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