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. 2018 Aug 1;11(1):99.
doi: 10.1186/s13045-018-0641-1.

Targeting WEE1 to enhance conventional therapies for acute lymphoblastic leukemia

Andrea Ghelli Luserna Di Rorà  1 , Neil Beeharry  2   3 , Enrica Imbrogno  4 , Anna Ferrari  4 , Valentina Robustelli  4 , Simona Righi  4 , Elena Sabattini  4 , Maria Vittoria Verga Falzacappa  5 , Chiara Ronchini  5 , Nicoletta Testoni  4 , Carmen Baldazzi  4 , Cristina Papayannidis  4 , Maria Chiara Abbenante  4 , Giovanni Marconi  4 , Stefania Paolini  4 , Sarah Parisi  4 , Chiara Sartor  4 , Maria Chiara Fontana  4 , Serena De Matteis  6 , Ilaria Iacobucci  4   7 , Pier Giuseppe Pelicci  5 , Michele Cavo  4 , Timothy J Yen  2 , Giovanni Martinelli  8
Affiliations

Targeting WEE1 to enhance conventional therapies for acute lymphoblastic leukemia

Andrea Ghelli Luserna Di Rorà et al. J Hematol Oncol. .

Abstract

Background: Despite the recent progress that has been made in the understanding and treatment of acute lymphoblastic leukemia (ALL), the outcome is still dismal in adult ALL cases. Several studies in solid tumors identified high expression of WEE1 kinase as a poor prognostic factor and reported its role as a cancer-conserving oncogene that protects cancer cells from DNA damage. Therefore, the targeted inhibition of WEE1 kinase has emerged as a rational strategy to sensitize cancer cells to antineoplastic compounds, which we evaluate in this study.

Methods: The effectiveness of the selective WEE1 inhibitor AZD-1775 as a single agent and in combination with different antineoplastic agents in B and T cell precursor ALL (B/T-ALL) was evaluated in vitro and ex vivo studies. The efficacy of the compound in terms of cytotoxicity, induction of apoptosis, and changes in gene and protein expression was assessed using different B/T-ALL cell lines and confirmed in primary ALL blasts.

Results: We showed that WEE1 was highly expressed in adult primary ALL bone marrow and peripheral blood blasts (n = 58) compared to normal mononuclear cells isolated from the peripheral blood of healthy donors (p = 0.004). Thus, we hypothesized that WEE1 could be a rational target in ALL, and its inhibition could enhance the cytotoxicity of conventional therapies used for ALL. We evaluated the efficacy of AZD-1775 as a single agent and in combination with several antineoplastic agents, and we elucidated its mechanisms of action. AZD-1775 reduced cell viability in B/T-ALL cell lines by disrupting the G2/M checkpoint and inducing apoptosis. These findings were confirmed in human primary ALL bone marrow and peripheral blood blasts (n = 15). In both cell lines and primary leukemic cells, AZD-1775 significantly enhanced the efficacy of several tyrosine kinase inhibitors (TKIs) such as bosutinib, imatinib, and ponatinib, and of chemotherapeutic agents (clofarabine and doxorubicin) in terms of the reduction of cell viability, apoptosis induction, and inhibition of proliferation.

Conclusions: Our data suggest that WEE1 plays a role in ALL blast's survival and is a bona fide target for therapeutic intervention. These data support the evaluation of the therapeutic potential of AZD-1775 as chemo-sensitizer agent for the treatment of B/T-ALL.

Keywords: Acute lymphoblastic leukemia; Chemo-sensitizer agent; G2/M checkpoint; WEE1 inhibitor.

PubMed Disclaimer

Conflict of interest statement

The study was approved by the Ethical Committee at Policlinico-Universitario S. Orsola Malpighi. Full informed consent was obtained from all patients.

Not applicable

GM has competing interests with Novartis, BMS, Roche, Pfizer, ARIAD, MSD.

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
WEE1 overexpression in ALL samples. a WEE1 transcript levels in samples isolated from adult ALL (n = 58) and in MNCs (n = 7) from peripheral blood of healthy donors. One-way ANOVA test was performed to confirm the statistical significance of the differences. Results are expressed as Log10 2exp[−(ΔΔCt). b WEE1 transcript levels in samples isolated from adult BCR-ABL1-positive (Ph+) ALL at diagnosis (n = 17), adult BCR-ABL1-negative (Ph−) ALL at diagnosis (n = 27), adult BCR-ABL1-positive ALL at relapse (unpaired, n = 8), adult BCR-ABL1-negative ALL at relapse (unpaired, n = 6), and in MNCs (n = 7) from the peripheral blood samples of healthy donors. Results are expressed as Log10 2exp[−(ΔΔCt). c Immunohistochemistry analysis of a reactive bone marrow sample; WEE1 is positive at moderate to strong intensity in morphologically typical myeloid precursors (× 20). d Immunohistochemistry analysis of leukemic blasts scattered in the interstitium and positive for WEE1 (× 20). e Leukemic blasts are widely positive for the B cell marker CD79a (red) while those positive for WEE1 (brown) are much fewer, as shown by the few scattered double-stained blasts (× 20)
Fig. 2
Fig. 2
AZD-1775 overrides the G2/M checkpoint and induces mitosis in B/T-ALL cell lines. a Viability analyses in ALL cell lines incubated for 24 h with AZD-1775 (6 to 5000 nM). The percentage of viable cells is depicted as a percentage of untreated controls. b Apoptosis analyses in BV-173, NALM-6, MOLT-4, and CCRF-CEM cells after 24 h of incubation with AZD-1775 (for each cell line: IC50, IC25, and IC12.5). The percentage of apoptotic cells was detected after Annexin V/propidium iodide staining. c Cell cycle analysis of BV-173 and CCRF-CEM cell line incubated for 24 h with increasing concentration of AZD-1775. d Representative immunoblots showing the expression of key proteins of the WEE1 pathway after treatment with AZD-1775 (IC50 for each cell line) for 24 h of the indicated cells lines. β-actin was used for loading normalization. e Quantitative mRNA analysis of six representative genes of 24 G2/M checkpoint genes analyzed. The white columns represent the controls and the gray columns represent the samples treated with AZD-1775 (IC50) for 12 h of the indicated cell lines. Results are expressed as Log10 2exp[−(ΔΔCt)
Fig. 3
Fig. 3
AZD-1775 reduces the cell viability of primary leukemic samples. a Cell viability analysis on primary leukemic cells isolated from 13 adult ALL patients treated with AZD-1775 (2.5, 5, and 10 uM) for 24 h. Viable cells are depicted relative to the untreated controls. b Quantitative mRNA analyses of 24 genes of the G2/M checkpoint. The basal gene expression of MNCs samples (n = 3) was compared with the basal gene expression of poor (n = 3), intermediate (n = 2), and high (n = 2) responders to AZD-1775 (ex vivo). Clustergram with a color indicative of the degree of upregulation (red) or downregulation (green). Targets with similar regulation cluster together. c Immunoblotting analyses of primary leukemic cells (n = 2, samples #1 and #6) and MNCs (n = 1, donor 4) treated with AZD-1775 (2.5, 5, and 10 uM) for 24 h and then stained for markers of WEE1 functional inhibition (phospho-CDC2) and induction of DNA damages (phospho-CHK1 and phospho-γH2AX). β-actin was used for loading normalization. d Light microscopy analysis of normal MNCs and primary leukemic cells treated with AZD-1775 (10uM) for 24 h and then stained with May-Grünwald Giemsa solutions. In the figure, the yellow arrows indicate DNA bridges induced by the treatment. Controls in all panels are cells treated with DMSO 0.1%. Representative images are shown at × 100 magnification
Fig. 4
Fig. 4
AZD-1775 enhances the toxicity of antineoplastic compounds on ALL cell lines and primary cells. a Cell viability analyses of NALM-6, NALM-19, and REH cell lines incubated with AZD-1775 (6 to 5000 nM, dilution rate 1:3) and clofarabine (2.5, 5, and 10 nM) for 72 h. Viable cells are depicted relative to the untreated controls. Data were used to determine the CI values. b Growth curve of REH, NALM-6, CCRF-CEM, MOLT-4, and RPMI-8402 treated for 4 days with AZD-1775 (185 nM) and clofarabine (REH, NALM-6, and RPMI-8402 10 nM; CCRF-CEM and MOLT-4 20 nM). The number of viable cells was evaluated in the different groups every 24 h. c Apoptosis analyses of NALM-6 and MOLT-4 cell lines after 24 h of incubation with AZD-1775 (185 nM) and clofarabine (MOLT-4 20 nM and NALM-6 10 nM). The percentage of apoptotic cells was detected after Annexin V/propidium iodide staining. d Cell viability analysis on primary leukemic cells isolated from eight adult ALL patients treated with AZD-1775 (5 uM) and clofarabine (500 nM) for 24 h. Viable cells are depicted as percentage of the untreated controls. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001
Fig. 5
Fig. 5
AZD-1775 enhances the toxicity of tyrosine kinase inhibitors in ALL cell lines and leukemia primary samples. a Cell viability analyses of BV-173 cell line incubated simultaneously with AZD-1775 (6 to 5000 nM, dilution rate 1:3) and with bosutinib authentic (50 nM) or bosutinib isomer (50 nM) for 48 h. In the graph: AZD-1775 (control, white columns), bosutinib (Bos, gray columns), and bosutinib isomer (Bos-I, black columns). The percentage of viable cells is depicted relative to the untreated controls. Data were used to determine CI values. b Cell viability analyses of NALM-6 cell line incubated with AZD-1775 (6 to 5000 nM, dilution rate 1:3) and with bosutinib authentic (2 uM) or bosutinib isomer (2 uM) for 72 h. In the graph: AZD-1775(control, white columns), bosutinib (Bos, gray columns), and bosutinib isomer (Bos-I, black columns). The number of viable cells is depicted as a percentage of the untreated controls. Data were used to determine CI values. c Apoptosis analyses of BV-173 and NALM-6 cells after 24 h of incubation with AZD-1775 (185 nM) and bosutinib authentic/isomer (BV-173 50 nM; NALM-6 2 uM). The percentage of apoptotic cells was detected after Annexin V/propidium iodide staining. d Growth curve of BV-173 and NALM-6 treated for 4 days with AZD-1775 (185 nM) and bosutinib authentic/bosutinib isomer (BV-173 50 nM; NALM-6 2 uM). e Immunoblotting analysis of BV-173 treated with AZD-1775 (185 nM) and bosutinib (50 nM) for 24 h. β-actin was used for loading normalization. f Cell viability analysis in primary leukemic cells isolated from two adult BCR-ABL1-positive ALL patients treated with AZD-1775 (5 uM) and bosutinib authentic (2 uM) for 24 h. The percentage of viable cells is depicted relative to the untreated controls. Controls in all panels are cells treated with DMSO 0.1%. *p ≤ 0.05
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