Hyperthermia Synergizes With WEE1 Inhibitor AZD1775 By CDK1 Super-activation In Ovarian Cancers

Objective: The role of hyperthermic intraperitoneal chemotherapy(HIPEC)in epithelial ovarian cancer(EOC)with widespread peritoneal metastasis(PM)is still controversial partly because of the poorly understood mechanism of action and simplex combination mode.The main purpose of this study is to comprehensively elucidate the multi-omics panorama of hyperthermia-induced molecular and signaling reprogramming,identify acquired vulnerabilities,and optimize hyperthermic combination therapeutic strategies.Methods: Here,we conduct the first comprehensive multi-omics study(transcriptiome,proteome,and phosphoproteome)upon hyperthermia(HT)in ovarian cancer cells.Unbiased trans-omics approach deciphered a unique HT-induced molecular panorama and identified the accompanying vulnerabilities which could be employed as combinational therapy with synthetic lethal effects to enhancing the efficacy of HT.Then through lentiviral transfection,Western blot,DNA fiber,immunofluorescence,flow cytometry and other experimental methods to explore the mechanism of HT-induced CDK1 kinases hyperactivation.The drug screening in ovarian cancer cells was conducted using CCK8 in vitro.The effect of HT combined with WEE1 inhibitor AZD1775 on the cytotoxicity of ovarian cancer cells was analyzed by CCK8,clone formation and flow cytometry.Then through Western blot,DNA fiber,immunofluorescence,flow cytometry and other experimental methods to explore the synergetic mechanism of HT and AZD1775.Finally,using a murine ovarian cancer PM model and in-house developed devise mimicking the clinical HIPEC treatment protocol,proof-of-concept was delivered that combination of HT with AZD1775 leads to dramatic anti-tumor responses in vivo and provides a novel perspective about WEE1 inhibition as a promising combination therapy to improve the effectiveness of HIPEC in EOC.Results: Our trans-omics approach demonstrated rapid alterations in protein phosphorylation represented as the primary cell response upon HT,as opposed to changes in protein or RNA abundance.Based on the phospho-signature,we identified CDK1 kinases to be hyperactivated rapidly,and we verified the activation of CDK1 by the decreases of the inhibitory phosphorylation of CDK1 on Thr14 and Tyr15 in western blots.The replication pressure of tumor cells after HT was analyzed by DNA fiber,the increasing non-replicating S and G2/M phase cell populations after HT were analyzed by flow cytometry,the DNA damage and accumulation of ss DNA of cells were studied by immunofluorescence,and the effect of HT was saved by reverse addition of CDK1 inhibitor and d NTPs.Notably,HTinduced CDK1 hyperactivation and seems dynamic and reversible.These reversible dynamic molecular changes not only explained the limited therapeutic effects of mono-HT in cancers,but also provides the limits of the “therapeutic window” for targeting the vulnerabilities induced by HT.Through candidate drug screening of synergistic potential using CCK8,we found that the combination of HT with AZD1775 displayed the most consistent and strongest synergism,which exceeded the DDP and TAX combination.Besides,we next explored the therapeutic window for the combinational therapy of HT and AZD1775.We further confirmed that Myt1 downregulation contributes to HT induced CDK1 hyperactivation and synergistic effect with WEE1 inhibition by proteomics,Western Blots and Myt1 overexpression reverse verification.So,the concurrent inhibition of Myt1 and WEE1 resulted in super-activation of CDK1 causing catastrophic genome instability and cell death.More importantly,the synergism was observed in 5 patient-derived primary cell cultures from HGSCO.Finally,using an in-house developed devise mimicking the clinical HIPEC treatment protocol,the combination of HT with AZD1775 leads to dramatic anti-tumor responses in vivo,which was consistent with the conclusion of in vitro experiments.Conclusions: Our study illustrates the mechanism by which HT induces CDK1 hyperactivation in ovarian cancer cells and describes its therapeutics utility and synergic mechanism of CDK1 super-activation in combination with AZD1775.It sheds light on the instant cell response upon HT in ovarian cancer and provides new insights about how to shift the usual paradigms to improve the effectiveness of HIPEC.