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The 5-year programme award will support research on the DNA repair mechanisms that protect cancer cells from therapy, informing the development of future therapies.
Phenotypic screen of sixty-eight colorectal cancer cell lines identifies CEACAM6 and CEACAM5 as markers of acid resistance.
Elevated cancer metabolism releases lactic acid and CO2 into the under-perfused tumor microenvironment, resulting in extracellular acidosis. The surviving cancer cells must adapt to this selection pressure; thus, targeting tumor acidosis is a rational therapeutic strategy to manage tumor growth. However, none of the major approved treatments are based explicitly on disrupting acid handling, signaling, or adaptations, possibly because the distinction between acid-sensitive and acid-resistant phenotypes is not clear. Here, we report pH-related phenotypes of sixty-eight colorectal cancer (CRC) cell lines by measuring i) extracellular acidification as a readout of acid production by fermentative metabolism and ii) growth of cell biomass over a range of extracellular pH (pHe) levels as a measure of the acid sensitivity of proliferation. Based on these measurements, CRC cell lines were grouped along two dimensions as "acid-sensitive"/"acid-resistant" versus "low metabolic acid production"/"high metabolic acid production." Strikingly, acid resistance was associated with the expression of CEACAM6 and CEACAM5 genes coding for two related cell-adhesion molecules, and among pH-regulating genes, of CA12. CEACAM5/6 protein levels were strongly induced by acidity, with a further induction under hypoxia in a subset of CRC lines. Lack of CEACAM6 (but not of CEACAM5) reduced cell growth and their ability to differentiate. Finally, CEACAM6 levels were strongly increased in human colorectal cancers from stage II and III patients, compared to matched samples from adjacent normal tissues. Thus, CEACAM6 is a marker of acid-resistant clones in colorectal cancer and a potential motif for targeting therapies to acidic regions within the tumors.
Dosimetric validation of SmART-RAD Monte Carlo modelling for x-ray cabinet radiobiology irradiators
Abstract Objective: Accuracy and reproducibility in the measurement of radiation dose and associated reporting are critically important for the validity of basic and preclinical radiobiological studies performed with kilovolt x-ray radiation cabinets. This is essential not only to enable results of radiobiological studies to be repeated, as well as enable valid comparisons between laboratories. In addition, the commonly used single point dose value hides the 3D dose heterogeneity across the irradiated sample. This is particularly true for preclinical rodent models, and is generally difficult to measure directly. Radiation transport simulations integrated in an easy to use application could help researchers improve quality of dosimetry and reporting. 
Approach: this paper describes the use and dosimetric validation of a newly-developed Monte Carlo (MC) tool, SmART-RAD, to simulate the x-ray field in a range of standard commercial x-ray cabinet irradiators used for preclinical irradiations. Comparisons are made between simulated and experimentally determined dose distributions for a range of configurations to assess the potential use of this tool in determining dose distributions through samples, based on more readily available air-kerma calibration point measurements. 
Main results: simulations gave very good dosimetric agreement with measured depth dose distributions in phantoms containing both water and bone equivalent materials. Good spatial and dosimetric agreement between simulated and measured dose distributions was obtained when using beam-shaping shielding. 
Significance: the MC simulations provided by SmART-RAD provide a useful tool to go from a limited number of dosimetry measurements to detailed 3D dose distributions through a non-homogeneous irradiated sample. This is particularly important when trying to determine the dose distribution in more complex geometries. The use of such a tool can improve reproducibility and dosimetry reporting in preclinical radiobiological research.
Data from VPRBP Functions Downstream of the Androgen Receptor and OGT to Restrict p53 Activation in Prostate Cancer
<div>Abstract<p>Androgen receptor (AR) is a major driver of prostate cancer initiation and progression. O-GlcNAc transferase (OGT), the enzyme that catalyzes the covalent addition of UDP-N-acetylglucosamine (UDP-GlcNAc) to serine and threonine residues of proteins, is often highly expressed in prostate cancer with its expression correlated with high Gleason score. In this study, we have identified an AR and OGT coregulated factor, Vpr (HIV-1) binding protein (VPRBP) also known as DDB1 and CUL4 Associated Factor 1 (DCAF1). We show that VPRBP is regulated by the AR at the transcript level, and stabilized by OGT at the protein level. VPRBP knockdown in prostate cancer cells led to a significant decrease in cell proliferation, p53 stabilization, nucleolar fragmentation, and increased p53 recruitment to the chromatin. In human prostate tumor samples, VPRBP protein overexpression correlated with AR amplification, OGT overexpression, a shorter time to postoperative biochemical progression and poor clinical outcome. In clinical transcriptomic data, VPRBP expression was positively correlated with the AR and also with AR activity gene signatures.</p>Implications:<p>In conclusion, we have shown that VPRBP/DCAF1 promotes prostate cancer cell proliferation by restraining p53 activation under the influence of the AR and OGT.</p></div>
Data from O-GlcNAc Transferase – An Auxiliary Factor or a Full-blown Oncogene?
<div>Abstract<p>The β-linked N-acetyl-d-glucosamine (GlcNAc) is a posttranslational modification of serine and threonine residues catalyzed by the enzyme O-GlcNAc transferase (OGT). Increased OGT expression is a feature of most human cancers and inhibition of OGT decreases cancer cell proliferation. Antiproliferative effects are attributed to posttranslational modifications of known regulators of cancer cell proliferation, such as MYC, FOXM1, and EZH2. In general, OGT amplifies cell-specific phenotype, for example, OGT overexpression enhances reprogramming efficiency of mouse embryonic fibroblasts into stem cells. Genome-wide screens suggest that certain cancers are particularly dependent on OGT, and understanding these addictions is important when considering OGT as a target for cancer therapy. The O-GlcNAc modification is involved in most cellular processes, which raises concerns of on-target undesirable effects of OGT-targeting therapy. Yet, emerging evidence suggest that, much like proteasome inhibitors, specific compounds targeting OGT elicit selective antiproliferative effects in cancer cells, and can prime malignant cells to other treatments. It is, therefore, essential to gain mechanistic insights on substrate specificity for OGT, develop reagents to more specifically enrich for O-GlcNAc–modified proteins, identify O-GlcNAc “readers,” and develop OGT small-molecule inhibitors. Here, we review the relevance of OGT in cancer progression and the potential targeting of this metabolic enzyme as a putative oncogene.</p></div>
Data from Attenuating Adaptive VEGF-A and IL8 Signaling Restores Durable Tumor Control in AR Antagonist–Treated Prostate Cancers
<div>Abstract<p>Inhibiting androgen signaling using androgen signaling inhibitors (ASI) remains the primary treatment for castrate-resistant prostate cancer. Acquired resistance to androgen receptor (AR)-targeted therapy represents a major impediment to durable clinical response. Understanding resistance mechanisms, including the role of AR expressed in other cell types within the tumor microenvironment, will extend the clinical benefit of AR-targeted therapy. Here, we show the ASI enzalutamide induces vascular catastrophe and promotes hypoxia and microenvironment adaptation. We characterize treatment-induced hypoxia, and subsequent induction of angiogenesis, as novel mechanisms of relapse to enzalutamide, highlighting the importance of two hypoxia-regulated cytokines in underpinning relapse. We confirmed AR expression in CD34<sup>+</sup> vascular endothelium of biopsy tissue and human vascular endothelial cells (HVEC). Enzalutamide attenuated angiogenic tubule formation and induced cytotoxicity in HVECs <i>in vitro</i>, and rapidly induced sustained hypoxia in LNCaP xenografts. Subsequent reoxygenation, following prolonged enzalutamide treatment, was associated with increased tumor vessel density and accelerated tumor growth. Hypoxia increased AR expression and transcriptional activity in prostate cells <i>in vitro</i>. Coinhibition of IL8 and VEGF-A restored tumor response in the presence of enzalutamide, confirming the functional importance of their elevated expression in enzalutamide-resistant models. Moreover, coinhibition of IL8 and VEGF-A resulted in a durable, effective resolution of enzalutamide-sensitive prostate tumors. We conclude that concurrent inhibition of two hypoxia-induced factors, IL8 and VEGF-A, prolongs tumor sensitivity to enzalutamide in preclinical models and may delay the onset of enzalutamide resistance.</p>Implications:<p>Targeting hypoxia-induced signaling may extend the therapeutic benefit of enzalutamide, providing an improved treatment strategy for patients with resistant disease.</p></div>
Nanomedicines for the Delivery of Biologics.
A special symposium of the Academy of Pharmaceutical Sciences Nanomedicines Focus Group reviewed the current status of the use of nanomedicines for the delivery of biologics drugs. This meeting was particularly timely with the recent approval of the first siRNA-containing product Onpattro™ (patisiran), which is formulated as a lipid nanoparticle for intravenous infusion, and the increasing interest in the use of nanomedicines for the oral delivery of biologics. The challenges in delivering such molecules were discussed with specific emphasis on the delivery both across and into cells. The latest developments in Molecular Envelope Technology® (Nanomerics Ltd, London, UK), liposomal drug delivery (both from an academic and industrial perspective), opportunities offered by the endocytic pathway, delivery using genetically engineered viral vectors (PsiOxus Technologies Ltd, Abingdon, UK), Transint™ technology (Applied Molecular Transport Inc., South San Francisco, CA, USA), which has the potential to deliver a wide range of macromolecules, and AstraZeneca's initiatives in mRNA delivery were covered with a focus on their uses in difficult to treat diseases, including cancers. Preclinical data were presented for each of the technologies and where sufficiently advanced, plans for clinical studies as well as early clinical data. The meeting covered the work in progress in this exciting area and highlighted some key technologies to look out for in the future.
Data from Tumor pH and Protein Concentration Contribute to the Signal of Amide Proton Transfer Magnetic Resonance Imaging
<div>Abstract<p>Abnormal pH is a common feature of malignant tumors and has been associated clinically with suboptimal outcomes. Amide proton transfer magnetic resonance imaging (APT MRI) holds promise as a means to noninvasively measure tumor pH, yet multiple factors collectively make quantification of tumor pH from APT MRI data challenging. The purpose of this study was to improve our understanding of the biophysical sources of altered APT MRI signals in tumors. Combining <i>in vivo</i> APT MRI measurements with <i>ex vivo</i> histological measurements of protein concentration in a rat model of brain metastasis, we determined that the proportion of APT MRI signal originating from changes in protein concentration was approximately 66%, with the remaining 34% originating from changes in tumor pH. In a mouse model of hypopharyngeal squamous cell carcinoma (FaDu), APT MRI showed that a reduction in tumor hypoxia was associated with a shift in tumor pH. The results of this study extend our understanding of APT MRI data and may enable the use of APT MRI to infer the pH of individual patients' tumors as either a biomarker for therapy stratification or as a measure of therapeutic response in clinical settings.</p>Significance:<p>These findings advance our understanding of amide proton transfer magnetic resonance imaging (APT MRI) of tumors and may improve the interpretation of APT MRI in clinical settings.</p></div>
Copper-based reactions in analyte-responsive fluorescent probes for biological applications.
Copper chemistry has been capitalized on in a wide spectrum of biological events. The central importance of copper in biology lies in the diverse chemical reactivity of the redox-active transition metal ranging from electron transfer, small molecule binding and activation, to catalysis. In addition to its many different roles in natural biological systems, the diverse chemical reactivity of copper also represents a rich opportunity and resource to develop synthetic bioanalytical tools for the study of biologically important species and molecules. In this mini-review, fluorescent probes featuring a specific copper-based chemical reaction to selectively detect a biologically relevant analyte will be discussed. In particular, fluorescent probes for sensing labile copper ions, amino acids and small reactive species will be highlighted. The chemical principles, advantages and limitations of the different types of copper-mediated chemical reactions in these fluorescent probes will be emphasized.
Imaging PARP with [18F]rucaparib in pancreatic cancer models.
PURPOSE: Rucaparib, an FDA-approved PARP inhibitor, is used as a single agent in maintenance therapy to provide promising treatment efficacy with an acceptable safety profile in various types of BRCA-mutated cancers. However, not all patients receive the same benefit from rucaparib-maintenance therapy. A predictive biomarker to help with patient selection for rucaparib treatment and predict clinical benefit is therefore warranted. With this aim, we developed [18F]rucaparib, an 18F-labelled isotopologue of rucaparib, and employed it as a PARP-targeting agent for cancer imaging with PET. Here, we report the in vitro and in vivo evaluation of [18F]rucaparib in human pancreatic cancer models. METHOD: We incorporated the positron-emitting 18F isotope into rucaparib, enabling its use as a PET imaging agent. [18F]rucaparib binds to the DNA damage repair enzyme, PARP, allowing direct visualisation and measurement of PARP in cancerous models before and after PARP inhibition or other genotoxic cancer therapies, providing critical information for cancer diagnosis and therapy. Proof-of-concept evaluations were determined in pancreatic cancer models. RESULTS: Uptake of [18F]rucaparib was found to be mainly dependent on PARP1 expression. Induction of DNA damage increased PARP expression, thereby increasing uptake of [18F]rucaparib. In vivo studies revealed relatively fast blood clearance of [18F]rucaparib in PSN1 tumour-bearing mice, with a tumour uptake of 5.5 ± 0.5%ID/g (1 h after i.v. administration). In vitro and in vivo studies showed significant reduction of [18F]rucaparib uptake by addition of different PARP inhibitors, indicating PARP-selective binding. CONCLUSION: Taken together, we demonstrate the potential of [18F]rucaparib as a non-invasive PARP-targeting imaging agent for pancreatic cancers.
Rhenium complexes of bidentate, bis-bidentate and tridentate N-heterocyclic carbene ligands.
A series of eight Rhenium(I)-N-heterocyclic carbene (NHC) complexes of the general form [ReCl(CO)3(C^C)] (where C^C is a bis(NHC) bidentate ligand), [ReCl(CO)3(C^C)]2 (where C^C is a bis-bidentate tetra-NHC ligand) and [Re(CO)3(C^N^C)](+)[X](-) (where C^N^C is a bis(NHC)-amine ligand and the counter ion X is either the ReO4(-) or PF6(-)) have been synthesised using a Ag2O transmetallation protocol. The novel precursor imidazolium salts and Re(I) complexes were characterized by elemental analysis, (1)H and (13)C NMR spectroscopy and the molecular structures for two imidazolium salt and six Re(I) complexes were determined by single crystal X-ray diffraction. These NHC ligand systems are of interest for possible applications in the development of Tc-99m or Re-186/188 radiopharmaceuticals and as such the stability of two complexes of the form [ReCl(CO)3(C^C)] and [Re(CO)3(C^N^C)][ReO4] were evaluated in ligand challenge experiments using the metal binding amino acids L-histidine or L-cysteine. These studies showed that the former was unstable, with the chloride ligand being replaced by either cysteine or histidine, while no evidence for transchelation was observed for the latter suggesting that bis(NHC)-amine ligands of this type may be suitable for biological applications.
Rhenium and technetium tricarbonyl complexes of N-heterocyclic carbene ligands.
A strategy for the conjugation of N-heterocyclic carbene (NHC) ligands to biomolecules via amide bond formation is described. Both 1-(2-pyridyl)imidazolium or 1-(2-pyridyl)benzimidazolium salts functionalized with a pendant carboxylic acid group were prepared and coupled to glycine benzyl ester using 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide. A series of 10 rhenium(I) tricarbonyl complexes of the form [ReX(CO)3(ĈN)] (ĈN is a bidentate NHC ligand, and X is a monodentate anionic ligand: Cl(-), RCO2(-)) were synthesized via a Ag2O transmetalation protocol from the Re(I) precursor compound Re(CO)5Cl. The synthesized azolium salts and Re(I) complexes were characterized by elemental analysis and by (1)H and (13)C NMR spectroscopy, and the molecular structures for one imidazolium salt and seven Re(I) complexes were determined by single-crystal X-ray diffraction. (1)H NMR and mass spectrometry studies for an acetonitrile-d3 solution of [ReCl(CO)3(1-(2-pyridyl)-3-methylimidazolylidene)] show that the monodentate chloride ligand is labile and exchanges with this solvent yielding a cationic acetonitrile adduct. For the first time the labeling of an NHC ligand with technetium-99m is reported. Rapid Tc-99m labeling was achieved by heating the imidazolium salt 1-(2-pyridyl)-3-methylimidazolium iodide and Ag2O in methanol, followed by the addition of fac-[(99m)Tc(OH2)3(CO)3](+). To confirm the structure of the (99m)Tc-labeled complex, the equivalent (99)Tc complex was prepared, and mass spectrometric studies showed that the formed Tc complexes are of the form [(99m/99)Tc(CH3CN)(CO)3(1-(2-pyridyl)-3-methylimidazolylidene)](+) with an acetonitrile molecule coordinated to the metal center.
Copper-Mediated Radiosynthesis of [18F]Rucaparib.
The poly(ADP-ribose) polymerase (PARP) inhibitor rucaparib is used in the clinic to treat BRCA-mutated cancers. Herein, we report two strategies to access the 18F-isotopologue of rucaparib by applying a copper-mediated nucleophilic 18F-fluorodeboronation. The most successful approach features an aldehydic boronic ester precursor that is subjected to reductive amination post-18F-labeling and affords [18F]rucaparib with an activity yield of 11% ± 3% (n = 3) and a molar activity (Am) up to 30 GBq/μmol. Preliminary in vitro studies are presented.
Rhenium(i) complexes of N-heterocyclic carbene ligands that bind to amyloid plaques of Alzheimer's disease.
A series of [Re(i)L(CO)3]+ complexes (where L is a bifunctional bis(NHC)-amine ligand) that are analogues of potential Tc-99m diagnostic imaging agents for Alzheimer's disease have been synthesised. One of the complexes bound to amyloid plaques in human frontal cortex brain tissue from subjects with Alzheimer's disease.
Optical Mie Scattering by DNA-Assembled Three-Dimensional Gold Nanoparticle Superlattice Crystals
Programmable assemblies of gold nanoparticles engineered with DNA have intriguing optical properties such as Coulomb-interaction-driven strong coupling, polaritonic response in the visible range, and ultralow dispersion dielectric response in the infrared spectral range. In this work, we demonstrate the optical Mie resonances of individual microcrystals of DNA-gold nanoparticle superlattices. Broadband hyperspectral mapping of both transmission and dark-field scattering reveal a polarization-insensitive optical response with distinct spectral features in the visible and near-infrared ranges. Experimental observations are supported by numerical simulations of the microcrystals under a resonant effective medium approximation in the regime of capacitively coupled nanoparticles. The study identifies a universal characteristic optical response which is defined by a band of multipolar Mie resonances, which only weakly depend on the crystal size and light polarization. The use of gold superlattice microcrystals as scattering materials is of interest for fields such as complex nanophotonics, thermoplasmonics, photocatalysis, sensing, and nonlinear optics.
Oncological outcomes of patients with oligometastatic oesophagogastric cancer.
Oligometastatic oesophagogastric cancer was recently defined by consensus as the presence of no more than two metastases and an 18-week period of oncological stability during chemotherapy. The number of patients who fit this criterion and whether their oncological outcome differs from those with multi-metastatic disease is unknown. We analysed a database of 497 patients from 2017 to 2021 with metastatic oesophageal cancer. In total, 36 (7.2%) had oligometastatic disease and significantly improved median overall survival (mOS) versus multi-metastatic disease. In synchronous OMD, mOS was 26.8 months versus 7.3 months and in metachronous OMD, 38.6 months versus 6.1 months (both p
Data from Targeting IGF Perturbs Global Replication through Ribonucleotide Reductase Dysfunction
<div>Abstract<p>Inhibition of IGF receptor (IGF1R) delays repair of radiation-induced DNA double-strand breaks (DSB), prompting us to investigate whether IGF1R influences endogenous DNA damage. Here we demonstrate that IGF1R inhibition generates endogenous DNA lesions protected by 53BP1 bodies, indicating under-replicated DNA. In cancer cells, inhibition or depletion of IGF1R delayed replication fork progression accompanied by activation of ATR–CHK1 signaling and the intra-S-phase checkpoint. This phenotype reflected unanticipated regulation of global replication by IGF1 mediated via AKT, MEK/ERK, and JUN to influence expression of ribonucleotide reductase (RNR) subunit RRM2. Consequently, inhibition or depletion of IGF1R downregulated RRM2, compromising RNR function and perturbing dNTP supply. The resulting delay in fork progression and hallmarks of replication stress were rescued by RRM2 overexpression, confirming RRM2 as the critical factor through which IGF1 regulates replication. Suspecting existence of a backup pathway protecting from toxic sequelae of replication stress, targeted compound screens in breast cancer cells identified synergy between IGF inhibition and ATM loss. Reciprocal screens of ATM-proficient/deficient fibroblasts identified an IGF1R inhibitor as the top hit. IGF inhibition selectively compromised growth of ATM-null cells and spheroids and caused regression of ATM-null xenografts. This synthetic-lethal effect reflected conversion of single-stranded lesions in IGF-inhibited cells into toxic DSBs upon ATM inhibition. Overall, these data implicate IGF1R in alleviating replication stress, and the reciprocal IGF:ATM codependence we identify provides an approach to exploit this effect in ATM-deficient cancers.</p>Significance:<p>This study identifies regulation of ribonucleotide reductase function and dNTP supply by IGFs and demonstrates that IGF axis blockade induces replication stress and reciprocal codependence on ATM.</p></div>
The AsiDNA™ decoy mimicking DSBs protects the normal tissue from radiation toxicity through a DNA-PK/p53/p21-dependent G1/S arrest.
AsiDNA™, a cholesterol-coupled oligonucleotide mimicking double-stranded DNA breaks, was developed to sensitize tumour cells to radio- and chemotherapy. This drug acts as a decoy hijacking the DNA damage response. Previous studies have demonstrated that standalone AsiDNA™ administration is well tolerated with no additional adverse effects when combined with chemo- and/or radiotherapy. The lack of normal tissue complication encouraged further examination into the role of AsiDNA™ in normal cells. This research demonstrates the radioprotective properties of AsiDNA™. In vitro, AsiDNA™ induces a DNA-PK/p53/p21-dependent G1/S arrest in normal epithelial cells and fibroblasts that is absent in p53 deficient and proficient tumour cells. This cell cycle arrest improved survival after irradiation only in p53 proficient normal cells. Combined administration of AsiDNA™ with conventional radiotherapy in mouse models of late and early radiation toxicity resulted in decreased onset of lung fibrosis and increased intestinal crypt survival. Similar results were observed following FLASH radiotherapy in standalone or combined with AsiDNA™. Mechanisms comparable to those identified in vitro were detected both in vivo, in the intestine and ex vivo, in precision cut lung slices. Collectively, the results suggest that AsiDNA™ can partially protect healthy tissues from radiation toxicity by triggering a G1/S arrest in normal cells.