Pathophysiological significance and therapeutic targeting of germinal center kinase in diffuse large B-cell lymphoma

Abstract

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma, yet 40% to 50% of patients will eventually succumb to their disease, demonstrating a pressing need for novel therapeutic options. Gene expression profiling has identified messenger RNAs that lead to transformation, but critical events transforming cells are normally executed by kinases. Therefore, we hypothesized that previously unrecognized kinases may contribute to DLBCL pathogenesis. We performed the first comprehensive analysis of global kinase activity in DLBCL, to identify novel therapeutic targets, and discovered that germinal center kinase (GCK) was extensively activated. GCK RNA interference and small molecule inhibition induced cell-cycle arrest and apoptosis in DLBCL cell lines and primary tumors in vitro and decreased the tumor growth rate in vivo, resulting in a significantly extended lifespan of mice bearing DLBCL xenografts. GCK expression was also linked to adverse clinical outcome in a cohort of 151 primary DLBCL patients. These studies demonstrate, for the first time, that GCK is a molecular therapeutic target in DLBCL tumors and that inhibiting GCK may significantly extend DLBCL patient survival. Because the majority of DLBCL tumors (∼80%) exhibit activation of GCK, this therapy may be applicable to most patients.

Figure 1

The MAPK family of proteins is upregulated in DLBCL cell lines. (A) An unsupervised heat map comprising the MAPK pathway proteins exhibiting up- or downregulation in 9 DLBCL cell lines, when compared with normal B cells from reactive tonsils. The displayed matrix represents the log₂ ratio of LC-MS/MS detected activity/expression of the kinases from each experimental DLBCL cell line relative to normal B lymphocytes, as depicted by the corresponding color scale. Each row represents a separate probe binding site for the indicated kinase, identified and quantified by LC-MS/MS, and each column represents a separate DLBCL cellular lysate. KiNativ data were clustered with Cluster software and visualized with Treeview. ABC-like DLBCLs are depicted in blue, and GCB-like DLBCLs are depicted in orange. Similar results were obtained in one additional KiNativ experiment. (B) Immunoblotting of GCK, p-MAP2K4, MAP2K4, p-JNK, and JNK total proteins in unmanipulated DLBCL cell lines. Similar results were obtained in 3 sets of independent blots. (C) Immunoblotting of GCK total protein in muscle cells, human germinal center (GC) B cells, tonsillar B cells, and unmanipulated DLBCL cell lines. Similar results were obtained in 3 sets of independent blots. (D) Normalized expression reflecting average densitometry readings of 3 independent experiments in which total GCK protein levels were quantified and normalized to GAPDH (glyceraldehyde-3-phosphate dehydrogenase) level for protein concentration. Quantification was performed using Image J software (NIH). (E) JNK kinase assay. JNK was immunoprecipitated from the indicated cell lines and tonsils and used to phosphorylate its target c-jun in the presence of ATP. p-c-jun is detected by immunobloting. (F) LC-MS/MS signal for JNK after pull-down with the KiNativ ATP-mimetic probe in B cells purified from human tonsils, and in DLBCL cell lines. KiNativ data (A) is representative of 2 independent experiments. Results in (B-C,E-F) are representative of 3 independent experiments. Results in (D) are the normalized average ± standard error of the mean of 3 independent experiments.

Figure 2

GCK and its downstream effectors are upregulated in de novo primary DLBCL, in a subtype-independent manner. (A) Summary of IHC results from primary DLBCL tumors. Representative staining for GCK negative (B), moderate (C), and strong (D) expressing de novo primary DLBCL tumors. (E) A noncancerous lymph node stained for GCK expression. (F) Fifty-seven primary DLBCLs were analyzed by IHC for GCK, pMAP3K1, pMAP2K7, pMAP2K4, pJNK, and pP38, and (G) 36 primary DLBCLs were stained for GCK, the germinal center markers BCL6, CD10, HGAL, and LMO2, and the ABC-like markers BCL2 and MUM1. Tumor samples were clustered with Cluster software and visualized with Treeview. Kaplan–Meier plots of (H) PFS (P = .04) and (I) overall survival of 151 DLBCL patients treated with R-CHOP grouped on the basis of IHC staining for GCK. The positive cases including GCK medium (n = 33) and high expressing cases (n = 98) were analyzed together. n.s., results that are not statistically significant.

Figure 3

GCK RNA interference leads to cell death and cell-cycle arrest of DLBCL cell lines. (A) DLBCL cells were infected with lentiviral vectors expressing both GFP and either shRNA#1 directed against GCK or a nontargeting (control) vector. The percentage of live cells in the GCK shRNA vector expressing cells at 72 hours postinfection are presented as a percentage of the control cells. The efficacy of these shRNAs is demonstrated by immunoblotting for V5-tagged GCK in 293T cells. (B) Cell-cycle analysis of GCK shRNA#1 vector or control infected cells, representative of results in 3 independent experiments. (C) DLBCL cells were infected with lentiviral vectors with a puromycin resistance cassette and either shRNA#2 directed against GCK or a nontargeting (control) vector. ATP turnover was measured using the ATPlite System, as described in “Methods,” to simultaneously assess growth and viability. A western blot showing representative knockdown in SU-DHL-6 cells is to the right. This experiment was performed 3 times. (D) ON-TARGETplus siRNA was transfected using AMAXA electroporation to knock down GCK expression in OCI-LY-10, OCI-LY-19, SU-DHL-6, or G452 cells. Survival of cells at 72 hours posttransfection was analyzed by flow cytometry. Western blots showing representative knockdown in the same cell line are beneath each survival graph. All experiments were performed at least 3 times. Data in (A,C-D) are represented as mean ± standard error of the mean.

Figure 4

HG6-64-1, a chemical GCK inhibitor. (A) Six DLBCL cell lines and 2 control, non-DLBCL cell lines, were treated with serial dilutions of HG6-64-1. 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl-2-(4-sulfophenyl)-2H-tetrazolium (MTS) readings were taken just prior to and after drug addition and compared with control, DMSO-treated cells. Cellular proliferation was calculated as the change in MTS reading for each experimental condition, divided by the increase in DMSO-treated cells. (B) The calculated EC₅₀ of HG6-64-1 for each listed cell line. (C) Asynchronously growing DLBCL cell lines were subject to media change followed by treatment with HG6-64-1 or the vehicle, DMSO. After treatment, cells were ethanol permeabilized and saturated with PI in PBS. Cell cycle was analyzed by flow cytometry, and results are depicted in a bar graph. (D) Time-dependent measurement of cellular death by flow cytometry in DLBCL cell lines. (E) Flow cytometry measurement of cellular death at 48 hours of tumor B cells isolated from fresh biopsies of DLBCL primary tumors, or normal B cells isolated from healthy tissue, and treated with HG6-64-1 or DMSO. In one primary sample, cells were derived from the leukemic phase (DLBCL2A) and from a spleenic tumor (DLBCL2B) of the same patient. (F) Measurement of cellular death, by flow cytometry, of DLBCL cells lines treated with HG6-64-1, doxorubicin, or both to measure potential additive effects of combination treatment. All cell-line experiments were performed at least 3 times.

Figure 5

HG6-64-1 inhibits the growth of DLBCL xenograft tumors and prolongs the survival of tumor-bearing mice. Mice bearing subcutaneous OCI-LY-19 xenograft tumors were treated with injections of HG6-64-1, R-CHOP, or PBS. Tumor volume (area in cubic millimeters) is depicted in mice treated with intratumoral injections of HG-6-64-1 (A) or intraperitoneal injections of HG6-64-1, intravenous R-CHOP with orally administered prednisone as described, or PBS (B). Overall survival of the mice is shown in (C-D). Similar results to those shown in (A-D) were observed in one additional independent experiment. The P value for survival of mice given R-CHOP treatment compared with control was P = .0082; HG6-64-1 compared with control was P = .0034, and HG6-64-1 compared with R-CHOP was P = .02. IT, intratumoral administration; IP, intraperitoneal administration; IV + oral, intravenous administration alongside oral prednisone.