Abstract 856: Biomarkers of sensitivity and resistance to the BTK inhibitor PCI-32765 in mantle cell lymphoma
Authors: Balasubramanian Sriram , Chen Jun , Crowley Richard , Buggy Joseph J. 
Published: August 2012

Abstract
Bruton's tyrosine kinase (Btk) is a central regulator of BCR signaling in B-cell lymphomas. PCI-32765 is a potent and selective inhibitor of Btk which has shown good clinical activity, particularly in mantle cell lymphoma. But its mechanism of action in this disease is poorly understood. In this study, we explored the role of Btk and the activity of PCI-32765 on BCR signaling in PBMC samples from patients, which contain a high percentage of tumor cells, as well as in several MCL lines, including Granta-519, Jeko-1, JVM-2, JVM-13, Maver-1, Mino, NCEB-1, Rec-1 and Z-138. In this work, we present a detailed characterization of signalling pathways inhibited by PCI-32765 downstream of the B-cell receptor in cell lines and tumor cells, studied by gene expression profiling, flow cytometry, and proteomic analyses of signaling pathways and effectors. Correlation with PCI-32765 activity in tumor cells and patients leads to the identification of biomarkers of biomarkers of sensitivity and resistance. Results: Btk and surface IgM protein expression was detected in all MCL lines at variable levels. In a 3-day proliferation assay, JVM-2 & MINO emerged as the most sensitive lines to PCI-32765 (GI50: 1.75-4.4uM). Rec-1 was resistant to PCI-32765 alone (11.3uM) but became much more sensitive (1.45uM) upon BCR stimulation using anti-IgM (10ug/mL). Other lines such as Maver-1, Granta-519 & Jeko-1 all required &gt;10uM of PCI-32765 for inhibition and BCR stimulation did not make much difference. When signaling pathways downstream of BCR activation were studied, intracellular calcium flux following stimulation with IgM was observed in all lines (except JVM-2) and was inhibited at &lt;100nM PCI-32765 in most of them, but no correlation between this and growth inhibition was observed. Constitutive BTK autophosphorylation was observed in all lines and was completely abolished by PCI-32765. BCR stimulation increased p-BTK which was also blocked by PCI-32765 in all lines. Mino and JVM-2 showed constitutive p-ERK activity, which was slightly increased upon BCR stimulation and could be blocked with PCI-32765, whereas the more resistant lines such as Maver-1 and Rec-1 had low endogeneous levels of p-ERK, but which was increased by BCR stimulation and only partially or not reversed by PCI-32765 at 5uM. Little change was observed in levels of p-PLCg1 or p-NF-kB p65. Additionally, in all cell lines stimulation with anti-IgM led to an increased secretion of the chemokines CCL3 and CCL4, which was inhibited by PCI-32765. Blockade of Btk function using PCI-32765 inhibits MCL cell proliferation, BCR signaling and chemokine secretion. These findings highlight the importance of BCR signaling and Btk in MCL, help explain the activity of the Btk inhibitor PCI-32765 in MCL patients, and provide biomarkers that may be of value in the clinic.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 856. doi:1538-7445.AM2012-856