New Treatment options. Virological approaches cccdna Inhibition

Transcription

1 New Treatment options. Virological approaches cccdna Inhibition Massimo Levrero Cancer Research Center of Lyon (CRCL) and INSERM U52, Lyon, France Hopital de la Croix-Rousse - Hospices Civils de Lyon, Lyon, France CLNS@SAPIENZA, Istituto Italiano di Tecnologia (IIT), Rome, Italy Dept of Internal Medicine - DMISM, Sapienza University, Rome, Italy DIPARTIMENTO DI MEDICINA INTERNA E SPECIALITA MEDICHE

2 Disclosures Advisory Committees or Review Panels: - Gilead - Jansen - BMS - Arbutus - Galapagos - Assembly Pharma - Sanofi/Aventis Speaking and Teaching: - MSD - Roche - BMS - Jansen - Gilead

3 Durantel & Zoulim, J Hepatol 26 HBV cure: targeting ccdna

4 HBV cure: targeting cccdna cccdna controls replication: template for transcription controls persistence: stability and latency not directly affected by current treatments Durantel & Zoulim, J Hepatol 26

5 Definition of HBV cure «Functional cure» Situation where antiviral therapy could be stopped with a minimal risk of viral reactivation HBsAg loss with anti-hbsab seroconversion cccdna inactivation and/or control by host mechanisms «Complete cure» In all cases, «HBV cure» associated with clinical benefit: regression in the risk of disease progression and HCC The impact of molecular damage and integrated viral sequences in infected hepatocytes will need to be addressed. HBsAg clearance and cccdna eradication Zeisel, Lucifora et al, Gut 25; Revill et al, Nature Reviews Gastroenterol Hepatol 26 Levrero, Testoni, Zoulim Curr Opin Virology 26 Durantel & Zoulim, J Hepatol 26

6 Targeting cccdna Functional cure Targeted silencing of transcription Ø HBx inhibitors Ø cccdna epigenetic modifiers Ø antisense RNA (post cccdna) Ø RNA interference (post cccdna) SILENCING DESTRUCTION FORMATION Ø Inhibit «entry» Ø Inhibit «capsid delivery» Ø Inhibit cccdna formation Ø Inhibit cccdna chromatin formation Cure Cytolytic elimination of cccdna Ø selective killing of infected hepatocytes Ø hepatocytes turnover Non-cytolytic cccdna purging Ø molecular scissors : - CRISPR-Cas, TALEN, NgAgo Ø cytokine induced purging Modified from Nassal, Gut 25

7 cccdna HBsAg. Cytolytic elimination of cccdna Ø selective killing of infected hepatocytes Ø hepatocytes turnover 2. Non-cytolytic cccdna purging Ø molecular scissors : - CRISPR-Cas, TALEN, NgAgo Ø cytokine induced purging 3. Targeted silencing of transcription Ø HBx inhibitors Ø cccdna epigenetic modifiers Ø antisense RNA (post cccdna) Ø RNA interference (post cccdna)

8 Do in vivo proliferation of hepadnavirus infected hepatocytes affect cccdna levels? Ducks (NAs + rapid liver growth): Reaiche-Miller, Virology 23 HuHep mice undergoing serial engrafments: No origin of replication No nuclear retaining signal Does cccdna survive mitosis? Lutgehetmann, Hepatology 2

9 Do in vivo proliferation of hepadnavirus infected hepatocytes affect cccdna levels? Ducks (NAs + rapid liver growth): Hepatocytes turn-over: limitations for cure Difficult to control No origin of replication No nuclear retaining signal Does cccdna survive mitosis? Reaiche-Miller, Virology 23 HuHep mice undergoing serial engrafments: May trigger clonal selection of hepatocytes (oncogenic?) Lutgehetmann, Hepatology 2

10 cccdna HBsAg. Cytolytic elimination of cccdna Ø selective killing of infected hepatocytes Ø hepatocytes turnover 2. Non-cytolytic cccdna purging Ø molecular scissors : - CRISPR-Cas, TALEN, NgAgo Ø cytokine induced purging 3. Targeted silencing of transcription Ø HBx inhibitors Ø cccdna epigenetic modifiers Ø antisense RNA (post cccdna) Ø RNA interference (post cccdna)

11 Direct targeting of cccdna stability a) Designer endonucleases for targeted disruption of cccdna I. ZNFs (Zinc finger nucleases) II. TALENs (Transcription activator-like effector nulceases) III. CRISPR/Cas (Cluster of regularly interspaced short palindromic repeats / Cas nucleases) IV. NgAgo? (Natronobacterium gregoryi Argonaute endonuclease) 5 p-ssdna guide & expression of NgAgo Cradick et al., Molecular Therapy, 2 Weber et al., PLoS ONE, 24 Seeger and Sohn, Mol Ther-Nucleic Acids, 24 Chen et al., Molecular Therapy, 24 Seeger and Sohn, Mol Ther-Nucleic Acids, 26 Swarts et al., Nature 24 Guo et al., Nature Biotech, 26 b) Activation of host defence: deaminases mediating degradation of cccdna Uracil excision DNA deamination Endonucleases Stenglein et al., Nat Struct Mol Biol., 2 Lucifora, Xia et al, Science 24

12 cccdna destruction: CRISPR/Cas9 Double strands breaks NHEJ DNA repair Deletions/large mutations Induction of deletions in cccdna Decreased number of cells expressing viral antigens Seeger, Mol Therapy Nucl Acids, 24; Lin, Mol Therapy Nucl Acids, 24; Kennedy, Virology, 25; Kennedy, Antiviral Res 25

13 cccdna destruction: CRISPR/Cas9 Targeted cccdna cleavage: limitations for cure Potential off-target effects Double strands breaks NHEJ DNA repair Deletions/large mutations Specific delivery to infected cells is lacking Effect of cccdna chromatin accessibility? Induction of deletions in cccdna Decreased number of cells expressing viral antigens Seeger, Mol Therapy Nucl Acids, 24; Lin, Mol Therapy Nucl Acids, 24; Kennedy, Virology, 25; Kennedy, Antiviral Res 25

14 cccdna destruction: deamination IFNα, Lymphotoxin β agonists, IFNγ, TNFα can induce APOBEC3A/B dependent degradation of HBV cccdna Lucifora, Science 24; Shlomai & Rice, Science 24; Xia, Gastroenterology 25

15 Dual activity of T cells: cccdna is removed cytolytic and non-cytolytic mock IFN-γ +TNF-α co-culture transwell +HBsAg transwell -HBsAg S-CAR T cell non-cytolytic cytolytic non-cytolytic non-cytolytic HBV infected HepaRG cells non-cytolytic T cells contribute to cytolytic and non-cytolytic elimination of cccdna cytolytic killing Xia, Stadler, Gastroenterology 26

16 Dual activity of T cells: cccdna is removed cytolytic and non-cytolytic S-CAR T cell mock IFN-γ +TNF-α co-culture T cells contribute to cytolytic and non-cytolytic elimination of cccdna transwell +HBsAg transwell -HBsAg non-cytolytic cytolytic non-cytolytic non-cytolytic non-cytolytic Cytokines-induced deamination: limitations for cure cytolytic killing HBV infected HepaRG cells Strong but partial effect on the established cccdna pool in vitro Specificity to be further confirmed Are they targeting only a subset of cccdna molecules? Xia, Stadler, Gastroenterology 26

17 cccdna HBsAg. Cytolytic elimination of cccdna Ø selective killing of infected hepatocytes Ø hepatocytes turnover 2. Non-cytolytic cccdna purging Ø molecular scissors : - CRISPR-Cas, TALEN, NgAgo Ø cytokine induced purging 3. Targeted silencing of transcription Ø HBx inhibitors Ø cccdna epigenetic modifiers Ø antisense RNA (post cccdna) Ø RNA interference (post cccdna)

18 cccdna is a minichromosome subjected to the histone code cccdna is associated to histones and non-histone proteins 6 nucleosomes with a spacing ~ 2bp (% < to cell chromatin) Bock, Virus Gen 994 and JMB 2; Newbold, JVI 995; Pollicini, 26, Belloni, PNAS 29; Tropberger, 25

19 cccdna is a minichromosome subjected to the histone code cccdna is associated to histones and non-histone proteins 6 nucleosomes with a spacing ~ 2bp (% < to cell chromatin) cccdna ChIP assay crosslink chromatinized cccdna detectable h post infection, PTMs at 2 hrs sonicate Immunoprecipitation reverse crosslink purify DNA real time PCR [cccdna primers] cccdna ChIP-Seq cccdna molecules from infected HepG2-NTCP, PHH and human liver share similar, but not identical, histone PTMs profiles HBc binds the cccdna [2 hrs after infection] input DNA NGS (ChIP-Seq) HBx binds the cccdna [4 hrs after infection] O. Floriot, unpublished M Locatelli and B Testoni, unpliblished Bock, Virus Gen 994 and JMB 2; Newbold, JVI 995; Pollicini, 26, Belloni, PNAS 29; Tropberger, 25

20 HBV replication is regulated by the acetylation status of cccdna-bound H3/H4 histones ChIP-Seq - HBV infected PHH cccdna ChIP CHB liver biopsies Tropberger, 25 Pollicino, 26 High Replication Low Replication Occult HBV CBP PCAF p3 PCAF p3 HDAC HDAC Ezh2 CBP Sirt Sirt MeCP2 HDAC Sirt HDAC Suv39 HP Sirt spontaneously iatrogenic iatrogenic immunosuppression ChIP Input IgG Pollicino, Belloni (unpublished)

21 Modulation of cccdna epigenetic control : IFNα In vivo HBV infection DHBV Tet-on cell line HBV-infected HepG2-NTCP Belloni, 22 Liu, 23 Tropberger, 25 Alpha-IFN: inhibits cccdna transcription (no reduction in cccdna levels) reduces the acetylation of cccdna-bound histones

22 Modulation of cccdna epigenetic control : IL6 A B C HepG HepG2 NT ril6 NT ril6 NT ril µg,25 µg mock 24h NT 24h 48h ril6 NT 48h ril6 24h NT 24h ril6 Fold induction.5.5 *** capsid associated HBV DNA Fold induction.5.5 *** 3.5 Kb/pgRNA Fold induction.5.5 *** Pre-S/S RNA S - 8S - D NT ril6 E NT ril6 STAT3 HNF HNF4 HNF STAT3 cccdna Fold induction.5.5 HBV cccdna % of input FI.5.5 *** AcH3 5 *** HDAC HDAC HDAC ril6 HDAC HDAC IL6: inhibits cccdna transcription (no reduction in cccdna levels) reduces the acetylation of cccdna-bound histones and HNF, HNF4, STAT3 occupancy increases HDAC recruitment Palumbo, 25

23 Can we target cccdna epigenetic modifications with compounds that affect chromatin modifiers?

24 Modulation of cccdna epigenetic control : small molecules PCAF/p3 inhibitor A B EML264 Sirt/2 stimulator Arbitrary Unit.5 Arbitrary Unit Capsid associated HBV DNA Days p.i. 2 Days p.i. ** * ** ** ** ** Kb RNA Arbitrary Unit.5 * ** * ** * *** Arbitrary Unit Capsid associated HBV DNA Kb RNA Ac Ac Active cccdna TFn2 TFn Ac Ac Suppressed cccdna MC279 JMD3 inhibitor MC39 C D ** * E * *** Arbitrary Unit.5 αach4 4 2 α3meh3k27 Arbitrary Unit Arbitrary Unit.5 αach4 NT EML264 MC279 MC39 F Arbitrary Unit 3 2 α3meh3k27 Ac Ac TFn2 TFn Ac Ac EMs modify chromatin remodelling enzymes recruitment onto HBV minichromosome Different classes of compounds acang as epidrugs: inhibit cccdna transcripaon modulate acetylaaon/methylaaon of cccdna- bound histones Palumbo ACS 26 submitted

25 Modulation of cccdna epigenetic control : small molecules The p3/cbp inhibitor C646: inhibits cccdna transcripaon reduces the acetylaaon of cccdna- bound histones Tropberger, 25

26 Modulation of cccdna epigenetic control : small molecules Endpoint Make active carriers true inactive and, eventually, over time occult carriers by locking the cccdna [ functional cure ] Stage of development Pre-clinical proof of concept

27 Modulation of cccdna epigenetic control : small molecules Endpoint Make active carriers true inactive and, eventually, over time occult carriers by locking the cccdna [ functional cure ] Stage of development Pre-clinical proof of concept cccdna epigenetic control: limitations for cure complexity of responses functional redundancy duration of response off target effects expected therapeutic margins unknown no virus specific / enriched histone PTM or chromatin modifier, so far should we silence cccdna?!generation of a protected pool? potential for rational associations mostly un-checked

28 Modulation of cccdna epigenetic control : small molecules Read-outs for screening and clinical development cccdna transcriptional activity - golden standard [pgrna / cccdna ratio] - requires biopsies and standardization of cccdna assays pgrna - also reduced if the drug reduces the cccdna pool - requires biopsies, technological advances for easier assays HBeAg - surrogate marker of cccdna transcription in cells and animal models - not applicacle in HBeAg negative patients HBcrAg - composite biomarker (HBcAg + HBeAg + p22crag/hbe precursors) - non validated in cell cultures yet - not restricted to HBeAg pos CHB pts - surrogate marker of cccdna transcription (promising results at AASLD 26) Circulating RNA particles - poor surrogate: may increase with NUCs, decrease with capsid interfering drugs qhbsag - poor surrogate: different promoter on cccdna, influence of integration, - secretion intefering drugs, PreS to S ratios, mutations with retention phenotype

29 AASLD 26 HepaAAs B core- related anagen correlates with intrahepaac covalently closed circular DNA (cccdna) levels and acavity in untreated chronic hepaaas B (CHB) paaents Testoni Barbara,2, Berby Françoise, Miaglia Clothilde,2,3, Lebossé Fanny,2,3, Lamper@co Pietro 4, Levrero Massimo,3,5, Zoulim Fabien,2,3 INSERM U52- Cancer Research Center of Lyon (CRCL), 698 Lyon, France 2 University of Lyon, UMR_S52, CRCL, 698 Lyon, France 3 Department of Hepatology, Croix Rousse hospital, Hospices Civils de Lyon, France 4 Division of Gastroenterology and Hepatology, Fondazione IRCCS Ca Granda Ospedale Maggiore Policlinico, Università degli Studi di Milano, Milan, Italy 5 Department of Internal Medicine - DMISM and the IIT Center for Life Nanoscience (CLNS), Sapienza University, Rome, Italy Not-treated CHB patients

30 cccdna HBsAg. Cytolytic elimination of cccdna Ø selective killing of infected hepatocytes Ø hepatocytes turnover 2. Non-cytolytic cccdna purging Ø molecular scissors : - CRISPR-Cas, TALEN, NgAgo Ø cytokine induced purging 3. Targeted silencing of transcription Ø HBx inhibitors Ø cccdna epigenetic modifiers Ø antisense RNA (post cccdna) Ø RNA interference (post cccdna)

31 HBx binds to and is required for cccdna minichromosome transcription HBx degrades the Smc5/6 restriction factor (DDB Cul4 E3 ligase complex) Smc5/6 Smc5/6 Decorsière, Nature 26 Decorsiere, Nature 26 Murphy, Cell Rep 26 Kim. JVI 26 HBx Transcription +++ HATs p3 SetA HATs p3 HBx TF HDAC TF HBx Sirt SetA TF HBx SETDB Suv39 HP PMTs H3K4me3 H3K27ac H3K22ac Belloni, PNAS 29; Benhenda, JVI 23; Ducroux, PlosPath 24; Riviere, J Hepatol 25; Decorsiere, Nature, 25; Alarcon, SciRep 26 HBx prevents cccdna epigenetic silencing

32 HBx inhibition Endpoint make active carriers true inactive and, eventually, over time occult carriers by locking the cccdna [ functional cure ] relation with cccdna pool / cccdna destabilization unclear [ HBx not required for establishing the cccdna ] Stage of development Pre-clinical / discovery Screening HBV-infected PHH or HepG2NTCP HTS assays (high throughput screening) of small compound libraries Target oriented screenings (DDB interaction?)

33 HBx inhibition Endpoint make active carriers true inactive and, eventually, over time occult carriers by locking the cccdna [ functional cure ] relation with cccdna pool / cccdna destabilization unclear [ HBx not required for establishing the cccdna ] Stage of development Pre-clinical / discovery HBx inhibition: limitations for cure Screening HBV-infected PHH or HepG2NTCP HTS assays (high throughput screening) of small compound libraries Target oriented screenings (DDB interaction?) a very attractive target for HBV cure (virus specific, off-cccdna effects beneficial) difficult task: HBx has no enzymatic activity cccdna transcription as screening phenotype sirna against viral RNAs are under evaluation HBx-host factors interactions for screening [DDB interaction, Smc5/6 degradation]

34 HBx protein, HBV replication and HBV pathogenesis DDB HBx PMRT SETDB HDAC HBx u u HBx b i n d s t o c e l l u l a r promoters and modulates the epigenome by relocating chromatin regulators Impact for viral replication and HCC development HBx proteasome VDAC3 cccdna Ras Raf MAPKK AP NF-kB I-kB NF-kB TF TF Transcriptional activation Ac Ac HBx Me Ac Ac HMT Me Me Transcription al repression Ca ++ csrc mitochondria cytochrome C caspases pgrna TFs activation Deregulation of gene expression Epigenetics Protein stability DNA Repair Genetic instability Apoptosis mir-38-2 mir-224 mir596 RabA Rab2B Rab5A Beclin- SREBP2 ATG4B Endocytosis Autophagy HBV replication HCC development/progression mir-33a Levrero & Zucman Rossi, J Hepatology 26 Guerrieri, BMC Genomics 26 Salerno (in preparation)

35 HBc and cccdna HBV capsid (2 HBc dimers) u u u HBc dimer Bock, 2 Arbitrary Units ahbc Belloni 29 HBc binds the cccdna and modifies cccdna nucleosome spacing HBc binds cccdna CpG islands and an epigenetic permissive state HBc binds to cellular promoters and regulates gene expression FI % Input FI % Input c-src IL29 FI % Input IL6 Ezh2 E2F Plating DMSO 2.5%.5 HAP2 affects cccdna -4-3 T MOI 2 cccdna nt HAP 2 T HepG2 NTCP.5 pgrna nt HAP 2 PHH HAP mm.5 T6 HBV tot nt HAP 2 HAP2 prevents cccdna formation HepG2 NTCP cccdna pgrna.5 HBV tot T Pla@ng DMSO 2.5% MOI 2 T HAP µm T2 nt HAP nt HAP HepG2 NTCP nt HAP 2.5 cccdna nt HAP pgrna nt Bock T. et al., JMB 2; Belloni L. et al. PNAS USA 29; Guo, BMC genomics, 23; Durantel D. et al., AASLD 23; Lupacchini and Floriot et al. unpublished HAP HBV tot nt HAP % of input.3.2. ChIP a-hbc mock NT HAP % of input ChIP a-ach4 mock NT HAP

36 HBc and cccdna HBV capsid (2 HBc dimers) u u u HBc dimer Bock, 2 Arbitrary Units ahbc Belloni 29 HBc binds the cccdna and modifies cccdna nucleosome spacing HBc binds cccdna CpG islands and an epigenetic permissive state HBc binds to cellular promoters and regulates gene expression Bock T. et al., JMB 2; Belloni L. et al. PNAS USA 29; Guo, BMC genomics, 23; Durantel D. et al., AASLD 23; Lupacchini and Floriot et al. unpublished FI % Input c-src IL29 HAP2 prevents cccdna formation Pla@ng DMSO 2.5%.5 HepG2 NTCP T cccdna nt MOI 2 HAP T pgrna nt HAP FI % Input HAP µm FI % Input T2 HBV tot nt HAP IL6 Ezh2 E2F % of input Plating DMSO 2.5% HAP2 affects cccdna -4-3 T MOI 2 cccdna nt HAP 2 cccdna nt HAP ChIP a-hbc T HepG2 NTCP.5 mock NT HAP pgrna nt HAP 2 PHH pgrna nt HAP HepG2 NTCP % of input HAP mm T6 HBV tot Hap2 CpAM - blocks new cccdna formation - repress residual cccdna transcription - leads to an inappropriate assembly of viral capsids - potential to reduce the cccdna pool virus specific mechanism NOT ALL ANTI-CAPSID HAVE FULL SPECTRUM ACTIVITIES nt HAP 2 HBV tot nt HAP 2 ChIP a-ach4 mock NT HAP

37 Conclusions cccdna targeting strategies include - targeting cellular epigenetic modifiers [preclinical proof of concepts] - inhibiting HBx (HBx interactome) [discovery] - inhibiting HBx synthesis (sirnas) [in clinics, MOF not investigated] - TFs binding sites mutagenesis on cccdna [theoretical] - cccdna mutagenesis [pre-clinical] Pros and Cons - Targeting cellular epigenetic modifiers off target effects low activity vs toxicity - HBx inhibitors targeting virus-related functions - cccdna destruction low activity vs off target effects Biomarkers for drug development and patients management - Epigenetic modifiers cccdna transcription serum markers need validation - cccdna destruction cccdna Current knowledge position HBx drugs as powerful agents for functional cure

38 Thanks to INSERM U52 - Equipe 23 Massimo Levrero Oceane Floriot Francesca Guerrieri Mohamed Ibachi INSERM U52 Equipe 5 Fabien Zoulim Barbara Testoni Judith Fresquet Maelle Locatelli David Durantel ValenAna D Arienzo Laboratory of Gene Expression Massimo Levrero Laura Belloni Gianna Aurora Palumbo Leonardo Lupacchini Francesca Guerrieri Natalia Pediconi Ludovica Calvo Debora Salerno Silvia di Cocco Giancarlo Ruocco LeAzia Chiodo Collaborations: Adam Zlotnick Lichun Li Indiana University, Bloomington, USA Uri LopaAn Assembly PharmaceuAcals Maura Dandri Lena Alweiss University Medical Hospital Hamburg Ulrike Protzer Julie Lucifora TUM - Helmholtz Zentrum München Stephan Urban Jessika Sonnabend Dept of Molecular Virology Heidelberg Univ Hospital Financial support Sabrina Strano Giovanni Blandino Regina Elena Cancer Center Rome