Spleen Tyrosine Kinase Inhibitor Fostamatinib Blocks B Cell Receptor Signaling and Reduces Viability of BCR Subtype Diffuse Large B Cell Lymphoma
Authors: Zhang Yue , Parmentier Julie , Lai Zhongwu , O'Connor Greg , Passino Melissa , Powell Francoise , Devereaux Erik , Byth Kate 
Published: October 2019

Abstract
Abstract 3720
Spleen tyrosine kinase (SYK) plays a key role in B cell receptor mediated survival in certain B cell malignancies including Diffuse Large B Cell Lymphoma (DLBCL). Therefore, targeting SYK represents an emerging therapeutic approach for the treatment of DLBCL. Indeed, fostamatinib, an orally available SYK inhibitor, has shown promising clinical activity in non-Hodgkin lymphoma (Friedberg et al. 2010 Blood 115: 2578). However, the overall response rate for novel targeted agents in unselected relapsed refractory DLBCL patients remains 25–30%, highlighting the opportunity for development of better treatment strategies. Here, we used preclinical models to study the mechanism underlining the efficacy of R406, the metabolic active form of fostamatinib. R406 was characterized in a heterogeneous panel of 17 DLBCL cell lines including both ABC and GCB subtypes. Overall, the cellular selectivity of R406, particularly in ABC-subtype DLBCL cell lines, was consistent with other BCR-targeted agents such as BTK inhibitor PCI-32765 (ibrutinib) and PI3 kinase δ inhibitor CAL-101. This strongly suggests that R406 functions through inhibition of BCR-mediated survival signaling. Furthermore, phospho-flow analysis and Western Blotting have demonstrated the effect of R406 on both basal and anti-IgG/M stimulated BCR signaling. Specifically, R406 decreases phosphorylation of proximal BCR pathway regulators BLNK, PLCγ2, as well as key players in downstream effect pathways such as ERK, AKT, RPS6, 4EBP and STAT3. To investigate whether the inhibition of BCR signaling by R406 affects cell viability, flow-cytometry based apoptosis analysis was employed. R406 induced apoptosis in sensitive cell lines in a dose-dependent manner. Mechanistically, treatment with R406 reduced MCL1 protein level and down-regulated Bfl-1 expression in sensitive, but not resistant, cell lines, which may partially contribute to the observed efficacy. Given the critical role of NFκB-dependent survival signaling in ABC subtype DLBCL, we then investigated the effect of R406 on this pathway by monitoring NF-κB target gene expression via quantitative real time PCR. Consistent with previous publication (Davis et al. 2010, Nature 463: 88), R406 significantly blocked chronic active BCR-induced NFκB signaling in sensitive cell lines with CD79A/B mutations, leading to downregulation of IL-6/IL-10 and subsequent suppression of JAK/STAT3 signaling. In contrast, we observed little effect of R406 in cell lines with downstream activating mutations of the NFκB pathway (such as CARD11, A20 mutations). Therefore, the molecular nature of NF-κB pathway lesions may serve as a predictive marker for R406 responsiveness in ABC subtype DLBCL cell lines. Furthermore, in exploration of a common indicator of sensitivity to R406 in both ABC and GCB subtypes, we have confirmed that response to R406 is dependent on surface expression of a functional BCR and presence of a intact BCR signaling cascade (Chen et al. 2008 Blood 111: 2230). These cells are thus characterized as “B-cell Receptor active” or BCR subtype. Work is currently underway to further elucidate the characteristics of the BCR subtype, which may serve as a general selective marker for R406's efficacy in DLBCL cell lines. In conclusion, we have demonstrated that R406 functions as a BCR antagonist and reduces viability in DLBCL through inhibition of NFκB-mediated survival signals and downregulation of MCL-1. In addition, we have confirmed in ABC subtype DLBCL cell lines, similar to other BCR-targeted agents, R406 sensitivity is correlated with CD79A/B mutations whereas A20 or CARD11 mutations render cells resistance. Eventually, we proposed that BCR classification may serve as a broader selection maker for all DLBCL cell lines.

Disclosures:
Zhang: AstraZeneca: Employment, Equity Ownership. Parmentier:AstraZeneca: Employment, Equity Ownership. Lai:AstraZeneca: Employment, Equity Ownership. O'Connor:AstraZeneca: Employment, Equity Ownership. Passino:AstraZeneca: Employment, Equity Ownership. Powell:AstraZeneca: Employment, Equity Ownership. Devereaux:AstraZeneca: Employment, Equity Ownership. Byth:AstraZeneca: Employment, Possible shareholder Other.