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We explore a novel strategy of activating immune signaling through increased micronuclei formation utilizing a cell cycle checkpoint inhibitor to drive cell cycle progression following ionizing radiation. The Chk1/2 inhibitor AZD7762 is used to abrogate radiation therapy (RT)-induced G2/M cell cycle arrest in multiple cell lines and, we find that this therapeutic combination promotes increased micronuclei formation in vitro and subsequently drives increased type I interferon signaling and cytotoxic T-cell activation. In vivo studies using B16-F10 melanoma cancer cells implanted in C57/BL6 mice demonstrate improved rates of tumor control at the abscopal (unirradiated) site, located outside of the radiation field, only in the AZD7762 + RT group, with a corresponding reduction in mean tumor volume, increase in the CD8 T-cell population, and immune activated gene signaling. Our results demonstrate that targeted inhibition of cell cycle checkpoint activation following ionizing radiation drives increased production of immunogenic micronuclei, leading to systemic tumor response with potential future clinical benefit.

Melanomas that result from mutations in the gene encoding BRAF often become resistant to BRAF inhibition (BRAFi), with multiple mechanisms contributing to resistance. While therapy-induced autophagy promotes resistance to a number of therapies, especially those that target PI3K/mTOR signaling, its role as an adaptive resistance mechanism to BRAFi is not well characterized. Using tumor biopsies from BRAF(V600E) melanoma patients treated either with BRAFi or with combined BRAF and MEK inhibition, we found that BRAFi-resistant tumors had increased levels of autophagy compared with baseline. Patients with higher levels of therapy-induced autophagy had drastically lower response rates to BRAFi and a shorter duration of progression-free survival. In BRAF(V600E) melanoma cell lines, BRAFi or BRAF/MEK inhibition induced cytoprotective autophagy, and autophagy inhibition enhanced BRAFi-induced cell death. Shortly after BRAF inhibitor treatment in melanoma cell lines, mutant BRAF bound the ER stress gatekeeper GRP78, which rapidly expanded the ER. Disassociation of GRP78 from the PKR-like ER-kinase (PERK) promoted a PERK-dependent ER stress response that subsequently activated cytoprotective autophagy. Combined BRAF and autophagy inhibition promoted tumor regression in BRAFi-resistant xenografts. These data identify a molecular pathway for drug resistance connecting BRAFi, the ER stress response, and autophagy and provide a rationale for combination approaches targeting this resistance pathway.

Ionizing radiation induces cell death in the gastrointestinal (GI) epithelium by activating p53. However, p53 also prevents animal lethality caused by radiation-induced acute GI syndrome. Through single-cell RNA-sequencing of the irradiated mouse small intestine, we find that p53 target genes are specifically enriched in regenerating epithelial cells that undergo fetal-like reversion, including revival stem cells (revSCs) that promote animal survival after severe damage of the GI tract. Accordingly, in mice with p53 deleted specifically in the GI epithelium, ionizing radiation fails to induce fetal-like revSCs. Using intestinal organoids, we show that transient p53 expression is required for the induction of revival stem cells and is controlled by an Mdm2-mediated negative feedback loop. Together, our findings reveal that p53 suppresses severe radiation-induced GI injury by promoting fetal-like reprogramming of irradiated intestinal epithelial cells. The tumor suppressor p53 is the guardian of the genome. Here, the authors use comprehensive approaches to demonstrate that transient p53 activity induces revival stem cells to promote the regeneration of severely irradiated intestinal epithelium in mice.

Aberrant vascularization is a hallmark of cancer progression and treatment resistance. Here, we have shown that endothelial cell (EC) plasticity drives aberrant vascularization and chemoresistance in glioblastoma multiforme (GBM). By utilizing human patient specimens, as well as allograft and genetic murine GBM models, we revealed that a robust endothelial plasticity in GBM allows acquisition of fibroblast transformation (also known as endothelial mesenchymal transition [Endo-MT]), which is characterized by EC expression of fibroblast markers, and determined that a prominent population of GBM-associated fibroblast-like cells have EC origin. Tumor ECs acquired the mesenchymal gene signature without the loss of EC functions, leading to enhanced cell proliferation and migration, as well as vessel permeability. Furthermore, we identified a c-Met/ETS-1/matrix metalloproteinase-14 (MMP-14) axis that controls VE-cadherin degradation, Endo-MT, and vascular abnormality. Pharmacological c-Met inhibition induced vessel normalization in patient tumor-derived ECs. Finally, EC-specific KO of Met inhibited vascular transformation, normalized blood vessels, and reduced intratumoral hypoxia, culminating in suppressed tumor growth and prolonged survival in GBM-bearing mice after temozolomide treatment. Together, these findings illustrate a mechanism that controls aberrant tumor vascularization and suggest that targeting Endo-MT may offer selective and efficient strategies for antivascular and vessel normalization therapies in GBM, and possibly other malignant tumors.

Glioblastoma is a highly heterogeneous disease, with variations observed at both phenotypical and molecular levels. Personalized therapies would be facilitated by non-invasive in vivo approaches for characterizing this heterogeneity. In this study, we developed unsupervised joint machine learning between radiomic and genomic data, thereby identifying distinct glioblastoma subtypes. A retrospective cohort of 571 IDH-wildtype glioblastoma patients were included in the study, and pre-operative multi-parametric MRI scans and targeted next-generation sequencing (NGS) data were collected. L21-norm minimization was used to select a subset of 12 radiomic features from the MRI scans, and 13 key driver genes from the five main signal pathways most affected in glioblastoma were selected from the genomic data. Subtypes were identified using a joint learning approach called Anchor-based Partial Multi-modal Clustering on both radiomic and genomic modalities. Kaplan–Meier analysis identified three distinct glioblastoma subtypes: high-risk, medium-risk, and low-risk, based on overall survival outcome ( p  < 0.05, log-rank test; Hazard Ratio = 1.64, 95% CI 1.17–2.31, Cox proportional hazard model on high-risk and low-risk subtypes). The three subtypes displayed different phenotypical and molecular characteristics in terms of imaging histogram, co-occurrence of genes, and correlation between the two modalities. Our findings demonstrate the synergistic value of integrated radiomic signatures and molecular characteristics for glioblastoma subtyping. Joint learning on both modalities can aid in better understanding the molecular basis of phenotypical signatures of glioblastoma, and provide insights into the biological underpinnings of tumor formation and progression.

The great preclinical promise of the pancreatic endoplasmic reticulum kinase (PERK) inhibitors in neurodegenerative disorders and cancers is marred by pancreatic injury and diabetic syndrome observed in PERK knockout mice and humans lacking PERK function and suffering from Wolcott-Rallison syndrome. PERK mediates many of the unfolded protein response (UPR)-induced events, including degradation of the type 1 interferon (IFN) receptor IFNAR1 in vitro. Here we report that whole-body or pancreas-specificPerkablation in mice leads to an increase in IFNAR1 protein levels and signaling in pancreatic tissues. Concurrent IFNAR1 deletion attenuated the loss of PERK-deficient exocrine and endocrine pancreatic tissues and prevented the development of diabetes. Experiments using pancreas-specific Perk knockouts, bone marrow transplantation, and cultured pancreatic islets demonstrated that stabilization of IFNAR1 and the ensuing increased IFN signaling in pancreatic tissues represents a major driver of injury triggered byPerkloss. Neutralization of IFNAR1 prevented pancreatic toxicity of PERK inhibitor, indicating that blocking the IFN pathway can mitigate human genetic disorders associated withPERKdeficiency and help the clinical use of PERK inhibitors.

Radiation quality and cellular oxygen concentration have a substantial impact on DNA damage, reproductive cell death and, ultimately, the potential efficacy of radiation therapy for the treatment of cancer. To better understand and quantify the effects of radiation quality and oxygen on the induction of clustered DNA lesions, we have now extended the Monte Carlo Damage Simulation (MCDS) to account for reductions in the initial lesion yield arising from enhanced chemical repair of DNA radicals under hypoxic conditions. The kinetic energy range and types of particles considered in the MCDS have also been expanded to include charged particles up to and including 56Fe ions. The induction of individual and clustered DNA lesions for arbitrary mixtures of different types of radiation can now be directly simulated. For low-linear energy transfer (LET) radiations, cells irradiated under normoxic conditions sustain about 2.9 times as many double-strand breaks (DSBs) as cells irradiated under anoxic conditions. New experiments performed by us demonstrate similar trends in the yields of non-DSB (Fpg and Endo III) clusters in HeLa cells irradiated by γ rays under aerobic and hypoxic conditions. The good agreement among measured and predicted DSBs, Fpg and Endo III cluster yields suggests that, for the first time, it may be possible to determine nucleotide-level maps of the multitude of different types of clustered DNA lesions formed in cells under reduced oxygen conditions. As particle LET increases, the MCDS predicts that the ratio of DSBs formed under normoxic to hypoxic conditions by the same type of radiation decreases monotonically toward unity. However, the relative biological effectiveness (RBE) of higher-LET radiations compared to 60Co γ rays (0.24 keV/μm) tends to increase with decreasing oxygen concentration. The predicted RBE of a 1 MeV proton (26.9 keV/μm) relative to 60Co γ rays for DSB induction increases from 1.9 to 2.3 as oxygen concentration decreases from 100% to 0%. For a 12 MeV 12C ion (681 keV/μm), the ‵predicted RBE for DSB induction increases from 3.4 (100% O2) to 9.8 (0% O2). Estimates of linear-quadratic (LQ) cell survival model parameters (α and β) are closely correlated to the Monte Carlo-predicted trends in DSB induction for a wide range of particle types, energies and oxygen concentrations. The analysis suggests α is, as a first approximation, proportional to the initial number of DSBs per cell, and β is proportional to the square of the initial number of DSBs per cell. Although the reported studies provide some evidence supporting the hypothesis that DSBs are a biologically critical form of clustered DNA lesion, the induction of Fpg and Endo III clusters in HeLa cells irradiated by γ rays exhibits similar trends with oxygen concentration. Other types of non-DSB cluster may still play an important role in reproductive cell death. The MCDS captures many of the essential trends in the formation of clustered DNA lesions by ionizing radiation and provides useful information to probe the multiscale effects and interactions of ionizing radiation in cells and tissues. Information from Monte Carlo simulations of cluster induction may also prove useful for efforts to better exploit radiation quality and reduce the impact of tumor hypoxia in proton and carbon-ion radiation therapy.

Cells adjust to hypoxic stress within the tumor microenvironment by downregulating energy-consuming processes including translation. To delineate mechanisms of cellular adaptation to hypoxia, we performed RNA-Seq of normoxic and hypoxic head and neck cancer cells. These data revealed a significant down regulation of genes known to regulate RNA processing and splicing. Exon-level analyses classified > 1,000 mRNAs as alternatively spliced under hypoxia and uncovered a unique retained intron (RI) in the master regulator of translation initiation, EIF2B5. Notably, this intron was expressed in solid tumors in a stage-dependent manner. We investigated the biological consequence of this RI and demonstrate that its inclusion creates a premature termination codon (PTC), that leads to a 65kDa truncated protein isoform that opposes full-length eIF2Bε to inhibit global translation. Furthermore, expression of 65kDa eIF2Bε led to increased survival of head and neck cancer cells under hypoxia, providing evidence that this isoform enables cells to adapt to conditions of low oxygen. Additional work to uncover -cis and -trans regulators of EIF2B5 splicing identified several factors that influence intron retention in EIF2B5: a weak splicing potential at the RI, hypoxia-induced expression and binding of the splicing factor SRSF3, and increased binding of total and phospho-Ser2 RNA polymerase II specifically at the intron retained under hypoxia. Altogether, these data reveal differential splicing as a previously uncharacterized mode of translational control under hypoxia and are supported by a model in which hypoxia-induced changes to cotranscriptional processing lead to selective retention of a PTC-containing intron in EIF2B5.

Background Recent advances in radiation biology and preclinical research have identified that high doses of radiation at ultra-high dose rate can lead to sparing of normal tissue, while maintaining tumor control. This has been termed the FLASH effect and has been extended from electrons to protons, heavy ions and photons. Lung cancer treatments, despite the advancements in radiotherapy with precise protons, are still associated with significant damage to the normal tissue. FLASH proton exposures have not been characterized yet with regards to lung tissue injury or sparing. Methods In this pilot study we used precision cut lung slices from human healthy non-smoker donors, two male and one female, and exposed this ex vivo model to a single dose of 12 Gy of standard or FLASH protons and analyzed them with untargeted metabolomics at 24 h after exposure. Results We identified 22 metabolites of interest, with primary enrichment in β-alanine metabolism, sphingolipid metabolism, primary bile acid biosynthesis, and one carbon pool by folate, while chemical classes showed that sphingoid bases and eicosanoids were two of the most enriched chemical classes after radiation exposure. Classification analysis with receiver operating characteristic curves based on the 22 metabolites indicated the presence of individual variability to responses, that warrant future studies with a larger cohort. Conclusions Although this is a pilot study, we show the utility of ex vivo models in radiation research and particularly with standard and FLASH protons, and that further research in this area should include both male and female subjects. This ex vivo human model should therefore be investigated further to identify early responses to proton exposures.

Tumor cell adaptation to hypoxic stress is an important determinant of malignant progression. While much emphasis has been placed on the role of HIF‐1 in this context, the role of additional mechanisms has not been adequately explored. Here we demonstrate that cells cultured under hypoxic/anoxic conditions and transformed cells in hypoxic areas of tumors activate a translational control program known as the integrated stress response (ISR), which adapts cells to endoplasmic reticulum (ER) stress. Inactivation of ISR signaling by mutations in the ER kinase PERK and the translation initiation factor eIF2α or by a dominant‐negative PERK impairs cell survival under extreme hypoxia. Tumors derived from these mutant cell lines are smaller and exhibit higher levels of apoptosis in hypoxic areas compared to tumors with an intact ISR. Moreover, expression of the ISR targets ATF4 and CHOP was noted in hypoxic areas of human tumor biopsy samples. Collectively, these findings demonstrate that activation of the ISR is required for tumor cell adaptation to hypoxia, and suggest that this pathway is an attractive target for antitumor modalities.