AntiCyclin D1 Gene Therapy for Mantle Cell Lymphoma

1
Anti-Cyclin D1 Gene Therapy for Mantle Cell
Lymphoma

• Joya Mukerji
• MOL 523
• April 30, 2004

2
Overview

• 1. What is mantle cell lymphoma (MCL)?
• (Epidemiology and lymphomagenesis)
• 2. How is MCL currently treated?
• 3. How could anti-cyclin D1 gene therapy be
  accomplished? How would it potentially benefit
  MCL patients?

3
What is mantle cell lymphoma? (I)

• Non-Hodgkins lymphomas rank 6th among the
  causes of cancer-related deaths in the US (23,400
  deaths in 2003).
• MCL is an aggressive malignancy of B-cells that
  comprises 5-10 of all non-Hodgkins lymphomas.
• MCL is typically diagnosed in individuals age 60
  or over, with disseminated disease
• Median survival 36 months
• No permanent remission

Bertoni et al., 2004. British Journal of
Haematology. v. 124. ACS Cancer Facts Figures,
2003
4
What is mantle cell lymphoma? (II)

• Aberrant B-cells are initially located in the
  mantle layer surrounding the germinal center of a
  lymph node.
• Tumor cells subsequently impinge on the structure
  of the lymph node and invade the germinal center.
• Common extranodal sites
• spleen, liver, and bone marrow

Bertoni et al., 2004. British Journal of
Haematology. v. 124. http//pleiad.umdnj.edu/hem
epath/lymph_intro/lymph_intro.html
5
Conventional Treatment for MCL

• Chemotherapy is the standard treatment for MCL.
• Surgery is rarely applicable
• (disseminated disease diffuse tumors)
• Radiation is a limited supplement to chemotherapy
  

• Stem cell transplantation following CHOP can
  extend progression-free survival, but patients
  relapse.

• R-CHOP chemotherapy
• Rituximab (Rituxan)
• anti-CD20 antibody
• Cyclophosphamide
• DNA alkylator
• Hydroxydoxorubicin (adriamycin)
• inhibits topoisomerase II
• Oncovin (vincristine)
• prevents microtubule polymerization
• Prednisone
• anti-inflammatory steroid

Lenz et al., 2004. Ann Hematol. v. 83.
6
Postulated Sequence of Molecular Changes in MCL

• Failure to recognize dsDNA breaks
• Impaired checkpoint activation following IR
• (G1/S and G2/M checkpoints via p53 and c-Abl)

Lack of ATM function ? improper recombination
during the process of generating antibody
diversity

• Juxtaposes CCND-1 with IgH heavy-chain promoter ?
  Constitutive expression of cyclin D1

• Various changes (ex inactivation or loss of
  p16, p21, p27, p53 overexpression of bcl-2)

Compiled from Decaudin, 2002. Leukemia and
Lymphoma. v. 43 (4).
7
Cell Cycle Deregulation in MCL
p16INK4a
Rb-E2F (inactive)
Cyclin D-CDK4/6
S phase
Rb-P E2F (active)
Myc
p19ARF
t(1114) (q13q32)
Cyclin E-CDK2
Mdm2
Apoptosis
p53
p27 p21
Bcl-2
Ub
Modified from Lowe and Sherr, 2003. Curr Opin
Genet Dev. v. 13.
8
Visualization of t(1114)(q13q32) by
fluorescence in situ hybridization
The t(1114)(q13q32) translocation is detectable
in 90-100 of MCLs.
Chr. 14
Chr. 11
CCND1 and IgH loci detected via FISH. Probe
signals are co-localized at translocation
breakpoints (arrows).
CCND1
IgH locus
Elnenaei et al., 2001. Leukemia Res. v. 25 (2)
Modified from Bertoni et al., 2004. British J
Hematol. v. 124
9
Novel Therapeutic Strategy
Treat MCL patients with B-cell-tropic
Epstein-Barr vector virions encoding small
interfering RNAs (siRNAs) against cyclin D1.
10
Hairpin siRNAs to be encoded in the B-cell
tropic vector

• Hairpin RNAs, with sequences specific for target
  regions of the cyclin D1 transcript, are
  transcribed from the vector.
• The hairpin, included for stability, is cleaved
  off.

Hairpin RNA

• The short duplex RNAs that remain are
  incorporated into RISC complexes (RNA-induced
  silencing complexes, triggered by dsRNA).
• As components of the complex, single strands of
  these short RNAs base-pair with the cyclin D1
  transcript, facilitating cleavage of the
  transcript into short RNA fragments (22nt in
  length) by the two dicer enzymes contained in
  each RISC complex.

Cleaved RNA

• Thus, siRNAs prevent translation of cyclin D1.

Brummelkamp et al., 2002. Cancer Cell. v. 2
Hannon, 2002. Nature. v. 418
11
Epstein-Barr virus (EBV), a B-cell-tropic
Herpesvirus

• Wild-type Epstein-Barr Virus (EBV) establishes
  latency in B-cells and can subsequently
  reactivate its lytic cycle.
• EBV is a herpesvirus that causes mononucleosis
  and is an etiological factor in Burkitts
  lymphoma.

Image courtesy of Prof. Enquist, MOL 459
12
Creating the vector to introduce hairpin siRNAs
against Cyclin D1 into MCLs
Strategy for Delivery of siRNAs

• Clone into a vector
• -- sequence encoding LacZ and siRNAs

• Transfect this vector construct into the Raji
  cell line to package in virions

• Deletions in the EBV genome of Raji
• -- packaging and lytic functions
• -- LMP-1 and EBNA-2 and -3

Robertson et al., 1996. PNAS. v. 93

• Assay for blue plaques on X-GAL

• Administer these recombinant vector virions to
  patients intravenously.

13
Effect of anti-cyclin D1 therapy

• MCL cells are overwhelmingly reliant upon cyclin
  D1-CDK4/6 complexes to mediate the G1?S
  transition.
• Therefore, depletion of cyclin D1 levels via
  siRNA would be expected to cause G1-arrest. The
  B-cells that comprise MCL tumors would not be
  able to pass the restriction point.

14
Assessment and Considerations

• Rationale
• Constitutive expression of cyclin D1 due to
  t(1114) is a driving force in 90-100 of MCL
  cases.
• Tumor cells could be arrested via anti-cyclin D1
  siRNA.

• Safety
• The EBV vector cannot reactivate, as it lacks
  lytic genes.
• Transformation-inducing genes are also absent.

• Specificity
• EBV capsid and membrane proteins confer B-cell
  tropism.

• Preventing resistance
• Employ multiple siRNA sequences, specific for
  regions of the cyclin D1 transcript that encode
  domains essential to the function of cyclin D1
  (ex CDK-binding domain).

15
Summary
Given the crucial role of cyclin D1
overexpression in MCL, the novel approach of
targeting anti-cyclin D1 siRNAs to B-cells could
represent an advance in the degree of molecular
specificity that is attainable in MCL therapy.
16
(No Transcript)
17
An alternative strategy Liposomal delivery to
mediate cell cycle arrest and apoptosis

• Co-administration of PS-341 (Bortezomib) could
  induce apoptosis of cells arrested by siRNAs
  against cyclin D1.
• Liposomes targeted to MCL cells via antibodies
  specific for CD5 could deliver siRNAs and PS-341,
  a proteasome inhibitor

18
Inducing apoptosis via proteasome inhibition

• PS-341 has been shown to induce G1 arrest and
  apoptosis in MCL cell lines
• PS-341 treatment leads to decreased expression of
  bcl-2 family members and increased caspase-3
  activation.

• PS-341 binds to the active site threonine residue
  of the ß subunit of the 26S proteasome
• Binding is reversible, and cancer cells are more
  sensitive to the effects of PS-341

Adams, 2002. Curr Opin Chem Biol. v.6 (4).
Millennium Pharmaceuticals, Inc.,
Pham et al., 2003. J Immunol. v.171 (1).