Systems biology drug screening identifies statins as enhancers of current therapies in chronic lymphocytic leukemia

Abstract

Chronic lymphocytic leukemia (CLL) is a B lymphoid malignancy highly dependent on the microenvironment. Despite new targeted therapies such as ibrutinib and venetoclax, disease progression and relapse remain an issue. CLL cell interactions with the supportive tissue microenvironment play a critical role in disease pathogenesis. We used a platform for drug discovery based on systems biology and artificial intelligence, to identify drugs targeting key proteins described to have a role in the microenvironment. The selected compounds were screened in CLL cell lines in the presence of stromal cells to mimic the microenvironment and validated the best candidates in primary CLL cells. Our results showed that the commercial drug simvastatin was the most effective and selective out of the tested compounds. Simvastatin decreased CLL cell survival and proliferation as well as cell adhesion. Importantly, this drug enhanced the antitumor effect of venetoclax and ibrutinib. We proposed that systems biology approaches combined with pharmacological screening could help to find new drugs for CLL treatment and to predict new combinations with current therapies. Our results highlight the possibility of repurposing widely used drugs such as statins to target the microenvironment and to improve the efficacy of ibrutinib or venetoclax in CLL cells.

Figure 1

Overall experimental procedure: (1) Data sources: Molecular description of CLL microenvironment effects retrieved from literature and mapping in a human protein functional network. Bioactive compounds and drugs with known protein target profile. (2) In silico analysis: Different metrics derived from network-based mathematical models have been used to select key molecular enclaves in the network around the known effector proteins of CLL microenvironment motive; to identify bioactive compounds with a potential effect around the key proteins and to identify antineoplastic agents that could have synergistic effect with simvastatin and (3) In vitro screening. Compound library and combination therapies with simvastatin were screened using an in vitro coculture CLL system.

Figure 2

Molecular description of CLL microenvironment and targets. (A) 139 proteins (effectors) selected as descriptors of the CLL microenvironment and the relations between them. (B) Extract of the network build around the selected effectors of the microenvironment that contains the targets for the 65 screened compounds. Dark green square: CLL microenvironment selected descriptors; Light green round: Proteins directly related with selected CLL microenvironment descriptors; Orange, round: Protein targets of the 65 screened compounds that lay within the network described by the microenvironment effector proteins and close neighbours; Red square: Proteins that are effectors and targets simultaneously. The images haven been developed with Cytoscape v3.7.2 software.

Figure 3

Compound library screening in HG3 and HS-5 cell lines. (A) Viability of cells treated with the compounds at the concentration of 15 µM in the HG3 and HS-5 cell lines. (B) Cytotoxicity of the compounds at 15 µM concentration in HG3 alone and in coculture with HS-5. (C) Heat maps showing viability of cells treated with the selected compounds (A12, A2, A1, F1, D1, C11, C7,C6) at the concentrations of 1–5–15 µM in the HG3 and HS-5 cell lines. (D) Heat maps showing cytotoxicity of the selected compounds (A12, A2, A1, F1, D1, C11, C7,C6) at the doses of 1–5–15 µM in HG3, HG3 in coculture with HS-5 and in HS-5. Cells were treated for 48 h with the compounds at concentrations from 1 to 15 µM. Each dot (A,B) and each box (C,D) represent the mean of 3 independent experiments. Dotted line in (A,B) indicates the threshold to discriminate the effect of the compounds. The cut off for MTT and cytotoxicity was 50% and 20%, respectively. Viability of CLL cells was measured using the MTT assay, and is depicted relative to untreated control. Cytotoxicity was defined as the increase in Annexin-V+/PI + cells compared to untreated control.

Figure 4

Screening of selected compounds in primary CLL cells. (A) Cytotoxicity of the compounds at 0.1–1–2.5 µM in primary CLL cells (n = 2–13). CLL cells were treated for 48 h with the compounds at concentrations from 0.1 to 2.5 µM. Dotted line indicates the threshold to discriminate the effect of the compounds. The cut off for cytotoxicity was 20%. Cytotoxicity was defined as the increase in Annexin-V⁺/PI⁺ cells compared to untreated control. (B) Cytotoxicity of the compounds at 0.1–1–2.5 µM concentration in primary CLL cells, primary CLL in coculture with HS-5 cells and HS-5 cells alone (n = 6–13). CLL cells were treated for 48 h with the compounds at concentrations from 0.1 to 2.5 µM. Dotted line indicates the threshold to discriminate the effect of the compounds. The cut off for cytotoxicity was 20%. Cytotoxicity was defined as the increase in Annexin-V + /PI + cells compared to untreated control. The IC50 has been calculated and presented for each condition. (C) Percentage of proliferating CD19⁺ CLL cells after ODN2006 + IL15 stimulation (30 min before treatment) and treatment with different compounds at the dose of 1 µM (C5, C7 and F1) or 0.25 µM (Ibrutinib) for 6 days measured by CFSE dilution (n = 10). (D) Cytotoxicity of the compounds at 0.1–1–2.5 µM concentration in primary CLL cells and in CD19 and CD3 positive cells from healthy donors (n = 8) after 48 h of incubation. Line into the bars represents the mean of all samples analyzed. Non-parametric Wilcoxon matched-pairs signed rank test was used for statistical analysis. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 5

Validation of selected targets. Cells were treated for 48 h with the compounds at concentrations from 1 to 15 µM. Each dot represents the mean of 3 independent experiments. Dotted line indicates the threshold to discriminate the effect of the compounds. The cut off for MTT and cytotoxicity was 50% and 20% respectively. Viability of CLL cells was measured using the MTT assay and is depicted relative to untreated control. Cytotoxicity was defined as the increase in Annexin-V +/PI+  cells compared to untreated control. (A) Viability of cells treated with A1, A12, C5 and C7 and NOD1/2 specific inhibitors in HG3, MEC1 and HS-5 cell lines. (B) Cytotoxicity of A1, A12, C5 and C7 and NOD1/2 specific inhibitors in HG3, HG3 in coculture with HS-5 and in HS-5 cells. (C) Viability of cells treated with the F1 compound (simvastatin) and different statins and specific LFA-1 inhibitors in the HG3, MEC and HS-5 cell lines. (D) Cytotoxicity of F1 compound and different statins and specific LFA-1 inhibitors in HG3 alone, HG3 in coculture with HS-5 and in HS-5 alone. Round dots: compounds that target NOD1/2; Square dots: compounds that target HMGCR and LFA-1; Grey dots: specific inhibitors for each target. (E) Schematic description of statins mechanism of action.

Figure 6

Statins reduce CLL migration, proliferation and viability. Cells were treated for 48 h with the compounds at concentrations of 1 µM. (A) Cell phase object confluence after F1 treatment which was monitored for 48 h by recording phase images using the IncuCyte Live Cell Analysis Imaging System in HG3, HS-5 and coculture of HG3 and HS5 cells (n = 3). Bars represent the ± SD of all samples analyzed. (B) CXCL12-induced adhesion/migration of CLL cells treated with statins or LFA-1 inhibitors for 3 h and analyzed by transwell assays. Values are presented as the ratio of migrating cells and total viable cells, relative to the untreated control. Dotted line indicates the untreated control reference. (C) CXCL13-induced adhesion/migration of CLL cells treated with statins or LFA-1 inhibitors for 3 h and analyzed by transwell assays. Values are presented as the ratio of migrating cells and total viable cells, relative to the untreated control. Dotted line indicates the untreated control reference. (D) Percentage of proliferating CD19⁺ CLL cells after ODN2006 + IL15 (ODN IL15) stimulation (30 min before treatment) and treatment with different drugs for 6 days measured by CFSE dilution (n = 7). (E) Flow cytometry histograms of a CLL representative case (#4) show the percentages of proliferating cells (gated on viable CD19+ cells) after 6 days of ODN2006 + IL15 stimulation. A decrease in CFSE signal is indicative of cells that have divided. FMO, fluorescence-minus-one. Non-parametric Wilcoxon matched-pairs signed rank test was used for statistical analysis. *P < 0.05. Lov: lovastatin, Fluv: fluvastatin, Rosu: rosuvastatin, Lifi: lifitegrast and B033: BDBM50199033B033.

Figure 7

Statins exert a cytotoxic effect in primary CLL cells alone or in coculture with stromal cells. CLL cells or PBMCs from healthy donors were treated for 48 h with the statins at concentrations from 0.1 to 2.5 µM. Cytotoxicity was defined as the increase in Annexin-V⁺/PI⁺ cells compared to untreated control. Cytotoxicity of statins in primary CLL cells alone, in coculture with HS-5 or in HS-5 alone (n = 6) and CD19⁺ B cells and CD3⁺ T cells from healthy donors (n = 8). Line into the bars represents the mean of all samples analyzed. Non-parametric Wilcoxon matched-pairs signed rank test was used for statistical analysis. *P < 0.05, **P < 0.01.

Figure 8

Combinatory effects of statins with venetoclax or ibrutinib. Primary CLL cells were treated with 0.1 µM statins, 1 nM venetoclax or 0.1 µM ibrutinib. (A) Viability of primary CD19⁺ CLL cells after incubation of cells with statins and ibrutinib for 6 days (n = 9–12). Percentage of viable cells was measured as CD19⁺Annexin-V⁻ cells by flow cytometry and normalized to untreated control samples. (B) Percentage of proliferating CD19⁺ CLL cells after ODN2006 + IL15 stimulation (30 min before treatment) and treatment with statins and ibrutinib for 6 days measured by CFSE dilution (n = 6). (C) Viability of CD19⁺ CLL cells after incubation with statins and venetoclax for 48 h (n = 7). Non-parametric Wilcoxon matched-pairs signed rank test was used for statistical analysis. *P < 0.05, **P < 0.01, ***P < 0.001.