Supplementary data

ATF4 Gene Network Mediates Cellular Response to the Anticancer PAD

Inhibitor YW3-56 in Triple Negative Breast Cancer Cells

Shu Wang, Xiangyun Amy Chen, Jing Hu, Jian-kang Jiang, Yunfei Li, Ka Yim Chan-Salis,

Ying Gu, Gong Chen, Craig Thomas, B. Frank Pugh, Yanming Wang

The supplementary data contain Supplementary Materials and Methods, Supplementary

Figures 1 through 8 with legends, and Supplementary Tables 1 to 5.

Supplementary Fig. 1. YW3-56 inhibits growth of p53 wild type and mutant cells.

Supplementary Fig. 2. PAD inhibitor YW3-56 inhibits PAD4 in HL-60 derived granulocytic

cells.

Supplementary Fig. 3. Effects of YW3-56 on the ATF4 and AMPK gene networks.

Supplementary Fig. 4. ATF4 directly binds to the SESN2 and DDIT4 promoters.

Supplementary Fig. 5. ChIP-exo analyses of ATF4 binding to its target sites.

Supplementary Figure 6. mTOR inhibition following thapsigargin induced ER stress.

Supplementary Figure 7. Effects of ATF4, SESN2, and DDIT4 depletion on cell growth and

YW3-56 mediated cell killing.

Supplementary Figure 8. Features of YW3-56 induced cell death.

Supplementary Table 1. List of qRT-PCR and ChIP-qPCR primers used in this research work.

Supplementary Table 2. Genes with altered expression after YW3-56 treatment. (see Excel

spreadsheet)

Supplementary Table 3. List of ATF4 associated genes. (see Excel spreadsheet)

Supplementary Table 4. List of CEBPB associated genes. (see Excel spreadsheet)

Supplementary Table 5. Expression changes of ATF4 and CEBPB candidate genes after YW3-

56 treatment.

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Supplementary Materials and Methods

MTT assays for cell viabilityCancer cells were treated with YW3-56 at different concentrations for 48 hr in 24-well plates. MTT reagent was added to a final concentration of 0.5 mg/ml and further incubated for 4 hr. The culture medium was then removed and 2 ml DMSO was added to dissolve formazan product reduced from MTT in living cells. Absorbance at 570 nm was measured using a BioMateTM 3 spectrophotometer (Thermo-Fisher).

PAD4 activity and histone citrullination analysesIn vitro PADs citrullination activity assays were preformed using His-PAD4 and GST-PAD4 proteins expressed and purified from E.coli BL-21, and acid extracted histones from Drosophila Kc cells. In a 20μl reaction, 0.1-0.2 μg His-PAD4 or GST-PAD4 protein was pre-incubated with compounds (Cl-amidine or YW3-56) or vehicle in a PAD assay buffer (50 mM Tris.HCl, pH7.6, 2 mM DTT, 2 mM CaCl2, 1 mM PMSF) at 37 °C for 0.5 hr. Then 1μg histones was added into the reaction for another 1.5 hr incubation at 37 °C. The reaction was stopped by adding in SDS buffer and boiling the samples. Histone H3 citrullination was analyzed using anti-histone H3Cit antibody (Abcam) in western blot experiment.

Colorimetric assay of citrullination by PAD42 μΜ His-PAD4 protein was pre-incubated with 50 μΜ YW3-56 or vehicle in at room temperature for 30min in the PAD assay buffer as described above. The protein samples were dialyzed in PBS buffer containing 10 nM DTT and 1 mM CaCl2 for 24 hours. 0.2 μg protein after dialysis was incubated with 5 mM BAEE in 100 μl PAD assay buffer for 1.5 hr at 37 °C. Reaction was stopped with the addition of 25 μl 5 M HClO4. The samples were briefly centrifuged at 12,000 rpm and the supernatant was mixed with equal volume of reagent A (0.5% w/v diacetyl monoxime and 15% w/v NaCl in water) and 2 volume of reagent B (1% w/v antipyrine, 0.15% w/v ferric chloride, 25% v/v H2SO4 and

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25% v/v H3PO4). Then the mixtures were boiled and absorbance at 464 nm was measured using a BioMateTM 3 spectrophotometer (Thermo-Fisher).

Chromatin immunoprecipitation (ChIP)ChIP experiments were carried out essentially as described previously (1). Anti-ATF4 antibody (Santa cruz, sc-200) was used. Primers used in quantitative ChIP-qPCR for SESN2, DDIT3 and DDIT4 were listed in Supplementary Table 5.

Flow cytometry analysesFlow cytometry analyses were performed using FC500 flow cytometer (Beckman Coulter Inc.) at the PSU Microscopy and Cytometry Facility. For cell death analyses, MDA-MB-231 cells treated with 16 μM YW3-56 for 12 hr were harvested and washed with PBS once. The cell pellet was resuspended in 1 ml pre-warmed DMEM containing 50 nM MitoTracker Deep Red (MDR, Molecular Probes) and incubated at 37 °C for 40 min. After incubation with MDR, cells were washed with PBS twice followed by staining with annexin V-FITC (BD Biosciences) and propidium iodide (PI, Sigma). Cells treated with vehicle were used as a control.

Supplemental References1. Li P, Yao H, Zhang Z, Li M, Luo Y, Thompson PR, et al. Regulation of p53 target geneexpression by peptidylarginine deiminase 4. Molecular and cellular biology. 2008;28:4745-58.

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Figure S1

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Supplementary Figure 1. YW3-56 inhibits growth of p53 wild type and mutant cells. (A)

The killing efficacy (IC50) of YW3-56 in a cohort of cancer cells. Values are presented as an

average of at least three repeat experiments. Standard deviations were within 10% of the total.

(B) YW3-56 inhibited the growth of the triple negative breast cancer MDA-MB-231 cells and its

derivative 1833 cells but not the non-tumorigenic MCF 10A cells. Averages and standard

deviations were shown (n=3).

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Figure S2

Supplementary Figure 2. PAD inhibitor YW3-56 inhibits PAD4 in HL-60 derived

granulocytic cells. (A) The structure of PAD4 inhibitor YW3-56. (B) YW3-56 inhibits histone

H3 citrullination mediated by PAD4 in HL-60 granulocytic cells as detected by Western blot.

(C) YW3-56 inhibits the activity of GST-PAD4 and His6-PAD4 expressed and purified from

E.coli as detected by Western blot. (D) YW3-56 inhibits PAD4 via an irreversible chemical

mechanism as indicated by colorimetric assays (n=3) before and after dialysis.

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Supplementary Figure 3. Effects of YW3-56 on the ATF4 and AMPK gene networks. Gene

network analyses by IPA found the ATF4-DDIT4-TRIB3 network genes (A) and SESN2-AMPK

network genes (B) were significantly changed after YW3-56 treatment.

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Supplementary Figure 4. ATF4 directly binds to the SESN2 and DDIT4 promoters. ChIP

analyses of ATF4 binding to SESN2 (A) and DDIT4 (B) promoters before and after YW3-56

treatment. **p<0.002. A distal region -16 kb upstream of the TSS of the SESN2 promoter (A)

was analyzed as a negative control.

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Supplementary Figure 5. ChIP-exo analyses of ATF4 binding to its target sites. (A) Screen

shots of ATF4 binding sites with increase after YW3-56 treatment. (B) Screen shots of ATF4

binding sites with no increase after YW3-56 treatment.

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Supplementary Figure 6. mTOR inhibition following thapsigargin induced ER stress.

Effects of ER stress inducer thapsigargin on the expression of ATF4 target genes (A and B) and

the phosphorylation of mTORC1 substrate (A). MDA-MB-231 cells treated with vehicle or 0.3

μM thapsigargin for 6 hr. *p<0.01, **p<0.002, n = 3.

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Supplementary Figure 7. Effects of ATF4, SESN2, and DDIT4 depletion on cell growth and

YW3-56 mediated cell killing. (A) Cell viability decreased after ATF4 siRNA transfection

compared with the control siRNA treated 1833 cells. p<0.001, n=6. (B) Effects of ATF4

depletion on YW3-56 mediated cell killing. The growth of control siRNA transfected 1833 cells

without YW3-56 treatment was normalized as 100%. At 16 mM of YW3-56, p=0.015, n=6. (C)

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Cell survival after SESN2 and DDIT4 siRNA double treatment compared with the control

siRNA treated cells. P<0.001, n=3. (D) Effects of SESN2 and DDIT4 depletion on YW3-56

mediated cell killing. The growth of control siRNA transfected cells without YW3-56 treatment

was normalized as 100%. p=0.02, n=3.

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Supplementary Figure 8. Features of YW3-56 induced cell death. (A) Flow cytometry

analyses of MDA-MB-231 cells treated with 16 μM YW3-56 for 12 hr. The coordination of two

of the three dyes (PI, annexin V and MitoTracker Deep Red) in the triple labeled cells is shown.

(B) Western blot of PARP cleavage in MDA-MB-231 cells treated with vehicle, 12 μM YW3-

56, or 4 μM doxorubicin for 24 hr. Star denotes the cleavage of PARP protein.

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Supplementary Table 1. List of qRT-PCR and ChIP-qPCR primers used in this research

work.

qRT-PCR primersActin-RT-F 5'-CATTGCCGACAGGATGCA-3'Actin-RT-R 5’-GCTGATCCACATCTGCTGGAA-3’ATF4-RT-F 5'-CAGCAAGGAGGATGCCTTCT-3'ATF4-RT-R 5'-TCCTTCAAATCCATTTTCTCCAA-3'CEBPB-RT-F 5'-CAAGAAGACCGTGGACAAGCA-3'CEBPB-RT-R 5'-CGCACGGCGATGTTGTT-3'DDIT3-RT-F 5'-CGCCTGACCAGGGAAGTAGA-3'DDIT3-RT-R 5'-TCATGCTTGGTGCAGATTCAC-3'DDIT4-RT-F 5'-ACGAGAAGCGGTCCCAAAG-3'DDIT4-RT-R 5'-CACTCTGAGTTCATCAGCAAAGG-3'GADD45-RT-F 5’-GAAGACCGAAAGGATGGATAAGG-3'GADD45-RT-R 5’-ACAGTGATCGTGCGCTGACT-3’GAPDH-RT-F 5'-TCTGGTAAAGTGGATATTGTTG-3'GAPDH-RT-R 5'-GATGGTGATGGGATTTCC-3'LC3B-RT-F 5'-CCATGCCGTCGGAGAAGA-3'LC3B-RT-R 5'-CTGCTCTCGAATAAGTCGGACAT-3'p21-RT-F 5’-GACAGCAGAGGAAGACCATGTG-3’p21-RT-R 5’-GGCGTTTGGAGTGGTAGAAATC-3’PUMA-RT-F 5’-GGGCCCAGACTGTGAATCCT-3’PUMA-RT-R 5’-ACGTGCTCTCTCTAAACCTATGCA-3SESN1-RT-F 5'-CATCCAGGAACTTGGCATTAGAA-3'SESN1-RT-R 5'-TCTGGGATGAATCTGCTTGGT-3'SESN2-RT-F 5'-AGAAGGTCCACGTGAACTTGCT-3'SESN2-RT-R 5'-TCAGGTCATGTAGCGGGTGAT-3'SESN3-RT-F 5'-TGCTGCGGAAGGATAAAAGAA-3'SESN3-RT-R 5'-AGGCACTTGGTCCTCTTGTCA-3'TRIB3-RT-F 5'-GGCACTGAGTATACCTGCAAG-3'TRIB3-RT-R 5'-TCCGAGTGAAAAAGGCGTAG-3'*Actin-RT-F 5'-AGAAAATCTGGCACCACACC-3'*Actin-RT-R 5'-CTCCTTAATGTCACGCACGA-3'*DDIT4-RT-F 5'-GTTCGCACACCCATTCAAG-3'*DDIT4-RT-R 5'-CTGATGAACTCAGAGTGCC-3'*SESN2-RT-F 5'-AGTATCTCATCTGTCCCCTCTC-3'

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*SESN2-RT-R 5'-AGAAAGTGCCTGGAATCGG-3'

ChIP-qPCR primersChIP-DDIT4- -1011F 5'-CTGCCAGGCCAGATTTCCT-3'ChIP-DDIT4- -1011R 5'-GCCATCCCGTGTTTCATCA-3'ChIP-SESN2- -138F 5'-TGGTGTTGCCAGGGATCTG-3'ChIP-SESN2- -138R 5'-TCGGGTGAATGCTGCAAA-3'ChIP-SESN2- -16K-F 5'-GCTGACTTTGGCCTGGTCTTA-3'ChIP-SESN2- -16K-R 5'-ACTAACACATTTGCTTGTTCACTCATT-3'

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Supplementary Table 5. Expression changes of ATF4 and CEBPB candidate genes after

YW3-56 treatment.

ATF4 candidategenes (73)

Fold ofchanges

CEBPB candidategenes (59)

Fold ofchanges

DDIT3 10.38 DDIT3 10.38SESN2 6.18 HMOX1 9.5CHAC1 5.77 CHAC1 5.77TSC22D3 4.6 TRIB3 3.89DDIT4 4.53 YPEL2 2.72TRIB3 3.89 NDRG1 2.63DLGAP1-AS1 3.84 TUFT1 2.46ALOXE3 3.45 RSPO3 2.43HERPUD1 3.28 CCNG2 2.39DNAJB1 3.04 CBX4 2.38GDF15 3.04 HSPA5 2.3JDP2 2.87 GADD45A 2.25JHDM1D 2.68 NCOA7 2.23PCK2 2.55 PTGS2 2.03FAM102A 2.51 IDH1 1.9MKNK2 2.47 SMIM14 1.9TUFT1 2.46 BHLHE40 1.79XBP1 2.39 UGDH 1.78CBX4 2.38 DUSP1 1.73PPP1R15A 2.36 DPYSL2 1.72HSPA5 2.3 GXYLT2 1.72GADD45A 2.25 TULP3 1.69NCOA7 2.23 RNF38 1.68ZFP69B 2.12 ATF3 1.67RHBDD2 2.09 BMP4 1.65DNAJB5 2.07 CPEB3 1.63ARNTL 2.05 ITPRIP 1.62VLDLR 2.04 CTTNBP2NL 1.61ALDH2 2.03 STOX1 1.61GPT2 2.03 ZBTB41 1.6WIPI1 2 CTSL 1.58MKX 1.97 HIPK3 1.57CLTCL1 1.9 NBR2 1.56SLC7A11 1.89 NFIL3 1.5ZNF682 1.88 CISD1 0.66ZNF419 1.86 FAM27C 0.66MALAT1 1.84 EID1 0.63TXNL4B 1.81 GRAMD1C 0.62CEBPB 1.78 ITPRIPL2 0.62TES 1.77 RUNX2 0.61AP3S2 1.74 AGPAT9 0.6BRF2 1.72 CDC7 0.6DPYSL2 1.72 RIN2 0.6RND3 1.72 EVI2A 0.59

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SLC3A2 1.71 SPRY1 0.59ASB1 1.66 TGFBR2 0.59STOX1 1.61 TGM2 0.56GFPT1 1.59 SLC25A19 0.54TXNRD1 1.58 FAM129B 0.53NBR2 1.56 FMNL1 0.53CRISPLD2 1.55 LGALSL 0.53WARS 1.54 SPRY4 0.53CCDC9 1.53 PCF11 0.52MTHFD2 1.52 HBEGF 0.5NFIL3 1.5 SMAD3 0.46FAM27C 0.66 IL4R 0.44GEMIN7 0.66 MYEOV 0.39CDCP1 0.65 ENC1 0.37KCTD14 0.65 ADORA2B 0.33RGMB 0.65WDR36 0.64BBS10 0.62CSTF2T 0.61RUNX2 0.61AGPAT9 0.6MOCS3 0.6EVI2A 0.59SNORD83B 0.56FMNL1 0.53MLPH 0.48SNORD96A 0.48TIPIN 0.48ADORA2B 0.33

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