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