Development of combination therapies with BTK inhibitors and dasatinib to treat CNS-infiltrating E2A-PBX1+/preBCR+ ALL

Abstract

The t(1;19) translocation, encoding the oncogenic fusion protein E2A (TCF3)-PBX1, is involved in acute lymphoblastic leukemia (ALL) and associated with a pre-B-cell receptor (preBCR+) phenotype. Relapse in patients with E2A-PBX1+ ALL frequently occurs in the central nervous system (CNS). Therefore, there is a medical need for the identification of CNS active regimens for the treatment of E2A-PBX1+/preBCR+ ALL. Using unbiased short hairpin RNA (shRNA) library screening approaches, we identified Bruton tyrosine kinase (BTK) as a key gene involved in both proliferation and dasatinib sensitivity of E2A-PBX1+/preBCR+ ALL. Depletion of BTK by shRNAs resulted in decreased proliferation of dasatinib-treated E2A-PBX1+/preBCR+ cells compared with control-transduced cells. Moreover, the combination of dasatinib with BTK inhibitors (BTKi; ibrutinib, acalabrutinib, or zanubrutinib) significantly decreased E2A-PBX1+/preBCR+ human and murine cell proliferation, reduced phospholipase C gamma 2 (PLCG2) and BTK phosphorylation and total protein levels and increased disease-free survival of mice in secondary transplantation assays, particularly reducing CNS-leukemic infiltration. Hence, dasatinib with ibrutinib reduced pPLCG2 and pBTK in primary ALL patient samples, including E2A-PBX1+ ALLs. In summary, genetic depletion and pharmacological inhibition of BTK increase dasatinib effects in human and mouse with E2A-PBX1+/preBCR+ ALL across most of performed assays, with the combination of dasatinib and BTKi proving effective in reducing CNS infiltration of E2A-PBX1+/preBCR+ ALL cells in vivo.

Graphical abstract

Figure 1.

BTK-depletion decreases the proliferation of human E2A-PBX1⁺/preBCR⁺ RCH-ACV cells treated with dasatinib. (A) Using a shRNA library screen approach, the BTK was identified as key gene involved to increase sensitivity to dasatinib in E2A-PBX1⁺/preBCR⁺ leukemia cells. (B) Gene ontology analysis of pathways enriched in genes increasing sensitivity to dasatinib. Only the top 10 KEGG pathways are shown. (C) RT-qPCR shows efficient shRNA-mediated knockdown of BTK2 and BTK3. (D) Western blot analysis (representative of 3 independent experiments) shows the protein levels following knockdown by sh-BTK2 and sh-BTK3 constructs in RCH-ACV cells. Messenger RNA (mRNA) and protein were extracted from the same stably transduced expanded sh-Luc control cells and sh-BTK2/sh-BTK3 knockdown cells used for (C) and (D) experiments. GADPH was used as loading control. Densitometry values were calculated using ImageJ software. (E) Graph shows the percentage of mCherry⁺ cells transduced with shRNAs for luciferase (control) or shRNA constructs of BTK2 or BTK3 and treated with dasatinib 20 nM for 24 days. Data represent the mean ± standard of error mean (SEM) of 3 independent experiments. Statistical analysis performed by nonparametric Mann-Whitney test. n.s., not significant; ∗∗∗P < .001. (F) Dot plot proportion of mCherry⁺ and GFP⁺ cells in flow cytometry at day 24 of culture of a representative experiment, in which RCH-ACV cells were transduced with control shRNA (shLuc, luciferase) or shRNA for BTK (shBTK2 and shBTK3) with a mCherry as fluorescence marker. KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 2.

In vitro effect of combined therapy with dasatinib and BTKi on human E2A-PBX1⁺/preBCR⁺ ALL cell proliferation and on PLCG2 and BTK phosphorylation. (A) Left panels, titration curves for E2A-PBX1⁺/preBCR⁺ human (RCH-ACV) cells with increasing concentrations of dasatinib and ibrutinib combination treatments. Viable RCH-ACV cells were counted with trypan blue exclusion assay after 3 days. Data represent IC₅₀ calculated using nonlinear regression analysis and curves were compared with the sum-of-squares F test. Dose-response curves of each BTKi concentration were compared with the dose-response curve of the vehicle-treated cells as controls. n.s., not significant; ∗∗P < .001; ∗∗∗P < .0001. Right panel, heat map representation of Bliss interaction index for RCH-ACV cells treated with dasatinib and ibrutinib. Three independent experiments were performed. (B) In vitro effects of the combined therapy with dasatinib and BTKi in human RCH-ACV cells on the phosphorylation status of the key pre-BCR⁺ pathway proteins PLCG2 and BTK, after 30 minutes of treatment. One representative graph per antibody is shown. Relative fold median fluorescence intensity (MFI) change of cells treated with inhibitors or vehicle are illustrated. Data represent mean of 3 independent experiments + SEM. (C) Western blot analysis (representative of 3 independent experiments) shows the protein levels of BTK, pBTK, PLCG2, and pPLCG2 following dasatinib (1 nM), ibrutinib (100 nM), and dasatinib (1 nM) + ibrutinib (100 nM) treatments compared with control, in human RCH-ACV ALL cells. GADPH was used as loading control. Densitometry values were calculated using ImageJ software.

Figure 3.

In vivo sensitivity of E2A-PBX1⁺/preBCR⁺ leukemia cells to the combination of dasatinib and BTKi. (A) Schematic representation of secondary transplantations of E2A-PBX1⁺/preBCR⁺ leukemia cells in healthy recipients and in vivo treatment with vehicle, dasatinib, BTKis, and combination of dasatinib with BTKis. (B) E2A-PBX1⁺ leukemia cells from preBCR⁺ leukemia were transplanted into sublethally irradiated recipient healthy C57/BL6 mice. Mice were treated for 20 days with vehicle (n = 10), dasatinib (n = 10), ibrutinib (n = 10), dasatinib + ibrutinib (n = 10), acalabrutinib (n = 5), dasatinib + acalabrutinib (n = 5), zanubrutinib (n = 5), and dasatinib + zanubrutinib (n = 5). In vivo dasatinib + BTKi treatments of pre-BCR⁺ leukemia in a secondary transplantation assay led to significantly prolonged disease-free survival compared with vehicle-treated mice. Statistical analysis was performed by log-rank test. (C) Immunohistochemical staining of bone marrow (BM) sections with anti-GFP antibody showing a decrease in GFP⁺ E2A-PBX1⁺/pre-BCR⁺ leukemic cells infiltration in double-treated mice compared with single- and vehicle-treated mice. Samples were collected from euthanized animals with signs of disease from vehicle- (mean = 18 days), dasatinib- (mean = 24 days), ibrutinib- (mean = 30 days), and dasatinib + ibrutinib– (mean = 37 days) treated animals. Objective lens: 20×; scale bar: 200 μm. GFP positivity was measured using the QuPath software after transformation of images to DAB and staining intensity quantified by a single cutoff threshold of 0.20. (D) The phosphorylation status of p-PLCG2 (Tyr 753) and p-BTK (Tyr 223) of BM murine cells by phospho-specific flow cytometry. The phosphorylation of both PLCG2 and BTK decrease significantly in the BM isolated from dasatinib + ibrutinib–treated mice. Fold MFI change of dasatinib-, ibrutinib-, and dasatinib + ibrutinib–treated mice compared with vehicle. Data represent mean of 3 independent experiments + SEM. n.s., not significant; ∗P < .01; ∗∗P < .001; ∗∗∗P < .0001.

Figure 4.

Decrease of CNS infiltration of leukemia cells by combination therapy with dasatinib and BTKi. (A) Upper panels, images of mouse brain rostral leptomeninges slices from WT C57/BL6 mice, vehicle-, dasatinib-, ibrutinib-, and dasatinib + ibrutinib–treated mice tissues, stained using the hematoxylin and eosin (H&E) method. Vehicle-treated mice show an increased level of leukemic infiltration (arrow) compared with the dasatinib-, ibrutinib-, and dasatinib + ibrutinib–treated mice. Samples were collected from sacrificed animals with signs of disease from vehicle- (mean = 18 days), dasatinib (mean = 24 days), ibrutinib (mean = 30 days), and dasatinib + ibrutinib (mean = 37 days) treated animals. Objective lens: 40×; scale bar: 50 μm. Lower panels, immunohistochemical staining of CNS sections with anti-GFP antibody showing less GFP⁺ E2A-PBX1⁺/preBCR⁺ leukemic cells infiltration in double- and single-treated mice compared with vehicle-treated mice. Arrows pointing at leukemic CNS infiltration. Unstained CNS sections are showed. Objective lens: 20×; scale bar: 200 μm. (B) GFP positivity from 3 representative mice of each group was estimated after transformation of images to DAB and GFP staining intensity quantified by a single cutoff threshold of 0.20. Statistical analysis was performed by 1-way analysis of variance (ANOVA), Dunnett multiple comparison test. (C) Single-cell suspensions from fresh tissue from both brain and spinal cord after cutting into small pieces by scalpel and resuspended in a cell dissociation solution were used for quantification of GFP⁺ cells in flow cytometry. Panels show the gating strategy for phospho-flow cytometry staining for GFP⁺ murine E2A-PBX1⁺/pre-BCR⁺ leukemia cells from vehicle-, dasatinib-, ibrutinib-, and dasatinib + ibrutinib–treated mice. Gating was performed on lymphocytes, single cells, and GFP⁺ cells. The frequency of GFP⁺ murine E2A-PBX1⁺/preBCR⁺ leukemia cells decrease in the combination treatment. (D-E) Frequency of GFP⁺ murine leukemia infiltrating (D) the brain and (E) the spinal cord of C57BL/6 WT, mice. Each symbol represents an individual mouse. Statistical analysis was performed by 1-way ANOVA, Dunnett multiple comparison test. Scatter dot plots represent mean ± SEM. ∗∗P < .01; ∗∗∗P < .0001. IHC, immunohistochemistry; WT, wild type.

Figure 5.

RNAseq of E2A-PBX1⁺/preBCR⁺ leukemia CNS-infiltrating cells compared across treatments. Bulk RNA sequencing of FACS-sorted CNS-infiltration E2A-PBX1-GFP⁺ leukemic cells from vehicle- (n = 3), dasatinib- (n = 3), ibrutinib- (n = 3), and dasatinib + ibrutinib–treated mice (n = 3) was performed. (A) MA plot displaying 349 most significant genes (P < .05) between all the treatments. (B) Heat map showing BCR–associated differential expressed (DE) genes. Myc (P = .06) is downregulated in the double-treated mice compared with vehicle-treated mice. A trend for decreased expression of BCR-associated genes Lck, Zap70, and Cd79a showed a trend for decreased expression under the treatment with BTKi alone or in combination with dasatinib. In contrast, Pten (P < .05) and Mapk1/ERK2 (P < .05) are upregulated in the double-treated mice compared with vehicle-treated mice. (C) Venn diagrams showing the most significant in-common up- and downregulated genes between the treatments. (D) Validation of Pten, Myc, and Mapk1 genes by RT-qPCR. Actb was used as a housekeeping gene. The experiment was performed in triplicate. Bars represent the mean and error bars, the SEM, of 3 independent experiments.

Figure 6.

In vitro effect of combined therapy with dasatinib and BTKi on primary ALL samples. (A) Flow cytometric analysis of primary samples aiming to detect the phosphorylation status of PLCG2 and BTK after 30 minutes of treatment with vehicle (DMSO), dasatinib, ibrutinib, and dasatinib + ibrutinib. Gating strategy on lymphocytes, single cells, 7AAD, CD19⁺, and CD117⁺. One representative histogram per antibody is showed. (B-C) Histogram represents the phosphorylation of (B) PLCG2 and (C) BTK in primary ALL samples with different karyotypes, after treatment with vehicle, ibrutinib, dasatinib, and dasatinib + ibrutinib treatment.