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| 1. | Mechanism of action of relatively low-dose radiation. The relationship between radiation dose rate and cytotoxicity is complex, with data indicating hypersensitivity at very low dose rates in both tumor and normal tissues (Hernandez MC and Knox SJ. Int J Radiat Oncol Biol Phys 59: 1274–87, 2004). The extent and mechanism remain to be fully elucidated. The initial observations of the inverse dose-rate effect were established by Mitchell JB and colleagues, Radiation Biology Branch (Radiat Res 79: 520–36, 1979). Microarray studies of low dose-rate gene induction are planned (Brechbiel M and Chuang E, Radiation Oncology Branch). | |
| 2. | Mechanisms of cell killing by radioisotopes. Lymphocytes undergo intermitotic cell death by apoptosis. Radiation modifiers can be used to enhance this. Indeed, radiosensitizers such as gemcitabine and taxol have been investigated in pre-clinical settings (DeNardo GL et al. Cancer 94: 1332–48, 2002; Gold DV et al. Clin Cancer Res 9: 3929s–37s, 2003). Other death mechanisms may be involved in epithelial cells. Thus, combined modality therapy with molecularly targeted drugs plus biologically targeted radiation provides new therapeutic opportunities. | |
| 3. | Optimization of radioisotope/carrier complex (Lin MZ et al. Clin Cancer Res 11: 129–38, 2005) including combined modality therapy. Selecting and designing the optimal molecule and linker and performing systematic pre-clinical studies and clinical trials are critical (Milenic DE et al. Nat Rev Drug Discov 3: 488–99, 2004; Milenic DE et al. Clin Cancer Res 10: 7834–41, 2004). [See also “Radioimmunotherapy of Disseminated Peritoneal Disease Targeting HER2” in this issue.] Non-radioactive antibody studies are ongoing (Waldmann TA, Metabolism Branch; Morris JC, Metabolism Branch; Pastan I, Laboratory of Molecular Biology) including combined chemotherapy and antibody therapy for lymphoma (Wilson W, Medical Oncology Branch). | |
| 4. | “Inverse Planning” for systemic radiotherapy. Currently, an antibody or fragment with a single radioisotope has been developed. The dose that a tumor receives depends on the biodistribution, targeting characteristics, and type of radioactive decay. A new concept is to better understand tumor physiology and to prepare a delivery vector with an isotope mix (α, β–) that delivers a dose specifically tailored to the disease. | |
| 5. | Radiation enhancement of vaccine therapy (Koski GK and Czerniecki BJ. Clin Cancer Res 11: 7–11, 2005). Radiation can enhance the ability to vaccinate tumors, as demonstrated by the Laboratory of Tumor Immunology and Biology (LTIB) (Chakraborty M et al. J Immunol 170: 6338–47, 2003; Chakraborty M et al. Cancer Res 64: 4328–37, 2004). Clinical trials are in progress using radiation therapy plus prostate-specific antigen (PSA) vaccine for patients with prostate cancer (Gulley J, LTIB; Arlen P, LTIB; Singh A, Radiation Oncology Branch; Camphausen KA, Radiation Oncology Branch; Schlom J, LTIB). | |
| 6. | Normal tissue toxicity from “moderate dose” radiation (1 to 10 Gy). The dose-limiting toxicity from STaRT is usually to bone marrow or kidney; lung injury is observed at moderate radiation doses with transplantation regimens. Clinical effects may occur months or even years later. Newer external beam radiation therapy techniques such as intensity-modulated radiotherapy (IMRT) enable the delivery of a higher dose to the tumor and dose escalation within the target. However, there is additional scattered dose to normal tissues, some within the 1 to 10 Gy range over a course of therapy. This moderate dose radiation is of interest in radiological/nuclear terrorism, with a major effort under way to develop countermeasures to radiation injury at these doses (Coleman CN et al. Radiat Res 159: 812–34, 2003; Stone HB et al. Radiat Res 162: 711–28, 2004), including establishing new Centers for Medical Countermeasures to Radiation (a National Institute of Allergy and Infectious Diseases [NIAID] and NCI program). | |
| 7. | Radiation-inducible molecular targets. Novel studies are in progress (Coleman CN, Chuang E, Mitchell JB, Radiation Oncology Branch and Radiation Biology Branch) investigating the potential role of radiation for inducing molecular changes to increase tumor susceptibility to molecularly targeted therapies (Hallahan D et al. Cancer Cell 3: 63–74, 2003). In this method, a radiation dose and schedule would be chosen for its desired molecular effect rather than just a toxicity-based regimen as is currently used. |
Because the technological and anatomical aspects of radiation oncology are critical to patient care, particularly with improved imaging and the ability to deliver highly targeted radiation with external beam or brachytherapy (radioactive implants), a great emphasis of the field has been on technology and imaging. STaRT advances this trend because a systemically administered isotope can be focused on the basis of pharmacology and physical properties. The biological effects of STaRT on cytotoxicity, induction of molecular processes, immune enhancement, sensitization to molecularly targeted therapies, and normal tissue toxicity require the skill and knowledge of radiation oncologists. Multimodality teams are required to optimize this approach, and it seems to us that the CCR is a great place for STaRT.
enetic
mutations that lead to cancer often result in the expression of novel tumor-associated
antigens (TAA) that can be targeted by the immune system. A successful immune
response to a tumor requires the cooperation of antigen-presenting cells that
process and present antigens to T cells as short peptide fragments bound to
the major histocompatability (MHC) molecules on their surface. These peptides
are recognized by receptors on specific T cells that are then activated to orchestrate
an immunological “attack” on the tumor. Dendritic cells (DC) are
powerful antigen-presenting cells. They express high levels of MHC and other
co-stimulatory molecules, and they secrete the required cytokines that sustain
and direct the immune response after the initial T-cell activation. As a result,
DC are potentially the ideal cells for presenting TAA to vaccinate against cancer.
Tumors, however, attempt to escape the immune response by secreting immunosuppressive
factors that inhibit DC maturation and decrease antigen presentation. These
defects can be overcome by growing DC in culture. DC can be pulse-loaded with
target antigen using peptides, or by incubating them with extracts made from
tumors, and used to vaccinate patients.
We hypothesized that the introduction of a gene encoding a TAA into DC using a recombinant viral vector might be beneficial. Potential advantages include the following: (1) There is no requirement for knowledge of the type of MHC molecule expressed, the peptide sequence, or its MHC binding affinity. (2) Gene transfer can provide larger antigenic sequences with more potential targets. (3) Natural antigen processing by DC may improve antigen presentation. (4) Constitutive expression of the antigen may allow for continuous replenishment of low-affinity TAA peptides as they are lost from MHC molecules, and (5) viral proteins expressed by the gene transfer vector may provide signals required for maturation, activation, and increased expression of co-stimulatory molecules on the DC, resulting in a stronger immune response.
We used the neu oncogene, the rodent homolog of the human HER-2/neu gene as our targeted TAA. HER-2/neu, an epidermal growth factor receptor family member is overexpressed in cancers of the breast, ovary, uterus, lung, and gastrointestinal tract and is associated with treatment resistance and a poorer clinical outcome. It is a therapeutically important immunological target as evidenced by traztuzumab (Herceptin), a humanized anti-HER-2/neu antibody approved for the treatment of breast cancer.
We generated a recombinant adenovirus expressing the extracellular and transmembrane domains of the neu oncogene (Ad.Neu). Studies using mouse bone marrow–derived DC showed that infection and significant neu antigen expression were achieved using our vector (Figure 1). In addition, viral infection increased the surface expression of MHC and co-stimulatory molecules, indicating maturation and activation of the DC. We studied the effectiveness of genetically modified DC vaccination using BALB-neu T transgenic mice. These mice express a neu gene controlled by a mammary-specific promoter. Female mice develop breast tumors at 14 to 15 weeks of age, and progress until all mammary glands are associated with tumors at 24 to 25 weeks. We found that three weekly vaccinations using one million DC modified with Ad.Neu (DCAd.Neu) prevented or delayed the onset of breast tumors compared with mice vaccinated with DC infected with a control vector (DCAd.null), or with unmodified DC alone. DCAd.Neu-vaccinated mice had significantly improved disease-free survival and a reduction in the average number of tumors that appeared. Vaccinated mice free of tumor at 28 weeks were challenged with injections of syngeneic neu+ or neu– tumor cell lines. The mice were protected from growth of the neu+ tumor cells, but not neu– breast cancer cell lines, indicating that immunity was specific for the target antigen. Mice vaccinated with DCAd.Neu had significant increases in anti-neu antibody titers. Surprisingly, induction of tumor-specific CD8+ cytolytic T lymphocytes (CTL) could not be demonstrated, suggesting that the protection was mediated by the development of antibodies and not CTL. On depleting specific immune cell populations, we found that CD4+ T cells, but not CD8+ T cells, played a critical role in the immune response to our vaccine, supporting our hypothesis. Since most adults have antibodies to adenoviruses that might influence vaccine efficacy, we hypothesized that our strategy presented tumor antigens as peptides that would be unaffected by circulating antibodies to adenovirus. Indeed, we found that DC vaccination was equally effective in mice with preexisting immunity to adenovirus.
Figure 1. Mouse bone marrow–derived dendritic cells demonstrating high levels of green fluorescence after infection with an adenovirus vector expressing enhanced green fluorescent protein (GFP).
Further collaborative work has shown that unlike traztuzumab, whose efficacy depends on the presence of the Fc receptor (FcR), the antibodies induced by the Ad.Neu vaccine protected mice by an FcR-independent mechanism and inhibited the growth of tumor cells in vitro. Therefore, we expect that these antibodies were acting directly through the HER-2/neu receptor on the tumor cells to inhibit growth. Several important questions remain to be answered. DCAd.Neu vaccination was effective in the BALB-neu T model when the mice were less than 7 to 8 weeks of age and prior to the appearance of tumors. Vaccination was less effective in older mice and ineffective once tumors appeared. Increasing the number of DCAd.Neu vaccinations only slightly increased survival once tumors developed. The reason for this is not clear, but could be the result of homeostatic or tumor-specific mechanisms that downregulate the immune response. We plan to examine strategies to overcome the resistance of older mice to anti-neu vaccination, including depletion of T regulatory cell populations and genetically modifying DC with immunostimulatory cytokines.
he
ends of linear eukaryotic chromosomes consist of telomeres that contain telomeric
DNA repeats, (TTAGGG)n hexanucleotide repeats
in mammalian chromosomes, and a number of associated proteins. This telomeric
structure is critical for distinguishing the chromosomal terminus from free
ends of damaged DNA, and thus, telomeres prevent the triggering of inappropriate
cell cycle arrest and/or apoptotic responses normally elicited by DNA damage.
In eukaryotic cells, the mechanism of chromosomal replication during cell division
results in incomplete terminal synthesis, so that in the absence of a compensatory
mechanism, 50–200 bases of terminal telomeric DNA are lost with each division.
Thus, successive cycles of cell proliferation can lead to progressive telomere
shortening, until a critically short length is reached at which telomere function
is compromised, with consequences that can include replicative senescence, apoptosis,
and tumorigenic chromosomal instability. A compensatory mechanism capable of
adding terminal telomeric repeats is mediated by the RNA-dependent DNA polymerase,
telomerase. This enzyme consists of two essential molecular components, the
telomerase RNA (TR) component, which includes a template for telomeric
DNA, and the catalytic telomerase reverse transcriptase
(TERT), which mediates telomere synthesis. Importantly, recent discoveries have
demonstrated that maintenance of telomere function is also dependent on the
influence of additional telomere-associated proteins, and elucidating the function
of these proteins is, therefore, an area of considerable interest.
TIN2 (TRF1-interacting protein 2) was recently identified as a telomere-associated
protein that interacts with TRF1, a molecule that binds directly to telomeric
DNA and functions as a negative regulator of telomere length. TIN2 contains
N-terminal basic and acidic regions, a central TRF1-binding domain, and a C-terminal
region. The basic and acidic regions are required for the regulation of TRF1
activity by TIN2. The TRF1-binding domain associates with the TRF1-homodimerization
domain, providing for the recruitment of TIN2 to the telomere. In vitro
studies have shown that overexpression of TIN2 inhibits telomere elongation
in human cell lines, whereas expression of dominant-negative mutants of TIN2
enhances telomere elongation. It has been suggested that the binding of wild-type
TIN2 induces changes in TRF1 conformation that in turn favor a telomeric structure
that is inaccessible to telomerase, thus preventing telomerase-mediated telomere
elongation. The absence of TIN2 would conversely favor telomerase accessibility
and telomere elongation.
The physiological role of TIN2 during in vivo development and in normal cell function had not previously been assessed. To better understand the in vivo function of TIN2, we have, therefore, studied the effect of TIN2 mutation on mouse development, using gene-targeting technology. No homozygous TIN2–/– mice were identified in the offspring of TIN2+/– mouse intercrosses. Furthermore, homozygous TIN2-deficient embryos were absent as early as day 7.5. This finding indicated that TIN2 is essential for mouse development and that homozygous inactivation of TIN2 is lethal before day 7.5 of embryonic development. However, day 3.5 TIN2–/– embryos were obtained in expected frequency (1/4) among offspring of TIN2+/– intercrosses. When day 3.5 TIN2–/– embryonic cells were cultured, it was striking that they were uniformly defective in their differentiation, in comparison to day 3.5 wild-type embryonic cultures. Wild-type embryonic cultures grew to form multilayered cell masses, whereas TIN2–/–embryonic cultures were flat and contained few viable cells. A growth and/or survival defect was thus apparent in TIN2–/– cells at an early stage of embryonic development.
The previously identified function of TIN2 was proposed to involve enhancing the activity of TRF1 in downregulating the telomerase elongation of telomeres. We asked whether the embryonic lethality observed in TIN2–/– mice might be telomerase dependent. To explore this possibility, TIN2+/– mice were bred to mTERT–/– mice that lacked telomerase activity. It was striking that no TIN2–/– mTERT–/– offspring were observed, whereas TIN2+/+ mTERT–/– and TIN2+/– mTERT–/– mice survived. Thus, embryonic lethality of TIN2–/– mTERT–/– mice indicated that the requirement for TIN2 in mouse development reflects a previously unappreciated telomerase-independent function of this molecule.
Recently, it was reported that inactivation of the mouse TRF1 gene results in embryonic lethality, and that TRF1 knockout blastocysts have a cell growth defect and increased apoptosis. The phenotype of TIN2 knockout mice thus appears to be similar to that of TRF1-deficient mice. These observations imply that, in addition to the telomerase-dependent functions played by TIN2/TRF1 complexes, both TIN2 and TRF1 also function in telomerase-independent roles. To understand the telomerase-independent roles of TIN2 and TRF1 in embryonic development and in adult animals, studies of inducible TIN2 or TRF1 conditional knockout mice will be informative. We have in fact generated TIN2 conditional knockout constructs using cre/loxP techniques and will use these constructs in studies of inducible and tissue-specific TIN2 inactivation. Additional telomere-associated proteins may be involved in the potentially complex functions of TIN2 and TRF1, and we are currently pursuing genetic approaches to analyze candidate components involved in these functions.
If you have scientific news of interest to the CCR research community, please contact one of the scientific advisors (below) responsible for your areas of research.
|
Biotechnology Resources David J. Goldstein, PhD David J. Munroe, PhD Carcinogenesis, Cancer and Cell Biology, Tumor Biology Joseph A. DiPaolo, PhD Stuart H. Yuspa, MD Clinical Research Frank M. Balis, MD Caryn Steakley, RN, MSW Immunology Jonathan D. Ashwell, MD Jay A. Berzofsky, MD, PhD |
Molecular Biology/ Carl Wu, PhD David L. Levens, MD, PhD Structural Biology/Chemistry Larry K. Keefer, PhD Christopher J. Michejda, PhD Sriram Subramaniam, PhD Translational Research Anita B. Roberts, PhD Elise C. Kohn, MD Leonard M. Neckers, PhD Virology Vinay K. Pathak, PhD John T. Schiller, PhD |
Center for Cancer Research
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r.
John Niederhuber, Acting Director, NCI, presented the 2006 Director’s Innovation
Awards to recipients at an award ceremony held during the NCI Intramural Scientific
Retreat on January 11. I worked closely with Drs. von Eschenbach and Niederhuber
to develop this award program as a means to stimulate highly innovative proposals
on cancer-related research problems from our junior staff. Based on the overwhelming
response and the high quality of proposals received, it is clear that the program
was a success, and is therefore likely to be repeated in the future. Of the
120 applicants in this round from CCR, 8 awards at the PI level (up to $50,000)
and 20 awards at the Career Development level (up to $10,000) were presented.
Please join with me in congratulating the recipients on their outstanding achievement.
adioimmunotherapy
(RIT)—the delivery of therapeutic radionuclides to cancer cells via
monoclonal antibodies (MAb)—has reemerged as a viable option for the treatment
and management of cancer patients. The cell surface antigen, HER2, provides
a molecular target to which site-specific, targeted radiation can be effectively
delivered via a well-defined, U.S. Food and Drug Administration (FDA)–approved
MAb (Herceptin). Monotherapy with Herceptin has resulted in a response rate
of 12% to 20% in metastatic breast cancer patients. A large percentage of eligible
patients, however, fail to respond to treatment and/or relapse. In addition
to breast cancer, HER2 is overexpressed in ovarian cancers and 35% to 45% of
all pancreatic adenocarcinomas. RIT offers an opportunity to complement and
enhance Herceptin’s intrinsic activity by direct incorporation of radiation
into the treatment regimen. 
mock-treated; 
