Poster Session I: Lymphoma biology & translational research
Use of targeted therapy for B-cell malignancies has resulted in significant improvements in patient overall- and progression-free-survival. These treatments have focused on inhibition of components of the B-cell receptor (BCR) such as BTK and PI3Kδ, targeted by ibrutinib and idelalisib respectively, and are often used in combination with other agents such as Microtubule Targeting Agents (MTA), alkylating agents and glucocorticoids. However, relapse and resistant disease remain a significant challenge. Thienopyridines, commonly used as anti-thrombotic therapy, have recently been identified as potential anti-cancer agents, with PLCγ2 (acting downstream of BTK in BCR signalling), Jak2 and Lck suggested as direct targets, while mitotic spindle inhibition via microtubule de-stabilisation has also been proposed. Theinopyridines may therefore have potential as a future treatment for B-cell malignancies.
This study aimed to investigate the anti-proliferative and microtubule-disrupting properties of ten novel thienopyridine compounds synthesised by our group, in B-cell Lymphoma cells in-vitro, to determine whether this class of compound has potential as future targeted therapy for B-cell malignancies.
DAUDI Burkit Lymphoma cells were cultured in the presence of novel thienopyridines for 24-72 h at a range of concentrations (100 μM to 1 nM). Synthesis of the thienopyridine compounds (DJ0014, DJ0021, DJ0041, DJ0081, DJ0109, DJ0171, DJ0199, DJ0206 and DJ0209[ND1] ) has been previously published. Cellular biochemical activity was assessed using the MTS assay, while apoptosis and necrosis were observed using the Annexin V/Propidium Iodide (AV/PI) flow cytometry assay. Cell cycle arrest was determined by flow cytometric Propidium Iodide cell cycle assay, and alpha-tubulin expression was visualised by confocal microscopy following staining with α-Tubulin (DM1A) Mouse mAb (Alexa Fluor® 488 Conjugate) antibody and DAPI nucleic acid stain. The rate of tubulin polymerisation in the presence of the compounds was assessed by Tubulin Polymerisation Assay, with the rate of polymerisation measured using spectrophotometry. Paclitaxel was used as a positive control for tubulin stabilisation.
Across all assays at 48 and 72 h, compounds DJ0014, DJ0041, DJ0081, DJ0199 and DJ0206 resulted in significantly reduced cell viability by MTS assay and increased early and late apoptosis by AV/PI, with corresponding reduction in live cell populations and absence of necrosis at 10 μM and 100 μM concentrations. Increased tetraploidy / G2/M arrest was also seen following treatment with the same compounds at 10 μM and 100 μM concentrations. These compounds showed a significant inhibitory effect on tubulin polymerisation at 10 μM dosage and signs of microtubule disruption via confocal microscopy.
The study demonstrates the anti-mitotic activity of these novel thienopyridine derivatives in malignant B-cells and their potential for use in the treatment of mature B-cell malignancies. Future work aims to clarify their effect on PLCγ2 and the mechanism by which this may result in the observed effect on microtubule assembly.