Gene p. Value - df6sxcketz7bb.cloudfront.net · m11s1 hipk3 acp2 ckap5 ddb2 rps3 pcf11 hbxap ube4a...
Transcript of Gene p. Value - df6sxcketz7bb.cloudfront.net · m11s1 hipk3 acp2 ckap5 ddb2 rps3 pcf11 hbxap ube4a...
Gene p. Value
PSMB5: proteasome subunit, type, 5 2.91E-02
PPP3CA: protein phosphatase 3, catalytic subunit, isozyme 6.50E-02
CDK5: cyclin-dependent kinase 5 1.34E-01
CYP1A1: cytochrome P450, family 1, subfamily A, polypeptide 1 8.45E-01
Supplementary Table 2. Statistical analyses of PPP3CA and several genes associated with
bortezomoib resistance in the data set from the millennium trial. Whether expression of
each gene in the data set from the millennium trial (58) was higher in non-responsive (no
change and progressive disease) than in responsive (minor response and better) patients
was analyzed using GEO2R.
0 1 2
1q21.1-1q22
MCL1IL6RPSMD4PSMB4UBE2QUBAP2LRBM8ARPS27PIAS3POLR3CHIST2H2AALASS2MRPL9JTBHAX1SHC1APH-1ABCL9ZNF364
1q22
SSR2MAPBPIPCCT3MEF2D
1q43-1q44FHHNRPUTFB2M
2p16.1 SMEK22q35 * BCS1L
3q27.1-3q27.2 * POLR2HEIF4G1
4p14-4p12
HIP2SCC-112RHOHUCHL1TMEM33
4q22.3-4q24 * PPP3CA
* H2AFZSLC39A8
5q13.2-5q13.3
TINP1HMGCRCOL4A3BPPOLK
5q14.1 SCAMP1ARSB
6p21.32 RDBPSTK19
6p21.1
CD2APMUTFBXO9TFAP2BTMEM14AICKELOVL5
6q13 SMAP1
7p14.3
LSM5FKBP9PTHB1KBTBD2
7q21.11 HGF
7q21.12MCFPDMTF1ASKTP53AP1
7q32.1-7q32.2
CALUTNPO3UBE2HARF5
* RBM28IRF5METTL2
8q24.12-8q24.13
DEPDC6
* MRPL13DERL1RNF139
8q24.21-8q24.3 MYCPVT1
9q34.11-9q34.3
ABLCIZ1NUP188SETENDOGSURF1SURF2CAMSAP1UBADC1SDCCAG3EDF1ANAPC2COBRA1
9q34.3MRPL41EHMT1
*
I (n=17)
II(n=16)
III(n=17)
GeneGenomic location
(Chromosome)
2
0 1 2
10p12.33-10p12.31 DNAJC1
11p13
CD59FBXO3M11S1HIPK3ACP2CKAP5DDB2RPS3PCF11HBXAPUBE4AATP5LRPS25HMBSCBLDDX6TRAPPC4HYOU1
15q24.2 IMP3
17p11.2 RPL13PAIP1
17q23.2 MTMR4RAD51C
18q12.1-18q12.2ZNF271P15RSSLC39A6
18q21.2-18q21.33MALT1BCL2FVT1
19p13.3-19p13.2
CDC34POLRMT
* WDR18ATP5DMUM1NDUFS7RPS15UQCRCSNK1G2MKNK2INSRTIMM13EEF2TICAM1RPL36CLPPSH2D3ANDUFA7RPS28RAB11BMRPL4ICAM1ICAM3CDC37ILF3QTRT1SMARCA4
19q13.11 PDCD5ANKRD27
19q13.12 ZNF146
19q13.2ZFP36RPS16SUPT5H
19q13.31 ZNF227
19q13.32-19q13.43
FLT3LGPSCD2RPL18BAXRUVBL2SNRP70RPL13ARPS11TBC1D17NUP62NDUFA3PRPF31RPS9HSPBP1RPL28UBE2SU2AF2
20q12-20q13.12
TDE1SFRS6YWHABPPIA
21q22.3MCM3APC21orf106HRMT1L1
11p11.2
11q14.1
11q23.3
11q113.4-11q14.1
GeneGenomic location
(Chromosome)
I (n=17)
II(n=16)
III(n=17)
Supplementary Figure 1. Expression analyses of the mRNA of candidate oncogenes in MM patients. mRNA expression data on 179 candidate oncogenes in MM patient samples obtained were analyzed using GEO (20). Each patient’ s mRNA amount normalized was displayed by heat maps. Differences between stage I and III patients were analyzed using Tukey-Kramer HSD (honestly significant difference) test. *, significant.
KMS-26PPP3CA
actin
KMS-12PEPPP3CA
actin
- +panobinostat
0.5
1.0
1.5
0Rel
ativ
e m
RN
A
DMSO
pano
binos
tat
romide
psin
ACY-1215
PPP3CA
BA
Supplementary Figure 2. PPP3CA is suppressed by HDAC inhibitors in MM cells. (A) Protein expression of PPP3CA in KMS-12PE and KMS-26 treated with 20 nM panobinostat for 48 h. Two biologically independent experiments were performed. (B) Relative mRNA expression of PPP3CA in U266 treated with 20 nM panobinostat, 2 nM romidepsin, or 10 mM ACY-1215 for 24 h (n=3). Two biologically independent experiments were performed.
KMS-11
0.4
0.8
1.2
0
Gro
wth
U266
- - -+ + +0 0.1 10FK506 (µM)
ACY-1215 - - -+ + +0 0.1 10
actin
PPP3CA
-- -
-+ ++ +
ACY-1215FK506
B
C
A
Gro
wth
KMS-11 KMS-12PEKMS-18
FK506 (µM)
panobinostat - - -+ + +
0 0.1 10
- - -+ + +
0 0.1 10
- - -+ + +
0 0.1 10
t(4;14) -+ +
U266 KMS-26
- +
- - -+ + +
0 0.1 10
- - -+ + +
0 0.1 10
0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Supplementary Figure 3. Analyses of MM cells treated with HDAC inhibitors and FK506. (A) PPP3CA protein expression in U266 treated with 0.75 µM ACY-1215, 10 µM FK506, or both panobinostat and FK506 as indicated for 48 h. Two biologically independent experiments were performed. (B) MTT assays in U266 treated with 0.75 µM ACY-1215, FK506 or both ACY-1215 and FK506 as indicated for 48 h (n=5). Two biologically independent experiments were performed. (C) MTT assays in U266, KMS-11, KMS-18, KMS-26 and KMS-12PE treated with 15 nM panobinostat, FK506, or both panobinostat and FK506 as indicated for 48 h (n=5). Two biologically independent experiments were performed. The presence or absence of t(4;14) in each cell lines is described.
0
0.5
1
1.5
2
2.5
panobinostat (-) FK506 (+)panobinostat (-) FK506 (+)
panobinostat (-) FK506 (-)
panobinostat (+) FK506 (-)
panobinostat (+) FK506 (+)Tum
or v
olum
e re
lativ
e to
day
1
A
day 1
1
B
1.25
1.00
0.75
0.50
0.25
day 8
day 15
0.2
0.4
0.6
0.8
day 1
1
day 81.2
1.0
0.8
0.6
0.40.2
0.2
0.4
0.6
0.8
0.20.4
0.6
0.8
1.0
Time (days)1 8 15 22
panobinostat (+)FK506(-)
panobinostat (+)FK506(+)
C
day 8
day 15
day 221.2
panobinostat (+)FK506(-)
panobinostat (+)FK506(+)
Supplementary Figure 4. Time-dependent variation of tumor volume in xenograft mouse model treated for 22 days and statistical analyses of time-dependent variation of tumor volume in vivo study. (A) NOD/SCID mice bearing U266 cells were treated with vehicle (n=4), panobinostat (n=5), FK506 (n=7), or both panobinostat and FK506 (n=9). Treatment was continued for 22 days. Average of the ratio of the tumor volume on days 8, 15, and 22 to that on day 1 is displayed for each condition. Error bars represent the standard errors. Difference between panobinostat (+) FK506 (-) and panobinostat (+) FK506 (+) on day 22 was analyzed using Scheffe test. *, significant. (B, C) Time-dependent variation of tumor volume in xenograft mouse model was analyzed by SAS ver. 9.4 using GLM procedure. The distribution of tumor volume on designated days is dyplayed by box plot. Diamonds correspond to mean. The difference between two groups on designated days were estimated by Scheffe test analyses. *, siginificant. (B) Panobinostat (+) FK506(-) and panobinostat (+) FK506(+), treatment for 15 days. (C) Panobinostat (+) FK506(-) and panobinostat (+) FK506(+), treatment for 22 days. The ratio of mean volume of panobinostat (+) FK506 (+)-treated mice to that of panobinostat (+) FK506 (-)-treated mice on day 22 (0.459) was less than that on day 15 (0.623).
*
*
*
*
*
*
100 101 102 103
51.8
100 101 102 103
100
101
102
103
7.4
100
101
102
103
5.4 39.8
18.0
7.3
3.1
1.7
4.982.4
3.771.0
44.014.6
38.86.3
Annexin V
PI
FK506(+)
FK506(-)
ACY-1215(-)
ACY-1215(+)
Supplementary Figure 5. Analyses of MM cells treated with ACY-1215 and FK506. (A) KMS-11 was treated with 0.5 µM ACY-1215, 10 µM FK506, or both ACY-1215 and FK506 as indicated for 36 h. Induced apoptosis was evaluated by flow cytometry. Four biologically independent experiments were performed.
actin
PPP3CA
0
0 0
01.5 1.5
2.5 2.5
ACY-1215 (µM)
bortezomib (nM)
0.4
0.8
1.2
0
Gro
wth
BA
0.4
0.8
1.2
0
Gro
wth
0
0 0.1
00 5
10 0
bortezomib (nM)
FK506 (µM) 0.1 10
5 5
actin
PPP3CA
Supplementary Figure 6. Synergistic inhibition of MM cell growth and PPP3CA expression by ACY-1215 and bortezomib or bortezomib and FK506. (A) Cell growth (n=5) and protein expression in U266 treated with ACY-1215, bortezomib, or both ACY-1215 and bortezomib for 48 h. Two biologically independent experiments were performed. (B) Cell growth (n=5) and PPP3CA expression in U266 treated with bortezomib, FK506, or both bortezomib and FK506 as indicated for 72 h. Two biologically independent experiments were performed.
Gro
wth
vector #1 #3 vector #1 #3FLAG-PPP3CA
FLAG-PPP3CA
1.561 1.75 1.531 1.52
Supplementary Figure 7. Ectopic overexpression of PPP3CA blocks the combination strategy with panobinostat and borterzomib. Cell growth of KMS-11 lentivirally transduced with control vector or FLAG-PPP3CA (clone #1, #3) treated with vehicle or 10 nM of panobinostat and bortezomib for 72 h is displayed (n=5). The growth ratio of FLAG-PPP3CA-transduced cells to that of control cells is displayed. Two biologically independent experiments were performed.
00.20.40.60.81.01.21.41.61.82.0
The growth ratio ofFLAG-PPP3CA-transduced cellsto that of control cells
0 nMpanobinostat,bortezomib 10 nM