Mechanisms Responsible For The Synergistic Antileukemic Interactions Between ATR Inhibition And Cytarabine In Acute Myeloid Leukemia Cells

Acute myeloid leukemia(AML)is the most common form of acute leukemia and has a guarded prognosis.Cytarabinehas been the mainstay induction therapy for most AML patients for the past 40 years.Although many patients respond to induction chemotherapy,the majority of patients relapse leading to overall survival rates of only 25% for adults and 65% for children.One major mechanism of resistance to chemotherapy is increased DNA damage response(DDR).Ataxia telangiectasia and Rad3 related(ATR)is one of the two chief regulators of the DDR.It is activated in response to single-stranded DNA structures,which can arise during repair of DNA double-strand breaks or stalled replication forks.Most tumor cells have a defective G1 cell-cycle checkpoint and rely heavily on the S and G2 checkpoints for cell survival from DNA damage.Thus,inhibition of ATR may represent a promising means to enhance the antileukemic activities of DNA damaging agents(e.g.cytarabine)in AML cells.ATR inhibitors have been tested in combination with DNA damaging agents such as gemcitabine,cisplatin and PARP inhibitors in preclinical solid tumor models,and have demonstrated promising preclinical results.Though,an in depth understanding of the mechanism of action when used in such combinations is lacking.ATR plays important roles in multiple cellular functions including cell cycle arrest,inhibition of replication origin firing,protection of stressed replication forks,and DNA repair.Determining which mechanism contributes in combination regimens will likely deepen our understanding of how ATR inhibitors enhance the antitumor effects of DNA damaging agents and will allow for rationally designed combination therapies for treating AML.In this study,we investigated the mechanism of action of the ATR selective inhibitors AZ20 and AZD6738 alone and in combination with cytarabine in AML cell lines and preclinical models of AML.We found that AZ20 abolishes the G2/M cell cycle checkpoint.AZ20 induced DNA damage and apoptosis,which were independent of CDK1 activity.It also induced DNA replication stress and caused down regulation of ribonucleotide reductase M1(RRM1)and M2(RRM2)subunits,which were not dependent on CDK1 activity.The combined treatment with cytarabine and AZ20 or AZD6738 caused increase in chromatin-bound RPA32 and increased γH2AX levels prior to induction of apoptosis,demonstrating that ATR inhibition and cytarabine treatment cooperate to induce DNA replication stress and DNA damage,leading to apoptosis.Cytarabine treatment led to S and G2/M cell cycle arrest,which was abrogated by the addition of AZ20.Our findings provide insight into the mechanism of action underlying the synergistic antileukemic activity of ATR inhibition in combination with cytarabine.