Page 202 - Haematologica May 2020

L. Chen et al.
(germinal center B- (GCB-) and activated B-cell like (ABC)-type tumors, respectively).3,5-7
In both types of BCR-dependent DLBCLs, inhibition of SYK or PI3K decrease the phosphorylation of AKT and Forkhead Box O1 (FOXO1) and increase the nuclear retention and associated activity of unphosphorylated FOXO.13,8 BCR-dependent DLBCLs with low baseline NF-κB (GCB tumors) frequently exhibit inactivating mutations or copy loss of Phosphatase and tensin homolog (PTEN) and decreased abundance of the PTEN protein.1,3,6 In these DLBCLs, proximal inhibition of BCR signaling primarily modulates the PI3K/AKT pathway.3,5- 7,9 In contrast, SYK/PI3K blockade additionally limits BTK/NF-κB signaling in BCR-dependent DLBCLs with high baseline NF-κB activity and frequent MYD88L265P and/or CD79B mutations (ABC tumors).1,3,7,9
We sought to identify an indicator of BCR dependence in DLBCLs with low or high baseline NF-κB and noted that C-X-C chemokine receptor 4 (CXCR4) transcripts were significantly more abundant in both DLBCL sub- types following the inhibition of proximal BCR signal- ing.3 In experimental model systems, BCR engagement promotes the internalization of CXCR4 and limits stro- mal cell-derived factor-1α) (SDF-1α)-induced chemo- taxis.10 For these reasons, we hypothesized that BCR blockade might increase CXCR4 expression and associ- ated tumor cell migration.
Chemical inhibition of SYK, PI3K or BTK
The chemical SYK inhibitor, R406, was a gift from Rigel Pharmaceuticals (San Francisco, CA, USA). R406 was dissolved in DMSO at a concentration of 10 mM and stored at -80°C. For immediate inhibition, cells were incubated with 1 mM R406 or vehicle alone (in PBS) in a 37°C water bath for 2 hours (h). For long-term inhibition, R406 was added to cell culture medium at a final concentration of 1 mM and cells were maintained in an incubator at 37°C for 24 h. The chemical pan-PI3K inhibitor, LY294002, was purchased from Sigma-Aldrich (Saint Louis, MO, USA), The chemical SYK inhibitor, GS-9973 (entospletinib), the PI3K isoform-predominant inhibitors, GDC-0941 (pictilisib, PI3K α/d>β/γ), CAL101 (idelalisib, d) and IPI145 (duvelisib, d/γ) and the BTK inhibitor, PCI-32765 (ibrutinib) were purchased from Selleckchem (Houston, TX, USA). DLBCL cell lines were treated with GS-9973 (2 mM), LY294002 (10 mM), GDC-0941 (0.5 mM), CAL101 (2 mM), IPI145 (1 mM), PC1-32765 (0.1 mM) or vehicle (DMSO) for 24 h as previously described.9 The doses of SYK, PI3K and BTK inhibitors used in these studies were deter- mined based on prior analyses of the respective agent EC50s of these agents;9 the LY294002 dose was chosen based on previous- ly reported studies.3,16 Following treatment with chemical SYK, PI3K or BTK inhibitors, cells were harvested for additional analyses (below).
Quantitative RT-PCR (qRT-PCR)
QRT-PCR was performed as previously described9 (Online Supplementary Materials and Methods).
Flow cytometry
A PE-conjugated mouse anti-human CD184 (CXCR4) anti- body (BD Bioscience, CA, USA) was used for flow cytometry analysis on a FACS Canto II flow cytometer (BD Biosciences) and the data were analyzed with FlowJo 10 software (Flowjo Data analysis software LLC, Ashland, OR, USA).
Lentiviral-mediated shRNA transduction
The shRNA knockdown of target genes was performed as previously described3 (Online Supplementary Materials and Methods).
Transduction with myristoylated AKT
DLBCL cell lines were retrovirally transduced with constructs encoding constitutively active (myristoylated) AKT (pMIG- mAKT1-IRES-GFP) or pMIG-IRES-GFP as previously described.3 After 72 h, GFP+ cells were sorted, treated with R406 or vehicle, and analyzed for CXCR4 expression by flow cytometry.
Chemotaxis assay
Physiologically, the CXCR4 chemokine receptor binds to SDF-1αand plays a critical role in the chemotaxis of normal germinal center (GC) B cells.11-13 CXCR4 is a known FOXO1 target gene that is induced in normal FOXO1-rich dark zone GC B-cells.13 In the GC, CXCR4+ B-cells migrate in response to a SDF-1α chemokine gra- dient.11
CXCR4 transduces SDF-1α signals via G-protein cou- pled activation of PI3K isoforms.14-18 As a consequence, CXCR4 is also considered to be a possible therapeutic target in multiple B-cell malignancies, including DLBCL.19-24 Herein, we assess CXCR4 modulation and signaling as both an indicator of sensitivity to BCR blockade and a potential resistance mechanism in DLBCL.
Methods
Cell lines and culture conditions
The DLBCL cell lines, SU-DHL4 (DHL4), SU-DHL6 (DHL6), OCI-LY7 (LY7), HBL1, TMD8, U-2932, Karpas 422 (K422), Toledo and OCI-LY4 (LY4), were cultured as previously described.25 The identities of the DLBCL cell lines used in this study were confirmed via STR profiling with PowerPlex ®1.2 system (Promega, Madison, WI, USA). DHL4, DHL6, LY7, HBL1 and U-2932 were previously characterized as BCR- dependent and K422, Toledo and LY4 were BCR-independent.3,9
Primary tumor specimens
Cryopreserved viable primary DLBCL samples were obtained according to the Institutional Review Board (IRB) – approved protocols from the Brigham and Women’s Hospital Department of Pathology. These anonymous primary tumor specimens were considered discarded tissues which did not require informed consent. The six primary DLBCLs were previously characterized for surface immunoglobulin (Ig) expression, BCR signaling and baseline NF-κB activity.3
DLBCL cell lines were treated with vehicle, R406 (1 mM), GDC-0941 (0.5 mM), Ibrutinib (0.1 mM), AMD3100 (10 mM) or R406 + AMD3100 for 24 h. Before the chemotaxis assay, Permeable Polycarbonate Membrane Inserts in the CorningTM TranswellTM 24-well plate (Fisher Scientific, pore size 8 mm) were pretreated by adding 600 mL of RPMI-1640 media contain- ing 0.5% bovine serum albumin (Sigma) with or without SDF-1α (25-100 ng/mL, R&D Systems) into the bottom chamber at 37°C for 1 h. Each lot of SDF-1α was individually titrated for activity in the chemotaxis assay prior to use. Treated cells were harvested and resuspended in RPMI-1640 media for a final den- sity of 2×106/mL. 100 mL of cell suspension was transferred to each top chamber and incubated at 37 °C for 2-4 h. Cells in the lower chambers were harvested and cell numbers were deter- mined by manual counting. Each condition was set up in tripli- cate.
1362
haematologica | 2020; 105(5)