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Ionizing radiation induces cell death in the gastrointestinal (GI) epithelium by activating p53. However, p53 also prevents animal lethality caused by radiation-induced GI injury. Through single-cell RNA-sequencing of the irradiated mouse intestine, we find that p53 target genes are specifically enriched in stem cells of the regenerating epithelium, including revival stem cells that promote animal survival after GI damage. Accordingly, in mice with p53 deleted specifically in the GI epithelium, ionizing radiation fails to induce revival stem cells. Using intestinal organoids, we show that transient p53 expression is required for the induction of revival stem cells that is controlled by an Mdm2-mediated negative feedback loop. These results suggest that p53 suppresses severe radiation-indued GI injury by promoting intestinal epithelial cell reprogramming.One-Sentence SummaryAfter severe radiation injury to the intestine, transient p53 activity induces revival stem cells to promote regeneration.

Thoracic radiation therapy can cause endothelial injury in the heart, leading to cardiac dysfunction and heart failure. Although it has been demonstrated that the tumor suppressor p53 functions in endothelial cells to prevent the development of radiation-induced myocardial injury, the key mechanism(s) by which p53 regulates the radiosensitivity of cardiac endothelial cells is not completely understood. Here, we utilized genetically engineered mice that express mutations in p53 transactivation domain 1 (TAD1) (p5325,26) or mutations in p53 TAD1 and TAD2 (p5325,26,53,54) specifically in endothelial cells to study the p53 transcriptional program that protects cardiac endothelial cells from ionizing radiation in vivo. p5325,26,53,54 loses the ability to drive transactivation of p53 target genes after irradiation while p5325,26 can induce transcription of a group of non-canonical p53 target genes, but not the majority of classic radiation-induced p53 targets critical for p53-mediated cell cycle arrest and apoptosis. After 12 Gy whole-heart irradiation, we found that both p5325,26 and p5325,26,53,54 sensitized mice to radiation-induced cardiac injury, in contrast to wild-type p53. Histopathological examination suggested that mutation of TAD1 contributes to myocardial necrosis after whole-heart irradiation, while mutation of both TAD1 and TAD2 abolishes the ability of p53 to prevent radiation-induced heart disease. Taken together, our results show that the transcriptional program downstream of p53 TAD1, which activates the acute DNA damage response after irradiation, is necessary to protect cardiac endothelial cells from radiation injury in vivo.

Nearly two-thirds of cancer patients are treated with radiation therapy (RT), often with the intent to achieve complete and permanent tumor regression (local control). RT is the primary treatment modality used to achieve local control for many malignancies, including locally advanced cervical cancer, head and neck cancer, and lung cancer. The addition of concurrent platinum-based radiosensitizing chemotherapy improves local control and patient survival. Enhanced outcomes with concurrent chemoradiotherapy may result from increased direct killing of tumor cells and effects on nontumor cell populations. Many patients treated with concurrent chemoradiotherapy exhibit a decline in neutrophil count, but the effects of neutrophils on radiation therapy are controversial. To investigate the clinical significance of neutrophils in the response to RT, we examined patient outcomes and circulating neutrophil counts in cervical cancer patients treated with definitive chemoradiation. Although pretreatment neutrophil count did not correlate with outcome, lower absolute neutrophil count after starting concurrent chemoradiotherapy was associated with higher rates of local control, metastasis-free survival, and overall survival. To define the role of neutrophils in tumor response to RT, we used genetic and pharmacological approaches to deplete neutrophils in an autochthonous mouse model of soft tissue sarcoma. Neutrophil depletion prior to image-guided focal irradiation improved tumor response to RT. Our results indicate that neutrophils promote resistance to radiation therapy. The efficacy of chemoradiotherapy may depend on the impact of treatment on peripheral neutrophil count, which has the potential to serve as an inexpensive and widely available biomarker.

Genetically engineered mouse models that employ site-specific recombinase technology are important tools for cancer research but can be costly and time-consuming. The CRISPR-Cas9 system has been adapted to generate autochthonous tumours in mice, but how these tumours compare to tumours generated by conventional recombinase technology remains to be fully explored. Here we use CRISPR-Cas9 to generate multiple subtypes of primary sarcomas efficiently in wild type and genetically engineered mice. These data demonstrate that CRISPR-Cas9 can be used to generate multiple subtypes of soft tissue sarcomas in mice. Primary sarcomas generated with CRISPR-Cas9 and Cre recombinase technology had similar histology, growth kinetics, copy number variation and mutational load as assessed by whole exome sequencing. These results show that sarcomas generated with CRISPR-Cas9 technology are similar to sarcomas generated with conventional modelling techniques and suggest that CRISPR-Cas9 can be used to more rapidly generate genotypically and phenotypically similar cancers., Site-specific recombination and CRISPR-Cas9 have been used to generate genetically engineered mouse models of cancer. Here the authors compare sarcomas generated using both systems and see similar genetic and cellular phenotypes, suggesting CRISPR-Cas9 can be used to rapidly generate sarcoma models.

3-Methyladenine (3-MA) is one of the most commonly used inhibitors in autophagy research today. However, rather than inhibiting class III PI3K that is involved in autophagy suppression, 3-MA might also interfere with class I PI3K and consequently augment autophagy flux. In this study, we aim to get a thorough understanding on the action mechanisms of 3-MA in TLR4-mediated inflammatory responses in RAW264.7 macrophages and, moreover, to decipher the action of 3-MA in modulation of autophagy. We found that 3-MA could enhance LPS-induced NF-κB activation and production of TNF-α, inducible NO synthase (iNOS), cyclooxygenase-2, IL-1β, and IL-12. In contrast, 3-MA suppressed LPS-induced IFN-β production and STAT signaling. Studies revealed that 3-MA can, through inhibition of Akt as a result of class I PI3K interference, positively regulate p38, JNK, and p65, but negatively regulate TANK-binding kinase 1 and IFN regulatory factor 3 mediated by TLR4. As glycogen synthase kinase 3β (GSK3β) is an important Akt substrate, we further explored its involvement in the actions of 3-MA. 3-MA was found to enhance LPS-induced NF-κB activation, iNOS, and pro–IL-1β expression, and these actions were reversed by either GSK3β inhibitors or small interfering GSK3β. Lastly, we demonstrated that 3-MA acts as an autophagy inducer in RAW264.7 macrophages, but the stimulating effects on NF-κB activation and iNOS and cyclooxygenase-2 expression were not affected in LPS-stimulated macrophages with small interfering autophagy protein-5 treatment. These results not only shed new light on the action mechanisms of 3-MA to differentially regulate inflammatory outcomes derived from TLR4-mediated MyD88 and Toll/IL-1R domain-containing adapter inducing IFN-β pathways, but also highlight the necessity to check autophagy status upon taking 3-MA as a general autophagy inhibitor.

Genetically engineered mouse models (GEMMs) that employ site-specific recombinase (SSR) technology are important tools for cancer research, and recently the CRISPR/Cas9 system has been increasingly utilized to model cancer in mice. Here, we used CRISPR/Cas9 to generate two primary mouse models of sarcoma, undifferentiated pleomorphic sarcoma (UPS) in a GEMM, and malignant peripheral nerve sheath tumor (MPNST) in wild-type mice, to demonstrate the versatility of the system to generate multiple soft-tissue sarcoma subtypes. Because CRISPR technology is becoming more prevalent in cancer modeling, it is critical to thoroughly evaluate if these models are indeed comparable as tools to study cancer biology compared to conventional GEMMs initiated by recombinase technology. We used two Kras-driven sarcoma models of UPS generated with either Cre recombinase technology or CRISPR/Cas9 technology and compared the mutational profiles, histology, and growth kinetics of these models. KrasLSL-G12D/+; Rosa26LSL-Cas9-EGFP/+ (KC) mice received intramuscular delivery of an adenovirus expressing Cre recombinase and a single guide RNA (sgRNA) targeting Trp53. Cre-mediated expression of oncogenic Kras and Cas9, in combination with CRISPR/Cas9-mediated knockout of Trp53, was sufficient to generate primary soft-tissue sarcomas. Compared to the Cre/loxP model, we determined that sarcomas generated with CRISPR/Cas9 had similar growth kinetics, histology, copy number variation, and mutational load as assessed by whole-exome sequencing. We also demonstrated that off-target mutations in the sarcomas initiated by the Cas9 endonuclease were rare in tumors. Finally, we analyzed the Cas9-mediated indels present in tumors as genetic barcodes, which will enable future studies of tumor heterogeneity and clonality. These results show that sarcomas generated with CRISPR/Cas9 technology are similar to sarcomas generated with conventional modeling techniques. Ultimately this work corroborates CRISPR/Cas9-generated mouse models with traditional GEMMs phenotypically and genotypically, and expands the range of sarcoma mouse models available for research. Citation Format: Jianguo Huang, Mark Chen, Melodi Javid Whitley, Hsuan-Cheng Kuo, Eric S. Xu, Andrea Walens, Yvonne M. Mowery, David Van Mater, William C. Eward, Diana M. Cardona, Lixia Luo, Yan Ma, Omar M. Lopez, Christopher E. Nelson, Jacqueline N. Robinson-Hamm, Anupama Reddy, Sandeep S. Dave, Charles A. Gersbach, Rebecca D. Dodd, David G. Kirsch. Generation and comparison of CRISPR/Cas9 and Cre-mediated genetically engineered mouse models of sarcoma [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr A17.

Genetically engineered mouse models (GEMMs) that employ site-specific recombinase (SSR) technology are important tools for pre-clinical studies, but this approach is costly and time-consuming. Here, we show that the CRISPR/Cas9 system can be used to efficiently complement existing GEMMs of sarcoma and generate primary sarcomas in wild type mice. Mice with the genotype KrasLSL-G12D/+; Rosa26LSL-Cas9-EGFP/+ received intramuscular delivery of an adenovirus expressing Cre recombinase and a single guide RNA (sgRNA) targeting Trp53. Cre-mediated expression of oncogenic Kras and Cas9, in combination with CRISPR/Cas9-mediated knockout of Trp53, was sufficient to generate primary soft tissue sarcomas. These tumors arose with kinetics similar to those generated using the Cre-loxP system to activate oncogenic Kras and delete Trp53 alleles. Additionally, we injected an adenovirus containing Cas9 and sgRNAs targeting Nf1 and Trp53 into the sciatic nerve of wild type mice. These mice formed malignant peripheral nerve sheath tumors (MPNSTs) in the same timeframe as MPNSTs generated using the Cre-loxP system to delete Nf1 and Ink4a/Arf alleles in GEMMs. These data demonstrate that CRISPR/Cas9 can be used to generate soft tissue sarcomas in wild type mice. Moreover, these results suggest that this technology can complement existing GEMMs for rapid assessment of tumor-modifying genes. These tools should decrease the time and expense associated with employing autochthonous mouse models of sarcoma for preclinical research. Citation Format: Jianguo Huang, Mark Chen, Melodi J. Whitley, Hsuan-Cheng Kuo, Andrea Walens, Yvonne M. Mowery, David V. Mater, William Eward, Diana M. Cardona, Lixia Luo, Yan Ma, Christopher E. Nelson, Jacqueline N. Robinson-Hamm, Charles A. Gersbach, Rebecca D. Dodd, David G. Kirsch. Using CRISPR/Cas9 to generate primary soft tissue sarcoma in genetically engineered and wild-type mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2810. doi:10.1158/1538-7445.AM2017-2810