Therapeutic Strategies for Ph Chromosome-Positive 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 for treatment of the chronic phase of CML but has been less effective for the blast crisis stage of CML or the initial treatment of Ph-positive ALL. In both of these aggressive leukemias, additional genetic changes complement the action of BCR-ABL and the malignancy continues to grow in the face of the kinase-targeted therapy. Although some of this lack of responsiveness is due to mutations in the kinase that render the drug less effective, in other cases the combination of genetic changes allows the bypass of cell killing by the drug.
We have recently concentrated on understanding the genetics of Ph-positive ALL, which is a deadly form of leukemia affecting children and adults. Previous work has determined that the Ikaros family of transcriptional regulators acting as tumor suppressors are major factors in Ph-positive ALL progression. In collaboration with Stephen Smale (University of California, Los Angeles), we are evaluating specific mutant alleles of the many spliced isoforms of Ikaros to define their role in developmental progression of immature B cells in the bone marrow and how their loss of function complements the action of the BCR-ABL tyrosine kinase. We hope that these studies will provide insights into pathways operative in such leukemias that may point to new therapeutic strategies.
Lymphoid Development and Immune Monitoring by Positron Emission Tomography
Protection against pathogens depends on the coordinated function of multiple immune cell types within the body. Imbalance of the diverse effector functions in the immune system results in either immune deficiency or autoimmunity.
My group previously identified Bruton's tyrosine kinase (BTK) as a critical regulator of B cell development. 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. BTK is now a target for therapeutic attack in a variety of B cell malignancies and autoimmune disorders.
Cancer also causes functional immune deficiency. My group is involved in a collaborative effort with several labs at UCLA, Caltech, and elsewhere to engineer lymphocytes from cancer patients so that they express T cell receptors specific for tumor antigens, arming them to fight the tumor. Transplant of engineered lymphocytes back into the patient has shown promise in fighting melanoma, but the determinants of therapeutic success are largely unknown. To monitor and predict the success of cell-based immunotherapies, my group has developed new imaging approaches using positron emission tomography (PET) to noninvasively monitor immune function throughout the body in mice and humans. These include PET reporter technology in which immune cells are engineered to express a reporter gene that mediates specific accumulation of a positron-labeled probe within the cell. We have shown that it is possible to use a viral-based PET reporter to track tumor-specific lymphocytes in mice during an anticancer immune response. Long-term expression of viral-based proteins in humans has been shown to be strongly immunogenic. To facilitate the translation of PET reporter imaging to clinical trials for cell-based immunotherapy, we have recently identified and tested PET reporters derived from human nucleoside kinases.
Immune cells modulate their metabolism according to the functions they need to perform. We have demonstrated that it is possible to measure immune function in vivo with PET probes specific for distinct metabolic pathways. 18F-FDG (fluorodeoxyglucose) accumulates in cells with high rates of glycolysis and is commonly used in the clinic to measure tumor burden and the response to therapy. Activated immune cells also demonstrate a shift in glycolysis, and we showed that FDG PET can evaluate the trafficking and expansion of inflammatory immune cells within the central nervous system during experimental autoimmune encephalitis, a commonly used model for multiple sclerosis.
In collaboration with Caius Radu (UCLA), we developed a PET probe specific for the nucleoside salvage pathway. 2'-Fluoro-2'-deoxy-(arabinofuranosyl)cytosine (18F-FAC) accumulates in cells expressing high levels of deoxycytidine kinase (DCK), an enzyme in the nucleoside salvage pathway that is preferentially expressed in immune cells. FAC PET can highlight areas of immune activation in vivo during anticancer immune responses and during autoimmunity. In comparison to FDG, which accumulates to high levels in innate immune cells such as macrophages, FAC labels proliferating adaptive immune cells. Genetic deletion of DCK in mice results in loss of FAC signal, and these animals are deficient in producing cells of the adaptive immune response. These combined data suggest DCK may be a useful target for certain leukemias and immune disorders.
A family of PET probes based on FAC has been developed, and these can be used to predict the uptake of closely related chemotherapy agents; this may aid in selecting specific patients for more-personalized chemotherapy regimens. Clinical testing of the FAC family probes has been initiated, in collaboration with Caius Radu and Johannes Czernin (UCLA). Additional studies on reporter gene strategies with Melissa McCracken to monitor lymphocyte function are also underway.
Prostate Cancer Pathogenesis
Studies in leukemia taught us that blood-related malignancies could be deconstructed to determine the cells that give rise to the disease, the alterations that transform target cells, and the mechanisms that drive and sustain cellular growth in cancer. Leukemia is a disease of stem and progenitor cells in which normal cellular pathways are diverted. Solid tumors such as prostate cancer have not typically been addressed in this way. We set out to understand the disease in terms of the target cells, the transforming oncogenes, and the mechanisms that promote prostate malignancy.
To develop a system where the prostate gland could be studied analogously to blood, we adapted a previously described tissue recombination model to reconstruct prostatic tissue from dissociated single cells when combined with an inductive stromal source and transplanted into immune-deficient mice. This system allows for the purification of cells that generate prostatic structures, and also provides a way to define specific oncogenic insults or combinations capable of transforming naïve prostate epithelium. We discovered that the stem cell antigen-1 (Sca-1) identified a subpopulation of prostate cells enriched for the ability to reconstitute prostate tissue. The Sca-1+ progenitor-enriched population was a target for transformation as activation of the PI3K pathway in this distinct subset of cells was sufficient to initiate prostate malignancy.
We went on to define additional oncogenes that transform prostate cells, and determined oncogenic combinations that preferentially synergize in the transformation process. Certain combinations of oncogenes, such as the androgen receptor (AR), AKT, and the ETS family transcription factor ERG, can drive different stages of disease progression from hyperplasia to the precursor lesion prostatic intraepithelial neoplasia (PIN) to frank carcinoma. In addition to the cell autonomous oncogenic signals that transform primary cells, we showed that sustained growth factor signals from the stroma can induce oncogenic changes in the epithelium. Fibroblast growth factor 10 (FGF10) is essential for formation of the prostate and initiates a signal through the epithelium to promote increased expression of AR and subsequent prostate malignancy. Such stromal-mediated paracrine signals may provide a partial explanation for the multifocal nature of human prostate cancer.
To purify subsets of cells from the mouse prostate representing varying stages of differentiation, we used new markers such as CD49f (integrin alpha6) and Trop2. These enabled us to highly enrich different populations of cells and defined a stem cell-enriched basal fraction with the capacity to self-renew and differentiate into all three lineages of the epithelium (basal, luminal, neuroendocrine). We introduced a variety of oncogenes into purified basal and luminal cells and found that basal cells are the preferred target for transformation, regardless of oncogenic insult, implicating these stem-like cells in disease initiation.
Building on our expertise in the mouse system, we asked if we could purify distinct subsets of cells from the human prostate, using surface markers CD49f and Trop2 to define basal and luminal cells isolated from primary benign human prostate tissue. Basal cells, defined as CD49fhiTrop2+, could self-renew in vitro and generate prostatic ducts in vivo. Introduction of three oncogenes -- AKT, ERG, and AR -- in basal cells, but not luminal cells, was sufficient to initiate prostate adenocarcinoma. The resulting cancer had a luminal phenotype, demonstrating that a stem-like basal cell can serve as a cell of origin and differentiate into a malignant cell with a mature marker profile.
With knowledge of the target population, we looked for mechanisms enriched in these cells that sustain their stem-like potential. The polycomb complex/protein Bmi-1, enriched for expression in the target basal cells, provides a mechanism that promotes their self-renewal. Targeting Bmi-1 through short hairpin RNA could partially block cancer initiation from several genetic sources. Future studies will be aimed at purifying basal and luminal fractions into discrete cell subsets and defining genetic alterations that target different populations. We plan to interrogate the cells that give rise to prostate cancer to identify pathways that can be targeted at an earlier stage in the disease. These principles of tissue reconstruction and defining stem cells as targets for oncogenesis are being applied for the study of other cancer types.
Recent work has concentrated on the definition of tissue and cancer stem cells, and their role in the generation of the most aggressive neuroendocrine/small cell variants of prostate cancer.
Partial support of these studies was provided by the Prostate Cancer Foundation, the California Institute for Regenerative Medicine, and the National Cancer Institute.
As of April 29, 2016