CXCR4 Antagonist BKT140 Synergizes with Rituximab, Targeting Non Hodgkin Lymphoma (NHL) In Vitro and In Vivo in a Xenograft Model with Bone Marrow Involvement
Authors: Beider Katia , Ribakovsky Elena , Abraham Michal , Wald Hanna , Eizenberg Orly , Avigdor Abraham , Peled Amnon , Nagler Arnon 
Published: October 2019

Abstract
Abstract 952

Background:
B-cell Non Hodgkin lymphoma (NHL) represents the most common malignant lymphoid neoplasm, with heterogeneous pathophysiological and clinical presentation. It may show systemic, nodal or extra nodal localization in various sites, including bone marrow (BM). Although anti-CD20 antibody rituximab significantly improved the outcome of NHL patients, the relapsed/refractory rates are still high. Chemokine receptor CXCR4 and its ligand CXCL12 are critically involved in the survival and trafficking of normal and malignant B lymphocytes. Taking together with the fact that interaction of malignant B cells with stromal cells via CXCR4/CXCL12 signaling may provide chemo-resistance, we hypothesized that blockade of CXCR4 may antagonize the survival and spreading of lymphoma cells and restore their chemo-sensitivity.

Results:
The effect of CXCR4 antagonist BKT140 on lymphoma cell growth and rituximab-induced cytotoxicity was tested in vitro and in vivo. Inhibition of CXCR4 with BKT140 in CD20-expressing lymphoma cell lines (BL-2, BJAB, Raji, Ramos, SUDHL4, OCI-Ly7) and primary lymphoma cells from patients with BM involvement resulted in significant inhibition of cell growth and in the induction of cell death, respectively. Combination of BKT140 with rituximab significantly enhanced the cytotoxic effect (apoptosis) against the lymphoma cells in a dose-dependent manner (p<0.01). This effect was concurrent with increase in Annexin V binding, loss of ΔΨm and caspase 3 cleavage. These findings indicate that mitochondrial membrane dysfunction and caspase 3 activation are involved in BKT140- and rituximab-mediated lymphoma cell death. Moreover, we were able to show that rituximab induced CXCR4 expression in lymphoma cell lines and primary lymphoma cells, both on mRNA and cell-surface levels, suggesting the possible interaction between CD20 and CXCR4 pathways in NHL. This interaction provides the rationale for CXCR4 targeting in combination with rituximab treatment. Indeed, highly-expressing CXCR4 BL-2 cells were protected by BM stromal cells from rituximab-induced apoptosis, whereas low-CXCR4 BJAB cells were not. The protective effect of stoma was abrogated by BKT140. To evaluate the in vivo anti-lymphoma effect of BKT140 we established a xenograft model of B-cell lymphoma with BM involvement in mice. Human CXCR4-expressing B NHL cell line, BL-2, was subcutaneously implanted into NOD/SCID mice, resulting in the development of aggressive local tumors which specifically spread to the bone marrow. Following the tumor establishment, mice were injected subcutaneously with BKT140, rituximab or with combination of both. BKT140 inhibited the local tumor progression of BL-2 generated tumors. Furthermore, BKT140 treatment significantly reduced the number of BL-2 tumor cells in the BM by 77% (p<0.01) compared to the untreated mice, while rituximab decreased this number only by 20%. Importantly, the combination treatment of BKT140 with rituximab further decreased the number of viable lymphoma cells in the BM, achieving 93% reduction (p<0.012). We found that BKT140 promoted lymphoma cell apoptosis within the bone marrow microenvironment. Furthermore, we found that the number of viable lymphoma cells in the peripheral blood of tumor-bearing animals was also reduced following the treatment with BKT140.

Conclusions:
Our results demonstrate potent anti-lymphoma effect of CXCR4-specific antagonist BKT140 in vitro and in vivo. The BKT140-mediated anti-lymphoma effect synergizes with that of Rituximab. Moreover, BKT140 effectively targets lymphoma cells in the bone marrow microenvironment, overcoming the stroma-induced resistance to rituximab. These findings suggest the possible interaction between CD20 and CXCR4 pathways in NHL, and provide the scientific basis for the development of novel combined CXCR4-targeted therapies for refractory NHL.

Disclosures:
No relevant conflicts of interest to declare.