Q
Expression (log2 normalized signal intensity)
DNMT1
DNMT3A
DNMT3B
co
*
4.5
**
7
6
0
++
3.5
6
5
m
o
intermediate
low
high
intermediate
intermediate
low
high
low
high
Expression (log2 normalized signal intensity)
TET1
TET2
TET3
0
1
o
4.5
1
o
6
o
+
0
5
6
3.5
+
8
5
O
low
intermediate
high
low
intermediate
high
low
intermediate
high
b
DNMT1
DNMT3A
1.0
1.0
High expression
Overall survival (%)
Moderate expression
0.8
0.8
Low expression
0.6
0.6
0.4
0.4
++
0.2
0.2
0.0
p-value = 1.22e-5
0.0
p-value = 0.146
0
50
100
150
0
50
100
150
Time (months)
Time (months)
(a) Expression levels of DNMT1, DNMT3A, DNMT3B, TET1, TET2 and TET3 in the ENSAT patients exhibiting ICIMP, iCIMP and hCIMP (two-sided t-test, *p< 0.05, ** p< 0.01, *** p <0.005).
(b) Kaplan-Meier estimates of overall survival for ACC patients, as a function of DNMT1 or DNMT3A.
DNMT1
DNMT3A
Expression (log2 normalized signal intensity
8.0
spearman rho = 0.718
normalized signal intensity
p-value < 1.6e-16
CIMP Status
7.5
5.5
spearman rho = 0.227
p-value < 0.17
7.0
Expression (log2
· High CIMP ☒
5.0
6.5
. Intermediate CIMP
6.0
4.5
. Low CIMP
5.5
4.0
5.0
3.5
4.5
3.0
-0.2
-0.1
0.0
0.1
0.2
0.3
-0.2
-0.1
0.0
0.1
0.2
0.3
Proliferation score
Proliferation score
G2M checkpoint
b Allograft rejection
Enrichment score (ES)
0.7
NES = 3.182
0.00
0.6
-0.05
0.5
p.value < 0
-0.10
0.4
-0.15
-0.20
0.3
-0.25
0.2
-0.30
NES = - 2.21
0.1
-0.35
-0.40
p.value < 0
0.0
-0.45
Cholesterol homeostasis
Fatty acid metabolism
Enrichment score (ES)
0.45
NES = 1.88
0.10
0.40
p.value < 4e-4
0.05
0.35
0.00
0.30
-0.05
0.25
-0.10
0.20
-0.15
0.15
-0.20
0.10
-0.25
NES = - 1.73
0.05
-0.30
p.value < 4e-3
0.00
-0.35
DNA repair
Protein secretion
Enrichment score (ES)
0.40
NES = 1.841
0.10
0.35
0.05
p.value < 3e-4
0.30
0.00
-0.05
0.25
-0.10
0.20
-0.15
0.15
-0.20
0.10
-0.25
NES = - 1.66
0.05
-0.30
p.value < 6e-3
0.00
-0.35
-0.40
(a) Representative example of gene sets enriched in hCIMP.
(b) Representative example of gene sets enriched in ICIMP.
T cells
CD8 T cells
Cytotoxic lymphocytes
Relative abundance
3.1
3.2
0
2.9
0
3.0
2.9
2.8
2.7
2.8
·
2.7
2.4
2.5
1
Low
Medium
High
Low
Medium
High
Low
Medium
High
NK cells
B lineage
Monocytic lineage
Relative abundance
3.0
3.6
8
0
5.0
0
3.4
2.8
3.2
4.5
2.6
3.0
4.0
2.4
2.8
3.5
Low
Medium High
Low Medium Hi
High
Low
Medium
High
Myeloid dendritic cells
Neutrophils
Fibroblasts
Relative abundance
4.2
3.4
7.0
3.8
3.3
6.0
·
3.4
3.2
5.0
3.0
3.1
4.0
Low
Medium
High
Low
Medium
High
Low
Medium
High
Cell growth rate 6 days
1.2
*
1
₸
* T
T
T
* T
T
*
% of control
0.8
T
0.6
T
0.4
T
0.2
0
H295R
MUC1
HUVEC
☒ Control
☒ ΑΖΑ 1μM
☐ ΑΖΑ 5 μΜ
☐ ΑΖΑ 10μΜ
Figure S5. Impact of demethylating agent on cell proliferation in ACC cell lines
Effect of a 6-day treatment with the DNA methylation inhibitor 5-azacytidine (AZA) at 1, 5, or 10uM in the H295R (n=4), MUC1 (n=5) cell lines and the non-tumor HUVEC cell line (n=5). Cell growth was estimated by neutral red proliferation assay. P-values of the Kruskal-Wallis test and Dunn’s test for stochastic dominance are reported: * p-value< 0.05; ** p-value < 0.01; *** p-value < 0.005
adjusted p-value (FDR)
Pathway in Cancer
MAPK Signaling Pathway
Focal Adhesion
Endocytosis
Neuroactive Lig. Receptor Interaction
Axon Guidance
Regulation of Actin Cytoskeleton
Leukocyte Transendothelial Migration
Hypertrophic Cardiomyopathy
Vascular Smouth Muscle Contraction
Calcium Signaling Pathway Viral Myocarditis
Dilated Cardiomyopathy
Hematopoietic Cell Lineage
Adherens Junction
Complement & Coagul. Cascade Junc.
Adipocytokine Signaling Pathway
Hedgehog Signaling Pathway
Glutathione Metabolism
Bladder Cancer
Amino Sugar & Nucleotide Sugar Met.
Renin Angiotensin System
Glycosphingolipid Biosynth. Ganglio
Glycosphingolipid Biosynthesis Globo
Glycosaminoglycan Biosynthesis
KEGG pathways
1.3e-2
4.8e-3
4.07e-3
2.9e-3
3.03e-2
5.76e-4
3.07e-2
4.82e-3
4.07e-3
1.4e-2
9.62e-2
4.82e-3
3.6e-2
3.42e-2
1.75e-2
3.6e-2
9.62e-2
5.63e-2
3.84e-2
9.32e-2
9.63e-2
4.77e-2
3.66e-2
3.6e-2
9.04e-2
0
5
10
15
Number of genes
Pathway enrichment analysis on genes that exhibit a significantly hypermethylated DMR in their promoter and significantly lower expression in H295R than in MUC-1 cells. Pathways related to the immune response are highlighted in red.