Supplementary Information

The histone methyltransferase and putative oncoprotein MMSET is overexpressed in a

large variety of human tumors

Heidi Rye Hudlebusch, Eric Santoni-Rugiu, Ronald Simon, Elisabeth Ralfkiær, Henrik Holm Rossing, Jens Vilstrup Johansen, Mette Jørgensen, Guido Sauter, and Kristian Helin

Legends to Supplementary Figures

Supplementary Figure S1. MMSET 9A6 mAb tests.

A) Immunoblotting of whole cell extracts comparing MMSET expression, H3K36 di- and

trimethylation levels in U2OS cells versus human myeloma cell lines. Immunoblotting with

H3 Ab was included as a loading control. * denotes t(4;14)+ myeloma cell lines. In the

t(4;14)+ myeloma KMS-11 cell line, the breakpoint of chromosome 4 is located upstream of

the MMSET translation start site and overexpressed MMSET is therefore detectable by the

9A6 mAb (recognizing the N-terminal part of MMSET). In the t(4;14)+ myeloma LP-1 and

NCI-H929 cell lines, the breakpoint of chromosome 4 is located intragenic resulting in

overexpression of N-terminal truncated MMSET protein, that is not detectable by 9A6 mAb.

Antibodies used: 9A6 mAb (1:4; hybridoma supernatant), H3 (Abcam 1791), H3K36me2

(Cell Signaling 2901), H3K36me3 (Cell Signaling 9763). B) The cells described in Fig. 1B

were embedded in paraffin and immunostained with the 9A6 mAb (central panels).

Corresponding haematoxylin-eosin staining (left panels) and negative control with unspecific

Ig (right panel) can be seen. 40x magnification. C) Correlation between MMSET expression

and t(4;14) translocation in myelomas. The translocation was determined by FISH analysis

using the Vysis LSI IGH/FGFR3 dual color, dual fusion translocation probe (Abbott) on

FFPE tissue. A minimum of 100 malignant plasma cells was counted. Cut-off: 10%. Yes:

translocation is present; No: no evidence for a translocation. D) MMSET immunostainings of

reactive lymph nodes with hyperplastic germinal centers were performed (left panel) and

negative control (right panel). 20x magnification.

Supplementary Figure S2. MMSET expression in tumors of female genitals

MMSET expression pattern in cancers of the ovary (A), corpus uteri (B), and cervix uteri (C)

are illustrated (left). Representative immunostainings of endometroid adenocarcinoma of

ovary, serous carcinoma of corpus uteri, and squamous carcinoma of cervix uteri are shown

to the right. 10x and 20x magnifications. If no p-value is noted, no significant difference in

MMSET expression was found between the tumor and the corresponding normal tissue. ACa:

adenocarcinoma, Ca: carcinoma, N: normal, Sq Ca: squamous carcinoma.

Supplementary Figure S3. MMSET expression in tumors of the skin

The MMSET expression pattern in tumors of the skin is illustrated in the diagram (top). The

30 normal skin samples included 22 skin samples and 8 cutaneous adnexa samples.

Representative MMSET immunostainings in squamous carcinoma, basal cell carcinoma,

benign nevus, and malignant melanoma are shown. 10x, 20x, and 40x magnifications. If no

p-value is noted, no significant difference in MMSET expression was found between the

tumor and the corresponding normal tissue. Ca: carcinoma, N: normal, Sq Ca: squamous

carcinoma.

Supplementary Figure S4. Lack of correlation between MMSET expression levels and global

levels of H3K36me2 and H3K36me3 in U2OS cells and in primary myelomas.

A) The levels of H3K36me2 and H3K36me3 were determined in whole cell extracts from

U2OS cells also shown in Fig. 1B. H3 and β-ACTIN levels were determined and used as

loading controls. IHC was performed with antibodies specific for H3K36 di- and

trimethylation for the panel of 39 myelomas. Immunostainings were scored as described for

MMSET. No correlation was found between MMSET expression and the level of B)

H3K36me2 and C) H3K36me3 in myelomas. Details of H3K36me2 and H3K36me3 IHC of

the 39 myelomas are shown in Supplementary table I. D) Representative MMSET,

H3K36me2, and H3K36me3 immunostainings of multiple myelomas are shown (MM 24 and

39 in Supplementary table I) with the IHC scoring noted in the images. Haematoxylin-eosin

stainings are also shown. Antibodies used: H3 (Abcam 1791), H3K36me2 (Cell Signaling

2901), H3K36me3 (Cell Signaling 9763), β-ACTIN (Abcam 6276).

Supplementary table I MMSET staining, t(4;14) translocation, and staining with H3K36me2/3 antibodies in 39 myeloma samples Myeloma MMSET

IHC t(4;14) FISH

H3K36me2 IHC

H3K36me3 IHC

MM 1 MM 2 MM 3 MM 4 MM 5 MM 6 MM 7 MM 8 MM 9

MM 10 MM 11 MM 12 MM 13 MM 14 MM 15 MM 16 MM 17 MM 18 MM 19 MM 20 MM 21 MM 22 MM 23 MM 24 MM 25 MM 26 MM 27 MM 28 MM 29 MM 30 MM 31 MM 32 MM 33 MM 34 MM 35 MM 36 MM 37 MM 38 MM 39

0 0 0 0 0 1 0 0 0 0 0 0 0 1 1 0 0 0 0 0 3 2 0 0 0 3 2 0 2 1 0 0 2 3 0 0 3 0 3

No No No No No No No No ND No No No No ND ND ND No Yes ND No ND Yes No Yes Yes ND Yes ND ND Yes ND ND Yes Yes ND ND Yes No Yes

0 2 2 0 1 0 1 2 3 1 0 1 0 0 0 0 0 0 1 3 2 0 2 3 2 0 2 2 0 1 1 0 0 0 0

ND 0 1 2

0 2 1 1 2 0 1 2 2 2 0 2 0 3 0 0 0 0 1 1 1 0 1 2 2 0 1 2 0 3 1 0 2 0 1

ND 0 1 1

MM: multiple myeloma. ND: Not determined. 13/39 myelomas were not informative in relation to t(4;14) FISH analysis (fewer than 100 malignant plasma cells in he sample or poor quality due to too much autofluorescence). H3K36me2 and H3K36me3 stainings were performed similar to staining with the 9A6 mAb using antibodies from Cell Signaling (#2901 and #9763, diluted 1:50). Antigen retrieval was performed in citrate buffer, pH 6.0.

Supplementary table II Clinico-pathological features of carcinomas of large intestine (colorectal + anal) in cohort 2, n=1206 MMSET expression 0

(n=563) 1

(n=350) 2

(n=182) 3

(n=111) Age group, years 1-40 41-50 51-60 61-70 71-80 >80

4

28 74

146 170 122

3

12 57 96

112 57

3

14 14 54 64 31

0 9

16 36 30 19

Sex F M

273 272

191 147

94 86

61 49

Tumor localization Caecum Ascending colon Transverse colon Descending colon Sigmoid colon Rectosigmoid junction Rectum Anal

93 56 50 26 99 40

169 6

44 28 31 18 60 24

129 2

26 18 18 8

38 10 61 0

12 14 10 5

18 5

39 3

pT category pT1 pT2 pT3 pT4

16 52

351 118

21 73

201 38

4 28

120 25

7 23 67 9

pN (regional lymph nodes) pN0 pN1 pN2

236 159 136

192 75 59

100 45 28

63 27 12

Grade G1 G2 G3

12 473 72

9 299 39

2 155 22

5 91 12

Invasive margin Infiltrating Pushing

380 157

188 144

108 68

58 47

Peritumoral lymphocytes Absent Present

307 232

184 148

97 79

56 49

Vascular invasion No Yes

290 249

192 140

93 83

70 35

Supplementary table III Clinico-pathological features of urinary bladder cancers in cohort 3, n=1293 MMSET expression 0

(n=773) 1

(n=361) 2

(n=116) 3

(n=43) Age group, years 1-40 41-50 51-60 61-70 71-80 >80

20 50

153 238 222 88

2 7

63 120 106 62

1 4

17 38 39 17

0 4 4

13 16 6

Sex F M

196 575

92 267

32 84

9 34

Smoking No Yes

106 188

39 81

11 16

2 10

Histological tumor type Urothelial carcinoma Sarcomatoid carcinoma Squamous carcinoma Adenocarcinoma Small cell carcinoma

689 7

33 9 5

332 3

10 2 6

98 2 3 2 6

35 1 0 1 3

pT category pTa pT1 pT2-4

348 142 255

132 102 104

17 44 48

7 12 21

Grade G1 G2 G3

124 368 281

22 161 178

0 25 91

0 7

36 Growth pattern Papillary Solid

575 195

277 80

77 39

24 18

Tumor distribution Unicentric Multicentric

148 95

60 50

8 15

7 3