Immunomodulatory Effects of CC-4047 on Hematopoietic Progenitors Are Associated with a Shift in the Cytokine Secretion Profile Favoring Myeloid Differentiation.

CC-4047 is an immunomodulatory analog of thalidomide with stronger anti-myeloma and anti-angiogenic activity than thalidomide. We could show that CC-4047 directly influences lineage commitment and differentiation of hematopoietic stem cells (Koh et al., Blood 2004 in press). We found that CC-4047 effectively inhibits erythroid cell colony formation from CD34+ cells and increases the frequency of myeloid colonies. We also demonstrated that development of both erythropoietin-independent and erythropoietin-dependent red cell progenitors was strongly inhibited by CC-4047, while terminal red cell differentiation was unaffected. However, there is little information regarding the mechanism by which CC-4047 affect hematopoiesis. Due to the fact that CC-4047 has been shown to influence secretion of proinflammatory cytokines of peripheral mononuclear cells after LPS stimulation we investigated the cytokine profile of hematopoietic progenitors treated with this drug. CD34+ cells were cultured with SCF, IL-3 and IL-6 in the presence of thalidomide (100μM) or CC-4047 (100μM) for 1, 3 or 6 days and cytokine gene expression was studied in these hematopoietic progenitor cells using gene array analyses. Furthermore, supernatants were collected and examined for IL-1b, IL-2, IL-4, IL-5, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, TNF-a, IFN-g, GM-CSF, G-CSF, MCP-1, and MIP-1b.

Our analysis revealed that cytokines supporting myelopoiesis increased very early after treatment with CC-4047. After CC-4047 stimulation, secretion of G-CSF increased within 24 hours 10-fold in comparison to control cells. MCP-1, which is known to support predominantly the granulocytic lineage and to augment the clonal expansion of hematopoietic progenitor cells, increased also up to 5-fold on day 1 under CC-4047 treatment in compared to control. Secretion of IL-10, a pro-inflammatory cytokine known to inhibit erythropoiesis, was also up regulated. In addition, IL-13, which favors the development of erythroid progenitors, decreased 3-fold by CC-4047 on day 1 compared to control. In contrast, thalidomide induced much weaker changes in cytokine secretion. This is in line with our observation that thalidomide has only weak effects on lineage commitment.

Cytokine analysis after 24 hours

In contrast to the previous findings that CC-4047 inhibits TNF-a, IL-12 and IL-1b synthesis in activated mononuclear cells are our results showing that secretion of TNF-a, IL-12, IL-1b and also of IL-2, IL-4, IL-7, IL-8, IL-17, IFN-g, GM-CSF and MIP-1b is not significantly affected by CC-4047 and thalidomide. Analyses of cytokine gene expression confirmed our results. In conclusion, these data indicate that CC-4047 might directly influence lineage commitment of hematopoietic cells by modulation of cytokine secretion increasing the propensity of stem and/or progenitor cells to undergo myeloid cell development and concomitantly inhibiting red cell development. The influence on cytokine secretion is an early event since these changes can observed within the first 24 hours of CC-4047 treatment and depends strictly on cell type and differentiation level. CC-4047 provides a valuable tool to study the mechanisms underlying lineage commitment.