A Drug Repositioning Approach Identifies a Combination of Compounds as a Potential Regimen for Chronic Lymphocytic Leukemia Treatment

Abstract

Drug repositioning is a promising and powerful innovative strategy in the field of drug discovery. In this study, we screened a compound-library containing 800 Food and Drug Administration approved drugs for their anti-leukemic effect. All screening activities made use of human peripheral blood mononuclear cells (PBMCs), isolated from healthy or leukemic donors. Compounds with confirmed cytotoxicity were selected and classified in three groups: i) anti-neoplastic compounds which are drugs used in leukemia treatment, ii) compounds known to have an anti-cancer effect and iii) compounds demonstrating an anti-leukemic potential for the first time. The latter group was the most interesting from a drug repositioning perspective and yielded a single compound, namely Isoprenaline which is a non-selective β-adrenergic agonist. Analysis of the cytotoxic effect of this drug indicated that it induces sustainable intracellular ATP depletion leading, over time, to necrotic cell death. We exploited the Isoprenaline-induced intracellular ATP depletion to sensitize primary leukemic cells to fludarabine (purine analogue) and Ibrutinib (Bruton's tyrosine kinase inhibitor) treatment. In-vitro treatment of primary leukemic cells with a combination of Isoprenaline/fludarabine or Isoprenaline/Ibrutinib showed a very high synergistic effect. These combinations could constitute a new efficient regimen for CLL treatment following successful evaluation in animal models and clinical trials.

Keywords: ATP depletion; BTKi; CLL; Ibrutinib; Isoprenaline; fludarabine; synergistic effect.

Figure 1

Hit compounds of the first round of screening. (A) List of Hit compounds selected in the phenotypic single-dose (10µM) HTS campaigns. Primary leukemic cells used in this screening were isolated from blood samples derived from patients with Acute Myeloid Leukemia (AML, n=4), Acute Lymphocytic Leukemia (ALL, n=4), Chronic Lymphoid Leukemia (CLL, n=4), Chronic Myeloid Leukemia (CML, n=4) and from four healthy donors (n=4). The overall screening results are displayed and expressed as mean of inhibition ± SD and normalized to DMSO (100%). (B) Chemical structure of Isoprenaline.

Figure 2

Primary leukemic cells deriving from CLL patients show the highest sensitivity to Isoprenaline. (A) Dose-response plots showing the cytotoxic effect of Isoprenaline on PBMC isolated from healthy donors (solid line) and leukemic patients (dashed line). Each dose-response curve represents the average of multiple samples testing; data are expressed as mean ± SD and normalized to DMSO (100%). Cell viability was determined by the CellTiter-Glo luminescence-based assay. CPCs are the only leukemic cells to show a significant higher sensitivity to Isoprenaline than the normal PBMCs. The p-values for each point (Isoprenaline concentration) of the plot were calculated with Student’s t test (ns, non-significant (P>0.05); *P < 0.05; **P < 0.01 and ****P < 0.0001). (B) The cytotoxic effect of a single dose (3µM) of Isoprenaline is compared for PBMC (n=8) and the four leukemia subtypes respectively [CLL (n= 23), CML (n=4), AML (n= 4), ALL (n=4)] (p values are indicated for each group). (C) Dose-dependent cytotoxic effect of Isoprenaline on different Leukemia cell lines.

Figure 3

Isoproterenol does not induce cell death in primary CLL cells but it induces a long-lasting dose-dependent intracellular ATP depletion. (A) Primary CLL cells were treated with 3µM Isoprenaline (IC₅₀ determined using the CellTiter-Glo assay) for 48h. Markers of apoptosis (cleaved PARP, cleaved caspase-3 and caspase-9) and autophagy (accumulation of LC3 II) were assessed by western blotting. The non-competitive selective phosphodiesterase inhibitor, IBMX, known to induce apoptosis in CLL B cells, was used as a positive control. Full-length blots/gels are presented in Supplementary Figure 5 . (B) Isoprenaline-induced cell death in normal PBMC (black bars) and in primary CLL cells (gray bars) was also assessed by propidium iodide staining followed by flow cytometry analysis. (C) Cytotoxic effect induced by a gradient of Isoprenaline concentrations (dose-response plots) was assessed with an ATP-dependent (CellTiter-Glo, dashed line) and independent (PI/flow cytometry) assays. (D) The level of expression of the different adrenergic receptors in primary leukemic cells (ALL, AML, CLL and CML), normal PBMCs and in the CLL cell line WA-C3CD5+ was measured by quantitative real time PCR (qPCR). (E) Isoprenaline-induced intracellular cAMP accumulation in normal PBMCs and primary CLL cells pre-treated for one hour with phosphodiesterase inhibitor (IBMX) than with 10µM Isoprenaline for 24h. (**P < 0.01). (F) Human phosphokinase array reveals alteration in phosphorylation of kinases upon in-vitro treatment of CPCs with 10µM Isoprenaline for 24h. In the array each kinase is spotted in duplicate. Hybridization signals at the corners serve as control. Relative levels of protein phosphorylation (normalized intensity for each antibody) were calculated for each untreated and treated sample. p-CREB (indicated by black arrow) was the only significantly up-regulated kinase upon Isoprenaline treatment.

Figure 4

Isoprenaline cytotoxic effect in CLL primary cells. (A) Cell viability assessment after the treatment of CPCs with either low dose of Isoprenaline (red bars) or high dose (green bars). The graph shows the relative cell-death rate to the control (basic spontaneous cell-death percentage observed in the control was subtracted from the rate observed in treated cells). Time course assessment of the cytotoxic effect of, low (B) and high (C) doses of Isoprenaline, through the quantification of apoptotic (PARP, caspase-9 and -3) and autophagic (accumulation of LC3 II) markers. [ns, non-significant (P > 0.05); *P < 0.05; **P < 0.01 and ***P < 0.001)]. Full-length blots/gels used to generate panels (B) and (C) are presented in Supplementary Figures 6 and 7 respectively.

Figure 5

Isoprenaline potentiates fludarabine effect to synergistically induce apoptotic cell death in CLL primary cells. CPCs were pre-treated with Isoprenaline (30μM) for 12h, to allow intracellular ATP depletion, prior to the addition of the second drug of the combination. (A) Effect of the combinatorial treatment of Isoprenaline with different anthracyclines (Doxorubicin and Mitoxantrone) on CPCs. Cytotoxic effects of the different treatment were assessed through the quantification of apoptotic cell death markers (caspase-3, -9 and cleaved PARP). All cut and reassembled bands belong to the same blot-images (see Supplementary Figure 8 for original blot images). (B) Cytotoxicity/cell-growth inhibition of primary CLL cells by the purine analogue fludarabine (10μM) alone or in combination with Isoprenaline (30µM). Induced cell death was assessed by PI staining followed by flow cytometry analysis. (C) Average cytotoxic effect of Isoprenaline/Fludarabine combination on leukemic cells isolated from 5 CLL patients. The Cooperation Index (CI) between Isoprenaline and fludarabine was calculated, and was far below 1 indicating a high synergism between these two drugs. (D) Fluorescent microscopic images showing CPCs treated with fludarabine (10μM) alone or in combination with Isoprenaline (30μM). The cells were stained with Hoechst (blue), AnnexinV (green), PI (red). (E) Assessment of Isoprenaline/fludarabine combination on apoptotic cell-death markers (caspase-3, -9 and PARP) in CPCs. Full-length blots/gels are presented in Supplementary Figure 8 .

Figure 6

Isoprenaline synergizes specifically with the purine analogue fludarabine in dose-dependent manner. (A) The cytotoxic effect of fludarabine (10μM) combined with an increasing concentration of Isoprenaline (1, 10, 30μM) was assessed through the quantification of the apoptotic marker (cleaved PARP). Full-length blots/gels are presented in Supplementary Figure 9 . (B) CPCs were treated with a gradient of fludarabine (red plots) or Doxorubicin (blue plots) concentrations combined with either 1μM or 10μM Isoprenaline. The cytotoxic effect of Fludarabine/Isoprenaline combination was also assessed in PBMC isolated from normal donors (gray scale plots).

Figure 7

Isoprenaline-effect on intracellular ATP content is irreversible in CLL primary cells. (A) Isoprenaline induced a long-lasting intracellular ATP depletion. The concentration of intracellular ATP in CLL primary cells was measured over 72 h in: i) non-treated cells (blue bars), ii) cells treated with Isoprenaline for only 24h then Isoprenaline was removed by cell wash and cells were maintained in Isoprenaline-free media (orange bars), or iii) in cells permanently treated with Isoprenaline (gray bars). Concentrations of intracellular ATP at the different time points were normalized to the respective negative control (non-treated cells). (B) Primary CLL cells were treated with Isoprenaline for only 24h then Isoprenaline was removed by cell wash and cells were maintained in Isoprenaline-free media, fludarabine (10μM) was added at different time points after Isoprenaline removal (0, 24, 48 and 72h). Primary CLL cells were incubated with fludarabine for 24h then fludarabine cytotoxic effect was assessed through quantification of the apoptotic marker (cleaved PARP). Full-length blots/gels are presented in Supplementary Figure 10 . (C) Normal PBMC were treated as in (A). (D) Normal PBMC were treated as in (A) except that media was renewed after 48 hours instead of 24 hours.

Figure 8

Synergistic effect between Isoprenaline and the Bruton’s tyrosine kinase inhibitor (Ibrutinib). (A) Combinatorial treatment of CLL primary cancer cells with Isoprenaline (20μM) and Ibrutinib (20 μM) shows that Isoprenaline sensitizes CLL primary cancer cells to the cytotoxic effect of Ibrutinib. (B) This synergistic effect between Isoprenaline and Ibrutinib was more pronounced (CI=0.48) in CPCs showing higher resistance to Ibrutinib than in more sensitive CPCs (CI=0.83).