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The role of bone marrow (BM) and BM-derived cells in radiation-induced acute gastrointestinal (GI) syndrome is controversial. Here we use bone marrow transplantation (BMT), total body irradiation (TBI) and abdominal irradiation (ABI) models to demonstrate a very limited, if any, role of BM-derived cells in acute GI injury and recovery. Compared with WT BM recipients, mice receiving BM from radiation-resistant PUMA KO mice show no protection from crypt and villus injury or recovery after 15 or 12 Gy TBI, but have a significant survival benefit at 12 Gy TBI. PUMA KO BM significantly protects donor-derived pan-intestinal haematopoietic (CD45+) and endothelial (CD105+) cells after IR. We further show that PUMA KO BM fails to enhance animal survival or crypt regeneration in radiosensitive p21 KO-recipient mice. These findings clearly separate the effects of radiation on the intestinal epithelium from those on the BM and endothelial cells in dose-dependent acute radiation toxicity. Ionizing radiation damages the gastrointestinal system, but the cell types involved in intestinal damage and repair are controversial. Here the authors use bone marrow transplantation models and various irradiation regimes to rule out a role of bone marrow-derived cells in acute gastrointestinal injury and recovery in mice.

To better understand a role of eIF4E S209 in oncogenic translation, we generated EIF4E S209A/+ 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 Apc Min/+ 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.

Exposure to high levels of ionizing radiation (IR) leads to debilitating and dose-limiting gastrointestinal (GI) toxicity. Using three-dimensional mouse crypt culture, we demonstrated that p53 target PUMA mediates radiation-induced apoptosis via a cell-intrinsic mechanism and identified the GSK-3 inhibitor CHIR99021 as a potent radioprotector. CHIR99021 treatment improved Lgr5+ cell survival and crypt regeneration after radiation in culture and mice. CHIR99021 treatment specifically blocked apoptosis and PUMA induction and K120 acetylation of p53 mediated by acetyl-transferase Tip60, while it had no effect on p53 stabilization, phosphorylation or p21 induction. CHIR99021 also protected human intestinal cultures from radiation by PUMA but not p21 suppression. These results demonstrate that p53 posttranslational modifications play a key role in the pathological and apoptotic response of the intestinal stem cells to radiation and can be targeted pharmacologically.

Multiplexed imaging technologies have made it possible to interrogate complex tissue 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. However, limited work has been done in developing such generalist methods within the unsupervised context. Here we present an easy-to-use unsupervised segmentation (UNSEG) method that achieves deep learning level performance without requiring any training data via leveraging a Bayesian-like framework, and nucleus and cell membrane markers. We show that UNSEG is internally consistent and better at generalizing to the complexity of tissue morphology than current deep learning methods, allowing it to unambiguously identify the cytoplasmic compartment of a cell, and localize molecules to their correct sub-cellular compartment. We also introduce a perturbed watershed algorithm for stably and automatically segmenting a cluster of cell nuclei into individual nuclei that increases the accuracy of classical watershed. Finally, we demonstrate the efficacy of UNSEG on a high-quality annotated gastrointestinal tissue dataset we have generated, on publicly available datasets, and in a range of practical scenarios. An unsupervised segmentation algorithm that achieves state-of-art deep learning performance for segmenting cells and their nuclei in complex biological tissue images without requiring any training data.

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.

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.

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.

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 APC Min/+ mice. Everolimus in diet strongly reduces p-4EBP1, p-S6, and Myc levels, and induces apoptosis of cells with activated β-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.

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 APC Min/+ 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.

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-β, 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-1β. 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.