Chemokine receptor CXCR4 is critically involved in the migration of hematopoietic cells to the stromal derived factor (SDF-1α)-producing bone marrow microenvironment. We and others have previously demonstrated that stroma/leukemia interactions mediate protection of leukemic cells from chemotherapy-induced apoptosis (

Konopleva, Leukemia 2002:1713
;
Burger Blood 2000: 2655
). Using AMD3465, the second-generation small-molecule CXCR4 inhibitor with a greater potency than AMD3100, we tested the hypothesis that CXCR4 inhibition interferes with stromal/leukemia cell interactions resulting in increased sensitivity to chemotherapy. Our results showed that AMD3465 inhibited surface expression of CXCR4 on AML cell lines in a dose dependent manner. AMD3465 (1μM) significantly inhibited SDF-1α and stromal (MS-5)-induced migration of OCI-AML2 cells (78% and 54% inhibition, respectively), U937 cells (71% and 41.3%) and diminished SDF-1α- or stromal-induced migration of leukemic blasts from four primary AML samples tested (SDF-1α, 43.4 ± 8.6%, MS-5, 38.4 ± 8.5% inhibition). In in vitro co-culture systems, stromal cells significantly protected leukemic cell lines and primary AML cells from spontaneous and chemotherapy induced apoptosis (p<0.01; p<0.001). Measurements of intracellular Ara-CTP levels determined by HPLC showed that stromal cells diminished incorporation of Ara-C into leukemic cells by 20%. AMD3465 enhanced AraC- and Busulfan-induced apoptosis by 44% and 69%, respectively. Western blot revealed that AMD3465 downregulated AKT signaling in AML cells. Most importantly, it decreased stroma-mediated protection from AraC-induced apoptosis in five out of ten primary AML samples with surface expression of functional CXCR4 (mean increase, 29.9±19.5% compared to chemotherapy alone). Curiously, the highest sensitization was observed in a sample from AML patient harboring Flt3/ITD mutation (Ara-C, 30.3% annexinV(+); Ara-C+AMD, 62.8%), confirming recently documented role for Flt3/ITD in modulation of CXCR4 signaling (
Fukuda, Blood 2005:3117
). Taken together, our data suggest that SDF-1α/ CXCR4 interactions contribute to the resistance of leukemic cells to chemotherapy-induced apoptosis. Disruption of these interactions by the potent CXCR4 inhibitor AMD3465 represents a novel strategy for targeting leukemia cell/bone marrow microenvironment interactions. A clinical trial testing this concept in patients with AML is in preparation.

Topics:
antagonists, apoptosis, cell communication, chemotherapy regimen, cxcr4 receptors, leukemia, leukemic cells, cytarabine, impedance threshold device, ms-like tyrosine kinase 3

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2005, The American Society of Hematology
2005
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