We are interested in understanding the genetic basis of human cancer, with a focus on hematopoietic malignancies. A detailed understanding of these cancers should provide insights not only into the molecular pathogenesis of carcinogenesis—and development of novel therapeutic approaches—but also into normal developmental processes that are dysregulated in cancers. Most recently we have focused on the role of cancer stem cells in disease pathogenesis, and the relationship between these cells and their normal tissue counterparts.

Our approach uses genome-wide screening strategies to identify cancer disease alleles in humans, characterizes disease alleles in cell culture and murine models of disease, develops molecularly targeted therapies based on insights derived from such analyses, and implements phase I/II clinical trials. Our successes include characterization of mutations that constitutively activate the FLT3 tyrosine kinase in acute myeloid leukemias (AMLs). Based on preclinical work from our lab and others, a phase III trial of the FLT3 inhibitor PKC412 is being initiated in the national cooperative oncology group setting for treatment of AML associated with activating mutations in FLT3.

In addition, our laboratory and others discovered the JAK2V617F allele as a cause of myeloproliferative diseases (MPDs) that include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (MF). These malignancies, which are collectively about 5 times more prevalent than BCR-ABL–positive chronic myelogenous leukemia, had been enigmatic from a genetic perspective for decades. We reasoned, based on analogy with other myeloproliferative diseases, that PV, ET, and MF might be caused by activating point mutations in tyrosine kinases. To test this, we first used an Internet-based Institutional Review Board–approved protocol to recruit 325 patients throughout the United States to provide DNA for analysis. Then, in collaboration with HHMI colleagues at the Broad Institute, we employed high-throughput, robotically driven DNA sequence analysis of all 90 of the fully annotated tyrosine kinases in the human genome. We discovered that a single allele, JAK2V617F, accounts for the majority of cases of PV, ET, and MF. We have subsequently demonstrated that JAK2V617F is a constitutively activated tyrosine kinase that transforms hematopoietic cells to factor-independent growth. Furthermore, expression of JAK2V617F in a murine model of disease recapitulates the human disease phenotype, validating it as a candidate for molecularly targeted therapy. We have gone on to screen for small-molecule JAK2 inhibitors, demonstrated efficacy of these inhibitors in our murine model of disease, and will enter the first of these into phase I clinical trials in MPD patients in 2007.

Although tyrosine kinase inhibitors have shown efficacy in a broad spectrum of tumor types associated with mutant tyrosine kinases, including hematopoietic malignancies, gastrointestinal tumors, and certain types of lung cancer, these targeted therapies are rarely, if ever, curative. An emerging concept in cancer treatment is that there is a small subpopulation of cells that have long-term self-renewing potential, are ultimately responsible for maintenance and propagation of tumors, and are resistant to current therapeutic modalities. These "cancer stem cells" have been demonstrated in the context of leukemias, but there is also evidence for their presence in breast and colon cancer and in tumors of the central nervous system.

We have focused on understanding the origins of cancer stem cells, the similarities and differences between cancer stem cells and their normal tissue counterparts, and the transcriptional programs that regulate their long-term self-renewing potential. For example, we have demonstrated that certain leukemia oncogenes, such as MOZ-TIF2, are able to transform hematopoietic cells with no potential for self-renewal, into leukemia stem cells. That is, granulocyte-macrophage progenitors (GMPs) isolated using high-speed multiparameter flow cytometry are terminally committed to apoptotic cell death and have no potential for long-term self-renewal. However, expression of MOZ-TIF2 initiates transcriptional instructions that convert GMPs into bona fide leukemia stem cells. These observations provide tools for comparing transcriptional programs that define normal hematopoietic stem cells (HSCs) and leukemia stem cells. This may enable identification of targets for therapeutic intervention that will discriminate between these two. Furthermore, insights into the transcriptional switches that can engender properties of long-term self-renewal may inform approaches for tissue regeneration.

We are also studying genes whose function is altered in cancer, with the goal of understanding their function in the normal hematopoietic compartment. For example, we have recently studied members of the FoxO family of forkhead transcription factors that are inactivated by constitutively activated tyrosine kinases in cancer, such as BCR-ABL. FoxO transcription factors influence quiescence of cells in both vertebrates and lower phylogenetic organisms, but their precise role in stem cell compartments was not understood. In collaboration with Ronald DePinho (Harvard Medical School), we characterized a mouse with homozygous conditional loss-of-function alleles for all three FoxO-family members expressed in the hematopoietic system (FoxO1, FoxO3, and FoxO4). We observed a phenotype that is restricted to the HSC compartment, in which loss of FoxOs results in rapid extinction of HSCs. Extinction of the HSCs was associated with markedly elevated levels of reactive oxygen species (ROS) in this compartment, and pharmacologic treatment with antioxidants in vivo resulted in reversion of the phenotype. Thus, FoxO transcription factors regulate HSC longevity, and do so through management of reactive oxygen. It will be of interest to determine whether FoxOs play a similar role in other tissue stem cells, but these findings have may have important implications for strategies to enhance the longevity of normal stem cells. In addition, as noted above, FoxOs are inactivated by mutant tyrosine kinases associated with a variety of cancers. It would be predicted that inactivation of FoxOs in cancer stem cells would result in increased ROS and be deleterious to their survival—but these cells are capable of long-term self-renewal. Cancer stem cells must in some way manage ROS in the absence of FoxOs, but it is plausible that they might nonetheless be susceptible to oxidative stress.

We will continue to study the relationship between normal and malignant stem cells. Future work will include the use of high-throughput short hairpin RNA (shRNA) and chemical genetic screens to characterize the interface between normal and leukemic stem cells and their stromal microenvironment, analysis of epigenetic influences on development of cancer stem cell phenotypes, and assessment of cancer stem cells in other tissue contexts based on insights gleaned from analysis of the hematopoietic system. These studies should provide insights into the developmental programs that regulate cancer stem cells and their normal tissue counterparts and may inform new therapeutic approaches to cancer, as well as strategies for tissue regeneration.

This work is also supported by grants from the National Institutes of Health, the Leukemia and Lymphoma Society, and the Doris Duke Charitable Foundation.