CXCR4 Up-Regulation by Imatinib Mesylate Induces CML Cell Migration to Bone Marrow Stroma and Promotes Survival of Chemoresistant Quiescent CML Cells.
Authors: Tabe Yoko , Jin Linhua , Leysath Clinton E. , Kimura Shinya , Kantarjian Hagop , Andreeff Michael , Konopleva Marina 
Published: October 2019

Abstract
Chronic myelogenous leukemia (CML) is driven by constitutively activated BCR-ABL tyrosine kinase which triggers impaired CML cells adhesion to BM stroma. Abnormal circulation and proliferation of CML hematopoietic progenitors has been attributed to the reduced expression or function of the CXC chemokine receptor 4 (CXCR4) (Cancer Research2005;65:2676). We investigated CXCR4 expression on CD34+ bone marrow cells from primary CML patients and its modulation by INF-α or imatinib mesylate. CXCR4 expression was significantly lower in untreated (n=12, 10.1 ± 2.8% CXCR4+ cells) or hydroxyurea-treated CML patients (n=16, 11.2 ± 2.4% CXCR4+ cells) compared to normal bone marrow CD34+ cells (n=12, 46.9 ± 6.2% CXCR4+, p<0.001). CXCR4 expression was increased in a group of patients treated with INF-α (32.4 ± 5.2%, n=15, p=0.001). Longitudinal analysis of CXCR4 expression on CD34+ cells from 4 CML-blast crisis patients treated with imatinib demonstrated upregulation of CXCR4 levels in peripheral blood CD34+ cells (pre-treatment 26.9 ± 20.8%; day 3–15 of STI-571, 52.2 ± 14.0% (n=4)). In the in vitro co-culture system with bone marrow-derived stromal cells (MSC) exposure of KBM-5 cells to Abl tyrosine kinase inhibitors imatinib increased CXCR4 membrane expression (% CXCR4 positive, KBM-5 control 16.2 ± 1.6, with MSCs 24.6 ± 3.8, with MSCs/imatinib 42.3 ± 6.3, p=0.04). In contrast, this effect was not seen in KBM-5/STI cells harboring T315 mutation in Bcr/Abl (CXCR4 positive %, KBM-5/imatinib control 14.7 ± 1.5, with MSCs 20.9 ± 1.1, with MSCs/imatinib 22.7 ± 2.2, p=0.35). A novel Bcr-Abl/Lyn dual inhibitor, INNO-406 (formerly NS-187) also increased CXCR4 membrane expression in KBM-5 but not in KBM-5/STI (data not shown). This increase in CXCR4 expression by Abl tyrosine kinase inhibitors was associated with enhanced migration to MSCs, which was inhibited by blocking CXCR4 with the pharmacological CXCR4 antagonist AMD3465 (AnorMED). In KBM-5 cells, 0.5μM imatinib induced a G0/1 cell cycle block which further increased in co-culture with MSC (% cells in G1/G0: KBM-5 control 38.8 ± 5.2, with MSC 53.7 ± 4.1, imatinib 59.8 ± 2.5, imatinib with MSC 77.9 ± 5.5, p=0.01). Most importantly, imatinib/MSC-mediated cell cycle arrest resulted in inhibition of Ara-C induced apoptosis, which was partially reversed by AMD3465 (% AnnexinV(+) cells: KBM-5 treated with Ara-C, 52.7 ± 4.1; KBM-5 treated with Ara-C in MSC co-culture, 49.4 ± 2.0; KBM-5 treated with Ara-C and imatinib in MSC co-culture, 29.9 ± 3.1; KBM-5 treated with Ara-C, imatinib plus AMD3465 in MSC co-culture, 39.4 ± 5.6, p=0.02). Taken together, these findings indicate that p210BCR-ABL de-regulates CXCR4 expression in CML cells which can be restored by Abl tyrosine kinase inhibitors or INF-α. Up-regulation of CXCR4 by imatinib within the BM microenvironment induces migration of CML cells to stroma and promotes G0/1 cell cycle arrest of CML cells, rendering them quiescent and drug resistant. Our results suggest that interfering with the protective effects of BM stroma cells through CXCR4 inhibition could be beneficial for the eradication of quiescent chemoresistant CML cells. This hypothesis is currently under investigation in primary CML samples.