Prostate-targeted radiosensitization via aptamer-shrna chimeras in human. tumor xenografts

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1 SUPPLEMENTARY DATA Prostate-targeted radiosensitization via aptamer-shrna chimeras in human tumor xenografts Xiaohua Ni 1, Yonggang Zhang 2, Judit Ribas 1, Wasim H. Chowdhury 1, Mark Castanares 1, Zhewei Zhang 2, Marikki Laiho 2, Theodore L. DeWeese 1,2,3, Shawn E. Lupold 1,2,3* 1 James Buchanan Brady Urological Institute, 2 Department of Radiation Oncology and Molecular Radiation Sciences, 3 Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD * Correspondence to S.E.L (slupold@jhmi.edu). Supplemental Experimental Procedures Materials. Unless otherwise noted, all DNA primers were purchased from Sigma-Aldrich, sirnas were purchased from IDT and all cell culture products were purchased from Gibco BRL/LifeTechnologies. Interferon assay. For evaluating of interferon β response, 2x10 5 LNCaP cells were either transfected with sirna DNA-PK or incubated with 400 nm A10-3-Con, A10-3-DNA-PK or Neg- DNA-PK, Poly(I:C) (invivogen) as a positive control, for 48 h before the secretion of interferon β into the cell culture supernatant was analyzed. Detection of interferon β was accomplished by using a commercially available sandwich interferon β ELISA kit (PBL) following manufacturer s

2 recommendations. The results obtained were compared to serial dilutions of an interferon β positive control provided with the kit. RNA secondary structure. M-fold was used to predict the structures of Aptamer-shRNAs. The most stable structures with the lowest energies for each RNA oligo were compared. PSMA cell-surface expression. PSMA cell-surface expression was determined by flow cytometry using antibodies specific to human PSMA (J591 from Neil Bander, Weill Medical College of Cornell University). PC3-PIP or PC3-Flu cells were trypsinized and washed three times in PBS. 1X10 6 cells were resuspended in100 µl cell sorting buffer (1x PBS, 0.5% bovine serum albumin (BSA), 2 mmol/l EDTA) with a 1:5000 dilution of Human PSMA antibody J591 and incubated at 4C 0 for 20 min. Cells were then washed in 1 ml cold cell sorting buffer and incubated at 4C 0 for 20 min with a 1:1,000 dilution of Alexa Fluor 488 F(ab') 2 fragment of antihuman IgG (A11013; Invitrogen) in cell sorting buffer. Cells were washed and incubated at 4C 0 for 20 min with 4% PFA(1ml). After fixation, cells were then resuspended in cell sorting buffer and analyzed by flow cytometry (Becton Dickson Calibur FACS Analytic cytometer). Supplemental Figure 1. Supplemental Figure 2. Supplemental Figure 3. Supplemental Figure 4. Supplemental Figure 5 Supplemental Table 1. Screen data of DNA repair sirna library

3 Supplemental Table 2. Primers for qpcr Supplemental Table 3. Primers and templates for aptamer-shrnas synthesis Supplemental Table 4. Aptamer-shRNAs sirna sequences Supplemental Table 5. sirna sequences

4 Supplemental Figure 1. sirna-mediated knock-down. DU145 cells were transfected with Candidate sirnas (5 nm), 48 hrs later the mrna level of target genes were evaluated by qrt- PCR. Data are normalized to GAPDH mrna. (A) DNA-PK, (B) MAD2L2, (C) BRCA2, (D) NBN, (E) Rad23B, and (F) RAD54L. Columns represent mean± S.E.M, n=3.

5 Supplemental Figure 2. sirna-mediated radiation sensitization. LNCaP cells were seeded in 6-well plates at 2x10 5 cells per well. After 24 hr, cells were either transfected with 100 nm of DNA-PK, BRCA2, NBN, MAD2L2, RAD23B, RAD54L sirnas or control sirnas separately using HiPerfectTransfection Reagent, 48 hr later, cells were seeded in 96 well plate at 2000 cells per well. 24hr later, cells were then irradiated with Gy IR using a Gammacell 40 (Nordion, Ottawa, ONT, Canada) 137Cs radiator at approximately 0.6 Gy/min. Cell viability was assessed after 12 days by MTS. means ± S.E.M, n=3

6 Supplemental Figure 3. Evaluation of interferon response. 2x10 5 LNCaP cells were either transfected with DNA-PK sirna or incubated with 400 nm A10-3-Con, A10-3-DNA-PK or Neg- DNA-PK for 48 h before the secretion of interferon β into the cell culture supernatant was analyzed. Cells were separately treated with 5 μg/ml Poly(I:C) (invivogen) as a positive control. Detection of interferon β was accomplished by using a commercially available sandwich interferon β ELISA kit (PBL) following manufacturer s recommendations. 25-2,000 pg/ml of interferon-beta standard was used as a positive control for assay integrity. means ± S.E.M, n=3

7 Supplemental Figure 4. PSMA cell-surface expression. PSMA positive PC3-PIP cells and PSMA negative PC3-Flu cells were incubated with Anti-PSMA (J591) antibody and stained with secondary antibody conjugated with Alexa488. PSMA expression was then determined by flow cytometry. 48.2% of PC3-PIP (A) cells expressed PSMA when compared to PC3-Flu (B) cells.

8 Supplemental Figure 5. Individual LNCaP tumor growth curves. (A-G), Established LNCaP tumors were intratumorally injected with 200 pmol Aptamer-shRNAs on days -3 and day -2. On day 0, animals were divided into groups that received either 6 Gy radiation (+R) or no radiation (-R). Tumors were measured every other day until reaching four times the volume at time of radiation. (A) PBS(-R) (n=6); (B) A10-3-Con (-R) (n=6);(c) A10-3-DNA-PK(-R) (n=6); (D) Neg- DNA-PK(-R) (n=6); (E)A10-3-Con(+R) (n=6); (F)Neg-DNA-PK(+R) (n=6); (G) A10-3-DNA- PK(+R) (n=8).

9 Supplemental Table 1 0 Gy Avg 0 Gy St dev 6 Gy Avg 6 Gy St dev Relative Sensitization control sirna % ACLY % ACLY % AHCY % AHCY % AKT % AKT % AKT % AKT % ALKBH % ALKBH % ALKBH % ALKBH % APEX % APEX % APEX % APEX % APEX % AR % AR % ARMET % ARMET % ATM % ATM % ATR % ATR % BIRC % BIRC % BIRC % BIRC % BIRC % BIRC % BLM % BLM % BRCA % BRCA % BRCA % BRCA % C18orf % C18orf % CANX % CANX % CARM % CARM %

10 CBX % CBX % CCNH % CCNH % CCT % CCT % CCT % CCT % CDC % CDC % CDK % CDK % CDKN1A % CDKN1A % CDKN2A % CDKN2A % CDKN2B % CDKN2B % CDKN2C % CDKN2C % CDKN2D % CDKN2D % CDKN % CDKN % CETN % CETN % CHEK % CHEK % CHEK % CHEK % CKS % CKS % CLU % CLU % COL1A % COL1A % COPB % COPB % CRIP % CRIP % DCLRE1A % DCLRE1A % DCLRE1B % DCLRE1B % DCLRE1C % DCLRE1C % DDB %

11 DDB % DDB % DDB % DMC % DMC % DNMT % DNMT % DNMT3A % DNMT3A % DNMT3A % DNMT3B % DNMT3B % DOT1L % DOT1L % DUT % DUT % DVL % DVL % E2F % E2F % EHMT % EHMT % EIF4A % EIF4A % ERCC % ERCC % ERCC % ERCC % ERCC % ERCC % ERCC % ERCC % ERCC % ERCC % ERCC % ERCC % ERCC % ERCC % EXO % EXO % EZH % EZH % FANCA % FANCA % FANCC % FANCC % FANCD %

12 FANCD % FANCE % FANCE % FANCF % FANCF % FANCG % FANCG % FAP % FAP % FEN % FEN % FLJ % FLJ % G3BP % G3BP % GTF2H % GTF2H % GTF2H % GTF2H % GTF2H % GTF2H % GTF2H % GTF2H % H2AFX % H2AFX % H2AFZ % H2AFZ % HDAC % HDAC % HDAC % HDAC % HDAC % HDAC % HDAC % HDAC % HDAC % HDAC % HDAC % HDAC % HLTF % HLTF % HNRNPA2B % HNRNPA2B % HSP90B % HSP90B % HSPD % HSPD %

13 HSPE % HSPE % HUS % HUS % IARS % IARS % IFNGR % IFNGR % IGF1R % IGF1R % ILF % ILF % ITGB % ITGB % KDELR % KDELR % KIAA % KIAA % KPNA % KPNA % LDHA % LDHA % LIG % LIG % LIG % LIG % LIG % LIG % MAD2L % MAD2L % MAPK % MAPK % MBD % MBD % MBD % MBD % MBD % MBD % MBD % MBD % MCL % MCL % MCM % MCM % MECP % MECP % MGMT %

14 MGMT % MLH % MLH % MLH % MLH % MLL % MLL % MMP % MMP % MMS % MMS % MNAT % MNAT % MPG % MPG % MRE11A % MRE11A % MRPL % MRPL % MRPS % MRPS % MSH % MSH % MSH % MSH % MSH % MSH % MSH % MSH % MSH % MSH % MTHFD % MTHFD % MUTYH % MUTYH % NBN % NBN % NCBP % NCBP % NEIL % NEIL % NEIL % NEIL % NEIL % NEIL % NFKB % NFKB %

15 NME % NME % NONO % NONO % NTHL % NTHL % NUDT % NUDT % NUP % NUP % OGG % OGG % OGT % OGT % PAFAH1B % PAFAH1B % PAICS % PAICS % PARP % PARP % PARP % PARP % PCNA % PCNA % PIK3CB % PIK3CB % PLK % PLK % PMS % PMS % PMS % PMS % PMS2L % PMS2L % PNKP % PNKP % POLB % POLB % POLD % POLD % POLE % POLE % POLG % POLG % POLH % POLH % POLH %

16 POLI % POLI % POLK % POLK % POLL % POLL % POLM % POLM % POLN % POLN % POLN % POLN % POLQ % POLQ % PPP2R5C % PPP2R5C % PRDX % PRDX % PRDX % PRDX % PRKDC % PRKDC % PRMT % PRMT % PSMA % PSMA % PSMC % PSMC % PSME % PSME % PTMA % PTMA % RAD % RAD % RAD % RAD % RAD % RAD % RAD23A % RAD23A % RAD23B % RAD23B % RAD % RAD % RAD % RAD % RAD51C %

17 RAD51C % RAD51L % RAD51L % RAD51L % RAD51L % RAD % RAD % RAD54B % RAD54B % RAD54L % RAD54L % RAD9A % RAD9A % RBM % RBM % RECQL % RECQL % REV % REV % REV3L % REV3L % RFC % RFC % RPA % RPA % RPA % RPA % RPA % RPA % RPA % RPA % RPL % RPL % RPL % RPL % RPL % RPL % RRM2B % RRM2B % SDHC % SDHC % SDHC % SETD % SETD % SETD % SETD % SMARCA %

18 SMARCA % SMUG % SMUG % SND % SND % SNRPE % SNRPE % SNRPF % SNRPF % SOX % SOX % SPO % SPO % SSBP % SSBP % SSR % SSR % SSR % SUV39H % SUV39H % SUV39H % SUV39H % TARS % TARS % TDG % TDG % TDP % TDP % TGIF % TGIF % TMEM30A % TMEM30A % TOP2A % TOP2A % TP53BP % TP53BP % TPX % TPX % TRAF % TRAF % TRDMT % TRDMT % TREX % TREX % TREX % TREX % TSTA %

19 TSTA % TUBB % TUBB % UBE2A % UBE2A % UBE2A % UBE2B % UBE2B % UBE2N % UBE2N % UBE2S % UBE2S % UBE2V % UBE2V % UNG % UNG % WRN % WRN % XAB % XAB % XPA % XPA % XPC % XPC % XRCC % XRCC % XRCC % XRCC % XRCC % XRCC % XRCC % XRCC % XRCC % XRCC % XRCC % XRCC % ZDHHC % ZDHHC %

20 Ni et al Supplementary Table 2 Ni et al Supplementary Table 3

21 Ni et al Supplementary Table 4 Ni et al Supplementary Table 5

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TFDP2 TFDP1 SUMO1 SERTAD1 RPA3 RBBP8 RAD9A RAD17 RAD1 NBN MRE11A MNAT1 KPNA2 KNTC1 HUS1 HERC5 GTSE1 GTF2H1 GADD45A E2F4 DNM2 DDX11 CUL3 SKP2 RBL1 RB1

TFDP2 TFDP1 SUMO1 SERTAD1 RPA3 RBBP8 RAD9A RAD17 RAD1 NBN MRE11A MNAT1 KPNA2 KNTC1 HUS1 HERC5 GTSE1 GTF2H1 GADD45A E2F4 DNM2 DDX11 CUL3 SKP2 RBL1 RB1 -8-6 -4-2 CHEK2 MKI67 RB1 RBL1 SKP2 Fold-Change Fold-Change -3-2 -1 CCND1 CCNE1 CDK4 CDK5R1 5 1 15 ATM ATR BAX BCCIP BRCA1 BRCA2 BL1AT ABL1A ABL1 CCNG1 CCNG2 CCNH CCNT1 CCNT2 CDC2 CDC34 CDKN1A CDKN1B CDKN2A