1 SCIENTIFIC REPORTS | (2019) 9:3617 | https://doi.org/10.1038/s41598-019-40218-0 www.nature.com/scientificreports Efect of CHK1 Inhibition on CPX- 351 Cytotoxicity in vitro and ex vivo Nicole D. Vincelette 1 , Husheng Ding 2 , Amelia M. Huehls 2 , Karen S. Flatten 2 , Rebecca L. Kelly 1 , Mira A. Kohorst 3 , Jonathan Webster 4 , Allan D. Hess 4 , Keith W. Pratz 4 , Larry M. Karnitz 1,2 & Scott H. Kaufmann 1,2 CPX-351 is a liposomally encapsulated 5:1 molar ratio of cytarabine and daunorubicin that recently received regulatory approval for the treatment of therapy-related acute myeloid leukemia (AML) or AML with myelodysplasia-related changes based on improved overall survival compared to standard cytarabine/daunorubicin therapy. Checkpoint kinase 1 (CHK1), which is activated by DNA damage and replication stress, diminishes sensitivity to cytarabine and anthracyclines as single agents, suggesting that CHK1 inhibitors might increase the efectiveness of CPX-351. The present studies show that CPX-351 activates CHK1 as well as the S and G2/M cell cycle checkpoints. Conversely, CHK1 inhibition diminishes the cell cycle efects of CPX-351. Moreover, CHK1 knockdown or addition of a CHK1 inhibitor such as MK-8776, rabusertib or prexasertib enhances CPX-351-induced apoptosis in multiple TP53-null and TP53-wildtype AML cell lines. Likewise, CHK1 inhibition increases the antiproliferative efect of CPX-351 on primary AML specimens ex vivo, ofering the possibility that CPX-351 may be well suited to combine with CHK1-targeted agents. Despite advances in the molecular understanding of acute myeloid leukemia (AML), improvements in therapy are still needed. Most current regimens rely on treatment with the nucleoside analog cytarabine and an anthra- cycline for induction therapy. Depending on the subtype of AML, the driving oncogenic changes and the doses administered, these regimens have a 40–70% complete remission rate 1,2 . However, certain groups of patients, including those with antecedent hematological conditions or therapy-related AML, do particularly poorly with cytarabine/anthracycline-based induction therapy 3–5 . A number of mechanisms of resistance to current AML induction therapy have recently been targeted, includ- ing increased expression of anthracycline exporters and increased activity of antiapoptotic BCL2 family members. However, resistance mechanisms involving the DNA damage and replication stress checkpoint pathways have received less attention. We and others previously reported that cytarabine activates the replication checkpoint kinases ATR and CHK1, which regulate a coordinated series of cellular responses that facilitate survival during replication stress 6–10 . Importantly, alterations that prevent activation of the replication checkpoint such as inhi- bition or downregulation of CHK1 sensitize AML cells to cytarabine 7–10 . CHK1 downregulation has also been reported to sensitize solid tumor cell lines to the anthracycline doxorubicin 11,12 , but this has not been tested using selective checkpoint kinase inhibitors in AML. CPX-351 (Vyxeos ® ) is a liposomal formulation containing a synergistic 5:1 ratio of cytarabine and dauno- rubicin 13 . Tis formulation has a number of appealing properties, including the ability to kill AML cells har- boring certain resistance mechanisms 14 and a longer serum half-life than either cytarabine or daunorubicin 15 . CPX-351, which showed promising activity in AML clinical trials 16,17 , is now FDA approved for the treatment of therapy-related AML and AML with myelodysplasia-related changes 18 . Here we have assessed the impact of CHK1 inhibition on the cell cycle efects and cytotoxicity of CPX-351 in AML cell lines and clinical isolates. 1 Department of Molecular Pharmacology, Mayo Clinic, Rochester, MN, USA. 2 Division of Oncology Research, Mayo Clinic, Rochester, MN, USA. 3 Department of Pediatrics, Mayo Clinic, Rochester, MN, USA. 4 Sidney Kimmel Cancer Center at Johns Hopkins, Baltimore, MD, USA. Nicole D. Vincelette, Husheng Ding and Amelia M. Huehls contributed equally. Larry M. Karnitz and Scott H. Kaufmann jointly supervised this work. Correspondence and requests for materials should be addressed to L.M.K. (email: Karnitz.Larry@Mayo.edu) or S.H.K. (email: Kaufmann.Scott@ Mayo.edu) Received: 24 October 2018 Accepted: 11 February 2019 Published: xx xx xxxx OPEN