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Synergistic targeting of WEE1 and GLS1 in T-ALL
5D). These findings corroborate the anti-T-ALL efficacy of MK1775/BPTES combination in vivo.
We also evaluated the toxicity of the drug combination in healthy C57/BL6 mice using the same treatment strate- gy as shown in Figure 5A. Body weights of these mice were comparable to the control cohort (Online Supplementary Figure S8A). Analysis of blood parameters at the end of the treatment revealed a mild decrease in red and white cell counts (Online Supplementary Figure S8B), and the decline in blood cell counts vanished two weeks after treatment ended (Online Supplementary Figure S8C). Moreover, hematoxylin & eosin (H&E) staining revealed undetectable damage in various organs (Online Supplementary Figure S8D). Consistently, healthy human BM cells were not sensitive to dual treatment either (Figure 4C), arguing of manageable toxicity of this combi- nation strategy. This is probably due to lower WEE1 and MYC expression in the majority of normal cells. Therefore, therapeutic targeting of WEE1 may not induce significant inhibition of MYC and glycolysis as much as it does in tumor cells.
Combined WEE1/GLS1 inhibition prolongs overall survival in a T-cell acute lymphoblastic leukemia patient-derived xenograft
Given CB-839 is currently being evaluated in clinical tri- als,39 we next explored the translational potential of MK1775 in combination with CB-839 in a T-ALL PDX. Human CD45+ leukemia cell distributions in vivo were ana- lyzed to assess tumor burden and therapeutic responses. Again, the combination treatment induced synergistic tumor growth inhibition compared with the control or monotherapy, as evidenced by much lower human CD45+ tumor cell percentages (Figure 6A). Immunohistological (IHC) analysis of the spleen sections further confirmed reduced cell proliferation and massive intratumoral apop- tosis due to administration of both compounds, as quanti- fied by attenuated proliferating cell nuclear antigen (PCNA) and increased cleaved Caspase-3 staining, respec- tively (Figure 6B). Injection of MK1775 and CB-839 result- ed in much smaller spleen size and more reddish bones (Figure 6C). More importantly, dual treatments significant- ly prolonged the lifespans of the T-ALL PDX as compared to single treatment (Figure 6D). Together with the pre- clinical studies in HPB-ALL xenografts, these results strongly suggest the clinical potential of WEE1/GLS1 inhibitors as a promising T-ALL targeted therapeutics.
Discussion
Intensified T-ALL chemotherapies face the challenges of significant side effects, frequent relapses, and drug resist- ance. To improve the treatment of T-ALL and reduce asso- ciated toxicity, introduction of new targeted agents is des- perately needed. We here show that WEE1 kinase is a promising therapeutic target. Reduced cell viability upon WEE1 inhibitor MK1775 treatment is partly attributable to significant suppression of aerobic glycolysis, leading to T- ALL cells more addicted to glutaminolysis. Administration of WEE1 and GLS1 inhibitors induces synergistic lethality in T-ALL cells and leukemia xenografts. These results highlight a promising therapeutic strategy of dual target- ing of cell cycle kinase and metabolic enzymes in T-ALL treatments.
Loss of cell cycle control plays a prominent role in the pathogenesis of T-ALL. The tumor suppressors p16INK4A and p14ARF encoded by the CDKN2A locus are frequently lost in T-ALL due to chromosomal deletions.40 p16INK4A and p14ARF inactivates the cyclin D1-CDK4/6 and cyclin E- CDK2 complexes, respectively, leading to G1-S arrest for DNA repair.9 As such, loss of CDKN2A results in overacti- vation of these CDK complexes, enabling T-ALL cells to enter S phase for replication despite DNA damage. T-ALL cells, with a deficient G1-S checkpoint, are therefore more reliant on the G2-M cell cycle checkpoint to prevent excessive DNA damage that may lead to mitotic catastro- phe and cell death. In support of this notion, we and oth- ers have shown that WEE1 expression is significantly increased in a variety of T-ALL cell lines and patient- derived primary cells.13 We further delineate the molecular mechanism underlying WEE1 expression in T-ALL by identifying MYC as a prominent regulator directly activat- ing the WEE1 transcription. MYC-mediated WEE1 upreg- ulation is also found in Burkitt’s lymphoma cells, suggest- ing the MYC-WEE1 axis as a general regulatory mode governing WEE1 expression in human cancers.
Inhibition of WEE1 in cancer cells circumvents cell cycle arrest during the G2 phase and enables cell division despite accumulation of DNA damage. T-ALL cells crucial- ly depend on the G2 checkpoint in the presence of DNA- damage inducing drugs. WEE1 inhibitor manifested syner- gistic anti-leukemic activity in combination with cytara- bine or olaparib.13,41,42 We here identify an additional meta- bolic vulnerability of T-ALL cells in response to WEE1 inhibition after which they become particularly addicted to glutaminolysis for cell survival. The underlying mecha- nism involves MK1775-mediated glycolytic suppression at least in part via downregulation of MYC, a master regula- tor in controlling glucose metabolism in the majority of tumor contexts.37 Indeed, we demonstrate that overex- pression of MYC significantly rescued glycolysis defect due to MK1775 treatment, suggesting that MYC acts as an important downstream player mediating the role of WEE1 in the regulation of glycolysis. It is interesting to note that although a previous report suggests that MYC promotes glutaminolysis as well by activating GLS1 expression in B lymphoma and prostate cancer cells,43 neither MYC downregulation nor WEE1 inhibition affected GLS1 expression in the context of T-ALL (data not shown). Moreover, WEE1 inhibition switches T-ALL cells to a more glutamine-dependent state such that simultaneous suppression of glycolysis and glutaminolysis by MK1775 and GLS1 inhibitor respectively induced potent synergistic anti-T-ALL effects (Online Supplementary Figure S9).
Our findings reveal a molecular link between cell cycle and cancer metabolism by demonstrating the contribution of WEE1 to glycolysis. WEE1 inhibition, which unleashes the G2-M checkpoint and accelerates cell cycle, repro- grams the cellular metabolism such that tumor cells with decreased glycolysis become more addicted to glutaminol- ysis. These results are reminiscent of a recent finding that activation of cyclin D1-CDK4/6 by dysregulation of Fbxo4-cyclin D1 axis leads to cellular dependency on glu- tamine metabolism and sensitizes tumor cells to CB-839.44 As such, increased glutamine dependency could be a con- sequence of over-activated CDK complex, which provides a potential therapeutic opportunity in fast-dividing tumor cells. Loss of cell cycle checkpoints due to genetic muta- tions and/or utilizing checkpoint inhibitors in cancer treat-
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