Supplementary Figure S1.
B MCF7 Cas9 MCF7 p53 KO DMSO Nutlin-3 RITA DMSO Nutlin-3 RITA A MCF7 Cas9 MCF7 p53 KO p53
p53 β-actin DAPI
C RITA Nutlin-3 D 1 DMSO 0.1 µM 0.5 µM 1 µM 5 µM 10 µM * MCF7 Cas9 * 0.8 * RITA 0.05 µM MCF7 *** RITA 0.1 µM (parental) viability 0.6 * RITA 1 µM 1.00 0.47 0.35 0.22 0.65 0.46 *** MCF7 p53KO 0.4 RITA 0.05 µM MCF7 * RITA 0.1 µM Cas9 0.2 RITA 1 µM 0.91 0.51 0.35 0.15 0.58 0.32 cell Relative 0 MCF7 0 24 48 72 Time (h) p53KO 1.01 0.45 0.41 0.29 1.24 1.05 F MCF7 Cas9 MCF7 p53 KO RITA 0 8 16 24 0 8 16 24 h p53 E MCF7 cas9 MCF7 p53KO PARP 120 120 Cleav. PARP γH2AX 80 80 DMSO RITA β-actin
40 40 Cells (Count) Cells Cells (Count) Cells PUMA NOXA p21 MDM2PPM1D MCL1 MDMX 0 0 0 1 2 3 * 100 101 102 103 10 10 10 10 H 5 DCF-DA DCF-DA 4 G MCF7 Cas9 MCF7 p53 KO RITA 0 8 16 24 0 8 16 24 h 3 (log2*FC) *** * p21 2 MDM2 level 1 Wip1 MCL1 0
MDMX -1 β-actin MCF7 Cas9
Relative expression expression Relative -2 MCF7 p53KO J DMSO RITA Nutlin-3 -3
I A375 A375
1.00 0.62 0.20 A375 p53 p53shRNA β-actin 1.08 1.03 1.20 A375 p53KO 0.90 1.00 1.11 Supplementary Figure S2.
R² = 0.0006 R² = 0.0299 R² = 0.0264
A 3 3 3
(GI50) (GI50) 2 2 2
1 1 1
CDDP (GI50) CDDP
to Oxaliplatin
0 0 doxorubicin 0 to -1 -1 to -1
Sensitivity -2 -2 -2
-2 -1 0 1 2 3 Sensitivity -2 -1 0 1 2 3 -2 -1 0 1 2 3 Sensitivity to RITA (GI50) Sensitivity to RITA (GI50) Sensitivity Sensitivity to RITA (GI50) R² = 0.002 R² = 0.0029
3 3 R² = 0.025 3
(GI50)
(GI50) (GI50) 2 2 2
1 1 1 topotecan
0 irinotecan 0 0
to
to camptothecin
to -1 -1 -1
-2 -2 -2 Sensitivity -2 -1 0 1 2 3 Sensitivity -2 -1 0 1 2 3 -2 -1 0 1 2 3 Sensitivity to RITA (GI50) Sensitivity Sensitivity to RITA (GI50) Sensitivity to RITA (GI50)
B PUMA NOXA p21 MDM2 PPM1D MCL1 MDMX 6
5
4
3 (log2*FC)
2 level 1
0
-1
Relative expression expression Relative -2 RITA CDDP Dox Irino Campto Nutlin-3 -3 0.173 D 0.576 C 0.690 0.704 RITA Campto CDDP Dox Nutlin p53 p21 MDM2 Wip1 MCL1 MDMX
β-actin log2FC differentially (8995) RITAupon genes expressed differentially log2FC
High Low Supplementary Figure S3.
A T47D OVCAR-3 B
120 DMSO T47D p53 RITA 80 AF PARP Onc-1 Cleav. PARP 40
γH2AX (Count) Cells β-actin 0 100 101 102 103 DCF-DA C T47D OVCAR-3 120 DMSO OVCAR-3 RITA 80 AF MDM2 Onc-1 40
Wip1 (Count) Cells 0 MCL1 100 101 102 103 MDMX DCF-DA 120 DMSO β-actin U2OS RITA 80 AF DMSO RITA AF Onc1 Onc-1 D 40
MCF7 (Count) Cells Cas9 0 100 101 102 103 1.00 0.41 0.65 0.47 DCF-DA MCF7 p53KO 0.95 0.50 0.75 0.45
E GLI1 p-SMAD2 SMO p-mTOR SMAD2 β-actin mTOR p-SMAD3 IGF1-R β-actin precursor SMAD3 IGF1-R β-actin β-actin SupplementaryFigure S4. B A RING1 PTPN6 STX10 CTU1 PDHB PAK4 HSCB MAPKAPK2 EPHB4 Onc AF RITA AF Onc AF RITA AF Treatment GIT1 PDK3 PDHA1 PKN2 ONC RITA RAB9A - - NCK1 1 1 CTU2 Onc TGS1 TIMM44 RITA SYK - 1 C BCKDHA - SRGAP2 1 BAIAP2 PKN1 HSPA9 AKTIP CLNS1A Total 111 117 ARHGAP5 98 44 19 19 GEMIN5 8 CCDC97 ATP6V1B2 BCAR1 ATP6V1G1
Relative mtiochondrial potential (%) TRIP6 NUP62 HOOK1 ATP6V1E1 SPG RWDD POLR MDN PDE FAM RUNDC RPAIN CIAO RFC TMPO PHF LRRC UFM PAK MSRA SF NACC KIAA COX TBC Proteins 100 120 80 20 40 60 3 0 PFN2 B CHMP4C 2 1 23 4 20 4 5 PRKCD 83 1524 1 2 SLBP 1 40 ELAVL1 D EIF3G 1 DIP 2 A 1 AAR SNRPC K 4 NAA CTU 24 STX TRMT TMEM SIK RALBP DLG MAGOH H 1 RBM8A CFAP RDH FECH EPN DMSO PACSIN UBLCP with IQGAP DYNC TRIP SUGP1 MRPL 2 3 UBAP1 PAN2 HSPB 10 2 EIF4A3 38 3 CSTF2T KLHDC LONP 13 NXF1 1 OLA 36 12 SF RBM22 CHMP7 RABGEF 1 263 FAM CCDC 6 NUF PFAS SYK CHMP5 ∆ TRMT10C 1 POLR EIF4H 1 1 CSTF1 PAN 10 DHX38 8 CEP PFN 1 2 퐓 SPECC H 1 SF3B2 EIF SF3B4 HNRNPM CPSF1 퐦 PROSC 1 MIPEP ALKBH 1 DYNLL FCCP 2 21 WWP MRPS CUL 3 88 PSMC 2 250 CASP2 NARS > 2 *** 4 TSTD 3 EIF4E HSPA TANK A HNRNPM A 1 TP G ZC C ퟎ 1 7 3 HSD17B10 PAPOLA SH ALDH7A1 ETFA MAGOH L HADHA TBCEL . 53 LGALSL 2 OPLAH SSU72 1 3 2 RBM STK38 PCBP2 26 BPHL SF1 ퟓ MYCBP CASP8 1 4 RIPK1 HAV BLM PTBP1 3 RAD 9 GIGYF LIMD NQO RK ° RGL BGRL SNRPC MRPS IDH RITA BCKDHA 퐂 PSMC TIAL1 HADH SF3B1 NS 8 GGA 1 PPP1CB LAMTOR1 PAIP 50 LYSMD A 2 NCK 1 2 1 HID KIF CCDC URI1 2 PHKG CIRBP TSC1 TOR PPP GSK3B RING HADHB STK LAMTOR 15 EPS SMU PPP1R12A 2 FAM 2 2 13 1 1 HARS POLR2A ETFB PIPSL B PSMD ATP 2 10 NUDT2 1 15 SLBP 1 B GPATCH 97 POLR2K AF PRKCI R 2 AIP 1 ANKRD 1 * 117 TIAL HIBCH 1 EPS L DDX SSU SKA POLR2L PDZD RFC 1 B 6 HARS PPP2R5C 2 PSMC4 2 V TRIP 1 CRCP POLR3G NARS B LYSMD 8 PDHA 15 CCND 1 1 ACAD 72 3 PDK 19 5 EYA E MRPS Onc-1 NUMB SMARCB NCOA3 52 11 1 NABP2 CCDC 8 PEA GSK3A GMEB 13 A NS PSMD5 RBPJ NT TRNT 2 3 3 ANP RDBP CEP NBN 1 ZFP PSMD8 1 9 RFC2 EPHB 2 PSMD 15 AMMECR 5 RIPK RFC4 21 TIPIN RBM PAPSS C TGS 174 295 METTL 1 91 32 3 1 BCAR NUF2 SPR SMARCB1 PPP2R1B 1 A ETFA SUCLA NT 1 4 E PSMD14 POLE4 1 FAM NXF 42 PSMC2 11 RFC5 ARRB CCNT1 RASSF CHTF18 NUDT PKN ADCK GCLC VPS ATP 2 5 PSMD3 LYRM CCND1 1 DC JMY MRPL 2 SRGAP 1 HSD 1 PSMD11 96 BOP B 2 L 2 41 6 2 2 4 CHTF V CCNT 8 BLM 2 UNG B CDK2 GEMIN GBAS DTL HSCB NCOA 1 17 9 C CASP CDC HIBCH 1 28 GUK B 21 ELAVL 2 ARID2 2 B PSMD PHKG2 DCAF11 18 PPP 10 KLHL orf 1 DPF TIPIN MAPKAPK 27 UBLCP1 RBL2 1 MRPS 2 CDC27 RAB 3 D URI GALE 5 SRPK 59 PRR SART NBN NIPSNAP 2 MTHFD DDX RAB 2 EIF 1 3 RAD50 22 R 1 MRPL 28 IARS BAIAP EIF5A NUBP2 5 CLNS RBPJ RABEP2 14 3 DBNDD 27 C 2 POLG 9 G 3 3 FAM96B PRKCI A SMAD2 SUGP X L TYW EIF LRRC MRPS 2 ; 2 TIMM TRIM21 1 DDX MRPL 1 53 KEAP1 2 CPSF 1 A NUBP1 TANK POLR 4 EYA3 ATP ZNHIT 5 PRPF POLE H 1 TRAFD 1 59 UGDH SUMF 3 NELFE 18 EEF FAM PAIP KLHL22 DOHH Y RABGEF1 46 44 1 CUL7 6 SAR1A 2 ZC B CCNL V GMPS CRCP PRKAR 38 L 4 CIAO1 1 2 NARFL 1 OXNAD GRHPR GSK 3 B ASNA PRKAR1A 1 2 118 RABEP 2 G TUFM B H 2 NARFL HAUS 1 MRPS31 HAUS PRMT GNB2 14 SGSM 2 GATAD 3 C A DCAF B MRPL27 1 GIT 8 MRPS30 DMSO GUK1 1 HADHB CELF A orf GPD 1 2 NUDT SF MRPL 6 4 AF 1 MRPS6 1 PDLIM IRS NT5C3 NUMB ALKBH 3 RRM1 37 UBAP 11 3 APAF IQGAP MRPL45 MRPS5 2 LENG 1 TUBA B 1 IQGAP1 2 B 1 L ARHGAP BRD ZBTB 8 18 RABL WDR PRKCD VPS MTHFS MRPL44 1 1 MRPL22 7 GSK MRPS27 5 IQGAP2 1 NUSAP CCT5 1 NUBP MRPL53 3 1 EIF RNF IFT UGDH C 8 8 DTL MRPS21 STK DHX 2 26 REPS1 OS 3 MRPS26 CHMP PHYH B CMPK1 5 22 A 5 149 VRK A ; MRPS18B RANBP PDHB RABL 38 2 AP SARS EPN1 ; GRIPAP PTBP 38 1 EIF UMPS RILPL MRPS15 UMPS HOOK CDK MRPL46 4 FLII 1 RANBP MRPL28 4 KEAP MRPS 5 B TUBA1C 2 TRAP C AL 2 1 1 SAR A 6 1 2 FAM PKN MRPS ARRB2 IFT CHMP MRPS34 1 MRPL18 NABP 1 BLOC 1 ESYT NSMCE NBEAL 1 TCEAL EPS15L1 5 1 1 UBR 1 81 MRPL10 EPN BDH A 1 PACSIN1 EPS15 98 CMPK SNX NAE TUFM MRPL DYNC 6 2 CSTF 1 2 7 C 3 3 S PSMD SHQ 2 2 ESD PTPN 1 3 4 COASY 5 1 TRMT CRYL CDC A KIF13B CMTR KIF3A FLAD 1 FTSJ IRAK CHMP 1 1 27 PFAS IFT81 1 CBLL LI ENOSF RABIF 6 5 123 NUBP 2 1 1 MRPL 10 4 1 MNX SNX SMYD 1 DTWD REPS TSC DYNC1LI2 1 FGD PSMD 5 C GALE SYTL ARL Onc KIF5B MYL RITA EXOC 1 17 TARS FAM 1 1 22 1 RBL 4 1 HADHA 2 2 DGCR XPNPEP 2 2 PPP RBM GRSF 6 14 AF AKTIP AAGAB OSBPL - PAPOLA B GNB RFC 195 3 2 1 2 RITA RABL5 MRPL GMPPA 1 FHOD ALDH GMPS DYNC1H1 R 22 ARF4 14 B 4 1 2 12 DYNLL1 CCT POLR CASP 9 YARS ZNF 1 NUP DOHH CSTF A RRM Onc1 44 7 1 ETFB SNX SNX4 ARL A ACOT 5 131 PCBP KIF 1 8 2 62 SAMHD 2 EIF 1 A 4 TAX MRPS T 6 5 APPBP ZADH ADCK MRPL DPH MRPS SMAD PIP 4 B PTGES 9 HAUS4 2 E HAUS6 1 HADH HSDL SLIRP BP CTU 4 1 31 K 1 2 3 45 2 30 3 2 CEP250 2 2 MRPS DLGAP C PPP TRIM 1 KDM MARK 2 PRUNE GALK ARID LARS PATL SF KIF ARF 1 3 3 CB 2 B 21 34 A A 4 2 2 4 2 5 1 1 Supplementary Figure S5.
T47D B T47D OVCAR-3 A DMSO 16h RITA 16h AF 16h Onc-1 24hDMSO 24h
EU RNA pol II p-RNA pol II (S2) sc35 β-actin
C DAPI RNA pol II p-RNA pol II (S5) β-actin D MCF7 DMSO 8h RITA 8h AF 8h Onc-1 24 h DMSO 24h p53KO
EU
DAPI
E 0.645 F 0.653 G 0.810 0.810 THZ531 ActD 0.846 0.846 DMSO DMSO 0.1 1 0.01 2.5 µM
AF Onc-1 RITA THZ531 AF Onc-1 RITA ActD RNA pol II 1
- p-RNA pol II (S2)
1 - p21 MDM2 Wip1
RITA, AF and and Onc AF RITA, MCL1 RITA, AF and and Onc AF RITA,
MDMX upon
upon β-actin
DEGs DEGs
H
common
common
log2FC 3154 3154 log2FC log2FC 3154 3154 log2FC
High Low High Low
ActD RITA RITA I 5 nM 0.1 μM 1 μM Nutlin-3 DMSO MCF7 p53KO
p53
RPL11
β-actin Supplementary Figure S6. A U2OS synchronized in mitosis (nocodazole) 0h 4h 8h 12h 16h 24h
103 BrdU 102 DMSO 101
100 0 200 400 600 103
Active S-phase 102 Arrested S-phase RITA G1 101 G2 100 0 200 400 600 0 200 400 600 0 200 400 600 0 200 400 600 0 200 400 600 PI
B MCF7 synchronized in early G1 (lovastatin) EU sc35 DAPI C MCF7 synchronized in G1/S (double thymidine block)
EdU DAPI EU sc35 DAPI γH2AX DAPI
DMSO 8h DMSO
phase
-
DMSO 8h DMSO
RITA 8h RITA
RITA 8h RITA Released in S Released
EdU DAPI
DMSO 8h DMSO
8h
No release No
RITA 8h RITA 24h Supplementary Figure S7. A 140 120 100 80 60 40
DNA interactaltion DNA 20
0
(% of non interaclated DNA) of non interaclated (%
0.08 0.04 0.31 0.62 1.25
15.62 10.00 15.62 31.25 62.50 15.62 31.25 62.50 31.25 62.50 DNA PI (μM) RITA (μM) AF (μM) Onc-1 (μM) B RITA AF Onc-1
NES = 2.406 NES = 2.887 NES = 2.666 FDR < 10E-6 FDR < 10E-6 FDR < 10E-6
C DMSO RITA AF Onc-1 Dox
λH2AX E * 70 * p=0.066 * 60 DAPI 50 * * *
5 ASCC2 foci ASCC2 5 40 ≥ D DMSO RITA AF Onc-1 MMS 30
20 ASCC2-HA
% of cells with with cells of % 10
0 DAPI DMSO RITA AF ONC1 MMS Resveratrol SupplementaryFigure S8. E NDGA
Relative expression level (log2*FC) - C - - - 3 2 1 0 1 2 3 4 5 DMSO A 0.99 1.03 1.00 PUMA Onc-1 AF RITA
8 Relative fluorescence - Merge oxodG D RITA DAPI
0.96 1.06 0.41 intensity of nuclear 8-oxodG 1,000 1,500 2,000 500 MCF7 p - RNA pol II (S2) DMSO 1.00 1.08 0.50 8h NOXA AF p53KO MCF7 MCF7 RNA polII Onc-1 +NDGA AF NDGA + RITA + NDGA β - actin H Onc1 8h 0.88 0.84 0.37 2 O 2 DMSO RITA p21 0.83 0.99 1.00 8h RITA 0.87 0.95 0.49 AF 8h U2OS MDM2 Onc1 0.93 0.98 0.57 16h AF Resveratrol Onc DMSO 16h 0.86 0.94 0.72 - 1 PPM1D DMSO 0.91 0.90 1.00 B RITA 0.91 0.70 0.41 MCL1 T47D 120 120 Cells (Count) 40
Cells (Count) 80 40 80 0 0 10 10 0 0.94 0.65 0.37 0 AF 10 10 DCF DCF 1 Onc 1 0.75 0.64 0.47 MDMX - - - DA DA 1 10 10 DMSO 2 2 0.89 0.98 1.00 Resveratrol RITA + RITA RITA RITA + NDGA DMSO RITA DMSO F 10 10 3 3 240
Cells (Count)160 RITA 80 1.08 0.60 0.20 0 OVCAR 10 0 BJ BJ - 0.95 1.00 0.22 3 AF - - 10 hTert hTert DCF 1 - DA + HRas Onc 0.97 0.89 0.76 10 - 2 1 V12 10 3 Supplementary Figure S9.
A B U2OS T47D OVCAR-3 CtIP MDC1 MRE11 RNF168 RPA32 RNF8 WRAP53 RAD51 RNF168 β-actin RNF8 D RAD51 DMSO RITA RAD51-GFP - + - - + - + - - + β-actin RNF8-HA - - + - + - - + - + RNF168-GFP - - - + + - - - + + DMSO C RITA MDM2 CHX 0 2 4 6 8 10 0 2 4 6 8 10 h p21 RNF168 MDMX RNF8 Wip1 RAD51 MCL1 β-actin β-actin 1 0.8 E RNF8-HA 0.6 +RNF168-GFP DMSO peGFP +Rad51-GFP 0.4 RITA
0.2 DMSO Relative RNF168 level RNF168 Relative 0 1.00 0.95 0 2 4 6 8 10 Time of CHX (h) 1 RITA 0.8 0.27 0.32 0.6 DMSO F RITA 0.4 1.2 *** *** 0.2 1 ***
Relative level RNF8 Relative 0 0 2 4 6 8 10 Time of CHX (h) 0.8
1 viability 0.6 0.8 0.4 0.6 DMSO 0.4 RITA cell Relative 0.2 0.2 0
Relative RAD51 level RAD51 Relative 0 Olaparib - + - + - + - + 0 2 4 6 8 10 Time of CHX (h) RITA AF Onc-1
G RITA AF Onc-1 H DMSO 0.05 µM 0.1 µM 1 µM 1.2 1.2 DMSO NS 1 1 NS 1.00 0.79 0.98 0.97 *** 0.8 0.8 ***
Olaparib viability 10 µM viability 0.6 0.6 0.76 0.48 0.71 0.39 0.4 RITA AF Onc-1 0.4
DMSO 0.05 µM 0.1 µM 1 µM Relative cell Relative 0.2 cell Relative 0.2 DMSO 0 0 1.00 0.65 0.97 0.86 RITA - - + + - - + + RITA - - + + - - + + Talazoparib Olaparib - + - + - + - + Talazoparib - + - + - + - + 0.1 µM +cMyc +cMyc 0.81 0.36 0.81 0.43 Supplementary Figure S10.
PARPi RITA DNA repair AF ROS factors PARP1 Onc-1 Stalled Fork RNA pol II RNA pol II RNA polymerases stalling and degradation
Oncogenes and pro-survival factors CANCER CELL DEATH G1 phase S phase SUPPLEMENTARY FIGURE LEGENDS
Supplementary Figure S1. A-B, Validation of p53 expression after CRISPR/Cas9 mediated knock-out of p53 in MCF7 breast cancer cell line. p53 was stabilized by 8h treatment with MDM2 inhibitor Nutlin-
3 (10 μM) or p53-reactivating compound RITA, and p53 protein level was assessed by western blot (A) or immunofluorescence (B). C, Crystal violet staining of surviving MCF7 cells after 5 days of treatment with RITA or Nutlin-3 (10 μM). Relative quantification is indicated below the pictures. D, Cell viability of wt and p53KO MCF7 cells after 1-3 days treatment with RITA. E, Induction of ROS in wt and p53KO
MCF7 cells upon RITA (8h) measured by DCF-DA. F, Induction of apoptosis (PARP cleavage) and
DNA damage response (γH2AX) in in RITA-treated wt and p53KO MCF7. G, Protein levels of selected
RITA-downregulated oncogenes and pro-survival factors in RITA-treated wt and p53KO MCF7. H, mRNA level of p53 target genes (PUMA, NOXA, p21) and RITA-downregulated oncogenes after 8h treatment in RITA-treated wt and p53KO MCF7 (n=3, * p<0.05, *** p<0.01). I, Control of p53 protein level after CRISPR/Cas9 mediated knock-out of p53 or transduction with p53 shRNA in A375 melanoma cell line. J, Crystal violet staining of surviving A375 cells after 5 days of treatment with
RITA or Nutlin-3 (10 μM). Relative quantification is indicated below the pictures.
Supplementary Figure S2. A, Absence of correlation of sensitivity (GI50) between RITA and different genotoxic agents (CDDP, oxaliplatin), ROS inducing compound (doxorubicin) or topoisomerase I inhibitors (camptothecin, irinotecan, topotecan) in the NCI-60 cell lines panel. B, Comparison of mRNA level of p53 target genes (PUMA, NOXA, p21) and known RITA-downregulated oncogenes of MCF7 cells treated 16h with RITA (1 μM), cisplatin (CDDP; 10 μM), doxorubicin (Dox; 1 μM), irinotecan
(Irino; 100 μM), camptothecin (Campto; 10 μM) and Nutlin-3 (10 μM). C, Protein levels of selected
RITA-downregulated oncogenes and pro-survival factors levels of MCF7 cells treated 24h with RITA, irinotecan, doxorubicin, and cisplatin. D, Heatmap comparing DEGs (p ≤ 0.05) in MCF7 cells upon treatment with RITA, with Nutlin-3 (10 μM) and in published data of MCF7 treated with cisplatin
(CDDP; 50 μM), doxorubicin (Dox; 2 μM) and camptothecin (Campto; 1 μM).
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Supplementary Figure S3. A, Detection of p53 level, PARP cleavage (indicating apoptosis) and
γH2AX (induction of DNA damage response) after treatment of T47D (36h) and OVCAR-3 cells (24h) with RITA, AF or Onc-1. B, Induction of ROS by RITA, AF or Onc-1 in T47D, OVCAR-3 and U2OS cells. C, Protein levels of selected RITA-downregulated oncogenes after treatment of T47D (36h) and
OVCAR-3 (24h) cells with RITA, AF or Onc-1. D, Surviving cells after 3 days treatment of wt and p53KO MCF7 cells with RITA, AF or Onc-1 assessed by crystal violet staining. Relative quantification is indicated below the pictures. E, Inhibition of TGF-β, Sonic Hedgehog, IGF and mTOR oncogenic signaling pathways at protein level in MCF7 cells upon RITA, AF or Onc-1 treatment.
Supplementary Figure S4. A, Repartition of the TPP hits between treatments with RITA, AF and Onc-
1. B, STRING network of the combination of TPP hits from RITA, AF and Onc-1. Each ball represents a protein with significantly altered thermal stability upon at least one of the compounds. Only proteins with connections in the network are displayed. The color code indicates which compound affect which protein in the network. C, Mitochondrial potential of MCF7 cells treated with RITA, AF or Onc-1 measured by TMRE assay. FCCP is used as positive control. (n=3, * p<0.05, *** p<0.01). D,
Transmission electron microscopy pictures of MCF7 cells treated with RITA, AF or Onc-1 for 8h. Scale bar 0.1 μm.
Supplementary Figure S5. A, RNA synthesis (detected by EU staining) and modifications of SFCs
(detected by sc35 immunostaining) in T47D cells treated with RITA, AF or Onc-1. B, Total and phospho-Ser2 RNA Pol II CTD protein levels in T47D cells (left panel) or OVCAR-3 cells (right panel) treated with RITA, AF or Onc-1 for 24h (OVCAR-3) or 36h (T47D). C, Total and phospho-Ser5 RNA
Pol II CTD protein levels in MCF7 treated with RITA, AF or Onc-1 for 24h. D, RNA synthesis monitored by EU incorporation in MCF7 p53KO cells treated with RITA, AF, Onc-1. E, Heatmap comparing DEGs (p ≤ 0.05) in MCF7 cells upon treatment with RITA, AF, Onc-1 and in Jurkat T-ALL
2 cells upon treatment with CDK12/CDK13 inhibitor THZ5531 (0.5 μM). F, Heatmap comparing DEGs
(p ≤ 0.05) in MCF7 cells upon treatment with RITA, AF, Onc-1 and ActD (2.5 μM). G, Protein levels of RNA pol II and selected RITA-downregulated oncogenes and pro-survival factors in MCF7 cells treated with THZ531 or ActD for 16h. H, 45S pre-rRNA or MYC mRNA detected by qPCR in MCF7 p53KO cells treated with RITA for 8h. RNA level (ΔCt) is relative to the untreated control, RPL13A is used as housekeeping gene. I, p53 level in MCF7 cells treated with RITA, ActD (5 nM) and Nutlin-3
(10 μM) upon inhibition of RPL11 by specific siRNA.
Supplementary Figure S6. A, Cell cycle analysis of U2OS cells synchronized in mitosis by nocodazole and then treated with RITA. B, RNA synthesis (detected by EU incorporation and labelling) and modifications of SFCs (detected by sc35 immunostaining) in MCF7 cells synchronized in early G1 by lovastatin and then treated with RITA. Lower panel: progression into S-phase was monitored by following EdU incorporation. C, RNA synthesis (detected by EU incorporation and labelling), modifications of SFCs (detected by sc35 immunostaining) and γH2AX phosphorylation in MCF7 cells synchronized in G1/S by double thymidine block. Cells were released in S-phase (upper panel) or kept arrested at G1/S transition (lower panel) and then treated with RITA. Progression into S-phase was monitored by following EdU incorporation.
Supplementary Figure S7. A, Absence of DNA intercalation by RITA, AF and Onc-1 assessed in-vitro by thiazole orange (ThO) displacement assay. Propidium iodide (PI) is used as positive control. B, Gene set enrichment analysis for UV response in MCF7 upon RITA, AF or Onc-1 treatment. C, γH2AX immunostaining upon treatment by RITA, AF, Onc-1 or Doxorubicin (Dox, 1 μM). D, ASCC2 foci formation in HA-ASCC2 overexpressing MCF7 cells upon treatment by RITA, AF, Onc-1 or the alkylating agent methyl methanesulfonate (MMS). E, Quantification of the percentage of cells with 5 or more ASCC2 foci (n > 100 cells for each condition).
3
Supplementary Figure S8. A, Oxidative DNA damage measured by of 8-oxodG immunostaining in
MCF7 cells treated with H2O2, RITA, AF or Onc-1. Upper panel, quantification of 8-oxodG fluorescence intensity per nucleus (n=200); lower panel, representative images. B, Control of antioxidant activity of resveratrol (1 μM) or NDGA (10 μM) in RITA-treated MCF7 cells, as assessed by DCF-DA. C, Rescue of RITA-, AF- and Onc-1-induced cell death by resveratrol or NDGA in MCF7,
U2OS, T47D and OVCAR-3 cells, monitored by crystal violet staining. Relative quantification is indicated below the pictures. D, RNA Pol II CTD total and phospho-Ser2 levels in MCF7 p53KO cells treated with RITA, AF or Onc-1 for 24h in presence of resveratrol. E, qPCR for p53 target genes and
RITA-downregulated oncogenes in MCF7 cells upon RITA, AF or Onc-1 in presence of NDGA. F,
Induction of ROS after 48h of RasV12 activation in BJ-hTert HRasV12ER-Tam measured by DCF-DA.
Supplementary Figure S9. A, Western blots for DNA repair factors in MCF7 cells treated 24h with
RITA, AF or Onc-1. B, Western blots for RNF8, RNF168 and RAD51 after treatment with RITA, AF or Onc-1 for 24h in U2OS and OVCAR-3 cells or for 36h in T47D cells. C, CHX chase analysis of
RNF8, RNF168 and RAD51 protein degradation. Upper panel: Western blot for protein levels in CHX- treated (10 μM) MCF7 cells upon RITA treatment. Lower panel: densitometric quantification of the bands normalized to β-actin. D, Western blots for selected RITA-downregulated oncogenes and pro- survival factors in MCF7 transiently overexpressing RAD51-GFP, RNF8-HA and RNF168-GFP treated with RITA for 16h. E, Crystal violet staining of MCF7 transiently overexpressing RAD51-GFP, RNF8-
HA and RNF168-GFP treated with RITA for 2 days. Relative quantification is indicated below the pictures. F, Cell viability of MCF7 cells treated with sub-lethal doses of RITA (0.1 μM), AF (0.5 μM) or Onc-1 (1 μM) for 24h in presence of 1 μM olaparib (n=3, *** p<0.01). G, Synthetic lethality in
OVCAR-3 cells treated with sub-lethal doses of RITA, AF or Onc-1 in presence of PARP inhibitor olaparib (upper panel) or talazoparib (lower panel), as assessed by crystal violet staining. Relative quantification is indicated below the pictures. H, Synthetic lethality in cMyc-inducible U2OS cells
(U2OS cMycER-Tam) treated with sub-lethal dose of RITA (0.05 μM), in presence of olaparib 2.5 μM (left panel) or talazoparib 0.1 μM (right panel), with or without induction of cMyc.
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Supplementary Figure S10. Model for RITA, AF and Onc-1 anticancer MoA. The three compounds cause a ROS-dependent RNA pol II stalling and degradation in G1 phase of the cell cycle, which lead to the inhibition of transcription of major oncogenic pathways as well as critical DNA repair factors such as Rad51. In S-phase, RITA, AF and Onc-1 increase replication stress and impair stalled forks processing due to the lack of key repair factors, sensitizing to PARP inhibition. The resulting imbalance between the increased oxidative and replicative stress burden and the downregulation of critical prosurvival factors leads to cancer-selective cell death.
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