Therapeutic Strategies for 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. 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 inhibited by a rationally designed compound. We determined that myeloid cell transformation driven by BCR-ABL is codependent on the c-KIT cellular receptor.
Multiple groups have demonstrated that point mutants in the BCR-ABL kinase domain 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). Recently we have applied such techniques to mouse models of CML and hematopoietic stem cells, to serve as a preclinical test of efficacy.
Lymphoid Development and Immune Monitoring
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 BTK is introduced into hematopoietic stem cells from such mice, the deficiency in B cell development and immune response is largely corrected.
Selected G protein–coupled receptors (GPCRs) can 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. 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, while TDAG determines the efficiency of certain antitumor responses.
To better study the role of the cellular immune response in mice and humans, we have several approaches that use positron emission tomography (PET). These include reporter gene strategies to mark populations of lymphocytes reactive to antigens on solid tumors and follow lymphocyte homing and expansion in living mice. PET is also suitable for evaluation of human antitumor responses. 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. In mouse models of experimental autoimmune encephalitis (EAE) 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 class of fluoropyrimidine compounds (called the FAC family), which can be used as effective PET probes for detection of activated lymphocytes through uptake and trapping by the DNA salvage pathway. Members of this probe family can be used to predict the uptake of closely related chemotherapy agents; this may aid in selecting specific patients for more-personalized chemotherapy regimens. Early-phase clinical testing of the FAC family probes has been initiated, in collaboration with Caius Radu and Johannes Czernin (both at the University of California, Los Angeles).
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 therapeutic target is PSCA. This protein is highly 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 and are using them to fractionate normal murine prostate cell populations to define an active stem cell population for the murine prostate. Other markers, such as Trop2, have been used to refine the cell purification for both mouse and human prostate stem cells. 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 NKX3.1. These pathways control cell expansion, as well as 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. We recently evaluated a large series of genes associated with human prostate cancer, including the Ets family of transcription factors, genes like Bmi that mediate self-renewal pathways, androgen receptor, and many members of the protein kinase family. Our results support a model in which a diverse range of pathway combinations can progress normal epithelium to carcinoma. The range of combinatorial stimuli that can produce prostate cancer presents a difficult therapeutic challenge.
