Supplementary Information for A recurrent inactivating ... · 1 Supplementary Information for A recurrent inactivating mutation in RHOA GTPase in angioimmunoblastic T-cell lymphoma

1

Supplementary Information for

A recurrent inactivating mutation in RHOA GTPase

in angioimmunoblastic T-cell lymphoma

Hae Yong Yoo1,2*

, Min Kyung Sung3*

, Seung Ho Lee1, Sangok Kim

4,5, Haeseung Lee

4,5, Seongjin Park

3, Sang

Cheol Kim3, Byungwook Lee

3, Kyoohyoung Rho

6, Jong-Eun Lee

6, Kwang-Hwi Cho

7, Wankyu Kim

4,5,

Hyunjung Ju2, Jaesang Kim

4,5, Seok Jin Kim

2,8, Won Seog Kim

2,8, Sanghyuk Lee

4,5 & Young Hyeh Ko

2,9

1Department of Health Sciences and Technology, Samsung Advanced Institute for Health Sciences and

Technology, Sungkyunkwan University, Seoul 135-710, Korea

2Samsung Biomedical Research Institute, Research Institute for Future Medicine, Samsung Medical Center,

Seoul 135-710, Korea

3Korean Bioinformation Center (KOBIC), Korea Research Institute of Bioscience and Biotechnology, Daejeon

305-806, Korea

4Ewha Research Center for Systems Biology (ERCSB), Ewha Womans University, Seoul 120-750, Korea

5Department of Life Science, Ewha Womans University, Seoul 120-750, Korea

6DNA Link Inc., Seoul 138-736, Korea

7School of Systems Biomedical Science, Soongsil University, Seoul 156-743, Korea

8Division of Hematology and Oncology, Department of Medicine, Samsung Medical Center, Sungkyunkwan

University School of Medicine, Seoul 135-710, Korea

9Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 135-

710, Korea

*These authors contributed equally to this work.

Correspondence should be addressed to S.L. ([email protected]) or Y.H.K. ([email protected]).

Nature Genetics: doi:10.1038/ng.2916

mailto:[email protected]

mailto:[email protected]

2

Table of Contents

Supplementary Tables

Supplementary Table 1. Mapping summary of Exome-seq data ......................................... 3

Supplementary Table 2. Mapping summary of RNA-seq data ........................................... 4

Supplementary Table 3. Clinical information of patients for deep sequencing .................. 5

Supplementary Table 4. Full list of somatic SNVs

Supplementary Table 5. List of nonsynonymous somatic SNVs ........................................ 6

Supplementary Table 6. List of somatic indels (supported by RNA-seq evidence) .......... 10

Supplementary Table 7. Association of RHOA G17V mutation with clinical parameters in

AITL patients........................................................................................................ 11

Supplementary Table 8. Full list differentially expressed genes

Supplementary Table 9. Primer and oligo sequences ....................................................... 12

Supplementary Table 10. List of genes affected by copy number variations

Supplementary Figures

Supplementary Fig. 1. Representative histologic evaluation of the tumor tissue ............. 13

Supplementary Fig. 2. Frozen section of the tumor sample ............................................. 14

Supplementary Fig. 3. Sanger sequencing traces of RHOA G17V mutation in T-cell

lymphoma patients ..................................................................................................... 15

Supplementary Fig. 4. Kaplan-Meier survival plot according to RHOA mutation ........... 16

Supplementary Fig. 5. In vitro proliferation assays .......................................................... 17

Supplementary Fig. 6. Inhibition of the RHOA-ROCK pathway downregulates AKT

phosphorylation and promotes cell proliferation ....................................................... 18

Supplementary Fig. 7. Structural models of wild-type and mutant RHOA proteins ........ 19

Supplementary Fig. 8. Heat map from the hierarchical clustering of DEGs .................... 20

Supplementary Fig. 9. Statistically significant KEGG pathways from GAGE gene set

enrichment analysis ................................................................................................... 21

Supplementary Fig. 10. Comparison of gene expression according to RHOA mutation

status .......................................................................................................................... 22


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Supplementary Table 1. Mapping summary of Exome-seq data

Raw Data Mapping information

Pat No. Raw reads

% of

>=Q30

Bases (PF)

Mean

Quality

Score (PF) QC-passed reads Mapped reads

Mapping

rate

Mean

depth

% bases

above 15

1 blood 61,283,566 0.85 34.15 53,381,183 52,909,911 99.12% 55.82 82.30%

tissue 66,869,132 0.86 34.14 53,531,301 53,059,930 99.12% 57.33 83.50%

2 blood 65,008,414 0.86 34.2 53,707,167 53,271,212 99.19% 59.82 82.60%

tissue 70,942,580 0.87 34.22 60,272,665 59,868,249 99.33% 68.58 84.50%

3 blood 87,213,304 90.29 35.44 69,776,540 68,971,842 98.85% 76.15 87.80%

tissue 180,699,808 89.93 35.33 125,668,538 124,117,767 98.77% 138.72 93.30%

4 blood 90,492,992 90.86 35.61 73,118,305 72,217,348 98.77% 82.6 88.20%

tissue 178,219,562 90.75 35.55 134,493,532 132,857,990 98.78% 162.72 93.20%

5 blood 105,511,824 89.42 35.18 82,089,637 81,111,144 98.81% 88.6 89.50%

tissue 182,598,358 89.05 35.05 118,088,184 116,523,954 98.68% 134.67 93.30%

Average 108,883,954 54.37 34.89 82,412,705 81,490,935 98.94% 92.501 87.82%


4

Supplementary Table 2. Mapping summary of RNA-seq data

RNA-Seq 9 patients Mapping information

Pat No. Raw reads

% of

>=Q30

Bases

Mean

Quality

Score QC-passed reads Mapped reads

Mapping

rate

Mean

depth

1 76,232,830 83.66 33.28 74,783,379 55,613,975 74.37% 39.01

2 67,626,174 85.08 33.75 66,444,218 52,799,086 79.46% 37.03

3 93,631,482 90.21 35.21 92,925,993 63,313,998 68.13% 44.41

4 96,893,070 90.79 35.4 96,222,402 75,749,873 78.72% 53.13

5 91,702,468 89.72 35.05 90,987,093 60,439,135 66.43% 42.39

6 59,876,636 83.51 33.23 58,695,147 40,062,652 68.26% 28.10

7 54,832,758 88.43 34.71 54,194,570 37,698,202 69.56% 26.44

8 72,563,038 83.96 33.37 71,132,134 52,428,999 73.71% 36.77

9 61,191,174 84.57 33.58 60,098,281 45,532,531 75.76% 31.94

Average 74,949,959 86.66 34.18 73,942,580 53,737,606 72.71% 37.69


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Supplementary Table 3. Clinical information of patients for deep sequencing.

Patient

ID

Age/

Sex

Tumor

Tissue Stage Treatment

Follow

up

(months)

Outcome

Exome-Seq RNA-

Seq

Normal

(buffy

coat)

Tumor

(fresh

tissue)

Tumor

(fresh

tissue)

PAT1 38/M LN IV CHOP +

PBST 26 DOD O O O

PAT2 41/F LN III CHOP +

PBST 29 CR O O O

PAT3 75/M LN IV CHOP 18 AWD O O O

PAT4 73/M LN III CHOP 16 CR O O O

PAT5 70/M LN IV CHOP Lost O O O

PAT6 67/M LN IV CHOP 5 DOD X X O

PAT7 65/M LN IV Steroid 2 DOD X X O

PAT8 77/M LN IV CHOP 23 CR-Recur X X O

PAT9 75/M LN IV CHOP 24 CR X X O

PAT: patient, LN: lymph node, CHOP: chemotherapy regimen. C: cyclophosphamide, H: hydroxydaunorubicin, O:

oncovin, P: prednisone. PBST: peripheral blood stem cell transplantation, DOD: died of disease, CR: complete

remission, AWD: alive with disease


6

Supplementary Table 5. List of nonsynonymous somatic SNVs

Exome-seq RNA-seq

Normal Tumor Tumor

No. Gene ID RNA

support

CHR POS Transcript ID REF ALT AA Change REF ALT REF ALT REF ALT PAT

No.

Other patients of

RNA support TYPE*

Sanger

Valid.**

1 ABCB10 FALSE 1 229676372 NM_012089 C A R395L 104 0 90 4 87 0 2 NS

2 ADAMTS7 TRUE 15 79058445 NM_014272 G A P1270S 14 0 13 4 0 2 2 NS

3 ALDH2 FALSE 12 112221046 NM_000690 G T G102C 31 0 18 5 123 0 2 NS

4 ALPP

FALSE 2 233245026 NM_001632 G A R263H 66 1 84 11 0 0 3 NS O

5 FALSE 2 233246249 NM_001632 A G E451G 24 0 46 3 0 0 4 NS X

6 ANKRD11 FALSE 16 89349909 NM_001256183* C A G1014V 112 0 133 6 55 0 1 NS

7 AQP5 FALSE 12 50355970 NM_001651 C T A57V 23 0 22 4 0 0 1 NS

8 ASPH FALSE 8 62596650 NM_001164755* C G W67C 124 0 129 19 71 0 2 NS

9 BMP5 FALSE 6 55638913 NM_021073 G A R321X 190 1 346 17 3 0 4 SG

10 CCNL2 FALSE 1 1333667 NM_030937* C T R140H 62 0 64 4 162 0 2 NS

11 CD28

TRUE 2 204591454 NM_006139 T G F51V 129 1 186 16 235 34 5 NS X

12 TRUE 2 204599555 NM_001243077* A C T195P 53 0 38 8 136 60 1 4,7 NS O

13 CEACAM3 FALSE 19 42312901 NM_001277163* G C G159R 101 0 78 16 3 0 2 NS

14 CEL FALSE 9 135946015 NM_001807 T C I488T 57 1 99 6 21 0 3 NS

15 CHIT1 TRUE 1 203186950 NM_003465* C T W358X 22 0 28 6 776 2 1 SG

16 CNOT4 FALSE 7 135048804 NM_001190850* C T V548I 198 0 220 8 12 0 1 2,4,5,8 NS X

17 CNPPD1 TRUE 2 220041022 NM_015680 C T R34Q 41 0 103 7 199 8 4 NS

18 CRYBA2 FALSE 2 219855683 NM_057093* C T G113R 76 0 80 8 0 0 1 NS

19 CSF1R FALSE 5 149459846 NM_005211 G A Q121X 17 0 23 7 61 0 2 SG

20 CSPP1 FALSE 8 68024292 NM_024790 C A N402K 100 0 85 4 10 0 1 NS

21 CTNNB1 TRUE 3 41266137 NM_001904* C A S45Y 49 0 67 5 337 14 1 NS

22 CXorf22 FALSE X 35985846 NM_152632 C A H571N 60 0 50 4 0 0 2 NS

23 CYP2A7 FALSE 19 41381771 NM_030589* T A N387Y 17 1 22 5 0 0 1 NS

24 DAB1 FALSE 1 57537223 NM_021080 T A D177V 112 0 115 18 0 0 2 NS

25 DEFB116 FALSE 20 29891127 NM_001037731 T G K66T 187 0 218 19 0 0 2 NS

26 DENND1B TRUE 1 197515066 NM_001195215 T C T446A 51 0 32 4 62 5 4 NS O

27 DFNA5 FALSE 7 24756986 NM_001127453* G A A195V 31 0 14 4 41 0 1 NS

28 DPY19L2 TRUE 12 63964599 NM_173812 T C I647V 119 1 92 11 1 1 5 NS

29 EEPD1 FALSE 7 36324428 NM_030636 A C K392T 38 0 31 6 8 0 2 NS

30 EIF3A TRUE 10 120810098 NM_003750 C T R803K 240 0 235 7 312 1 1 7 NS X


7

31 EPC1 TRUE 10 32594852 NM_001272004* T A H54L 62 0 71 8 30 9 1 NS X

32 EZH2 TRUE 7 148525895 NM_004456* C T D188N 122 0 159 9 173 14 5 NS X

33 FABP2 FALSE 4 120241902 NM_000134 T C T55A 79 0 93 6 0 0 1 NS

34 FAM47A FALSE X 34148082 NM_203408 T C I772V 103 0 153 9 0 0 5 NS

35 FAT4 FALSE 4 126242449 NM_024582 T G V1628G 116 0 220 7 5 0 4 NS

36 FGF18 FALSE 5 170863155 NM_003862 C A A43D 18 0 45 3 1 0 5 NS

37 FKBP9 FALSE 7 33039753 NM_007270 T C I418T 140 0 297 17 67 0 4 NS

38 GLE1 FALSE 9 131289735 NM_001499* G A A415T 40 0 35 3 81 0 2 NS

39 GNAQ

FALSE 9 80537095 NM_002072 G T Y101X 42 0 43 3 58 0 1 SG

40 FALSE 9 80537112 NM_002072 T A T96S 50 1 47 3 68 0 1 NS

41 GOLGA6L6 FALSE 15 20739739 NM_001145004 A G W671R 12 0 22 7 0 0 3 NS

42 GPAT2 FALSE 2 96688961 NM_207328 G A S681L 33 0 22 7 5 0 1 NS

43 GPD2 TRUE 2 157426609 NM_001083112* A G H496R 58 1 53 14 47 4 1 NS O

44 GRID2 FALSE 4 94137973 NM_001510 C T Q292X 147 0 255 10 0 0 4 SG

45 H6PD TRUE 1 9324122 NM_004285 C G Q524E 88 1 130 15 36 1 5 NS

46 HEYL FALSE 1 40092744 NM_014571 C T R141H 15 0 26 4 2 0 2 NS

47 HGF FALSE 7 81335621 NM_000601* A T V580D 106 0 175 8 3 0 4 NS

48 HIVEP1 TRUE 6 12124184 NM_002114 A G K1386E 160 0 326 15 45 7 4 NS X

49 IDH2 TRUE 15 90631837 NM_002168 C G R172S 57 0 79 4 798 144 5 NS X

50 IQSEC3 FALSE 12 176089 NM_001170738 A G Y14C 19 1 52 5 0 0 4 NS

51 KIF7 TRUE 15 90176073 NM_198525 C A S958I 11 0 10 10 1 3 1 8 NS O

52 KRT4 FALSE 12 53207583 NM_002272 C G G87A 111 0 196 10 0 0 5 NS

53 KRT8

FALSE 12 53298675 NM_001256293* A C S31A 25 0 79 4 10 0 3 NS

54 FALSE 12 53298699 NM_001256293* G A R23C 33 0 77 5 10 0 3 NS

55 KRTAP4-3 FALSE 17 39324333 NM_033187 T A Q31L 77 0 64 22 0 0 2 NS

56

LILRB1

TRUE 19 55147987 NM_006669* G C E564Q 27 0 60 6 106 2 2 NS O

57 FALSE 19 55147987 NM_001081639* G C E565Q 54 0 138 15 294 0 4 NS O

58 TRUE 19 55147988 NM_006669* A C E564A 27 0 60 6 104 2 2 NS O

59 FALSE 19 55147988 NM_001081639* A C E565A 52 1 140 15 287 0 4 NS O

60 LRRK2 FALSE 12 40681333 NM_198578 A T D894V 39 1 38 11 27 0 1 NS

61 LYZL4 FALSE 3 42448394 NM_144634 C T R79H 25 0 22 6 0 0 1 NS

62 MAGEB6 FALSE X 26213093 NM_173523 C T A377V 44 0 38 3 0 0 1 NS

63 MLL3 TRUE 7 151848624 NM_170606 C G S4190T 33 0 21 5 23 3 1 NS

64 MPRIP TRUE 17 17078670 NM_201274* G A G885S 17 0 28 10 84 16 1 NS X

65 MUC12 FALSE 7 100634964 NM_001164462 C A P374T 164 0 166 6 0 0 1 NS O


8

66 MUC17 FALSE 7 100682139 NM_001040105 T C V2481A 286 1 233 50 0 0 1 NS O

67

MUC2

FALSE 11 1092827 NM_002457 C A T1549N 41 1 105 8 0 0 4 NS X

68 FALSE 11 1092947 NM_002457 C A T1589N 72 1 177 8 0 0 5 NS X

69 FALSE 11 1093004 NM_002457 c A P1608H 72 1 142 7 0 0 3 NS X

70 NEIL3 FALSE 4 178274835 NM_018248 A C Q471H 90 0 99 5 33 0 1 2,3,8,9 NS O

71 NT5C3A

FALSE 7 33054388 NM_016489* T C D283G 56 0 64 5 119 0 1 NS X

72 FALSE 7 33054388 NM_016489* T C D283G 77 0 111 6 210 0 4 NS X

73 OR1B1 FALSE 9 125391127 NM_001004450 C T A230T 27 0 31 3 0 0 1 NS

74 PAPLN TRUE 14 73718820 NM_173462 G A W286X 11 0 8 6 18 8 1 SG O

75 PAPPA FALSE 9 119115101 NM_002581 C T R1361W 32 0 35 10 1 0 1 NS

76 PARD3 TRUE 10 34671609 NM_001184793* T C R420G 50 0 54 4 24 6 2 NS X

77 PARP4 FALSE 13 25052393 NM_006437 G T H490Q 55 1 90 9 107 0 3 NS

78 PCDHA12 FALSE 5 140255657 NM_031864* G T L200F 124 0 130 6 0 0 1 NS

79 PCDHGB7 FALSE 5 140798759 NM_018927* G A V445I 166 0 363 11 7 0 4 NS

80 PDE4DIP FALSE 1 144856817 NM_001198834* T C E2223G 24 0 26 5 57 0 1 NS O

81 PHIP TRUE 6 79675701 NM_017934 A G L1093P 145 0 184 6 55 1 1 4,7,8,9 NS O

82 PLCG1 TRUE 20 39792584 NM_002660* C T S345F 45 0 29 8 118 44 1 NS O

83 PLEKHD1 FALSE 14 69993469 NM_001161498 T C S327P 15 1 22 5 3 0 1 NS

84 POM121 FALSE 7 72409928 NM_001257190* G A R209K 105 1 142 9 97 0 5 NS

85 PRKRIR

FALSE 11 76072076 NM_004705 G A A81V 61 0 74 4 162 0 3 NS

86 TRUE 11 76072085 NM_004705 C T R78Q 57 0 71 4 133 1 3 NS

87 PSORS1C1 FALSE 6 31106516 NM_014068 C T P43S 106 0 101 5 0 0 1 NS

88 PTPRU FALSE 1 29606644 NM_001195001* C T A620V 17 0 19 8 0 0 1 NS

89 RASA2 FALSE 3 141305584 NM_006506 C A H641Q 45 0 54 4 39 0 5 NS

90 RFC2 FALSE 7 73663432 NM_002914* C T G81D 81 0 115 5 142 0 4 NS

91 RGL4 FALSE 22 24034987 NM_153615 C A H169N 35 0 67 4 20 0 1 NS

92 RHOA

TRUE 3 49412973 NM_001664 C A G17V 87 0 65 8 1171 271 2 4,5,6,7,8,9 NS O

93 TRUE 3 49412973 NM_001664 C A G17V 107 1 257 17 1527 123 4 2,5,6,7,8,9 NS O

94 ROPN1L TRUE 5 10461378 NM_031916* C T T167M 73 1 64 11 2 1 1 NS

95 SHROOM4 FALSE X 50339869 NM_020717 A C F1436L 10 1 7 4 3 0 1 NS

96 SLC4A8 TRUE 12 51856122 NM_001258401* A C K324T 82 1 74 10 5 3 1 NS

97 SMARCD1 TRUE 12 50484328 NM_139071* C T A363V 58 1 104 14 145 27 5 NS O

98 SPRY2 FALSE 13 80911723 NM_005842 C A V40F 39 0 52 3 15 0 2 NS

99 SSX5 FALSE X 48054519 NM_021015 A C L39R 55 0 39 3 0 0 1 NS

100 STAB2 FALSE 12 104140461 NM_017564 G A G2075S 109 1 103 20 11 0 1 NS


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101 TATDN2 TRUE 3 10318105 NM_014760 C T R632X 86 0 75 8 110 11 2 SG O

102 TCF4 FALSE 18 52927188 NM_001243230* G T S352Y 155 0 150 6 115 0 5 NS

103 TET2

TRUE 4 106156729 NM_017628* C T R544X 57 1 55 16 21 2 1 SG X

104 FALSE 4 106194075 NM_001127208 G C E1513Q 43 0 40 16 30 0 2 NS X

105 TM4SF19 FALSE 3 196054377 NM_001204898* C T A29T 68 0 109 5 1 0 5 NS

106 TMEM14B FALSE 6 10756728 NM_030969* C T R108C 53 1 39 5 252 0 2 NS

107 TMPRSS7 FALSE 3 111775937 NM_001042575 G T M297I 57 0 50 12 0 0 1 NS

108 TSG101 FALSE 11 18528459 NM_006292 G A P165S 66 0 67 4 264 0 2 NS

109 TUB FALSE 11 8115674 NM_003320* C T A167V 26 0 35 11 7 0 2 NS

110 UNC5C FALSE 4 96171704 NM_003728 A C Y237D 209 0 240 8 1 0 5 NS

111 USP8 FALSE 15 50784955 NM_001128610* C A N764K 42 0 56 8 106 0 2 NS X

112 VAV1 TRUE 19 6833596 NM_001258206* G T E556D 47 0 43 11 189 40 1 NS O

113 VSIG2 FALSE 11 124620772 NM_014312 A T S89T 70 0 116 5 1 0 5 NS

114 VWA7 FALSE 6 31733811 NM_025258 G A S783L 101 0 111 6 2 0 2 NS

115 WDR64 FALSE 1 241929579 NM_144625 T A Y659X 170 0 326 10 0 0 4 SG

116 XRCC6BP1 TRUE 12 58335626 NM_033276 A T S48C 14 0 9 19 18 5 2 1,3,4,5,6,7,8,9 NS O

117 ZNF133 TRUE 20 18297101 NM_003434* G A G535S 25 0 18 4 12 1 1 NS

118 ZNF717 FALSE 3 75787049 NM_001128223 C A K575N 9 0 10 5 2 0 4 NS

119 ZNF814 TRUE 19 58385748 NM_001144989 G A A337V 123 0 105 51 2 3 2 3,4,5 NS

- Asterisk in transcript ID indicates that other overlapping transcripts with the same characteristics exist.

- A full list, including synonymous SNVs, is provided in Supplementary File 1.

*NS = nonsynonymous; SG = stopgain

**Sanger validation indicates unambiguous results only.


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Supplementary Table 6. List of somatic indels (supported by RNA-seq evidence)

Exome-seq RNA-seq

Normal Tumor Tumor

No. Gene ID CHR POS Transcript ID REF ALT AA Change REF ALT REF ALT REF ALT PAT TYPE

**

Sanger

Valid.***

1 C19orf33 19 38795550 NM_033520 G

GAAGAAGGAG

AAGGGC K89delinsKKKEKG 31 1 37 7 5 1 4 IF

2 CNPY3 6 42897357 NM_006586 TTGC T 17_18del 19 0 20 3 199 4 5 IF

3 FNBP4 11 47788663 NM_015308 CGGTGGT C 58_59del 5 0 0 3 22 8 1 IF O

4 HLA-DPB1 6 33048688 NM_002121 G GA G114fs 29 0 65 2 1494 4 3 FS O

5 HLA-DPB1 6 33048688 NM_002121 G GA G114fs 33 0 70 3 2105 10 4 FS O

6 HRCT1 9 35906583 NM_001039792 TCCA T 100_101del 1 0 4 2 0 1 5 IF

7 LOC100132247 16 22545743 NM_001135865

TTCCACCC

TCAGC T 480_484del 856 1 1259 746 271 1 3 IF

8 LOC283710 15 31521505 NM_001243538 CGG C 25_26del 0 0 0 3 10 30 3 FS X

9 LOC283710 15 31521505 NM_001243538 CG C R26fs 0 0 0 3 12 14 4 FS X

10 LSR 19 35758275 NM_205835* G GGGA G450delinsGR 2 0 4 5 20 15 2 IF O

11 MAZ 16 29821435 NM_002383* AGCGGCG A 440_441del 4 0 6 4 213 44 2 IF O

12 NCOA3 20 46279836 NM_181659* A ACAGCAG Q1254delinsQQQ 68 1 59 64 146 10 3 IF O

13 NEFH 22 29885567 NM_021076 A

AAAGTCCCCT

GAGAAGGCC

A646delinsAKSPEK

A 156 1 2 133 7 1 2 IF X

14 PDCD6 5 271857 NM_001267559* CCCGGCCCTGGGG C 8_12del 7 0 3 2 98 39 1 IF

15 SCAF1 19 50155361 NM_021228 G GCCGCTC R572delinsRRS 1 0 1 5 75 22 4 IF

16 SCAF1 19 50155361 NM_021228 GCCGCTC G 572_574del 7 0 1 2 40 11 5 IF

17 SCARF2 22 20779973 NM_182895* C CG E764fs 0 0 0 3 2 1 4 FS

18 ZNF579 19 56089908 NM_152600 C CCCG G366delinsGG 6 0 4 2 1 5 4 IF O

19 ZNF579 19 56089908 NM_152600 C CCCG G366delinsGG 3 0 5 3 3 3 5 IF O

*Asterisk in transcript ID indicates that other overlapping transcripts with the same characteristics exist.

**IF = in-frame indels; FS = frame-shift indels

***Sanger validation indicates unambiguous results only.


11

Supplementary Table 7. Association of RHOA G17V mutation with clinical parameters in AITL

patients

All patients (n=45)

RHOA wild-type (n=21)

RHOA G17V mutant (n=24)

P-value

Median age, years (range) 64 (35–83) 63 (35-77) 64.5 (38-83)

Age, no. (%) 60 years or less Older than 60 years

16 (36%) 29 (64%)

8 (38%) 13 (62%)

8 (33%) 16 (67%)

0.765

Sex, no. (%) Male Female

32 (71%) 13 (29%)

15 (71%) 6 (29%)

17 (71%) 7 (29%)

1.0

Ann Arbor stage, no. (%) Limited, I–II Advanced, III–IV

2 (5%) 42 (95%)

0 (0%) 21 (100%)

2 (10%) 21 (90%)

0.491

Extranodal involvement, no. (%) 0 or 1 2 or more

31 (72%) 12 (28%)

14 (67%) 7 (33%)

17 (77%) 5 (23%)

0.855

Lactic dehydrogenase, no. (%) Upper limit or below Over upper limit

10 (22%) 35 (78%)

5 (24%) 16 (76%)

5 (21%) 19 (79%)

0.711

IPI risk groups, no. (%) Low/low intermediate High intermediate/high

13 (30%) 30 (70%)

8 (38%) 13 (62%)

5 (23%) 17 (77%)

0.762

Bone marrow involvement, no. (%) Negative Positive

29 (64%) 16 (36%)

13 (62%) 8 (38%)

16 (67%) 8 (23%)

0.765

Primary treatment, no. (%) CHOP Others

37 (84%) 8 (16%)

19 (90%) 2 (10%)

18 (75%) 6 (25%)

0.401

Response to frontline Tx, no. (%) CR Non-CR

25 (56%) 20 (44%)

12 (57%) 9 (43%)

13 (54%) 11 (46%)

0.617

Death, no. (%) Alive, no. (%)

24 (56%) 19 (44%)

10 (50%) 10 (50%)

14 (61%) 9 (39%)

0.547

Median survival (months) 4513.26 45.2323.03 21.9015.09 0.509 (LogRank)

* Data were missing as follows: Ann Arbor stage (n = 1), no. of extranodal involvement (n = 2), IPI risk

groups (n = 2), death (n=2), IPI, International Prognostic Index; CHOP, cyclophosphamide, doxorubicin,

vincristine, and prednisone; Tx, treatment; CR, complete response


12

Supplementary Table 9. Primer and oligo sequences

Objective Sequences

PCR amplification

of RHOA mutation

5'- GTATACTCACCTGCTTTCCATCCACC- 3'

and 5'-CAGCAATGGCTGCCATCCGGAAG-3'

Sanger sequencing

of RHOA 5'- CCTGCTTTCCATCCACCTCGATATC- 3'

siRNA for RHOA

inhibition GGGCUAAGUAAAUAGGAAU


13

Supplementary Fig. 1. Representative histologic evaluation of the tumor tissue. (a) Classical

morphology with effacement of normal architecture and marked vascular proliferation

associated with aggregates of atypical lymphoid cells (H&E staining). (b) The majority of

atypical cells express CD3. (c) CD21 immunostaining showing hyperplastic follicular dendritic

cell meshwork. (d) A portion of tumor cells express CD10. (e) Some tumor cells express

CXCL13. (f) PD-1 is expressed in some neoplastic cells.


14

Supplementary Fig. 2. Frozen section of the tumor sample showing hypercellular lymphoid

tissue without necrosis (H&E staining).


15

Supplementary Fig. 3. Sanger sequencing traces of RHOA G17V mutation in T cell lymphoma

patients. Patient ID with RHOA G17V mutation is indicated by red text and arrows.

Representative traces of wild-type and G17V mutant RHOA are shown in the top left panel. The

horizontal red arrow shows the direction of genomic DNA sequencing. The mutant sequence

shows a clear heterozygous mutation. Traces from tumor samples obtained from 45 AITL, 20

NK/T cell and 13 PTCL-NOS patients are shown in the rectangles. Patients 5, 6, 9, 10, 29, 30

and 43 in the AITL group showed the mutant as the dominant peak. The other 17 AITL patients

showed the heterozygous mutation clearly. All three NK/T cell patients with somatic mutation

showed the heterozygous genotype. Only one PTCL-NOS patient showed a heterozygous

mutation. Sequencing trace chromatograms are representative of at least three independent

experiments.


16

Supplementary Fig. 4. Kaplan-Meier survival plot according to RHOA mutation.


17

Supplementary Fig. 5. In vitro proliferation assays in (a) the SUP-T1 cell line and (b) the

MOLT-4 cell line. Cells expressing inactive RHOA (G17V and T19N) displayed enhanced cell

proliferation. P < 0.01 compared with the cells expressing WT. Each condition with three

replicates were repeated three times and expressed as mean ± s.d.


18

Supplementary Fig. 6. Inhibition of the RHOA-ROCK pathway downregulates AKT

phosphorylation and promotes cell proliferation. (a) After transfection of Jurkat cells with

control siRNA or RHOA siRNA, AKT phosphorylation levels and cell proliferation were

assessed. Cell lysates were prepared 48 h after transfection and processed for immunoblotting

with the indicated antibodies. The extent of RHOA depletion was determined by

immunoblotting with anti-RHOA antibodies. As a loading control, -tubulin was used. Cells

transfected with siRNA were incubated for 48 h, and relative cell proliferation was determined

using a cell counting kit (CCK-8). Five replicates of each condition were repeated three times;

the data are expressed as mean ± s.d. (b) After treatment with the ROCK inhibitor, Y-27632 (30

M), AKT phosphorylation levels and cell proliferation were measured. Cell lysates were

prepared 48 h after treatment and processed for immunoblotting with the indicated antibodies.

Cells treated with Y-27632 were incubated for 48 h, and relative cell proliferation was

determined using a cell counting kit (CCK-8). Five replicates of each condition were repeated

three times, and the data are expressed as mean ± s.d.


19

Supplementary Fig. 7. Structural models of wild-type and mutant RHOA proteins. Homology

modeling was based on the PDB structure of 3LXR. GTP substrate and its ribose moiety are

indicated in yellow and pink, respectively. The glycine and valine residues are shown in green

and red, respectively.


20

Supplementary Fig. 8. Heat map from the hierarchical clustering of differentially expressed

genes. AITL patients from our study (PAT1-PAT9) and GSE6338 (AITL1-AITL6) were

included. Normal control samples are indicated as CD4+. Gene clusters from hierarchical

classification were subjected to the DAVID web server for Gene Ontology analysis, and the

most enriched term for each cluster was determined using the q value from the FDR test.


21

Supplementary Fig. 9. Statistically significant KEGG pathways from GAGE gene set

enrichment analysis. Pathways are grouped into functional categories. Up- and downregulated

pathways are indicated in pink and green backgrounds. Numbers of SNVs, indels, CNVs, and

DEGs indicate the number of genes affected in each pathway.


22

Supplementary Fig. 10. Comparison of gene expression according to RHOA mutation status.

(a) Patients of wild-type RHOA (PAT1 and PAT3). (b) Patients with G17V RHOA mutation

(PAT2 and PAT4-PAT9).


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Supplementary Information for A recurrent inactivating ... · 1 Supplementary Information for A recurrent inactivating mutation in RHOA GTPase in angioimmunoblastic T-cell lymphoma