Ibrutinib significantly inhibited Bruton's tyrosine kinase (BTK) phosphorylation, in-vitro proliferation and enhanced overall survival in a preclinical Burkitt lymphoma (BL) model

Abstract

Pediatric and adult patients with recurrent/refractory Burkitt lymphoma (BL) continue to have poor outcomes, emphasizing the need for newer therapeutic agents. Bruton's tyrosine kinase (BTK) is activated following B-cell receptor stimulation and in part regulates normal B-cell development. Ibrutinib, a selective and irreversible BTK inhibitor, has been efficacious in chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), Waldenström's macroglobulinemia, and marginal zone lymphoma. In this study, we investigated the efficacy of ibrutinib alone and in selective adjuvant combinations against BL in-vitro and in a human BL xenografted immune-deficient NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ (NSG) mouse model. Our data demonstrated that phospho-BTK level was significantly reduced in BL cells treated with ibrutinib (p < 0.001). Moreover, we observed a significant decrease in cell proliferation as well as significant decrease in IC₅₀ of ibrutinib in combination with dexamethasone, rituximab, obinutuzumab, carfilzomib, and doxorubicin (p < 0.001). In-vivo studies demonstrated ibrutinib treated mice had a significantly prolonged survival with median survival of mice following ibrutinib treatment (32 days) (24 days) (p < 0.02). In conclusion, our findings demonstrate the significant in-vitro and preclinical in-vivo effects of ibrutinib in BL. Based on our preclinical results in this investigation, there is an on-going clinical trial comparing overall survival in children and adolescents with relapsed/refractory BL treated with chemoimmunotherapy with or without ibrutinib (NCT02703272).

Keywords: Bruton’s Tyrosine Kinase; Burkitt Lymphoma; Cell Proliferation; Ibrutinib; Xenografted NSG Mice.

Figure 1.

Significant inhibition of BTK phosphorylation in ibrutinib treated BL cell lines. Raji (A) and Ramos (B) BL cell lines were treated with ibrutinib at varying doses (0, 0.1, 0.2, 0.5, 1.0, 5.0, and 10 μM) for five days. Ibrutinib was dissolved in DMSO. DMSO (ibrutinib dose at 0) was used as control. The total levels of BTK protein and phosphorylated BTK protein (p-BTK) was examined by western blot analysis with specific antibody against BTK and phospho-BTK (Tyr223). GAPDH was used as loading control. Representative Western blot results are shown in the left panels of (A) and (B). Intensities of immunoreactive phospho-BTK (Tyr223) bands on Western blots shown in (A) were quantified by densitometric analysis as shown in the middle panels of (A) and (B). Intensities of immunoreactive BTK bands on Western blots shown in (A) were quantified by densitometric analysis as shown in the right panels of (A) and (B). n = 3, ***p < 0.00001, **p < 0.0005, *p < 0.005.

Figure 2.

Inhibition of BL cell proliferation by ibrutinib alone or in combination with dexamethasone. Raji (A) and Ramos (B) BL cell lines were treated with ibrutinib at varying doses (0, 0.1, 0.2, 0.5, 1.0, 5.0, and 10 μM) for five days. Ibrutinib was dissolved in DMSO. DMSO (ibrutinib dose at 0) was used as control. Cell proliferation was examined by CellTiter 96® Aqueous One solution cell proliferation assays. Fold change in cell proliferation was calculated by the equation: treated A490 reading/DMSO A490 reading. DMSO A490 reading was set as 1. Significant inhibition of cell proliferation was observed in ibrutinib-treated Raji (A) and Ramos (B) BL cell lines following 5 days treatment. Raji (n = 3) IC₅₀ = 5.2μM, Ramos (n = 3) IC₅₀ = 0.87μM. *p < 0.05.Raji (C) and Ramos (D) BL cell lines were treated with ibrutinib at varying doses (0, 0.25, 0.5, 1.0, 5.0, and 10 μM) in combination with 1μM dexamethasone for five days. Both Ibrutinib and dexamethasone were dissolved in DMSO. DMSO (ibrutinib dose at 0 without dexamethasone) was used as control. Cell proliferation was examined by CellTiter 96® Aqueous One solution cell proliferation assays. Cell proliferation fold change was calculated by the equation: treated A490/DMSO A490. DMSO A490 was set as 1. IC₅₀ was determined with CompuSyn software. Raji (C) and Ramos (D) cell proliferations were significantly decreased with the combination of ibrutinib and dexamethasone. IC₅₀ of ibrutinib and dexamethasone against Raji is 0.34 μM compared to ibrutinib alone 5.2μM (n = 3, p < 0.01) and IC₅₀ of ibrutinib and dexamethasone against Ramos is 0.13 μM compared to ibrutinib alone 0.87μM (n = 3, p < 0.01). *p < 0.01, **p < 0.005, ***p < 0.0001.

Figure 3.

Inhibition of BL cell proliferation by ibrutinib in combination with rituximab or with obinutuzumab. Raji (A) and Ramos (B) BL cell lines were treated with ibrutinib (Ibr) with varying doses (0, 0.25, 0.5, 1.0, 5.0, and 10 μM) in combination with rituximab (RTX) (20ug/ml) or obinutuzumab (ObiN) (20ug/ml) for five days. Ibrutinib was dissolved in DMSO. DMSO (ibrutinib dose at 0 without rituximab or obinutuzumab) was used as control. Cell proliferation was examined by CellTiter 96® Aqueous One solution cell proliferation assays (MTS). Cell proliferation fold change was calculated by the equation: treated A490 reading/DMSO A490. DMSO A490 was set as 1. IC₅₀ was determined with CompuSyn software. The second and third grey bars with ibrutinib dose at 0 represent the cells treated with 20ug/ml rituximab or 20ug/ml obinutuzumab alone. Raji (A) and Ramos (B) cell proliferations were significantly decreased with the combination of Ibr and RTX or Ibr and ObiN. IC₅₀ of Ibr and RTX against Raji is 0.67 μM compared to Ibr alone 4.55μM (n = 3, p < 0.0005) (A) and IC₅₀ of Ibr and RTX against Ramos is 0.16 μM compared to Ibr alone 1.41μM (n = 3, p < 0.0001) (B). IC₅₀ of Ibr and ObiN against Raji is 0.16 μM compared to Ibr alone 4.55μM (n = 3, p < 0.00005) (A) and IC₅₀ of Ibr and ObiN against Ramos is 0.01 μM compared to Ibr alone 1.41μM (n = 3, p < 0.001) (B).

Figure 4.

Inhibition of BL cell proliferation by ibrutinib in combination with carfilzomib, idelalisib or doxorubicin. Raji cells were treated with ibrutinib (Ibr) with varying doses (0, 0.25, 0.5, 1.0, 5.0, and 10 μM) in combination with carfilzomib (CFZ) (A), idelalisib (B), or doxorubicin (dox) (C) for five days. The doses of CFZ, idelalisib, and dox were indicated in the figures. Ibrutinib was dissolved in DMSO. DMSO (ibrutinib dose at 0 without any other drug) was used as control. The second and the third grey bars in A with ibrutinib dose at 0 represent the cells treated with carfilzomib alone. The second grey bar in B with ibrutinib dose at 0 represents the cells treated with idelalisib alone. The second and the third grey bars in C with ibrutinib dose at 0 represent the cells treated with doxorubicin alone. Cell proliferation was examined by CellTiter 96® Aqueous One solution cell proliferation assays. Cell proliferation fold change was calculated by the equation: treated A490 reading/DMSO A490 reading. DMSO A490 reading was set as 1. IC₅₀ was determined with CompuSyn software.

Figure 5.

Survival of Raji BL xenografted mice after ibrutinib treatment 1 × 10⁶ Raji-Luc cells were administered by sterile subcutaneous (s.c) injections in the lower flank of 6–8 week old female NSG mice. 5 days later after tumor xenografted. Ibrutinib (12.5 mg/kg) or PBS (control) was given to mice by oral gavage once a day for 10 continuous days. (A) Kaplan–Meier curves representing survival of mice from the beginning of tumor implantation. p < 0.0001. (B) Experimental schema showing ibrutinib treatment for different groups of irradiated BL xenografted NSG mice. (C) Bioluminescence imaging of representative mice at 3, 10, 15, 20 and 25 days post tumor cells injection are shown and mice were then monitored for quantified bioluminescence (D). (p < 0.001 at day 20, p < 0.05 at day 25, respectively).

Figure 6.

Comparison of tumor progression and survival following ibrutinib versus cyclophosphamide and obinutuzumab in BL xenografted mice. Experimental schema showing ibrutinib, cyclophosphamide or obinutuzumab treatment for different groups of irradiated BL xenografted NSG mice (A). Significantly reduced bioluminescence signal was observed in ibrutinib, cyclophosphamide (CTX) or obinutuzumab versus control treated groups at day 15 or day 20 (B and C) (p < 0.01 at day 15, p < 0.05 at day 20, respectively). Furthermore, there was equal effect of survival between ibrutinib vs CTX vs obinutuzumab. Ibrutinib significantly prolonged the survival versus control but was similar in survival when compared to single agent of CTX and obinutuzumab (D and E).