John Mcwhirter

CD200 Expression on Tumor Cells Suppresses Antitumor Immunity: New Approaches to Cancer Immunotherapy

Although the immune system is capable of mounting a response against many cancers, that response is insufficient for tumor eradication in most patients due to factors in the tumor microenvironment that defeat tumor immunity. We previously identified the immune-suppressive molecule CD200 as up-regulated on primary B cell chronic lymphocytic leukemia (B-CLL) cells and demonstrated negative immune regulation by B-CLL and other tumor cells overexpressing CD200 in vitro. In this study we developed a novel animal model that incorporates human immune cells and human tumor cells to address the effects of CD200 overexpression on tumor cells in vivo and to assess the effect of targeting Abs in the presence of human immune cells. Although human mononuclear cells prevented tumor growth when tumor cells did not express CD200, tumor-expressed CD200 inhibited the ability of lymphocytes to eradicate tumor cells. Anti-CD200 Ab administration to mice bearing CD200-expressing tumors resulted in nearly complete tumor growth inhibition even in the context of established receptor-ligand interactions. Evaluation of an anti-CD200 Ab with abrogated effector function provided evidence that blocking of the receptor-ligand interaction was sufficient for control of CD200-mediated immune modulation and tumor growth inhibition in this model. Our data indicate that CD200 expression by tumor cells suppresses antitumor responses and suggest that anti-CD200 treatment might be therapeutically beneficial for treating CD200-expressing cancers.

The human leukemia oncogene bcr-abl abrogates the anchorage requirement but not the growth factor requirement for proliferation.

Proliferation of normal cells in a multicellular organism requires not only growth factors but also the proper attachment to the extracellular matrix. A hallmark of neoplastic transformation is the loss of anchorage dependence which usually accompanies the loss of growth factor requirement. The Bcr-Abl tyrosine kinase of human leukemias is shown here to abrogate only the anchorage, not the growth factor, requirement. Bcr-Abl-transformed cells grow in soft agar but do not proliferate in serum-free media. Bcr-Abl does not activate the mitogenic pathway, as indicated by its inability to induce enhancers such as the serum response element or the tetradecanoyl phorbol acetate response element (TRE). However, Bcr-Abl can alleviate the anchorage requirement for the induction of the TRE enhancer; i.e., it allows serum to activate the TRE in detached cells. This activity is dependent on the association of an active Bcr-Abl tyrosine kinase with the actin filaments. Despite its association with the adapter protein Grb2, Bcr-Abls effect on the TRE enhancer is not blocked by dominant negative Ras or Raf. The finding that Bcr-Abl tyrosine kinase abrogates only anchorage dependence may have important implications on the pathogenesis of chronic myelogenous leukemia.

Effect of Bcr sequences on the cellular function of the Bcr-Abl oncoprotein

In Philadelphia chromosome (Ph1)-positive human leukemia, the c-Abl tyrosine kinase is activated by fusion to sequences encoded by the breakpoint cluster region (bcr) gene. Two major types of Bcr-Abl fusion proteins have been found in human leukemia. Fusion of the N-terminal 426 amino acids of Bcr generates p190Bcr-Abl which is mostly found in acute lymphocytic leukemia (ALL), whereas fusion of the N-terminal 902 or 927 amino acids of Bcr generates p210Bcr-Abl mostly found with chronic myelogenous leukemia (CML). Previous studies have demonstrated that both the Bcr and the Abl functional domains contribute to the oncogenic activity of Bcr-Abl proteins. Present in both p190 and p210 is the N-terminal coiled-coil of Bcr (aa 1 – 63), which is shown here to be functionally replaceable with the leucine zipper of the yeast transcription factor GCN4. The ZIP-Bcr-Abl protein transforms Rat-1/myc cells, is autophosphorylated on tyrosine and localized predominantly to actin filaments. Thus, formation of homo-oligomers through either Bcr or GCN4 coiled-coil can activate the tyrosine kinase and F-actin binding functions of Abl. We also found that a Bcr-Abl fusion containing only Bcr amino acids (1 – 191) can efficiently transform Rat-1/myc cells. Fusion of additional Bcr sequences (aa 192 – 923) did not affect the transformation of Rat-1/myc cells but progressively reduced the disruptive effect on the actin cytoskeleton. In particular, the Dbl homology domain present in p210Bcr-Abl but not in p190Bcr-Abl contributes to the stabilization of actin fibers. The modulatory effect of Bcr sequences on actin structure may underlie the apparent pathogenic variations between the different Bcr-Abl fusion proteins.

Tyrosine-kinase-dependent signaling pathways.

Protein tyrosine kinases are important transducers of a variety of extracellular signals that regulate proliferation, differentiation, and specific functions of differentiated cells. Most of the known protein tyrosine kinases are associated with cell surface receptors. Ligand binding to the receptor activates one or more protein tyrosine kinases to initiate a network of signaling pathways that regulate metabolism and gene expression. Recently, a focal adhesion tyrosine kinase (FAK) has been shown to respond to the assembly of adhesion junctions, indicating a role for tyrosine kinase in the transduction of cell adhesion signal. The discovery of a DNA-binding tyrosine kinase, c-Abl, and the identification of RNA polymerase II as a substrate of nuclear tyrosine kinases demonstrate that protein tyrosine kinases may also directly regulate transcription. Highlights of these recent developments in tyrosine-kinase-dependent signaling pathways are summarized here.