Date Published: June 19, 2012
Publisher: Hindawi Publishing Corporation
Author(s): Yiyun Zhang, J.-R. Joanna Yeh.
In vivo chemical screening is a broadly applicable approach not only for dissecting genetic pathways governing hematopoiesis and hematological diseases, but also for finding critical components in those pathways that may be pharmacologically modulated. Both high-throughput chemical screening and facile detection of blood-cell-related phenotypes are feasible in embryonic/larval zebrafish. Two recent studies utilizing phenotypic chemical screens in zebrafish have identified several compounds that promote hematopoietic stem cell formation and reverse the hematopoietic phenotypes of a leukemia oncogene, respectively. These studies illustrate efficient drug discovery processes in zebrafish and reveal novel biological roles of prostaglandin E2 in hematopoietic and leukemia stem cells. Furthermore, the compounds discovered in zebrafish screens have become promising therapeutic candidates against leukemia and included in a clinical trial for enhancing hematopoietic stem cells during hematopoietic cell transplantation.
Zebrafish has been used effectively as a vertebrate model for studying blood cell development and function (for reviews see [1–5]). It is an advantageous model because the optical clarity of its embryos, and their ex utero development enables easy and real-time detection of hematopoietic cells during development. A wide variety of tools and reagents have been developed for in vivo labeling and imaging of blood cells and for investigating blood cell function (for reviews of these methods and protocols, see [6–10]). In addition, transient and stable genetic manipulation can link hematopoietic genes to their functions [11–16]. Added to this arsenal of research tools available in zebrafish is in vivo chemical screening [17–20]. By exposing zebrafish embryos to a chemical library, bioactive compounds that affect any complex developmental and physiological processes may be identified. Furthermore, in vivo chemical screening may be used for uncovering chemical agents that modify a disease phenotype in a whole animal. The compounds that induce a unique biological effect may serve as invaluable probes for identifying critical components of biological pathways, and compounds that can reverse a disease phenotype in vivo may have therapeutic potential or shed light on an effective therapeutic target. This innovative approach has created a unique utility for the zebrafish model in chemical biology and contributed to its emerging role in drug discovery (for additional reviews see [21–24]).
Both genetic and in vivo chemical screens may be used to dissect genetic pathways that regulate specific biological processes. However, an in vivo chemical screen offers the advantage of temporal control that a traditional genetic screen does not. In a genetic screen, gene function is affected from conception. Thus, the role of a gene in early embryonic development may preclude characterization of its roles during later stages. On the other hand, in a chemical screen, compounds that affect the function of a gene can be administered at specific time points and for fixed durations chosen by the investigator so that its roles at different developmental stages may be distinctly determined. In addition, in a genetic screen, the roles of a protein family may sometimes be masked by functional redundancy of its family members. However, chemical modulators may exhibit similar activities against multiple members of a protein family and can, therefore, reveal their in vivo cumulative roles. It should be noted that some compounds may affect multiple cellular proteins and thus their on-target effects should be carefully verified using additional chemical agents as well as genetic manipulations. Taken together, in vivo chemical screens may complement traditional genetic approaches and uncover hematopoietic genes that cannot be identified in genetic screens.
Currently, the most common approach for identifying potential therapeutics is the target-driven approach (for reviews see [25, 26]). This approach relies on a priori understanding of disease mechanisms to the point of knowing a specific cellular component to be targeted. Thereafter, lead compounds may be obtained using in vitro or cell-based assays to determine binding to or modulation of target activity. Typically, these leads will be further optimized using these assays again before being assessed for their in vivo efficacy and toxicity. Targets employed by this approach are often enzymes such as kinases that are likely to have small-molecule binding pockets (for more discussions on target druggability, see reviews [26, 27]). Proteins that do not have an obvious pocket, such as transcription factors that often act by recruiting other cofactors, are sometimes dubbed undruggable targets.
Some of the model organisms that may be used for in vivo chemical screening are Drosophila, C. elegans and embryonic/larval zebrafish (Danio rerio) (for a review see ). All of these models have the scalability required for high-throughput screening. Among them, zebrafish is the only vertebrate model and thus possesses the closest physiological similarities to humans.
Compounds that can augment HSC formation and function may exert therapeutic benefits to patients undergoing hematopoietic cell transplantation. North et al. performed a chemical screen to identify small molecules regulating HSC formation in zebrafish embryos . In this study, embryos were exposed between 11 and 36 hpf to 2,357 compounds from three chemical libraries of known bioactive compounds. As mentioned above, HSCs are cmyb+ and runx1+ and both transcription factors are indispensable for HSC development. By examining cmyb and runx1 expression using RNA in situ hybridization, the authors found 35 compounds that increased HSC numbers and another 47 compounds that decreased them. Based on their known bioactivities, they found that 10 of these compounds affect prostanoid biosynthesis. Prostanoids, including prostaglandins, prostacyclins, and thromboxanes, are lipid mediators that play major roles in inflammation and other physiological responses. The cyclooxygenases (COXs), including COX-1 and COX-2 (also known as prostaglandin G/H synthase 1 and 2), convert arachidonic acid into prostaglandin H2, which can then be metabolized into other prostanoids by additional enzymes . Interestingly, the authors found that while exposure to COX inhibitors such as celecoxib and sulindac reduced cmyb/runx1 expression in the hemogenic aorta, exposure to linoleic acid, a precursor of arachidonic acid, enhanced it. Previously it had been shown that prostaglandin E2 (PGE2) is the major prostanoid produced in zebrafish embryos . Thus, North et al. confirmed the involvement of the prostaglandin pathway in HSC formation by incubating zebrafish embryos with PGE2 or selective inhibitors of COX-1 and COX-2, as well as by genetic knockdown of ptgs1 and ptgs2 that encode COX-1 and COX-2 proteins, respectively. Subsequently, the authors investigated the expression patterns of ptgs1 and ptgs2 and found that both genes were upregulated at the onset of definitive hematopoiesis. While both genes were expressed in the HSCs, ptgs1 was also expressed in the neighboring endothelium. These results strongly suggest that COX-1 and COX-2 promote HSC formation through functions in both the HSCs and their niche. Furthermore, Goessling et al. showed that PGE2 promotes HSC formation by activating the Wnt/β-catenin signaling pathway .
Hematopoietic cell transplantation (HCT) is frequently used in the treatment of hematological malignancies. HSCs not only self-renew but also give rise to all blood lineages and can repopulate an entire hematopoietic system. Patients about to receive HCT need to undergo myeloablation and are treated simultaneously with immunosuppressants to prevent transplant rejection. It is essential that the transplanted HSCs effectively and efficiently engraft in the bone marrow. Various methods aiming to enhance the in vitro and in vivo expansion of stem/progenitor cells and their homing efficiency to bone marrow are currently under intensive investigation [104–107]. The chemical modulators of HSCs identified by North et al. in zebrafish represent another new therapeutic opportunity.
Since COX inhibitors scored as hits in our screen, we investigated the genes coding for COX proteins and found that ptgs2 but not ptgs1 expression was significantly upregulated in the hematopoietic cells of Tg(hsp:AML1-ETO) zebrafish . At the time of this discovery, very little was known about the potential contribution of the COX enzymes in AML leukemogenesis, although overexpression of COX-2 had been reported in various types of epithelial tumors, including colorectal carcinoma and breast cancers [125, 126]. Moreover, PGE2 had been shown to promote colon cancer cell growth via a β-catenin-dependent signaling pathway [127, 128]. As in zebrafish, we found that AML1-ETO induced ptgs2 but not ptgs1 expression in the K562 human myeloid leukemia cell line . AML1-ETO induced the activity of a β-catenin reporter and inhibited erythroid differentiation in these cells, and both effects could be abrogated by NS-398. Subsequently, we found that genetic knockdown of β-catenin rescued AML1-ETO’s effects in zebrafish embryos . Thus, AML1-ETO affects hematopoietic differentiation through the COX-2/β-catenin pathway in both zebrafish and human leukemia cells.
In this paper, we presented two specific studies on hematopoiesis that appropriately exemplify the general utility of embryonic zebrafish and phenotypic in vivo chemical screening in discovering potential new therapeutics. In these cases, the use of an in vivo screening platform allowed the identification of compounds that may act in a noncell autonomous fashion such as hemodynamic forces, bypassed the well-known technical difficulties involved in culturing hematopoietic or leukemia stem cells, and also circumvented the obstacles conferred by undruggable targets or unknown disease mechanisms. Both of the studies uncovered novel biological mechanisms as well as strong candidates for clinical therapeutic use. It is important to note that most of the advantageous features of the zebrafish model occur at its embryonic and larval stages. Thus, a disease phenotype under investigation must manifest during these stages in order to be most effectively exploited for chemical screening. Since multitudinous signaling pathways acting together in zebrafish during early development are also likely to play important roles in maintaining homeostasis in adults and may be disrupted or reactivated during disease progression, a surrogate embryonic phenotype can often be very useful for identifying potential disease modulators. For example, compounds that suppress T-cell development in embryonic zebrafish may demonstrate potent inhibitory effects against T-cell leukemia . Overall, drug discovery in zebrafish benefits from the feasibility of high-throughput chemical screening, closer physiological similarities to human than invertebrate screening strategies, and the ability to create complex disease models not achievable in vitro. The possibility of detecting a wider range of hematopoietic phenotypes using innovative assays promises an ever-increasing role for zebrafish in future drug discovery processes.