Global Phosphoproteomics Analysis Of Burkitt Lymphoma (BL) Demonstrates Abnormal Protein Phosphorylation In BCR, Spliceosome, mTOR and Chemokine Signaling Pathways In Rituximab Induced Resistant BL
Authors: Awasthi Aradhana , Rolland Delphine , Barth Matthew J. , Ayello Janet , Ven Carmella van de , Czuczman Myron S. , Elenitoba-Johnson Kojo S.J. , Lim Megan , Cairo Mitchell S. 
Published: October 2019

Abstract

Background
Burkitt Lymphoma (BL) is the most common non-Hodgkin lymphoma (NHL) in children and adolescents and has an excellent prognosis (≥80% 5years, EFS) following short but intense multi-agent chemotherapy (Cairo et al JCO, 2012). The prognosis has improved following the addition of targeted immunotherapy with rituximab (Goldman/Cairo et al, Leukemia, 2013). However, a subset of patients with chemoimmunotherapy resistant disease have a dismal prognosis (≤ 10% 5 years, EFS) (Miles/Cairo et al. BJH, 2012 and Cairo et al Blood, 2007). Deregulation of signaling pathways controlled by protein phosphorylation underlies the pathogenesis of B-cell lymphomas, however the extent to which they contribute to rituximab resistance is largely unknown (Barth et al. BJH, 2013). Recently, Czuczman et al have generated a chemoimmunotherapy resistant BL cell line Raji4RH from the native Raji BL line after serial exposures to rituximab (Czuczman et al Clin Cancer Res, 2008).

Objective
We therefore sought to compare the in-vitro cytotoxicity of rituximab to Raji vs. Raji4RH and the resulting phosphoproteomics changes between untreated and rituximab treated Raji vs. Raji4RH by global phosphoproteomics.

Methods
In order to identify deregulated signaling proteins/pathways that play a role in BL rituximab resistance, we performed a mass spectrometry-based label-free quantitative phosphoproteomic profiling of the BL cell line Raji, (CD20+,ATCC, Manhassas, VA) and its rituximab resistant derivative Raji 4RH in the presence or absence of Rituximab (100µg/ml for 24 hr) or isotype control. 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 using a cocktail of 3 anti-phosphotyrosine antibodies. An LTQ Orbitrap XL in-line with a Paradigm MS2 HPLC was employed for acquiring high-resolution MS and MS/MS data which were searched with the Swissprot Human taxonomic protein database. Quantitation of identified phosphopeptides was based on spectral counts of phosphorylated peptides. The accuracy of the phospho-site localization was predicted using Luciphor program. Pathway analyses were performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID)

Results
Our in-vitro results shows that rituximab induced significantly more cell death against Raji vs. Raji4RH (32.7±6.8% vs.5.5±2.2%, p=0.001) compared to isotype control (9.35±0.75% vs.8.9±1.1%, p=NS) at 24 hrs. Furthermore, 2272 unique phosphopeptides were identified and quantified with a probability P> 0.7 and a false localization rate (FLR) < 0.01. These unique phosphopeptides corresponded to 850 unique phosphorylated proteins. Six hundred and nine proteins were differentially phosphorylated (>1.5-fold change) between Raji and Raji 4RH (Figure 1A). Out of these proteins, 374 were more phosphorylated in Raji4RH than in Raji and 235 were more phosphorylated in Raji than in Raji 4RH. Proteins in the spliceosome and the BCR signaling pathway were significantly represented.
Changes in the phosphorylation of 430 proteins were induced by Rituximab in Raji cells and involved those in the B-cell receptor (BCR) signaling pathway (LYN, LCK, SLAMF1, PAG1, CD20), the spliceosome (SF3A1, SF3B1, SRSF6), the mTOR pathway (RICTOR, PDPK1, RPS6KA1, BRAF) and the chemokine signaling pathway (CXCR5, PTK2B, CCL1, PRKCD).
After rituximab treatment, 559 proteins were differentially phosphorylated between Raji and Raji 4RH (Figure 1B). Rituximab treatment increased the phosphorylation of 262 proteins in Raji 4R while it increased the phosphorylation of 299 proteins in Raji.

Conclusions
The data indicate that Rituximab results in global changes in proteins in BL involved in not just BCR signaling but also in the spliceosome, the mTOR and the chemokine signaling pathway. Furthermore, aberrant phosphorylation-induced signaling events in the spliceosome and the BCR signaling pathways may contribute to resistance to rituximab. These data revealed the utility of unbiased phosphoproteome interrogation of rituximab mediated signaling events as well as characterizing signaling networks that may provide insights into pathogenetic mechanisms of rituximab resistance.
AA and DR considered co-primary first authors, and ML and MC considered co-senior authors.

Disclosures: Cairo:
Roche/Genentech : advisory board Other.