Hsu-Ping Kuo

Combination of Ibrutinib and ABT-199 in Diffuse Large B-Cell Lymphoma and Follicular Lymphoma

Diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma are the most prevalent B-lymphocyte neoplasms in which abnormal activation of the Bruton tyrosine kinase (BTK)–mediated B-cell receptor signaling pathway contributes to pathogenesis. Ibrutinib is an oral covalent BTK inhibitor that has shown some efficacy in both indications. To improve ibrutinib efficacy through combination therapy, we first investigated differential gene expression in parental and ibrutinib-resistant cell lines to better understand the mechanisms of resistance. Ibrutinib-resistant TMD8 cells had higher BCL2 gene expression and increased sensitivity to ABT-199, a BCL-2 inhibitor. Consistently, clinical samples from ABC-DLBCL patients who experienced poorer response to ibrutinib had higher BCL2 gene expression. We further demonstrated synergistic growth suppression by ibrutinib and ABT-199 in multiple ABC-DLBCL, GCB-DLBCL, and follicular lymphoma cell lines. The combination of both drugs also reduced colony formation, increased apoptosis, and inhibited tumor growth in a TMD8 xenograft model. A synergistic combination effect was also found in ibrutinib-resistant cells generated by either genetic mutation or drug treatment. Together, these findings suggest a potential clinical benefit from ibrutinib and ABT-199 combination therapy. Mol Cancer Ther; 16(7); 1246–56. ©2017 AACR.

Abstract 3072: Combination of ibrutinib and corticosteroids in B-cell non-Hodgkin lymphomas (NHL)

Introduction: The B-cell receptor (BCR) signaling pathway is a major driver in the pathogenesis of B-cell malignancies. A vast array of BCR-associated kinases have emerged as rational therapeutic targets, including Bruton9s tyrosine kinase (BTK), which plays a pivotal role in BCR signaling. Ibrutinib is a first-in-class, oral, covalent BTK inhibitor approved in the US for patients with mantle cell lymphoma and chronic lymphocytic leukemia (CLL) who have received at least 1 prior therapy, CLL patients with 17p deletion, and patients with Waldenstrom9s macroglobulinemia. While ibrutinib is efficacious as a single agent, combinations with other drugs may further enhance efficacy and increase response rates in patients with NHLs. Corticosteroids are included in nearly all combination regimens for NHL treatment, showing promising results when combined with chemotherapy and antibodies (Cunningham, Lancet 2013). We therefore evaluated corticosteroids in combination with ibrutinib in preclinical models of activated B cell-like (ABC) and germinal center B cell-like (GCB) diffuse large B-cell lymphoma (DLBCL), and follicular lymphoma (FL). Methods: ABC-DLBCL, GCB-DLBCL, and FL cell lines were used in this study. Drug effect on cell growth was evaluated with CellTiter-Glo luminescent cell viability assay (Promega) following treatment with ibrutinib or the combinations for 3 days. Combination index (CI) was determined using CalcuSyn. Synergy score (SS) was calculated using the Chalice Analyzer (Horizon CombinatoRx). Mutation profiles were extracted from the Catalogue of Somatic Mutations in Cancer database. Results: Synergistic growth suppression of dexamethasone and ibrutinib was identified in 7 of 14 GCB-DLBCL cell lines. Dexamethasone had a stronger effect on cells with lower EC50 on ibrutinib treatment. Intriguingly, 4 of these 7 cell lines that showed synergy of the 2 compounds had BCL-2 nonsynonymous mutations, whereas only 1 of the cell lines with no combination effect had BCL-2 silent mutation. In addition to dexamethasone, other corticosteroids, including hydrocortisone, prednisolone, and mometasone, also showed synergy with ibrutinib in GCB-DLBCL (SU-DHL-4, CI = 0.219-0.668, SS = 5.08-15.15; SU-DHL-10, CI = 0.008-0.224, SS = 7.27-11.24), ABC-DLBCL (TMD-8, CI = 0.001-0.016, SS = 21.1), and FL cell lines (DoHH2, CI = 0.020-0.467, SS = 12.3-30.86; WSU-FSCCL, CI = 0.017-0.022, SS = 18.24). The in vivo effect and underlying mechanisms of the combinations are currently under investigation. Conclusions: We identified synergistic inhibitory effects of ibrutinib and corticosteroids on ABC-DLBCL, GCB-DLBCL, and FL cell growth, providing a rationale for the design of combination clinical trials in these NHL types. Further understanding of the mechanisms contributing to the synergy may help to stratify patient populations and expand treatment to other lymphoid neoplasms. Citation Format: Hsu-Ping Kuo, Sidney Hsieh, Karl J. Schweighofer, Leo WK Cheung, Mutiah Apatira, Mint Sirisawad, Shiquan Wu, Karl Eckert, Yu Liang, Jeff Hsu, Chun-Te Chen, Darrin Beaupre, Betty Y. Chang. Combination of ibrutinib and corticosteroids in B-cell non-Hodgkin lymphomas (NHL). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3072.

Abstract 4860: The BTK inhibitor ibrutinib modulates T cell immunity in mouse models and in differentiated human T cells

Ibrutinib inhibits Bruton9s tyrosine kinase (BTK) and effectively treats B-cell malignancies. Ibrutinib also targets IL-2-inducible T-cell kinase (ITK) and modulates the T-cell receptor signaling pathway. Because ibrutinib enhanced antitumor immunity when combined with anti-PD-L1 in various syngeneic solid tumor and lymphoma models (Sajiv-Barfi, PNAS 2015), we sought to determine the effect of ibrutinib on T-cell function and activation. Using in vitro human primary T-cell activation assays, we found that ibrutinib did not suppress T-cell proliferation but slightly inhibited IFNa production in CD4 and CD8 effector T cells when human peripheral blood mononuclear cells (PBMCs) were stimulated with anti-CD3 and anti-CD28 antibodies. Ibrutinib also inhibited PD-1 expression on human CD8 effector T cells. More strikingly, ibrutinib significantly suppressed naive murine CD4 T-cell differentiation in vitro into Foxp3+ regulatory T cells (Treg) in the presence of TGF-â. Further, we evaluated the immunomodulatory effect of ibrutinib in tumor-bearing mice. In pancreatic (Pan02) and renal cell carcinoma (Renca) models, we found that single-agent ibrutinib, as well as in combination with standard-of-care agents, effectively inhibited tumor growth. In the syngeneic Renca tumor model, combination ibrutinib treatment significantly reduced CD4+Foxp3+ Treg cells both in the spleen (P Citation Format: Yujun Huang, Jeff Hsu, Mint Sirisawad, Hsu-Ping Kuo, Betty Y. Chang. The BTK inhibitor ibrutinib modulates T cell immunity in mouse models and in differentiated human T cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4860.

Abstract 2598: Synergistic effect of ibrutinib and inhibitors targeting TLR signaling in ABC subtype of diffuse large B-Cell lymphoma

Introduction: Diffuse large B-cell lymphoma (DLBCL) is the most prevalent B-lymphocyte neoplasm (Morton, Blood 2006). The activated B-cell (ABC) subtype is associated with poorer prognosis (Lenz, NEJM 2008). Over 20% of ABC-DLBCL cases have mutations in the CD79B or CD79A subunits of the B-cell receptor (BCR), generating chronically active BCR signaling (Staudt, ASH 2012). Ibrutinib is a first-in-class, once-daily, oral, covalent inhibitor of Bruton9s tyrosine kinase, an essential BCR pathway enzyme. Phase 2 trial results of single-agent ibrutinib in DLBCL showed efficacy in the ABC subtype, with an overall response rate of 40% and 60% in CD79B-mutant ABC-DLBCL (Wilson, ASH 2012). Identification of MYD88 L265P mutations in ABC-DLBCL revealed a link between Toll-like receptor (TLR) signaling and ABC-DLBCL (Ngo, Nature 2011). The findings that a TLR 7/8/9 antagonist selectively inhibits cell survival of MYD88 L265P-mutated ABC-DLBCL cells (Bhagat, AACR 2014) emphasize the importance of TLR signaling in ABC-DLBCL. We hypothesized that TLR-related kinases/pathways important to ABC-DLBCL may be unknown and tested for synergy between ibrutinib and inhibitors targeting these pathways in ABC-DLBCL cells. Methods: Combination effects of ibrutinib and TLR9 antagonists, a TAK1 inhibitor, or an endosomal TLR inhibitor were tested in ABC-DLBCL cell lines with MYD88 mutations. TMD-8, HBL-1, and OCI-LY10 cell lines were treated with inhibitors or antagonists alone or in combination with ibrutinib for 3 days; cell growth effects were determined by the CellTiter-Glo luminescent cell viability assay (Promega). The combination index (C.I.), a drug interactivity measurement, was calculated with CalcuSyn. Synergy scores were calculated by the Chalice Analyzer (Horizon CombinatoRx). Results: We identified synergistic inhibitory effects on ABC-DLBCL cell growth between ibrutinib and TLR9 antagonists as well as the TAK1 inhibitor. Different TLR9 antagonist combinations significantly enhanced ibrutinib killing effects in ABC-DLBCL cells in the presence or absence of class A, B, and C TLR9 agonists. Stimulation with TLR9 agonists increased resistance of ABC-DLBCL cells. Strong synergy was also identified between ibrutinib and an inhibitor of endocytic TLRs (TLR3, 7, 8, 9) with an average C.I. of 0.11 and synergy score of 4.22. Our assays demonstrated that an endosomal TLR inhibitor possesses similar ibrutinib synergistic effects as other TLR9 antagonists. Similar results were observed for inhibition of the downstream TLR signaling molecule TAK1. Conclusions: We provide evidence of strong synergistic effects between ibrutinib and inhibitors targeting TLR signaling. Survival of ABC-DLBCL cell lines with MYD88 mutations requires signals through both TLR and BCR. Combined, these results provide rationale for targeting both pathways simultaneously in MYD88-mutated ABC-DLBCL. Citation Format: Hsu-Ping Kuo, Sidney Hsieh, Betty Chang. Synergistic effect of ibrutinib and inhibitors targeting TLR signaling in ABC subtype of diffuse large B-Cell lymphoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2598. doi:10.1158/1538-7445.AM2015-2598