Therapeutic Targeting Of CHK1 And MTOR In T-Cell Acute Lymphoblastic Leukemia And Neuroblastoma

Purpose: MYC contributes to almost every aspect of tumorigenesis.MYC-induced tumors in established experimental models regress or even fully reverse when MYC was arbitrarily switched off,thus offering a promising therapeutic opportunity in cancer treatment on targeting of MYC.However,directly targeting oncogenic of MYC remains unsuccessful.Therefore,indirectly inhibiting MYC seems to be an alternative approach.Overactivation of MYC promotes defective G1 cell cycle checkpoints and lead to genomic instability and tumorigenesis,but also contribute to endogenous replication stress.To avoid apoptosis induced by excessive replicative stress,tumor cells depend on more on G2/M checkpoints,which are activated by ATR/CHK1 signaling.Checkpoint kinase 1(CHK1)is such a protein kinase that coordinates the G2/M cell cycle checkpoint and protects cancer cells from excessive replicative stress.Using c-MYC-mediated T-cell acute lymphoblastic leukemia(T-ALL)and N-MYC-driven neuroblastoma as model systems,we attempt to reveal the functional interplay between MYC and CHK1.Meanwhile,clinical treatment of kinase inhibitor is often associated with short-lived response and rapid drug resistance.We attempt to develop a metabolism-related synergistic therapy to further enhance the replicative stress induced by CHIR-124 and decipher the underlying mechanism for this synergy.Method: We applied pharmacological or short hairpinRNA-mediated inhibition to assess the role and interaction of multiple genes in loss-of-function assays.mRNA and protein levels were analyzed by real-time PCR(q RT-PCR)and immunoblot to analyze the effect of shRNAs and inhibitors in the T-ALL and neuroblastoma cells.The rates of apoptotic cell death after administration of inhibitors were determined by the flow cytometry.In order to improve the anti-tumor efficiency of CHK1 inhibitors,we performed a small-scale drug screening of metabolic inhibitors.Immunofluorescence was applied to directly visualize the recruitment of RPA32 and?H2AX as the signal of endogenous single-stranded DNA damage and replicative stress.Anti-tumor effect of drug combination of CHK1 and mTOR inhibitors was further assessed in vitro and in vivo to demonstrate therapeutic efficacy.Results: We herein revealed that CHK1 is highly expressed in T-ALL patient cohort compared with normal bone marrow,and that CHK1 is much higher in high-risk neuroblastoma patient samples and predicts poor prognosis.Both c-MYC and N-MYC directly bind to the CHK1 locus and activate its transcription,so MYC-mediated regulation of CHK1 may be the primary mechanism underlying its dysregulation in tumors.The recruitment of RPA32 induced by N-MYC nuclear translocation also demonstrated the increased ss DNA damage and elevated replicative stress induced by MYC.Tumors require high levels of CHK1 protein to protect cancer cells from excessive replication stress,so CHK1 inhibitor is synthetic lethal with MYC overexpression.Based on a small-scale drug screening of metabolic inhibitors,we found that CHK1 inhibitor CHIR-124 induced remarkable synergistic lethality with mTOR inhibitor rapamycin in T-ALL and MYCN-amplified cells,while imposed minimal effect on isogenic SHEP cells exhibiting very low MYCN expression.Mechanistically,rapamycin inhibited pyrimidine synthesis by inactivation of the carbamoyl-phosphate synthetase 2,aspartate transcarbamoylase,and dihydroorotase(CAD),the essential enzyme for the first three steps of de novo pyrimidine synthesis,thus reducing d CTP/d TTP abundance and decreasing DNA repair efficiency.Therefore,rapamycin significantly increased the replication stress induced by CHIR-124.Importantly,the drug combination manifested great efficacy in both human T-ALL and neuroblastoma xenografts,and significantly prolonged animal survival.Conclusion: Deregulation of MYC in cancer cells contributes to not only tumorigenesis but also DNA replication stress.To keep replicative stress compatible with cell survival,c-MYC/N-MYC activates CHK1 transcription to protect cancer cells from excessive replication stress.mTOR inhibitors sensitize T-ALL and neuroblastoma cells to CHK1 inhibitors in vitro and in vivo,suggesting simultaneous targeting of CHK1 and mTOR as a novel and powerful strategy for MYC-mediated tumors.