Nemeth Lab

Background

There is a fundamental gap in understanding how adult stem cells decide to undergo self-renewal or differentiation. Self-renewal is the process by which a stem cell gives rise to daughter progeny that retain the parent stem cell phenotype and is the means by which stem cell numbers are maintained throughout life. Nowhere is this gap more relevant than the study of cancer stem cells which bear some of the characteristics of normal adult stem cells, such as nearly limitless self-renewal, and are hypothesized to be the cells that maintain tumors and promote relapse. Our long-term goal is to identify the mechanisms that regulate the development and maintenance of adult stem cells and determine how similar mechanisms operate in cancer stem cells. Lack of such knowledge is an important problem, because without it, it is unlikely that researchers will be able to design therapies that can target the rare cancer stem cell population.

Figure 1. A simplified schematic diagram of the hematopoietic hierarchy. The hematopoietic stem cell (HSC) sits at the top of the hierarchy. Upon activation, the HSC is capable of undergoing self-renewal or differentiating into clonal progenitors that can expand exponentially as well as continue the process of differentiation. Hematopoietic cells are broadly divided into myeloid and lymphoid cells. Myeloid cells include monocytes/macrophages, granulocytes, red blood cells (RBC) and platelets (as well as other cell types such as eosinophils, mast cells, and basophils). Lymphoid cells include T cells, B cells, natural killer cells (NK) and dendritic cells.

Our model system for this work is the hematopoietic stem cell (HSC) and its malignant counterpart, the leukemia stem cell (LSC). The HSC initiates hematopoiesis, the process by which blood cells of all lineages are continuously generated throughout life (Figure 1). During development, HSCs are highly proliferative before entering a predominantly quiescent state in adult mammals. The HSC is the best characterized stem cell and much of our knowledge of stem cells in general comes from applying the lessons learned in HSC biology.

We have focused on signaling pathways that have proven to be important in development and stem cell biology. The Wnt signaling pathways are induced by the Wnt family of ligands and are categorized as canonical, which utilizes the beta-catenin protein as a signaling mediator (Figure 2), and non-canonical, which do not. Wnt signaling pathways are critical for normal development and have been shown to regulate the normal function of multiple types of stem cells, including embryonic, intestinal, epithelial, and hematopoietic.

Figure 2. A schematic diagram of selected components of the canonical Wnt signaling pathway. (A) The absence of Wnt ligand binding to the Frizzled receptor and the LRP5/6 co-receptor enables the formation of a multi-protein complex (which includes APC, Axin, GSK-3b, and CK1) that promotes the phosphorylation and subsequent degradation of b-catenin. In the absence of b-catenin translocation, repressor proteins bind to TCF/LEF transcription factors and prevent transcription from occurring. (B) In the presence of Wnt ligand, the LRP co-receptors are phosphorylated by membrane-bound casein-kinase 1g and GSK-3b (not depicted), which recruits Axin to the cell membrane, disrupting the multi-protein complex. The Dishevelled protein (Dvl) is necessary for this process to occur but the mechanism is undefined. The disintegration of the multi-protein complex ultimately results in the accumulation and translocation of b-catenin to the nucleus. b-catenin interacts with TCF/LEF factors and transcription occurs.

Furthermore, abnormal regulation of Wnt signaling pathways is present in both solid and liquid tumors, such as leukemias. Therefore, determining the function of Wnt signaling pathways in HSC and LSCs may lead to the development of novel therapeutics specifically targeted to LSCs and cancer stem cells in general.

A. Non-canonical Wnt signaling

Previously, we have shown that induction of non-canonical Wnt signaling pathways by the ligand Wnt5a induces quiescence of HSCs and enhances their engraftment. Wnt5a also suppresses development of leukemia and hypermethylation of its promoter correlates with decreased rates of relapse-free and overall survival in acute lymphoid leukemia. Our research into the role of non-canonical Wnt signaling is HSC and LSC biology is focused on testing the central hypothesis that non-canonical Wnt signaling regulates the development and maintenance of the adult hematopoietic system by maintaining HSCs within a quiescent state and that the loss of this regulatory pathway leads to increased risk of developing leukemia. We plan to investigate the validity of this model by testing the following working hypotheses:

1. Non-canonical Wnt signaling pathways initiate and maintain the adult phenotype of HSCs by inducing them to enter into a quiescent G0 state.

2. Deficiencies in non-canonical Wnt signaling induces the initiation and progression of leukemia by increasing the probability of transformation of the HSC.

From this work, we expect to contribute to a detailed understanding of how Wnt5a-induced signaling pathways regulate the development and maintenance of adult and malignant hematopoiesis. This contribution is significant because it is expected to provide the knowledge necessary for development of pharmacologic strategies that will either promote or inhibit the induction of non-canonical Wnt signaling pathways.

B. Canonical Wnt signaling

We have shown that the activation of the canonical Wnt signaling pathway correlates with enhanced HSC self-renewal. Given that abnormal activation of canonical Wnt signaling is observed in hematopoietic malignancies, such as acute and chronic myeloid leukemia and chronic lymphoid leukemia, we have hypothesized that the canonical Wnt pathway regulates identical functions in both HSCs and LSCs, specifically stem cell self-renewal. To test this hypothesis, our first step in pursuit of that goal is to determine the common target genes of the canonical Wnt signaling pathway in HSCs and LSCs. We are currently doing so using newly available technologies that enables us to map where -catenin binds to DNA and what genes it activates across the whole genome. By combining these data with in silico biological pathway analysis, we expect to identify novel canonical Wnt target genes that regulate HSC and LSC function. This is significant because it is expected to provide the knowledge needed to determine the mechanisms by which canonical Wnt signaling regulates development of hematologic malignancies. Once these mechanisms are known, there is the potential for development of therapies to target the malignant aspects of canonical Wnt signaling. Furthermore, such knowledge could advance the field of in vitro manipulation of adult stem cells. Finally, these findings can be applied to cancers beyond the hematopoietic organ.