CXCR4 Inhibition as a Therapeutic Strategy in Leukemia.
Authors: Zeng Zhihong , Evans Randall L. , Huang Ziwei , Andreeff Michael , Konopleva Marina 
Published: April 2020

Abstract
The chemokine receptor CXCR4 is critically involved in the migration of hematopoietic cells to the stroma derived factor (SDF-1α)-producing bone marrow microenvironment. We and others have previously demonstrated that stromal-leukemic interactions mediate protection of leukemic cells from chemotherapy-induced apoptosis. (Konopleva et al, Leukemia 2002; Tabe, Konopleva, et al, Blood 2004; Burger JA et al., Blood 2000). Using peptide based CXCR4 inhibitors, derived from the chemokine viral macrophage inflammatory protein II (vMIP II), we tested the hypothesis that CXCR4 inhibition interferes with stromal/leukemia cell interactions resulting in increased sensitivity to chemotherapy. CXCR4 was highly expressed on the cell surface of CML myeloid blood crisis cells (KBM5), KBM5/STI-resistant cells, lymphoid CEM and Jurkat cells, myeloid leukemic OCI-AML3 and U937 cells. In contrast, NB4 and TF-1 cells expressed low-levels surface CXCR4, and no surface expression was detected on KG-1 and HL-60 leukemic cells. Among CXCR4(+) cell lines, Jurkat cells demonstrated the highest chemoattractive response to SDF-1α(23 +/− 0.03% migration at SDF-1α50ng/ml, and 54 +/− 0.01% at 100ng/ml). The ability of three CXCR4 inhibitors to inhibit chemotaxis of Jurkat cells was examined in a standard migration assay. Results indicate that D10-vMIP-II, a polypeptide with the first 10 amino acids substituted by the D isoform, exhibits the strongest antagonistic effect on SDF-1α or stromal cell induced chemoattraction. D10-vMIP-II also decreases CXCR4 surface expression in a concentration-dependent manner: flow cytometry and live cell confocal microscopy revealed that within 30min of exposure D10-vMIP-II causes CXCR4 internalization that persisted for at least 4 hrs at 0.01μM and for 24 hrs at 0.1μM. Analysis of SDF-1α-mediated signaling demonstrated that D10-vMIP-II inhibits AKT and ERK phosphorylation. Finally, we examined the effects of D10-vMIP-II on the response to chemotherapy of leukemic cells co-cultured with MS5 stromal cells. Pre-treatment of Jurkat cells enhanced doxorubicin-induced apoptosis: Doxorubicin alone (10μM) 75 +/− 0.07% viable cells compared to control; Doxorubicin and D10-vMIP-II: 53 +/− 0.04% viable cells. Furthermore, D10-vMIP-II enhanced the sensitivity of primary CLL cells to Fludarabine in the in vitro stromal co-culture system. CLL samples with high surface expression of CXCR4 (n=3) co-cultured with stromal MS-5 cells were pre-treated with 0.1μM D10-vMIP II followed by 10μM Fludarabine (9-β-D-arabinofuranosyl-2-fluoroadenine). Stromal cells prevented Fludarabine-induced killing (64%±16.2 viable cells in stromal co-culture compared to 31% viable cells in medium only). Inhibition of CXCR4 signaling abrogated this protective effect and diminished CLL cell survival (26.9±7.1% viable cells, p=0.03 compared to Fludarabine-treated CLL cells co-cultured with MS-5). This growth inhibition was mediated by apoptosis induction as determined by CD45/annexinV flow cytometry (DMSO, 14.49±5.3% annexinV(+) leukemic cells; Fludarabine, 47.2±24.9%; D10-vMIP II followed by Fludarabine, 61.3±18.9%). 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 D10-vMIP-II represents a novel strategy for the targeting leukemic cells within their bone marrow microenvironment.