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Synergistic targeting of WEE1 and GLS1 in T-ALL
geted therapies are needed to improve the outcomes of those patients with a poor prognosis and reduce the side effects associated with chemotherapies.
Uncontrolled proliferation is one of the hallmarks of cancer. Many cancer cells possess a deficient G1 cell cycle checkpoint, for example due to p53 loss, and this impairs the ability of cells to halt the cell cycle and repair DNA damage before replication (S-phase).8 It provides cancer cells with a means to accumulate mutations and propagate irregularities that are favorable for proliferation. Meanwhile, tumor cells become more reliant on the G2-M cell cycle checkpoint to prevent mitotic entry with exces- sive DNA damage, which may lead to apoptosis due to mitotic catastrophe. WEE1 is a tyrosine kinase that plays a crucial role as the gatekeeper of the G2-M checkpoint.9 When DNA damage occurs, it leads to activation of WEE1 which phosphorylates CDK1 and maintains the CDK1- cyclin B complex in an inactive form, preventing entry into mitosis.10 To limit excessive genomic instability in tumor cells, it is not surprising that WEE1 is highly expressed in a variety of cancer types.11-13 Moreover, high WEE1 expression has been associated with poor rates of disease-free survival.11,14,15 Despite considerable studies on the role of WEE1 in cell cycle checkpoints, it remains unclear how expression of WEE1 is increased and how increased WEE1 expression promotes neoplastic pheno- types.
Cellular metabolism is at the foundation of all biological activities, and altered tumor cell metabolism is now firmly established as another hallmark of human cancer. Normal cells primarily rely on aerobic respiration/oxidative phos- phorylation to meet their energy requirements, yet fast- growing, poorly differentiated tumor cells typically exhib- it increased aerobic glycolysis by converting a majority of glucose-derived pyruvate to lactate.16 Because of this, tumor cells depend on glutamine anaplerosis to replenish tricarboxylic acid (TCA) cycle intermediates (e.g., a-ketoglutarate) to sustain the metabolic integrity and produce nicotinammide adenina dinucleotide (NADH).17 Reprogramming of glucose and glutamine metabolism not only provides tumor cells with building blocks for macro- molecule biosynthesis but also rescues them from a stressed cellular microenvironment by maintaining proper redox homeostasis.16 In T-ALL, both glycolysis and gluta- minolysis play crucial roles in mediating leukemia cell pro- liferation, survival and drug resistance.18,19
In this study, we show that the elevated expression of WEE1 kinase in T-ALL results from oncogenic MYC-medi- ated transcriptional activation, and this WEE1 upregula- tion significantly contributes to efficient aerobic glycoly- sis. Pharmacological inhibition of WEE1 leads to a marked decrease in glycolytic flux, rendering T-ALL cells particu- larly vulnerable to glutamine deficiency. Based on these findings, dual targeting of WEE1 and glutaminase (GLS1), the key rate-limiting enzyme in the glutaminolysis path- way,20 shows great promise in anti-T-ALL targeted thera- pies.
Methods
Cell culture and reagents
T-ALL cells were maintained in RPMI-1640 (Hyclone, Logan, UT, USA) supplemented with 10% FBS (Hyclone) as described.21,22 Human primary specimens were obtained with
informed consent from Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences and Zhongnan Hospital, Wuhan University, China. Polymerase chain reaction (PCR) primer sequences and antibodies used in this study are listed in Online Supplementary Tables S1 and S2, respectively.
Metabolomic analysis
HPB-ALL cells were treated with mock (DMSO) or selective WEE1 inhibitor MK177523 (200 nM) for 20 hours (h). Ten million cells in each treatment were collected and quenched in liquid nitrogen. Metabolite samples were prepared for analysis using standard solvent extraction methods and then subjected to the gas chromatograph system (Agilent Technologies, Santa Clara, CA, USA) coupled with a Pegasus HT gas chromatography time- of-flight mass spectrometer (GC-TOF-MS; LECO Corporation, St Joseph, MI, USA).24,25 Identification of chemical entities was based on comparison to Fiehn metabolomics library. Chroma TOF 4.3x software and the LECO-Fiehn Rtx5 database were used for raw peak identification and integration of the peak area. Both mass spectrum match and retention index match were taken into consideration.26 Normalized data were uploaded using the SIMCA software package (V14.1, Sartorius Stedim Data Analytics AB, Umea, Sweden) for principal component analysis (PCA) and orthogonal projections for latent structures- discriminant analysis (OPLS-DA). Differential metabolites between experimental groups were determined by variable importance in the projection (VIP) values (VIP>1) and Student t- test. The metabolic pathway enrichment analysis was per- formed using Kyoto Encyclopedia of Genes and Genomes (KEGG, http://www.genome.jp/kegg/) and MetaboAnalyst 4.0 (http://www.metaboanalyst.ca).
Mouse studies
HPB-ALL xenografts were carried out as previously described.21,27 NOD-Prkdcscid IL2Rγnull NPG mice (4-6 weeks old, Beijing Vitalstar Biotechnology Co., Beijing, China) were injected with five million cells infected with lentiviruses expressing the green fluorescent protein (GFP) and luciferase (pWPXLd- Luciferase-GFP). Mice were subjected to bioimaging at day 6 post engraftment with IVIS Lumina II (Waltham, MA, USA) to ensure equivalent tumor onset in vivo. These animals were then random- ly divided into four groups undergoing treatments in a three days on and three days off mode for four cycles. MK1775 (20 mg/kg) was administered twice daily by oral gavage and BPTES (25 mg/kg) was intraperitoneally injected once daily. Disease progres- sion and therapy response were evaluated by bioimaging.
For drug synergy studies in the patient-derived xenograft (from primary T-ALL sample #1, Online Supplementary Table S3), T-ALL cells were injected into irradiated 4-6 week old NPG mice (2 Gray), which were subjected to treatment at day 6 post engraftment. Control, MK1775 (20 mg/kg), CB-839 (200 mg/kg) or both were administrated by oral gavage every other day for two consecutive weeks. Leukemia burden was assessed by flow cytometry analysis of human CD45+ cells. All animal experi- ments were performed under animal ethical regulations and the study protocol was approved by the Institutional Animal Care and Use Committee of Wuhan University.
Statistical analysis
Spearman rank correlation test was used to analyze the WEE1 and MYC expression in primary T-ALL samples (Figure 2 F-H). Log-rank analysis was used to evaluate differences in Kaplan- Meier survival curves. Student t-test or one-way ANOVA was used in other statistical analysis. P<0.05 was considered statistical- ly significant.
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