Activity of the Wee1 inhibitor MK1775 plus cytarabine in AML through interference with DNA repair capacity at the DNA level.

Abstract

7106 Background: Cytarabine (AraC) resistance is a fundamental characteristic of refractory/relapsed AML. In previous RNAi screens we identified WEE1 kinase as top candidate target whose inhibition sensitized to AraC in AML. The WEE1 inhibitor MK1775 potently synergized with AraC ex vivo and in vitro and clinical trials are in preparation. However, a mechanistic understanding has remained elusive. Methods: In siRNA rescue screens of 44 genes involved in WEE1 and DNA damage repair (DDR) pathway regulation were conducted and below described experiments conducted. Results: Several RNAi hits converged on DDR repair genes with a focus on the MRN (MRE11, Rad51, NBS1) complex. Interfering with MRN complex members significantly altered the response of AML cells to combined AraC+MK1775 vs. single agent MK1775 or AraC. Unexpectedly the ATM-CHEK1 pathway was not activated, and Homologous recombination (HR)-mediated repair was compromised as shown by a DR-GFP expression vector. Consistently other HR markers decreased as well. The cell cycle was globally dysregulated by slower S-phase kinetics (progression), an expected abrogated G2/M checkpoint as well as de-regulated DNA replication origin formation and firing as evidenced by Cdt1 and Mus81. As a consequence high-single and double strand breaks (H2AX) occurred with early mitotic entry (increased phospho-histone H3) induced by AraC + MK1775 vs. single agents. Changes were followed by massive induction of apoptosis, with specific anti-apoptotic members decreasing and pro-apoptotic BH3 members increasing. Finally, AraC+MK1775 dramatically reduced colony formation vs. single agent MK1775 or even high-dose AraC incubations indicating a stem cell targeting combination, which was p53 independent. Conclusions: Herein we propose combined AraC+MK1775 as a potent AML regimens by directly interfering with DDR via compromising the MRN complex with subsequent reduced HR, early abortive mitotic entry subsequent massive ÒdefaultÓ apoptosis, in a p53 independent manner. These data provides a rationale to clinically evaluate AraC+MK1775 in patients with AML, points towards a mechanistic understanding as well as offers suggestions for biomarkers.