Pharmacological modulation of CXCR4 cooperates with BET bromodomain inhibition in diffuse large B-cell lymphoma

Abstract

Constitutive activation of the chemokine receptor CXCR4 has been associated with tumor progression, invasion, and chemotherapy resistance in different cancer subtypes. Although the CXCR4 pathway has recently been suggested as an adverse prognostic marker in diffuse large B-cell lymphoma, its biological relevance in this disease remains underexplored. In a homogeneous set of 52 biopsies from patients, an antibody-based cytokine array showed that tissue levels of CXCL12 correlated with high microvessel density and bone marrow involvement at diagnosis, supporting a role for the CXCL12-CXCR4 axis in disease progression. We then identified the tetra-amine IQS-01.01RS as a potent inverse agonist of the receptor, preventing CXCL12-mediated chemotaxis and triggering apoptosis in a panel of 18 cell lines and primary cultures, with superior mobilizing properties in vivo than those of the standard agent. IQS-01.01RS activity was associated with downregulation of p-AKT, p-ERK1/2 and destabilization of MYC, allowing a synergistic interaction with the bromodomain and extra-terminal domain inhibitor, CPI203. In a xenotransplant model of diffuse large B-cell lymphoma, the combination of IQS-01.01RS and CPI203 decreased tumor burden through MYC and p-AKT downregulation, and enhanced the induction of apoptosis. Thus, our results point out an emerging role of CXCL12-CXCR4 in the pathogenesis of diffuse large B-cell lymphoma and support the simultaneous targeting of CXCR4 and bromodomain proteins as a promising, rationale-based strategy for the treatment of this disease.

Figure 1.

Design of a new potent inhibitor of CXCR4. (A) Skeletal structure of IQS-01.01. *chiral carbons. (B) Inhibition of CXCL12-mediated intracellular cAMP release was determined in the presence of a racemic mixture of IQS-01.01 and its three individually purified stereoisomers, using AMD3100 blocking activity as a reference control. (C) Ball-and-stick representation of IQS-01.01RS. * chiral carbons. (D) MTT assay showing superior antitumor effect of IQS-01.01RS compared to that of the IQS-01.01 racemic mixture after 48 h. The graph shows mean values obtained from three GCB-DLBCL cell lines (SUDHL6, SUDHL-16, and WSU-DLCL2) and three ABC-DLBCL cell lines (OCI-LY3, OCI-LY10 and SUDHL-2). (E) Inhibition of CXCL12-induced migration upon DLBCL cell treatment with a 100 μM dose of IQS-01.01 racemic mixture or IQS-01.01RS. Mean values for the SUDHL-6 and OCI-LY3 cell lines are shown. *P<0.05, **P<0.01, ***P<0.001.

Figure 2.

IQS-01.01RS has better pharmacological properties than those of AMD3100. (A) Predicted docking of IQS-01.01RS (red) and AMD3100 (blue) on their target, CXCR4 (orange). CXCL12 is represented in green. (B) A CXCR4 occupancy assay shows the competition between IQS-01.01RS or AMD3100 (100 μM) with a phycoerythrin-labeled anti-CXCR4 antibody for binding to the receptor. A blocking antibody (30 μg/mL, R&D Systems) was used as a control. (C) Inhibition of CXCL12-induced migration upon DLBCL cell treatment with increasing doses of IQS-01.01RS or AMD3100. The graphs show mean values from three GCB-DLBCL cell lines (SUDHL8, Toledo and SUDHL-6) and two ABC-DLBCL cell lines (U2932 and OCI-LY3). Data are representative of at least three independent experiments. (D) Mean percentage of tumor B cells detected in blood samples of NSG mice injected intravenously with SUDHL-6-GFP-LUC cells and treated for 27 days with IQS-01.01RS, AMD3100, or vehicle (n=4 animals/group). (E) Time-dependent antitumor effect of IQS-01.01RS (100 μM) and AMD3100 (100 μM) in a panel of 13 DLBCL cell lines; the effect was determined by a MTT assay. Representative results from three experiments are shown. (F) Relative induction of apoptosis in CD19⁺ tumor B cells upon treatment of primary DLBCL biopsies (n=5) with the indicated doses of IQS-01.01RS for 48 h. The mean viability of untreated primary cells was 79±8%. (G) Western blot analysis of CXCR4 downstream signaling in SUDHL6 and U2932 cells upon 2 h starvation, followed by exposure to recombinant CXCL12 for 1 min, with or without pretreatment with the indicated doses of IQS-01.01RS or AMD3100. β-actin was used as a loading control. (H) CXCR4 downstream signaling and MYC modulation in SUDHL-6 cells at different time points, after 2 h starvation followed by receptor triggering in the presence or absence of 100 μM IQS-01.01RS. β-actin was used as a loading control. Ab: antibody: TM: transmembrane. *P<0.05, ***P<0.001.

Figure 3.

IQS-01.01RS synergizes with the BET bromodomain inhibitor CPI203 in vitro. (A) The relative antitumor effect of IQS-01.01RS (100 μM), CPI203 (0.5 μM) and the combination of both was determined by a MTT assay, after 48 h. The data shown are the mean results of the 13 DLBCL cell lines. (B) Cooperation between IQS-01.01RS and CPI203 in the inhibition of CXCR4 downstream signaling, as assessed by western blot analysis of p-AKT and MYC. SUDHL-6 and U2932 cells were starved for 2 h, and treated for 1 h with 100 μM IQS-01.01RS and/or CPI203 (0.5 μM) prior to a 1 min stimulation with 200 ng/mL recombinant CXCL12. α-tubulin was used as a loading control. (C) Relative MYC transcript levels in SUDHL-6 and U2932 cells upon 6 h treatment with 100 μM IQS-01.01RS, 0.5 μM CPI203 and the combination of both. Control untreated cells were used as a reference. (D) Time-dependent determination of MYC protein levels in SUDHL-6 cells treated with the translational blocker cycloheximide, as previously described, in the presence or absence of 100 μM IQS01.01-RS. β-actin was used as a loading control. CI: combination index; CHX: cycloheximide; COMBO: combination treatment with IQS-01.01RS and CPI203.

Figure 4.

IQS-01.01RS and CPI203 cooperate to reduce tumor growth in a subcutaneous mouse model of diffuse large B-cell lymphoma. NSG mice were subcutaneously injected with SUDHL6-GFP⁺Luc⁺ cells and tumor-bearing mice were randomly assigned to one of the following treatment arms (4 mice per group): IQS-01.01RS 2 mg/kg daily (per os), CPI203 1.5 mg/kg BID (intraperitoneally), both agents or equal volume of vehicle, for 2 weeks. (A) Tumor volume was evaluated twice a week using external calipers. (B) Tumor burden was evaluated at week 3 and week 4 by analysis of the bioluminescence signal. Left panel: color maps of two representative animals per group. Right panel: quantification of luciferase activity using Image J software. (C) Mean tumor weight in each treatment group at the final time point. (D) Immunohistochemical labeling of p-histone H3, activated caspase-3, MYC and p-AKT in consecutive tissue sections from four representative tumor specimens (magnification ×200). act casp3: activated caspase 3; COMBO/combo: combination treatment with IQS-01.01RS and CPI203; SEM: standard error of mean; ns=not significant; *P<0.05.