[1437] BTK Inhibition and Response Prediction in Mantle Cell Lymphoma Cells

Jiao Ma, Ailin Guo, Shuhua Cheng, Pin Lu, Peter Martin, Richard R Furman, Morton Coleman, Lynn Y Wang. Weill Cornell Medical College, New York, NY

Background: Ibrutinib is a potent, selective Bruton tyrosine kinase (BTK) inhibitor that binds covalently to cysteine 481 with an IC50 of 0.5 nM. Despite its promising clinical activity in a recent phase 2 clinical trial in patients with mantle cell lymphoma (MCL), the mechanism by which it induces clinical responses remains unclear.
Design: In the present study, we analyzed MCL patient samples and three MCL cells lines for cellular and molecular events affected by ibrutinib. 1) Western blot analysis was performed to compare levels of total BTK and phospho-BTK (Y223) in primary MCL cells versus resting B-cells. 2) MTT assays were used to evaluate the relative sensitivity of the three MCL cell lines to ibrutinib. 3) Intracellular staining for downstream B-cell receptor (BCR) signaling proteins in MCL cell lines and primary MCL samples was performed to assess the signaling pathway(s) that may determine sensitivity to ibrutinib treatment.
Results: Immunoblotting demonstrated that primary MCL cells expressed higher levels of p-BTK (Y223) compared to resting B-cells from normal donors, suggesting that lymphoma cells may be more susceptible to BCR inhibition than normal B-cells. Jeko-1 was sensitive to ibrutinib while Granta-519 and Mino had an IC50 above 400 nM (the maximum clinically achievable concentration). Both growth and survival of Jeko-1 cells were inhibited by the drug at a low concentration of 200 nM. Suppression of cell proliferation was achieved by cell cycle arrest at G1-S transition, which was not observed in either Mino or Granta-519 cells. Following ibrutinib treatment, Jeko-1 cells displayed a reduction in several cell cycle regulatory proteins. In BCR-stimulated lymphoma cells, ibrutinib induced a considerable reduction in the activity of ERK1/2 and AKT in Jeko-1 cells only. In addition, using MCL primary cells we found that ERK1/2 and AKT activity was inhibited by ibrutinib in a dose-dependent manner. Moreover, a good correlation was found between the reduction in AKT activity and the pre-treatment Ki67 index in patient specimens, suggesting that highly proliferative MCL cells may be particularly sensitive to BTK inhibition. Genetic knockdown of BTK effectively reduced the growth of Jeko-1 cells suggesting the anti-tumor activity of ibrutinib was mainly mediated by specific BTK inhibition.
Conclusions: Our data may provide insights into the mechanisms of ibrutinib, and suggest potential biomarkers that may determine cellular sensitivity to the inhibitor.
Category: Hematopathology

Tuesday, March 5, 2013 9:30 AM

Poster Session III # 163, Tuesday Morning


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