Targeting the wee1 kinase for treatment of pediatric Down syndrome acute myeloid leukemia

Abstract

Background: Most Down syndrome children with acute myeloid leukemia (DS-AML) have an overall excellent prognosis, however, patients who suffer an induction failure or relapse, have an extremely poor prognosis. Hence, new therapies need to be developed for this subgroup of DS-AML patients. One new therapeutic approach is preventing cell cycle checkpoint activation by inhibiting the upstream kinase wee1 with the first-in-class inhibitor MK-1775 in combination with the standard genotoxic agent cytarabine (AraC).

Procedure: Using the clinically relevant DS-AML cell lines CMK and CMY, as well as ex vivo primary DS-AML patient samples, the ability of MK-1775 to enhance the cytotoxicity of AraC was investigated with MTT assays. The mechanism by which MK-1775 enhanced AraC cytotoxicity was investigated in the cell lines using Western blots to probe CDK1 and H2AX phosphorylation and flow cytometry to determine apoptosis, cell cycle arrest, DNA damage, and aberrant mitotic entry.

Results: MK-1775 alone had modest single-agent activity, however, MK-1775 was able to synergize with AraC in causing proliferation arrest in both cell lines and primary patient samples, and enhance AraC-induced apoptosis. MK-1775 was able to decrease inhibitory CDK1(Y15) phosphorylation at the relatively low concentration of 100 nM after only 4 hours. Furthermore, it was able to enhance DNA damage induced by AraC and partially abrogate cell cycle arrest. Importantly, the DNA damage enhancement appeared in early S-phase.

Conclusions: MK-1775 is able to enhance the cytotoxicity of AraC in DS-AML cells and presents a promising new treatment approach for DS-AML.

Keywords: AraC; DNA damage; Down syndrome; MK-1775; wee1.

Fig.1

MK-1775 has single agent effect against DS-AML. A: AraC IC50s for CMK and CMY after 72-hour treatment as determined by MTT assay. B: Response of CMK and CMY to increasing concentrations of MK-1775 after 72-hour treatment as determined by MTT assay. C: Apoptosis induction in CMK and CMY cells after indicated 48-hour treatment, as determined by AnnexinV/PI staining and flow cytometry analysis. D and E: Expression of wee1 and p-CDK1(Y15), as determined by Western blots and qRT-PCR, after stable knockdown of wee1 using a lentivirus shRNA delivery system. F: Sensitivity of CMK and CMY cells to MK-1775 after knockdown of wee 1, as determined by a 72-hour MTT assay.

Fig. 2

Pharmacodynamic changes in p-CDK1(Y15) after MK-1775 treatment. Top: Expression of CDK1 and p-CDK1(Y15) in CMK and CMY (A and B) cells treated for 24 hours with increasing concentrations of MK-1775, as determined by Western blots. Bottom: CMK and CMY (A and B) cells were treated with 100 nM MK-1775 for 4 hours followed by drug washout. Expression of CDK1 and p-CDK1(Y15) was determined by Western blots.

Fig. 3

MK-1775 synergizes with AraC in both cell lines and primary patient samples. A and B: CMK and CMY cells were treated with different combinations of AraC and MK-1775 for 72-hour and viability was determined by MTT assay. Standard, normalized isobolograms demonstrate the synergistic inhibition of cell viability by the combination of AraC and MK-1775. C: CMK and CMY-ntc and -shwee1 cells were treated for 72 hours with AraC and viability was determined by MTT. D and E: CMK (D) and CMY (E), were treated with varying doses of AraC in the presence or absence of 100 nM MK-1775 for48 hours and apoptosis was measured using AnnexinV/PI staining. F: Various combinations of AraC and MK-1775 were tested in two primary DS-AML samples ex vivo using 48-hour MTT. The CI-Fa plot shows the combination index versus the fraction affected, with CI < 1 representing synergy.

Fig. 4

MK-1775 can abrogate AraC-induced CDK1(Y15) phosphorylation and enhance AraC-induced DNA damage. CMK and CMY cells were treated for 24 hours with the indicated drugs and levels of CDK1(Y15), γH2AX, and cleaved PARP were determined by Western blots.

Fig. 5

Effects of MK-1775 and AraC on cell cycle, mitosis, and DNA damage determined by flow cytometry analysis. CMY cells were treated with the indicated drugs for 24 hours and analyzed by flow cytometry. Top: The effects of AraC and MK-1775 on cell cycle were determined using PI staining. Middle: The effects of AraC and MK-1775 on mitosis versus cell cycle were determined using dual pH3/PI staining. Bottom: The effects of AraC and MK-1775 on DNA damage versus cell cycle were determined using dual γH2AX/PI staining. * Indicates P < 0.05 compared to AraC treatment.

Fig. 6

Schematic presentation of the effects of MK-1775 and AraC on cell survival. A: In untreated cells, DNA replication is successful and cell survival is primary endpoint. B: In AraC treated cells, S-phase arrest, and DNA damage occur, but damage can be repaired and cells can survive. C: In MK-1775 treated cells, some DNA damage occurs but cells primarily survive. D: In combination treated cells, MK-1775 overrides AraC induced checkpoint activation, resulting primarily in cell death.