BCR Signalosome-Oriented Phosphotyrosine Profiling of CLL

Introduction

There is strong evidence that B-cell receptor (BCR) signaling has a critical role in the pathogenesis of B-cell malignancies such as non-Hodgkin’s lymphoma (NHL) and chronic lymphocytic leukemia (CLL). The BCR signalosome, B-cell receptor signaling protein complex, is therefore a rational therapeutic target. This has already been proven by success in clinical trials of B-cell signaling inhibitors such as Ibrutinib and Idelalisib. It is becoming more important to know what combinations of new and traditional agents is best for each patient. Although genetic profiles may help to predict sensitivity to treatment to some degree, it is ideal to profile the BCR signaling state of each case to select most effective B-cell signaling inhibitors. Our goal is to develop a BCR signalosome-oriented molecular marker and investigate its clinical value. We focus on protein-protein interactions in the B-cell signalosome that are regulated by tyrosine kinases, their substrates, and SH2 domains. We hypothesized that the global tyrosine phosphorylation state determined by SH2 profiling, an SH2 domain-based molecular diagnostic approach, may meaningfully represent the B-cell signaling state of B-cell malignancies. 

Here we conducted SH2 profiling of 1) BCR signalosome peptides to determine specificity of BCR SH2 domain probes and 2) CLL patient samples to determine the presence of patient specific BCR SH2 profiles.   

Methods

For microarrays, phosphorylation site databases were extensively searched and 368 tyrosine phosphorylation sites from the core 38 proteins which make up the BCR-signalosome were selected for peptide synthesis. Replicated peptide microarrays containing pairs of phosphorylated and unphosphorylated tyrosine motifs were separately probed with a set of BCR SH2 domains including BTK, BLNK, LYN, PI3K, SYK, and PLCg2. 

For clinical sample experiments, PBMC samples were collected from 35 CLL patients who visited UConn Health (UCH) and Saint Francis Hospital (SFH) between 2008 and 2014. The median age at study enrollment was 67 (46-98 years). Male patients constituted 63%. Binet stage A disease was present in 76% of the patients. Reverse-phase SH2 domain binding assay was performed as previously described using the BCR SH2 domains. 

Results

According to the microarray results, 94% of BCR signalosome phosphotyrosines were bound by at least one SH2 domain (median 5 domains). A group of proteins including CD22, CD79A, CD45, and PLCg2 protein harbour tyrosine sites that can bind to more than 10 SH2 domains suggesting competition between these SH2 domains may exist in the cell. Specificity of BCR SH2 domains could be grouped into three levels: very specific (BTK, BLNK), medium (Lyn, PLCg2, SHP-1, etc) and broad (SHIP). We found a number of previously undocumented SH2-ligand interactions that may be involved in specific downstream signaling pathways. 

A clustering analysis of CLL samples revealed the presence of different patient groups such as BLNK-dominant and PLC-dominant. Of those clusters, we observed that a cluster with low BLNK signal and high LYN signal was enriched with clinically progressive type CLL cases. To test the prognostic impact of the BLNK/LYN profile, the CLL cases were divided into four groups by high (+) and low (-) BLNK and LYN SH2 binding and compared with PFS. There was a significant difference between the groups in a log-rank test, indicating that patients with the BLNK-low & LYN-high profile progressed more rapidly. Of note, in the microarray experiments we identified that a group of BCR signaling proteins/peptides such as CD19, CD79A, and Dok1 show a similar BLNK-low & LYN-high profile. Their involvements in the SH2 profile of CLL samples remain to be determined. 

Conclusion

Aiming to develop a new molecular marker based on the BCR signaling state of B-cell malignancies, we applied SH2 profiling to BCR peptide microarrays and CLL/NHL patient samples. We confirmed that BCR signalosome-oriented SH2 probes have sufficient specificity to distinguish various signalosome tyrosine sites. SH2 profiling of CLL indicated that the BCR SH2 probes are able to distinguish CLL subgroups, one of which was correlated with a poor PFS. Further efforts are underway to determine the clinical marker value of the BCR signalosome profile, such as its utility in risk stratification, early detection of disease progression, and prediction or assessment of response to B-cell signaling inhibitor therapy.