Pharmacologic ATM but not ATR kinase inhibition abrogates p21-dependent G1 arrest and promotes gastrointestinal syndrome after total body irradiation

Frank P Vendetti,Elaine Cadogan,Mark J O’Connor,Sandy Schamus,Brian J Leibowitz,Christopher J Bakkenist,Jennifer Barnes,David Andy Clump,Thomas Conrads,Jan H Beumer,Jian Yu,Shira Abberbock,Brian F Kiesel

doi:10.1038/srep41892

Abstract

We show that ATM kinase inhibition using AZ31 prior to 9 or 9.25 Gy total body irradiation (TBI) reduced median time to moribund in mice to 8 days. ATR kinase inhibition using AZD6738 prior to TBI did not reduce median time to moribund. The striking finding associated with ATM inhibition prior to TBI was increased crypt loss within the intestine epithelium. ATM inhibition reduced upregulation of p21, an inhibitor of cyclin-dependent kinases, and blocked G1 arrest after TBI thereby increasing the number of S phase cells in crypts in wild-type but not Cdkn1a(p21CIP/WAF1)−/− mice. In contrast, ATR inhibition increased upregulation of p21 after TBI. Thus, ATM activity is essential for p21-dependent arrest while ATR inhibition may potentiate arrest in crypt cells after TBI. Nevertheless, ATM inhibition reduced median time to moribund in Cdkn1a(p21CIP/WAF1)−/− mice after TBI. ATM inhibition also increased cell death in crypts at 4 h in Cdkn1a(p21CIP/WAF1)−/−, earlier than at 24 h in wild-type mice after TBI. In contrast, ATR inhibition decreased cell death in crypts in Cdkn1a(p21CIP/WAF1)−/− mice at 4 h after TBI. We conclude that ATM activity is essential for p21-dependent and p21-independent mechanisms that radioprotect intestinal crypts and that ATM inhibition promotes GI syndrome after TBI.

Highlights

The rapid and continuous division of labile stem cells to replace the short-lived functionally mature cells in the epidermis, hematopoietic system, and gastrointestinal (GI) tract renders them sensitive to genotoxic stress, and the irreversible loss of these stem cells and their descendants from an organism results in acute radiation disease within days of exposure to ionizing radiation (IR)[1]
The Ataxia telangiectasia-mutated (ATM) kinase-dependent phosphorylation and stabilization of p53 after IR may contribute to different phenotypic outcomes observed in different cells31,32. p53 is required for the upregulation of PUMA which induces Bax/Bak and mitochondrial-dependent apoptosis in hematopoietic stem cells leading to hematopoietic syndrome after irradiation3,31–35. p53 is required for the upregulation of p21, an inhibitor of several cyclin-dependent kinases (CDKs), which induces G1 cell cycle arrest in intestinal crypt stem cells and this prevents GI syndrome after irradiation[3,35,36,37,38,39]
There was no difference in survival detected between the cohort treated with both AZ31 and AZD6738 and the cohort treated with AZ31 alone

Summary

Introduction

The rapid and continuous division of labile stem cells to replace the short-lived functionally mature cells in the epidermis, hematopoietic system, and gastrointestinal (GI) tract renders them sensitive to genotoxic stress, and the irreversible loss of these stem cells and their descendants from an organism results in acute radiation disease within days of exposure to ionizing radiation (IR)[1]. Since ataxia telangiectasia (A-T) patients who express no ATM protein are the most radiosensitive patients identified, pharmacologic ATM kinase inhibitors may increase the efficacy of targeted radiotherapy[6,7,8]. Consistent with this premise, ATM inhibitors sensitize cancer cells to IR in vitro[9,10,11]. Knockin mouse models causes early embryonic lethality[14,15] This suggests that ATM inhibition does not phenocopy ATM protein disruption in vivo[16]. We explore the physiological consequences of a single dose of first orally active and bioavailable ATM kinase inhibitor, AZ3140, or the orally active and bioavailable ATR kinase inhibitor, AZD673826, prior to 9 or 9.25 Gy total body irradiation (TBI)

Methods

Results

Conclusion