CLICK
FOR ABSTACT
James Herman, M.D. || Marie
Hardwick, Ph.D. ||Robert
Brodsky, M.D.
Yiping Yang, M.D., Ph.D.
Methylation PCR in Serum from Lymphoma Patients
Principal Investigator: James Herman, M.D.
Background
And Preliminary Data:
Transcriptional Silencing and Methylation: Genetic alterations are a
hallmark of human cancer. Changes in DNA methylation, an epigenetic
modification present in mammalian cells, are also characteristic of
human cancer. The CpG dinucleotide is clustered in the promoter regions
of some genes. These promoter regions are called "CpG islands";
CpG islands are protected from methylation in normal cells with the
exception of genes on the inactive X chromosome and imprinted genes.
Methylation of promoter region CpG islands is associated with loss of
expression of these genes. Promoter hypermethylation commonly silences
tumor suppressor genes in human cancer, serving as an alternative mechanism
for loss of tumor suppressor gene function. The p16 gene (CDKN2A) is
a cyclin dependent kinase inhibitor that functions in the regulation
of the phosphorylation status of the Rb protein which is frequently
hypermethylated in carcinomas1-4 and lymphomas5-7. p15 (CDKN2B) also
regulates the phosphorylation of Rb. Its proximity to CDKN2A leads to
frequent co-deletion with p16 in the homozygous deletions observed at
this locus. However, unlike CDKN2A, it is rarely inactivated by promoter
region hypermethylation in most epithelial tumors with the exception
of many forms of hematological malignancies including acute myelogenous
leukemia and acute lymphoblastic leukemia5,8-11, Burkitt's lymphoma6,8,
and in the progression of myelodysplasia to acute leukemia12,13. p15
and p16 alterations in hematological malignancy have been recently reviewed7.
Utility
of determining methylation of individual genes: Genes involved in apoptotic
pathways, are inactivated by promoter region methylation in cancer.
We found that the death associated protein kinase (DAP kinase) is methylated
in the majority of B-cell lymphomas14 and other malignancies15. Hypermethylation
of the DAP kinase promoter was associated with decreased patient survival,
suggesting an important role in determining the biologic aggressiveness
of early-stage NSCLC16.
The DNA-repair
enzyme O6-methylguanine-DNA methyltransferase (MGMT) inhibits the killing
of tumor cells by alkylating agents. Methylation of the MGMT promoter
silences this gene in cancer. We recently examined whether methylation
of the MGMT promoter in gliomas is related to the responsiveness of
the tumor to alkylating agents17. MGMT methylation was an independent
and stronger prognostic factor than age, stage, tumor grade, or performance
status. The pathogenesis of diffuse large B-cell lymphoma (B-DLCL) is
a heterogeneous process involving multiple molecular pathways. MGMT
inactivation by transcriptional silencing due to promoter hypermethylation
is a mechanism for loss of MGMT activity in B-DLCL18. 30 of 84 (36%)
B-DLCL had MGMT promoter hypermethylation. The presence of MGMT methylation
in lymphomas was associated with a significant increase in overall survival
(hazard ratio 2.8, 95% CI, 1.2 to 7.5, p=0.01) and progression-free
survival (hazard ratio 2.6, 95% CI, 1.3 to 5.8, p=0.005). MGMT promoter
hypermethylation was both independent of and stronger than established
prognostic factors, such as age, stage, serum LDH and performance status.
The effects of MGMT promoter methylation on survival were also independent
from the international prognostic indicator (IPI). (JNCI, in press,
Appendix).
Promoter region methylation is a powerful molecular tool: We have recently
shown that aberrant methylation of gene promoter regions is a common
event in all forms of malignancy15. We analyzed a series of promoter
hypermethylation changes in 12 genes (CDKN2a/p16INK4a, p15INK4b, p14ARF,
p73, APC, BRCA1, hMLH1, GSTP1, MGMT, CDH1, TIMP3, and DAPK) in DNA from
> 600 primary tumor samples representing 15 major tumor types. The
genes play important roles in tumor suppression, cell cycle regulation,
apoptosis, DNA repair, and metastasis. The hypermethylation of these
genes occurs independently to the extent that a panel of three to four
markers defines an abnormality in 70-90% of each cancer type. Promoter
methylation represents a powerful set of markers to outline the disruption
of critical pathways in tumorigenesis and for derivation of sensitive
molecular detection strategies for virtually every human tumor type.
In lymphoma, the most frequently hypermethylated genes are: DAP-kinase,
MGMT, p16, p14 and p15.
Promoter
methylation as a tumor detection marker: DNA is released and can be
detected in the serum of patients with cancer. The level of this DNA
has been somewhat correlated to tumor burden. Tumor specific alterations
in DNA can be detected and the specificity of these changes allows the
separation of DNA from tumor and normal tissue. We have developed and
sensitive and specific method of detection for methylation changes19.
Promoter region methylation has been detected in the serum of patients
with lung, head and neck, breast, and colon cancer20-22. The presence
or level of this tumor specific change may correlate with disease status
and a reduction in tumor DNA may reflect tumor response. In a recent
study of esophageal cancer, hypermethylated APC DNA was observed in
the plasma of 25% adenocarcinoma patients and high plasma levels of
methylated APC DNA were statistically significantly associated with
reduced patient survival23. Similar results have been reported for non-small
cell lung cancer24.
Multi-gene
analysis: To analyze a number of genes and to work with samples with
limited amounts of DNA from formalin-fixed paraffin-embedded DNA we
have developed a multiplex nested MSP approach. The sensitivity of this
approach has been enhanced approximately 50 fold. The specificity of
MSP is maintained during this modification. We have already combined
the external primers for 17 candidate genes and tested the specificity
of MSP for these genes. All genes retained the specificity of the original
MSP procedure (example Figure 1).
Figure
1. Methylation specific PCR for the p15 and p16 genes following muliplex
nested approach. Normal lymphocyte DNA (Nl) shows no methylation of
these genes, as shown by the lack of PCR product in lanes labeled M,
while visible signal is present in the U lane. Control DNA demonstrates
amplification in the M lane. Methylated control DNA was mixed with normal
DNA at the ratio shown above, and M signal is positive for all dilutions,
even to 1:100000 (that is 10 pg methylated DNA mixed in 1 microgram
of normal lymphocyte DNA), demonstrating the sensitivity and specificity
of this approach.
Overall Approach/Study Design: This will be prospective cohort study.
Detection of aberrant methylation of promoter regions by methylation-specific
PCR will be used. A multiplex-nested MSP analysis as described above
will be employed. Tumor specimens will be used to determine a methylation
profile of each tumor. Serum will be collected during routine blood
sampling during clinic visits. CT scans will be used to determine response
to therapy or relapse. DNA extracted from serum or plasma assayed to
determine the presence or absence of tumor specific DNA alterations
in the patient’s blood. Molecular results will be correlated with
disease state
Top
of Page
Bcl-2 Point Mutations
Principal Investigator: Marie Hardwick, Ph.D.
Introduction
and Rationale
The bcl-2 oncogene was first identified at t(14;18) translocation breakpoints
that occur in the majority of individuals with follicular B cell lymphomas
(22,23). This translocation event results in the overexpression of bcl-2
that allows B cells to survive when they would normally die by apoptosis
(12,14). Overexpression of the Bcl-2 protein protects a wide variety
of cell types from many death-inducing stimuli including growth factor
withdrawal/axotomy, treatment with calcium ionophores, glucose withdrawal,
membrane peroxidation, glucocorticoid treatment, chemotherapeutic agents,
and virus infection, implying that BCL-2 functions at the point of convergence
of many different signals (1). A role for Bcl-2 as a true physiologic
inhibitor of cell death in animals is supported by studies with knockout
mice. In Bcl-2-deficient mice, the immune system starts to develop normally,
but massive apoptosis in the spleen and thymus occurs subsequently (10,16,17,24).
More recently, altered expression of the BCL-2 protein has been described
in a wide range of human cancers, without the occurrence of chromosome
translocations. Additionally, a body of evidence indicates that elevated
BCL-2 expression causes resistance to chemotherapeutic treatments while
suppressed BCL-2 expression correlates with apoptosis in response to
anticancer agents (18). Therefore, anticancer strategies that target
BCL-2 by anti-sense mechanisms or peptides/drugs that bind in the cleft
of BCL-2 are being explored. How does Bcl-2 block apoptosis? The biochemical
mechanism by which BCL-2 inhibits apoptosis is still not fully understood
but BCL-2 is generally believed to work at the mitochondrial membrane
where it prevents mitochondrial damage that otherwise leads to leakage
of prodeath mitochondrial factors into the cytosol and eventual disruption
of mitochondrial membrane potential (3).
We have
shown that endogenous BCL-2 is cleaved in the loop domain near its N-terminus
by caspase-3 during programmed cell death in a variety of cell types
(2). The resulting C-terminal cleavage product localizes to mitochondria,
induces the release of cytochrome c and is potently pro-apoptotic (2,11).
It has been suggested that cleavage of BCL-2 is important in chemotherapy-induced
death of leukemia/lymphoma cells (7,8). This phenomenon where prodeath
activity is induced or enhanced following proteolysis is now known to
occur for many BCL-2 family members including BCL-xL, BID, BAX and BAD
(4,9,13,20). Sequencing of six cell lines and tumors derived from non-Hodgkin’s
lymphomas revealed that one of these contained a point mutation in the
caspase cleavage site at Asp34 as well as other mutations (19,21). The
aspartate residue at the P1 position (amino acid 34) is absolutely required
for caspase cleavage. Therefore, the BCL-2 protein in this tumor cell
would not be susceptible to caspase cleavage. Mutation of the caspase
cleavage site in BCL-2 is known to significantly enhance the antiapoptotic
function of BCL-2 rendering these cells less susceptible to a variety
of death stimuli including ionizing radiation (2,4). Therefore, we propose
to examine the sequence of BCL-2 found in a bank of lymphoma samples
to explore the possibility that modified susceptibility of BCL-2 to
proteolysis contributes to lymphoma. In particular we will focus on
relapsed patients are these are deemed most likely to contain such mutations.
Top
of Page
Augmentation of antibody-mediated complement dependent cytotoxicity
in lymphoma.
Principal
Investiagtor: Robert Brodsky, M.D.
Monoclonal antibodies are effective in the treatment of a wide variety
of lymphoproliferative disorders and autoimmune diseases. It is now
recognized that monoclonal antibodies kill cells through a combination
of complement-mediated cytotoxicity, antibody-dependent cellular toxicity,
and apoptosis.1-3 Recent data demonstrate that a major cause of resistance
to monoclonal antibody therapy is an upregulation of the complement-regulatory
proteins, CD59 and CD55.2;4
The human
disease paroxysmal nocturnal hemoglobinuria (PNH) is clonal hematopoietic
stem cell disorder that leads to a marked deficiency or absence of the
glycosylphosphatidylinositol (GPI) anchored complement regulatory proteins,
CD55 and CD59.5 Our preliminary data demonstrates that EBV-transformed
lymphocytes from PNH patients are markedly more sensitive to monoclonal
antibody therapy and that selective biochemical removal of GPI-anchored
proteins greatly increases complement-mediated cytotoxicity in non-PNH
lymphoma cell lines. Thus, we propose to investigate molecular, biochemical,
and pharmacologic strategies to interrupt GPI-anchored proteins in order
to augment antibody-mediated complement dependent cytotoxicity in lymphomas.
Specific
Aims:
1. Disruption
of GPI-anchor protein expression will greatly increase the sensitivity
of lymphomas to monoclonal antibody therapy.
2. Establish preclinical models to test the efficacy of GPI-achor ablation
in treating lymphomas.
Hypothesis 2.1: Lymphomas devoid of GPI-anchors will be more vulnerable
to monclonal antibody therapy than GPI-anchor replete lymphomas.
Hypothesis 2.2: PIPLC can safely and transiently remove GPI-anchored
proteins in bone marrow specimens resulting in more effective purging
with monoclonal antibodies.
Top
of Page
Antigen-defined Immunotherapy for Lymphoma: The Use of Lentivirus transduced
Dendritic Cells Matured ex vivo or in vivo
Principle Investigator: Yiping Yang, M.D., Ph.D., Co-Investigator: Drew
Pardoll, M.D., Ph.D.
Background,
Rationale And Specific Aims
The major barrier to cancer immunotherapy has been immune tolerance
to tumor antigens (1). A potent vaccine capable of inducing effective
anti-tumor immunity is desired to reverse immune tolerance to these
tumor antigens. Dendritic cell (DC)-based vaccines have emerged as a
promising approach for eliciting effective anti-tumor immune responses.
To be effective, these peptide, protein or RNA loaded DC, or virally
transduced DC have to be administered before or a few days after tumor
challenge (2-5). This appears to be due, in part, to inefficient loading
of tumor antigens (via retrovirus or RNA), or lack of CD4 T helper function
in the case of CD8 peptide loading or lack of sustained antigen presentation
by DC (via protein loading). These factors limit the effective and sustained
stimulation of antigen specific T cells by DC. In an attempt to improve
DC-based vaccine strategies, we have developed a different approach
to allow more efficient tumor antigen presentation by lentivirus-transduced
DC matured ex vivo or in vivo in a murine model of A20 B cell lymphoma
using HA as a model tumor antigen. HA behaves like a natural tumor antigen
in a number of tumor models such as the A20 lymphoma, in that moderate
levels of HA expression do not alter the biology, immunogenicity, or
in vivo growth characteristics of the tumor (6-9). In particular, the
groups of Dr. Pardoll and Dr. Levitsky at Johns Hopkins have extensively
utilized the HA antigen and the pair of MHC class I (Kd) and II (I-Ed)
restricted, HA specific TCR transgenic mice to address fundamental mechanisms
of immunoregulation.
The goal of this application is to explore the utility of lentivirus-HA
transduced DC matured ex vivo or in vivo in eliciting effective sustained
antigen-specific anti-tumor immunity. In particular, we will evaluate
whether vaccination with mature DC transduced with self-inactivating
(SIN) lentiviral vectors (ref. 10) expressing HA generates effective
antigen-specific T responses to “break tolerance” in an
HA tolerant transgenic mouse model. In parallel, we will explore a novel
approach to allow efficient tumor antigen presentation by DC derived
from hematopoietic stem cells (HSC) transduced with lentivirus HA and
selectively activated in vivo with a drug inducible DC activator. Success
in enhancement of antigen specific T cell activation against tumor cells
by these novel approaches will lead to clinical trials in treating antigen-defined
tumors such as EBV (+) Hodgkin’s lymphoma. We will focus on the
following specific aims:
1) To evaluate the ability of lentivirus-HA transduced DC vaccine to
“break tolerance” in HA tolerant transgenic mouse models.
2) To investigate whether lentivirus-HA transduced HSC-derived DC selectively
activated in vivo with a drug inducible DC activator, induce antigen
specific T cell activation in mature T cells.
3) To assess anti-tumor efficacy of these different approaches in murine
prevention and treatment models using the well characterized HA-expressing
A20 lymphoma
Top
of Page
