Interleukin-2-Inducible T-Cell Kinase (ITK) Inhibitors for the Treatment of T-Cell Lymphoproliferative Disorders
Authors: Reneau John C. , Hwang Steven R. , Murga-Zamalloa Carlos A. , Buggy Joseph J. , Janc James W. , Wilcox Ryan A. 
Published: October 2019

Abstract
Introduction: We previously demonstrated that engagement of the T-cell receptor (TCR) in malignant T-cells leads to ITK-dependent activation of the transcription factors NF-κB and GATA3, thus promoting chemotherapy resistance. TCR-mediated chemotherapy resistance was reversed upon knockdown or pharmacologic inhibition of ITK (Wang, 2017). ITK and resting lymphocyte kinase (RLK) are partially redundant TEC family kinase members involved in TCR signaling (Dubovsky, 2013). Here, we report results from preclinical studies evaluating the use of the ITK-specific inhibitor CPI-818 and the dual ITK/RLK inhibitor CPI-893 in the treatment of T-cell lymphoproliferative disorders.
Methods/Results: Kinome screening was performed for CPI-818 and CPI-893. CPI-818 had high specificity for ITK over RLK (IC50 2.3 nM and 260 nM, respectively). In contrast CPI-893 had a high affinity for both ITK and RLK (IC50 0.36 nM and 0.4 nM, respectively). Normal and malignant T-cells from primary patient samples were evaluated for both ITK and RLK by western blot. As anticipated, normal CD3+ T-cells isolated from healthy donors expressed both ITK and RLK (n=3/3). In contrast, malignant T-cells preferentially expressed ITK in 3 of 3 cases and low levels of RLK in a single case. Tissue microarray analysis of cutaneous T-cell lymphoma and peripheral T-cell lymphoma cases further demonstrated ITK expression across multiple subtypes.
To confirm on target effects of CPI-818 and CPI-893, Jurkat cells (which express ITK but not RLK) were stimulated with anti-CD3/CD28 beads for 5 minutes in the presence or absence of either agent. Auto-phosphorylation of ITK Y180 and PLC-γ Y783, an ITK substrate, were significantly reduced in the presence of either agent, confirming on target effects. We next evaluated the effect of both drugs on GATA3 expression. Cells were stimulated for 24 hours with anti-CD3/CD28 beads in the presence or absence of these agents (or vehicle control). ITK inhibition with either agent significantly reduced GATA3 expression in these experiments.
We next evaluated the effect of CPI-818 and CPI-893 on cell viability in normal T-cells. TCR stimulation for 24 hours increased viability by 359% in the presence of vehicle control when normalized to unstimulated cells (n=3). Treatment with 1 µM CPI-893 reduced the cell viability to only 133% when compared to unstimulated controls (n=3, p = 0.0045). In contrast, treatment with the ITK-specific inhibitor CPI-818 at 1 µM had a numerically smaller, but still statistically significant effect (n=3, average viability 278%, p = 0.0056). We next quantified IL-10 expression by ELISA in cell culture supernatants. Consistent with the observed effect on cell proliferation and viability, only CPI-893 significantly reduced IL-10 production upon TCR stimulation (886 pg/mL vs 11.7 pg/mL, p <0.0001).
In order to assess TCR-mediated resistance to chemotherapy in malignant T-cells, T8ML-1 cells (PTCL, NOS cell line) were incubated in the presence or absence of CPI-818 or CPI-893 (both 1 µM) with varying concentrations of vincristine. When compared to TCR-stimulated cells alone, both agents displayed single agent activity, but also sensitized the cells to the effects of vincristine. The IC50 for vincristine in unstimulated cells, TCR-stimulated cells, CP-818 treated stimulated cells, and CPI-893 treated stimulated cells was 0.049, 0.443, 0.066, and 0.091 nM, respectively (n=4 per group). Similar results were observed in primary patient samples treated with romidepsin. Consistent with the observed effect on proliferation and viability, IL-10 production was decreased when compared to TCR stimulated controls (15.97 pg/mL, n=3 for each group) with both CPI-818 (3.47 pg/mL, p <0.0001) and CPI-893 (2.3 pg/mL, p <0.0001).
Conclusions: Our results demonstrate that TCR signaling inhibition with the selective ITK inhibitor CPI-818 significantly impaired malignant T-cell growth and proliferation, increased chemotherapy sensitivity, and decreased GATA3 expression. In contrast, this agent had minimal effect on normal T cells; this is likely explained by the expression of both ITK and RLK in most conventional T-cells. Collectively, our data support further preclinical and clinical studies with CP-818 in T-cell lymphoproliferative disorders.

Disclosures
Buggy: Corvus Pharmaceuticals, Inc.: Employment, Equity Ownership. Janc:Corvus Pharmaceuticals, Inc.: Employment, Equity Ownership. Wilcox:Incyte, Corp: Research Funding.