67 kDa Laminin Receptor (67LR) Involvement in the Trafficking of Normal and Leukemic Hematopoietic Stem Cells; Computer-Aided Identification of a Small Inhibitory Molecule

The 67 kDa laminin receptor (67LR) is a cell-surface receptor with high affinity for laminin (LM), which plays a key role in tumour progression. 67LR is also involved in normal human T lymphocytes homing and in acute myeloid leukaemia (AML), lymphoma and myeloma cell adhesion and migration. Recently, we demonstrated 67LR involvement in granulocyte colony-stimulating factor (G-CSF)-induced mobilization of CD34+ hematopoietic stem cells (HSCs), in human healthy donors. 67LR has also been shown to contribute to HSC homing to BM after transplantation, in mice. We now investigated the role of 67LR in the trafficking of leukemic CD34+ HSCs and identified a 67LR inhibitory small molecule. Flow cytometric analysis showed a strong upregulation of 67LR expression in BM as well as in peripheral blood (PB) CD34+ cells from acute myeloid leukemia (AML) patients, as compared to normal BM and PB CD34+ cells. Then, we investigated whether 67LR upregulation could modulate CD34+ leukemic cell adhesion and migration to LM and stromal derived factor 1 (SDF1), the key chemokine in HSC trafficking. 67LR-transfected CD34+ cells from the KG1 cell line showed increased migration toward LM and SDF1, as compared to untransfected cells; on the contrary, 67LR overexpression did not increase CD34+ KG1 cell adhesion to LM. 67LR activation by LM determined increased phosphorylation of p38 MAP Kinase, protein Kinase C and calcium/calmodulin-dependent protein kinase II. Using the high resolution crystal structure of 67LR (PDB code: 3BCH), we identified 46 compounds that were predicted to interact with a previously identified LM-binding site on 67LR. Of these 46 chemical hits, compound 1-((4-methoxyphenylamino)methyl)naphthalen-2-ol, termed NSC 47924, specifically inhibited 67LR-mediated cell adhesion, migration and proliferation to LM. Leukemic cells undergo changes in adhesive properties that allow their migration into the circulation, leading to development of extramedullary disease. Our data show that 67LR overexpression is associated with a migratory phenotype both in cytokine-stimulated normal CD34+ HSCs and in leukemic CD34+ HSCs. Moreover, we demonstrated that 67LR function can be specifically blocked by a newly-identified small molecule, making 67LR a promising therapeutical target.