Anti-DLL4, a cancer therapeutic with multiple mechanisms of action

Cancer can be considered as an aberrant and mutant recapitulation of normal tissue development and homeostasis. Tumors are typically highly heterogeneous at the cellular level, and this heterogeneity frequently mirrors the cellular heterogeneity of the normal tissue. Normal tissue development and homeostasis is driven by an organized hierarchy of stem and progenitor populations which give rise to various differentiated cell types with specialized functions. Long term tissue maintenance is enabled by the unique ability of the stem cell to exhibit self-renewal, which is defined as the ability to proliferate while maintaining pluripotency. Similarly, cancer cells inappropriately activate self-renewal pathways and this enables their ability to grow indefinitely. Thus, the potential connections between normal stem cells and cancer have great importance for understanding tumor biology and also for developing new therapeutic strategies. In the past decade it has become increasingly clear that this self-renewal property is not possessed by all cells within a tumor, but that there exists a subpopulation of cells, often referred to as "cancer stem cells" or "tumor initiating cells" which possesses the ability to undergo self-renewal and thereby drive the growth of the tumor [1‐3]. These cells also possess the ability to initiate the growth of new tumors that recapitulate the heterogeneity of the parent tumor. Thus, these cells have hallmark capabilities analogous to normal stem cells. This relationship has been strengthened by genetic studies which have shown that normal stem cells can be the cell of origin for tumors [4]. Additionally, cancer initiating mutations can originate in more differentiated cells and confer stem-like properties on the tumor cells. These connections have led to a careful examination of stem cell signaling pathways and their role in cancer. Intriguingly, several of these pathways, including the Notch and Wnt pathways, have long been recognized to be activated by oncogenic mutations and disregulated in cancer.

In addition to cancer's need to achieve the stem cell-like property of self-renewal, a cancer must also recruit a support system of stroma and vasculature. The development of vasculature is a complex developmental process, analogous to the development of organs, and so it is not surprising to note that here too signaling pathways important to stem cells, including Notch, have important role in cell fate decisions. There are roles recognized for multiple Notch receptors and multiple Notch ligands within this process [5‐8]. These molecules play roles both within the endothelial cell layer, where they are involved in vessel branching and maturation, and in the surrounding pericyte and smooth muscle layers. Jagged1 and Notch3 are of particular importance in pericyte function [9‐11]. DLL4 acting through Notch1 and Notch4 appears to play key roles regulating endothelial cells and bone marrow-derived endothelial cell progenitors during normal and tumor angiogenesis [12, 13].

These two lines of research, the role of the Notch pathway in the maintenance of cancer stem cells, and the activity of Notch in tumor vasculature, have led to intense research interest in targeting components of this pathway for the development of novel therapeutics. Through this effort, DLL4 has emerged as a compelling target. Indeed, an antibody to DLL4, OMP-21M18, was the first therapeutic entity that selectively targeted the Notch pathway to enter human clinical trials. Gamma-secretase inhibitors (GSIs) that inhibit the ligand-dependent cleavage of Notch receptors have also been developed as anti-cancer therapies. Treatment with GSIs has been found to result in severe gastrointestinal toxicity, limiting their therapeutic utility, due to the combined inhibition of both Notch1 and Notch2 within the stem-progenitor compartment of the intestinal crypt [14, 15]. In addition to processing Notch proteins, gamma-secretase cleaves many other membrane proteins and is involved in a large number of signaling pathways apart from Notch, and these pleiotropic effects are also likely to contribute to the toxicity of GSIs [16, 17].

DLL4 is one of three delta-like ligands in the mammalian genome [18]. Similar to Drosophila Delta, it acts as an agonist ligand to modulate the activity of Notch receptors. Binding studies and in vitro signal transduction studies indicate that DLL4 is readily able to signal through each of the four human Notch receptors, demonstrating its potential to participate in many of the diverse functions that have been described for the Notch family. Elucidating the full range of its activities is an ongoing area of investigation, and is somewhat complicated by the potential for compensatory action by other Notch ligands. Further complicating this analysis is the potential for DLL4 to be expressed in minor subsets of cells, such as stem cells and progenitors, which may lead to an under-appreciation of DLL4 expression and its relevance to a particular tissue. LacZ reporter studies in developing mouse embryos have revealed expression with the vascular system, multiple structures within the nervous system, the gastrointestinal system, the glomeruli of the kidney, and the thymus [19] Gene disruption studies indicate that DLL4 plays an important role in angiogenesis - haploinsufficiency results in embryonic lethality due to angiogenic defects [20‐22]. Conditional gene disruption studies have also demonstrated critical roles for DLL4 in T cell development [23, 24]. In addition, DLL4 is also expressed by myeloid cells including macrophage and dendritic cells and plays a role in modulating adaptive immune response [25‐27]. DLL4 is one of the ligands important for lineage commitment within the epithelial cell lining of the gastrointestinal tract. Interestingly, DLL1 and DLL4 appear to have redundant functions in normal intestinal development and homeostasis [28], which accounts for the fact that selective inhibition of DLL4 has minimal impact on the function of the GI tract.