Key Points Ibrutinib is the first clinically viable irreversible ITK inhibitor. Ibrutinib inhibits the formation of Th2 but not Th1 immunity.

Key Points Persistent CLL cells during ibrutinib therapy show evidence of biochemical activation, but inhibited BCR and no proliferation. Long lymphocytosis during ibrutinib therapy is not associated with adverse progression-free survival.

Chronic lymphocytic leukemia (CLL) is characterized by constitutive activation of the B-cell receptor (BCR) signaling pathway, but variable responsiveness of the BCR to antigen ligation. Bruton's tyrosine kinase (BTK) shows constitutive activity in CLL and is the target of irreversible inhibition by ibrutinib, an orally bioavailable kinase inhibitor that has shown outstanding activity in CLL. Early clinical results in CLL with other reversible and irreversible BTK inhibitors have been less promising, however, raising the question of whether BTK kinase activity is an important target of ibrutinib and also in CLL. To determine the role of BTK in CLL, we used patient samples and the Em-TCL1 (TCL1) transgenic mouse model of CLL, which results in spontaneous leukemia development. Inhibition of BTK in primary human CLL cells by small interfering RNA promotes apoptosis. Inhibition of BTK kinase activity through either targeted genetic inactivation or ibrutinib in the TCL1 mouse significantly delays the development of CLL, demonstrating that BTK is a critical kinase for CLL development and expansion and thus an important target of ibrutinib. Collectively, our data confirm the importance of kinase-functional BTK in CLL. (Blood. 2014; 123(8):1207-1213 IntroductionChronic lymphocytic leukemia (CLL) is a common adult leukemia that is currently incurable outside of stem cell transplantation. Although response to IgM ligation is variable, the B-cell receptor (BCR) signaling pathway is aberrantly active in this disease, with antigendependent 1,2 or -independent autonomous activation, 3 leading to constitutive activation of kinases inducing cell survival and proliferation. [4][5][6][7] One BCR pathway kinase that is uniformly overexpressed at the transcript level 8 and constitutively phosphorylated in CLL is Bruton's tyrosine kinase (BTK). Ibrutinib, an orally bioavailable irreversible inhibitor of BTK, has recently been shown to have outstanding clinical activity in CLL with extended durable remissions in both untreated and relapsed disease. 9 BTK is a critical mediator of B-lymphocyte signaling and development. Mutations in various domains are responsible for X-linked agammaglobulinemia, 10,11 a disorder characterized by developmental arrest of B cells and profound humoral immune deficiency in humans. A point mutation in the Pleckstrin homology domain is responsible for the milder X-linked immunodeficiency (XID) phenotype in the mouse, 12,13 which is characterized by reduced numbers of circulating B cells and reduced serum immunoglobulins. BTK is also a critical mediator in B-cell signaling. It is recruited to the membrane-bound signalosome in the early stages of B-cell activation, and, following phosphorylation by Syk and Lyn, participates in the phosphorylation of phospholipase C, gamma 2 (PLCg2), which leads to production of the second messengers diacylglycerol and inositol-1,4,5-triphosphate. This pathway is amplified in CLL and leads to prosurvival signals through its effects on phosphatidylinositol 3-kinase (PI3K), PL...

Key Points Ibrutinib combined with ofatumumab in relapsed CLL had had an ORR of 83% with median time to response of <3 months in all groups. All 3 sequences of administration were acceptably tolerated and active; responses were durable, and median PFS was not yet reached.

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. Disclosures: Jaglowski: Pharmacyclics: Research Funding. Chang:Pharmacyclics, Inc.: Employment. Maddocks:Pharmacyclics: Research Funding. Buggy:Pharmacyclics: Employment, Equity Ownership. Byrd:Pharmacyclics: Research Funding.