Abstract 4881: Targeting the CXCR4 chemokine receptor thwarts stromal cell mediated drug resistance in B-cell acute lymphoblastic leukemia (B-ALL)
Authors: Randhawa Shubhchintan , Ghosh Dipanjan , Peled Amnon , Davis Richard E. , Burger Jan A. 
Published: May 2015

CXCR4 inhibitors Plerixafor (small molecule) and BKT140 (peptide) interfere with CXCR4 activation, signaling and bone marrow homing of leukemia cells. Adhesion of B-ALL cells to bone marrow stromal cells (BMSC) through CXCR4/CXCL12 protects leukemia cells from cytotoxic drugs, and causes cell adhesion-mediated drug resistance (CAM-DR). Due to the critical role of the CXCR4/CXCL12 axis in B-ALL cell adhesion to bone marrow stromal cells (BMSC), we hypothesize that targeting CXCR4 will attenuate CAM-DR in B-ALL.

All tested B-ALL cell lines and xenografts were found to be positive for surface expression of CXCR4, using 12G5 mAbs. Pre-treatment with Plerixafor (10μg/ml) or BKT140 (10μg/ml) significantly inhibited the spontaneous migration of B-ALL cells beneath BMSCs in vitro (Pseudoemperipolesis/PEP) to levels that were 61.4 ± 3.3% or 53.2 ± 5.6%, of respective controls (mean ± SEM, p&lt;0.05). B-ALL cell line and xenograft B-ALL cell chemotaxis towards CXCL12 (100ng/ml) was also abrogated by pre-incubation with 10μg/ml Plerixafor (mean ± SEM, 26.7 ± 4.9% of control, p&lt;0.05) and 10μg/ml BKT140 (mean ± SEM, 18.3 ± 4.7% of control, p&lt;0.05). In order to better dissect CXCR4 function in B-ALL, we established two CXCR4 knock out cell lines, TANOUE-CXCR4-KO and NALM6-CXCR4-KO, using Zinc Finger Nuclease and CRISPR Cas9 technologies respectively. Chemotaxis to 100ng/ml CXCL12 and PEP beneath BMSCs for TANOUE-CXCR4-KO was 10.3 ± 0.48%, n=3 (p&lt;0.05) and 50.06 ± 2.5%, n=4 (p&lt;0.05) respectively, in comparison to TANOUE wild type control without any drug treatment. For NALM6-CXCR4-KO chemotaxis and PEP was 43.1%, n=1 and 41.8 ± 1.9%, n=4 (p&lt;0.05) respectively in comparison to NALM6 wild type control without any drug treatment.

CAM-DR experiments with ICN12 xenograft-BMSC co-culture showed significantly enhanced cytotoxicity when cells were treated with a combination of 100nM Dexamethasone (DEX) and 10μg/ml Plerixafor (66.1 ± 5.6% viability, n=3, p&lt;0.05) or 10μg/ml BKT140 (59.7 ± 6.07% viability, n=3, p&lt;0.05) as compared to DEX alone (87.3 ± 2.6% viability) at 48 hours. Similarly, pre-incubation of NALM6 cells with 10μg/ml Plerixafor/BKT140 showed significantly enhanced cytotoxicity (57.93±3.26%, n=3, p&lt;0.05 and 54.2±3.26%, n=3, p&lt;0.05 respectively) in comparison to DEX alone (72.38±0.25%, n=3). BMSCs rescued NALM6 cells from DEX induced cytotoxicity but failed to rescue NALM6-CXCR4-KO cells indicating that CXCR4 is the central mechanism used by stromal cells to provide drug resistance to ALL cells. Collectively, these findings provide a rationale for clinical targeting of CXCR4 in patients with B-ALL.

Citation Format: Shubhchintan Randhawa, Dipanjan Ghosh, Amnon Peled, Richard E. Davis, Jan A. Burger. Targeting the CXCR4 chemokine receptor thwarts stromal cell mediated drug resistance in B-cell acute lymphoblastic leukemia (B-ALL). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4881. doi:10.1158/1538-7445.AM2014-4881