PCI-31523: An irreversible inhibitor of Bruton’s tyrosine kinase (Btk) that disrupts B cell receptor (BCR) signaling, is cytotoxic to B cell lymphomas, and is active in an animal arthritis model

5398 Many lymphomas require an intact BCR for survival and, therefore, should be uniquely sensitive to drugs that target the BCR pathway. The kinase Btk is activated by stimulation of the BCR and it in turn phosphorylates and activates Phospholipase C-gamma (PLCg) to transmit BCR signaling further downstream. We have designed PCI-31523, a selective and irreversible inhibitor that covalently binds to Cys-481 in the active site of Btk. In purified enzyme assays, PCI-31523 inhibits Btk with an IC50 of 200-fold selectivity over the structurally related kinase Lck. In normal human B cells, PCI-31523 inhibits anti-IgM induced signaling and proliferation at concentrations as low as 10nM. To confirm that PCI-31523 acts as a selective inhibitor in cells, we used phospho-specific antibodies that detect known phosphorylation events downstream of BCR stimulation. We found that PCI-31523 does not significantly inhibit the Src-family proximal kinases that trans-phosphorylate Btk at Tyr-551. In contrast, at 10nM, PCI-31523 completely blocks the Btk-dependent phosphorylation of PLCg. In addition, PCI-31523 leads to a significant decrease in Ca++ flux and phosphorylation of Erk. Lastly, PCI-31523 has no effect on PMA/Ionomycin induced phosphorylation of Erk. Thus, as predicted for a selective Btk inhibitor, PCI-31523 acts downstream of the Src-family kinases and upstream of PLCg. We tested the effects of PCI-31523 on a variety of BCR expressing lymphoma cell lines. The diffuse large B cell lymphoma lines DHL-4 and DHL-6 as well as the follicular lymphoma line DoHH2 are sensitive to PCI-31523, with growth arrest and apoptosis occurring at concentrations as low as 0.5uM. In contrast, the lymphoma cell lines Ramos, Raji, and DB are resistant to PCI-31523 cytotoxicity. To investigate this differential sensitivity, we used phospho-flow cytometry to quantitate basal and anti-IgM-induced phosphorylation of Erk. PCI-31523 is more potent at reducing phospho-Erk in a sensitive cell line (DHL-6) than in a resistant cell line (Ramos). Thus the amount of inhibition of phospho-Erk appears to be correlated with the degree of cytotoxicity and this biomarker may function as a predictor of clinical responsiveness. PCI-31523 has been tested in vivo in a mouse collagen antibody arthritis model. Oral dosing of PCI-31523 (10mg/kg qd) results in almost complete inhibition of inflammation, indicating that this compound may be effective in both autoimmune disease and lymphoma.