Abstract

The chemokine receptor CXCR4 is critically involved in migration of hematopoietic cells to the stromal derived factor (SDF-1α)-producing bone marrow microenvironment. CXCR4 is regulated in part by mutant FLT3 signaling, but in a series of 122 AML samples with diploid karyotype and lack of FLT3 mutation (ITD), high CXCR4 expression negatively correlated with DFS and OS (p=0.03 and p=0.04, respectively), after multivariate analysis (Konoplev, ASH 2006). We hypothesized that inhibition of SDF-1α-/CXCR4 interactions would result in mobilization of leukemic blasts from the bone marrow into circulation. The in vivo effect of the CXCR4 antagonist AMD3100 was studied in three patients with AML, who had insufficient mobilization of CD34+ cells for autologous stem cell transplantation with G-CSF and/or cytoxan. The combination of G-CSF (10 μg/kg QD) and AMD3100 (240 μg/kg QD SC starting on d4 and repeated for 3–4 days) resulted in massive mobilization of leukemic cells into the circulation in a time-dependent fashion, as determined by flow cytometry and interphase FISH analysis of their respective cytogenetic abnormalities.

Patient #Cytogenetics% (+) cells% (+) cellsApheresis
FCMDay 2Day 4/5CD34x106/kg
Trisomy 21  22.6 57.0 
 FCM CD7/33  22.0  
Trisomy 9 28.6 68.6  
 Inv 16 29.0 75.8 4.8 
 FCM CD13/33  74.0  
Mono 17 40.4 53.4  
 5q31 37.5 49.6 8.7 
 FCM CD13/33  50.0  
Patient #Cytogenetics% (+) cells% (+) cellsApheresis
FCMDay 2Day 4/5CD34x106/kg
Trisomy 21  22.6 57.0 
 FCM CD7/33  22.0  
Trisomy 9 28.6 68.6  
 Inv 16 29.0 75.8 4.8 
 FCM CD13/33  74.0  
Mono 17 40.4 53.4  
 5q31 37.5 49.6 8.7 
 FCM CD13/33  50.0  

We and others have previously demonstrated that stroma/leukemia interactions mediate protection of leukemic cells from chemotherapy-induced apoptosis (

Konopleva et al,
Leukemia
2002
:
1713
). We then tested the hypothesis that CXCR4 inhibition would result in increased sensitivity to chemotherapy, using AMD3465, the second generation small-molecule CXCR4 inhibitor with greater potency than AMD3100. Results demonstrate inhibition of surface expression of CXCR4 and of SDF-1α-, and stroma(MS-5)-induced migration of AML cells. In vitro co-culture systems with stromal cells significantly protected leukemic cells (p < 0.01), while AMD3465 decreased stroma-mediated protection from AraC and Busulfan apoptosis and downregulated AKT signaling in AML cells. In a murine model of luciferase labeled Baf-FLT3ITD leukemias, AMD3465 induced massive dissemination of leukemia, which was abrogated by treatment with Sorafenib, a potent FLT3ITD inhibitor (Zhang, ASH 2006). 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 CXCR4 inhibition results in leukemia dissemination and chemosensitization. Our results in leukemia patients provide first in man proof-of principle for a novel strategy of targeting the leukemia cell/bone marrow microenvironment interactions. A clinical trial testing this concept in patients with AML is under development.

Disclosures: Drs. Gary Calandra and Gary Bridger are employees of AnorMED.; Drs. Calandra and Bridger own stocks in AnorMED.

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