The Wee1 Inhibitor, MK-1775, Sensitizes Leukemic Cells to Different Antineoplastic Drugs Interfering with DNA Damage Response Pathway
Authors: Ghelli Luserna Di Rora Andrea , Iacobucci Ilaria , Beeharry Neil , Soverini Simona , Papayannidis Cristina , Martinelli Giovanni , Yen Timothy J 
Published: October 2019

Abstract
Due to inadequate treatments, the survival rate of adult Acute Lymphoblastic Leukemia (ALL) patients with the exclusion of patients with particular genetic alterations, like the Philadelphia positive patients, is still very low. Moreover even the rate of patient that responds to specific treatment develops relapses during their life. Thus there is a need to improve the efficacy of conventional therapy and to discover novel specific targets. In eukaryotic cells Wee1, ATR/Chk1 and ATM/Chk2 are three pathways involved in cell cycle regulation, DNA damages response and DNA repair. Wee1 is a checkpoint kinase, involved mainly in the regulation of G2/M transition through the inhibitory phosphorylation of both Cyclin-dependent kinase 1 (CDK1) and 2 (CDK2) respectively. This study evaluates the effectiveness of MK-1775, a selective Wee1 inhibitor, as a monotherapy and as chemosensitizer agent for the treatment of B-/T-Acute Lymphoblastic Leukemia. Human B (BV-173, SUPB-15, NALM-6, NALM-19 and REH) and T (MOLT-4, RPMI-8402 and CEM) ALL cell lines were tested in this study. MK-1775 alone strongly reduced the cell viability in a dose and time-dependent manner in all the cell lines treated. The anti-proliferative activity of MK-1775 was accompanied by an increase in apoptotic cells (AnnexinV/Pi staining) and by DNA damage markers (gH2AX and Parp-1 cleavage). Moreover the inhibition of Wee1 disrupted the cell cycle profile by arresting the cells in late S and in G2/M phase. We hypothesized that targeting Chk1, a kinase upstream, of Wee1, would be more effective in reducing cell proliferation. Indeed, the concomitant inhibition of Chk1 and Wee1 kinases, using the PF-0477736 in combination with MK-1775, synergized in the reduction of the cell viability, inhibition of the proliferation index and induction of apoptosis. Moreover the immunofluorescence staining for the DNA damage marker gH2AX and the mitotic marker phosphor-Histone H3 showed that co-treatment with MK-1775 and PF-0477736 induced cell death by mitotic catastrophe. We undertook further studies to understand the immediate clinical potential of the compound, thus MK-1775 was combined with different drugs (Clofarabine, Bosutinib Authentic, and a particular isomer of this compound).The combination between MK-1775 and clofarabine showed an additive effect in terms of reduction of the cell viability and induction of apoptosis. Finally the Wee1 inhibitor was combined with the tyrosine kinase inhibitors Bosutinib and Bos-isomer (Bos-I). Both the isomers in combination with MK-1775 showed an additive effect in term of reduction of the cell viability. Interestedly the cytotoxic effect of Bos-I was stronger on the Philadelphia-negative cell lines in comparison to the positive counterpart. Western blot analysis highlighted that this compound, but not the Bosutinib authentic, interfered with the Chk1/Chk2 and Wee1 pathway. This supported our previous studies showing that Bosutinib and its isomer possess off-target effects against both Wee1 and Chk1 kinases and thus maybe used as a chemosensitizer (Beeharry et al. Cell Cycle 2014). The results of this study in our opinion identify the Wee1 kinase as a promising target for the treatment of ALL not only as a monotherapy but also as chemosensitizer agent to increase the cytotoxicity of different kind of drugs already used in clinical trials.

Disclosures
Soverini: Novartis, Briston-Myers Squibb, ARIAD: Consultancy. Martinelli:Novartis: Consultancy, Speakers Bureau; Ariad: Consultancy; AMGEN: Consultancy; ROCHE: Consultancy; BMS: Consultancy, Speakers Bureau; MSD: Consultancy; Pfizer: Consultancy.