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Gualtiero Alvisi, Henna Kuusisto, Ivan K.H. Poon, Kylie M. Wagstaff and David A. Jans

TUMOR CELL-SPECIFIC NUCLEAR TARGETING BY CAV VP3 (APOPTIN): PROMISES FOR ANTI- CANCER THERAPY ? Nuclear Signalling Lab. Dept. Biochem. & Mol. Biol. Monash University (Melbourne, Australia). Gualtiero Alvisi, Henna Kuusisto, Ivan K.H. Poon, Kylie M. Wagstaff and David A. Jans.

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Gualtiero Alvisi, Henna Kuusisto, Ivan K.H. Poon, Kylie M. Wagstaff and David A. Jans

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  1. TUMOR CELL-SPECIFIC NUCLEAR TARGETING BY CAV VP3 (APOPTIN): PROMISES FOR ANTI- CANCER THERAPY ?Nuclear Signalling Lab. Dept. Biochem. & Mol. Biol.Monash University(Melbourne, Australia) Gualtiero Alvisi, Henna Kuusisto, Ivan K.H. Poon, Kylie M. Wagstaff and David A. Jans

  2. Anti-Cancer Approaches • Cancer is a growing problem world-wide • We have drugs that kill tumor (but also normal) cells efficiently (ultimately the patient) • There is an urgent need to be able to deliver drugs specifically to tumor cells, and yet retain efficiency in anti-cancer therapy

  3. Viral/non-viral mechanisms of drug delivery: Cellular barriers to bioactive molecules Alvisi & Jans (2006) Drug Resistance Updates6, 40-50

  4. Anti-Cancer Approaches • Cancer is a growing problem world-wide • We have drugs that kill tumor (but also normal) cells • There is an urgent need to be able to deliver drugs specifically to tumor cells, and yet retain efficiency in anti-cancer therapy • Cancer cells derive from normal cells; the idea of a magic bullet to combat cancer (a single property of a cancer cell that can be targeted specifically) may be naïve (Alvisi & Jans (2006) Drug Resistance Updates6, 40-50) • VP3/apoptin– a viral gene-product with tumor cell-specific activity

  5. Chicken Anemia Virus (circovirus) • ssDNA virus • Causes immunosuppression /severe anaemia in young chicks by inducing apoptosis in thymocytes/ erythroid progenitors • 3 viral proteins (VPs): VP1, VP2 andVP3 http://www.tanglewoodfarms.net/img/poulet-rouge/Cou-nus-young1.jpg

  6. VP3/Apoptin Tumor/transformed cells Normal/untransformed cells • Nuclear localization • Induces apoptosis Cytoplasm Nucleus VP3 • Cytoplasmic localization • No apoptosis • Non-isogenic cell comparisons • (eg. SAOS-2 human osteosarcoma line v. VH10 foreskin fibroblasts • Transformed human embryonic kidney v. Rat primary hepatocytes)

  7. N L S NLS c. 12 distinct EXPs N E S N E S Ran Ran GTP GTP MULTIPLE SIGNAL-DEPENDENT PATHWAYS FOR TRANSPORT THROUGH THE NPC NUCLEAR ENVELOPE CYTOPLASM NUCLEUS Ran GTP  Importin a/b1-Mediated Nuclear Import N L S a  a NLS, nuclear localisation signal (generally Lys/Arg-rich) IMPa/b IMPa NPC Ran GTP  Importin b1-Mediated Nuclear Import NLS b1 IMPb1 IMPbx (exportin)-Mediated Nuclear Export Exportin Nuclear Pore Complex (nups – nucleoporins) Exportin

  8. N L S NLS N E S N E S Ran GTP MULTIPLE SIGNAL-DEPENDENT PATHWAYS FOR TRANSPORT THROUGH THE NPC NUCLEAR ENVELOPE CYTOPLASM NUCLEUS Ran GTP  Importin a/b1-Mediated Nuclear Import N L S a  a NLS, nuclear localisation signal (generally Lys/Arg-rich) IMPa/b IMPa NPC Ran GTP  Importin b1-Mediated Nuclear Import NLS b1 IMPb1 Exportin 1 (CRM1)-Mediated Nuclear Export NES, nuclear export signal (generally hydrophobic) Exportin Ran Exportin GDP

  9. N L S NLS N E S N E S LMB, leptomycin B: CRM1-specific inhibitor X Ran GTP MULTIPLE SIGNAL-DEPENDENT PATHWAYS FOR TRANSPORT THROUGH THE NPC NUCLEAR ENVELOPE CYTOPLASM NUCLEUS Ran GTP  Importin a/b1-Mediated Nuclear Import N L S a  a NLS, nuclear localisation signal (generally Lys/Arg-rich) IMPa/b IMPa NPC Ran GTP  Importin b1-Mediated Nuclear Import NLS b1 IMPb1 Exportin 1 (CRM1)-Mediated Nuclear Export Exportin X Ran Exportin GDP

  10. VP3/Apoptin Tumor/transformed cells Normal/untransformed cells • Nuclear localization • Induces apoptosis Cytoplasm Nucleus VP3 • Cytoplasmic localization • No apoptosis

  11. Nuclear Targeting Signals in VP3 97 1 88 33 46 82 105 111 121 N C LRS NLS1 NES NLS2 IRIGIAGITITLSL KPPSKKR SCDPSEYR VSKLKESLI TT T108 PS RPRTAKRRIKL Is VP3 recognised by Importins or Exportins ? NLS: Nuclear Localization Sequence NES: Nuclear Export Sequence

  12. .O2 Excitation: 680nm Emission: 620nm 680nm .O2 AlphaScreen Binding Assay (Amplified Luminescence Proximity Homogeneous Assay) bImps: biotinylated Imps VP3-His6: hexa-His-tagged GFP-VP3-HMG S: streptavidin b: biotin Ni2 Donor beads Acceptor beads VP3-H6 bIMP S Binding 200 nm No Emission VP3-H6 Ni2 Donor beads Acceptor beads bIMP S No binding Wagstaff and Jans (2005) Anal. Biochem. 348, 49-56

  13. GFP-VP3(74-121) + IMPb Kd = 3.7 nM 100 80 60 + IMPa 40 + IMPa/b 20 0 0 10 20 30 40 50 60 IMP (nM) 74-121 NLS1 NES NLS2 GFP VP3 is recognised with high affinity by IMPb1; modulated by intramolecular masking GFP-VP3(1-121) + IMPb Kd = 9.7 nM 100 80 60 % Maximal Binding + IMPa/b 40 + IMPa 20 0 0 10 20 30 40 50 60 IMP (nM) 97 1 88 33 46 82 105 111 121 1-121 LRS NLS1 NES NLS2 GFP Wagstaff and Jans (2005) Anal. Biochem. 348, 49-56

  14. Poon et al. (2005) Cancer Res. 65, 7059-7064 Exportin 1 1 Importin 1 Wagstaff and Jans (2006) Anal. Biochem. 348, 49-56 Nuclear Targeting Signals in VP3 97 1 88 33 46 82 105 111 121 N C LRS NLS1 NES NLS2 IRIGIAGITITLSL KPPSKKR SCDPSEYR VSKLKESLI TT T108 PS RPRTAKRRIKL Thr108: phosphorylated specifically in tumor cells Rohn et al. (2002) 277, 50820 NLS: Nuclear Localization Sequence NES: Nuclear Export Sequence

  15. ISOGENIC CELL LINES Untransformed Transformed A) Isogenic cell line pair African Green Monkey Kidney CV-1 COS-7 (CV-1 transformed with SV40) B) Human osteosarcoma isogenic cell pair* SR40SAOS-2 (SAOS-2 with WT Rb) (Rb mutant) C) Human skin fibroblast isogenic cell pair** 1br3 1br3N (1br3 transformed using SV40 T-ag) * Goodrich et al. (1991) Cell 67:293-302 ** From A. Lehmann (Uni. Sussex) (1999) Curr. Biol. 9, 699-704

  16. VP3 nuclear localization is dependent on the C-terminus SAOS-2 (Transformed) GFP GFP-VP3(1-121) Cytoplasm Nucleus Poon et al. (2005) Cancer Res. 65, 7059-7064 97 1 88 33 46 82 105 111 121 1-121 LRS NLS1 NES NLS2 GFP

  17. GFP-VP3(74-121) GFP-VP3(1-73) GFP-VP3(1-89) GFP-VP3(1-111) VP3 nuclear localization is dependent on the C-terminus SAOS-2 (Transformed) GFP GFP-VP3(1-121) Cytoplasm Nucleus Poon et al. (2005) Cancer Res. 65, 7059-7064 1-73 LRS GFP 1-89 LRS NLS1 GFP 1-111 LRS NLS1 NES GFP 74-121 NLS1 NES NLS2 GFP

  18. GFP-VP3(74-121) GFP-VP3(1-73) GFP-VP3(1-89) GFP-VP3(1-111) GFP-VP3(NLS1m) GFP-VP3(NLS2m) VP3 nuclear localization is dependent on NLS1 and NLS2 SAOS-2 (Transformed) GFP GFP-VP3(1-121) Cytoplasm Nucleus 97 Poon et al. (2005) Cancer Res. 65, 7059-7064 1 88 33 46 82 105 111 121 LRS NLS1 NES NLS2 GFP NLS1m LRS NES NLS2 GFP NLS2m LRS NLS1 NES GFP

  19. VP3 can accumulate in non-tumor cells dependent on NLS1 & NLS2 SR40 (Untransformed) GFP GFP-VP3(1-121) GFP-VP3(74-121) GFP-VP3(1-73) GFP-VP3(1-89) GFP-VP3(1-111) GFP-VP3(NLS1m) GFP-VP3(NLS2m) Poon et al. (2005) Cancer Res. 65, 7059-7064 97 1 88 33 46 82 105 111 121 LRS NLS1 NES NLS2 GFP

  20. SR40 cells – VP3(LRSm) Fn Fn Fn Fb Fc IMAGE ANALYSIS USING A “WORK WINDOW”: Nuclear localization of VP3(LRSm) in SR40 cells (Fn – Fb) Fn/c = (Fc – Fb) • SOFTWARE (Public Domain): “IMAGE J” (NIH) • http://rsb.info.nih.gov/ij/ Trapani et al. (1996) J. Biol. Chem. 271, 4127-4133

  21. p < 0.0001 p < 0.0001 Dependent on NLS1 & NLS2, VP3 accumulates to a greater extent in tumor than in non-tumor cells SAOS-2 (Transformed) SR40 (Untransformed) Nuclear to cytoplasmic fluorescence (Fn/c) Poon et al. (2005) Cancer Res. 65, 7059-7064 97 1 88 33 46 82 105 111 121 1-121 LRS NES NLS2 NLS1 GFP 82 97 74 88 105 111 121 74-121 NLS1 NES NLS2 GFP

  22. Subcellular Localization of VP3 is Independent of Duration of Expression SAOS-2 p = 0.0083 SR40 80 80 DAY 1 DAY 6 Fn/c p = 0.0176 70 70 60 60 50 50 40 40 30 30 20 20 10 10 0 0 GFP GFP GFP-VP3(1-121) GFP-VP3(1-121) Is the increased nuclear accumulation in tumor cells due to more efficient nuclear import or reduced nuclear export ?

  23. GFP GFP-VP3(1-121) Lack of VP3 Nuclear Export Activity in Transformed Cells LMB, leptomycin B: exportin 1 (CRM1) inhibitor SAOS-2 (Transformed) - LMB + LMB GFP-Rev(2-116)

  24. p < 0.0001 Lack of VP3 Nuclear Export Activity in Transformed Cells (Transformed) SAOS-2 - LMB +LMB Nuclear to cytoplasmic fluorescence (Fn/c) 1-121 LRS NLS1 NES NLS2 GFP

  25. Differential Nuclear Export Activity in Transformed and Untransformed Cells SAOS-2 (Transformed) SR40 (Untransformed) - LMB + LMB - LMB + LMB GFP-Rev(2-116) GFP GFP-VP3(1-121)

  26. p < 0.0001 p < 0.0001 p < 0.0001 Differential Nuclear Export Activity in Transformed and Untransformed Cells (Untransformed) (Transformed) SAOS-2 SR40 - LMB - LMB + LMB + LMB Nuclear to cytoplasmic fluorescence (Fn/c) Nuclear to cytoplasmic fluorescence (Fn/c) 1-121 LRS NLS1 NES NLS2 GFP

  27. Exportin 1 p < 0.0001 p < 0.0001 p < 0.0001 VP3 nuclear export is mediated via the C-terminal NES not the LRS (Untransformed) (Transformed) SAOS-2 SR40 - LMB - LMB 60 60 50 50 40 40 Nuclear to cytoplasmic fluorescence (Fn/c) Nuclear to cytoplasmic fluorescence (Fn/c) 30 30 20 20 10 10 0 0 GFP-VP3(1-121) GFP-VP3(1-121) GFP-VP3(NESm) GFP-VP3(NESm) GFP-VP3(LRSm) GFP-VP3(LRSm) Poon et al. (2005) Cancer Res. 65, 7059-7064 1-121 LRS NLS1 NES NLS2 GFP LRSm NLS1 NES NLS2 GFP NESm LRS NLS1 NLS2 GFP

  28. Localization of VP3 to PML NBs Anti-PML Anti-PML GFP-VP3(1-121) Merge SAOS-2 (Transformed) SAOS-2 SR40 (Untransformed) SR40 1-121 LRS NLS1 NES NLS2 GFP Poon et al. (2005) Cancer Res. 65, 7059-7064; Janssen et al. (2006) Oncogene 26, 1557-1566

  29. GFP-VP3(74-121) GFP-VP3(LRSm) 1 1 88 88 33 33 46 46 82 82 97 97 105 105 111 111 121 121 N N C C LRS LRS NLS1 NLS1 NES2 NES2 NLS2 NLS2 Deletion/mutation of the LRS abolishes PML NB localisation GFP-VP3(1-121) Saos2 SR40 VP3 FL wt VP3 74-121 VP3 FL LRSm 88 74 82 97 105 111 121 C NLS1 NES2 NLS2 X

  30. Exportin 1 Promyelocytic leukemia (PML) nuclear bodies p < 0.0001 p < 0.0001 p < 0.0001 VP3 nuclear export is mediated via the C-terminal NES not the LRS (Untransformed) (Transformed) SAOS-2 SR40 - LMB - LMB 60 60 50 50 40 40 Nuclear to cytoplasmic fluorescence (Fn/c) Nuclear to cytoplasmic fluorescence (Fn/c) 30 30 20 20 10 10 0 0 GFP-VP3(1-121) GFP-VP3(1-121) GFP-VP3(NESm) GFP-VP3(NESm) GFP-VP3(LRSm) GFP-VP3(LRSm) Poon et al. (2005) Cancer Res. 65, 7059-7064 1-121 LRS NLS1 NES NLS2 GFP LRSm NLS1 NES NLS2 GFP NESm LRS NLS1 NLS2 GFP

  31. Tumor cell CRM1 CRM1 Normal cell GTP GTP Ran Ran RanGAP1 GDP Ran Differential Nuclear Export Activity Mediated by the VP3 NES in Normal and Tumor Cells Nucleus Cytoplasm Nuclear envelope Importin β1 VP3 NPC β1 β1 GTP LRS NES NLS2 NLS1 Ran NPC

  32. Thr108 1-121 LRS NLS1 NES NLS2 GFP Ala108 Ala108 LRS NLS1 NES NLS2 GFP Glu108 Glu108 LRS NLS1 NES NLS2 GFP Testing the role of Thr108 in modulating VP3 nuclear transport Thr108: phosphorylated specifically in tumor cells Rohn et al. (2002) 277, 50820

  33. p < 0.0001 p < 0.0001 p < 0.0001 p = 0.032 p < 0.0001 Phosphorylation at Thr108 Inhibits VP3 Nuclear Export in Tumor Cells (Untransformed) (Transformed) SAOS-2 SR40 - LMB - LMB +LMB +LMB Nuclear to cytoplasmic fluorescence (Fn/c) Nuclear to cytoplasmic fluorescence (Fn/c) Poon et al. (2005) Cancer Res. 65, 7059-7064 GFP-VP3(Ala108) - non-phosphorylatable VP3 GFP-VP3(Glu108) - mimic phosphorylated VP3

  34. PML Nuclear Body Cytoplasm Importin b1 NPC b1 PML b1 VP3 GTP P Ran PML Nuclear envelope LRS NES NLS2 NLS1 Tumor cell Normal cell Exportin 1 NPC Exportin 1 GTP GTP Ran Ran GDP Nucleus Ran RanGAP1 Poon et al. (2005) Cancer Res. 65, 7059-7064 Tumor cell-specific nuclear targeting of VP3 is effected by a phosphorylation-regulated NES

  35. Viral/non-viral mechanisms of drug delivery Alvisi & Jans (2006) Drug Resistance Updates6, 40-50

  36. p6 % Viable cells MRT-p6 MRT + DTox - DTox Sites of photoactivation (DHFA) Concentration (nM) MRT DTox HMP T-ag NLS MSH DNA damage Nucleus Cytoplasm New MRT DTox HMP VP3 tNLS MSH Dtox, Diphtheria toxin T-domain (endosomolytic) HMP, drug (PS) binding domain MSH, α-Melanocyte-stimulating hormone (Melanoma cell specific targeting/uptake) Normal/untransformed cells Tumor/transformed cells Tumour Cell-specific Drug Targeting ? Modular Recombinant Transporter Rosenkranz et al. (2003) FASEB J. 17, 1121-1124

  37. The VP3 tNTS (aa 74-121) is sufficient for tumour cell-enhanced nuclear accumulation SR40 SAOS-2 - LMB + LMB :dependent on NES activity as regulated by negative charge at Thr108

  38. p6 % Viable cells MRT-p6 MRT + DTox - DTox Sites of photoactivation (DHFA) Concentration (nM) MRT DTox HMP T-ag NLS MSH DNA damage Nucleus Cytoplasm New MRT DTox HMP VP3 tNLS MSH Dtox, Diphtheria toxin T-domain (endosomolytic) HMP, drug (PS) binding domain MSH, α-Melanocyte-stimulating hormone (Melanoma cell specific targeting/uptake) Normal/untransformed cells Tumor/transformed cells Tumour Cell-specific Drug Targeting ? Modular Recombinant Transporter Rosenkranz et al. (2003) FASEB J. 17, 1121-1124

  39. Anti-Cancer Approaches • Growing problem world-wide, with a need for efficient and specific anti-cancer therapies • Cancer cells derive from normal cells; the idea of a magic bullet to combat cancer (a single property of a cancer cell that can be targeted specifically) is naïve (Alvisi & Jans (2006) Drug Resistance Updates6, 40-50) • VP3/apoptin– tumor cell-hyperactivity (NOT all or nothing) • Need to combine multiple tumor cell-hyperactive components/strategies to be able to deliver drugs to cancer cells specifically (MULTIPLICATIVE)

  40. Cancer cell-specific signalling Phosphorylation • VP3 tNTS Non-viral approaches to achieve tumor cell-specific drug delivery ? • MSH • Her2 • c-Met Alvisi & Jans (2006) Drug Resistance Updates6, 40-50

  41. Anti-Cancer Approaches • Growing problem world-wide, with a need for efficient and specific anti-cancer therapies • Cancer cells derive from normal cells; the idea of a magic bullet to combat cancer (a single property of a cancer cell that can be targeted specifically) is naïve (Alvisi & Jans (2006) Drug Resistance Updates6, 40-50) • VP3/apoptin– tumor cell-hyperactivity (NOT all or nothing) • Need to combine multiple tumor cell-hyperactive components/strategies to be able to deliver drugs to cancer cells specifically (MULTIPLICATIVE) • VP3/apoptin– tumor cell-specific anti-cancer activities

  42. Nuclear Signalling Lab., Dept. Biochem. & Mol. Biol., Monash University, Clayton IVAN POON Cristina Oro Gualtiero Alvisi Henna Kuusisto Manisha Dias Kylie Wagstaff Jingpu Zhang (Beijing) Dept. of Mol. Genetics of Intracellular Transport, Institute of Gene Biology, Russian Academy of Sciences AND Dept. Biophysics, Moscow State University Alex Sobolev Andrey Rosenkranz

  43. MRT + DTox - DTox Sites of photoactivation (DHFA) MRT-p6 % Viable cells p6 Concentration (nM) Enhancement of cytotoxicity through cell-specific intranuclear delivery of the photosensitiser bacteriochlorin p6 in B16-F1 melanoma cells MRT DTox HMP T-ag NLS MSH Dtox, Diphtheria toxin T-domain (endosomolytic) HMP, drug (PS) binding domain MSH, α-Melanocyte-stimulating hormone (Melanoma cell specific targeting/uptake) Rosenkranz et al. (2003) FASEB J. 17, 1121-1124

  44. The distinct subcellular localization of VP3 is determined by differential nuclear import/export of VP3 in tumor and normal cells VP3/Apoptin Tumor/transformed cells Normal/untransformed cells • Nuclear localization • Induces apoptosis Cytoplasm Nucleus VP3 • Cytoplasmic localization • No apoptosis • Non-isogenic cell comparisons • (eg. human osteosarcoma line versus foreskin fibroblasts)

  45. VP3 Nuclear Accumulation is Independent of Cellular Expression Levels GFP-VP3(1-121) GFP-VP3(74-121) Fn/c Poon et al. (2005) J. Virol. 79, 1339-1341

  46. - LMB + LMB 1br3 1br3/n (transf.) 220 • LMB • + LMB Fn/c 165 110 55 0 1br3 1br3/n (transf.) Differential Nuclear Export Activity in Isogenic Transformed and Untransformed Cells (Fibroblasts) Alvisi & Jans (2006) Drug Resistance Updates6, 40-50

  47. VP3-Induced Apoptosis Cytoplasm Nucleus Apoptosis facilitating NE products IMP β 1 DNA IMP Transcriptional machinery α / β 1 β 1 α α P PML * HipK2 NPC NPC VP3 P NB * PML VP3 APC1 Hippi APC/C Downstream NPC NPC caspases Hippi Hip - 1 Cytochrome c release Apoptosis Mitochondria

  48. Domain Organization of VP3 Apoptosis induction Apoptosis induction 1 69 80 121 APC1 binding 82 121 DNA binding 121 66 DNA binding 1 80 Hippi binding 1 59 PML NB localization 1 73 Multimerization 29 69 1 88 33 46 82 97 105 111 121 LRS NLS1 NES NLS2 N-terminus C-terminus VP3 IRIGIAGITITLSL KPPSKKR SCDPSEYR VSKLKESLI TTT108PS RPRTAKRRIKL

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