The RNA-binding protein La/SSB associates with radiation-induced DNA double-strand breaks in lung cancer cell lines

First published: 12 October 2021Abstract Background: Platinum-based chemotherapy and radiotherapy are standard treatments for non-small cell lung cancer, which is the commonest, most lethal cancer worldwide. As a marker of treatment-induced cancer cell death, we have developed a radiodiagnostic imaging antibody, which binds to La/SSB. La/SSB is an essential, ubiquitous ribonuclear protein, which is over expressed in cancer and plays a role in resistance to cancer therapies. Aim: In this study, we examined radiation-induced DNA double strand breaks (DSB) in lung cancer cell lines and examined whether La/SSB associated with these DSB. Method: Three lung cancer lines (A549, H460 and LL2) were irradiated with different X-ray doses or X-radiated with a 5 Gy dose and examined at different time-points postirradiation for DNA DSB in the form of γ-H2AX and Rad51 foci. Using fluorescence microscopy, we examined whether La/SSB and γ-H2AX co-localise and performed proximity ligation assay (PLA) and co-immunoprecipitation to confirm the interaction of these proteins. Results: We found that the radio-resistant A549 cell line compared to the radiosensitive H460 cell line showed faster resolution of radiation-induced γ-H2AX foci over time. Conversely, we found more co-localised γ-H2AX and La/SSB foci by PLA in irradiated A549 cells. Conclusion: The co-localisation of La/SSB with radiation-induced DNA breaks suggests a role of La/SSB in DNA repair, however further experimentation is required to validate thisAlexander H. Staudacher, Yanrui Li, Vasilios Liapis, Michael P. Brow

Iconoscope : Designing a Game for Fostering Creativity

This paper introduces Iconoscope, a game aiming to foster the creativity of a young target audience in formal or infor- mal educational settings. At the core of the Iconoscope de- sign is the creative, playful interpretation of word-concepts via the construction of visual icons. In addition to that, the game rewards ambiguity via a scoring system which favors icons that dichotomize public opinion. The game is played by a group of players, with each player attempting to guess which of the concepts provided by the system is represented by each opponent's created icon. Through the social interac- tion that emerges, Iconoscope prompts co-creativity within a group of players; in addition, the game o ers the poten- tial of human-machine co-creativity via computer-generated suggestions to the player's icon. Experiments with early prototypes, described in this paper, provide insight into the design process and motivate certain decisions taken for the current version of Iconoscope which, at the time of writing, is being evaluated in selected schools in Greece, Austria and the United Kingdom.peer-reviewe

Fc gamma receptor is not required for in vivo processing of radio- and drug-conjugates of the dead tumor cell-targeting monoclonal antibody, APOMAB®

The Fc region of a monoclonal antibody (mAb) can play a crucial role in its biodistribution and therapeutic activity. The chimeric mAb, chDAB4 (APOMAB®), which binds to dead tumor cells after DNA-damaging anti- cancer treatment, has been studied pre-clinically in both diagnostic and therapeutic applications in cancer. Given that macrophages contribute to the tumor accumulation of chDAB4 and its potency as an antibody drug con- jugate in vivo, we next wanted to determine whether the Fc region of the chDAB4 mAb also contributed. We found that, regardless of prior labeling with chDAB4, dead EL4 lymphoma or Lewis Lung (LL2) tumor cells were phagocytosed equally by wild-type or Fcγ knock-down macrophage cell lines. A similar result was seen with bone marrow-derived macrophages from wild-type, Fcγ knock-out (KO) and NOTAM mice that express Fcγ but lack immunoreceptor tyrosine-based activation motif (ITAM) signaling. Among EL4 tumor-bearing wild-type, Fcγ KO or NOTAM mice, no differences were observed in post-chemotherapy uptake of 89Zr-labeled chDAB4. Similarly, no differences were observed between LL2 tumor-bearing wild-type and Fcγ KO mice in post-chemotherapy uptake of 89Zr-chDAB4. Also, the post-chemotherapy activity of a chDAB4-antibody drug conjugate (ADC) directed against LL2 tumors did not differ among tumor-bearing wild-type, Fcγ KO and NOTAM mice, nor did the proportions and characteristics of the LL2 tumor immune cell infiltrates differ significantly among these mice. In conclusion, Fc-FcγR interactions are not essential for the diagnostic or therapeutic applications of chDAB4 conjugates because the tumor-associated macrophages, which engulf the chDAB4-labelled dead cells, respond to endogenous ‘eat me’ signals rather than depend on functional FcγR expression for phagocytosis.Alexander H. Staudacher, Vasilios Liapis, Nicole L. Wittwer, William Tieu, Hiu Chun Lam, Jeanette Leusen, Michael P. Brow

Improved non-invasive positron emission tomographic imaging of chemotherapy-induced tumor cell death using Zirconium-89-labeled APOMAB®

Purpose: The chimeric monoclonal antibody (mAb) chDAB4 (APOMAB®) targets the Lupus associated (La)/Sjögren Syndrome-B (SSB) antigen, which is over-expressed in tumors but only becomes available for antibody binding in dead tumor cells. Hence, chDAB4 may be used as a novel theranostic tool to distinguish between responders and nonresponders early after chemotherapy. Here, we aimed to ascertain which positron emitter, Zirconium-89 ([⁸⁹Zr]Zr(IV)) or Iodine-124 ([¹²⁴I]I), was best suited to label chDAB4 for post-chemotherapy PET imaging of tumor-bearing mice and to determine which of two different bifunctional chelators provided optimal tumor imaging by PET using [⁸⁹Zr]Zr(IV)-labeled chDAB4. Methods: C57BL/6 J mice bearing subcutaneous syngeneic tumors of EL4 lymphoma were either untreated or given chemotherapy, then administered radiolabeled chDAB4 after 24 h with its biodistribution examined using PET and organ assay. We compared chDAB4 radiolabeled with [⁸⁹Zr] Zr(IV) or [¹²⁴I] I, or [⁸⁹Zr]Zr-chDAB4 using either DFO-NCS or DFOSq as a chelator. Results: After chemotherapy, [⁸⁹Zr]Zr-chDAB4 showed higher and prolonged mean (± SD) tumor uptake of 29.5 ± 5.9 compared to 7.8 ± 1.2 for [¹²⁴I] I -chDAB4. In contrast, antibody uptake in healthy tissues was not affected. Compared to DFO-NCS, DFOSq did not result in significant differences in tumor uptake of [⁸⁹Zr]Zr-chDAB4 but did alter the tumor:liver ratio in treated mice 3 days after injection in favour of DFOSq (8.0 ± 1.1) compared to DFO-NCS (4.2 ± 0.7). Conclusion: ImmunoPET using chDAB4 radiolabeled with residualizing [⁸⁹Zr] Zr(IV) rather than [¹²⁴I] I optimized post-chemotherapy tumor uptake. Further, PET imaging characteristics were improved by DFOSq rather than DFO-NCS. Therefore, the radionuclide/chelator combination of [⁸⁹Zr] Zr(IV) and DFOSq is preferred for the imminent clinical evaluation of chDAB4 as a selective tumor cell death radioligand.Vasilios Liapis, William Tieu, Stacey E. Rudd, Paul S. Donnelly, Nicole L. Wittwer, Michael P. Brown, and Alexander H. Staudache

Calcium oxalate crystals induce renal inflammation by NLRP3-mediated IL-1β secretion

Nephrocalcinosis, acute calcium oxalate (CaOx) nephropathy, and renal stone disease can lead to inflammation and subsequent renal failure, but the underlying pathological mechanisms remain elusive. Other crystallopathies, such as gout, atherosclerosis, and asbestosis, trigger inflammation and tissue remodeling by inducing IL-1β secretion, leading us to hypothesize that CaOx crystals may induce inflammation in a similar manner. In mice, intrarenal CaOx deposition induced tubular damage, cytokine expression, neutrophil recruitment, and renal failure. We found that CaOx crystals activated murine renal DCs to secrete IL-1β through a pathway that included NLRP3, ASC, and caspase-1. Despite a similar amount of crystal deposits, intrarenal inflammation, tubular damage, and renal dysfunction were abrogated in mice deficient in MyD88; NLRP3, ASC, and caspase-1; IL-1R; or IL-18. Nephropathy was attenuated by DC depletion, ATP depletion, or therapeutic IL-1 antagonism. These data demonstrated that CaOx crystals trigger IL-1β–dependent innate immunity via the NLRP3/ASC/caspase-1 axis in intrarenal mononuclear phagocytes and directly damage tubular cells, leading to the release of the NLRP3 agonist ATP. Furthermore, these results suggest that IL-1β blockade may prevent renal damage in nephrocalcinosis

Anticancer efficacy of the hypoxia-activated prodrug evofosfamide (TH-302) in osteolytic breast cancer murine models

Tumor hypoxia is a major cause of treatment failure for a variety of malignancies. However, hypoxia offers treatment opportunities, exemplified by the development of compounds that target hypoxic regions within tumors. Evofosfamide (TH-302) is a prodrug created by the conjugation of 2-nitroimidazole to bromo-isophosphoramide mustard (Br-IPM). When evofosfamide is delivered to hypoxic regions, the DNA cross-linking effector, Br-IPM, is released. This study assessed the cytotoxic activity of evofosfamide in vitro and its antitumor activity against osteolytic breast cancer either alone or in combination with paclitaxel in vivo. A panel of human breast cancer cell lines were treated with evofosfamide under hypoxia and assessed for cell viability. Osteolytic MDA-MB-231-TXSA cells were transplanted into the mammary fat pad, or into tibiae of mice, allowed to establish and treated with evofosfamide, paclitaxel, or both. Tumor burden was monitored using bioluminescence, and cancer-induced bone destruction was measured using micro-CT. In vitro, evofosfamide was selectively cytotoxic under hypoxic conditions. In vivo evofosfamide was tumor suppressive as a single agent and cooperated with paclitaxel to reduce mammary tumor growth. Breast cancer cells transplanted into the tibiae of mice developed osteolytic lesions. In contrast, treatment with evofosfamide or paclitaxel resulted in a significant delay in tumor growth and an overall reduction in tumor burden in bone, whereas combined treatment resulted in a significantly greater reduction in tumor burden in the tibia of mice. Evofosfamide cooperates with paclitaxel and exhibits potent tumor suppressive activity against breast cancer growth in the mammary gland and in bone.Vasilios Liapis, Irene Zinonos, Agatha Labrinidis, Shelley Hay, Vladimir Ponomarev, Vasilios Panagopoulos, Aneta Zysk, Mark DeNichilo, Wendy Ingman, Gerald J. Atkins, David M. Findlay, Andrew C. W. Zannettino, Andreas Evdokio

Positron emission tomographic imaging of tumor cell death using zirconium-89-labeled APOMAB(R) following cisplatin chemotherapy in lung and ovarian cancer xenograft models

Published online 06 July 2021Purpose Early detection of tumor treatment responses represents an unmet clinical need with no approved noninvasive methods. DAB4, or its chimeric derivative, chDAB4 (APOMAB®) is an antibody that targets the Lupus associated antigen (La/SSB). La/SSB is over-expressed in malignancy and selectively targeted by chDAB4 in cancer cells dying from DNA-damaging treatment. Therefore, chDAB4 is a unique diagnostic tool that detects dead cancer cells and thus could distinguish between treatment responsive and nonresponsive patients. Procedures In clinically relevant tumor models, mice bearing subcutaneous xenografts of human ovarian or lung cancer cell lines or intraperitoneal ovarian cancer xenografts were untreated or given chemotherapy followed 24h later by chDAB4 radiolabeled with [⁸⁹Zr]ZrIV. Tumor responses were monitored using bioluminescence imaging and caliper measurements. [⁸⁹Zr]Zr-chDAB4 uptake in tumor and normal tissues was measured using an Albira SI Positron-Emission Tomography (PET) imager and its biodistribution was measured using a Hidex gamma-counter. Results Tumor uptake of [⁸⁹Zr]Zr-chDAB4 was detected in untreated mice, and uptake significantly increased in both human lung and ovarian tumors after chemotherapy, but not in normal tissues. Conclusion Given that tumors, rather than normal tissues, were targeted after chemotherapy, these results support the clinical development of chDAB4 as a radiodiagnostic imaging agent and as a potential predictive marker of treatment response.Vasilios Liapis, William Tieu, Nicole L. Wittwer, Tessa Gargett, Andreas Evdokiou, Prab Takhar, Stacey E. Rudd, Paul S. Donnelly, Michael P. Brown, Alexander H. Staudache

Cytotoxicity of crystals involves RIPK3-MLKL-mediated necroptosis

Crystals cause injury in numerous disorders, and induce inflammation via the NLRP3 inflammasome, however, it remains unclear how crystals induce cell death. Here we report that crystals of calcium oxalate, monosodium urate, calcium pyrophosphate dihydrate and cystine trigger caspase-independent cell death in five different cell types, which is blocked by necrostatin-1. RNA interference for receptor-interacting protein kinase 3 (RIPK3) or mixed lineage kinase domain like (MLKL), two core proteins of the necroptosis pathway, blocks crystal cytotoxicity. Consistent with this, deficiency of RIPK3 or MLKL prevents oxalate crystal-induced acute kidney injury. The related tissue inflammation drives TNF-alpha-related necroptosis. Also in human oxalate crystal-related acute kidney injury, dying tubular cells stain positive for phosphorylated MLKL. Furthermore, necrostatin-1 and necrosulfonamide, an inhibitor for human MLKL suppress crystal-induced cell death in human renal progenitor cells. Together, TNF-alpha/TNFR1, RIPK1, RIPK3 and MLKL are molecular targets to limit crystal-induced cytotoxicity, tissue injury and organ failure

Cytotoxicity of crystals involves RIPK3-MLKL-mediated necroptosis

Crystals cause injury in numerous disorders, and induce inflammation via the NLRP3 inflammasome, however, it remains unclear how crystals induce cell death. Here we report that crystals of calcium oxalate, monosodium urate, calcium pyrophosphate dihydrate and cystine trigger caspase-independent cell death in five different cell types, which is blocked by necrostatin-1. RNA interference for receptor-interacting protein kinase 3 (RIPK3) or mixed lineage kinase domain like (MLKL), two core proteins of the necroptosis pathway, blocks crystal cytotoxicity. Consistent with this, deficiency of RIPK3 or MLKL prevents oxalate crystal-induced acute kidney injury. The related tissue inflammation drives TNF-alpha-related necroptosis. Also in human oxalate crystal-related acute kidney injury, dying tubular cells stain positive for phosphorylated MLKL. Furthermore, necrostatin-1 and necrosulfonamide, an inhibitor for human MLKL suppress crystal-induced cell death in human renal progenitor cells. Together, TNF-alpha/TNFR1, RIPK1, RIPK3 and MLKL are molecular targets to limit crystal-induced cytotoxicity, tissue injury and organ failure

Критика Юркевичем утилітаризму

This work focuses on studying players behaviour in interactive narratives with the aim to simulate their choices. Besides sub-optimal player behaviour due to limited knowledge about the environment, the difference in each player's style and preferences represents a challenge when trying to make an intelligent system mimic their actions. Based on observations from players interactions with an extract from the interactive fiction Anchorhead, we created a player profile to guide the behaviour of a generic player model based on the BDI (Belief-Desire-Intention) model of agency. We evaluated our approach using qualitative and quantitative methods and found that the player profile can improve the performance of the BDI player model. However, we found that players self-assessment did not yield accurate data to populate their player profile under our current approach.Comment: CHI Play 201