Targeting The Wee1 Kinase With MK-1775 For Treatment Of Acute Myeloid Leukemia In The Down Syndrome Population
Authors: Caldwell J. Timothy , Buck Steven A , Ge Yubin , Taub Jeffrey W 
Published: October 2019

Abstract
Approximately one seventh of pediatric acute myeloid leukemia (AML) patients also have Down Syndrome (DS), or constitutional trisomy 21. DS AML is considered to be a unique disease from non-DS AML, due in part to a high prevalence of somatic GATA1 mutations and a megakaryocytic phenotype (AMKL). In contrast to the non-DS population, DS AML is generally associated with a very favorable prognosis. However, treatment of this disease is not without complications. Adverse drug effects are significant within this population, especially with up to half of patients possessing congenital heart abnormalities. Furthermore, for those patients who suffer either an induction failure or relapse, prognoses are dismal (Taga T. et al., Blood, 120: 1810-1815, 2012) including the outcomes after stem cell transplant (Hitzler JK. et al., Biol Blood Marrow Transplant, 19:893-897, 2013). Therefore, less toxic and more efficacious therapies are needed for this population.
Recently, chemotherapeutic targeting of cell cycle checkpoints has emerged as an exciting possibility, with many clinical trials underway. The wee1 kinase has been identified as a potential target in many malignancies, including leukemia. It is responsible for inhibiting CDK1 and CDK2 by adding phosphate groups at amino acid residue tyrosine 15 (Y15), which is necessary for subsequent checkpoint activation by checkpoint proteins (e.g. CHK1). The development of a wee1-specific inhibitor, MK-1775, has allowed investigations into its use alone or in combination with classical chemotherapeutics as treatment for many types of cancer, with favorable results thus far. However, there is some controversy with regards to the mechanism by which it enhances cell death.
In this preclinical work we used DS AML cell lines to investigate the potential use of MK-1775, both alone and in combination with cytarabine, to improve outcomes in DS AML patients. The two cell lines, CMK and CMY, were derived from DS AMKL patients and both harbor GATA1 mutations. CMK was derived from a patient who relapsed after initial treatment and CMY was derived from a patient who experienced an induction failure. The CMY line displays significant resistance to multiple different classes of chemotherapy drugs including nucleoside agents (e.g. cytarabine) and anthracyclines. We found that MK-1775 alone was able to cause growth inhibition and apoptosis at clinically relevant doses in both cell lines, independent of sensitivity to cytarabine or other agents. This effect was greatly enhanced by the addition of cytarabine. Addition of 250nM MK-1775 was able to reduce the cytarabine IC50 9- and 45-fold in CMK and CMY cells, respectively. Combination indices (CI) for this combination ranged from 0.66-0.95 and 0.50-0.94 in CMK and CMY, respectively.The combination of MK-1775 and cytarabine was able to synergistically induce apoptosis as measured by annexinV/propidium iodide (PI) staining. Addition of MK-1775 was able to decrease inhibitory CDK1 and CDK2 Y15 phosphorylation induced by cytarabine treatment. Interestingly, treatment with MK-1775 increased DNA damage induced by cytarabine as measured by γH2AX signal on western blots. Furthermore, this damage was induced primarily in S-phase, as measured by γH2AX/PI dual-staining and flow-cytometry. Finally, to aid with the clinical translation of this work, the dose- and time-dependency of MK-1775 treatment on CDK1 and CDK2 Y15 phosphorylation was determined. Briefly, even low doses (100nM) of MK-1775 were able to reduce CDK1 and CDK2 Y15 phosphorylation after only four hours. However, this inhibitory phosphorylation was able to return after only 1 hour, suggesting that treatment with MK-1775 must be continued during cytarabine therapy.
In conclusion, these data support the combination of MK-1775 and cytarabine for the treatment of DS AML, including possible relapsed/refractory cases which have a very poor prognosis. Combining MK-1775 with cytarabine was able to synergistically induce apoptosis in both cell lines, possibly by increasing DNA damage and decreasing inhibition of CDK1 and CDK2.

Disclosures:
No relevant conflicts of interest to declare.