DOD Prostate Cancer Consortium

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DOD Prostate Cancer Consortium
TEAM 2 Report
Grand Hyatt Washington

• Kickoff Meeting
• July 11, 2003

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Team 2

• Team Members
• 5 members, 3 institutions
• Ron Rodriguez, M.D., Ph.D. (JHU)
• Leland Chung, Ph.D. (Emory)
• Ted DeWeese, M.D. (JHU)
• JT Hsieh, Ph.D. (UTSW)
• Ken Koeneman, M.D. (UTSW)

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Project 3 Overview Enhancing Adenoviral Gene
Therapy Efficacy by Altering the Therapeutic
Potency

• SA1 RADIOSENSITIZATION
• To develop radiosensitizing adenoviral vectors
  and test the efficacy of these constructs when
  combined with radiation therapy, both in vitro
  and in vivo.
• SA2 PROSTATE CA CRAD
• To develop next generation prostate specific
  conditionally replication competent adenoviral
  (CRAd) vectors, which circumvent E1a-mediated AR
  inhibition.
• SA3 COMBINATION THERAPIES
• To evaluate current and future CRAd vectors
  (e.g., Ad-PSE-E1a, Ad-BSP-E1a, Ad-OC-E1a, etc) in
  combination with anti-telomerase antisense
  oligonucleotide (ASO) therapy, taxotare-based
  chemotherapy and radiation using subcutaneous,
  orthotopic and intraosseous models of androgen
  independent prostate cancer.

TD
RR
KK
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Project 4 Overview Enhancing Adenoviral Gene
Therapy Efficacy by Altering Transcriptional and
Cellular Targeting.

• SA1 HDACIs AS SENSITIZERS TO AD
• To develop methods for rapid screening of
  clinical specimens of prostate cancer for overall
  CAR expression levels (as a means of enhancing
  patient selection in clinical trials) and to
  assess various differentiating agents for their
  ability to re-express the adenoviral receptor
  (CAR) in preclinical models of AIPCa.
• SA2 TARGETING TO PSMA
• To utilize phage display to screen for peptide
  ligands capable of binding to prostate cancer
  cell surface membrance proteins and utilize such
  ligands to generate fiber modified adenoviral
  constructs.
• SA3 CHARACTERIZE PROSTATE TFs
• To evaluate the roles of prostate cancer and bone
  stroma-derived factors in upregulating the
  transcription of genes that control the
  expression of human osteocalcin promoter(hOC).
  Through the study of regulation of hOC promoter
  in prostate cancer and bone stroma cells, we can
  expect to accomplish two goals 1) to devise a
  more efficient means by delivering transgenes and
  improve adenoviral replication in prostate cancer
  and bone stromal cells in vitro and in chiceric
  prostate tumor models in vivo 2) to interrupt
  the levels of key transcription factors via
  antisense or ribozyme approach so that the
  proliferation and apoptotic programs of the
  prostate cancer and bone stomal cells can be
  altered.

JTH
RR
LC
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PROJECT 3 TIMELINE

Task 1

Task 5

Task 2

Task 3

Task 7

Task 11

Task 4

Task 6

Task 9

Task 8

Task 10

Year 1

Year 2

Year 3

Year 1

Year 2

Year 3

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PROJECT 3 TIMELINEYear 1 Progress To Date

• Task 1 TD
• Generate AD5E4orf6 adenoviral replication
  deficient vectors.
• Status Task abandoned after AD5E4orf6 failed to
  demonstrate radiosensitization as anticipated.
  siRNA approach taken, paper published Can Res 4
  months ago.
• Task 4 RR
• Complete the generation of E1a-AR chimera.
• Status Cloning of all chimera completed.
• Task 5 RR
• Screen all of the fusions for optimal activity
  and specificity in transient transfections with
  expression plasmids.
• Status Screening completed. Manuscript in
  preparation. Viruses generated, but await
  characterization.
• Task 9 KK
• Evaluate combination therapies of the current
  vectors with anti-Telomerase ASO (antisense
  oligonucleotide), and Docetaxel in models of
  prostate cancer.
• Status Combination therapies evaluated with
  Ad5BMP-E1A, manuscript accepted to Cancer Research

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Summary Project 3

• All milestones met ahead of schedule
• Year 2 milestones
• Task 2 Test viral vectors for radiation
  sensitization in prostate and non-prostate cancer
  cell lines.
• Status Performed with siRNA and U6 promoter,
  which failed to function in adeno. Now working
  on VA1 promoter. Initial results promising.
• Task 6 Generate conditionally replication
  competent adenoviruses based on the most active
  and specific chimera.
• Status All viruses made, large scale preps in
  progress. Characterization to follow.

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PROJECT 4 TIMELINE

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PROJECT 4 TIMELINEYear 1 Progress To Date

• Task 1 JTH
• Screen multiple cell lines for CAR expression
  after induction with a variety of differentiating
  agents (e.g., Trichostatin, Phenylbutryate, SAHA
  FR901228) and confirm mechanism of
  differentiating agent action as histone
  deacetylase inhibition by western blot analysis,
  looking for acetylated H3 and H4 histones.
• Status.Completed. Revised manuscript just
  submitted to Can Res.
• Task 6 RR
• Generate phage display ligands against
  established prostate specific membrane markers,
  such as PSMA.
• Status. Ligands identified. Manuscript in
  preparation.
• Task 10 LC
• Identify the trans-acting factors responsible for
  regulation gene expression by the human OC
  promoter.
• Status Identified a gene on 8q which encodes a
  TF involved in regulation of OC and PSA
  promotersgt PrLZ, patent submitted.

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Summary Project 4

• All Year 1 milestones met ahead of schedule.
• Year 2 milestones
• No year 2 milestones are completed yet.
  Currently all are still in early phase, but ahead
  of schedule.
• There are no anticipated problems with year 2
  milestones.

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Targeting Tumor-Stroma InteractionLeland Chung
and Rouxiang Wang

• Progress Report
• Identified a new cis-DNA element within human OC
  promoter that is highly responsive to soluble
  factor-induced OC expression in prostate cancer
  cells (Huang, et al. 2003).
• Identified an AR and NF-kB cis-DNA element, XBE,
  within PSA promoter that confers negative
  regulatory function to AR-mediated gene
  regulation (Cinar, Zhau et al. 2003).

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Targeting Tumor-Stroma InteractionLeland Chung
and Rouxiang Wang

• Progress Report (Continue)
• Isolated and cloned a new prostate-specific
  protein, PrLZ, and its promoter, from human
  prostate cancer cell lines. PrLZ was found to be
  overexpressed in clinical prostate cancer
  specimens and was found to reside at chromosome
  8q, the most frequently amplified chromosome in
  human prostate cancer (Wang, et al. 2003).

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Progress in Prostate Specific Conditionally
Replication Competent Adenoviruses

• Ron Rodriguez, M.D., Ph.D.
• Shawn Lupold, Ph.D.
• Ying Li, Ph.D.
• Wasim Chowdhury
• Spensor Collis, Ph.D.
• Ted DeWeese, M.D.

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Introduction

• Prostate Specific Replication Competent
  Adenovirus (Rodriguez et al, 1997)

• Replication depends on E1a and E1b
• Vector CV706 (Calydon, Inc)

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• CV706 Prostate Specific CRAD

E1a
PSA Pro/Enh

• Prostate Specific lytic replication cycle
• Achieve higher dose at site of interest
• Single injection decreases likelihood of immune
  response limiting efficacy

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CV706 cont

• Obstacles identified with CV706
• PSA pro/enh requires androgen for maximal
  induction, but CV706 was not inducible with
  androgen
• Target population of patients usually actively
  treated with anti-androgen
• Efficacy, while encouraging, was still modest
• ? Why?

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AR and E1A Functions
AR Activated by binding ligand Chromatin
remodeling (Swi/Snf) Binds DNA at
AREs Complexes with p300/CBP, other
HATs Complexes with co-activator
complexes Promotes Pol II transcription E1A C
onstitutively active Chromatin remodeling
(Swi/Snf) DOES NOT Bind DNA (mostly) Complexes
with p300/CBP, other HATs Is a
co-activator Promotes or represses Pol II
transcription
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E1A Inhibition of AR Targeted Gene
Mock (-R1881) Control vector (-R1881) Control
vector (R1881) E1A plasmid (R1881)
- - - - - - - - -
- - - - - - - - - -
- - - - - - - - -
- - - - - - - - - - -
- - - - - - - - - -
- - - - - - - - -
- -
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E1A Inhibition of Various Prostate Specific
Promoters
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Model of E1A Inhibition of AR
p400
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Oncolytic Effect of E1A Inhibition of AR
E2-4
L1-5
E1B
Attenuated Replication
A
AR
Prostate Specific TFs
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Dilemma

• E1A and AR mutually inhibit each other
• AR required for prostate specific promoters
• ALL Prostate specific oncolytic adenoviruses
  attenuated
• Net Effects
• Lack of androgen induction of oncolytic activity
  of CV706.
• Decreased potency/efficacy

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Androgen Receptor E1A Chimera
E1A-Androgen Receptor Chimera
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RLU ( Renilla Activity)
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Additional Issue

• Most patients with locally advanced or metastatic
  prostate cancer are treated with androgen
  withdrawal (e.g., non-steroidal anti-androgen)
• Can the E1A-AR chimera be made inducible by an
  anti-androgen?
• ? Single point mutation in LBD

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(No Transcript)
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AR is functional in E1A-AR chimera (reporter
PSE-PBN-luc in PC3 cells)
R1881 - - -
- -
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E1A is Functional in E1A-AR Chimera (reporter
pE1B-luc in PC3 Cells)
R1881 - - -
- -
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Summary

• Projects 3 and 4 are progressing well
• We have met all of our Year 2 milestones
• Multiple manuscripts are either in preparation,
  submission, or already accepted
• Initial Ad5E4orf6 paradigm not tenable, but a
  better alternative, in the same spirit of
  radiosensitization has been identified and
  progressing nicely
• DOD consortium allows flexibility to change SAs
  to fit the group needs.
• Funding not really to pay for experiments (not
  nearly enough for that), but rather to foster the
  strong interactions leading to facilitated
  therapeutic development and testing.