Germinal Center Kinase Regulates The Proliferation and Survival Of Diffuse Large B-Cell Lymphoma

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma (NHL). The pathogenesis of DLBCL represents a multi-step process that involves the accumulation of multiple genetic and molecular lesions. Marked advances in the understanding of DLBCL pathobiology have been made by the application of gene expression arrays, comparative genomic hybridization arrays and “next” generation sequencing. This led to the identification of previously unrecognized DLBCL subtypes (germinal center-like (GCB) and activated B cell-like (ABC)) as well as type specific-deregulation of particular signaling pathways. These approaches focused on genetic aberrations and mRNA expression profiles, whereas the crucial events transforming normal cells are executed by proteins. Kinases play an important role in neoplastic transformation. Herein, we have undertaken the task of profiling kinase activity in DLBCL to further delineate potential mechanisms of DLBCL pathogenesis and develop novel therapeutic agents.

A comprehensive analysis of global kinase activity/protein expression was performed using KiNativ technology. Kinomic analysis of 8 DLBCL cell lines, as compared to non-cancerous primary B-cells, led to the discovery of 13 members of the MAPK cascade which were activated and/or overexpressed in DLBCL. Only three of the detected MAPK members were inactive or had reduced expression compared to their non-cancerous counterparts. To determine whether these findings could be extended to de novo primary human DLBCL tumors, we performed immunohistochemistry (IHC) of the proximally activated kinase, MAP4K2 or “Germinal Center Kinase” (GCK) and the phosphorylated forms of its downstream targets: MAP3K1, MAP2K4, MAP2K7, and C-jun N-terminal Kinase 1 (JNK1). Analyzed kinases were expressed and activated in more than 80% of primary DLBCL tumors, confirming the KiNativ cell line data. The kinase array data was further corroborated with classical immunoprecipitation-based JNK and p38 assays. Hierarchical clustering analysis of 36 DLBCL specimens stained for GCB and ABC markers demonstrated that GCK expression/activation is not DLBCL subtype specific. Notably, in a cohort of 151 primary DLBCL cases, we found that patients whose tumors did not express GCK had an estimated progression free survival (PFS) of 85% at 10 years of follow up, whereas those tumors expressing GCK were associated with significantly reduced PFS of 53% (p=0.04). While there was a similar trend in overall survival, it did not reach statistical significance, which may be due to the relatively small number of DLBCL cases not expressing GCK and the potential rescue of these patients with second line treatments.

RNA interference studies in DLBCL cell lines confirmed the importance of GCK for the survival of these tumors, resulting in reduced viability and G0/G1 arrest. We next developed a small molecule inhibitor, HG6-64-1. KiNativ, Ambit and Invitrogen profiling of HG6-64-1 targets revealed that it potently inhibited GCK. In vitro treatment with the novel GCK inhibitor, HG6-64-1, led to cell cycle arrest and the induction of apoptosis in DLBCL cell lines and primary DLBCL tumors. G452, a DLBCL cell line minimally expressing GCK, was not affected by HG6-64-1. In vivo treatment with HG6-64-1, via intratumoral and intraperitoneal injections, significantly decreased the tumor growth rate resulting in a significantly extended lifespan of DLBCL xenograft mouse models. Overall our results identified a previously unrecognized activation of the GCK pathway which contributes to the proliferation and survival of DLBCLs and can be used as a therapeutic target using novel GCK inhibitors.