Molykutty J. Aryankalayil

DNA exonuclease Trex1 regulates radiotherapy-induced tumour immunogenicity

Radiotherapy is under investigation for its ability to enhance responses to immunotherapy. However, the mechanisms by which radiation induces anti-tumour T cells remain unclear. We show that the DNA exonuclease Trex1 is induced by radiation doses above 12–18 Gy in different cancer cells, and attenuates their immunogenicity by degrading DNA that accumulates in the cytosol upon radiation. Cytosolic DNA stimulates secretion of interferon-β by cancer cells following activation of the DNA sensor cGAS and its downstream effector STING. Repeated irradiation at doses that do not induce Trex1 amplifies interferon-β production, resulting in recruitment and activation of Batf3-dependent dendritic cells. This effect is essential for priming of CD8+ T cells that mediate systemic tumour rejection (abscopal effect) in the context of immune checkpoint blockade. Thus, Trex1 is an upstream regulator of radiation-driven anti-tumour immunity. Trex1 induction may guide the selection of radiation dose and fractionation in patients treated with immunotherapy.

Defining molecular signature of pro-immunogenic radiotherapy targets in human prostate cancer cells.

To understand the impact of clinically relevant radiation therapy (RT) on tumor immune gene expression and to utilize the changes that occur during treatment to improve cancer treatment outcome, we examined how immune response genes are modulated in prostate cancer cells of varying p53 status. LNCaP (p53 wild-type), PC3 (p53 null) and DU145 (p53 mutant) cells received a 10 Gy single dose or 1 Gy × 10 multifractionated radiation dose to simulate hypofractionated and conventionally fractionated prostate radiotherapy. Total RNA was isolated 24 h after multifractionated radiation treatment and single-dose treatments and subjected to microarray analysis and later validated by RT-PCR. RT-PCR was utilized to identify total-dose inflection points for significantly upregulated genes in response to multifractionated radiation therapy. Radiation-induced damage-associated molecular pattern molecules (DAMPs) and cytokine analyses were performed using bioluminescence and ELISA. Multifractionated doses activated immune response genes more robustly than single-dose treatment, with a relatively larger number of immune genes upregulated in PC3 compared to DU145 and LNCaP cells. The inflection point of multifractionated radiation-induced immune genes in PC3 cells was observed in the range of 8–10 Gy total radiation dose. Although both multifractionated and single-dose radiation-induced proinflammatory DAMPs and positively modulated the cytokine environment, the changes were of higher magnitude with multifractionated therapy. The findings of this study together with the gene expression data suggest that cells subjected to multifractionated radiation treatment would promote productive immune cell–tumor cell interactions.

Exploiting radiation-induced signaling to increase the susceptibility of resistant cancer cells to targeted drugs: AKT and mTOR inhibitors as an example

Implementing targeted drug therapy in radio-oncologic treatment regimens has greatly improved the outcome of cancer patients. However, the efficacy of molecular targeted drugs such as inhibitory antibodies or small molecule inhibitors essentially depends on target expression and activity, which both can change during the course of treatment. Radiotherapy has previously been shown to activate prosurvival pathways, which can help tumor cells to adapt and thereby survive treatment. Therefore, we aimed to identify changes in signaling induced by radiation and evaluate the potential of targeting these changes with small molecules to increase the therapeutic efficacy on cancer cell survival. Analysis of “The Cancer Genome Atlas” database disclosed a significant overexpression of AKT1, AKT2, and MTOR genes in human prostate cancer samples compared with normal prostate gland tissue. Multifractionated radiation of three-dimensional–cultured prostate cancer cell lines with a dose of 2 Gy/day as a clinically relevant schedule resulted in an increased protein phosphorylation and enhanced protein–protein interaction between AKT and mTOR, whereas gene expression of AKT, MTOR, and related kinases was not altered by radiation. Similar results were found in a xenograft model of prostate cancer. Pharmacologic inhibition of mTOR/AKT signaling after activation by multifractionated radiation was more effective than treatment prior to radiotherapy. Taken together, our findings provide a proof-of-concept that targeting signaling molecules after activation by radiotherapy may be a novel and promising treatment strategy for cancers treated with multifractionated radiation regimens such as prostate cancer to increase the sensitivity of tumor cells to molecular targeted drugs. Mol Cancer Ther; 17(2); 355–67. ©2017 AACR. See all articles in this MCT Focus section, “Developmental Therapeutics in Radiation Oncology.”

Radiation-Induced Long Noncoding RNAs in a Mouse Model after Whole-Body Irradiation

Long noncoding RNAs (lncRNAs) are emerging as key molecules in regulating many biological processes and have been implicated in development and disease pathogenesis. Biomarkers of cancer and normal tissue response to treatment are of great interest in precision medicine, as well as in public health and medical management, such as for assessment of radiation injury after an accidental or intentional exposure. Circulating and functional RNAs, including microRNAs (miRNAs) and lncRNAs, in whole blood and other body fluids are potential valuable candidates as biomarkers. Early prediction of possible acute, intermediate and delayed effects of radiation exposure enables timely therapeutic interventions. To address whether long noncoding RNAs (lncRNAs) could serve as biomarkers for radiation biodosimetry we performed whole genome transcriptome analysis in a mouse model after whole-body irradiation. Differential lncRNA expression patterns were evaluated at 16, 24 and 48 h postirradiation in total RNA isolated from whole blood of mice exposed to 1, 2, 4, 8 and 12 Gy of X rays. Sham-irradiated animals served as controls. Significant alterations in the expression patterns of lncRNAs were observed after different radiation doses at the various time points. We identified several radiation-induced lncRNAs known for DNA damage response as well as immune response. Long noncoding RNA targets of tumor protein 53 (P53), Trp53cor1, Dino, Pvt1 and Tug1 and an upstream regulator of p53, Meg3, were altered in response to radiation. Gm14005 (Morrbid) and Tmevpg1 were regulated by radiation across all time points and doses. These two lncRNAs have important potential as blood-based radiation biomarkers; Gm14005 (Morrbid) has recently been shown to play a key role in inflammatory response, while Tmevpg1 has been implicated in the regulation of interferon gamma. Precise molecular biomarkers, likely involving a diverse group of inducible molecules, will not only enable the development and effective use of medical countermeasures but may also be used to detect and circumvent or mitigate normal tissue injury in cancer radiotherapy.

Long-term Tumor Adaptation after Radiotherapy: Therapeutic Implications for Targeting Integrins in Prostate Cancer

Adaptation of tumor cells to radiotherapy induces changes that are actionable by molecular targeted agents and immunotherapy. This report demonstrates that radiation-induced changes in integrin expression can be targeted 2 months later. Integrins are transmembrane cell adhesion molecules that are essential for cancer cell survival and proliferation. To analyze the short- and long-term effects of radiation on the integrin expression, prostate cancer cells (DU145, PC3, and LNCaP) were cultured in a 3D extracellular matrix and irradiated with either a single dose of radiation (2–10 Gy) or a multifractionated regimen (2–10 fractions of 1 Gy). Whole human genome microarrays, immunoblotting, immunoprecipitation assays, and immunofluorescence staining of integrins were performed. The results were confirmed in a prostate cancer xenograft model system. Interestingly, β1 and β4 integrins (ITGB1 and ITGB4) were upregulated after radiation in vitro and in vivo. This overexpression lasted for more than 2 months and was dose dependent. Moreover, radiation-induced upregulation of β1 and β4 integrin resulted in significantly increased tumor cell death after treatment with inhibitory antibodies. Combined, these findings indicate that long-term tumor adaptation to radiation can result in an increased susceptibility of surviving cancer cells to molecular targeted therapy due to a radiation-induced overexpression of the target. Implications: Radiation induces dose- and schedule-dependent adaptive changes that are targetable for an extended time; thus suggesting radiotherapy as a unique strategy to orchestrate molecular processes, thereby providing new radiation-drug treatment options within precision cancer medicine.

Microarray analysis of miRNA expression profiles following whole body irradiation in a mouse model

Abstract Context: Accidental exposure to life-threatening radiation in a nuclear event is a major concern; there is an enormous need for identifying biomarkers for radiation biodosimetry to triage populations and treat critically exposed individuals. Objective: To identify dose-differentiating miRNA signatures from whole blood samples of whole body irradiated mice. Methods: Mice were whole body irradiated with X-rays (2 Gy–15 Gy); blood was collected at various time-points post-exposure; total RNA was isolated; miRNA microarrays were performed; miRNAs differentially expressed in irradiated vs. unirradiated controls were identified; feature extraction and classification models were applied to predict dose-differentiating miRNA signature. Results: We observed a time and dose responsive alteration in the expression levels of miRNAs. Maximum number of miRNAs were altered at 24-h and 48-h time-points post-irradiation. A 23-miRNA signature was identified using feature selection algorithms and classifier models. An inverse correlation in the expression level changes of miR-17 members, and their targets were observed in whole body irradiated mice and non-human primates. Conclusion: Whole blood-based miRNA expression signatures might be used for predicting radiation exposures in a mass casualty nuclear incident.

Abstract C146: Radiation-inducible molecular targets in a human prostate cancer mouse model

Background: In order to understand the changes induced in tumor cells following multi-fraction (MF) radiation therapy, we have previously studied molecular changes using prostate cancer cells and endothelial cells treated in vitro with MF doses of 0.5 Gy/1 Gy x 10 and 2 Gy x 5 and single-dose (SD) of 5 Gy and 10 Gy. The hypothesis being tested is that the response and adaptation to radiation-induced stress will produce a druggable phenotype. This might increase the utility of molecularly targeted therapeutics and also help address tumor cell heterogeneity. The data indicate more genes and pathways are induced by MF compared to SD and that the change in phenotype is more stable following MF. In this report, the focus is on new data from PC-3 cells irradiated in vivo and comparing it to MF and SD in vitro using MF 1 Gy x 10 and SD 10 Gy. Methods: PC-3 prostate cancer cells were implanted subcutaneously into the lateral aspect of rear leg of nude mice. Mice were divided into three groups (n = 3), based on radiation dose/schedule- control, SD, and MF. SD and MF employed similar dose/schedule as used for the in vitro studies, 10 Gy x 1 and 1 Gy x 10 respectively. RNA was isolated 24 h after radiation treatment. mRNA microarray analysis was performed using Agilent Technologies Human Gene Expression 4 × 44 K V2 microarrays. The data was generated and analyzed with GeneSpring® software (Agilent Technologies, Santa Clara, CA) and IPA software (IPA, QIAGEN, Redwood City, CA). Results: 6,374 genes were significantly altered by MF, with a cohort of genes, based on the > 250 gene ontology categories, involved in DNA response to stimulus, DNA repair, mitosis, cell cycle, and metabolism. In contrast, only 453 genes were significantly altered by SD, with ontological categories associated with cell morphology, assembly and organization such as actin filament-based process, extracellular matrix organization and biogenesis, fibril organization and biogenesis and collagen catabolism. Further bioinformatics analysis of the gene expression data with IPA, identified multiple pathways with functions correlated with the ontological categories. Significantly altered MF-induced genes are members of pathways which play a central role in DNA replication, recombination, and repair, cell proliferation and metabolism such as HIPPO Signaling, Protein Ubiquitination Pathway, JNK/SAPK Signaling, ERK/MAPK Signaling, G2/M DNA Damage Checkpoint Regulation, ATM Signaling, PI3K/AKT Signaling and Oxidative Phosphorylation. These pathways were uniquely up-regulated by MF treatment, as none of these changes were identified with SD radiation exposure. Conclusion: Our result show the differential expression pattern between SD and MF, with MF inducing changes in “targetable” molecular pathways. Ongoing studies: Currently we are in the process of evaluating radiation-induced targets in ATM signaling, DNA damage and repair, and multiple metabolic targets, and their potential for using radiation to prime cells for molecular-targeted drug therapy. Citation Format: Adeola Y. Makinde, Iris Eke, Molykutty J. Aryankalayil, Mansoor Ahmed, C. Norman Coleman. Radiation-inducible molecular targets in a human prostate cancer mouse model. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C146.

Abstract 3046: Differential microRNA patterns in prostate carcinoma cells after single and fractionated radiation

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Our previous mRNA microarray analysis showed that fractionated radiation induced more differentially expressed genes compared to single dose radiation in PC3 and DU145 cells. MicroRNAs (miRNAs) are an important class of non-coding small RNAs capable of regulating gene expression at the translational level. To understand the role of miRNAs in regulation of radiation-induced gene expression patterns, we studied the expression levels of miRNAs by microarray analysis in prostate cancer cells. Methods: PC3 and DU145 cells were exposed to 5 Gy and 10Gy either as a single dose radiation or multi-fractionated (0.5Gyx10 and 1Gyx10) radiation. RNA was extracted at 24h after the final dose of radiation and miRNA microarray analysis was done using Agilent human miRNA Microarray Kit (V2). The chip contains probes for 723 human and 76 human viral microRNAs from the Sanger database v.10.1. Data were analyzed using GeneSpring software (Agilent technologies). Results: Of the total 723 miRNAs represented in the array, 141 miRNAs were differentially expressed (> 1.5 fold change) by the 4 radiation protocols. Significant difference in the miRNA expression pattern was noted between the two cell lines. The number of radiation - induced miRNAs was higher in PC3 cells than in DU145 cells. In PC3 cells fractionated radiation resulted in more down regulated miRNAs compared to single dose radiation. In our previous study immune response genes were significantly up regulated by fractionated radiation in PC3 cells but not in DU145 cells. In the present study Mir-146a was significantly up regulated in DU145 cells after fractionated radiation but not in PC3 cells suggesting an inverse correlation between the differential expression of mir-146a and immune response genes in both cell lines. This study also demonstrated significant differences in let-7 family of miRNAs in the two cell lines and also between single and fractionated radiation. In PC3 cells, 8 of the let-7 miRNAs were upregulated after fractionated radiation whereas in DU145 cells they were either downregulated or not changed. Conclusion: Fractionated radiation resulted in expression of more number of miRNAs compared to single dose radiation. We are currently in the process of evaluating radiation-induced differential gene expression changes by a combined approach of mRNA, miRNA and protein array analysis to identify radiation induced molecular targets for cancer therapy. This work was supported by the Intramural Research Program of Center for Cancer Research, National Cancer Institute, National Institutes of Health. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3046.