The Checkpoint Kinase 1 Inhibitor Prexasertib Induces Regression of Preclinical Models 1 of Human Neuroblastoma 2 Running Title : Prexasertib induces regression of neuroblastoma models 3

47 Purpose: Checkpoint kinase 1 (CHK1) is a key regulator of the DNA damage response and a 48 mediator of replication stress through modulation of replication fork licensing and activation of S 49 and G2/M cell cycle checkpoints. We evaluated prexasertib (LY2606368), a small molecule 50 CHK1 inhibitor currently in clinical testing, in multiple preclinical models of pediatric cancer. 51 Following an initial assessment of prexasertib activity, this study focused on preclinical models 52 of neuroblastoma. 53 Experimental Design: We evaluated the antiproliferative activity of prexasertib in a panel of 54 cancer cell lines; neuroblastoma cell lines were among the most sensitive. Subsequent western 55 blot and immunofluorescence analyses measured DNA damage and DNA repair protein 56 activation. Prexasertib was investigated in several cell line-derived xenograft mouse models of 57 neuroblastoma. 58 Results: Within 24h, single agent prexasertib promoted γH2AX-positive double-strand DNA 59 breaks and phosphorylation of DNA damage sensors ATM and DNA-PKcs, leading to 60 neuroblastoma cell death. Knockdown of CHK1 and/or CHK2 by siRNA verified that the double61 strand DNA breaks and cell death elicited by prexasertib were due to specific CHK1 inhibition. 62 Neuroblastoma xenografts rapidly regressed following prexasertib administration independent of 63 starting tumor volume. Decreased Ki67 and increased immunostaining of endothelial and 64 pericyte markers were observed in xenografts after only 6 days of exposure to prexasertib, 65 potentially indicating a swift reduction in tumor volume and/or a direct effect on tumor 66 vasculature. 67 Conclusions: Overall, these data demonstrate that prexasertib is a specific inhibitor of CHK1 in 68 neuroblastoma and leads to DNA damage and cell death in preclinical models of this 69 devastating pediatric malignancy. 70