Inhibitors of SRC kinases impair antitumor activity of anti-CD20 monoclonal antibodies

Abstract

Clinical trials with SRC family kinases (SFKs) inhibitors used alone or in a combination with anti-CD20 monoclonal antibodies (mAbs) are currently underway in the treatment of B-cell tumors. However, molecular interactions between these therapeutics have not been studied so far. A transcriptional profiling of tumor cells incubated with SFKs inhibitors revealed strong downregulation of MS4A1 gene encoding CD20 antigen. In a panel of primary and established B-cell tumors we observed that SFKs inhibitors strongly affect CD20 expression at the transcriptional level, leading to inhibition of anti-CD20 mAbs binding and increased resistance of tumor cells to complement-dependent cytotoxicity. Activation of the AKT signaling pathway significantly protected cells from dasatinib-triggered CD20 downregulation. Additionally, SFKs inhibitors suppressed antibody-dependent cell-mediated cytotoxicity by direct inhibition of natural killer cells. Abrogation of antitumor activity of rituximab was also observed in vivo in a mouse model. Noteworthy, the effects of SFKs inhibitors on NK cell function are largely reversible. The results of our studies indicate that development of optimal combinations of novel treatment modalities with anti-CD20 mAbs should be preceded by detailed preclinical evaluation of their effects on target cells.

Keywords: ADCC, antibody-dependent cell-mediated cytotoxicity; BCR, B-cell receptor; CD20; CDC, complement-dependent cytotoxicity; CFSE, carboxyfluorescein succinimidyl ester; CLL, chronic lymphocytic leukemia; DLBCL, diffuse large B-cell lymphoma; NHL, non-Hodgkin lymphoma; PBMC, peripheral blood mononuclear cells; SFKs, SRC family kinases; SRC family kinases; dasatinib; ofatumumab; rituximab.

Figure 1.

Transcriptional profiling of Raji cells incubated for 24 h with dasatinib or PP2. (A) Total RNA from control Raji cells or from cells incubated for 24 h with either 100 nM dasatinib or 10 μM PP2 was used to generate cRNA for hybridization to human-specific AMADID Release GE 8x60K microarrays. The volcano plot shows changes in expression of all the Agilent microarray transcripts in cells incubated with dasatinib or PP2. Red spots represent genes for which expression changed significantly (p < 0.01, Fold Change > 3, unpaired t test and Benjamin-Hochberg FDR < 5% correction) due to dasatinib or PP2 treatment, gray spots represent genes for which expression was not significantly changed. Arrow indicates MS4A1 gene (blue spot). The plot was made using GeneSpring (Agilent) software. (B) GeneSpring (Agilent, USA) cluster of genes (containing MS4A1 gene) for which expression changed similarly in cells upon incubation with dasatinib or PP2. All genes with a fold change cut-off > 3.0 (p value < 0.01 in unpaired t test and Benjamin-Hochberg FDR < 5% correction) were considered as significantly regulated and are presented in a matrix format: each row represents a single gene, and each column represents an experimental sample. In each sample, the ratio of the abundance of transcripts of each gene to the median abundance of the gene's transcript across all sample, is represented by the color of the corresponding cell in the matrix.

Figure 2.

For figure legend, see next page.Figure 2 (See previous page). SFKs inhibitors downregulate surface CD20 levels and impair antitumor activity of rituximab and ofatumumab. (A) The surface CD20 level was determined with FITC-conjugated anti-CD20 antibody (clone L27, BD) in Raji cells pre-incubated for 48 h with increasing concentrations of multi-SRC family kinases inhibitors. Results are presented as a percentage of MFI of control cells (± SD). (B–C) Raji cells pre-incubated for 48 h with increasing concentrations of multi-SRC kinases inhibitors were washed and incubated for 1 h with rituximab (B) or ofatumumab (C) (1–100 μg/ml) and 10% human AB serum. Cell viability in CDC assay was measured with PI. The survival of cells is presented as a percentage of control cells without antibody (± SD). (D) CFSE-stained Raji cells were co-incubated for 4 h with either rituximab or ofatumumab (100 μg/ml) and NK cells at E:T ratio 6:1 in presence of dasatinib or PP2. Raji cell survival in ADCC assay was determined with flow cytometry after staining with PI and is presented as a percentage of control cells without rituximab/ofatumumab. (E) NK and Raji cells were incubated simultaneously at effector to target ratio 1:1 with rituximab (100 μg/ml) and either dasatinib (100 nM) or PP2 (10 μM) for 4 h at 37°C. For degranulation assay NK cells were co-incubated with GolgiStop and anti-CD107a antibody. Cells were washed, stained with anti-CD56, anti-CD3 and Fixable Viability Dye. To determine cytokines production, cells were washed, permeabilized with Cytoperm/Cytofix and stained with anti-interferon (IFN)-γ or anti-tumor necrosis factor (TNF) antibodies followed by flow cytometry analysis. Results are presented as a percentage of CD107a, TNF or IFN-γ positive NK cells (± SD) within the whole NK cell population. Shown is one representative of at least 3 independent experiments.

Figure 3.

Dasatinib impairs antitumor activity of rituximab in a murine model. Mice were inoculated with 5x10⁵ of EL4-hCD20 cells intravenously and randomized to groups of 6–8. Dasatinib was given once a day i.p. (50 mg/kg) for 4 consecutive days. Rituximab was given i.p. (10 mg/kg) at days 3 and 6. Control mice were injected with equal volume of PBS. Luminescence was followed from day 1 until termination of the experiment at day 9. Data are presented as mean ± SEM, statistical significance was reached by comparison between groups rituximab vs rituximab+dasatinib, p = 0.0045, t test. Shown is one representative of 2 independent experiments.

Figure 4.

Influence of dasatinib on CD20 levels in primary CD20-positive leukemia/lymphoma cells. Primary human CD20-positive B-CLL/lymphoma cells (total n = 30; CLL n = 25, MCL n = 4, FL n = 1) were incubated for 48 h with increasing concentrations of dasatinib. After washing, cells were incubated with saturating amount of FITC-conjugated anti-CD20 mAb (clone L27, BD) for 30 min at room temperature in the dark. Prior to flow cytometry analysis, cells were washed with PBS and resuspended in PBS with PI. Results are presented as a percentage of MFI of control cells (± SD). The differences in CD20 MFI between control and drug-treated groups were statistically significant (p < 0.0001) as measured using Wilcoxon signed-rank test.

Figure 5.

Dasatinib modulates CD20 levels at a transcriptional level. (A) Western blotting analysis of CD20 and β-actin in protein extracts from Raji cells pre-incubated for 48 h with increasing concentrations of dasatinib. (B) cDNA from Raji cells pre-incubated for 48 h with increasing concentrations of dasatinib was used for qRT-PCR amplification of CD20 and B2M products with corresponding probes labeled with FAM and DABCYL. (C) cDNA from Raji cells pre-incubated for 24 h with dasatinib (100 nM) and/or cycloheximide (10 nM) was used for qRT-PCR amplification of CD20, RPL29 and ACTB products. (D) In actinomycin D chase mRNA decay assay cDNA from Raji cells pre-incubated for 24 h with dasatinib (100 nM) and/or actinomycin (10 μg/ml) was used for qRT-PCR amplification of CD20, RPL29 and ACTB products. Shown is one representative of at least 3 independent experiments.

Figure 6.

Modulation of CD20 expression by dasatinib requires CD20 promoter. (A) HeLa cells stably transduced to express CD20 (HeLa pLVX-CD20-IRES-PURO) were incubated for 48 h with increasing concentrations of dasatinib. Surface CD20 levels were determined as described earlier. (B) Protein lysates from Raji cells modified to express CD20-Histag fusion protein pre-incubated for 48 h with increasing concentrations of dasatinib were separated in polyacrylamide gel. CD20 and CD20-Histag proteins were detected with anti-CD20 antibody. (C) Relative luciferase activity was measured in lysates from Raji cells transfected with either empty vector or pGL4-wild type CD20 promoter or with pGL4-truncated CD20 promoters and further incubated with dasatinib (100 nM) for subsequent 24 h. (D) Scheme of truncated CD20 promoters used for reporter assays. (E) Relative luciferase activity was measured in lysates from Raji cells transfected with either pGL4-wild type or mutated (BAT-box mut) CD20 promoters incubated with dasatinib (100 nM) for subsequent 24 h. (F) Nuclear lysates from Raji cells either control or incubated for 24 h with dasatinib (100 nM) were mixed with biotinylated BAT-box probe in presence or absence of specific competitor. Formed DNA-protein complexes were analyzed for the binding of the proteins to the putative Oct-2 binding site in the CD20 promoter. Shown is one representative of at least 2 independent experiments.

Figure 7.

Effect of SFKs inhibitors on CD20 levels is rescued by activation of SFKs and BCR downstream signaling pathways. (A) Raji cells pre-incubated with 100 nM dasatinib for 48 h were washed and subsequently incubated without dasatinib for indicated times. CD20 levels were determined as described earlier. (B) Raji cells pre-incubated for 48 h with increasing concentrations of AKT inhibitor (MK-2206) were analyzed for surface CD20 levels as previously described. (C) p-AKT (Ser473), pan AKT and p-SRC (Tyr416) were detected with corresponding antibodies in protein lysates from Raji cells pre-incubated with dasatinib. (D) Relative luciferase activity was measured in protein lysates of Raji cells co-transfected with pGL4-wt CD20 promoter and either pMIG or pMIG-myrAKT and incubated with dasatinib for 48 h. (E) Relative luciferase activity was measured in protein lysates of Raji cells co-transfected with pGL4-wt CD20 promoter and either empty vector, wt PTEN or C124S PTEN and incubated with dasatinib for 48 h. (F) Raji cells stably transduced with either pMIG or pMIG-myrAKT were sorted based on GFP expression. AKT, p-AKT (Thr308, Ser473), CD20, β-actin were detected with corresponding antibodies in protein lysates from unsorted and sorted cells. (G) p-AKT, AKT, CD20 and β-actin were detected with corresponding antibodies in protein lysates from sorted Raji cells (Raji pMIG or Raji pMIG-myrAKT) pre-incubated with dasatinib for 24 h. (H) Surface CD20 levels in Raji pMIG or Raji pMIG-myrAKT cells pre-incubated with dasatinib for 48 h were determined as described earlier. Results are presented as PE MFI (± SD) of GFP-positive cells. (I) cDNA from Raji pMIG and Raji pMIG mAkt cells pre-incubated for 24 h with dasatinib was used for qRT-PCR amplification of CD20, ACTB and RPL29 products. (J) CDC assay was performed using Raji pMIG or Raji pMIG-myrAKT cells as previously described. Shown is one representative of at least 2 independent experiments.