Lenalidomide Upregulates CXCR4 on CD34⁺ Hematopoietic Cells Resulting In Increased Binding to Bone Marrow Niche and Inhibiting Mobilization Into Peripheral Blood In Multiple Myeloma Patients

In multiple myeloma (MM), lenalidomide has impressive clinical activity in patients with both relapsed/refractory and newly diagnosed disease. Nevertheless hematopoietic stem cell transplantation (HSCT) is still the “backbone” in the treatment of newly diagnosed MM patients and very often lenalidomide treatment (as induction therapy) is combined with HSCT in order to achieve high response rates. Clinical data have shown that in up to 43% of those patients, standard mobilization of CD34⁺ cells with G-CSF alone failed to mobilize significant numbers of hematopoietic progenitors into peripheral blood, raising concerns about potential stem cell toxicity of lenalidomide (Mazumder et al, Leukemia 2008). Interestingly mobilization of hematopoietic progenitors with AMD-3100 (Plerixafor) overcomes mobilization failures in almost all patients previously treated with lenalidomide. The fact that AMD-3100 antagonizes the binding of chemokine stromal- cell–derived factor-1α (SDF-1α) to CXC chemokine receptor 4 (CXCR4) suggests a potential role of the CXCR4/SDF-1α axis in mediating mobilization failure after lenalidomide treatment. Subsequently, the findings noted above raised questions on: 1) the stem cell toxicity of lenalidomide; 2) the underlying mechanism that mediates the development of G-CSF resistance; and 3) the mechanism of the modulation of the CXCR4/SDF-1α axis by lenalidomide. In our previous work we showed the following. 1) Lenalidomide neither inhibited colony formation in standard colony assays nor the development of cobble stone area forming cells (CAFC) in LTC-IC assays, suggesting that lenalidomide is not stem cell toxic (Koh et al, Blood 2005). 2) We further showed that lenalidomide significantly upregulated G-CSF secretion of CD34⁺ cells (600%), suggesting that the high levels of G-CSF may mediate a relative resistance towards G-CSF-induced mobilization (Pal et al, Blood, 2010).

We analyzed why blocking CXCR4 by AMD-3100 overcomes mobilization failure to G-CSF. We first examined the CXCR4 expression profile of CD34⁺ cells treated with lenalidomide. Lenalidomide treatment significantly upregulated the expression of CXCR4 on cell surface after 48h treatment measured by flow cytometry. Increased expression of CXCR4 onCD34⁺ cells remained high with continuous treatment. Western blot assay of the hydrophobic (membrane) and the hydrophilic (cytosol) cell fraction confirmed our data showing that lenalidomide increases the expression of CXCR4 on CD34⁺ cell surface. Confocal microscopy showed that lenalidomide inhibited SDF-1α induced CXCR4 internalization. In accordance with the increased CXCR4 surface expression, transwell migration assay revealed that the SDF-1α-induced migration of CD34⁺ cells in the presence of lenalidomide significantly increased by 52% in comparison to control. Quantitation of SDF-1α showed that CXCR4 had no significant effect on the secretion of SDF-1α by CD34⁺ cells and in human stromal cells.

In conclusion, our data show that lenalidomide is not toxic to hematopoietic progenitors. The strong increase of G-CSF secretion by CD34⁺ cells might contribute to a desensitization of CD34⁺ cells to G-CSF mobilization. Primarily our data indicate that increased expression of CXCR4 followed by blocked internalization, increases binding to SDF-1α secreted by the bone marrow niche. This subsequently prohibits the mobilization of CD34⁺ cells. These data suggest that blocking the CXCR4 receptor by AMD-3100 disrupts this circle and finally permits the mobilization of hematopoietic cells from the bone marrow niche into peripheral blood. This study provides novel insights into the effects of lenalidomide on CD34⁺ cells relevant for HSCT in MM.

Roodman:Amgen: Consultancy; Celgene Corp: Consultancy; Acceleron: Consultancy; Millennium: Consultancy. Mapara:Gentium: Equity Ownership. Lentzsch:Celgene Corp: Research Funding.