Jiao Li, Ning Ding,Lixia Feng, Yalu Liu, Weiwei Song, Yuqin Song, Jun Zhu

Department of Lymphoma, Beijing Cancer Hospital, Beijing.

Objective：Mantlecell lymphoma (MCL) is an aggressive and incurable malignant lymphoma with amedian survival of 5 years that accounts for approximately 6% to 8% of B-NHLpatients. Given the lack of successful treatment options for MCL, thedevelopment of new combination strategies for the treatment of thisdebilitating disease is urgently needed. The B-cell antigen receptor (BCR)signal pathway has gained significant attention as a potential therapeutictarget in B-cell lymphoma. We recently developed a novel covalent irreversibleBtk inhibitor PLS-123, which exhibits a dual-action mode of inhibition for boththe catalytic activity of Btk and its own activation. This study aims toinvestigate whether PLS-123 potentiates the growth inhibitory effect ofeverolimus, a specific mTOR inhibitor, in human MCL cells. Method：Invitro cell viability was analyzed using Cell Titer-Glo Luminescent CellViability Assay. Induction of apoptosis and cell cycle arrest were measured byflow cytometry. Western Blotting analysis was used to detect the essentialregulatory enzymes with in related signaling pathways post combination ofPLS-123 and everolimus treatment. Result: Co-administration of PLS-123and everolimus synergistically induced anti-proliferative effect in MCL celllines (Granta519，Minoand Z138). Exposure of these MCL cells to minimally toxic concentration ofeverolimus and PLS-123 resulted in more significant inhibitory activity thaneach agent alone. Interestingly, combined treatment of MCL cells witheverolimus and PLS-123 resulted in marked induction of apoptosis monitoredbyflow cytometry compared to single agent treatment, which were accompanied bymarked up-regulation of apoptosis-related proteins (cleaved-caspase3, caspase-8,caspase-9 and PARP) and pro-apoptotic proteins Bax and Bad. The combination ofPLS-123 and everolimus also substantially increased the cell population of G1phase and decreased that of G2 in cell cycle, which indicted that combinedtreatment blocked cell cycle progression at G1-S phase. The expression of CDK4and CDK6, which regulated G1/S phase cell cycle transition, were reduced postcombination treatment. Furthermore, the possible impacts of combinationtreatment towards related signaling cascades were next investigated byimmunoblotting analysis. Combined exposure of MCL cells to everolimus andPLS-123 resulted in marked inactivation of the mTOR/AKT pathway compared tosingle drug treatment. The activation of p-ERK and p-AKT was also moresignificantly diminished after treatment of PLS-123/everolimus. Conclusion：Thecombination of novel Btk inhibitor PLS-123 and everolimus synergisticallyinduced anti-tumor activity in MCL cell lines, suggested a new combinationdirection for the treatment of MCL patients.

Key Words：Mantlecell lymphoma  Btk inhibitor  mTOR inhibitor