angiogenin and Leukemia--Myelogenous--Chronic--BCR-ABL-Positive

Angiogenin (ANG) is a secreted ribonuclease (RNase) with cell-type- and context-specific roles in growth, survival, and regeneration. Although these functions require receptor-mediated endocytosis and appropriate subcellular localization, the identity of the cell surface receptor remains undefined. Here, we show that plexin-B2 (PLXNB2) is the functional receptor for ANG in endothelial, cancer, neuronal, and normal hematopoietic and leukemic stem and progenitor cells. Mechanistically, PLXNB2 mediates intracellular RNA processing that contribute to cell growth, survival, and regenerative capabilities of ANG. Antibodies generated against the ANG-binding site on PLXNB2 restricts ANG activity in vitro and in vivo, resulting in inhibition of established xenograft tumors, ANG-induced neurogenesis and neuroprotection, levels of pro-self-renewal transcripts in hematopoietic and patient-derived leukemic stem and progenitor cells, and reduced progression of leukemia in vivo. PLXNB2 is therefore required for the physiological and pathological functions of ANG and has significant therapeutic potential in solid and hematopoietic cancers and neurodegenerative diseases.

Topics: Animals; Breast Neoplasms; Cell Proliferation; Female; Glioblastoma; Hematopoietic Stem Cells; Heterografts; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Mice; Mice, Inbred C57BL; Neoplasm Transplantation; Nerve Tissue Proteins; Neurogenesis; Ribonuclease, Pancreatic

Angiogenesis is critical for the clinical progression of haematopoietic malignancies and depends on angiogenic factors. Angiogenin is a powerful factor produced by neoplastic cells and host microenvironment. High levels of soluble angiogenin (sAng) correlate with a poor prognosis in patients affected by acute myeloid leukaemia and myelodysplastic syndromes, but no data are available on sAng in chronic myeloproliferative disorders (CMD). Therefore, in this study we investigated the clinical significance of the angiogenin in sera of patients with chronic myeloid leukaemia (CML) (n = 14) or essential thrombocythaemia (ET) (n = 20), and correlated them with those of soluble transforming growth factor-beta(1) (sTGF beta(1)). Enzyme-linked immunosorbent assay detected (P < 0.05) higher levels of sAng in CMD compared with healthy subjects (1026.74 +/- 464.60 pg/mL and 196.00 +/- 39.90 pg/mL, respectively). The highest levels of sAng were detected in CML patients (1349.23 +/- 549.55 pg/mL). Interestingly, CML patients who achieved haematological remission after interferon therapy showed circulating levels of angiogenin significantly (P < 0.05) decreased when compared with those at diagnosis. In ET patients, levels of angiogenin (889.34 +/- 267.66 pg/mL) and sTGF beta(1) (76.69 +/-6.08 pg/mL) were higher (P < 0.05) compared with healthy controls (57.93 +/- 19.39 pg/mL). No correlation was found between levels of sAng and levels of sTGF beta(1) or platelet count among ET patients. Our results show for the first time that elevated blood levels of angiogenin feature chronic myeloid malignancies, suggesting a role of angiogenin in the pathogenesis of these diseases.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Case-Control Studies; Chronic Disease; Female; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Middle Aged; Myeloproliferative Disorders; Neoplasm Proteins; Prognosis; Remission Induction; Ribonuclease, Pancreatic; Solubility; Thrombocythemia, Essential; Transforming Growth Factor beta

The construction and expression of a chimeric gene encoding a mouse/human antibody to the human transferrin receptor fused to the gene for angiogenin, a human homolog of pancreatic RNase, are described. F(ab')2-like antibody-enzyme fusions were prepared by linking the gene for human angiogenin to a chimeric anti-transferrin receptor heavy chain gene. The antibody-enzyme fusion gene was introduced into a transfectoma that secretes the chimeric light chain of the same antibody, and cell lines were cloned that synthesize and secrete the antibody-enzyme fusion protein of the expected size at a concentration of 1-5 ng/ml. Culture supernatants from clones secreting the fusion protein caused inhibition of growth and protein synthesis of K562 cells that express the human transferrin receptor but not toward a non-human-derived cell line that lacks this receptor. Whereas excess antibody to the same receptor did not itself inhibit protein synthesis, it was able to completely prevent the protein synthesis inhibition caused by the fusion protein. These results indicate that the cytotoxicity is due to a transferrin receptor-mediated mechanism involving the angiogenin portion of the fusion protein and demonstrate the feasibility of constructing recombinant antibody-RNase molecules capable of killing tumor cells bearing the transferrin receptor. The significance of the acquired cytotoxicity of a mouse/human chimeric antibody linked to a human protein may bear importantly in human therapeutic strategies that use mouse antibodies linked to toxins from plants or bacteria to target tumor cells. It is expected that the humanization of immunotoxins will lead to less toxicity and immunogenicity than currently available reagents.

Topics: Animals; Antibodies; Cell Division; Cell Line; Humans; Immunotoxins; Kinetics; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mice; Neoplasm Proteins; Plasmids; Proteins; Receptors, Transferrin; Recombinant Fusion Proteins; Restriction Mapping; Ribonuclease, Pancreatic; Transfection