Abstract 2854: Targeting constitutive NF-kB activation through Bruton's tyrosine kinase (Btk) and the proteasome in mantle cell lymphoma
Zhishuo OuArchito T. TamayoLiang ZhangJ. Jack LeeKejie ZhangChangping LiJacqueline WangJames P. PhamLuhong SunChristopher J. KirkR. FordLan V. Pham
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Abstract Mantle cell lymphoma (MCL) is an aggressive B-cell non-Hodgkin lymphoma (NHL) with poor prognoses; novel agents are needed for its therapy. Bruton's tyrosine kinase (Btk) has been identified as an essential kinase for B-cell survival and it is activated through the B-cell receptor (BCR) pathway. Btk has recently emerged as a promising target in MCL, as demonstrated by recent clinical trials on the Btk inhibitor PCI-32765 (Pharmacyclics, Sunnyvale, CA), suggesting that elucidating critical signaling pathways emanating through Btk will hold an important key to deciphering the pathogenesis of MCL that can lead to the development of more effective targeted therapies. In this study, we showed that Btk is constitutively phosphorylated in most MCL cell lines except Rec1 and DB SP53 and is variable among primary cells from patients. We demonstrated that knockdown of Btk by siRNA diminished Btk expression, reduced constitutive NF-kB activation by luciferase assays, leading to the cell growth inhibition and induction of apoptosis in MCL cell lines. MCL cells treated with the Btk inhibitor PCI-32765 effectively inhibits Btk activity, leading to reduced MCL cell growth with IC50 values range from 2-6 uM in Mino, Jeko1, Z138 and JMP1. Interestingly, the IC50 value in DB SP53, which lacks both phosphorylated Btk and membrane immunoglobulins, is 35 uM, suggesting that BCR signaling is not active in these cell lines. PCI-32765 also induced caspase-dependent apoptosis in a dose- and time-dependent manner in representative MCL cell lines and in patient primary cells. We further demonstrated that PCI-32765 down-regulates NF-kB activity through both, the canonical and alternative NF-kB pathways. Since previous studies have indicated synergy between proteasome inhibitors and tyrosine kinase inhibitors, we evaluated whether there is synergism between PCI-32765 and the next generation proteasome inhibitor Carfilzomib (Onyx Pharmaceuticals, South San Francisco, CA). Our data suggest potential synergy between Carfilzomib and PCI-32765 in represented MCL cell lines in terms of inhibiting cell growth and induction of apoptosis. Both compounds also synergize to inhibit NF-kB activation in MCL cells. In summary, our data suggest that Btk is a key survival kinase in MCL and strategic targeting of growth/survival Btk-mediated NF-kB pathways with novel therapeutic agents such as PCI-32765 should provide a novel therapy regimen for MCL patients. Combining PCI-32765 with the proteasome inhibitor Carfilzomib can synergize its effect in MCL and may be a useful therapeutic strategy, particularly for patients with relapsed/refractory MCL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2854. doi:1538-7445.AM2012-2854breakpoint cluster region
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Bruton tyrosine kinase (BTK) is a critical effector molecule for B cell development and plays a major role in lymphoma genesis. Ibrutinib is the first-generation BTK inhibitor. Ibrutinib has off-target effects on EGFR, ITK, and Tec family kinases, which explains the untoward effects of ibrutinib. Resistance to ibrutinib was also reported. The C481S mutation in the BTK kinase domain was reported to be a major mechanism of resistance to ibrutinib. This review summarizes the clinical development of novel BTK inhibitors, ACP-196 (acalabrutinib), ONO/GS-4059, and BGB-3111.
Hematology
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Inhibition of Bruton's Tyrosine Kinase (BTK) by ibrutinib, an irreversible inhibitor, has dramatically improved the outcomes in both previously treated and naïve chronic lymphocytic leukemia (CLL) patients. Ibrutinib inactivates BTK through covalently binding to the cysteine 481 residue (C481) which then leads to the inhibition of autophosphorylation of BTK and the inactivation of downstream survival nexus. Although ibrutinib demonstrated >90% overall and event-free survival, about 25% of patients discontinue ibrutinib due to leukemia progression and intolerance. Patients that initially respond to treatment may develop resistance and the most prevalent resistance mechanism of ibrutinib is described as the point mutation affecting the C481 residue of BTK that results in disruption of ibrutinib binding and acquired ibrutinib resistance. Most common mutations are C481S and C481R. Vecabrutinib is a potent reversible BTK inhibitor that binds to BTK through noncovalent interactions. As vecabrutinib does not require binding to C481 residue, it retains its efficacy with mutant BTK in vitro. To better understand differential biology of Wild-Type (WT) and serine and arginine substituted BTK, we labelled MEC-1 cell line with green fluorescence protein (GFP) and overexpressed either BTKWT, BTKC481Sor BTKC481R that contributes to ibrutinib resistance. We selected MEC-1 cell line as it represents CLL disease and the phenotype of the cells share several characteristics of ex vivo CLL cells. We performed Reverse Phase Protein Array (RPPA) and mRNA-sequencing to determine and compare the proteomic and transcriptomic profiles of the MEC-1 cells harboring WT and mutant BTK. Ingenuity Pathway Analysis (IPA) of RPPA data revealed that overexpression of BTK WT leads to the enrichment of protein alterations involved in cell cycle regulation, B cell receptor signaling, PI3K/AKT signaling, PTEN signaling and ERK/MAPK signaling. IPA of RNAseq data upon BTK WT overexpression unraveled the top canonical pathways that include signaling through axonal guidance, ephrin receptor, c-AMP mediated, CXCR4 signaling and PTEN signaling. Comparative analysis of MEC-1 cells with mutant BTK (C481S vs C481R) using IPA distinguished the activated pathways in BTKC481Sharboring cells from cells that express BTKC481R. These results are being validated by western blot and qRT-PCR assays. Immunoblotting results showed that following 24, 48, and 72 hour of vecabrutinib (at 1 µM) treatment reduced p-BTK (Tyr223), p-PLCG2 (Tyr759) and p-ERK (Thr 202/Tyr 204) levels in MEC-1 cells with mutant BTK. These data indicate that vecabrutinib effectively inhibits BTK and its downstream signaling in the presence of mutant BTK, suggesting that vecabrutinib treatment may be a rational approach to overcome ibrutinib resistance.Citation Format: Burcu Aslan, Mikhila Mahendra, Michael D Peoples, Joe R. Marszalek, Christopher P Vellano, Xiaofeng Zheng, Jing Wang, Pietro Taverna, Varsha Gandhi. Vecabrutinib inhibits C481 mutated Bruton's tyrosine kinase and its downstream signaling in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 317.
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Abstract Ibrutinib (Imbruvica ™ ) is an irreversible, potent inhibitor of Bruton's tyrosine kinase ( BTK ). Over the last few years, ibrutinib has developed from a promising drug candidate to being approved by FDA for the treatment of three B cell malignancies, a truly remarkable feat. Few, if any medicines are monospecific and ibrutinib is no exception; already during ibrutinib's initial characterization, it was found that it could bind also to other kinases. In this review, we discuss the implications of such interactions, which go beyond the selective effect on BTK in B cell malignancies. In certain cases, the outcome of ibrutinib treatment likely results from the combined inhibition of BTK and other kinases, causing additive or synergistic, effects. Conversely, there are also examples when the clinical outcome seems unrelated to inhibition of BTK . Thus, more specifically, adverse effects such as enhanced bleeding or arrhythmias could potentially be explained by different interactions. We also predict that during long‐term treatment bone homoeostasis might be affected due to the inhibition of osteoclasts. Moreover, the binding of ibrutinib to molecular targets other than BTK or effects on cells other than B cell‐derived malignancies could be beneficial and result in new indications for clinical applications.
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Research over the role of Bruton's agammaglobulinemia tyrosine kinase (BTK) in B-lymphocyte development, differentiation, signaling and survival has led to better understanding of the pathogenesis of B-cell malignancies. Down-regulation of BTK activity is an attractive novel strategy for treating patients with B-cell malignancies. Ibrutinib (PCI-32765), a potent inhibitor of BTK induces impressive responses in B-cell malignancies through irreversible bond with cysteine-481 in the active site of BTK (TH/SH1 domain) and inhibits BTK phosphorylation on Tyr223. This review discussed in details the role of BTK in B-cell signaling, molecular interactions between B cell lymphoma/leukemia cells and their microenvironment. Clinical trials of the novel BTK inhibitor, ibrutinib (PCI-32765), in B cell malignancies were summarized.
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Multiple myeloma (MM) is characterized by the over-production of monoclonal plasma cells that eventually become malignant in the bone marrow. MM remains as an incurable cancer, but it can be treated through chemotherapy. Nonetheless, research on novel therapies for effective treatment of MM is ongoing and in this case the involvement of Bruton’s tyrosine kinase (Btk) in B-cell malignancies has made it one of the new therapeutic targets. In MM patients, it has been reported that the expression of Btk was elevated and this could potentially contribute to chemoresistance indirectly via enhancement of cell proliferation and survival. Ibrutinib is a highly selective irreversible Btk inhibitor commonly used as treatment for B-cell malignancies such as Mantle Cell Lymphoma (MCL) and Chronic Lymphocytic Leukemia (CLL). With reference to the potential involvement of Btk in MM and current treatment of MCL and CLL using ibrutinib, researchers have begun to examine the effect of ibrutinib treatment on MM. This review provides information on the association of MM and Btk in conjunction with the treatment using ibrutinib. To date, clinical trials of ibrutinib as therapeutic alternative for MM have produced promising results, particularly as combination therapy with other agents such as dexamethasone and carfilzomib. However, there is limited evidence on the Btk mechanisms involved in MM, hence, it is important to further investigate the Btk oncogenic signalling pathway(s) in MM cells in order to establish successful and improved treatment of MM.
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Abstract The Tec family kinase, Bruton’s Tyrosine Kinase (BTK), is one of the key intracellular signaling components proximal to the B cell receptor (BCR). In this study, we utilized an orally-accessible BTK inhibitor (BTKi) to study the functional effects blocking this signaling pathway has on antigen-driven B cell activation in vivo. We report that BTK is required for anti-IgD driven B cell activation in vivo as evidenced by suppression of the surface activation markers CD86 and mRNA transcripts (e.g. c-Myc, Bcl-xL, CCL3, CD98, EBI2, EGR1, EGR2 and IRF4) otherwise rapidly induced upon engagement of the BCR with antigen. Using the T-dependent protein PE and SRBCs as model antigens, we tested for a requirement of BTK in generating antigen-specific IgM and IgG antibody titers and germinal centers, and find that BTK is required for germinal center maintenance. Lastly, we tested for a role of BTK in long-lived PE-specific memory B cell re-activation and report that BTKi significantly suppressed the induction of PE-specific IgM and IgG titers upon antigen re-challenge. The use of BTKi allowed us to bypass the impact a genetically inactivated BTK gene has on B cell development and to assess BTK inactivation in both a wild-type setting and in a temporal manner. These results provide insight into the role of BTK in BCR-driven B cell activation in various antigen naïve and experienced B cell populations.
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B-cell receptor (BCR)-mediated signals provide the basis for B-cell differentiation in the BM and subsequently into follicular, marginal zone, or B-1 B-cell subsets. We have previously shown that B-cell-specific expression of the constitutive active E41K mutant of the BCR-associated molecule Bruton's tyrosine kinase (Btk) leads to an almost complete deletion of immature B cells in the BM. Here, we report that low-level expression of the E41K or E41K-Y223F Btk mutants was associated with reduced follicular B-cell numbers and significantly increased proportions of B-1 cells in the spleen. Crosses with 3-83 mu delta and VH81X BCR Tg mice showed that constitutive active Btk expression did not change follicular, marginal zone, or B-1 B-cell fate choice, but resulted in selective expansion or survival of B-1 cells. Residual B cells were hyperresponsive and manifested sustained Ca(2+) mobilization. They were spontaneously driven into germinal center-independent plasma cell differentiation, as evidenced by increased numbers of IgM(+) plasma cells in spleen and BM and significantly elevated serum IgM. Because anti-nucleosome autoantibodies and glomerular IgM deposition were present, we conclude that constitutive Btk activation causes defective B-cell tolerance, emphasizing that Btk signals are essential for appropriate regulation of B-cell activation.
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