ARTICLE Non-Hodgkin Lymphoma Ferrata Storti Foundation Pharmacological modulation of CXCR4 cooperates with BET bromodomain inhibition in diffuse large B-cell lymphoma Clara Recasens-Zorzo,1 Teresa Cardesa-Salzmann,2 Paolo Petazzi,3 Laia Ros-Blanco,4 Anna Esteve-Arenys,1 Guillem Clot,1 Martina Guerrero-Hernández,1 Vanina Rodríguez,1 Davide Soldini,2 Alexandra Valera,2 Alexandra Moros,1 Fina Climent,5 Eva González-Barca,6 Santiago Mercadal,6 Leonor Arenillas,7 Xavier Calvo,7 José Luís Mate,8 Haematologica 2019 Gonzalo Gutiérrez-García,9 Isolda Casanova,3,10 Ramón Mangues,3,10 Volume 104(4):778-788 Alejandra Sanjuan-Pla,11 Clara Bueno,3 Pablo Menéndez,3,12 Antonio Martínez,1,2 Dolors Colomer,1,2 Roger Estrada Tejedor,4 Jordi Teixidó,4 Elias Campo,1,2 Armando López-Guillermo,1,9 José Ignacio Borrell,4 Luis Colomo,2,7 Patricia Pérez-Galán1 and Gaël Roué1,13 1 Division of Hemato-Oncology, Institut d'Investigacions Biomediques August Pi I Sunyer (IDIBAPS), CIBERONC, Barcelona; 2Hematopathology Unit, Department of Pathology, Hospital Clinic, Barcelona; 3Josep Carreras Leukemia Research Institute, Department of Biomedicine, School of Medicine, University of Barcelona; 4Grup d’Enginyeria Molecular, IQS School of Engineering, Universitat Ramon Llull, Barcelona; 5Pathology Department, Hospital Universitari de Bellvitge, L’Hospitalet de Llobregat; 6Institut Catalá d’Oncología, Hospital Duran I Reynals, L’Hospitalet de Llobregat; 7Pathology Department, IMIM, Hospital del Mar, Barcelona; 8Pathology Department, Hospital Universitari Germans Trias i Pujol, Badalona; 9Department of Hematology, Hospital Clinic, Barcelona; 10Grup d’Oncogènesi i Antitumorals, lnstitut d’Investigacions Biomèdiques Sant Pau (IIB-Sant Pau) and Centro de Investigación Biomédica en Red CIBER-BBN, Barcelona; 11 Hematology Research Group, Health Research Institute La Fe, Valencia; 12Institucio Catalana de Recerca I Estudis Avançats (ICREA), CIBERONC, Barcelona and 13Laboratory of Experimental Hematology, Department of Hematology, Vall d'Hebron Institute of Oncology, Vall d’Hebron University Hospital, Barcelona, Spain LC, PP-G and GR. share senior authorship. Correspondence: GAËL ROUÉ groue@vhebron.net ABSTRACT C PATRICIA PÉREZ-GALÁN onstitutive activation of the chemokine receptor CXCR4 has pperez@clinic.ub.es been associated with tumor progression, invasion, and chemotherapy resistance in different cancer subtypes. Although Received: September 8, 2017. the CXCR4 pathway has recently been suggested as an adverse prognos- Accepted: June 25, 2018. tic marker in diffuse large B-cell lymphoma, its biological relevance in Pre-published: June 28, 2018. this disease remains underexplored. In a homogeneous set of 52 biopsies from patients, an antibody-based cytokine array showed that tissue lev- doi:10.3324/haematol.2017.180505 els of CXCL12 correlated with high microvessel density and bone mar- row involvement at diagnosis, supporting a role for the CXCL12-CXCR4 Check the online version for the most updated axis in disease progression. We then identified the tetra-amine IQS- information on this article, online supplements, 01.01RS as a potent inverse agonist of the receptor, preventing CXCL12- and information on authorship & disclosures: www.haematologica.org/content/104/4/778 mediated chemotaxis and triggering apoptosis in a panel of 18 cell lines and primary cultures, with superior mobilizing properties in vivo than ©2019 Ferrata Storti Foundation those of the standard agent. IQS-01.01RS activity was associated with Material published in Haematologica is covered by copyright. downregulation of p-AKT, p-ERK1/2 and destabilization of MYC, allow- All rights are reserved to the Ferrata Storti Foundation. Use of ing a synergistic interaction with the bromodomain and extra-terminal published material is allowed under the following terms and conditions: domain inhibitor, CPI203. In a xenotransplant model of diffuse large B- https://creativecommons.org/licenses/by-nc/4.0/legalcode. cell lymphoma, the combination of IQS-01.01RS and CPI203 decreased Copies of published material are allowed for personal or inter- tumor burden through MYC and p-AKT downregulation, and enhanced nal use. Sharing published material for non-commercial pur- poses is subject to the following conditions: the induction of apoptosis. Thus, our results point out an emerging role https://creativecommons.org/licenses/by-nc/4.0/legalcode, of CXCL12-CXCR4 in the pathogenesis of diffuse large B-cell lym- sect. 3. Reproducing and sharing published material for com- phoma and support the simultaneous targeting of CXCR4 and bromod- mercial purposes is not allowed without permission in writing from the publisher. omain proteins as a promising, rationale-based strategy for the treatment of this disease. 778 haematologica | 2019; 104(4) CXCR4 and MYC dual targeting in DLBCL Introduction Methods Diffuse large B-cell lymphoma (DLBCL) is the most Patients’ samples common type of non-Hodgkin lymphoma among adults, Fifty-two biopsy specimens from untreated patients with de accounting for 30-40% of newly diagnosed cases.1 novo DLBCL from the Catalan lymphoma-study group (GEL- Although the introduction of rituximab into clinical prac- CAB) were included in this study (see details in Online tice has increased the survival of affected patients by 10- Supplementary Table S1 and the Online Supplementary Materials 15%,2 60% of patients with high-risk DLBCL are still not and Methods). For functional studies, primary tumor cells from cured by immunochemotherapy and have a dismal out- five patients were isolated, cryopreserved, and conserved within come. For this subgroup, the development of more effec- the hematopathology collection of our institution (Hospital tive salvage strategies remains an important objective. Clínic-IDIBAPS Biobank R121001-094), as previously Gene expression profiling studies have confirmed the described.28 Primary cultures were maintained in complete medi- heterogeneity of DLBCL, not otherwise specified, and um in the presence of the mesenchymal cell line StromaNKTert have recognized two major subtypes according to the (Riken BioResource Center)29 at a 4:1 ratio, to prevent sponta- putative cell of origin, i.e. activated B-cell (ABC) and ger- neous ex vivo apoptosis. The ethical approvals for this project, minal center B-cell (GCB).3 These studies have also evi- including informed consent from the patients, were granted fol- denced the role of the stromal microenvironment in the lowing the guidelines of the Hospital Clínic Ethics Committee pathogenesis of the disease, as well as in environment- (Institutional Review Board, registration number 2012/7498). mediated resistance of DLBCL cells to chemotherapeu- tics.4 As normal B cells are strongly dependent on soluble Cell lines cytokines for their development and throughout their Thirteen DLBCL cell lines from both GCB and ABC subtypes whole lifespan, it is not surprising that malignant B cells were used in this study. SUDHL-4, SUDHL-6, SUDHL-8, exploit their microenvironment interaction properties for SUDHL-16, NUDHL-1 and U2932 cell lines were purchased their own selective advantage.5 The CXCR4 chemokine from the Deutsche Sammlung von Mikroorganismen und Zellkulturen receptor (fusin, CD184) has a well-known function in (DSMZ). SUDHL-2 and WSU-DLCL2 were obtained from the normal B-cell development, including homing of American Type Culture Collection (ATCC) cell bank (LGC hematopoietic stem cells to the bone marrow, B-cell and Standards). OCI-LY8 and Toledo were kindly provided by Dr M. T-cell lymphopoiesis, leukocyte trafficking, and B-cell Raffeld (National Cancer Institute, Bethesda, MD, USA) and Dr positioning in the germinal center, among others.6-11 MA Piris (Fundación Jiménez Díaz, Madrid, Spain). OCI-LY3 CXCR4 overexpression has been linked to metastasis in and OCI-LY10 cells were provided by Dr A. Staiger (Dr. a variety of cancers and recently identified as an adverse Margarete Fischer-Bosch Institute of Clinical Pharmacology, prognostic factor in DLBCL.12,13 Accordingly, the CXCR4 Stuttgart, Germany). HBL-1 was provided by Dr E Valls ligand, CXCL12 (SDF-1α), is among the genes included (Transcriptional regulation of gene expression group, IDIBAPS, in the proangiogenic “stromal 2 gene signature” associat- Barcelona, Spain). Cell lines were authenticated upon reception ed with an unfavorable outcome in DLBCL. 4 This by short tandem repeat profiling, using an AmpFlSTR identifier cytokine is secreted by normal and tumor stroma and is kit (Thermo Fisher Scientific), and based on available short tan- a major regulator of cell chemotaxis.14 Leukemia stem dem repeat profiles. All cell lines were cultured routinely at 37ºC cells and other CXCR4-expressing tumors utilize the in a humidified atmosphere with 5% carbon dioxide in RPMI CXCL12-CXCR4 signaling axis to localize to vascular 1640 or Iscove modified Dulbecco medium supplemented with and endosteal niches normally restricted to hematopoiet- 10% to 20% heat-inactivated fetal bovine serum, 2 mM gluta- ic stem cells,15 thus obtaining protection from the effects mine and 50 mg/mL penicillin-streptomycin (Thermo Fisher). of cytotoxic chemotherapy and making these niches look Mycoplasm infection was routinely tested for by polymerase like a reservoir for minimal residual disease and relaps- chain reaction. SUDHL-6 cells expressing green fluorescent pro- es.16-18 CXCR4 expression allows tumor cell migration, tein (GFP) and Luciferase reporter genes, were generated as pre- and homing of the neoplastic cells to sites where non- viously reported.30 malignant stromal cells express CXCL12.15 This latter promotes tumor progression by recruiting CD31+ Immunohistochemistry endothelial progenitor cells and consequent tumor angio- Fifty-two DLBCL samples were included in tissue microarrays genesis.19-21 CXCR4 is expressed in hematologic tumors using duplicated cores of 1 mm per tumor sample. CD31+ as diverse as B-cell acute lymphoblastic leukemia, acute microvascular density was stained and quantified as previously myeloid leukemia, chronic lymphocytic leukemia and described.31 Microvascular density values were grouped in quar- multiple myeloma, and various ongoing clinical trials for tiles and considered high or low when above or below the 50th patients with relapsed/refractory hematologic malignan- percentile. Xenograft tumor samples were stained for phospho- cies and recurrent high-grade glioma are evaluating the histone H3, cleaved caspase-3 and MYC, as previously benefit of targeting the tumor microenvironment described.32 Phospho-Akt was detected using a monoclonal anti- through CXCL12-CXCR4.22-27 phospho-Akt-Ser473 antibody (Cell Signaling Technology). Here we analyzed the clinical significance of CXCL12 Preparations were evaluated on a DP70 or a BX51 microscope expression level in a homogeneous series of patients using Cell B Basic Imaging Software (Olympus). with de novo DLBCL. We further characterized a new, potent CXCR4 inhibitor showing in vitro and in vivo com- Western blot analysis binational activity with a BET bromodomain inhibitor, Whole cell proteins were extracted from 107 DLBCL cells as thereby demonstrating that dual targeting of CXCR4 and previously described.28 Proteins (30-50 mg/lane) were subjected MYC represents a promising therapeutic strategy for to 10-12% sodium dodecylsulfate-polyacrylamide gel elec- DLBCL. trophoresis, and transferred onto polyvinylidene difluoride haematologica | 2019; 104(4) 779 C. Recasens-Zorzo et al. membranes (Immobilon-P; Millipore), or nitrocellulose mem- For the systemic DLBCL model, 8-week old NSG female mice branes (Amersham Biosciences). Membranes were then incubat- (n=12) were inoculated intravenously with 107 SUDHL6-GFP- ed with primary and secondary antibodies (Online Supplementary Luc cells, randomly assigned into three equivalent cohorts, and Materials and Methods) and visualized on a mini-LAS4000 device treated daily with 5 mg/kg AMD3100 (intraperitoneally), 10 (Fujifilm) by enhanced chemiluminescence (ECL, Amersham mg/kg IQS-01.01RS salt (per os) or vehicle. After 27 days, ani- Life Science). mals were sacrificed and peripheral blood was collected by sub- mandibular puncture. Erythrocytes were lysed using ACK buffer Flow cytometry analysis (Quality Biological) and the percentage of SUDHL-6 cells was For the detection of surface CXCR4, DLBCL cells (2x105) were evaluated by detection of a GFP signal on an Attune cytometer. stained with a phycoerythrin (PE)-labeled anti-CXCR4 or iso- For the subcutaneous DLBCL model, SUDHL6-GFP-Luc cells type control antibody (BD Biosciences). For the CXCR4 occu- were inoculated subcutaneously in 8-week old NSG female mice pancy assay, cells were pretreated for 1 h with IQS-01.01, as previously.28,32 Animals were randomly assigned into four AMD3100 (Sigma-Aldrich) or an anti-human CXCR4 control cohorts of four mice each and were given a twice daily dose of antibody, followed by sCXCR4 staining as above. For quantifi- 1.5 mg/kg CPI203 (intraperitoneally), a daily dose of 2 mg/kg cation of apoptosis, cells were labeled with Pacific Blue-conju- IQS-01.01RS (per os), both agents, or an equal volume of vehicle. gated annexin V (Thermo Fisher). A total of 10,000 events were Tumor engraftment was determined weekly following mice acquired and analyzed on an Attune acoustic focusing cytometer injection with 75 mg/kg D-luciferine (AnaSpec) and biolumines- (Thermo Fisher). The mean fluorescence intensity ratio was cal- cence imaging (BLI) using an Aequoria Luxiflux device equipped culated as the ratio between the median fluorescence intensity with an ORCA-ER camera (Hamamatsu). The bioluminescence of the CXCR4-labeled sample and that of the isotype. imaging signal was quantified using Image J software. Tumor volume was measured by external calipers twice a week, up to Chemotaxis assay 28 days. Animals were then sacrificed and tumors were harvest- DLBCL cells (5x106 cells/mL) were cultured for 1 h in culture ed and weighed. Animals were handled following protocols medium not containing fetal bovine serum but supplemented approved by the Animal Ethics Committee of the University of with 0.5% bovine serum albumin (Sigma-Aldrich), in the pres- Barcelona (protocol #154/16). ence or absence of 100 mM AMD3100 or IQS-01.01RS, and ana- lyzed for CXCL12-dependent chemotaxis, as previously Statistical analysis described.28 Values are referred to cells cultured without A Wilcoxon rank-sum test, Fisher exact test, Spearman rank CXCL12. correlation or t-test was used to examine the statistical signifi- cance of associations between clinico-pathological data and Cell proliferation assay CXCL12-CXCR4 positivity or cytokine expression level, as DLBCL primary cultures and cell lines (5x104 cells) were incu- appropriate. Survival curves were estimated using the Kaplan- bated for 24-48 h with IQS-01.01RS, AMD3100 and/or CPI203 Meier method. A log-rank test was used to compare survival (kindly provided by Constellation Pharmaceuticals) at the indi- curves between groups. For in vitro and in vivo functional assays, cated doses. MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenylte- unpaired and paired t-tests were employed to obtain the statisti- trazolium bromide) reagent (Sigma-Aldrich) was added for 1–5 cal significance. The Benajmini-Hochberg method was used to additional hours before spectrophotometric measurements. adjust P-values for multiple testing. Results were considered sta- Each measurement was made in triplicate. Values are represent- tistically significant when the P value was less than 0.05. ed using untreated control cells as the reference. Combination indexes were calculated using Calcusyn software version 2.0 (Biosoft). The interaction between two drugs was considered Results synergistic when the combination index was less than 0.8. CXCL12 expression correlates with microvascular In vivo assays density and confers an unfavorable prognosis Prior to efficacy testing, an acute toxicity assay was conducted to patients with diffuse large B-cell lymphoma in NSG mice exposed to IQS-01.01RS salt or AMD3100. Gene expression profiling studies have highlighted the Animals (2 adult males and 2 adult females per dose) received a prognostic value of the microenvironment and compo- single administration of vehicle, IQS01.01-RS (per os) or plerix- nents of several immune regulatory pathways in DLBCL.4 afor (intraperitoneally) at doses ranging from 2 to 10 mg/kg, and To characterize which cytokines may have a significant were monitored during the first 4 h after administration, and impact on the pathogenesis of DLBCL, we performed a daily for 2 weeks, for viability/mortality and vital parameters. fluorescence-based cytokine antibody array using frozen This toxicity assay defined a maximum non-lethal dose of 5 tissue from 12 DLBCL cases, out of an initial cohort of 52 mg/kg for AMD3100, while the maximum lethal dose of IQS- patients (Online Supplementary Table S1), and correlated 01.01RS remained unreached. expression data with clinico-biological characteristics of Table 1. Top cytokines associated with high microvessel density in diffuse large B-cell lymphoma biopsies. Cytokine Mean Mean Difference t statistic P Adjusted P (MVD=low) (MVD=high) SDF1α/CXCL12 5.207 5.637 0.430 -3.758 0.000 0.032 IGFBP2 4.467 4.946 0.479 -3.744 0.000 0.032 IGFBP1 5.085 5.601 0.516 -3.666 0.001 0.032 MVD: microvessel density. 780 haematologica | 2019; 104(4) CXCR4 and MYC dual targeting in DLBCL the patients. Among the 175 cytokines included in this phenylene group harboring two chiral carbons. The com- array, we found that only CXCL12, IGFBP2 and IGFBP3 pound thus comports three stereoisomers, namely IQS- correlated significantly with high microvascular density 01.01RR, IQS-01.01SS and IQS-01.01RS (Figure 1A). (P<0.05) (Table 1), a parameter associated with an unfa- Nitrogen atoms on each site are intercalated in carbon vorable prognosis in DLBCL.31,33 Additionally, high chains at a similar distance as those of the cyclam in expression of CXCL12 correlated significantly with bone AMD3100 providing IQS-01.01 with positively charged marrow involvement at diagnosis (P=0.02, data not nitrogen atoms at physiological pH which, as with shown), thus supporting a role for the CXCL12-CXCR4 AMD3100, will interact with the lateral chains of acidic axis in the progression of DLBCL. amino acids of CXCR4, but with greater flexibility. We first assessed the inhibitory activity of IQS-01.01 (racemic IQS-01.01RS is a novel CXCR4 inhibitor with improved mixture), and its three individually purified stereoiso- pharmacological properties mers, using a CXCR4 antagonist screening study. To evaluate the therapeutic potential of targeting AMD3100 was used as a reference drug. As shown in CXCR4 in DLBCL, we took advantage of a novel family Figure 1B, the three stereoisomers had superior CXCR4 of CXCR4 inhibitors with improved pharmacodynamic antagonist activity to that of AMD3100, with IQS- properties and lower cardiotoxicity than the standard 01.01RS (see structure in Figure 1C) being the most CXCR4 inhibitor AMD3100.34,35 Similarly to AMD3100, potent agent. This compound led to a 181% inhibition of IQS-01.01 is a symmetric molecule with a central p- receptor activity, thus resembling an inverse receptor ago- A B C D E 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 mM 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. haematologica | 2019; 104(4) 781 C. Recasens-Zorzo et al. A D C D E F G H 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 mM) with a phycoerythrin-labeled anti-CXCR4 antibody for binding to the receptor. A blocking antibody (30 mg/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 mM) and AMD3100 (100 mM) 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 mM IQS-01.01RS. β-actin was used as a loading control. Ab: antibody: TM: transmembrane. *P<0.05, ***P<0.001. 782 haematologica | 2019; 104(4) CXCR4 and MYC dual targeting in DLBCL Table 2. Sensitivity of diffuse large B-cell lymphoma cell lines to CXCR4 and BET bromodomain inhibition. CXCR4 expression IQS-01.01RS CPI203 Combination (MFI-R) cytotoxic effect cytotoxic effect index Subtype Cell line (100 µM) (0.5 µM) OCI-LY8 36 32% 38% 1.1 SUDHL-4 25 37% 59% 0.6 SUDHL-6 41 42% 47% 0.4 GCB SUDHL-16 1 36% 86% 0.5 WSU-DLCL2 3 35% 55% 0.4 Toledo 52 9% 43% 0.5 SUDHL-8 91 38% 14% 1.1 NUDHL-1 1 36% 60% 0.8 OCI-LY3 4 48% 75% 0.7 ABC OCI-LY10 1 40% 40% 0.8 SUDHL-2 1 53% 95% 0.8 U2932 46 43% 31% 0.9 HBL-1 1 63% 39% 0.6 ABC: activated B-cell; GCB: germinal center B-cell; MFI-R: median fluorescence intensity ratio. nist. We then analyzed the antitumor activity of this AKT and the AKT downstream kinase GSK3-β, was highly active stereoisomer, together with IQS-01.01 superior to that of AMD3100 (Figure 2G). Of special (racemic mixture) in six representative DLBCL cell lines interest, IQS-01.01RS, rather than AMD3100, allowed of both GCB and ABC subtypes. As shown in Figure 1D, strong downregulation of the MYC proto-oncogene in IQS-01.01RS was significantly more active than the ABC- and GCB-DLBCL cells (Figure 2H). This down- racemic mixture at all the doses tested, with the greatest stream target of CXCR4 with a well-established role in effect being observed at the 100 mM dose (mean cytotox- the progression of DLBCL,37 has been reported to depend icity: 44%; range, 42-49%). This effect was mainly attrib- on AKT phosphorylation level for its stabilization, medi- uted to proliferation blockade, as IQS-01.01RS-treated ated by a GSK3-β-dependent phosphorylation of its cultures showed low (< 5%) levels of apoptosis (data not Thr58 residue and consequent proteosomal degrada- shown). In agreement with this, migration experiments tion.38,39 Supporting the hypothesis that the downregula- using a CXCL12 gradient further demonstrated that IQS- tion of MYC may be consequent to inhibition of the 01.01RS was a more potent inhibitor of cell chemotaxis CXCR4-AKT axis in IQS-01.01RS-treated cells, the than was the racemic mixture, with a 2-fold improve- expression of both p-AKT and MYC was almost com- ment of cell migration blockade (Figure 1E). Based on pletely abrogated within a few minutes after the addition these results, we proceeded to a deeper study of the of IQS-01.01RS (Figure 2H) and this phenomenon could stereoisomer of choice, IQS-01-01RS. be maintained for at least 3 h (data not shown). A predictive docking model showed that this stereoiso- Collectively, these results indicate that the new CXCR4 mer localized into the extracellular CXCL12 binding inhibitor IQS-01.01RS has a unique structure that enables domain of CXCR4, close to the position occupied by its binding to two distinct domains of the receptor, con- AMD3100, and simultaneously interacted with an inner, ferring improved mobilizing properties in vivo, as well as transmembrane domain of the receptor. This unique fea- superior antitumor activity in vitro. ture potentially provides IQS-01.01RS with the ability to inhibit CXCR4 in the presence of the ligand, and to inter- IQS-01.01RS cooperates with BET bromodomain fere with the catalytic activity of the receptor (Figure 2A). inhibition in vitro and in vivo Supporting this model, using the representative cell line Based on our observation that IQS-01.01RS had the SUDHL-6 we observed that IQS-01.01RS and AMD3100 capacity to downregulate MYC, we further investigated had similar CXCR4 occupancy activity (Figure 2B), and in our 13 DLBCL cell lines whether the compound could had similar anti-migratory properties against a CXCL12 cooperate with the BET bromodomain antagonist gradient in vitro, both in ABC- and GCB-DLBCL cell lines CPI20332 to block cell proliferation. The combination of (Figure 2C). However, IQS-01.01RS triggered superior each agent induced up to 78% cytoxicity at the optimal mobilization of DLBCL cells into the circulating blood of doses of 100 mM IQS-01.01RS and 500 nM CPI203, with the animals (Figure 2D) and was able to block the prolif- a mean CI of 0.67, indicative of a synergistic drug interac- eration of a panel of 13 GCB/ABC-DLBCL cell lines in a tion in both ABC- and GCB-DLBCL cells (Figure 3A and time-dependent manner, contrasting with the modest Table 2). As expected, a dramatic reduction in MYC pro- antitumor activity of AMD3100 (40% versus 12% mean tein levels was observed after exposure to the combina- cell growth inhibition at 48 h) (Figure 2E and Table 2). In tion (Figure 3B). We confirmed by quantitative poly- addition, in a set of five DLBCL primary cultures, a dose- merase chain reaction analysis and cycloheximide protein dependent induction of apoptosis was observed upon stability assays that this phenomenon was consequent to exposure to IQS-01.01RS (Figure 2F), contrasting with the a simultaneous CPI203-evoked transcriptional repression reported inability of AMD3100 to induce cell death.36 of MYC gene and an IQS-01.01RS-mediated destabiliza- Accordingly, the analysis of CXCR4 downstream signal- tion of MYC protein (Figure 3C,D). Confirming that this ing showed that the capacity of IQS-01.01RS to inhibit effect was not due to a BRD4 inhibitor-mediated dimin- basal and CXCL12-induced phosphorylation of ERK1/2, ished production of CXCR4, as previously described in T haematologica | 2019; 104(4) 783 C. Recasens-Zorzo et al. lymphocytes following JQ1 treatment,40 Figure 3B shows completely lack CXCR4 molecules on their surface. The that CPI203 addition did not alter CXCR4 protein levels main difference between these two cell lines was the either in the presence or absence of IQS-01.01RS. Flow response to CPI203 as a single agent, which was substan- cytometry analysis further confirmed that surface levels tially weaker in NALM6-CXCR4-KO cells. This may of CXCR4 were unmodified by BET bromodomain inhi- illustrate the recent finding that, in acute myeloid bition (data not shown). To further ascertain the role of leukemia cells, CXCR4 signaling is involved in the regula- CXCR4 expression in this drug interaction, we estab- tion of MYC transcription mediated by the downregula- lished genetic depletion of CXCR4 in SUDHL-6 cells tion of the miRNA let-7a,41 while in DLBCL cells no vari- employing a CRISPR/Cas9 gene editing tool to generate ation in MYC mRNA levels are observed following CXCR4 knocked out (KO) cell lines, prior to treatment CXCR4 ligation (Online Supplementary Figure S2). Another with CPI203, IQS-01.01RS and the combination of both explanation may come from the lower membrane expres- agents. In parallel, and as a control of non-lymphomatous sion (about one log10) of the receptor observed in parental B malignancy, we used the recently described NALM6- acute myeloid leukemia cells when compared to parental CXCR4-KO cell line (kindly provided by Dr Jan Burger, DLBCL cells, suggesting that NALM6 cells are less Department of Leukemia, The University of Texas MD dependent on CXCR4 and less susceptible to CXCR4- Anderson Cancer Center, Houston, TX, USA). As shown dependent cellular stress than are SUDHL-6 cells. Most in Online Supplementary Figure S1, CXCR4-KO cell lines importantly, in the acute myeloid leukemia cell line the A B C D Figure 3. IQS-01.01RS synergizes with the BET bromodomain inhibitor CPI203 in vitro. (A) The relative antitumor effect of IQS-01.01RS (100 mM), CPI203 (0.5 mM) 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 mM IQS-01.01RS and/or CPI203 (0.5 mM) 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 mM IQS-01.01RS, 0.5 mM 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,62 in the presence or absence of 100 mM 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. 784 haematologica | 2019; 104(4) CXCR4 and MYC dual targeting in DLBCL loss of CXCR4 was associated with 30% and 24% Thus, these results strongly support a crucial role for decreases in the cytotoxic effect of CPI203 and the CXCR4 in the cooperation between CPI203 and IQS- CPI203/IQS-01.01RS combination, respectively, confirm- 01.01RS in malignant B cells. ing that CXCR4 expression was required for the activity To assess the efficacy of the drug combination in vivo, of the bromodomain inhibitor alone, as well as for its NSG mice were subcutaneously injected with SUDHL6- synergistic activity with IQS-01.01RS. In the case of GFP-Luc cells, and given IQS-01.01RS, CPI203, the com- SUDHL6, although the combination was still active in bination of both agents, or the equivalent volume of vehi- CXCR4-depleted cells, the loss of the receptor induced a cle, for 13 days. Tumor burden was evaluated weekly by 13% reduction in the synergistic activity of the two bioluminescence signal recording and twice a week by agents when compared with the parental CXCR4+ cells. external calipers. At the final time point, tumors were A B C D 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 subcuta- neously 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 x200). 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. haematologica | 2019; 104(4) 785 C. Recasens-Zorzo et al. extracted and weighed. Figure 4A shows that CPI203 and disease.12 Nonetheless, AMD3100 presents serious limita- IQS-01.01RS as single agents induced 27% and 4% reduc- tions that could preclude its use as an anticancer drug, tions in tumor growth, respectively, while the combina- such as some degree of cardiotoxicity and adverse phar- tion of both drugs induced a 38% decrease in tumor bur- macodynamic properties including a positive charge at den. Accordingly, reduced luciferase activity as well as a physiological pH and a limited half-life, impairing its oral significant (38%) decrease in tumor weight was detected bioavailability.52 Thus, the lack of a CXCR4 inhibitor suit- in mice given combination treatment compared to the able for administration in a standard regimen supports values in the group administered the vehicle (Figure the development of less toxic and more stable CXCR4- 4B,C). Histological analysis of the corresponding tumors targeting agents.53 confirmed a greater reduction of the mitotic index togeth- Here, we describe the biological activity of IQS- er with an accumulation of apoptotic cells by the combi- 01.01RS, a new potential CXCR4 inhibitor with a higher nation therapy, as assessed by phospho-histone H3 and lethal dose in vivo and better pharmacodynamic proper- activated-caspase-3 staining (Figure 4D). In agreement ties than AMD3100.35 We report that by interacting with with the in vitro results, greater reductions in MYC and a different CXCR4 domain than that with which phospho-Akt were observed in animals treated with the AMD3100 interacts, this IQS-01.01RS acts as a putative combination of drugs (Figure 5D). Collectively, these allosteric CXCR4 inhibitor, impeding the activation of results suggest that the combination of IQS-01.01RS with the receptor upon CXCL12 ligation and allowing a more the BET inhibitor CPI203 enhances the antitumor proper- sustained inhibition of the pathway. In vivo, this effect, ties of each single agent through the blockade of CXCR4 together with better pharmacokinetic properties, was signaling, followed by the abrogation of MYC expression linked to an improved capacity of the compound to and the induction of apoptosis. mobilize DLBCL cells. Furthermore, we show that IQS- 01.01RS-mediated blockade of CXCR4 signaling led to the post-transcriptional downregulation of MYC. Discussion Overexpression of this oncogene is associated with shorter survival in DLBCL patients,54 and is known to be The chemokine receptor CXCR4 has a prominent role stabilized by CXCR4 in cancer cells.55 MYC was consid- in homing and retention of tumor cells in their microen- ered to be an undruggable factor until (+)-JQ1, a highly vironment, and in the promotion of drug resistance.42 It potent, selective and cell-permeable inhibitor of BRD4, a has been previously reported that CXCR4/CXCL12 member of the BET family of chromatin adaptors, was expression by tumor cells confers an adverse prognosis to first shown to have antitumor activity in multiple myelo- DLBCL patients.12,43 In agreement with this, we observed ma xenograft models.56 Further studies with (+)-JQ1 and that CXCL12 expression in tumor biopsies correlated CPI203, a molecule characterized by a superior bioavail- with bone marrow involvement at diagnosis, as well as ability after oral or intraperitoneal administration,32,57 highly vascularized tumors. These observations support have validated BET bromodomain targeting and subse- the hypothesis that the CXCL12-CXCR4 axis may play a quent blockade of MYC, NF-kB or BCL-2 protein family significant role in neo-angiogenesis in the DLBCL signaling as a promising therapeutic strategy in different microenvironment. Accordingly, various reports have subtypes of aggressive B-cell lymphoma, including highlighted a correlation between CXCR4 expression and DLBCL.58-60 BRD4 is a global regulator of gene transcrip- high serum levels of VEGF in different cancer sub- tion which selectively recognizes and binds to acetylated types.19,21,44 CXCL12 is also one of the genes included in lysine residues in histones to activate transcription and the DLBCL pro-angiogenic and unfavorable “stromal-2 mitosis. In DLBCL it preferentially localizes in super- signature”,4 while activation of the CXCL12-CXCR4 axis enhancers associated with key transcription factors is related to tumor angiogenesis and recruitment of implicated in lymphomagenesis, such as MYC, which endothelial progenitor cells to tumors in myelodysplastic are specifically sensitive to BRD4 inhibition thus explain- syndrome, glioma and pancreatic cancer.20,45,46 ing the selective anti-tumor effect of BRD4 inhibitors.61 It The CXCL12-CXCR4 axis may, therefore, represent a is worth pointing out that BET bromodomain inhibition, new therapeutic target for DLBCL, as inhibition of as a consequence, downregulates other tumor-related CXCR4 and tumor cell mobilization is bound to increase genes apart from MYC.61 Nevertheless, in this work, we the accessibility of these lymphomas to anticancer thera- focused on the capacity of CPI203 to inhibit MYC, con- pies. Validating this strategy, the Food and Drug firming that ABC- and GCB-DLBCL cells are highly sen- Administration-approved drug AMD3100 has been sitive to CPI203 monotherapy, and that the transcription- shown to potentiate the tumor growth inhibition afford- al downregulation of MYC achieved by this compound ed by standard chemotherapeutics and/or irradiation in allows almost complete abrogation of the protein when preclinical models of glioblastoma,26,47 to inhibit the the compound is combined with IQS-01.01RS. This dual engraftment of B-cell acute lymphoblastic leukemia,48 to approach underlies the synergistic interaction of the two mobilize and sensitize acute promyelocytic leukemia compounds and consequent sensitization to apoptosis in cells to cytosine arabinoside,49 and to enhance the vitro and in vivo. response to rituximab and to the anti-CD52 monoclonal In conclusion, the present work shows that, besides antibody alemtuzumab in a mouse model of disseminat- CXCR4, the level of CXCL12 may also have an impact on ed lymphoma.50 In the clinical setting, encouraging pre- the progression of DLBCL, and describes a new potent liminary data have been obtained from a phase II study orally available CXCR4 inhibitor with antitumor proper- using AMD3100 and standard chemotherapy in patients ties. 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