CD34+ Cells Mobilized with the New CXCR4 Antagonist AMD3100 + G-CSF Show Increased Anti-Apoptotic, Cell Cycle, DNA Repair and Cell Motility-Associated Gene Expression When Compared to G-CSF Mobilization Alone.

AMD3100 is a new CXCR4 antagonist which induces a rapid release of hematopoietic progenitors from the bone marrow to the peripheral blood. We conducted a clinical study where patients with multiple myeloma and Non-Hodgkin’s lymphoma were treated with AMD3100 (A) to increase the number of peripheral blood progenitor cells (PBPC) when given an mobilization regimen of granulocyte colony-stimulating factor (G-CSF, G). Since experimental data suggest that A+G-mobilized PBPC are functionally different from G-mobilized PBPC we were interested in an intraindividual comparison of the gene expression profile of CD34+ cells in the two different settings.

To this end peripheral blood CD34+ cells of 3 patients (3 G, 3 A+G samples) were isolated by immunomagnetic followed by flow cytometric sorting to a purity of >99%.

Total RNA was purified. Differentially expressed genes were analysed by using the GeneChip Human Genome U133 Plus2.0 (Affymetrix). The expression analysis was performed with the SAS-MAS 1.0.3 software (SAS Institute Inc.). We found a pattern of unanimously higher (81 genes, fold-change > 2e^0.5, p<10e⁻⁴) or lower (29 genes, fold-change > 2e^−0.4, p<10e⁻⁴) expressed genes in the G+A-mobilized vs. G-mobilized CD34+ PBPC. The significant changes noted in the microarray analysis were validated by representative quantitative real-time PCR of four genes in these three patients and in further four independent patients. Genes were grouped according to gene function. Only increased expression was found in the categories anti-apoptosis (e.g. MPO, HSPA1B), cell cycle (e.g. MS4A3, RRM2), replication/DNA repair (e.g. DNTT RRM2), cell motility (e.g. TNFSF4, HMMR) and oxygen transport. Only decreased expression occurred in the pro-apoptosis gene group (e.g. MDA5 BCL10). A mixed pattern of decreased and increased gene expression was found for the categories transcription, cytoskeleton, immune response, metabolism and signalling.

CXCR4 receptor gene expression itself was significantly increased 1.5-fold in the A+G vs. G group. Interestingly also regulating molecules upstream of CXCR4 such as Id1, MYC, CREBP1, CEBP were increased in their expression while molecules downstream of CXCR4, e.g. in the MAP kinase pathway were significantly decreased (EGR1, EGR3) or increased (DUSP1, DUSP6), suggesting activity of feedback loops. We conclude that A+G mobilizes CD34+ PBPC with increased gene expression in categories which potentially promote superior engraftment after myeloablative therapy than G-mobilized CD34+ PBPC.