Panoply™ Human CD79B Over-expressing Stable Cell Line

B-cell antigen receptor (BCR) downstream kinases are popular targets for non-Hodgkin lymphoma (NHL) therapy. Here, researchers show that lymphoma cells from patients with diffuse large B-cell lymphoma have high basal phosphorylation levels of most measured signaling nodes, while follicular lymphoma cells, in contrast, have no or very low basal phosphorylation levels. Patients with mantle cell lymphoma (MCL) have widely varying basal phosphorylation levels, with elevated phosphorylation levels of AKT, extracellular signal-regulated kinase, p38, STAT1, and STAT5 associated with poor patient prognosis. Chronic lymphocytic leukemia (CLL) tumors show elevated phosphorylation levels of BCR signaling nodes (Src family tyrosine kinases, spleen tyrosine kinase [SYK], phospholipase Cγ), but low levels of α-BCR-induced signaling. This is in contrast to MCL tumors, where α-BCR-induced signaling, although variable, is significantly enhanced compared with other tumor types. Overexpression of CD79B, combined with a gating strategy that directly quantifies the signal output of each cell as a function of CD79B levels, demonstrated a direct relationship between surface CD79B, immunoglobulin M (IgM), and IgM-induced signal levels. Furthermore, the intensity of the α-BCR-induced signal varied across patient samples and correlated with expression of the BCR subunit CD79B but negatively correlated with sensitivity of MCL to inhibitors of Bruton's tyrosine kinase (BTK) and SYK. Individual differences in BCR levels and signaling may be associated with differences in therapeutic response to BCR pathway inhibitors.

The intensity of α-IgM-induced signals in mantle cell lymphoma (MCL) may affect the inhibitory efficacy of BCR signaling inhibitors. To test this, CD79B overexpressing Granta 519 cells were pre-incubated with different concentrations of fostamatinib or ibrutinib or left to rest for 60 minutes before being activated with α-IgM for 4 minutes. Comparison of CD79B expression with p-BTK or p-PLCγ using dot plots showed a direct relationship between CD79B levels and the efficacy of ibrutinib in inhibiting α-IgM-induced phosphorylation (Figure 1A). Similarly, a gating strategy was used to measure signals in cells expressing different levels of CD79B (from low to high, L1-L6) to test how different levels of CD79B affect drug efficacy. This approach revealed a direct dose-response relationship between CD79B levels and the efficacy of ibrutinib (Figure 1B). Compared with cells with low CD79B expression and low IgM-induced signaling, cells with high CD79B expression (L5, L6) and strong α-IgM-induced signaling required higher concentrations of ibrutinib for effective inhibition (Figure 1B-C). A similar relationship was observed between the level of CD79B/α-IgM-induced signaling and the efficacy of fostamatinib (Figure 1D).

Figure 1. Higher concentrations of ibrutinib and fostamatinib are required to efficiently suppress α-BCR–induced signaling in CD79Bhi cells as compared with CD79Blow cells. (Myklebust J H, et al., 2017)