Inhibition of IL2-Inducible T-Cell Kinase (ITK)-Mediated Chemoresistance By Ibrutinib in T-Cell Lymphoproliferative Disorders

Background : Most (≈ 95%) T-cell lymphomas (TCL) express an intact T-cell receptor (TCR), suggesting that malignant T cells, like their B-cell counterparts, may benefit from antigen-receptor signaling. TCR engagement culminates in the activation of pathways required for T-cell proliferation and survival. The Tec family kinase and BTK homologue ITK is required for optimal TCR-dependent signaling. Therefore, we sought to understand whether TCR activation promotes chemotherapy resistance in TCL, and whether this may be overcome upon inhibition of ITK. Methods : TCL cell lines, mouse models and primary patient specimens were utilized. TCR signaling was engaged by anti-CD3/CD28 beads. ITK and GATA3 were inhibited by lentiviral-mediated shRNA knockdown and by ibrutinib, an ITK inhibitor. The chemoresistance of TCL cells was investigated in vitro and in vivo. Results : We have previously shown by gene expression profiling, proliferation, cytokine release, and signaling pathway analysis that TCR signaling remains intact in TCL [Blood, 2014, 124(21), 2959]. To further investigate the effect of TCR on chemoresistance, T8ML1 (a PTCL, NOS cell line) and primary cells from TCL patients (n=4) were treated with either vincristine or romidepsin in vitro . For T8ML1, the viability of cells treated with either vincristine or romidepsin increased by 2.9+/-0.14 fold or 1.4 +/- 0.035 fold respectively (p<0.01) upon TCR engagement. The enhanced viability of T8ML1 by TCR engagement under either vincristine or romidepsin treatment was abolished by shRNA-mediated ITK knockdown and significantly inhibited by ibrutinib (p<0.01). Similarly for primary TCL patient cells, the viability of cells treated with romidepsin increased by 2.4 +/- 0.35 fold upon TCR engagement, which was also significantly inhibited by ibrutinib treatment (p<0.01). To study the mechanism of TCR signaling in TCL chemoresistance, downstream targets of TCR signaling (NFκB, GATA3) were examined. Upon TCR engagement, NFκB activity increased by 1.7 +/- 0.22 fold in T8ML1 and 1.8 +/- 0.36 fold in primary cells from TCL patients (p<0.01) as shown by DNA-binding and nuclear localization. The activation of NFκB in T8ML1 and primary TCL patient cells was significantly inhibited by ibrutinib (p<0.01). We and others have previously shown that GATA3 identifies a distinct subset of PTCL, NOS that is characterized by inferior progression-free survival following anthracycline-based chemotherapy. [Blood, 2014, 123 (19), 3007-3015; Blood, 2014, 123(19), 2915-2923]. Furthermore, GATA-3 regulates the homeostatic survival of normal T cells following TCR engagement. In addition to NFκB, GATA3 protein increased by 3.0 +/- 0.45 fold in T8ML1 (p<0.01) and by 3.6 +/- 3.0 fold in primary TCL patient cells (p<0.03) upon TCR engagement, which was also inhibited by ibrutinib treatment. Furthermore, in T8ML1 cells, GATA3 upregulation by TCR engagement was abolished by shRNA-mediated ITK knockdown. To study the effect of GATA3 on chemoresistance, GATA3 was knocked down by lentiviral-mediated shRNAs in TCL cell lines (T8ML1, H9 and MyLa). The viability of TCL lines following GATA3 knockdown decreases by 2-4 fold (p<0.01) following treatment with either vincristine, romidepsin or 4-hydroxycyclophosphamide. In comparison to tumor xenografts generated fromTCL lines transduced with a non-targeting shRNA, GATA3 deficient tumor xenografts were significantly more sensitive to vincristine alone or combined vincristine/cyclophosphamide (p<0.01). Conclusions : TCR engagement promotes resistance to chemotherapy in T-cell lymphomas in an ITK- and GATA-3-dependent manner. Furthermore, chemotherapy resistance following TCR engagement is significantly impaired by ibrutinib. Therefore, ibrutinib may warrant further investigation in the T-cell lymphomas. Disclosures Off Label Use: We used ibrutinib to inhibit T-cell receptor signaling and discussed its clinical implication in T-cell lymphomas..