Quartz 4

40229247

8 min read

A

H295R

Neutral Lipids /DAPI

basal

DZNep

GSK343

TAZ

FFA/DAPI

B

MUC-1

Figure S1. EZH2i affect lipid content in ACC cells. A, B. Confocal images of LDs stained by BODIPY 493/503 fluorescent dye (Neutral lipids) and BODIPY™ 558/568 C12 (free fatty acids, FFA) in H295R (A) and MUC-1 (B) cells treated for 48h with DZNep (5 uM), GSK343 (10 uM) and Tazemetostat (TAZ, 5 uM). Nuclei were stained by DAPI. (scale bar 50 um)

Neutral Lipids /DAPI

basal

DZNep

GSK343

TAZ

FFA/DAPI

8

B

A

25 µM

5 µM

SLC2A1

HK1

HK2

H295R

PFKL

GAPDH

LDHA

-1.2041

-0.6021

0

0.6021

1.2041

H295R

GSK343 DZNep

NR5A1

NR5A2

SREBP1

SREBP2

PPARa

Myc

HIF1a

SLC2A1

HK1

HK2

PFKL

GAPDH

LDHA

CPT1

HADHA

HADHB

ACLY

ACACA

FASN

SCD1

ELOVL1

ELOVL5

PLIN1

PLIN2

ATGL

LIPE

MAGL

GOS2

CD36

SLC27A2

SLC27A3

SLC27A4

SCARB1

LDLR

GLS1

ACAT1

HMGCR

IDI

SQLE

CYP27A1

-1.2742

-0.6371

0

0.6371

1.2742

0

GSK343 DZNep

CPT1

HADHA

HADHB

ACOX1

ACLY

MUC-1

FASN

SCD1

ATGL

LIPE

-1

-0.5

0

0.5

1

Figure S2. Effects of EZH2i on the expression of metabolic genes. and GSK343 (10 uM) (B, C). Data are the values from 3 separate RNA samples presented real-time RT-PCR after 48 h treatment with GSK126 (5 and 25 uM) (A, D), DZNep (5 uM) A-D. Heatmaps of mRNA expression of metabolic genes in ACC cells evaluated by

D

25 uM 5 µM

GLS1

SLC7A11

SLC1A5

MUC-1

SLC6A9

GSS

GPX4

-1.05

-0.525

0

0.525

1.05

as log10 of fold-change.

Figure S3. EZH2i increase ROS in ACC cells. A-D. Flow cytometry analysis of reactive oxygen species (ROS) in ACC cells treated with GSK126 for 48 h. Cells treated with H2O2 for 2 h were used as positive control (B, D). ROS- positive cells are shown in red. Histograms represent the fold change in the number of ROS-positive cells. Data are expressed as mean + SEM. n=3 independent experiments. ** p <0.01, *** p <0.001, **** p <0.0001.

A

basal

GSK126 5μM

B

Positive control

100

M1 76.93%

M2 23.03%

M1 67.03%

M1 35.70%

M2 32.97%

M2 64.30%

0

1

2

3

4

0

1

2

3

4

0

1

2

3

4

GSK126 25uM

H295R

10

ROS-positive cells (fold over 0uM)

2.0-

M1 63.27%

M2 36.73%

1.5

*

1.0

0.5-

0

1

2

3

4

GSK126 (μM)

0.0

0

5

25

MUC-1

C

D

basal

GSK126 5uM

Positive control

100

10

M1 82.03%

M2 17.97%

M1 66.63%

M2 33.37%

M1 48.47%

M2 51.53%

0

1

2

3

4

0

1

2

3

4

0

1

2

3

4

GSK126 25µM

19

MUC-1

ROS-positive cells (fold over 0uM)

2.5-

M1 60.79%

M2 39.11%

2.0-

1.5-

1.0-

0.5

0

1

2

3

4

GSK126 (MM)

0.0

0

5

25

Groups + EZH2.high_GPX4.high + EZH2.high_GPX4.low + EZH2.low_GPX4.high + EZH2.low_GPX4.Jow

Survival probability

A

1.00-

0.75-

0.50

0.25-

p = 0.0015

0.00-

0

50

100

150

Time

Time
Number at risk
EZH2.high_GPX4.high EZH2.high_GPX4.low EZH2.low_GPX4.high EZH2.low_GPX4.low Groups15500
24420
24931
15711
050100150
compar.P-valueEZH2.low_GPX4.highEZH2.high_GPX4.lowEZH2.high_GPX4.highEZH2.low_GPX4.low
EZH2.low_GPX4.highNA0.00130.00030.1602
EZH2.high_GPX4.low0.0013NA0.33950.1018
EZH2.high_GPX4.high0.00030.3395NA0.0677
EZH2.low_GPX4.low0.16020.10180.0677NA
C Groups + EZH2.high_ACSL4.high + EZH2.high_ACSL4.Jow + EZH2.low_ACSL4.high + EZH2low_ACSL4.low

1.00-

0.75-

Survival probability

0.50-

0.25-

p < 0.0001

0.00-

0

50

100

150

Time

Number at risk
EZH2.high_ACSL4.high15720
EZH2.high_ACSL4.low EZH2.low_ACSL4.high Groups24200
241110
EZH2.low_ACSL4.low15532
050100150

Time

compar.P-valueEZH2.low_ACSL4.lowEZH2.high_ACSL4.lowEZH2.high_ACSL4.highEZH2.low_ACSL4.high
EZH2.low_ACSL4.lowNA0.00120.97760.1415
EZH2.high_ACSL4.low0.0012NA0.00330.0000
EZH2.high_ACSL4.high0.97760.0033NA0.1784
EZH2.low_ACSL4.high0.14150.00000.1784NA

Groups + EZH2high_SLC7A11.high + EZH2.high_SLC7A11.low + EZH2.low_SLC7A11.high + EZH2.low_SLC7A11.Jow

1.00

0.75-

Survival probability

0.50-

0.25-

p < 0.0001

0.00-

0

50

100

150

Time

Number at risk

Groups

EZH2.high_SLC7A11.high

26

4

0

0

EZH2.high_SLC7A11.low

13

5

2

0

EZH2.low_SLC7A11.high

13

6

1

0

EZH2.low_SLC7A11.low

26

10

3

2

0

50

100

150

Time

compar.P-valueEZH2.low_SLC7A11.lowEZH2.high_SLC7A11.highEZH2.low_SLC7A11.highEZH2.high_SLC7A11.J
EZH2.low_SLC7A11.lowNA0.00000.12310.1789
EZH2.high_SLC7A11.high0.0000NA0.00730.0112
EZH2.low_SLC7A11.high0.12310.0073NA0.7610
EZH2.high_SLC7A11.low0.17890.01120.7610NA

D

Groups + EZH2.high_SLC1A5.high + EZH2.high_SLC1A5.low + EZH2.low_SLC1A5.high + EZH2.low_SLC1A5.Jow

1.00-

0.75-

Survival probability

0.50

0.25-

p = 0.00018

0.00-

0

50

100

150

Time

Number at risk

Groups

EZH2.high_SLC1A5.high

21

5

0

0

EZH2.high_SLC1A5.low

18

4

2

0

EZH2.low_SLC1A5.high

18

7

2

1

EZH2.low_SLC1A5.low

21

9

2

1

0

50

100

150

Time

compar.P-valueEZH2.low_SLC1A5.lowEZH2.high_SLC1A5.highEZH2.high_SLC1A5.lowEZH2.low_SLC1A5.high
EZH2.low_SLC1A5.lowNA0.00700.35110.0345
EZH2.high_SLC1A5.high0.0070NA0.11910.0001
EZH2.high_SLC1A5.low0.35110.1191NA0.0070
EZH2.low_SLC1A5.high0.03450.00010.0070NA

F

Groups + EZH2high_SLC8A9.high + EZH2.high_SLO8AQ.Jow + EZH2.low_SLC8A9.high + EZH2.low_SLC8AQ.Jow

Groups + EZH2high_ELOVL5.high + EZH2.high_ELOVL5.low + EZH2.low_ELOVL5.high + EZH2.low_ELOVL5.low

1.00-

0.75-

Survival probability

0.50-

0.25-

p < 0.0001

0.00-

0

50

100

150

Number at riskTime
EZH2.high_ELOVL5.high23820
GroupsEZH2.high_ELOVL5.low16100
EZH2.low_ELOVL5.high16820
EZH2.low_ELOVL5.low23822
050100150

Time

compar.P-valueEZH2.low_ELOVL5.highEZH2.high_ELOVL5.lowEZH2.high_ELOVL5.highEZH2.low_ELOVL5.low
EZH2.low_ELOVL5.highNA0.00010.19420.3901
EZH2.high_ELOVL5.low0.0001NA0.06010.0000
EZH2.high_ELOVL5.high0.19420.0601NA0.0233
EZH2.low_ELOVL5.low0.39010.00000.0233NA

1.00-

0.75

Survival probability

0.50-

0.25-

p < 0.0001

0.00-

0

50

100

150

Time

Number at risk

Groups

EZH2.high_SLC6A9.high

23

3

0

0

EZH2.high_SLC6A9.low

16

6

2

0

EZH2.low_SLC6A9.high

16

9

2

1

EZH2.low_SLC6A9.low

23

7

2

1

0

50

100

150

Time

compar.P-valueEZH2.low_SLC6A9.highEZH2.high_SLC6A9.highEZH2.low_SLC6A9.lowEZH2.high_SLC6A9.Jov
EZH2.low_SLC8A9.highNA0.00030.95730.2012
EZH2.high_SLC6A9.high0.0003NA0.00010.0289
EZH2.low_SLC8A9.low0.95730.0001NA0.2272
EZH2.high_SLC6A9.low0.20120.02890.2272NA

Figure S4. Risk stratification according to the expression of EZH2 and ferroptosis-related genes in ACC patients. A-F. Kaplan-Meier curves showing overall survival (in months) of subgroups of patients stratified according to the median expressions of EZH2 and other ferroptosis-related genes. The total number of patients in each subgroup is shown in the middle panel. The p-value on top of the plot represents the overall result of the log-rank test including all groups. Tables at the bottom panels of each figure indicate the p-values of pairwise log-rank tests between each subgroup.

C11 Non-oxidized

C11-Oxidized

Merge

BasalGSK126RSL3RSL3 + GSK126

Figure S5. EZH2i sensitize MUC-1 cells to ferroptosis inducers.

A. Confocal images of lipid peroxidation detected by BODIPY C11 fluorescent dye in MUC-1 cells treated for 48 h with GSK126 (5 uM) in the presence or absence of RSL3 (1 µM, for the last 4h) (scale bar 50 um).

A

Figure S6. EZH2i increase lipid content and GPX4 expression in a cell model of TNBC. A. Confocal images of lipid droplets stained by BODIPY 493/503 fluorescent dye (Neutral lipids) and BODIPY™ 558/568 C12 (free fatty acids, FFA) in MDA-MB231 cells treated for 48 h with TAZ (5 uM) and GSK126 (5 UM and 25 uM). Nuclei were stained by DAPI. Scale bar 50 um. B, C, E. Cell viability by MTT assay of MDA-MB231 cells treated for 24, 48 and 72 h with the indicated doses of GSK126, TAZ and RSL3. n=3 independent experiments. D. Western blot of GPX4 in MDA-MB231 after 48 h of TAZ treatment. Actin was used as a loading control. Data are expressed as means + SEM. * p <0.05; ** p <0.01 **** p <0.0001.

BASAL

TAZ 5M

GSK126 5UM

GSK126 25uM

Neutral Lipids /DAPI

FFA/DAPI

B

C

D

100

100

0

5

20

ΤΑΖ (μM)

Cell viability (% of OuM)

75

**

*

Cell viability (% of OuM)

**

**

75

GPX4

50

50

25.

25

Actin

0

0

GSK126 [uM]

0

2,5

5

10

25

50

TAZ [uM]

0

2,5

5

10

20

E

100

Cell viability (% of OuM)

75-

Aeskeskeske

50

I

25.

0

RSL3 [uM]

0

0,1

1

10

100

A

5 µM GSK126

25 μM GSK126

5

55

120

Figure S7. GSK126-treated TNBC cells are enriched in lipid and antioxidant genes and metabolites.

Joint Pathway Analysis GSK126 25 UM

B

.

5

AMPK signaling pathway

alpha-Linolenic acid metabolism

4

Adipocytokine

-log10(p)

signaling pathway

3

☒

Fatty acid degradation

☒ Glutathione metabolism

2

☒

Argine biosynthesis

Insulin resistance

1

0

0.0

0.5

1.0

1.5

Pathway Impact

A. Untargeted metabolomics analysis was performed on MDA-MB231 cells treated with GSK126 (5 UM and 25 uM) for 48 h. Venn diagram represents statistically significant differentially abundant metabolites across 5 and 25 uM GSK126-treated cells. B. Overview of the pathway enrichment analysis based on both transcripts and metabolites with statistically significant difference in 25 uM GSK126-treated MDA-MB231 cells. Only pathways with - log10(p) > 2 are shown.

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