|
Growth Regulation of Hematopoietic and Epithelial Cancers and the Immune Response
Summary: Owen Witte is interested in the development of the immune response, growth regulation mechanisms used in leukemias and during metastasis of epithelial cancers to the bone marrow, and the application of new quantitative processes of whole-animal imaging.
The Role of the BCR-ABL Oncogene in Leukemias
Human chronic myelogenous leukemia (CML) is associated with the Philadelphia chromosome, which produces the BCR-ABL tyrosine kinase oncogene. Our laboratory demonstrated that this kinase is critical for the leukemic phenotype of CML and related types of leukemia. Targeted drug therapy for CML (Gleevec and other drugs) that inhibits the enzymatic activity of BCR-ABL has been very successful.
One remaining issue is to define the intracellular signaling pathways used by BCR-ABL to control abnormal growth. Our results show that many pathways implicated from the study of established cell lines are not activated when BCR-ABL is first expressed in progenitor cells. We have exploited an approach pioneered by Kevan Shokat (HHMI, University of California, San Francisco) in which selected mutations created in the ATP-binding pocket of a tyrosine kinase can render it uniquely inhibitable by a rationally designed compound. We determined that myeloid cell transformation driven by BCR-ABL is codependent on the c-KIT cellular receptor.
Recent work from multiple groups demonstrated that point mutants in the BCR-ABL kinase domain and elsewhere in the molecule render it resistant to inhibition by Gleevec and related drugs. We recently determined that destruction of the mRNA for BCR-ABL by stable introduction of tandem arrays of microRNA mimics dramatically suppresses expression of this oncogene for prolonged therapeutic effect in mouse models of ALL (acute lymphocytic leukemia).
Lymphoid Growth Regulation and Development
A critical regulator of B cell development is Bruton's tyrosine kinase (BTK), identified by my group. Loss of BTK function results in X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (xid) in mice.
In collaboration with David Rawlings (now at the University of Washington), we developed a successful test for the preclinical evaluation of retrovirally delivered BTK to correct the genetic deficiency found in human XLA. Mice deficient for BTK and the closely related gene tec have a severe block in B cell development. When full-length and kinase-active BTK is introduced into hematopoietic stem cells from such mice, the deficiency in B cell development and immune response is largely corrected.
We have identified G protein–coupled receptors (GPCRs) that appear to participate in both general inflammatory reactions and programmed immune responses. G2A serves as a negative regulator of lymphocyte growth during postnatal life. Mice deficient in G2A can develop a slowly progressive failure of peripheral lymphocyte homeostasis, with resulting lymphoid hyperplasia, and autoimmunity. G2A is used indirectly in lysophosphatidylcholine-mediated chemotaxis of T lymphocytes and macrophages. The receptor can be found on the cell surface but spontaneously cycles to an internal endosomal compartment. When lipid is added externally, the receptor accumulates to a higher level on the cell surface. Other members of this GPCR family, such as TDAG and GPR4, can be activated by acidic pH. Loss of function of GPR4 results in developmental defects in the vascular system.
We have used positron-emission tomography (PET) reporter gene strategies to mark populations of lymphocytes reactive to antigens on solid tumors and follow lymphocyte homing and expansion in living mice. Many of these techniques may be suitable for evaluation of human antitumor responses. We have developed a series of vectors that can transduce proteins to simultaneously report PET, bioluminescence, and fluorescent signals. PET technology can be used to visualize the activation and expansion of myeloid and lymphoid cell types within the local draining lymph nodes and tumor bed during response to an antigenic sarcoma.
Oncologists commonly use PET with 18F-FDG as a probe to monitor tumor burden and response to therapeutic agents. The same shift in glycolysis by activated immune and inflammatory cell populations can be exploited to follow active immune responses and autoimmune states with 18F-FDG PET. We used this technology to study murine models of experimental autoimmune encephalitis (EAE), and we can visualize specific populations of immune cells as they migrate into and expand within the spinal cord. Such approaches may be useful in a wide variety of human autoimmune disorders. Recent work defined a new fluoropyrimidine compound called FAC, which can be used as an effective PET probe for detection of activated lymphocytes through its uptake and use by the DNA salvage pathway.
Metastasis of Prostate Cancer to the Bone Marrow
Epithelial cancers are major public health concerns. Prostate cancer is unique in its highly regularized pattern of metastasis to the bone marrow, where it causes an osteoblastic response. One possible therapeutic target is PSCA (prostate homolog of hematopoietic stem cell antigen). This protein is expressed on a subset of normal prostate cells during active growth and is expressed on prostate cancers as they develop more aggressive behavior. Monoclonal antibodies reactive with human PSCA block establishment and metastatic progression of human prostate cancer xenographs in immunodefective mice. In clinical trials for prostate and other cancers, the pharmaceutical industry is evaluating human monoclonal antibodies reactive with PSCA.
We identified surface markers such as Sca1 and CD49F integrins and are using them to fractionate normal murine prostate cell populations to define an active stem cell population for the prostate. The prostate-regenerating cell populations are enriched in the proximal/urethral area of the adult gland. We developed a dissociated cell reconstitution system, in which prostate epithelial stem and progenitor cells can be induced to form glandular tissue structures by embryonic urogenital sinus mesenchyme tissue when implanted under the kidney capsule. In collaboration with Hong Wu (University of California, Los Angeles), we determined that the prostate basal layer stem cell population is regulated tightly by the lipid phosphatase PTEN and the homeobox protein NKX 3.1. These pathways control the amount of cell expansion of this population as well as its organization within the prostatic tubule.
The prostate-regenerating system can be used to assess combinations of oncogenes and tumor suppressors in cancer progression. Enhancement of the AKT serine kinase pathway in concert with increased levels of the androgen receptor is sufficient to drive normal epithelial development to frank carcinoma. Such models will be useful in assessing the relative importance of other pathways to generate prostate cancer and for the evaluation of new therapeutics. Recent work has demonstrated that prolonged expression of certain stimuli, such as fibroblast growth factor 10 from the surrounding stroma, can drive the epithelial compartment to a cancerous phenotype. These results may help explain the common presentation of prostate cancer as a multifocal disease. (Partial support for these studies was provided by grants from the Prostate Cancer Foundation and the National Cancer Institute.)
Last updated: April 23, 2008
|
