Targeting Myc-driven stress addiction in colorectal cancer.

MYC is a proto-oncogene that encodes a powerful regulator of transcription and cellular programs essential for normal development, as well as the growth and survival of various types of cancer cells. MYC rearrangement and amplification is a common cause of hematologic malignancies. In epithelial cancers such as colorectal cancer, genetic alterations in MYC are rare. Activation of Wnt, ERK/MAPK, and PI3K/mTOR pathways dramatically increases Myc levels through enhanced transcription, translation, and protein stability. Elevated Myc promotes stress adaptation, metabolic reprogramming, and immune evasion to drive cancer development and therapeutic resistance through broad changes in transcriptional and translational landscapes. Despite intense interest and effort, Myc remains a difficult drug target. Deregulation of Myc and its targets has profound effects that vary depending on the type of cancer and the context. Here, we summarize recent advances in the mechanistic understanding of Myc-driven oncogenesis centered around mRNA translation and proteostress. Promising strategies and agents under development to target Myc are also discussed with a focus on colorectal cancer.

Intestinal Radiation Protection and Mitigation by Second-Generation Probiotic Lactobacillus-reuteri Engineered to Deliver Interleukin-22.

(1) Background: The systemic administration of therapeutic agents to the intestine including cytokines, such as Interleukin-22 (IL-22), is compromised by damage to the microvasculature 24 hrs after total body irradiation (TBI). At that time, there is significant death of intestinal microvascular endothelial cells and destruction of the lamina propria, which limits drug delivery through the circulation, thus reducing the capacity of therapeutics to stabilize the numbers of Lgr5+ intestinal crypt stem cells and their progeny, and improve survival. By its direct action on intestinal stem cells and their villus regeneration capacity, IL-22 is both an ionizing irradiation protector and mitigator. (2) Methods: To improve delivery of IL-22 to the irradiated intestine, we gavaged Lactobacillus-reuteri as a platform for the second-generation probiotic Lactobacillus-reuteri-Interleukin-22 (LR-IL-22). (3) Results: There was effective radiation mitigation by gavage of LR-IL-22 at 24 h after intestinal irradiation. Multiple biomarkers of radiation damage to the intestine, immune system and bone marrow were improved by LR-IL-22 compared to the gavage of control LR or intraperitoneal injection of IL-22 protein. (4) Conclusions: Oral administration of LR-IL-22 is an effective protector and mitigator of intestinal irradiation damage.

Immunogenic cell death in colon cancer prevention and therapy.

Colorectal cancer (CRC) is a leading cause of cancer-related death worldwide. The colonic mucosa constitutes a critical barrier and a major site of immune regulation. The immune system plays important roles in cancer development and treatment, and immune activation caused by chronic infection or inflammation is well-known to increase cancer risk. During tumor development, neoplastic cells continuously interact with and shape the tumor microenvironment (TME), which becomes progressively immunosuppressive. The clinical success of immune checkpoint blockade therapies is limited to a small set of CRCs with high tumor mutational load and tumor-infiltrating T cells. Induction of immunogenic cell death (ICD), a type of cell death eliciting an immune response, can therefore help break the immunosuppressive TME, engage the innate components, and prime T cell-mediated adaptive immunity for long-term tumor control. In this review, we discuss the current understanding of ICD induced by antineoplastic agents, the influence of driver mutations, and recent developments to harness ICD in colon cancer. Mechanism-guided combinations of ICD-inducing agents with immunotherapy and actionable biomarkers will likely offer more tailored and durable benefits to patients with colon cancer.

γPNA FRET Pair Miniprobes for Quantitative Fluorescent In Situ Hybridization to Telomeric DNA in Cells and Tissue.

Measurement of telomere length by fluorescent in situ hybridization is widely used for biomedical and epidemiological research, but there has been relatively little development of the technology in the 20 years since it was first reported. This report describes the use of dual gammaPNA (γPNA) probes that hybridize at alternating sites along a telomere and give rise to Förster resonance energy transfer (FRET) signals. Bright staining of telomeres is observed in nuclei, chromosome spreads and tissue samples. The use of FRET detection also allows for elimination of wash steps, normally required to remove unhybridized probes that would contribute to background signals. We found that these wash steps can diminish the signal intensity through the removal of bound, as well as unbound probes, so eliminating these steps not only accelerates the process but also enhances the quality of staining. Thus, γPNA FRET pairs allow for brighter and faster staining of telomeres in a wide range of research and clinical formats.

FAK Drives Resistance to Therapy in HPV-Negative Head and Neck Cancer in a p53-Dependent Manner.

PURPOSE: Radiation and platinum-based chemotherapy form the backbone of therapy in human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC). We have correlated focal adhesion kinase (FAK/PTK2) expression with radioresistance and worse outcomes in these patients. However, the importance of FAK in driving radioresistance and its effects on chemoresistance in these patients remains unclear. EXPERIMENTAL DESIGN: We performed an in vivo shRNA screen using targetable libraries to identify novel therapeutic sensitizers for radiation and chemotherapy. RESULTS: We identified FAK as an excellent target for both radio- and chemosensitization. Because TP53 is mutated in over 80% of HPV-negative HNSCC, we hypothesized that mutant TP53 may facilitate FAK-mediated therapy resistance. FAK inhibitor increased sensitivity to radiation, increased DNA damage, and repressed homologous recombination and nonhomologous end joining repair in mutant, but not wild-type, TP53 HPV-negative HNSCC cell lines. The mutant TP53 cisplatin-resistant cell line had increased FAK phosphorylation compared with wild-type, and FAK inhibition partially reversed cisplatin resistance. To validate these findings, we utilized an HNSCC cohort to show that FAK copy number and gene expression were associated with worse disease-free survival in mutant TP53, but not wild-type TP53, HPV-negative HNSCC tumors. CONCLUSIONS: FAK may represent a targetable therapeutic sensitizer linked to a known genomic marker of resistance.

Genetically Engineered Probiotic Limosilactobacillus reuteri Releasing IL-22 (LR-IL-22) Modifies the Tumor Microenvironment, Enabling Irradiation in Ovarian Cancer.

Despite recent advances in cancer therapy, ovarian cancer remains the most lethal gynecological cancer worldwide, making it crucial and of the utmost importance to establish novel therapeutic strategies. Adjuvant radiotherapy has been assessed historically, but its use was limited by intestinal toxicity. We recently established the role of Limosilactobacillus reuteri in releasing IL-22 (LR-IL-22) as an effective radiation mitigator, and we have now assessed its effect in an ovarian cancer mouse model. We hypothesized that an LR-IL-22 gavage would enable intestinal radioprotection by modifying the tumor microenvironment and, subsequently, improving overall survival in female C57BL/6MUC-1 mice with widespread abdominal syngeneic 2F8cis ovarian cancer. Herein, we report that the LR-IL-22 gavage not only improved overall survival in mice when combined with a PD-L1 inhibitor by inducing differential gene expression in irradiated stem cells but also induced PD-L1 protein expression in ovarian cancer cells and mobilized CD8+ T cells in whole abdomen irradiated mice. The addition of LR-IL-22 to a combined treatment modality with fractionated whole abdomen radiation (WAI) and systemic chemotherapy and immunotherapy regimens can facilitate a safe and effective protocol to reduce tumor burden, increase survival, and improve the quality of life of a locally advanced ovarian cancer patient.

IGF binding protein-3 mediates stress-induced apoptosis in non-transformed mammary epithelial cells.

Mammary epithelial cell (MEC) number is an important determinant of milk production in lactating dairy cows. IGF-I increases IGF binding protein-3 (IGFBP-3) production in these cells, which plays a role in its ability to enhance proliferation. In the present study, we show that the apoptotic factor anisomycin (ANS) also increases IGFBP-3 mRNA and protein in a dose- and concentration-dependent manner that mirrors activation of caspase-3 and -7, with significant increases in both IGFBP-3 protein and caspase activation observed by 3 h. Knock-down of IGFBP-3 with small interfering (si) RNA attenuated the ability of ANS to induce apoptosis, while knock-down of IGFBP-2, the other major IGFBP made by bovine MEC, had no effect. Reducing IGFBP-3 also decreased the ability of ANS to induce mitochondrial cytochrome c release, indicating its involvement in the intrinsic apoptotic pathway. In contrast, transfection with IGFBP-3 in the absence of ANS failed to induce apoptosis. Since both the mitogen IGF-I and the apoptotic inducer ANS increase IGFBP-3 production in MEC, we proposed that cellular localization might determine IGFBP-3 action. While both IGF-I and ANS stimulated the release of IGFBP-3 into conditioned media, only ANS induced nuclear localization of IGFBP-3. A pan-caspase inhibitor had no effect on ANS-induced nuclear localization of IGFBP-3, indicating that nuclear entry of IGFBP-3 precedes caspase activation. Treatment with IGF-I had no effect on ANS-induced nuclear localization, but did block ANS-induced apoptosis. In summary, our data indicate that IGFBP-3 plays a role in stress-induced apoptosis that may require nuclear localization in non-transformed MEC.

mTOR inhibition suppresses Myc-driven polyposis by inducing immunogenic cell death.

Myc is a key driver of colorectal cancer initiation and progression, but remains a difficult drug target. In this study, we show that mTOR inhibition potently suppresses intestinal polyp formation, regresses established polyps, and prolongs lifespan of APCMin/+ mice. Everolimus in diet strongly reduces p-4EBP1, p-S6, and Myc levels, and induces apoptosis of cells with activated ß-catenin (p-S552) in the polyps on day 3. The cell death is accompanied by ER stress, activation of the extrinsic apoptotic pathway, innate immune cell recruitment, and followed by T-cell infiltration on day 14 persisting for months thereafter. These effects are absent in normal intestinal crypts with physiologic levels of Myc and a high rate of proliferation. Using normal human colonic epithelial cells, EIF4E S209A knockin and BID knockout mice, we found that local inflammation and antitumor efficacy of Everolimus requires Myc-dependent induction of ER stress and apoptosis. These findings demonstrate mTOR and deregulated Myc as a selective vulnerability of mutant APC-driven intestinal tumorigenesis, whose inhibition disrupts metabolic and immune adaptation and reactivates immune surveillance necessary for long-term tumor control.

Release of Interferon-ß (IFN-ß) from Probiotic Limosilactobacillus reuteri-IFN-ß (LR-IFN-ß) Mitigates Gastrointestinal Acute Radiation Syndrome (GI-ARS) following Whole Abdominal Irradiation.

Irradiation can be an effective treatment for ovarian cancer, but its use is limited by intestinal toxicity. Thus, strategies to mitigate toxicity are important and can revitalize the current standard of care. We previously established that LR-IL-22 protects the intestine from WAI. We now hypothesize that LR-IFN-ß is an effective radiation protector and mitigator and is rapidly cleared from the digestive tract, making it an option for intestinal radioprotection. We report that the gavage of LR-IFN-ß during WAI provides improved intestinal barrier integrity and significantly preserves the numbers of Lgr5+GFP+ intestinal stem cells, improving survival. The rapid clearance of the genetically engineered probiotic from the digestive tract renders it a safe and feasible radiation mitigator. Therefore, the above genetically engineered probiotic is both a feasible and effective radiation mitigator that could potentially revolutionize the management of OC patients. Furthermore, the subsequent addition of platinum/taxane-based chemotherapy to the combination of WAI and LR-IFN-ß should reduce tumor volume while protecting the intestine and should improve the overall survival in OC patients.

UNSEG: unsupervised segmentation of cells and their nuclei in complex tissue samples.

Multiplexed imaging technologies have made it possible to interrogate complex tumor microenvironments at sub-cellular resolution within their native spatial context. However, proper quantification of this complexity requires the ability to easily and accurately segment cells into their sub-cellular compartments. Within the supervised learning paradigm, deep learning based segmentation methods demonstrating human level performance have emerged. Here we present an unsupervised segmentation (UNSEG) method that achieves deep learning level performance without requiring any training data. UNSEG leverages a Bayesian-like framework and the specificity of nucleus and cell membrane markers to construct an a posteriori probability estimate of each pixel belonging to the nucleus, cell membrane, or background. It uses this estimate to segment each cell into its nuclear and cell-membrane compartments. We show that UNSEG is more internally consistent and better at generalizing to the complexity of tissue samples than current deep learning methods. This allows UNSEG to unambiguously identify the cytoplasmic compartment of a cell, which we employ to demonstrate its use in an example biological scenario. Within the UNSEG framework, we also introduce a new perturbed watershed algorithm capable of stably and accurately segmenting a cell nuclei cluster into individual cell nuclei. Perturbed watershed can also be used as a standalone algorithm that researchers can incorporate within their supervised or unsupervised learning approaches to replace classical watershed. Finally, as part of developing UNSEG, we have generated a high-quality annotated gastrointestinal tissue dataset, which we anticipate will be useful for the broader research community. Segmentation, despite its long antecedents, remains a challenging problem, particularly in the context of tissue samples. UNSEG, an easy-to-use algorithm, provides an unsupervised approach to overcome this bottleneck, and as we discuss, can help improve deep learning based segmentation methods by providing a bridge between unsupervised and supervised learning paradigms.

Targeting Myc-driven stress vulnerability in mutant KRAS colorectal cancer.

Mutant KRAS is a key driver in colorectal cancer (CRC) and promotes Myc translation and Myc-dependent stress adaptation and proliferation. Here, we report that the combination of two FDA-approved drugs Bortezomib and Everolimus (RAD001) (BR) is highly efficacious against mutant KRAS CRC cells. Mechanistically, the combination, not single agent, rapidly depletes Myc protein, not mRNA, and leads to GCN2- and p-eIF2α-dependent cell death through the activation of extrinsic and intrinsic apoptotic pathways. Cell death is selectively induced in mutant KRAS CRC cells with elevated basal Myc and p-eIF2α and is characterized by CHOP induction and transcriptional signatures in proteotoxicity, oxidative stress, metabolic inhibition, and immune activation. BR-induced p-GCN2/p-eIF2α elevation and cell death are strongly attenuated by MYC knockdown and enhanced by MYC overexpression. The BR combination is efficacious against mutant KRAS patient derived organoids (PDO) and xenografts (PDX) by inducing p-eIF2α/CHOP and cell death. Interestingly, an elevated four-gene (DDIT3, GADD45B, CRYBA4 and HSPA1L) stress signature is linked to shortened overall survival in CRC patients. These data support that Myc-dependent stress adaptation drives the progression of mutant KRAS CRC and serves as a therapeutic vulnerability, which can be targeted using dual translational inhibitors.

Lactobacillus reuteri Releasing IL-22 (LR-IL-22) Facilitates Intestinal Radioprotection for Whole-Abdomen Irradiation (WAI) of Ovarian Cancer.

Oral administration (gavage) of a second-generation probiotic, Lactobacillus reuteri (L. reuteri), that releases interleukin-22 (LR-IL-22) at 24 h after total-body irradiation (TBI) mitigates damage to the intestine. We determined that LR-IL-22 also mitigates partial-body irradiation (PBI) and whole-abdomen irradiation (WAI). Irradiation can be an effective treatment for ovarian cancer, but its use is limited by intestinal toxicity. Strategies to mitigate toxicity are important and can revitalize this modality to treat ovarian cancer. In the present studies, we evaluated whether LR-IL-22 facilitates fractionated WAI in female C57BL/6 mice with disseminated ovarian cancer given a single fraction of either 15.75 Gy or 19.75 Gy or 4 daily fractions of 6 Gy or 6.5 Gy. Mice receiving single or multiple administrations of LR-IL-22 during WAI showed improved intestinal barrier integrity (P = 0.0167), reduced levels of radiation-induced intestinal cytokines including KC/CXCL1 (P = 0.002) and IFN-γ (P = 0.0024), and reduced levels of plasma, Eotaxin/CCL11 (P = 0.0088). LR-IL-22 significantly preserved the numbers of Lgr5+GFP+ intestinal stem cells (P = 0.0010) and improved survival (P < 0.0343). Female C57BL/6MUC-1 mice with widespread abdominal syngeneic 2F8cis ovarian cancer that received LR-IL-22 during 6.5 Gy WAI in 4 fractions had reduced tumor burden, less intestinal toxicity, and improved 30-day survival. Furthermore, LR-IL-22 facilitated WAI when added to Paclitaxel and Carboplatin chemotherapy and further increased survival. Oral administration (gavage) of LR-IL-22 is a potentially valuable intestinal radioprotector, which can facilitate therapeutic WAI for widespread intra-abdominal ovarian cancer.

Interferon b drives intestinal regeneration after radiation.

The cGAS-STING cytosolic DNA sensing pathway is critical for host defense. Here, we report that cGAS-STING­dependent type 1 interferon (IFN) response drives intestinal regeneration and animal recovery from radiation injury. STING deficiency has no effect on radiation-induced DNA damage or crypt apoptosis but abrogates epithelial IFN-ß production, local inflammation, innate transcriptional response, and subsequent crypt regeneration. cGAS KO, IFNAR1 KO, or CCR2 KO also abrogates radiation-induced acute crypt inflammation and regeneration. Impaired intestinal regeneration and survival in STING-deficient mice are fully rescued by a single IFN-ß treatment given 48 hours after irradiation but not by wild-type (WT) bone marrow. IFN-ß treatment remarkably improves the survival of WT mice and Lgr5+ stem cell regeneration through elevated compensatory proliferation and more rapid DNA damage removal. Our findings support that inducible IFN-ß production in the niche couples ISC injury and regeneration and its potential use to treat acute radiation injury.

Non-steroidal anti-inflammatory drugs induce immunogenic cell death in suppressing colorectal tumorigenesis.

Use of non-steroidal anti-inflammatory drugs (NSAIDs) is associated with reduced risk of colorectal cancer (CRC). However, the mechanism by which NSAIDs suppress colorectal tumorigenesis remains unclear. We previously showed that NSAIDs selectively kill emerging tumor cells via death receptor (DR) signaling and a synthetic lethal interaction mediated by the proapoptotic Bcl-2 family protein BID. In this study, we found NSAIDs induce endoplasmic reticulum (ER) stress to activate DR signaling and BID in tumor suppression. Importantly, our results unveiled an ER stress- and BID-dependent immunogenic effect of NSAIDs, which may be critical for tumor suppression. NSAID treatment induced hallmarks of immunogenic cell death (ICD) in CRC cells and colonic epithelial cells upon loss of APC tumor suppressor, and elevated tumor-infiltrating lymphocytes (TILs) in the polyps of APCMin/+ mice. ER stress inhibition or BID deletion abrogated the antitumor and immunogenic effects of NSAIDs. Furthermore, increased ER stress and TILs were detected in human advanced adenomas from NSAID-treated patients. Together, our results suggest that NSAIDs induce ER stress- and BID-mediated ICD to restore immunosurveillance and suppress colorectal tumor formation.

eIF4E S209 phosphorylation licenses myc- and stress-driven oncogenesis.

To better understand a role of eIF4E S209 in oncogenic translation, we generated EIF4ES209A/+ heterozygous knockin (4EKI) HCT 116 human colorectal cancer (CRC) cells. 4EKI had little impact on total eIF4E levels, cap binding or global translation, but markedly reduced HCT 116 cell growth in spheroids and mice, and CRC organoid growth. 4EKI strongly inhibited Myc and ATF4 translation, the integrated stress response (ISR)-dependent glutamine metabolic signature, AKT activation and proliferation in vivo. 4EKI inhibited polyposis in ApcMin/+ mice by suppressing Myc protein and AKT activation. Furthermore, p-eIF4E was highly elevated in CRC precursor lesions in mouse and human. p-eIF4E cooperated with mutant KRAS to promote Myc and ISR-dependent glutamine addiction in various CRC cell lines, characterized by increased cell death, transcriptomic heterogeneity and immune suppression upon deprivation. These findings demonstrate a critical role of eIF4E S209-dependent translation in Myc and stress-driven oncogenesis and as a potential therapeutic vulnerability.

Second-generation Probiotics Producing IL-22 Increase Survival of Mice After Total Body Irradiation.

BACKGROUND/AIM: Intestinal damage induced by total body irradiation (TBI) reduces leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5)-expressing stem cells, goblet, and Paneth cells, breaching the epithelial lining, and facilitating bacterial translocation, sepsis, and death. MATERIALS AND METHODS: Survival was measured after TBI in animals that received wild-type or recombinant bacteria producing interleukin-22 (IL-22). Changes in survival due to microbially delivered IL-22 were measured. Lactobacillus reuteri producing IL-22, or Escherichia coli-IL-22 were compared to determine which delivery system is better. RESULTS: C57BL/6 mice receiving IL-22 probiotics at 24 h after 9.25 Gy TBI, demonstrated green fluorescent protein-positive bacteria in the intestine, doubled the number of Lgr5+ intestinal stem cells, and increased 30-day survival. Bacteria were localized to the jejunum, ileum, and colon. CONCLUSION: Second-generation probiotics appear to be valuable for mitigation of TBI, and radiation protection during therapeutic total abdominal irradiation.

Endogenous IGFBP-3 is required for both growth factor-stimulated cell proliferation and cytokine-induced apoptosis in mammary epithelial cells.

TNF-alpha and IGF-I exert opposing effects on mammary epithelial cell (MEC) growth and survival. However, both increase IGF binding protein-3 (IGFBP-3) expression, a multifunctional protein that plays both IGF-dependent as well as independent roles in these processes. We have reported that IGF-I utilizes the PI3-K and MAPK pathways to induce IGFBP-3 expression in bovine MEC. Here we show that TNF-alpha requires the SAPK pathway p38, but not JNK, to induce IGFBP-3 expression. Contrary to reports in cancer cell lines, TNF-alpha retained its ability to decrease DNA synthesis in cells transfected with IGFBP-3 siRNA. It also retained its ability to inhibit IGF-I-stimulated DNA synthesis in these cells. In contrast, the ability of IGF-I to increase DNA synthesis was attenuated with IGFBP-3 knockdown. IGFBP-3 knockdown also decreased basal DNA synthesis, indicating that a certain level of IGFBP-3 may be required for cell proliferation. While TNF-alpha alone failed to induce apoptosis, it increased cell death when added with the JNK agonist anisomycin (ANS). TNF-alpha and ANS were unable to induce apoptosis when either IGFBP-3 or JNK-2 was knocked-down, suggesting that both JNK and IGFBP-3 may interact with a downstream molecule central to apoptosis. There are reports that IGFBP-3 promotes either cell proliferation or apoptosis in different cell systems. However, this is the first report that endogenous IGFBP-3 is required for the action of both stimulatory and inhibitory factors within the same cell line. Therefore, the actions of IGFBP-3 are not pre-determined, but instead governed by cellular context such as JNK activation.

The GS-nitroxide JP4-039 improves intestinal barrier and stem cell recovery in irradiated mice.

Total body irradiation (TBI) leads to dose- and tissue-specific lethality. In the current study, we demonstrate that a mitochondrion-targeted nitroxide JP4-039 given once 24 hours after 9-10 Gy TBI significantly improves mouse survival, and the recovery of intestinal barrier, differentiation and stem cell functions. The GI-protective effects are associated with rapid and selective induction of tight junction proteins and cytokines including TGF-ß, IL-10, IL-17a, IL-22 and Notch signaling long before bone marrow depletion. However, no change was observed in crypt death or the expression of prototypic pro-inflammatory cytokines such as TNF-α, IL-6 or IL-1ß. Surprisingly, bone marrow transplantation (BMT) performed 24 hours after TBI improves intestinal barrier and stem cell recovery with induction of IL-10, IL-17a, IL-22, and Notch signaling. Further, BMT-rescued TBI survivors display increased intestinal permeability, impaired ISC function and proliferation, but not obvious intestinal inflammation or increased epithelial death. These findings identify intestinal epithelium as a novel target of radiation mitigation, and potential strategies to enhance ISC recovery and regeneration after accidental or medical exposures.

Targeting p53-dependent stem cell loss for intestinal chemoprotection.

The gastrointestinal (GI) epithelium is the fastest renewing adult tissue and is maintained by tissue-specific stem cells. Treatment-induced GI side effects are a major dose-limiting factor for chemotherapy and abdominal radiotherapy and can decrease the quality of life in cancer patients and survivors. p53 is a key regulator of the DNA damage response, and its activation results in stimulus- and cell type-specific outcomes via distinct effectors. We demonstrate that p53-dependent PUMA induction mediates chemotherapy-induced intestinal injury in mice. Genetic ablation of Puma, but not of p53, protects against chemotherapy-induced lethal GI injury. Blocking chemotherapy-induced loss of LGR5+ stem cells by Puma KO or a small-molecule PUMA inhibitor (PUMAi) prevents perturbation of the stem cell niche, rapid activation of WNT and NOTCH signaling, and stem cell exhaustion during repeated exposures. PUMAi also protects human and mouse colonic organoids against chemotherapy-induced apoptosis and damage but does not protect cancer cells in vitro or in vivo. Therefore, targeting PUMA is a promising strategy for normal intestinal chemoprotection because it selectively blocks p53-dependent stem cell loss but leaves p53-dependent protective effects intact.

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

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.