The Role Of ATR In The Antileukemic Activity Of RNA Pol Ⅰ Transcription Inhibitor CX-5461 In Acute Myeloid Leukemia Cells

Acute myeloid leukemia(AML)is a hematological malignancy.The annual incidence of AML in China is 1.62/100000,accounting for 60%-70% of adult acute leukemias.Although chemotherapy,hematopoietic stem cell transplantation,and small molecule targeted drugs have improved the disease-free survival(DFS),AML has a high rate of relapase,and once relapsed,it loses sensitivity to chemotherapy drugs and has a five-year survival rate of only 26%.Consequently,there is still an urgent need for new therapies to treat AML.CX-5461 is a RNA polymerase I(Pol)transcription inhibitor,which exerts potent antileukemic activity by targeting both bulk AML cells and AML stem cells.CX-5461 outperforms standard chemotherapy regimens and has no toxic effects on normal cells,representing a promising new drug for treating AML.Recent studies have shown that CX-5461 stabilizes the DNA G4-quadroplex structure,impedes the progression of DNA polymerase,and causes stalled replication forks,resulting in single stranded DNA(ssDNA)and increased replication stress.Replication pressure continues to accumulate,which eventually triggers a replication disaster that causes cell death.However,replication stress activates ATR,which activates CHK1.Activated CHK1 phosphorylates and inhibits Cdc25 phosphatases that regulate cell cycle transition at G2/M,resulting in cell cycle arrest.Cell cycle arrest would give the cells time to repair damaged DNA and lead to subsequent cell survival and proliferation.Thus,we hypothesized that targeting ATR would synergize with CX-5461 to induce apoptosis in AML cells.AZD6738 is an orally active ATR inhibitor(ATRi)currently in phase I clinical trials.AZD6738 has been tested in combination with DNA damaging agents such as gemcitabine,cisplatin,and cytarabine in preclinical solid tumor models,and has demonstrated promising preclinical results.In this study,we investigated the mechanism of CX-5461 in combination with AZD6738 in AML cell lines and preclinical models of AML.We found that CX-5461 can induce apoptosis in AML cells and when combined with AZD6738 can synergistically kill both bulk AML cells and AML progenitor cells.CX-5461 can induce DNA replication stress and DNA damage in AML cells,causing cell cycle arrest and inhibiting the proliferation of AML cells.AZD6738 can abolish CX-5461-induced G2/M cell cycle arrest and enhance CX-5461-induced DNA damage.CX-5461 can induce the expression of ribonucleotide reductase subunit RRM2,which increases the synthesis of dNTPs and provides material for DNA synthesis,alleviating DNA replication stress and providing opportunities for survival of AML cells.However,AZD6738 downregulates the expression of RRM2,enhancing the DNA replication stress and damage induced by CX-5461,leading to cell death.In summary,AZD6738 enhances the anti-AML activity of CX-5461 by enhancing CX-5461-induced DNA replication stress and DNA damage,and preventing CX-5461-induced G2/M cell cycle arest.