Increased Expression of CD47 Is a Constant Marker in Mouse and Human Myeloid Leukemias.

Abstract CD47, also known as integrin associated protein, is a ubiquitously expressed cell surface glycoprotein that interacts with a number of integrins, modulating leukocyte adhesion, migration, cell motility and platelet activation. CD47 is also the ligand for the macrophage inhibitory receptor signal regulatory protein α (SIRP α) and thus, impairs macrophage-mediated phagocytosis. Recent reports suggest that increased CD47 expression may play a role in the pathogenesis of lymphoproliferative disorders such as CLL (Mateo et al, Nat Med. 1999; 5:1277) and multiple myeloma, and that a bivalent single-chain antibody fragment against human CD47 induces apoptosis of myeloma cells (Kikuchi et al, Leukemia Research 2005; 29:445). However, little is known about the role of CD47 in the pathogenesis of myeloid leukemias or the stage of hematopoiesis at which CD47 is expressed. In order to identify the stage of hematopoiesis at which alterations in CD47 arise and its role in the pathogenesis of myeloid leukemias, we analyzed CD47 expression in hematopoietic stem cells (HSC), progenitors, and lineage-positive cells derived from three mouse transgenic models of myeloid leukemia including: 1) mice homozygous for overexpression of human bcl-2 in the myeloid lineage (hMRP8 bcl-2 x bcl-2); 2) mice deficient in Fas together with myeloid targeted overexpression of bcl-2 (Faslpr/lpr hMRP8 bcl-2); and 3) mice with both human bcl-2 and BCR-ABL targeted to the myeloid lineage (hMRP8-BCR-ABLxhMRP8-bcl-2). Quantitative RT-PCR analysis demonstrated a 3.01+/− 1.54 fold increase in CD47 transcripts in leukemic compared with control (bcl2+) bone marrow (normalized to b-actin) while FACS analysis revealed approximately a 10-fold increase in CD47 protein expression, as measured by mean fluorescence intensity (MFI), in leukemic GMP compared with wild type GMP. Moreover, transplantation experiments revealed that all mice with both primary (n=14 mice) and secondary (n=19 mice) leukemic transplantation potential had an expansion of granulocyte-macrophage progenitors (GMP) with high level CD47 expression. Human CD47 expression analysis was performed via FACS on human normal, pre-leukemic myeloproliferative disorder (MPD) or AML HSC, progenitors, and lineage positive cells derived from marrow or peripheral blood. MPD samples (n=63) included polycythemia vera (PV; n=15), post-polycythemic myeloid metaplasia/myelofibrosis (PPMM/MF; n=5), essential thrombocythemia (ET; n=8), atypical chronic myelogenous leukemia (aCML; n=2), CML (n=7), chronic eosinophilic leukemia (CEL; n=1), chronic myelomonocytic leukemia (CMML; n= 13) and acute myelogenous leukemia (AML; n=12). As noted with the transgenic leukemic mouse models, progression of human myeloproliferative disorders to AML (n=12) was associated with an expansion of the GMP pool (70.6% +/− S.D. 2.15) compared with normal bone marrow (14.7% +/− S.D. 2.3). Furthermore, FACS analysis revealed that CD47 expression first increased 1.7 fold in AML compared with normal HSC and then increased to 2.2 fold greater than normal with commitment of AML progenitors to the myeloid lineage. CD47 was over-expressed by AML primitive progenitors and their progeny but not by the majority of MPD (MFI 2.3+/−S.D. 0.43) compared with normal bone marrow (MFI 1.9 +/−S.D. 0.07). Thus, increased CD47 expression may serve as a useful diagnostic marker for progression to AML and may represent a novel therapeutic target.