Intratumoral IRIF imaging demonstrates similar patterns for GFP-IBD (green) and TagRFP-IBD (red) foci in tumor at 24 h after irradiation with 6 Gy

Intratumoral IRIF imaging demonstrates similar patterns for GFP-IBD (green) and TagRFP-IBD (red) fluorescence in tumor at 3 h after irradiation with 6 Gy.Control tumor

Three-dimensional imaging of irradiated MCF7 tumor xenografts with dual fluorescent reporters for IRIF (TagRFP, red) and shRNAmir expression (ZsGreen, green)

Time-lapse live cell imaging of caspase-3/7 activation in irradiated Panc02 cells treated with PUGNAc or alloxan

Supplementary Methods and Supplementary Figure, Movie and Table Legends

intratumoral imaging of Hoechst 33342 stained nuclei (blue), GFP-IBD (green) and TagRFP-IBD (red) reporters in untreated control tumor xenograft.

S1. Validation of TagRFP-IBD as IRIF reporter in vitro. S2. Effects of radiation on MCF7 cells with altered O-GlcNAcylation. S3. Targeting O-GlcNAc modification by small molecule inhibitors affects kinetics of IRIF resolution in different cell lines. S4. Cellular apoptosis assays using fluorescent dyes and a caspase substrate probe. S5. Alloxan potentiates senescence in Panc02 cells in a radiation dose-dependent manner. S6. Blocking O-GlcNAcylation in vivo by inducing shOGT or alloxan treatment qualitatively reduces EZH2 immunoreactivity after irradiation.

Heatmap and Reactome pathway proteomic data

O-GlcNAc proteomic hits of significance

To augment HIV-1 pox-protein vaccine immunogenicity using a next generation adjuvant, a prime-boost strategy of recombinant modified vaccinia virus Ankara and multimeric Env gp145 was evaluated in macaques with either aluminum (alum) or a novel liposomal monophosphoryl lipid A (MPLA) formulation adsorbed to alum, ALFA. Binding antibody responses were robust and comparable between arms, while antibody-dependent neutrophil and monocyte phagocytotic responses were greatly enhanced by ALFA. Per-exposure vaccine efficacy against heterologous tier 2 SHIV mucosal challenge was 90% in ALFA-adjuvanted males (P = 0.002), while alum conferred no protection. Half of the ALFA-adjuvanted males remained uninfected after the full challenge series, which spanned seven months after the last vaccination. Antibody-dependent monocyte and neutrophil phagocytic responses both strongly correlated with protection. Significant sex differences in infection risk were observed, with much lower infection rates in females than males. In humans, MPLA-liposome-alum adjuvanted gp120 also increased HIV-1-specific phagocytic responses relative to alum. Thus, next-generation liposome-based adjuvants can drive vaccine elicited antibody effector activity towards potent phagocytic responses in both macaques and humans and these responses correlate with protection. Future protein vaccination strategies aiming to improve functional humoral responses may benefit from such adjuvants.

Abstract The metabolic reprogramming associated with characteristic increases in glucose and glutamine metabolism in advanced cancer is often ascribed to answering a higher demand for metabolic intermediates required for rapid tumor cell growth. Instead, recent discoveries have pointed to an alternative role for glucose and glutamine metabolites as cofactors for chromatin modifiers and other protein posttranslational modification enzymes in cancer cells. Beyond epigenetic mechanisms regulating gene expression, many chromatin modifiers also modulate DNA repair, raising the question whether cancer metabolic reprogramming may mediate resistance to genotoxic therapy and genomic instability. Our prior work had implicated N-acetyl-glucosamine (GlcNAc) formation by the hexosamine biosynthetic pathway (HBP) and resulting protein O-GlcNAcylation as a common means by which increased glucose and glutamine metabolism can drive double-strand break (DSB) repair and resistance to therapy-induced senescence in cancer cells. We have examined the effects of modulating O-GlcNAcylation on the DNA damage response (DDR) in MCF7 human mammary carcinoma in vitro and in xenograft tumors. Proteomic profiling revealed deregulated DDR pathways in cells with altered O-GlcNAcylation. Promoting protein O-GlcNAc modification by targeting O-GlcNAcase or simply treating animals with GlcNAc protected tumor xenografts against radiation. In turn, suppressing protein O-GlcNAcylation by blocking O-GlcNAc transferase activity led to delayed DSB repair, reduced cell proliferation, and increased cell senescence in vivo. Taken together, these findings confirm critical connections between cancer metabolic reprogramming, DDR, and senescence and provide a rationale to evaluate agents targeting O-GlcNAcylation in patients as a means to restore tumor sensitivity to radiotherapy. Implications: The finding that the HBP, via its impact on protein O-GlcNAcylation, is a key determinant of the DDR in cancer provides a mechanistic link between metabolic reprogramming, genomic instability, and therapeutic response and suggests novel therapeutic approaches for tumor radiosensitization.

Radioresistance is a major hurdle in the treatment of head and neck squamous cell carcinoma (HNSCC). Here, we report that concomitant treatment of HNSCCs with radiotherapy and mevalonate pathway inhibitors (statins) may overcome resistance. Proteomic profiling and comparison of radioresistant to radiosensitive HNSCCs revealed differential regulation of the mevalonate biosynthetic pathway. Consistent with this finding, inhibition of the mevalonate pathway by pitavastatin sensitized radioresistant SQ20B cells to ionizing radiation and reduced their clonogenic potential. Overall, this study reinforces the view that the mevalonate pathway is a promising therapeutic target in radioresistant HNSCCs.

Abstract The elevation of glucose and glutamine metabolism characteristic of cancer has been ascribed to increased demand for metabolic intermediates that provide building blocks for rapid cell growth. However, recent discoveries have highlighted complementary effects mediated by metabolites serving as cofactors for chromatin modifying enzymes and providing metabolic intermediates for proteins posttranslational modification. Along with epigenetic regulation of gene expression, multiple chromatin modifiers modulate DNA damage repair, raising the question of whether metabolic reprogramming may thereby promote genomic instability and cell immortality. In our prior work, we uncovered a role for the glycolysis hexosamine biosynthetic pathway (HBP) in DNA repair, ionizing radiation induced foci (IRIF) resolution, and prevention of radiation induced cancer cell senescence. Here we report that the targeting of key enzymes in the HBP pathway, O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) or O-GlcNAcase (OGA/ MGEA5), have contrary effects on the DNA damage response in tumors. shRNA knockdown of OGA resulted in increased GlcNAc protein modification, protection of cancer cells from irradiation through improved double strand break (DSB) repair and IRIF resolution in tumor cells, increased proliferation, and prevented radiation-induced senescence. In turn, silencing of OGT diminished O-GlcNAc modification, prevented DSB repair, increased IRIF persistence, reduced proliferation, and promoted radiation-induced senescence in tumor xenografts. These findings reveal an important relationship between cancer cell metabolism, DSB repair, and senescence and provide a rationale for targeting metabolic reprogramming, specifically O-GlcNAc modification, to block cancer cell immortality and overcome resistance to genotoxic stress. Citation Format: Elena V. Efimova, Oliver K. Appelbe, Steve Seung-Young Lee, Natalia Ricco, Aishwarya Ramamarthy, Nicolas Rymut, Tamica Collins, Donald J. Wolfgeher, Sara Warrington, Stephen J. Kron. Tumor radiosensitization by suppression of hexosamine biosynthetic pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3505.

Along with vaccines and checkpoint blockade, immune adjuvants may have an important role in tumor immunotherapy. Oligodeoxynucleotides containing unmethylated cytidyl guanosyl dinucleotide motifs (CpG ODN) are TLR9 ligands with attractive immunostimulatory properties, but intratumoral administration has been required to induce an effective anti-tumor immune response. Following on recent studies with radiation-targeted delivery of nanoparticles, we examined enhanced tumor-specific delivery of amphiphile-CpG, an albumin-binding analog of CpG ODN, following systemic administration 3days after tumor irradiation. The combination of radiation and CpG displayed superior tumor control over either treatment alone. Intravital imaging of fluorescently labeled amphiphilic-CpG revealed increased accumulation in irradiated tumors along with decreased off-target accumulation in visceral organs. Within 48h after amphiphile-CpG administration, immune activation could be detected by increased Granzyme B and Interferon gamma activity in the tumor as well as in circulating monocytes and activated CD8+ T cells. Using radiotherapy to enhance the targeting of CpG to tumors may help advance this once promising therapy to clinical relevance.

The excitement surrounding the potential of gene therapy has been tempered due to the challenges that have thus far limited its successful implementation in the clinic such as issues regarding stability, transfection efficiency, and toxicity. In this study, low molecular weight linear polyethyleneimine (2.5 kDa) was modified by conjugation to a lipid, lithocholic acid, and complexed with a natural polysaccharide, dermatan sulfate (DS), to mask extra cationic charges of the modified polymer. In vitro examination revealed that these modifications improved complex stability with plasmid DNA (pDNA) and transfection efficiency. This novel ternary polyplex (pDNA/3E/DS) was used to investigate if tumor-targeted radiotherapy led to enhanced accumulation and retention of gene therapy vectors in vivo in tumor-bearing mice. Imaging of biodistribution revealed that tumor irradiation led to increased accumulation and retention as well as decreased off-target tissue buildup of pDNA in not only pDNA/3E/DS, but also in associated PEI-based polyplexes and commercial DNA delivery vehicles. The DS-containing complexes developed in this study displayed the greatest increase in tumor-specific pDNA delivery. These findings demonstrate a step forward in nucleic acid vehicle design as well as a promising approach to overall cancer gene therapy through utilization of radiotherapy as a tool for enhanced delivery.

Current strategies to target tumors with nanomedicines rely on passive delivery via the enhanced permeability and retention effect, leveraging the disorganized tumor microvasculature to promote macromolecule extravasation and the reduced lymphatic and venous drainage that favor retention. Nonetheless, FDA approvals and clinical use of nanomedicines have lagged, reflecting failure to display superiority over conventional formulations. Here, we have exploited image-guided X-irradiation to augment nanoparticle accumulation in tumors. A single 5 Gy dose of radiation, below that required to significantly delay tumor growth, can markedly enhance delivery of macromolecules and nanoparticles. The radiation effect was independent of endothelial cell integrity, suggesting a primary role for damage to microvascular pericytes and/or interstitial extracellular matrix. Significantly, radiation-guided delivery potentiated the therapeutic effects of PEGylated liposomal doxorubicin on experimental tumors. Applied to patients, these results suggest repurposing image-guided radiotherapy as a tool to guide cancer nanomedicine delivery, enhancing local control for primary tumors and metastatic disease while limiting systemic toxicity.

Presented are a set of procedures to produce water-soluble AgInS2/ZnS near-infrared emitting quantum dots for use as biological imaging agents. The known difficulty of producing near-infrared core/shell materials is resolved by overcoating the AgInS2 cores at a low temperature using highly reactive precursors. Several methods are explored to impart water solubility of the hydrophobic as-prepared materials. Insofar as achieving aqueous dispersion of quantum dots has only limited biological utility, several methods to further functionalize them are examined. In vivo studies are conducted using these quantum dots to demonstrate the ability to model delivery of nanoparticles to the tumour microenvironment.

Numerous studies have implicated Delta-like 1 (DLK1), a transmembrane protein that shares homology with Notch ligands, in embryonic growth and differentiation. Dlk1 expression is widespread, though not ubiquitous, during early development, but is confined to a few specific cell types in adults. Adult Dlk1-expressing tissues include the Insulin-producing β-cells of the pancreas and the Growth hormone-producing somatotrophs of the pituitary gland. Previously generated Dlk1 null mice (Dlk1(Sul-pat)), display a partially penetrant neonatal lethality and a complex pattern of developmental and adult phenotypes. Here we describe the generation of a conditional Dlk1 mouse line (Dlk1(flox)) to facilitate cell type-specific deletion of the Dlk1 gene, providing a powerful system to explore each aspect of the Dlk1 null phenotype. Four tissue-specific Cre mouse lines were used to produce individual Dlk1 deletions in pancreatic β-cells, pituitary somatotrophs and the endothelial cells of the embryo and placenta, key candidates for the Dlk1 phenotype. Contrary to expectations, all of these conditional mice were fully viable, and none recapitulated any aspect of the Dlk1(Sul-pat) null mice. Dlk1 expression is therefore not essential for the normal development of β-cells, somatotrophs and endothelial cells, and the tissues responsible for the Dlk1 null phenotype remain to be identified. Dlk1(flox) mice will continue to provide an important tool for further research into the function of Dlk1.

Congenital hydrocephalus, the accumulation of excess cerebrospinal fluid (CSF) in the ventricles of the brain, affects one of every 1000 children born today, making it one of the most common human developmental disorders. Genetic causes of hydrocephalus are poorly understood in humans, but animal models suggest a broad genetic program underlying the regulation of CSF balance. In this study, the random integration of a transgene into the mouse genome led to the development of an early onset and rapidly progressive hydrocephalus. Juvenile hydrocephalus transgenic mice (Jhy(lacZ)) inherit communicating hydrocephalus in an autosomal recessive fashion with dilation of the lateral ventricles observed as early as postnatal day 1.5. Ventricular dilation increases in severity over time, becoming fatal at 4-8 weeks of age. The ependymal cilia lining the lateral ventricles are morphologically abnormal and reduced in number in Jhy(lacZ/lacZ) brains, and ultrastructural analysis revealed disorganization of the expected 9+2 microtubule pattern. Rather, the majority of Jhy(lacZ/lacZ) cilia develop axonemes with 9+0 or 8+2 microtubule structures. Disruption of an unstudied gene, 4931429I11Rik (now named Jhy) appears to underlie the hydrocephalus of Jhy(lacZ/lacZ) mice, and the Jhy transcript and protein are decreased in Jhy(lacZ/lacZ) mice. Partial phenotypic rescue was achieved in Jhy(lacZ/lacZ) mice by the introduction of a bacterial artificial chromosome (BAC) carrying 60-70% of the JHY protein coding sequence. Jhy is evolutionarily conserved from humans to basal vertebrates, but the predicted JHY protein lacks identifiable functional domains. Ongoing studies are directed at uncovering the physiological function of JHY and its role in CSF homeostasis.