a well-defined structure in addition, they could rescue the nucleosome assembly reaction the presence of partners. In addition, the phosphorylation by adding excess of ASfl proteins to the reaction (data not imposed on the C-terminal domain of Asfl isoforms could shown). also participate in the regulation of their specific functions, as 0181. In conclusion, these data show that the use of the suggested recently for a differential regulation of the stability B-domain peptide can inhibit chromatin assembly indepen of Asfl isoforms (Pilyugin et al., 2009). One could envisage dent of DNA synthesis by inhibiting the interaction of Asf1 that these divergences in the sequence/structure of ASfl iso with HIRA. They hypothesize that the excess of B-domain forms on the C-terminus could also provide the specificity peptide titrates Asfl proteins away which can then no longer towards new interaction partners, as was the case for the interact with HIRA, resulting in an inhibition of chromatin Asfla-HIRA interaction or for the recently identified inter assembly. This is consistent with the fact that addition of an action between the Varicella-Zoster Virus (VZV) immediate excess of ASfl proteins can rescue the nucleosome assembly. early 63 protein (1E63) and Asfla isoform. A similar strategy could therefore be used to inhibit Asf1 Inhibition of Chromatin Assembly with the B-Domain of interaction with CAF-1. In particular, given the preferential HIRA interaction of Asf1b with CAF-1 (FIG. 21 C) and given the 0179 Among its conserved functions, the histone chaper existence of specific structural regions that could mediate this one Asfl synergizes with CAF-1 and HIRA in the replication preferential interaction of Asflb with CAF-1 (FIG.21B), one coupled and replication-independent nucleosome assembly could imagine that the B-domain peptide could be refined to pathways, respectively, rather than acting as a deposition specifically inhibit the interaction of Asflb with CAF-1. In factor on its own in vivo. Asf1 interacts with the B-domain of addition, the peptide could also be designed to inhibit the HIRA or CAF-1 p60 through a conserved hydrophobic interaction with histones together with the interaction with groove at a site located on the opposite side of the one CAF-1, potentially leading to major proliferation defects involved in its interaction with H3-H4 (English et al., 2006; (FIGS. 7, 18 and 19). Natsume et al., 2007). This explains how the histones can be handed over from one histone chaperone to another in order to Discussion promote chromatin assembly. One could hypothesize that a 0182 While yeast presents a single form of the Asf1 his peptide of the B-domain present in excess could block the tone H3-H4 chaperone, in many multicellular organisms, interaction of Asf1 with HIRA and/or CAF-1 pé0, and thus including plants or mammals, there are two distinct isoforms inhibit the function of the histone chaperone Asfl in chroma whose specific individual functions have remained unex tin assembly. plored. The inventors investigated their respective implica 0180. The inventors therefore decided to investigate if the tion in cell proliferation. In cultured cells, they reveal a unique B-domain of HIRA could inhibit chromatin assembly pro proliferation-dependent expression pattern of Asf1b, not moted by HIRA and Asfl. For this, they decided to use a shared by ASfla, that enables to distinguish tumoral from non cell-free system enabling chromatin assembly derived from tumoral derived breast cancer cells. Depletion of Asflb shows Xenopus laevis eggs. These egg extracts contain all factors the prominent role of this isoform for cell proliferation, with essential for chromatin assembly during the rapid rounds of a distinct transcriptional impact genome-wide and cellular DNA replication that occur in early development. When defects reminiscent of aging phenotypes. Moreover, using a depleted of a putative histone chaperone or with an excess of selection of samples from early stage breast tumors derived a putative inhibitory peptide, they lose their ability to support from patients, they demonstrate for the first time the clinical nucleosome assembly, which can be restored with add-back relevance of ASflb as a proliferation marker of prognostic experiments. This approach was successfully used to demon value in early stage breast cancers. strate that CAF-1 and HIRA are true deposition factors in A Distribution of Labour between Asfl Isoforms for Distinct Vivo, promoting chromatin assembly dependent and indepen Proliferation Status dent of DNA synthesis respectively (Quivy et al., 2001; Ray 0183. Using various model cell lines, the inventors found Gallet et al., 2002). The inventors therefore cloned the B-do that the two ASfl isoforms are expressed in a distinct manner. main of X. laevis HIRA in a vector allowing its expression as While ASfla levels remain unchanged in cycling, quiescent or a peptide with a GST tag at its end. In addition, they cloned a senescent cells. Asf1b levels directly reflect the proliferation mutated form of the B-domain mutated at position I461D capacity of the cells both at the protein and RNA levels. which has been shown to no longer interact with Asfl (Tang Remarkably, when exploring data from the Gene Expression et al., 2006). They verified by GST pull-down that the GST Omnibus database (GEO, NCBI) they found that the prolif B-domain of HIRA interacts in vivo with Asfl from X. laevis eration-dependent expression of human ASflb was conserved (FIG.22A). Importantly, the mutated form of the B-domain in other human cell types, such as in the human T98G glio of HIRA (I461D) does not interact with Asf1, consistent with blastoma cancer cells arrested by serum deprivation (GEO previous data (Tang et al., 2006). They then wondered if accession number: GDS911), and also extends to other organ addition of an excess of the B-domain of HIRA could inhibit isms such as mouse (GDS575), thus opening interesting chromatin assembly independent of DNA replication in the X. avenues for genetic studies. Interestingly, the levels of ASfl laevis system. They therefore added the GST-B-domain of isoforms may also vary in a distinct manner upon differentia HIRA to the nucleosome assembly reaction. They observed a tion (GDS586), as another form of cell cycle exit. Thus, while striking decrease in the amount of Supercoiled plasmid (1 ASfl isoforms share molecular and biochemical properties as form) (FIG. 22B) Suggesting that nucleosome assembly is histone H3-H4 chaperones, as exemplified by their overlap impaired in the presence of the B-domain of HIRA. Impor ping functions during replication (FIGS. 7A and 7B), the tantly, this dominant negative effect of the B-domain is work difference between these isoforms could reside partly in their ing through its interaction with ASfl since addition of the capacity to be uniquely regulated. In this respect, the finding mutated GST-B-domain I461D which does not interact with that Asflb is a direct transcriptional target of E2F1 would find Asfl had no effect on nucleosome assembly (FIG. 22B). In a physiological application in the present results to explain US 2013/O 14932O A1 Jun. 13, 2013 20 the proliferation-dependent regulation of Asflb. However, aberrant nuclear structures which resemble those observed in given that both ASfla and ASflb genes possess putative bind Hutchinson-Gilford progeria syndrome (HGPS) cells and in ing sites for the transcription factors E2F, additional aspects cells depleted of the histone chaperone RbAp48. RbAp48 is a should impart on this regulation. The present study in multi histone chaperone found in a complex together with ASfla cellular organisms showing the specific regulation of ASfla and ASflb and belongs to several chromatin-related com and Asflb levels in distinct cellular contexts as a means for a plexes such as CAF-1, NURD or PRC2 complexes. Interest distribution of labour between the two Asfl isoforms, opens ingly, the downregulation of RbAp48 expression in HGPS up avenues to examine how common properties can be cells was found to participate in the formation of ageing exploited in different physiological contexts. associated chromatin defects via loss of the integrity of the NURD complex. Intriguingly, in Asf1b depleted cells, but not Functional Importance of Asf1 Isoforms ASfla, Rb Ap48 gets downregulated. It is thus tempting to 0184 The genome-wide transcriptome analysis coupled speculate that Asflb may also act as a chaperone of Rb Ap48 with the analysis of the effects caused by the depletion of Asf1 to maintain levels required to prevent the appearance of pre isoforms shows that, while Asfla and Asf1b can partly com mature aging-related chromatin defects. Having high levels pensate for each other (FIG. 7A), these two isoforms have a of Asflb would therefore confer a growth advantage to pro distinct role in mammalian cells. Importantly, most of the liferating cells, in agreement with our observations that Asf1b effects observed in the transcriptome analysis were relatively levels are highly downregulated in primary senescent fibro Small compared to the depletion of transcription factors. This blasts (FIG. 3A). Thus, not only is Asflb expressed in a together with the preliminary ChIP-on-chip data supports the proliferation-dependent manner, but it is required for prolif view according to which ASfl iso forms may rather play an eration, and possibly prevents aging. These two aspects are auxiliary role in transcriptional regulation, while their main both relevant for cancer progression. function is to regulate the dynamics of the histone pool. The latter one could however show potential specificities and Asflb as a Marker of Diagnostic and Prognostic Value for regulations depending on cellular context. It is tempting to Breast Cancer consider that despite the high degree of similarity between the 0186. Using samples from 86 early-stage breast tumors two human Asf1 isoforms (71% of homology as shown in with a >10 years follow-up, the inventors demonstrate for the FIG. 1A), the divergence observed in the first 30 amino-acids first time the clinical relevance of Asflb as a new prognostic of the sequence, together with the different C-terminal part factor in breast cancer. High Asf1b levels correlate with a (156-202 amino-acids) could contribute to the distinct regu poor overall survival rate, a decreased disease free interval lations and functions of the two Asfl iso forms. In this and a higher occurrence of metastasis. By analyzing pub respect, given that the available information on structural lished transcriptomic data of the Oncomine database (Rhodes organization of ASfl focus on the conserved N-terminus, et al., 2004), they confirmed the prognostic value of Asf1b in future studies aiming to characterize the 3-dimensional orga breast cancer (FIG. 12A). Their current analysis extended to nization of the C-terminus should be revealing. This may a second, independent set of patient samples further confirm provide a particular docking site for new, yet to be identified, the highly prognostic value of Asflb and CAF-1 pé0 com interaction partners, as it is the case for the Asfla-HIRA pared to other markers. In addition, Asf1b levels also identify interaction. the aggressivity of breast tumor Subtypes, being higher in the 0185. Asflb appears to be most critical for proliferation basal-like cancers (FIG. 13B), and also in the Erbb2 positive (FIGS. 7B to 7F). In proliferating cells, Asflb would be best tumors. Multivariate analyses demonstrate that Asf1b levels able to handle the pool of replicative histone H3.1 thereby predict the occurrence of metastasis better than any standard acting as the prominent histone acceptor? donor during DNA prognostic markers, while CAF-1 pé0 proved here to be replication. This would be consistent with its major contribu another prognostic factor with a betterprediction value for the tion to the defects observed upon knockdown of both Asfl iso disease free interval and survival rates, consistent with recent forms (FIG. 7B). While we cannot formally exclude that data. Furthermore, the inventors found that beside a signifi ASflb could potentially directly upregulate genes related to cant and consistent overexpression in breast cancers, ASflb DNA replication (FIG.9, list for Asf1b depleted cells), such stands out in other types of malignancies. Such as skin cancer, transcriptional changes could simply represent a indirect liver cancer, ovarian cancer, lung cancer, liposarcoma, gastric effect enabling to compensate replication defects. Further cancer, pancreatic cancer, bladder cancer, Vulvar cancer, more, when cells enter into a non-dividing state, the amount colon cancer and brain cancer (FIGS. 12B and 25). Asf1b was of the H3.1 replicative histone variant available drops. Asfla also found in a cervical cancer proliferation cluster of 163 would then suffice to handle the remaining pool of histones highly correlated transcripts which were overexpressed in consisting mainly of H3.3 during all DNA metabolic pro cervical tumors with an unfavourable disease outcome. In cesses. While some preferential interactions with a given H3 contrast, ASfla was not retrieved as a significant gene in these variant are plausible, structural studies will be needed to studies. The present results therefore lead us to propose that explore whether this is dictated by the binding properties of Asflb represents a new proliferation marker of interest in a the partners or simply a reflection of availability of given H3 wide range of cancers which can be used as a classifier with a variants. Interestingly, ASflb depletion led to a slight upregu powerful prognostic value for metastasis occurrence. lation of Asfla at the RNA level, and did not give any obvious 0187 Interestingly, proliferation appears to be a common phenotype during S phase by flow cytometry analysis (FIG. driving force of several gene expression prognostic signa 7A), which would favor the latter option. In this context, the tures such as MammaPrint (vant Veer et al., 2002), or the upregulation of Asfla in cells depleted of Asflb could repre genomic-grade signature (Sotiriou et al., 2006), which aim at sent a compensatory mechanism to allow normal S phase providing better predictions of clinical outcome than the tra progression. Notably, in addition to its effect on cell prolif ditional clinical standards (Wirapati et al., 2008). The inven eration, Asflb depletion also led to an increased number of tors found that Asflb correlates with prognosis in breast US 2013/O 14932O A1 Jun. 13, 2013
cancer transcriptomic data validating the Mammaprint signa free medium for 72 h, and MCF7 cells in medium containing ture (FIG. 12A) and that Asflb belongs to a set of co-ex 10 nM of the anti-estrogen IC 1182780 (Fisher Bioblock Sci pressed proliferative genes with prognosis value in breast entific) (Carroll et al., 2000) for 48 h. HeLa cells were syn cancer. Remarkably, while most genes of this proliferation chronized with a double thymidine block, as follows: 16 h module are directly related to progression through cell cycle block in 2.5 mMthymidine (Sigma-Aldrich), 9h release in 30 Such as cyclins, replication factors, aurora kinases etc. ASflb mM 2'-deoxycytidine (Sigma-Aldrich), and 16 h block in 2.5 stands out as an interesting proliferation marker related to mM thymidine. The G1/S, S, S/G2, and G1 samples were chromatin organization and histone dynamics. One could collected after a 0-, 4-, 8-, 14-h release in 30 mM 2'-deoxy imagine that high ASflb levels would confer an important cytidine respectively. HeLa cells were treated with 100 chromatin plasticity, which is essential for the survival of ng/mL nocodazole for 15 h to obtain mitotic samples. cancer cells in a selective environment. The prognostic value 0191 Cell synchronization was verified by flow cytom of Asflb could therefore bring some complementary infor etry, using cells fixed in 70% ethanol (-20°C.) and stained mation compared to the previously known prognostic mark with propidium iodide (50 mg/mL in PBS containing 0.04 ers. Combination of Asf1b with other selected markers mg/mL RNaseA). A BDFACScalibur (BD Biosciences) was related to chromatin organization Such as HP1C. (De Koning used for signal analysis and analysis was carried with Flow Jo et al., 2009), and CAF-1 pé0 may have a stronger clinical (Tree Star Inc.) software. value in the most aggressive breast cancer Subtypes such as basal-like tumors. Plasmid Constructions for the Asfl Recombinant Proteins 0188 In conclusion, the present study enables to ascribe to and Antibodies the distinct Asf1 isoforms specific roles associated with dif ferent proliferation states. On the one hand, the proliferative 0.192 N-terminal fusions of the C-Terminal part of Asf1b predominent role of Asf1b would ensure to handle replicative (amino-acids 156-202) to a GST-tag and to a His-tag were histones via a replication-oriented function which could pre generated by PCR cloning of the C-terminus of Asflb (prim vent aging. On the other hand, Asfla would rather contribute CS 5'AGGTGCTAGAATTCA ACATGGACAG to processes in which handling histones would connect to GCTGGAGGCCATAG (SEQ ID No. 23), 3'CAGGC transcription/silencing or senescence. The proliferative role TATCTCGAGTTATTAGATGCAGTCCATGGAGTTCTCAG of Asf1b, culminates with a validation as a new proliferation (SEQ ID No. 24)), insertion into the EcoRI/XhoI site of marker of interest both in the context of model cell lines and pGEX-4T-1 (Novagen) and plT30a (Novagen) respectively tumor samples. Furthermore, the high ASflb expression cor followed by verification by sequencing. N-terminal fusion of relating with increased rates of disease progression and the C-Terminal part of Asfla (amino-acids 156-204) to the metastasis occurrence in Small breast cancer, defines ASflb as His-tag was generated by PCR cloning of the C-terminus of a new prognostic factor of clinical value. Future work should ASfla (primers: 5'AGGTGCTAGAATTCA ACACA explore how to exploit these findings that highlight Asf1b as GAAAAACTGGAAGATG (SEQ ID No 25), 3'CAGGC an attractive target for cancer treatment. TATCTCGAGTTATCACATGCAGTCCATGTGGGATTC (DEQ ID No. 26)), insertion into the EcoRI/XhoI site of Materials and Methods pET-30a (Novagen) and Verification by sequencing.
Cell Lines and Cell Culture Plasmid Constructions for the Recombinant HIRA B-Domain Proteins (0189 DMEM medium (GIBCO) was used for U-2-OS osteosarcoma (gift from J. Bartek, Copenhagen), HeLa cer 0193 The HIRA B-domain from Xenopus laevis corre vical carcinoma (gift from M. Bornens, Paris), MCF7 and sponding to amino acid 435 to 480 from HIRA (Accession MDA-MB-231 breast adenocarcinoma cancer cell lines, number: AJ404369) was cloned into pGEX4T-1 (GE health MEMC medium (GIBCO) was used for BJ primary foreskin care) and transformed in BL21 (DE3) (callbiochem) in order to fibroblasts (CRL-2522, ATCC), RPMI medium (GIBCO) purify bacterially expressed GST HIRA B-domain recombi Supplemented with 10 g/mL insulin (Sigma) was used for nant protein (see below). The DNA sequence of the HIRA HS478T breast cancer cells (gift from 0. Delattre, Paris) B-domain (1303-1440 pb) is the following: 5'-GGG (Hackett et al., 1977), and DMEM medium (GIBCO) con GAAAGCTTGGAGGACATAAGAAAGAAC taining 3Ong/mL Epidermal Growth Factor (TEBU) was CTCTTGAAAAAGCAAGTGGA GACACGAACAGCT used for HS478Bst healthy mammary cells (ATCC) (Hackett GATGGACGGCGAAGGATCACTCCACTCTGCATTGCTC et al., 1977). All media contain 10% FCS (Eurobio) and AGCTAGACACTGGGGACTTTTCCA 10mg/mL penicillin and streptomycin (GIBCO). Glutamax CAGCGTTTTTCAAT-3' (SEQ ID No. 27) and the protein DMEM (Invitrogen) supplemented with 15% FCS was used sequence of the HIRA B-domain Protein (aa 435-480) is the to grow early passage (PD25) IMR90 human primary fibro following: GESLEDIRKNLLKKQVETRTADGRRRIT blasts (ATCC) at 7.5% CO, and 3% 0. To obtain old (PD72) PLCIAQLDTGDFSTAFFN (SEQID No. 28). and senescent (PD80) cells, cells were passaged in a 1:4 (0194 The DNA sequence of the mutated HIRAB-domain regimen for additional population doublings where the new I461D from Xenopus laevis that does not interact with Asf1 PD was calculated as PD=PD at plating+In (iharvested/ anymore is the following: 5'-GGGGAAAGCTTGGAGGA CATAAGAAAGAACCTCTTGAAAAAG iseeded)/In2. Cells were counted with the Z1 Coulter Parti CAAGTGGAGAC ACGAACAGCTGATGGACGGC cule Counter (Beckman coulter). GAAGGGACACTCCACTCTGCATTGCTCAGCTAGA Synchronization of Cells CACTGGGGACTTTTCCACAGCGTTTTTCAAT-3 (SEQ ID No 29) and the protein sequence of the mutated HIRA 0.190 BJ primary cells and U-2-OS osteosarcoma cells B-domain I461D protein (aa 435-480) is the following: were synchronized in quiescence by incubation in a serum GESLEDIRKNLLKKQVETRTADGRRRDT US 2013/O 14932O A1 Jun. 13, 2013 22
PLCIAQLDTGDFSTAFFN (SEQ ID No 30). Of note, the optimem 1 medium (GIBCO) according to manufacturers protein sequence for the B-domain of human HIRA is the instructions. siRNA sequences were used against ASfla (GT same (100% homology) with the one of Xenopus laevis. GAAGAATACGATCAAGTdTdT, SEQ ID No. 13), Asf1b (CAACGAGTACCTCAACCCTdTdT, SEQ ID No. 14), Antibodies siControl (GCGCGCTTTGTAGGATTCGdTdT, SEQID No 0.195 Rabbit polyclonal antibody raised against the full 15) and siCFP length GST-Asfla (antibody #28134) was described previ (0198 (CACTTGTCACTACTTTCTCdTaT, SEQ ID No ously (Mello et al., 2002). An additional specific antibody 16) (Dharmacon) as in (Groth et al., 2007: Groth et al., 2005) against Asflb was produced. For this, the C-terminal part of siRNAs against Asfla and Asfbat a final concentration of (amino-acids 156-202) of Asf1b was cloned in a pCEX-4T-1 50 nM each for the double depletion of Asfl(a+b). vector (Novagen) (see above). For the immunization of two rabbits (#18130 and #18143) (Agrobio), bacterially Colony Formation Assay expressed GST-C-Term-Asf1b recombinant protein in E. (0199 Hela US-2-OS, Hs578T and MDA-MB-231 cells Coli BL21 (DE3, Novagen) was used (Moggs et al., 2000), were transfected with siRNA against Asfla, Asf1b, Asf1(a+b) purified on glutathione beads (17-0756-01, GE Healthcare) or GFP or with a control siRNA as above. 24 h after transfec and eluted with 10 mM glutathione according to the manu tion, 1000-2000 cells were plated as a single-cell suspension facturers instructions. The specificity of both Asfla and in 6 cm dishes, allowed cells to grow under normal conditions Asflb antibodies was confirmed by Western blotting and for 8-12 days before staining with 0.1% Coomassie Brilliant immunofluorescence microscopy (FIG. 1). Since Asfla and b Blue R-250 (Bio-Rad) dissolved in 50% methanol, 15% ace migrate at different positions, a mix of the specific ASfl tic acid and counted colonies using an automatic counting antibodies was used for simultaneous detection of the two colony counter pen. The mean plating efficiency (0.38) and isoforms by Western blotting. For immunofluorescence stud the Surviving fraction were determined as in (Franken et al., ies, either highly purified Asfla (#28134) or Asflb (#18143) 2006). antibodies were used from sera (Agrobio). Table 3 compiles all primary antibodies with their source, reference, and dilu Western Blotting tions for western blotting or immunofluorescence. In addi tion, the inventors used Rabbit polyclonal antibody against 0200 For total extracts, lysed cells were processed in HIRA (Ray-Gallet et al., 2002) and against GST (Abcam Laemmlisample buffer (LSB) 1x (62.5mM Tris HCl pH=6.8, ab9085) to reveal these proteins on FIG. 22. 10% glycerol, 2% SDS, 0.002% bromophenol blue and TABLE 3 List of all primary antibodies used in this study. Company. Oder Lot WE IF Antibody Reference Number Number Species dilution dilution
Asf1a Mello et al., 2002 #28134 Rabbit polyciona mix (a + b) 1/2000 purified ASf1b This study i18143 Rabbit polyciona } 1 1000 each 1/500 purified Cu-Tubulin Sigma T9026 (DM1A) 104K4800 Mouse monocional 1,10 OOO 3-actin Sigma A5441 (AC-15) – Mouse monocional 1,2S OOO CAF-1 pé0 Quivy et al., 2008 #17019 Rabbit polyciona 1,1OOO CAF-1 p150 Abcam ab76SS 588276 Mouse monocional 1,1OOO CENP-A Abcam ab13939 628.216 Mouse monocional 1,2SO CyclinA Santa Cruz Sc-751 GO104 Rabbit polyciona 1,1OOO H4 Upstate OS-856 JBC1361839 Rabbit polyciona 1,1OOO HURP Kato et al., 2007 Rabbit polyciona 1,1OOO HP1c. Euromedex 2HP-2G9 Mouse monocional 1 SOO LaminAC Ceil signaling 2032 2 Rabbit polyciona 1/50 Mom2 BD Transduction BM28 39289 Mouse monocional 1,1OOO laboratories PCNA DAKO MO879 (PC-10) 00026418 Mouse monocional 1,2OOO RbAp48 Abcam ab1766 8829 Rabbit polyciona 1,1OOO
0196) Company, as well as the order number, the lot num- 100mMDTT) as in (Martini et al., 1998). Memcode Protein ber, the species and the dilutions for western blotting (WB) Stain Kit (Thermo Scientific) was used to detect proteins and immunofluorescence (IF) are provided for each antibody. transferred on nitrocellulose membranes. Table 3 lists pri Since Asfla and Asf1b migrate at different positions in west mary antibodies. Secondary antibodies conjugated with ern blot, a mix of the specific Asf1 antibodies has been used in Horseradish peroxidase (HRP) (Interchim) were used and Western blot to recognize simultaneously the two isoforms. revealed signal by chemiluminescence substrate from Pierce The specific purified antibodies against Asfla or Asf1b were (SuperSignal West Pico or SuperSignal West Femto). For used separately in immunofluorescence. quantification, the inventors performed acquisition of the chemiluminescence signal on a ChemiDoc XRS (BioRad) siRNA and Transfections geldoc, and quantification of the intensity of the bands with 0197) U-2-OS, Hs578T and MDA-MB-231 cells were Quantity One 4.6.6 software. It was checked that the signal transfected in an antibiotics-free medium for 48 h with 100 response is in a linear range using dilution series. Values nM siRNA using Oligofectamine reagent (Invitrogen) and obtained for ASfla or Asf1b levels were normalized to the US 2013/O 14932O A1 Jun. 13, 2013
levels of a-tubulin, or to the Memcode (Invitrogen) protein - Continued staining in the case of the mammary cell lines. CAF-1 p150 Forward: (SEQ ID No. 19) Immunofluorescence Microscopy CAGCAGTACCAGTCCCTTCC 0201 Cells grown on coverslips, fixed in 2% paraformal CAF-1 p150 Reverse: dehyde, and permeabilized in PBS containing 0.2% Triton (SEQ ID No. 20) X-100, were processed as in (Martini et al., 1998). For lamin TCTTTGCAGTCTGAGCTTGTTC A staining, a pre-extraction step was performed to remove GAPDH Forward: soluble proteins. Briefly, cells were washed with CSK, (SEQ ID No. 21) extracted with CSK 0.5% Triton X-100 and rinsed with CSK GAGTCAACGGATTTGGTCGT; and PBS before fixation as described above. Cross-absorbed GAPDH Rewerse : Alexa-488 or Alexa-594 conjugated secondary antibodies (SEQ ID No. 22) (Molecular probes-Invitrogen) were used to detect primary TTGATTTTGGAGGGATCTCG. antibodies (Table 3). Images were acquired with a DM600 (Leica) upright widefield epifluorescence microscope (63x RNA Extraction and Quantitative RT-PCR: Breast Tumor objective/NA 1.32 or 40x objective/NA 1.0) piloted with Samples from 1995 and 1996 Metamorph software and equipped with a chilled CCD cam (0203 The RNeasy mini kit (QIAGEN) was used for total era (CoolSnap Ha2, Photometrics). Identical settings and the RNA extraction for transcriptome analysis and the miRNeasy same contrast adjustment were applied for all images to allow mini kit (QIAGEN) for RNA extraction from frozen breast accurate data comparison, except for LaminA staining on cancer samples (1995 and 1996) (De Koning et al., 2009). FIG. 18C which was specifically enhanced in Asf1b depleted Reverse transcription and quantitative RT-PCR were per cells in order to visualize the DNA bridges. For brightness formed as described below. All reverse transcription was per and contrast adjustment, Adobe Photoshop CS3 (Adobe) was formed using SuperScript II reverse transcriptase (Invitrogen) used. For quantitative analysis, a minimum number of n=100 with 500 ng-1 lug of RNA and 300 ng-3 ug of random primers nuclei were counted per experiment. (Invitrogen) per reaction respectively. For quantitative PCR analysis, the 96-well plate Step One Plus system (Applied Primers Biosystems) was used and the SYBR Green PCR Master mix (Applied Biosystems) (transcriptome and breast tumor (0202 For analysis of the 86 and 71 breast tumor samples samples from 1995) or the QuantiTect SYBR Green RT-PCR from 1995 and 1996, the following primers were used: kit (QIAGEN) or the SYBR(R) FASTABI Prism(R) Master Mix (KAPA Biosystems) (breast tumor samples from 1996). As f1la Forward: Plates were filled using an EpMotion 5070 Robot (Eppen (SEQ ID No 1) dorf). Duplicates were measured in all experiments and the CAGATGCAGATGCAGTAGGC; efficiency of each primer pair was checked with three subse quent cDNA dilutions for each of the breast tumor samples. As fla Reverse : (SEQ ID No 2) For each gene, the quantity of mRNA was normalized to the CCTGGGATTAGATGCCAAAA; quantity of mRNA corresponding to the human acidic ribo somal phosphoprotein PO (RPLPO) (de Cremoux et al., As f1lo Forward: 2004) or to the Glyceraldehyde 3-phosphate dehydrogenase (SEQ ID No 3) (GAPDH). To compare with the transcriptome analysis, CGAGTACCTCAACCCTGAGC; mRNA levels relative to control siRNA levels were plotted As f1lo Reverse : and logarithmic 2 values were taken. This is referred to as the (SEQ ID No. 4) log2(fold change). CCATGTTGTTGTCCCAGTTG.;
RPLPO Forward: Transcriptomic Data Analysis (SEO ID No. 5) GGCGACCTGGAAGTCCAACT; 0204. In two independent experiments, mRNAs were pre
RPLPO Reverse : pared using U-2-OS cells treated with control. Asfla, Asf1b (SEQ ID No 6) or Asfl (a+b) siRNAs for 48 h, and hybridized them on CCATCAGCACCACAGCCTTC: Affymetrix HG-U133-Plus2 oligonucleotide microarrays. The inventors determined differentially expressed genes HP1C. Forward: using the Bioconductor package limma (Smyth, 2004). For (SEQ ID No. 11) each gene, they constructed a linear model that relates the GATCATTGGGGCAACAGATT; expression value of the gene in the eight samples to a common HP1C, reverse: intercept, an effect for the presence of the siRNA against (SEQ ID No. 12) Asfla (siAsfla) and an effect for the presence of the siRNA TGCAAGAACCAGGTCAGCTT; against ASflb (siASflb). For each gene, a one-sample t-test CAF-1 péO Forward: was used to determine if the effects of siASfla and/or siAsflb (SEO ID No. 17) were significantly different from Zero. For each test the vari CGGACACTCCACCAAGTTCT; ance of the gene term was shrunk towards an overall variance CAF-1 péO Reverse: using the Empirical Bayes procedure of the Bioconductor (SEQ ID No. 18) package limma. The inventors corrected the significant p-val CCAGGCGTCTCTGACTGAAT; ues of the t-tests for multiple testing by controlling the False Discovery Rate (Benjamini et al., 2001). They report an effect US 2013/O 14932O A1 Jun. 13, 2013 24 as significantly different from Zero if the corrected p-value of given sample by applying x=100/(E(Cp Gene Cp the test was less or equal 0.05. A Venn diagramm was drawn RPLPO)), where E is the mean efficiency of the primers. For using the lists of differentially expressed genes (up- and statistical analysis, data from 55 (1995) or 64 (1996) patients down-regulated) determined against the control siRNA with a for Asfla, and 85 (1995) or 69 (1996) patients for Asf1b, 75 p-value of 0.05. (1995) or 70 patients (1996) for CAF-1 p60 and 86 (1995) or 71 (1996) patients for CAF-1 p150, which fulfilled the ampli 0205 To investigate whether the resulting lists of differ fication criteria (reproducible duplicates, consistent primer entially expressed genes had significant association with efficiency between samples) were retained. Importantly, Gene Ontology terms (Ashburner et al., 2000), the inventors because of the difference in the number of patients with data used the Bioconductor package topGO (Alexa et al., 2006). for ASfla and for Asf1b, it was verified that there was no They obtained the Gene Ontology (GO) annotation of genes significant differences in the composition of the two popula on the microarray from the Ensemble database in March tions of patients (data not shown). 2009. They disregarded the associations between genes and 0209 Correlations were calculated between various fac GO terms which were solely inferred from electronic anno tors using the Pearson correlation coefficient method and tation (GO evidence terms: IEA, NAS, ND). To mitigate the analyzed differences between groups with the Kruskal-Wallis dependencies between the tests imposed by the structure of test for continuous variables. The disease-free interval is the GO, if a gene was counted for the annotation of a specific defined as the time from the diagnosis of breast cancer until gene, it was not counted again for any ancestor terms of this the occurrence of disease progression, meaning local recur term (“elim' method of the package topGO). For each term, rence in the treated breast, regional recurrence in lymph node they performed a Hypergeometric test to determine whether bearing areas, controlateral breast cancer or distant recur genes of that list showed a more frequent association with a rences. A cut-off value that is prognostic for the disease free certain term than would be expected by chance given the GO interval (DFI) by using a Cox proportional risks model was annotation of all genes represented on the microarray. When determined and used the Wald test to evaluate the prognostic the test resulted in a p-value inferior or equal to 0.001, they value of this variable on each event. The overall survival (OS), considered these terms as significantly over-represented for the metastasis-free interval and the DFI rates were estimated the given list. They used the statistical software R (2.5.0 using the Kaplan-Meier method and compared the values version) to visualize gene expression values. between groups using a log-rank test. The inventors carried Breast Cancer Samples from 1995 out a multivariate analysis to assess the relative influence of 0206 Used samples from patients with breast tumor clas certain prognostic factors (age, number of mitosis, grade, sified as non-palpable (TO) or small (T1-T2), lymph node estrogen and progesteron-receptor status as well as p60, negative (NO) and metastasis free (MO), were selected at the p150, HP1a, Asf1b, Ki67 expression levels) on OS, DFI and Institut Curie Biological Resources Center and treated with metastasis free interval using the Cox stepwise forward pro primary conservative tumorectomy. 92 patients diagnosed in cedure (Cox 1972). The significance level was 0.05. The 1995 granted permission to use their sample and data for statistical software R (2.5.0 version) was used for the analy research purposes. Table 1A provides patients and tumor SCS. characteristics. RNA extracted from 86 cryofrozen tissue of Production of the Recombinant HIRA B-Domain Proteins sufficient quality were selected for further analysis by RT and GST Pull-Down QPCR and carried out statistical analyses. The median fol 0210. The inventors carried out the induction with IPTG low-up of the patients was 146 months (range: 30-161 0.4 mM at 30° C. during 3 h. Bacteria were centrifuged at 5 months). Recurrence-free and alive patients were censored to 000 g 15 min at 4°C. and washed with cold water. Bacteria the date of their last known contact. At the date of the analysis, pellets were then frozen in liquid nitrogen and stored at -80° 11% of the patients were no longer alive, with cause of death C. For the purification of recombinant proteins, the pellets being the initial breast cancer in 70% of these cases. 10% of were thawed on ice and resuspended in cold PBS containing patients developed loco-regional recurrence and 15% devel protease inhibitor cocktail (Complete without EDTA, Roche oped metastasis. Diagnostic) and 1 mg/ml lysosyme (Sigma Aldricht). After 10 Breast Cancer Samples from 1996 min incubation on ice, bacteria Suspensions were Sonicated 0207 Samples from patients with breast tumor classified using a Branson sonifier. 1% (final) tritonX100 was added to as non-palpable (TO) or small (T1-T2), lymph node negative the extract that was incubated another 30 minonice. Extracts (NO) and metastasis free (MO), were selected at the Institut were then cleared at 20 000 g for 15 minat 4°C. Supernatants Curie Biological Resources Center and treated with primary were incubated with GST beads (GE Healthcare) equilibrated conservative tumorectomy (median tumor size: 17 mm, in PBS, protease inhibitor and Triton X 100 (binding buffer) range: 8-35 mm). 71 patients diagnosed in 1996 granted for 2 hat 4°C. on a wheel. Beads were washed in the binding permission to use their sample and data for research purposes. buffer supplemented with 150 mMNaCl and stored at 4°C. or Table 2 provides patients and tumor characteristics. Recur the GST recombinant proteins were eluted by adding 3 times rence-free and alive patients were censored to the date of their some Tris HCl 50 mM pH8, 10 mM Gluthatione (Sigma last known contact. At the date of the analysis, 17% of the Aldricht). Elutions were dialized against the same buffer patients were no longer alive, with cause of death being the without the gluthatione and the GST recombinant proteins initial breast cancer in 58% of these cases. 9% of patients were Stored at -80° C. developed loco-regional recurrence and 18% developed 0211 GST pull-downs were performed by mixing 6 ml of metastasis. RNA extracted from 71 cryo frozen tissue of High speed Xenopus egg extract (HSE prepared as described sufficient quality were selected for further analysis by RT in (Almouzni, 1998)), 1X nucleosome assembly buffer, 300 QPCR. ngp', 4 mMATP 100 mg/ml creatine kinase and 3 mg Breast Tumor Samples from 1995 and 1996: Statistics of recombinant protein on beads during 3 hat 23°C. The flow 0208 For each gene, the quantity X of the gene mRNA through was recovered and beads were washed 3 times with relative to the quantity of RPLPO mRNA was expressed in PBS supplemented with 150 mM NaCl and 0.5% NP40. US 2013/O 14932O A1 Jun. 13, 2013 25
Different fractions were loaded on a NuPAGE 4-12% (Invit - Continued rogen) and ran in MES buffer. Gels were transferred for 1 h, at 15 V on a nitrocellulose membrane (0.22 mm, Whatman) CAF-1 péO Reverse: blocked with PBS, 0.1% tween20 and blotted 2 hat room (SEQ ID No. 18) temperature with specified antibody. CCAGGCGTCTCTGACTGAAT; Mcm2 Forward: Nucleosome Assembly Reaction (SEQ ID No 31) 0212. In vitro nucleosome assembly reactions indepen ACCAGGACAGAACCAGCATC; dent of DNA synthesis were performed for 3 hours at 23°C. with 10 ul of HSE (Almouzni, 1998) and non-irradiated p3S Mcm2 Reverse: plasmid as described in (Ray-Gallet and Almouzni, 2004). (SEQ ID No. 32) For the dominant negative assay, 2, 4 or 8 mg of the recom CAGGATGTCAAAGCGTGAGA; binant protein were added directly in the nucleosome assem HJURP Forward: bly reaction mix without any prior incubation. At the end of (SEQ ID No. 33) the assembly reaction, a small aliquot was removed for analy GCTGGAAGGGATGTACGTGT sis by western blotting. The supercoiling of the purified DNA plasmid was examined by agarose gel electrophoresis in 1X HJURP Rewerse : TAE buffer and visualized by staining with ethidium bromide (SEQ ID No. 34) (Ray-Gallet and Almouzni, 2004). TGGGTCACCAGGACTCTTTC; Example 2 RPLPO Forward: (SEQ ID No 35) 0213 Materials and methods AACTCTGCATTCTCGCTTCC RNA Extraction and QPCR Analysis RPLPO Reverse : (SEQ ID No. 36) 0214 RNA extraction and QPCR analysis were per TCGTTTGTACCCGTTGATGA. formed as disclosed in example 1. Primers Ovarian Tumor Samples and Statistics 0215 For analysis of the 122 ovarian tumor samples, the following primers were used: 0216. The inventors used samples from patients of 1989 to 2008 with ovarian tumor selected at the Institut Curie Bio
As f1la Forward: logical Resources Center and treated with primary conserva (SEQ ID No 1) tive tumorectomy. 122 patients granted permission to use CAGATGCAGATGCAGTAGGC; their sample and data for research purposes. Recurrence-free As fla Reverse: and alive patients were censored to the date of their last (SEQ ID No 2) known contact. At the date of the analysis, 38% of the patients CCTGGGATTAGATGCCAAAA; were no longer alive, with cause of death being the initial As f1lo Forward: ovarian cancer in 94% of these cases. 44% of patients devel (SEQ ID No 3) oped loco-regional recurrence and 70% developed metasta CGAGTACCTCAACCCTGAGC; sis. Patients and tumor characteristics are Summarized in Table 4 below TABLE 4 Description of the samples from patients with ovarian cancer Age Median: 58 (Range: 31-86) Histological I 5% Chemotherapy No 12% Menopaused Yes: 23% grade II 32% Yes 88% No: 779, III 63% Recidivism No 56% Histological serious 74% Histological I 1796 Yes 44% type mucinous 59 Stage II 11% Metastasis No 30% endometrioid 11% IIIa 79% Yes 70% other 11% IIIb-IV 66%
- Continued 0217. The inventors selected RNA extracted from 122 cryofrozen tissue of sufficient quality for analysis by RT As f1lo Reverse: QPCR. For each gene, the quantity X of the gene mRNA was (SEQ ID No. 4) expressed relative to the quantity of RPLPO mRNA in a given CCATGTTGTTGTCCCAGTTG.; sample by applying X=100*(E) (Cp RPLPO Cp Gene)), where E is the mean efficiency of the primers. For statistical CAF-1 péO Forward: analysis, the inventors retained data from all 122 patients for (SEO ID No. 17) Asfla, Asf1b, CAF-1 pé0, Mcm2 and HJURP which fulfilled CGGACACTCCACCAAGTTCT; their amplification criteria (reproducible duplicates, consis tent primer efficiency between samples). US 2013/O 14932O A1 Jun. 13, 2013 26
0218 Correlations between various factors were calcu 0235 De Koning et al., 2009, EMBO Mol Med 1, 178 lated using the Pearson correlation coefficient method and 191. differences between groups were analysed with the Kruskal 0236 D'Errico et al. 2009. EurJ Cancer 45, 461–469. Wallis test for continous variables. The significance level was 0237) Elbashir et al., 2001a, Embo J 20, 6877-88. 0.05. The statistical software R (2.5.0 version) was used for 0238 Elbashir et al., 2001b, Nature 411, 494-8. these analyses. 0239) Elston and Ellis, 2002. Histopathology 41, 151-3. Results 0240 English et al., 2006, Cell 127,495-508. 0241 Fanning and Symonds, 2006, RNA Towards Medi 0219. Asflb Correlates with Proliferation in Ovarian cine (Handbook of Experimental Pharmacology), ed. Tumor Samples Springer p. 289-303 0220 To assess the relevance of findings connecting 0242 Franken et al., 2006, Nat Protoc 1, 2315-2319. Asflb with proliferation in a physiological context, the inven tors analysed a selection of cryopreserved ovarian carcinoma 0243 Groth et al., 2005, Mol Cell 17, 301-311 samples collected between 1898 and 2008 at the Institut 0244 Groth et al., 2007, Science 318, 1928-1931. Curie. Table 4 provides the patients and tumor characteris 0245 Hackett et al., 1977, J Natl Cancer Inst 58, 1795 tics. The standard treatment received by the patients at the 1806. Institut Curie for ovarian cancers was tumor excision with 0246 Hu et al., 2010, Breast Cancer Res. 12, R18. 88% of patients receiving chimiotherapy. Asf1a, Asf1b, 0247 Jasencakova et al., 2010, Mol. Cell. 37, 736-43. CAF-1 pé0, Mcm2 and HJURP mRNA expression levels 0248 Kurdistani, 2007, Br J Cancer 97, 1-5. were measured by quantitative RT-PCR in 122 ovarian tumor 0249 Malay et al., 2008, J Biol Chem 283, 14022-31. samples and the expression levels were normalized to the 0250 Martini et al., 1998, J Cell Biol 143, 563-575. known reference gene RPLPO (de Cremoux et al., 2004). For 0251 Mello et al. 2002. EMBO Rep 3, 329-334. statistical analysis, only data that fulfilled amplification qual (0252 Moggs et al. 2000. Mol Cell Biol 20, 1206-1218. ity criteria were kept. 0253 Mousson et al. 2005. Proc Natl Acad Sci USA 102, 0221 First, the correlation between the levels of Asfl iso 5975-5980. forms and that of other proliferation markers such as CAF-1 p60 (Polo et al., 2004) or HJURP and Mcm2 was studied. 0254 Natsume et al., 2007, Nature 446, 338-341. Asfla levels only weakly correlated with that of Asf1b. (0255 Osborne et al., 1997, Curr Opin Chem Biol. 1, 5-9. CAF-1 p60, Mcm2 and HJURP (FIG.23 and data not shown). 0256 Pei et al. 2009. Cancer Cell 16, 259-266. In contrast, Asf1b levels significantly correlated with p60 0257 Polo et al., 2004, Cancer Res 64, 2371-2381. (r=0.7; p<10'), Mcm2 (r–0.8; p.<10) and HJURP (r–0.8; 0258 Pilyugin et al 2009. PLoS ONE 4, e8328. p<10'), which again demonstrated its important link with (0259 Quivy et al. 2001. Embo J20, 2015-2027. Ransomet cell proliferation (FIG. 23). The inventors then investigated al., 2010, Cell 140, 183-195. the correlation of Asf1b levels with clinical parameters to 0260 Ray-Gallet et al. 2002. Mol Cell 9, 1091-1 100. evaluate a potential diagnostic value. They found a high sig 0261 Ray-Galletet al., 2004, Methods Enzymol375, 117 nificant positive correlation of Asflb levels with the histo 131 logical grade of the tumor (p=2e-04), the histological stage of 0262 Reyalet al., 2008, Breast Cancer Research 10, R93 the tumor (p=0.043), the tumor resection (p=0.035) and the family antecedents of breast cancer (p=0.049) (FIG. 24). 0263 Rhodes et al., 2004, Neoplasia 6, 1-6. Taken together, these observations put forward Asf1b as a 0264 Rosty et al., 2005, Oncogene 24, 7094-7104. new proliferation marker of clinical interest in the context of 0265 Sabates-Bellveretal. 2007 Mol Cancer Res 5, 1263 ovarian cancer. 1275 0266 Sanchez-Carbayo et al. 2006. J. Clin Oncol 24, 778 REFERENCES 789 0222 Alexa et al., 2006, Bioinformatics 22, 1600-7 0267 Santegoets et al. 2007. Int J Cancer 121,759-766 0223 Almouzni, G. 1998. Assembly of chromatin and 0268 Schonk et al., 1989, Hum Genet 83, 297-299. nuclear structures in Xenopus egg extracts. HGould, Editor, 0269 Smyth, 2004, Stat Appl Genet Mol Biol3, Article3. Chromatin: A Practical Approach, Oxford University Press, 0270 Sotiriou et al., 2006, J Natl Cancer Inst 98,262-272. Oxford, 195-218. 0271 Sotiriou and Piccart, 2007, J Natl Cancer Inst 98, 0224 Ashburner et al., 2000, Nat Genet. 25, 25-9. 262-272. 0225. Barretina et al. 2010. Nat Genet 42, 715-721. 0272 Sotirou and Pusztai, 2009, N Engl J Med 360:790. 0226 Benjamini et al., 2001, Behav Brain Res. 125, 279 0273 Sun et al. 2006. Cancer Cell 9, 287-300 84. (0274 Tamburini et al., 2005, Eukaryot Cell 4, 1583-1590. 0227 Bernstein, et al., 2001, Nature 409,363-6. (0275 Tang et al., 2006, Nat Struct Mol Biol 13,921-929. 0228 Carrollet al., 2000, J Biol Chem 275,38221-38229. Epub 2006 September 2017. 0229 Corpet et al., 2009, Trends Cell Biol 19, 29-41. 0276 Umehara et al., 2003, J Biol Chem 278, 35660 0230 Corpet and Almouzi, 2009, EMBOJ 28, 1828-30. 35667. 0231 Daganzo et al., 2003, Curr Biol 13, 2148-2158. 0232 Das et al., 2009, Nature 459, 113-119. (0277 van 't Veer et al., 2002, Nature 415, 530-536. 0233 de Cremoux et al., 2004, Endocr Relat Cancer 11, 0278 Wirapati et al., 2008, Breast Cancer Res 10, R65. 489-495. 0279 Yuan et al., 2009, Cell Cycle 8, 1747-53. 0234 De Koning et al., 2007, Nat Struct Mol Biol 14, 0280 Zamore et al., 2000, Cell 101, 25-33. 997-1007. (0281 Zhang et al., 2005, Dev Cell 8, 19-30. US 2013/O 14932O A1 Jun. 13, 2013 27
SEQUENCE LISTING
<16O is NUMBER OF SEO ID NOS: 36
<210s, SEQ ID NO 1 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: artificial sequence 22 Os. FEATURE: 223s OTHER INFORMATION: Primer
<4 OOs, SEQUENCE: 1 Cagatgcaga tigcagtaggc
<210s, SEQ ID NO 2 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 2 Cctgggatta gatgccaaaa
<210s, SEQ ID NO 3 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 3 cgagtacctic aaccctgagc
<210s, SEQ ID NO 4 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 4 c catgttgtt gtcc.cagttg
<210s, SEQ ID NO 5 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 5 ggcgacctgg aagttccaact
<210s, SEQ ID NO 6 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 6 c catcagdac cacago ctitc US 2013/O 14932O A1 Jun. 13, 2013 28
- Continued <210s, SEQ ID NO 7 &211s LENGTH: 2O4 212. TYPE: PRT <213> ORGANISM: homo sapiens
<4 OO > SEQUENCE: 7 Met Ala Lys Val Glin Val Asn. Asn Val Val Val Lieu. Asp Asin Pro Ser 1. 5 1O 15 Pro Phe Tyr Asn Pro Phe Glin Phe Glu Ile Thr Phe Glu. Cys Ile Glu 2O 25 3O Asp Lieu. Ser Glu Asp Lieu. Glu Trp Llys Ile Ile Tyr Val Gly Ser Ala 35 4 O 45 Glu Ser Glu Glu Tyr Asp Glin Val Lieu. Asp Ser Val Lieu Val Gly Pro SO 55 6 O Val Pro Ala Gly Arg His Met Phe Val Phe Glin Ala Asp Ala Pro Asn 65 70 7s 8O Pro Gly Lieu. Ile Pro Asp Ala Asp Ala Val Gly Val Thr Val Val Lieu. 85 90 95 Ile Thr Cys Thr Tyr Arg Gly Glin Glu Phe Ile Arg Val Gly Tyr Tyr 1OO 105 11 O Val Asn Asn Glu Tyr Thr Glu Thr Glu Lieu. Arg Glu Asn Pro Pro Val 115 12 O 125 Llys Pro Asp Phe Ser Llys Lieu. Glin Arg Asn. Ile Lieu Ala Ser Asn Pro 13 O 135 14 O Arg Val Thir Arg Phe His Ile Asn Trp Glu Asp Asn Thr Glu Lys Lieu 145 150 155 160 Glu Asp Ala Glu Ser Ser Asn. Pro Asn Lieu. Glin Ser Lieu. Lieu. Ser Thr 1.65 17O 17s Asp Ala Lieu Pro Ser Ala Ser Lys Gly Trip Ser Thr Ser Glu Asn. Ser 18O 185 19 O Lieu. Asn Val Met Lieu. Glu Ser His Met Asp Cys Met 195 2OO
<210s, SEQ ID NO 8 &211s LENGTH: 2O2 212. TYPE: PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 8 Met Ala Lys Val Ser Val Lieu. Asn. Wall Ala Val Lieu. Glu Asn Pro Ser 1. 5 1O 15 Pro Phe His Ser Pro Phe Arg Phe Glu Ile Ser Phe Glu. Cys Ser Glu 2O 25 3O Ala Lieu Ala Asp Asp Lieu. Glu Trp Llys Ile Ile Tyr Val Gly Ser Ala 35 4 O 45 Glu Ser Glu Glu Phe Asp Glin Ile Lieu. Asp Ser Val Lieu Val Gly Pro SO 55 6 O Val Pro Ala Gly Arg His Met Phe Val Phe Glin Ala Asp Ala Pro Asn 65 70 7s 8O Pro Ser Lieu. Ile Pro Glu Thr Asp Ala Val Gly Val Thr Val Val Lieu. 85 90 95 Ile Thr Cys Thr Tyr His Gly Glin Glu Phe Ile Arg Val Gly Tyr Tyr 1OO 105 11 O
Val Asn. Asn. Glu Tyr Lieu. Asn Pro Glu Lieu. Arg Glu Asn. Pro Pro Met 115 12 O 125 US 2013/O 14932O A1 Jun. 13, 2013 29
- Continued
Llys Pro Asp Phe Ser Glin Lieu. Glin Arg Asn. Ile Lieu Ala Ser Asn Pro 13 O 135 14 O Arg Val Thir Arg Phe His Ile Asn Trp Asp Asn. Asn Met Asp Arg Lieu 145 150 155 160 Glu Ala Ile Glu Thr Glin Asp Pro Ser Lieu. Gly Cys Gly Lieu Pro Lieu. 1.65 17O 17s Asn Cys Thr Pro Ile Lys Gly Lieu. Gly Lieu Pro Gly Cys Ile Pro Gly 18O 185 19 O Lieu. Lieu Pro Glu Asn. Ser Met Asp Cys Ile 195 2OO
<210s, SEQ ID NO 9 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 9 attgaacctg. c.ggaagagct g 21
<210s, SEQ ID NO 10 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 10 ggagcgcagg gatatt CCct t 21
<210s, SEQ ID NO 11 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 11 gat cattggg gcaa.ca.gatt 2O
<210s, SEQ ID NO 12 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 12 tgcaagaacc aggtoagctt 2O
<210s, SEQ ID NO 13 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: artificial sequence 22 Os. FEATURE: 223s OTHER INFORMATION: siRNA 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (2O) . . (21) <223> OTHER INFORMATION: desoxythymidine
<4 OOs, SEQUENCE: 13 US 2013/O 14932O A1 Jun. 13, 2013 30
- Continued gtgaagaata catcaagtt t 21
SEQ ID NO 14 LENGTH: 21 TYPE: DNA ORGANISM: artificial sequence FEATURE: OTHER INFORMATION: siRNA FEATURE: NAMEAKEY: misc feature LOCATION: (2O) . . (21) OTHER INFORMATION: deoxythymidin
SEQUENCE: 14 caacgagtac ct caac cctt t 21
SEO ID NO 15 LENGTH: 21 TYPE: DNA ORGANISM: artificial sequence FEATURE: OTHER INFORMATION: siRNA FEATURE: NAMEAKEY: misc feature LOCATION: (2O) . . (21) OTHER INFORMATION: deoxythymidin
SEQUENCE: 15 gcgc.gctttg taggatt.cgt t 21
SEQ ID NO 16 LENGTH: 21 TYPE: DNA ORGANISM: artificial sequence FEATURE: OTHER INFORMATION: siRNA FEATURE: NAMEAKEY: misc feature LOCATION: (2O) . . (21) OTHER INFORMATION: deoxythymidin
SEQUENCE: 16 cacttgtcac tactittct ct t 21
SEO ID NO 17 LENGTH: 2O TYPE: DNA ORGANISM: artificial sequence FEATURE: OTHER INFORMATION: primer SEQUENCE: 17 cgga cactico accaagttct
SEQ ID NO 18 LENGTH: 2O TYPE: DNA ORGANISM: artificial sequence FEATURE: OTHER INFORMATION: primer SEQUENCE: 18 cCaggcgt.ct Ctgactgaat
SEQ ID NO 19 US 2013/O 14932O A1 Jun. 13, 2013 31
- Continued
LENGTH: 2O TYPE: DNA ORGANISM: artificial sequence FEATURE: OTHER INFORMATION: primer SEQUENCE: 19 cagoagtacc agt ccct tcc
SEQ ID NO 2 O LENGTH: 22 TYPE: DNA ORGANISM: artificial sequence FEATURE: OTHER INFORMATION: primer SEQUENCE: 2O t ctittgcagt ctdagcttgt to 22
SEQ ID NO 21 LENGTH: 2O TYPE: DNA ORGANISM: artificial sequence FEATURE: OTHER INFORMATION: primer SEQUENCE: 21 gagt caacgg atttggtcgt
SEQ ID NO 22 LENGTH: 2O TYPE: DNA ORGANISM: artificial sequence FEATURE: OTHER INFORMATION: primer SEQUENCE: 22 ttgattittgg agggat ct cq
SEQ ID NO 23 LENGTH: 39 TYPE: DNA ORGANISM: Artificial FEATURE: OTHER INFORMATION: primer SEQUENCE: 23 aggtgctaga attcaa.catg gaCaggctgg aggccatag 39
SEQ ID NO 24 LENGTH: 43 TYPE: DNA ORGANISM: Artificial FEATURE: OTHER INFORMATION: primer SEQUENCE: 24 gact Cttgag gtacctgacg tagattattg agctictatcg gac 43
SEO ID NO 25 LENGTH: 36 TYPE: DNA ORGANISM: Artificial FEATURE: OTHER INFORMATION: primer US 2013/O 14932O A1 Jun. 13, 2013 32
- Continued
<4 OOs, SEQUENCE: 25 aggtgctaga attcaacaca gaaaaactgg aagatg 36
<210s, SEQ ID NO 26 &211s LENGTH: 41 &212s. TYPE: DNA <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: primer <4 OOs, SEQUENCE: 26 Cttagggtgt acctgacgta Cactattgag ctictatcgga C 41
<210s, SEQ ID NO 27 &211s LENGTH: 138 &212s. TYPE: DNA <213> ORGANISM: Xenopus laevis <4 OOs, SEQUENCE: 27 ggggaaagct taggacat aagaaagaac Ctcttgaaaa agcaa.gtgga gacacgalaca 6 O gctgatggac ggcgaaggat cactic cactic tec attgctic agctagacac toggactitt 12 O tccacagogt ttittcaat 138
<210s, SEQ ID NO 28 &211s LENGTH: 46 212. TYPE: PRT <213> ORGANISM: Xenopus laevis <4 OOs, SEQUENCE: 28 Gly Glu Ser Lieu. Glu Asp Ile Arg Lys Asn Lieu Lleu Lys Lys Glin Val 1. 5 1O 15 Glu Thir Arg Thr Ala Asp Gly Arg Arg Arg Ile Thr Pro Lieu. Cys Ile 2O 25 3O Ala Glin Lieu. Asp Thr Gly Asp Phe Ser Thr Ala Phe Phe Asn 35 4 O 45
<210s, SEQ ID NO 29 &211s LENGTH: 138 &212s. TYPE: DNA <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: DNA sequence of the mutated HIRA B-Domain I461D <4 OOs, SEQUENCE: 29 ggggaaagct taggacat aagaaagaac Ctcttgaaaa agcaa.gtgga gacacgalaca 6 O gctgatggac ggcgaaggga cactic cactic tec attgctic agctagacac toggactitt 12 O tccacagogt ttittcaat 138
<210s, SEQ ID NO 3 O &211s LENGTH: 46 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Mutated HIRA B-Domain I461D protein <4 OOs, SEQUENCE: 30 Gly Glu Ser Lieu. Glu Asp Ile Arg Lys Asn Lieu Lleu Lys Lys Glin Val 1. 5 1O 15 US 2013/O 14932O A1 Jun. 13, 2013 33
- Continued Glu Thir Arg Thr Ala Asp Gly Arg Arg Arg Asp Thr Pro Lieu. Cys Ile
Ala Glin Lieu. Asp Thr Gly Asp Phe Ser Thr Ala Phe Phe Asn 35 4 O 45
<210s, SEQ ID NO 31 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial 22 Os. FEATURE: 223s OTHER INFORMATION: Primer
<4 OOs, SEQUENCE: 31 accaggacag aaccagcatc
<210s, SEQ ID NO 32 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: 223s OTHER INFORMATION: Primer
<4 OOs, SEQUENCE: 32 Caggatgtca aag.cgtgaga
<210 SEQ ID NO 33 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial 22 Os. FEATURE: 223s OTHER INFORMATION: Primer
<4 OOs, SEQUENCE: 33 gctggaaggg atgtacgtgt
<210s, SEQ ID NO 34 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial 22 Os. FEATURE: 223s OTHER INFORMATION: Primer
<4 OOs, SEQUENCE: 34 tgggtcacca ggactictitt C
<210s, SEQ ID NO 35 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial 22 Os. FEATURE: 223s OTHER INFORMATION: Primer
<4 OOs, SEQUENCE: 35 aactctgcat t ct cqcttico
<210s, SEQ ID NO 36 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial US 2013/O 14932O A1 Jun. 13, 2013 34
- Continued
22 Os. FEATURE: 223s OTHER INFORMATION: Primer
<4 OOs, SEQUENCE: 36 tcqtttgtac ccgttgatga 2O
1-28. (canceled) method comprises determining the expression level of Asf1b 29. An invitro method for predicting or monitoring clinical in a cancer sample from said subject before the administration outcome of a subject affected with a cancer, wherein the of the treatment, and in a cancer Sample from said subject method comprises the step of determining the expression after the administration of the treatment, a decreased expres level of ASflb (anti-silencing function lb) in a cancer sample sion level of Asflb in the sample obtained after the adminis from said subject, a high expression level of Asflb being tration of the treatment indicating that the Subject is respon indicative of a poor clinical outcome for said Subject. sive to the treatment. 30. The method according to claim 29, said method further 40. A method for treating a cancer by administering a comprising the administration of a cancer therapy, preferably therapeutic effective amount of a compound inhibiting the an adjuvant therapy, to a Subject having a high expression ASflb. level of ASflb. 41. The method according to claim 40, wherein the com 31. The method according to claim 29, wherein the expres pound is selected from the group consisting of a small mol sion level of Asflb is determined by measuring the quantity of ecule, an aptamer, an antibody, a nucleic acid and a molecule Asf1b protein or Asf1b mRNA. preventing the interaction Asflb with an Asflb interacting 32. The method according to claim 31, wherein the quan partner. tity of Asf1b protein is measured by immunohistochemistry, semi-quantitative Western-Blot, or protein or antibody 42. The method according to claim 40, wherein the mol arrays. ecule is selected from the group consisting of an antibody 33. The method according to claim 31, wherein the quan against ASflb and a nucleic acid molecule interfering specifi tity of Asf1b mRNA is measured by quantitative or semi cally with Asf1lb expression. quantitative RT-PCR, or by real time quantitative or semi 43. The method according to claim 42, wherein the mol quantitative RT-PCR or by transcriptome approaches. ecule is a nucleic acid molecule interfering specifically with 34. The method according to claim29, wherein the method ASflb expression and is selected from the group consisting of further comprises the step of comparing the expression level an antisense against ASflb and a siRNA against ASflb. of Asf1b to a reference expression level of AsflB. 44. The method according to claim 42, wherein the anti 35. The method according to claim 29, wherein a poor body, antisense or siRNA is specific of Asf1b in comparison clinical outcome for said Subject is decreased patient Survival to Asfla. and/or an early disease progression and/or an increased dis 45. The method according to claim 40, wherein, the com ease recurrence and/or increased metastasis formation. pound is a fragment of the B-domain of HIRA or is a fragment 36. The method according to claim 29, said method further of the B-domain of HIRA comprising SEQID NO: 28. comprising assessing at least one another cancer or prognosis 46. The method according to claim 40, wherein the admin marker selected from tumor grade, hormone receptor status, istration of a therapeutic effective amount of a compound mitotic index, tumor size, HJURP expression level or expres inhibiting the Asflb is combined with radiotherapy or a treat sion of proliferation markers selected from Ki67, MCM2, ment with an anti-tumoral agent. CAF-1 pé0 or CAF-1 p150 or a prognosis marker. 47. A method for selecting or identifying a molecule useful 37. The method according to claim 29, wherein the cancer is selected from the group consisting of breast cancer, for the treatment of cancer comprising testing a molecule for osteosarcoma, skin cancer, ovarian cancer, lung cancer, liver its ability to inhibit Asflb interaction with HIRA or CAF-1 cancer, cervix cancer, liposarcoma, gastric cancer, pancreatic p60 and selecting the molecule capable of inhibiting Asf1b cancer, bladder cancer, Vulvar cancer, colon cancer and brain interaction with HIRA or CAF-1 pé0. CaCC. 48. A combined preparation product or kit containing (a) a 38. The method according to claim 37, wherein the cancer compound inhibiting ASflb and (b) an anti-tumoral agent as a is breast cancer or early stage breast cancer without local or combined preparation for simultaneous, separate or sequen systemic invasion. tial use in the treatment of cancer with a high expression level 39. An in vitro method for monitoring the response to a of ASflb. treatment of a subject affected with a cancer, wherein the