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CXCR4 and MYC dual targeting in DLBCL
Introduction
Methods
Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma among adults, accounting for 30-40% of newly diagnosed cases.1 Although the introduction of rituximab into clinical prac- tice has increased the survival of affected patients by 10- 15%,2 60% of patients with high-risk DLBCL are still not cured by immunochemotherapy and have a dismal out- come. For this subgroup, the development of more effec- tive salvage strategies remains an important objective.
Gene expression profiling studies have confirmed the heterogeneity of DLBCL, not otherwise specified, and have recognized two major subtypes according to the putative cell of origin, i.e. activated B-cell (ABC) and ger- minal center B-cell (GCB).3 These studies have also evi- denced the role of the stromal microenvironment in the pathogenesis of the disease, as well as in environment- mediated resistance of DLBCL cells to chemotherapeu- tics.4 As normal B cells are strongly dependent on soluble cytokines for their development and throughout their whole lifespan, it is not surprising that malignant B cells exploit their microenvironment interaction properties for their own selective advantage.5 The CXCR4 chemokine receptor (fusin, CD184) has a well-known function in normal B-cell development, including homing of hematopoietic stem cells to the bone marrow, B-cell and T-cell lymphopoiesis, leukocyte trafficking, and B-cell positioning in the germinal center, among others.6-11 CXCR4 overexpression has been linked to metastasis in a variety of cancers and recently identified as an adverse prognostic factor in DLBCL.12,13 Accordingly, the CXCR4 ligand, CXCL12 (SDF-1α), is among the genes included in the proangiogenic “stromal 2 gene signature” associat- ed with an unfavorable outcome in DLBCL.4 This cytokine is secreted by normal and tumor stroma and is a major regulator of cell chemotaxis.14 Leukemia stem cells and other CXCR4-expressing tumors utilize the CXCL12-CXCR4 signaling axis to localize to vascular and endosteal niches normally restricted to hematopoiet- ic stem cells,15 thus obtaining protection from the effects of cytotoxic chemotherapy and making these niches look like a reservoir for minimal residual disease and relaps- es.16-18 CXCR4 expression allows tumor cell migration, and homing of the neoplastic cells to sites where non- malignant stromal cells express CXCL12.15 This latter promotes tumor progression by recruiting CD31+ endothelial progenitor cells and consequent tumor angio- genesis.19-21 CXCR4 is expressed in hematologic tumors as diverse as B-cell acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia and multiple myeloma, and various ongoing clinical trials for patients with relapsed/refractory hematologic malignan- cies and recurrent high-grade glioma are evaluating the benefit of targeting the tumor microenvironment through CXCL12-CXCR4.22-27
Here we analyzed the clinical significance of CXCL12 expression level in a homogeneous series of patients with de novo DLBCL. We further characterized a new, potent CXCR4 inhibitor showing in vitro and in vivo com- binational activity with a BET bromodomain inhibitor, thereby demonstrating that dual targeting of CXCR4 and MYC represents a promising therapeutic strategy for DLBCL.
Patients’ samples
Fifty-two biopsy specimens from untreated patients with de novo DLBCL from the Catalan lymphoma-study group (GEL- CAB) were included in this study (see details in Online Supplementary Table S1 and the Online Supplementary Materials and Methods). For functional studies, primary tumor cells from five patients were isolated, cryopreserved, and conserved within the hematopathology collection of our institution (Hospital Clínic-IDIBAPS Biobank R121001-094), as previously described.28 Primary cultures were maintained in complete medi- um in the presence of the mesenchymal cell line StromaNKTert (Riken BioResource Center)29 at a 4:1 ratio, to prevent sponta- neous ex vivo apoptosis. The ethical approvals for this project, including informed consent from the patients, were granted fol- lowing the guidelines of the Hospital Clínic Ethics Committee (Institutional Review Board, registration number 2012/7498).
Cell lines
Thirteen DLBCL cell lines from both GCB and ABC subtypes were used in this study. SUDHL-4, SUDHL-6, SUDHL-8, SUDHL-16, NUDHL-1 and U2932 cell lines were purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ). SUDHL-2 and WSU-DLCL2 were obtained from the American Type Culture Collection (ATCC) cell bank (LGC Standards). OCI-LY8 and Toledo were kindly provided by Dr M. Raffeld (National Cancer Institute, Bethesda, MD, USA) and Dr MA Piris (Fundación Jiménez Díaz, Madrid, Spain). OCI-LY3 and OCI-LY10 cells were provided by Dr A. Staiger (Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany). HBL-1 was provided by Dr E Valls (Transcriptional regulation of gene expression group, IDIBAPS, Barcelona, Spain). Cell lines were authenticated upon reception by short tandem repeat profiling, using an AmpFlSTR identifier kit (Thermo Fisher Scientific), and based on available short tan- dem repeat profiles. All cell lines were cultured routinely at 37oC in a humidified atmosphere with 5% carbon dioxide in RPMI 1640 or Iscove modified Dulbecco medium supplemented with 10% to 20% heat-inactivated fetal bovine serum, 2 mM gluta- mine and 50 mg/mL penicillin-streptomycin (Thermo Fisher). Mycoplasm infection was routinely tested for by polymerase chain reaction. SUDHL-6 cells expressing green fluorescent pro- tein (GFP) and Luciferase reporter genes, were generated as pre- viously reported.30
Immunohistochemistry
Fifty-two DLBCL samples were included in tissue microarrays using duplicated cores of 1 mm per tumor sample. CD31+ microvascular density was stained and quantified as previously described.31 Microvascular density values were grouped in quar- tiles and considered high or low when above or below the 50th percentile. Xenograft tumor samples were stained for phospho- histone H3, cleaved caspase-3 and MYC, as previously described.32 Phospho-Akt was detected using a monoclonal anti- phospho-Akt-Ser473 antibody (Cell Signaling Technology). Preparations were evaluated on a DP70 or a BX51 microscope using Cell B Basic Imaging Software (Olympus).
Western blot analysis
Whole cell proteins were extracted from 107 DLBCL cells as previously described.28 Proteins (30-50 mg/lane) were subjected to 10-12% sodium dodecylsulfate-polyacrylamide gel elec- trophoresis, and transferred onto polyvinylidene difluoride
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