BCR::ABL1 tyrosine kinase inhibitors hamper the therapeutic efficacy of blinatumomab in vitro

Abstract

Purpose: Acute B-lymphoblastic leukemia (B-ALL) is a malignant disease characterized by accumulation of clonal immature lymphocytes in the bone marrow and peripheral blood. The approval of BCR::ABL1 tyrosine kinase inhibitors (TKI) such as imatinib, dasatinib, nilotinib and ponatinib marked a milestone in targeted therapy only for a subset of patients carrying the translocation t(9;22)(q34;q11). Immunotherapy with the bispecific antibody (bsAb) blinatumomab targeting CD19xCD3 revolutionized treatment of all B-ALL cases. The combination of both TKI and bsAb, so-called "dual targeting", is currently under clinical investigation, although TKI might influence T cell effects.

Methods: We here investigated the combination of different TKI and blinatumomab in BCR::ABL1⁺ and BCR::ABL1^- B-ALL cell lines and primary samples regarding T cell proliferation, differentiation, cytokine release and killing of tumor cells.

Results: In vitro analysis revealed profound reduction of T cell proliferation, differentiation, cytokine release and killing of tumor cells upon application of BCR::ABL1 TKI with blinatumomab. Inhibition was more pronounced with dasatinib and ponatinib compared to nilotinib and imatinib. T cell signalling after CD3 stimulation was impaired by TKI mirrored by inhibition of LCK phosphorylation. This known off-target effect might influence the efficacy of bsAb therapy when combined with BCR::ABL1 TKI.

Conclusion: In conclusion, we propose that nilotinib and imatinib might also be suitable substances for combination with blinatumomab and suggest evaluation in clinical trials.

Keywords: Acute lymphoblastic leukaemia; BCR::ABL1; Blinatumomab; Tyrosine kinase inhibitors.

Fig. 1

Impact of BCR::ABL1 TKI on T cell activation induced by blinatumomab and B-ALL cells. CD69 expression on CD4⁺ and CD8⁺ T cells was analyzed by flow cytometry in 3 day coculture assays (n = 4 donors) with 100,000 PBMC, 100,000 ALL cells and blinatumomab at 1 ng/ml. The following ALL cell lines were utilized: A BCR::ABL1⁺ TOM-1, B BCR::ABL1⁺ SD-1, C BCR::ABL1⁻ Nalm-6, D BCR::ABL1⁻ Nalm-16. The dotted lines correspond to the CD69 expression levels with PBMC, ALL cell line and blinatumomab. Statistical analysis with Mann–Whitney U test. *p < 0.05

Fig. 2

Impact of BCR::ABL1 TKI on T cell proliferation induced by blinatumomab and B-ALL cells. Proliferation of CD4⁺ and CD8 T cells (depicted as fold increase) was analyzed by flow cytometry in 3 day coculture assays (n = 4 donors) with 100,000 PBMC, 100,000 ALL cells and blinatumomab at 1 ng/ml. The following ALL cell lines were utilized: A BCR::ABL1⁺ TOM-1, B BCR::ABL1⁺ SD-1, C BCR::ABL1⁻ Nalm-6, D BCR::ABL1⁻ Nalm-16. The dotted lines correspond to the proliferation seen with PBMC, ALL cell line and blinatumomab. Statistical analysis with Mann–-Whitney U test. *p < 0.05

Fig. 3

Impact of TKI on blinatumomab-induced differentiation of T cells and T cell signaling. A Proportion of different CD8⁺ T cell subsets after 3 day coculture assays (n = 4 donors) with 100,000 PBMC, 100,000 BCR::ABL1⁺ SD-1 cells and blinatumomab at 1 ng/ml. B Phosphorylation of LCK (p-Y394) on T cells after 0, 5 and 15 min of stimulation with plate-coated blinatumomab was determined by phosphoflow. Pretreatment with TKI was performed overnight prior to bsAb exposure. Exemplary data from one out of three experiments is depicted. *p < 0.05

Fig. 4

Blinatumomab-induced T cell-mediated target cell lysis in the presence of TKI. Lysis of different ALL cells during 3 day coculture assays with 100,000 PBMC, 100,000 B-ALL cells and blinatumomab at 1 ng/ml was determined by flow cytometry. The following ALL cell lines were utilized: A BCR::ABL1⁺ TOM-1, B BCR::ABL1⁺ SD-1, C BCR::ABL1⁻ Nalm-6, D BCR::ABL1⁻ Nalm-16. Statistical analysis with Mann–Whitney U test. *p < 0.05

Fig. 5

Autologous lysis of B-ALL patient samples in the presence of blinatumomab and TKI. A Exemplary microscopic documentation of T cell cluster formation after 3 day coculture of ALL patient samples treated with blinatumomab and different TKI at peak levels. Scale bar = 10 µm. B, C Flow cytometric analysis CD8⁺ T cell CD69 expression (B) and CD8⁺ T cell proliferation (C) after 3 day coculture assays using 200,000 cells/well (n = 4) and blinatumomab at 1 ng/ml. D Inhibition of lysis of ALL blasts by autologous T cells in the presence of TKI during 3 day coculture assays using 200,000 cells/well (n = 4) and blinatumomab at 1 ng/ml. *p < 0.05