Membrane-bound enzyme relocates to the cell nucleus to modify chromatin, inducing cancer resistance to radiotherapy.

DNA repair pathways are aberrant in cancer, enabling tumor cells to survive standard therapies-chemotherapy and radiotherapy. Our group previously reported that, upon irradiation, the membrane-bound tyrosine kinase receptor TIE2 translocates into the nucleus and phosphorylates histone H4 at Tyr51, recruiting ABL1 to the DNA repair complexes that participate in the nonhomologous end-joining pathway. However, no specific molecular mechanisms of TIE2 endocytosis have been reported. Here, we show that irradiation or ligand-induced TIE2 trafficking is dependent on caveolin-1, the main component of caveolae. Subcellular fractionation and confocal microscopy demonstrated TIE2/caveolin-1 complexes in the nucleus, and using inhibitor or small interfering RNAs (siRNAs) against caveolin-1 or Tie2 inhibited their trafficking. TIE2 was found in caveolae and directly phosphorylated caveolin-1 at Tyr14 and This modification regulated the generation of TIE2/caveolin-1 complexes and was essential for TIE2/caveolin-1 nuclear translocation. Our data further demonstrate that the combination of TIE2 and caveolin-1 inhibitors resulted in significant radiosensitization of malignant glioma cells, which will guide the development of combinatorial treatment with radiotherapy for patients with glioblastoma.

The Random Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) was applied to analyze the genetic variation of the Hilsa shad, Tenualosa ilisha Ham., from the two major inland rivers (Padma and Meghna) in Bangladesh. Twenty-eight random 10-mer primers were primarily scored in 8 individuals from each of the two locations. Fifteen primers, which gave polymorphism, were selected and used in the final analysis of 34 individuals from the two sites. Using these primers, 480 scorable DNA fragments were found, of which 98 (20.41%) were polymorphic. By comparing the RAPD banding patterns, variations were found between and within the populations. A dendrogram was constructed with the polymorphic fragments to analyze the genetic distances between the Hilsa shad populations. The results show two major clusters of Padma and Meghna, assuming different spawning populations with different stocks or races of Hilsa shad in the major Bangladesh rivers.

Glioblastoma is the most common and aggressive primary brain tumor in adults with a median survival of 9-15 months. This tumor invariably recurs after therapy, which is largely caused by the striking radioresistance. In gliomas, we found the tyrosine kinase receptor TIE2 is overexpressed in brain tumor stem cells (BTSCs) and in human surgical glioma specimens in relation to malignancy. Here we find that ionizing irradiation (IR) treatment of mice bearing intracranial BTSCs-derived xenografts resulted in an unexpected TIE2 nuclear localization and increased level of its natural ligand, ANG1. These data was corroborated in BTSCs and glioma cultures upon IR or Ang1 stimulus, using immunofluorescence and confocal analysis, and subcellular fractionation followed by Western blot. Of clinical interest, nuclear TIE2 is associated with radioresistance of glioma cells, which was reverted by using Tie2 soluble receptor (which inhibits ANG1/TIE2 interaction) and after genetic Tie2 mutation of a newly discovered nuclear localization signal. At the molecular level, we also found that after IR treatment, TIE2 localized in the DNA-repair foci and complexed with gH2AX, suggesting a role of TIE2 in the DNA repair machinery. To validate our hypothesis, we used a fluorescent reporter construct in which GFP gene was reconstituted following a nonhomologous end joining (NHEJ) event, and we observed that TIE2-expressing cells showed more efficient NHEJ repair than non-expressing TIE2 cells. Based on the recently reported role of ABL1 (cAbl) in the ATM and KAT5 mediated DNA damage repair, we explored the relationship between ABL1 and the TIE2-mediated radioresistance. Our data clearly showed that DNA repair efficiency significantly and specifically decreased by using ABL1 inhibitors or ABL1 siRNAs, but not after ABL2 inhibition. Collectively, our results should propel the development of preclinical studies on the combination of nuclear TIE2-targeting strategies and inhibition of ABL1 with radiotherapy for patients with glioblastomas.

Our approach involves a platform of killing cancer using more potent oncolytic viruses-based immunotherapy strategies. These replication competent adenoviruses are targeted to the Rb pathway to generate tumor-selectivity. The second generation of these therapeutic agents, Delta-24-RGD, was successfully translated to the clinical setting and is currently been tested in Phase I studies in several institutions in the USA and in Europe for the treatment of patients suffering from recurrent gliomas. Preliminary data from these clinical trials showed that 10 to 15% of Delta-24-RGD-treated tumors undergo complete regression. Agonistic treatments targeting co-stimulatory tumor necrosis factor receptor superfamily (TNFRSF), such as GITR (CD357), have been shown to enhance the proliferation and activation of T cells. Moreover, in preclinical tumor efficacy studies, these agonistic signals have shown potent tumoricidal activity. Different from antibodies, co-stimulatory ligands can be easily incorporated into replication competent oncolytic adenoviruses. Infection of cancer cells with these armed viruses will lead to the expression in their cell membranes of the co-stimulatory molecule that will directly interact with the tumor infiltrating lymphocytes to amplify and enhance the anti-tumor T-cell activity. In this study, we have developed an armed Delta-24-RGD carrying the cDNA of the mouse GITRL, Delta-24-GREAT. Treatment of glioma-bearing mice with intracranial injection of Delta-24-GREAT increased mice survival (P < 0.0001, long-rank test) and inhibited lung cancer growth in subcutaneous models. Infection of the tumor elicited an inflammatory response increasing the populations of CD4+ and CD8+ T cells versus treated controls. Importantly, 2 weeks after the adenovirus treatment a subset of brain hemispheric cells were positive for GITRL. In addition, co-culture experiments with tumor cells infected with viruses and splenocytes isolated from treated glioma-bearing mice demonstrated a response against the cancer cells as assessed by ELISA analyses of IFN-γ. Demonstrating the generation of anti-tumor memory, the surviving animals did not show evidence of tumor growth after re-challenging with GL261 glioma cell implantation in the contralateral hemisphere. However, survivors of GL261 tumors did not survive after the re-challenge was performed by intracranial implantation of B16/F10 melanoma cells, strongly indicating that the immune response was specific for GL261 glioma antigens. This is the first study with an oncolytic adenovirus expressing GITRL and our results strongly indicate that oncolytic adenoviruses armed with molecules of the TNFRSF may be of future clinical interest for the treatment of patients with cancer. Citation Format: Yisel A. Rivera-Molina, Francisco Puerta Martinez, Teresa Nguyen, Hong Jiang, Xuejun Fan, Rehnuma Shifat, Mohammad Belayat Hossain, Verlene K. Henry, Caroline C. Carrillo, Candelaria Gomez-Manzano, Juan Fueyo. Forced expression of GITRL in cancer cells enhances adenovirus-mediated in situ vaccination [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 4565. doi:10.1158/1538-7445.AM2017-4565