Inhibition of CDK4/6 protects against radiation-induced intestinal injury in mice
Liang Wei, Brian J. Leibowitz, Xinwei Wang, Michael Epperly, Joel Greenberger, Lin Zhang, Jian Yu
Liang Wei, Brian J. Leibowitz, Xinwei Wang, Michael Epperly, Joel Greenberger, Lin Zhang, Jian Yu
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Research Article Gastroenterology

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 6

Intestinal radioprotection by PD requires the p53 pathway.

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Intestinal radioprotection by PD requires the p53 pathway. Mice of the i...
Mice of the indicated genotypes were pretreated with vehicle or PD and subjected to 15 Gy TBI. The small intestine was analyzed at the indicated time points. (A) Representative images of BrdU IHC in PUMA-KO, p21-KO, and p53-KO crypts at 96 hours. Scale bar: 500 μm. Graph shows the quantification of surviving crypts at 96 hours in mice of the 4 genotypes. (B) Representative images of BrdU and TUNEL staining at 4 hours. Scale bars: 10 μm. Graph shows the quantification of BrdU+ and TUNEL+ crypt cells from mice of the 4 genotypes at 4 hours. (C) Top: Representative image of γH2AX/p-H3 double immunofluorescence in p21-KO crypts with higher-magnification images shown below. Green, γH2AX; red, p-H3; blue, DAPI. Scale bar: 50 μm, 20 μm (higher-magnification images). Graph shows the quantification of γH2AX/p-H3 double-positive crypt cells at 72 hours. (D) Representative images of 53BP1 immunofluorescence (red) in the crypts at 72 hours; blue, DAPI. Scale bar: 10 μm. Graph shows the quantification of 53BP1 foci at 72 hours. Values represent the mean ± SEM; N = 3 mice in each group. **P < 0.01 and ***P < 0.001, for vehicle versus PD treatment in mice of the same genotype, by unpaired, 2-tailed Student’s t test; ††P < 0.01 and †††P < 0.001, for WT versus the indicated genotypes or treatment, by 1-way ANOVA followed by Bonferroni’s multiple comparisons test.