A comparative global phosphoproteomics analysis of obinutuzumab (GA101) versus rituximab (RTX) against RTX sensitive and resistant Burkitt lymphoma (BL) demonstrates differential phosphorylation of signaling pathway proteins after treatment

Abstract

We recently demonstrated that obinutuzumab (GA101), a novel glycoengineered type II CD20 Ab compared to rituximab (RTX) mediates significantly enhanced antibody-dependent cell cytotoxicity (ADCC) in vitro and increased overall survival in a Burkitt lymphoma (BL) xenograft non-obese diabetic severe combined immunodeficiency gamma (NSG) model. In this study we compared the phosphoproteomic changes by pathway analysis following obinutuzumab vs RTX against RTX-sensitive (Raji) and -resistant BL (Raji4RH). Phosphoproteomic analyses were performed by mass-spectrometry (MS)-based label-free quantitative phosphoproteomic profiling. We demonstrated that 418 proteins in Raji and 377 proteins in Raji 4RH, were differentially phosphorylated (>1.5-fold) after obinutuzumab vs. RTX. Proteins that were significantly differentially phosphorylated included the B cell antigen receptor (BCR) (PLCG2, BTK and GSK3B), Fc gamma phagocytosis (FCRG2B, MAPK1, PLCG2 and RAF1), and natural killer cell-mediated cytotoxicity (MAPK1, RAF1, PLCG2 and MAPK3) signaling pathways. Differential phosphorylation of BCR or cytotoxicity pathway proteins revealed significant up-regulation of BTK, PLCY2 and ERK1/RAF1 after obinutuzumab compared to RTX. Silencing of PLCG2 in the BCR and MAPK1 in the cytotoxicity pathway significantly increased BL proliferation and decreased BL cytotoxicity after obinutuzumab compared to RTX. These results in combination with our previous results demonstrating a significant improvement in in vitro BL cytotoxicity and in vivo BL survival by obinutuzumab compared to RTX may in part be due to differential effects on selected BL protein signaling pathways.

Keywords: Burkitt lymphoma; obinutuzumab; proteomics; resistant; rituximab.

Figure 1. Identification and quantification of phosphorylated proteins in RTX-sensitive and -resistant BL cell lines after obinutuzumab vs. RTX

(A) CD20 expression: RTX-sensitive (Raji), RTX-resistant (Raji2R and Raji4RH) cell lines were stained with anti-CD20-PE antibody and surface expression of CD20 was determined by flow cytometry (52.3±8.3% vs. 23.9±1.4%, p=0.00003). (B) CD20 expression after obinutuzumab vs. RTX treatment: RTX-sensitive (Raji) and RTX-resistant (Raji2R and Raji4RH) cell lines were treated with 100ug/ml of obinutuzumab (obit), RTX and isotype control IgG for 24 hrs. Cells were lysed and total proteins (40 μg) were suspended in Laemmli sample buffer, boiled, and subjected to SDS polyacrylamide gel electrophoresis on 10% gels for CD20 expression (N=3). (C) Protein identification by phosphoproteomic analysis: Six milligrams of protein from each condition were digested by trypsin and peptides were subjected to phosphopeptide enrichment using metal oxide affinity chromatography (MOAC) and immunoprecipitation for phosphoproteiomics analysis. An LTQ Orbitrap XL in-line with a Paradigm MS2 HPLC was employed for acquiring high-resolution MS and MS/MS data that were searched with the Swissprot Human taxonomic protein database (N=3).

Figure 2. Differential phosphorylation of pathway proteins after RTX vs. obinutuzumab treatment

Swissprot human taxonomic protein database identify proteins phosphorylated at serine, threonine and tyrosine residues. Proteins with >1.5 fold increase in phosphorylation between anti-CD20 mAbs (RTX and Obinutuzumab) and IgG treatment in (A) Raji and (B) Raji4RH (P=0.05) were identified in multiple cellular pathways.

Figure 3. Phosphoproteomic analysis identifies proteins in BCR, phagocytosis and cytotoxicity pathways that are differentially altered after obinutuzumab vs. RTX

Mass spectrometry-based phosphoproteomic analysis identifies differences in phosphorylation of proteins that function in the BCR pathway (A) Raji (B) Raji4RH, phagocytosis (C) Raji, (D) Raji 4RH and cytotoxicity (E) Raji, (F) Raji4RH (N=3).

Figure 4. Phosphorylation of proteins involved in BCR signaling pathways are differentially expressed after obinutuzumab (Obit) vs. RTX

Differential phosphorylation of proteins was identified by MS-based phosphoproteomic analysis. Phosphorylation of proteins involved in BCR signaling pathways including (A) PLCG2, (B) BTK, (C) GSK3B, (D) LCK and (E) Lyn in Raji and Raji4RH BL cell lines. Obinutuzumab vs. rituximab treated cells were lysed and subjected to SDS polyacrylamide gel electrophoresis and western blot analyses were performed using antibodies as shown.

Figure 5. Silencing of PLCG2 significantly increased cell proliferation in Raji (RTX-sensitive) vs. Raji4RH (RTX-resistant) after obinutuzumab (Obit) vs. RTX

Silencing of (A) PLCG2 and in Raji and Raji4RH cell lines was carried out by SiRNA. Cell proliferation was determine by almar blue dye after PLCG2KD in Raji (B), p=0.0002 vs. 0.03, respectively) and (C) Raji 4RH (p=0.16 vs 0.00009, respectively). PLCG2KD cells were treated with (anti-IgM Ab for antigen stimulation with obinutuzumab or rituximab and analyzed by almar blue (D) Raji (E) Raji4RH. Data were shown in mean±SD. (N=3), (p=0.003 and 0.5, respectively).

Figure 5. Silencing of PLCG2 significantly increased cell proliferation in Raji (RTX-sensitive) vs. Raji4RH (RTX-resistant) after obinutuzumab (Obit) vs. RTX

Silencing of (A) PLCG2 and in Raji and Raji4RH cell lines was carried out by SiRNA. Cell proliferation was determine by almar blue dye after PLCG2KD in Raji (B), p=0.0002 vs. 0.03, respectively) and (C) Raji 4RH (p=0.16 vs 0.00009, respectively). PLCG2KD cells were treated with (anti-IgM Ab for antigen stimulation with obinutuzumab or rituximab and analyzed by almar blue (D) Raji (E) Raji4RH. Data were shown in mean±SD. (N=3), (p=0.003 and 0.5, respectively).

Figure 6. Differential phosphorylation of RAF/ERK1/2 in Raji and RajiRH may contribute to differential effect of ERK1 inhibition on NK-mediated cytotoxicity after obinutuzumab (Obit) vs. RTX

Raji vs. Raji4RH were treated with obinutuzumab vs. rituximab and analyzed by western blot for cytotoxicity signaling pathway proteins RAF and ERK1/2 (A) RAF total and p-RAF. (B) Total ERK total p-ERK1/2. Raji, Raji4RH cells were incubated with MAPK1 inhibitor (10 μM) and subsequently treated with obinutuzumab, rituximab and IgG isotype control (100 μg/ml) and incubated with expanded and activated NK cells for additional 4hrs at 37°C at 20:1 effector: targets ratio. Cell killing was determined by DELFIA cell cytotoxicity assay kit. (N=3) (C) Raji, p=0.0002 and (D) Raji4RH, p=0.1. Data were shown in mean ±SD. (N=3).