SDF-1 Signaling in Myeloid Malignancies,

Abstract 3824

The factor SDF-1 (stromal derived factor-1) was identified as an important chemoattractant factor produced by bone marrow cells. SDF-1 acts on its receptor CXCR4 and plays primordial function in migration, retention and development of hematopoietic progenitors in bone marrow. CXCR4 is expressed in leukemic cells and enables them to access marrow niches that normally are restricted to quiescent stem cells, thereby ensuring its protection from cell death resulting in a worse prognosis. In addition, it may induce activity of metalloproteinases (MMPs), which are enzymes that digest the extracellular matrix, making tumor cells more infiltrating. Moreover, higher levels of TIMP-2, an inhibitor of metalloproteinase 2 (MMP2), correlates with better prognosis in solid tumors. Recently, CXCR7 was identified as another SDF-1-binding receptor and the involvement of CXCR7 with tumor progression is well established in non-hematopoietic malignancies. Since it is well established that CD34 ⁺ progenitor cells from patients with myelodysplastic syndromes (MDS) are not attracted by gradient of SDF-1 despite of having CXCR4 normal expression, we addressed if MDS cells have an abnormal localization of CXCR4 or association with CXCR7. P39 and U937 cell line were used as a model of MDS and AML, respectively. Western blot analysis showed similar expression levels of CXCR4 and CXCR7 in both cell lines however we found, by confocal microscopy and flow cytometry, that CXCR4 was localized in the cytoplasm) of P39 cells while it was was in the membrane of U937 cells. Since the protein quinase C (PKC z) is related to the SDF-1/CXCR4 signaling by increasing CXCR4 expression and its membrane availability, we overexpressed HA-tagged PKCz in P39 cells.This procedure resulted in translocation of CXCR4 to the membrane of P39 cells but did not change the CXCR7 subcellular localization.

Transwell chemotaxis assay showed that P39 cells overexpressing PKCz displayed higher chemotactic ability upon SDF-1 treatment compared with control P39 (35 fold increase pcDNA3-PKCz-HA vs pcDNA3-HA transfected P39 cells, p=0.0032; x² test), suggesting that PKCz restored the chemotactic capacity of P39 cells.

RNA expression of CXCR7 and TIMP-2 was analyzed by Real-time PCR (normalized by GAPDH and HPRT) in bone marrow samples from 50 MDS (FAB= 22 RA, 8 RARS, 20 RAEB), 29 acute myeloid leukemia (AML) patients and 11 healthy donors. CXCR7 mRNA expression did not differ significantly comparing all groups: controls, MDS (low or high risk) and AML whereas the expression of TIMP- 2 mRNA was significantly decreased in high risk MDS (p=0.0033) and AML (p=0.0003), (Mann-Whitney test) compared with normal controls.

Taken together, our results suggest that a defect in the PKC ζ/CXCR4 pathway is involved with the unresponsiveness of MDS cells to SDF-1 and TIMP-2 downregulation could play a role in worse prognosis of myeloid malignancies. The role of CXCR7 is still undefined in myeloid malignancies.