Ibrutinib Is an Irreversible Molecular Inhibitor of Interleukin-2 Inducible Kinase: Expanding Therapeutic Potential and Modulating a Th1 Selective Pressure in CD4 T-Cells

Abstract ⁷⁷⁵

In chronic lymphocytic leukemia (CLL), mounting evidence points to an aberrant tumor associated Th2 bias that drives leukemic cell immune evasion, promotes formation of a supportive niche microenvironment, and functionally cripples innate and adaptive immunity. The end result is a high incidence of infections which is the primary cause of mortality in CLL. This same Th2 bias is induced by many other types of cancer. Th2 CD4 T-cells are singularly dependent upon IL-2-inducible T-cell kinase (ITK) for activation whereas Th1 CD4 and CD8 T-cells have compensatory resting lymphocyte kinase (RLK) which conducts T-cell receptor activation even in the absence of ITK. Thus, a clinically viable ITK inhibitor would be ideal for targeting immune suppression associated with CLL and potentially other types of cancer. Unfortunately, no such therapeutic is currently available. Ibrutinib, a confirmed inhibitor of the Bruton's tyrosine kinase (BTK) that irreversibly blocks downstream B-cell receptor activation, has demonstrated outstanding clinical activity in phase I/II clinical trials resulting in durable remissions in CLL. Our studies unveiled a previously uncharacterized Th1 cytokine switch in ibrutinib treated CLL patients which could not be attributed to B-lymphocytes. This ibrutinib-induced Th1 T-cell skewing was confirmed using the EμTCL1 mouse model of leukemia. Such alterations in cytokine patterns were reminiscent of mouse studies in which genetic ablation of ITK subverted Th2 immunity, thereby potentiating Th1-based adaptive immunity. The striking homology between BTK and ITK combined with intriguing in silico docking studies and promising in vitro kinase inhibition profiles with ibrutinib led to the hypothesis that this could be the first clinically viable irreversible ITK inhibitor. Cellular probe assays confirmed that the active site of ITK was covalently blocked by ibrutinib at pharmacologically relevant doses. Our comprehensive molecular analyses of T-cell signaling confirmed this in the Jurkat cell line. We further confirmed both molecular and functional outcomes in primary and in vitro polarized Th1 and Th2 CD4 T-cells. We found that mutation of the ITK-Cys442 covalent binding residue for ibrutinib alleviated molecular inhibition. We also demonstrated that Th1 and CD8 T-cell restricted expression of RLK provides a compensatory platform for T-cell activation offering a molecular explanation for the selective outgrowth of cytotoxic Th1 biased immunity. We further confirmed this effect using T-cells directly derived from CLL patients. To demonstrate that ibrutinib-induced ITK inhibition had direct clinical relevance in the setting of CLL we utilized a novel listeriosis/leukemia mouse model. In this model we clearly demonstrated complete recovery of functional immunity and all ibrutinib treated mice survived a potentially lethal Listeria monocytogenes infection. Our results expose novel molecular insights into the mechanism of action of ibrutinib in the context of Th2-biased immunosuppressive leukemia. We also postulate that ibrutinib's irreversible ITK inhibitory effects may prove effective in a number of other autoimmune, inflammatory, and viral diseases, including influenza A and HIV/AIDS.