Inhibition of CDK4/6 protects against radiation-induced intestinal injury in mice
Liang Wei, … , Lin Zhang, Jian Yu
Liang Wei, … , Lin Zhang, Jian Yu
Published October 4, 2016
Citation Information: J Clin Invest. 2016;126(11):4076-4087. https://doi.org/10.1172/JCI88410.
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Research Article Gastroenterology Stem cells

Inhibition of CDK4/6 protects against radiation-induced intestinal injury in mice

Abstract

Radiotherapy causes dose-limiting toxicity and long-term complications in rapidly renewing tissues, including the gastrointestinal tract. Currently, there is no FDA-approved agent for the prevention or treatment of radiation-induced intestinal injury. In this study, we have shown that PD 0332991 (PD), an FDA-approved selective inhibitor of cyclin-dependent kinase 4/6 (CDK4/6), prevents radiation-induced lethal intestinal injury in mice. Treating mice with PD or a structurally distinct CDK4/6 inhibitor prior to radiation blocked proliferation and crypt apoptosis and improved crypt regeneration. PD treatment also enhanced LGR5+ stem cell survival and regeneration after radiation. PD was an on-target inhibitor of RB phosphorylation and blocked G1/S transition in the intestinal crypts. PD treatment strongly but reversibly inhibited radiation-induced p53 activation, which blocked p53-upregulated modulator of apoptosis–dependent (PUMA-dependent) apoptosis without affecting p21-dependent suppression of DNA damage accumulation, with a repair bias toward nonhomologous end joining. Further, deletion of PUMA synergized with PD treatment for even greater intestinal radioprotection. Our results demonstrate that the cell cycle critically regulates the DNA damage response and survival of intestinal stem cells and support the concept that pharmacological quiescence is a potentially highly effective and selective strategy for intestinal radioprotection.

Authors

Liang Wei, Brian J. Leibowitz, Xinwei Wang, Michael Epperly, Joel Greenberger, Lin Zhang, Jian Yu

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Figure 3

PD blocks IR-induced crypt apoptosis.

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PD blocks IR-induced crypt apoptosis. Mice were pretreated with vehicle,...
Mice were pretreated with vehicle, PD, or LEE and subjected to 15 Gy TBI. The small intestine was analyzed at the indicated time points. (A) Representative images of TUNEL and cleaved caspase-3 (CASP-3) staining of intestinal sections at 4 hours. Scale bars: 20 μm. (B) Quantitation of TUNEL+ and cleaved caspase-3+ crypt cells at 4 and 24 hours. (C) Fractions of cleaved caspase-3+ crypt cells at 4 hours based on cell positions. (D) Quantification of TUNEL and BrdU index at 4 hours and regenerated crypts at 96 hours in LEE-treated and control (Ctrl) mice. (E) Intestinal expression of the indicated proteins analyzed by Western blotting. β-Actin was used as the loading control. Lysates were pooled from 3 mice per group. Replicate samples run on separate gels are presented. (F) Intestinal relative expression of the indicated mRNAs versus the nontreated mice. cDNA was synthesized from RNA pooled from 3 mice per group. Similar results were obtained in at least 3 independent experiments for Western blotting and RT-PCR. (B–D and F) Values represent the mean ± SEM; N = 3 mice in each group. *P < 0.05, **P < 0.01, and ***P < 0.001, vehicle versus PD or LEE treatment, by unpaired, 2-tailed Student’s t test.