Abstract A274: Novel SYK inhibitors have demonstrated potent antiproliferative effects in both ABC- and GCB-DLBCL cell lines via suppression of multiple pathways downstream of the B-cell receptor.

Background: Diffuse large B-cell lymphoma (DLBCL) has two major subtypes with distinct biological and clinical characteristics: activated B-cell like (ABC) and germinal center B-cell like (GCB) lymphoma. A Bruton's tyrosine kinase (BTK) inhibitor has shown clinical activity in a subset of ABC-DLBCL patients. Spleen tyrosine kinase (SYK) is an upstream regulator of BTK, but the effect of SYK inhibition versus BTK inhibition in DLBCL has not been elucidated. We developed potent and selective SYK inhibitors and evaluated mechanistic differences between SYK inhibition and BTK inhibition in DLBCL cell lines. Materials and Methods: GCB-DLBCL (Pfeiffer, SU-DHL-10, SU-DHL-6, SU-DHL-5, DOHH2, OCI-LY18, OCI-LY1, VAL) and ABC-DLBCL (OCI-LY10, OCI-LY3, TMD8) cell lines were cultivated under standard cell culture conditions. For the growth inhibition assay, cells were treated with compounds for 72 hr, and the number of living cells was determined by CellTiter-Glo (Promega). Cells were treated with the compounds for 24 hr and lysates were subjected to western blotting with antibodies specific for the components of PI3K, ERK, and NFkB pathways. Results: The selective SYK inhibitors, TAS-5567 and its analogue TAS-4335, inhibited SYK with IC50 values of 0.37 nM and 0.26 nM, respectively. The SYK inhibitors demonstrated antiproliferative activities in a wide range of DLBCL cell lines including both ABC and GCB subtypes (TAS-5567 IC50=298 nM in TMD8, 128 nM in SU-DHL-6, 134 nM in SU-DHL-5, and 68 nM in SU-DHL-10). On the other hand, a BTK inhibitor Ibrutinib exhibited antiproliferative activity only in the ABC subtype (IC50=2 nM in TMD8, 1468 nM in SU-DHL-6, 1240 nM in SU-DHL-5, and 1287 nM in SU-DHL-10). We further analyzed the effect of these inhibitors on BCR signaling in GCB-DLBCL cell lines SU-DHL-6, SU-DHL-5, and SU-DHL-10 and found that the SYK inhibitor but not the BTK inhibitor robustly suppressed the PI3K pathway as detected by phospho-AKT and phospho-PRAS40. In ABC-DLBCL cell line TMD8, which is known to be NFkB pathway dependent, the level of phospho-IkBα was reduced by both the SYK and BTK inhibitors. Conclusion: Our SYK inhibitors demonstrated a broad spectrum of antiproliferative efficacy in both ABC- and GCB-DLBCL cell lines, which may be explained by the blocking of multiple downstream pathways, including PI3K and NFkB signaling. Thus SYK inhibition may be a valuable approach in the treatment of heterogeneous DLBCL. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A274. Citation Format: Aki Kawagishi, Hiroki Irie, Yoshio Ogino, Hideya Komatani, Teruhiro Utsugi. Novel SYK inhibitors have demonstrated potent antiproliferative effects in both ABC- and GCB-DLBCL cell lines via suppression of multiple pathways downstream of the B-cell receptor. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A274.