Peter Johnson, MD
Honigberg LA, Smith AM, Sirisawad M, et al. The Bruton tyrosine kinase inhibitor PCI-32765 blocks B-cell activation and is efficacious in models of autoimmune disease and B-cell malignancy. Proc Natl Acad Sci USA. 2010;107:13075-13080.
Events downstream of the B-cell receptor (BCR) appear to be critical not only for the normal functions of humoral immunity but also in the life of many malignant B-cells. Circumstantial evidence of this comes from the constitutive activation of the BCR pathway in a variety of lymphomas. This has been highlighted recently by the observation in an interference screen that short hairpin RNAs directed against Bruton tyrosine kinase (Btk) were selectively lethal to lymphoma cell lines1 and the finding of specific patterns of glycosylation on the idiotype in follicular lymphoma, suggesting a selection pressure mediated by the interaction of the BCR with lectins.2 There is a growing sense that hijacking of the normal activation pathways in B cells, either endogenously by mutation or exogenously through cell-cell interactions, may be key events in lymphoma pathogenesis.
A research group at Stanford University and Pharmacyclics Inc. has previously identified potent inhibitors of Btk that act by binding covalently to a cysteine residue in the active site, and in this paper they present the results of applying this to B-cell lymphoma lines, mice with programmed autoimmunity, and dogs with spontaneous B-cell lymphoma. The selectivity of the lead compound, PCI-32765, was tested by synthesis of a fluorescent-tagged derivative, which was shown to bind to wild-type Btk or a catalytically inactive mutant but not to a version lacking the crucial cyteine residue. Immunoprecipitation from the DoHH-2 lymphoma cell line showed that the fluorescent derivative migrated with Btk with a high degree of specificity. Analysis of the effects upon signaling in DoHH-2 cells showed inhibition of phosphorylation of Btk and its substrate PLCγ, as well as of the downstream kinase ERK at nanomolar concentrations of PCI-32765. Normal, non-malignant B cells stimulated with anti-IgM demonstrated that the inhibitor blocked activation at doses as low as 10nM, while T-cell activation by CD3/CD28 beads was unaffected, even at high concentrations.
In vivo experiments showed suppression of collagen-induced arthritis in DBA/1 mice after 11 days oral dosing at 3.125 mg/kg, while the MRL-Fas(L) model of lupus showed partial reversal of the phenotype at similar dose levels with a reduction of proteinuria, anti-ds DNA antibody levels, and interstitial nephritis, although there was apparently less effect upon established glomerulonephritis.
Finally, studies in spontaneous canine B-cell lymphomas performed in collaboration with the Colorado State University Animal Cancer Center showed full and sustained occupancy of Btk in blood mononuclear cells and tumor biopsies following a daily oral dose of 2.5 to 20 mg/kg for one week, and three partial responses were seen among eight dogs treated.
It seems likely that one of the means by which B-cell lymphomas acquire a growth advantage is through activation of their “normal” signaling pathways, in particular the BCR, an extraordinarily potent mediator of proliferation and suppressor of apoptosis in the normal germinal center reaction. The sub-group of diffuse large B-cell lymphoma with patterns of gene expression corresponding to those of activated B cells has worse outcomes from conventional immuno-chemotherapy, and in this group a variety of mutations have been identified that result in constitutive signaling through the BCR pathway. It is an attractive strategy to develop small-molecule inhibitors to this pathway, and the compound described in this paper is interesting in that it shows durable activity at very low doses, through rapid and irreversible binding to the active site of Btk. It also appears to be highly selective and to have a relatively short half-life in vivo, allowing oral dosing with the promise of limited off-target effects. The first results of phase I clinical testing have been reported at the 2010 ASCO meeting, and there are high hopes for the application of this molecule in some of the more intractable types of B-cell malignancy.
- Davis RE, Ngo VN, Lenz G, et al. Chronic active B-cell-receptor signalling in diffuse large B-cell lymphoma. Nature. 2010;463:88-92.
- Coelho V, Krysove S, Ghaemmaghami AM, et al. Glycosylation of surface Ig creates a functional bridge between human follicular lymphoma and microenvironmental lectins. Proc Natl Acad Sci USA. 2010;107:18587-18592.
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