Generation of Sheep Induced Pluripotent Stem Cells With Defined DOX-Inducible Transcription Factors via piggyBac Transposition

Pluripotent stem cells (PSCs) have the potential to differentiate to all cell types of an adult individual and are useful for studying mammalian development. Establishing induced pluripotent stem cells (iPSCs) capable of expressing pluripotent genes and differentiating to three germ layers will not only help to explain the mechanisms underlying somatic reprogramming but also lay the foundation for the establishment of sheep embryonic stem cells (ESCs) in vitro. In this study, sheep somatic cells were reprogrammed in vitro into sheep iPSCs with stable morphology, pluripotent marker expression, and differentiation ability, delivered by piggyBac transposon system with eight doxycycline (DOX)-inducible exogenous reprogramming factors: bovine OCT4, SOX2, KLF4, cMYC, porcine NANOG, human LIN28, SV40 large T antigen, and human TERT. Sheep iPSCs exhibited a chimeric contribution to the early blastocysts of sheep and mice and E6.5 mouse embryos in vitro. A transcriptome analysis revealed the pluripotent characteristics of somatic reprogramming and insights into sheep iPSCs. This study provides an ideal experimental material for further study of the construction of totipotent ESCs in sheep.

RABL6A Promotes Pancreatic Neuroendocrine Tumor Angiogenesis and Progression In Vivo

Pancreatic neuroendocrine tumors (pNETs) are difficult-to-treat neoplasms whose incidence is rising. Greater understanding of pNET pathogenesis is needed to identify new biomarkers and targets for improved therapy. RABL6A, a novel oncogenic GTPase, is highly expressed in patient pNETs and required for pNET cell proliferation and survival in vitro. Here, we investigated the role of RABL6A in pNET progression in vivo using a well-established model of the disease. RIP-Tag2 (RT2) mice develop functional pNETs (insulinomas) due to SV40 large T-antigen expression in pancreatic islet β cells. RABL6A loss in RT2 mice significantly delayed pancreatic tumor formation, reduced tumor angiogenesis and mitoses, and extended survival. Those effects correlated with upregulation of anti-angiogenic p19ARF and downregulation of proangiogenic c-Myc in RABL6A-deficient islets and tumors. Our findings demonstrate that RABL6A is a bona fide oncogenic driver of pNET angiogenesis and development in vivo.

LGG-11. BH3-MIMETICS TARGETING BCL-XL SELECTIVELY IMPACT THE SENESCENT COMPARTMENT OF PILOCYTIC ASTROCYTOMA

Introduction Pilocytic astrocytoma (PA) is the most common brain tumor in children. Activation of the mitogen-activated protein kinase (MAPK) pathway is a hallmark of PA. Complete remission in non-resectable tumors is infrequently observed with current therapeutic approaches. Most PA tumors cells are in oncogene-induced senescence (OIS), which may explain the benign growth behavior of PAs but also account for resistance to therapy. Therefore, treatment of PA with senolytic agents such as BH3-mimetics is a promising new approach. Methods Three patient-derived PA cell lines, DKFZ-BT66, DKFZ-BT308 (both KIAA1549:BRAF-fusion positive) and DKFZ-BT314 (BRAF V600E-mutation positive) were used. Depending on inducible expression or repression of SV40 large T antigen all models can reflect both states of PA, proliferation and OIS. Cells in both states were treated with different BH3-mimetics. Inhibition of metabolic activity was measured after 72 hours. Target expression was assessed by RT-qPCR and Western blot. On-target activity of BH3-mimetics was determined by immunoprecipitation (IP) of Bcl-xL/BAK. Results BH3-mimetics with strong binding affinity for Bcl-xL (Navitoclax, A-1131852, A-1155463) showed selectivity for senescent cells in 2/3 models (DKFZ-BT66 and DKFZ-BT314) and acted in nanomolar ranges. IC50s for Navitoclax (Cmax 6600nM in patients) were 40nM (OIS) vs. 200nM (proliferation) and 170nM (OIS) vs. 3700nM (proliferation) in DKFZ-BT66 and DKFZ-BT314, respectively. Target engagement was evident in the Bcl-xL/BAK-IP, and target expression of Bcl-xL was similar in all models studied. The relative resistance of senescent DKFZ-BT308 despite on-target activity is currently being investigated. Conclusion Senolytic treatment of PA with BH3-mimetics targeting Bcl-xL is a promising new strategy directly targeting the major senescent part of the tumor in clinically archivable concentrations. However, our data suggests that not all PAs may respond to treatment. The analysis of comparative gene expression analysis and BH3-profiling is ongoing to define predictive biomarkers.

Acitretin and Retinoic Acid Derivatives Inhibit BK Polyomavirus Replication in Primary Human Proximal Renal Tubular Epithelial and Urothelial Cells

Small-molecule drugs inhibiting BK polyomavirus (BKPyV) represent a significant unmet clinical need in view of polyomavirus-associated nephropathy or hemorrhagic cystitis which complicate 5% to 25% of kidney and hematopoietic cell transplantations. We characterized the inhibitory activity of acitretin on BKPyV-replication in primary human renal proximal tubular epithelial cells (RPTECs). Effective inhibitory concentration 50% (EC50) and 90% (EC90) were determined in dilution series measuring BKPyV loads, transcripts and protein expression, using cell proliferation, metabolic activity, and viability to estimate cytotoxic concentrations and selectivity indices (SI). Acitretin EC50 and EC90 in RPTECs were 0.64 (SI50 250) and 3.25 μM (SI90 49.2), respectively. Acitretin effectively inhibited BKPyV-replication until 72 h post-infection when added 24 h before until 12 h after infection, but decreased to <50% at later timepoints. Acitretin did not interfere with nuclear delivery of BKPyV genomes, but decreased large T-antigen transcription and protein expression. Acitretin did not inhibit the initial round of BKPyV-replication following transfection of full-length viral genomes, but affected subsequent rounds of re-infection. Acitretin also inhibited BKPyV-replication in human urothelial cells and in Vero cells, but not in COS-7 cells constitutively expressing SV40-large T-antigen. Retinoic acid-agonists (all-trans-retinoic acid, 9-cis-RA, 13-cis-RA, bexarotene, tamibarotene) and the RAR/RXR-antagonist RO41-5253 also inhibited BKPyV-replication, pointing to as yet undefined mechanism. Importance Acitretin selectively inhibits BKPyV-replication in primary human cell culture models of nephropathy and hemorrhagic cystitis. Since acitretin is an approved drug in clinical use reaching BKPyV-inhibiting concentrations in systemically treated patients, further studies are warranted to provide data for clinical repurposing of retinoids for treatment and prevention of replicative BKPyV-diseases.

RABL6A promotes pancreatic neuroendocrine tumor angiogenesis and progression in vivo

Pancreatic neuroendocrine tumors (pNETs) are difficult-to-treat neoplasms whose incidence is rising. Greater understanding of pNET pathogenesis is needed to identify new biomarkers and targets for improved therapy. RABL6A, a novel oncogenic GTPase, is highly expressed in patient pNETs and required for pNET cell proliferation and survival in vitro. Here, we investigated the role of RABL6A in pNET progression in vivo using a well-established model of the disease. RIP-Tag2 (RT2) mice develop functional pNETs (insulinomas) due to SV40 large T-antigen expression in pancreatic islet β cells. RABL6A loss in RT2 mice significantly delayed pancreatic tumor formation, reduced tumor angiogenesis and mitoses, and extended survival. Those effects correlated with upregulation of anti-angiogenic p19ARF and downregulation of proangiogenic c-Myc in RABL6A-deficient islets and tumors. Our findings demonstrate that RABL6A is a bona fide oncogenic driver of pNET angiogenesis and development in vivo.

Comparison of Immunohistochemical Staining for Large T Antigen and Capsid Protein VP1 in BK Polyomavirus-Associated Nephropathy

<b><i>Aim:</i></b> Most transplant centres use SV40 large T antigen (TAg) staining for the diagnosis and assessment of BK polyomavirus-associated nephropathy (BKPyVAN). This study was performed to evaluate the significance of capsid protein VP1 expression in BKPyVAN. <b><i>Methods:</i></b> We performed immunohistochemical staining using anti-SV40 TAg and anti-BKPyV VP1 antibodies in 16 index biopsies and 12 re-biopsies of BKPyVAN and compared the patterns of positivity and the percentage of positive tubules by counting whole specimens. We investigated the correlation between serum creatinine increase from baseline and the percentage of positive tubules for both markers in 16 index biopsies. <b><i>Results:</i></b> In VP1 staining, positive findings were observed not only in the nuclei of tubular epithelial cells but also in the cytoplasm, cells shedding into the lumen, intra-tubular casts, and in the interstitium. Two of 28 biopsies (7.1%) showed TAg-positive and VP1-negative results, in which TAg-positive cells were detected only in a single tubule. The median (interquartile range) percentage of positive tubules was 2.8% (0.7–9.8%) for TAg and 1.4% (0.5–3.9%) for VP1 staining (<i>p</i> = 0.2). In 16 index biopsies, serum creatinine increases significantly correlated with the percentage of VP1-positive tubules (<i>r</i> = 0.49, <i>p</i> = 0.02), while this correlation revealed borderline significance with TAg-positive tubules. <b><i>Conclusions:</i></b> VP1 expression showed various patterns, but was detected in half as many tubules as TAg staining, which might lead to false negatives in the samples with minimal viral replication. However, increased VP1-positive tubules indicate advanced tubular damage and possible association with graft dysfunction.

TMOD-16. A NOVEL ADENOVIRAL-PERMISSIVE, IMMUNOCOMPETENT HAMSTER GLIOMA MODEL TO EVALUATE ONCOLYTIC ADENOVIRAL THERAPY

Oncolytic adenoviruses, including Delta-24-RGD, target tumors by direct tumor cell oncolysis and by activation of an anti-tumor immune response. Due to the species selectivity of oncolytic adenoviruses, there is currently no single preclinical animal model of glioma that supports viral replication, tumor oncolysis, and virus-mediated immune responses. To address this gap, we took advantage of the Syrian hamster to develop the first glioma model that is both adenovirus replication-permissive and immunocompetent. Hamster glioma stem-like cells (GSCs), transformed by forced expression of hTERT, SV40 large T antigen, and h-RasV12, reproducibly form intracranial tumors in hamsters. In vitro, electron microscopy and cytopathic effect assays demonstrated that hamster GSCs supported viral replication and were susceptible to Delta-24-RGD-mediated cell death. In vivo, hamster GSCs consistently developed into highly proliferative tumors resembling high-grade gliomas. Following intratumoral delivery of Delta-24-RGD, immunohistochemistry for viral proteins demonstrated viral infectivity and replication in hamster gliomas. Flow cytometry revealed increased T cell infiltration in Delta-24-RGD-infected tumors. Delta-24-RGD treatment of tumor-bearing hamsters led to significantly increased survival compared with hamsters treated with PBS. Using this model, we evaluated the effects of corticosteroid-mediated immunosuppression on Delta-24-RGD efficacy. Dexamethasone treatment significantly decreased peripheral blood lymphocytes, decreased tumor-infiltrating lymphocytes, and suppressed the levels of serum anti-adenovirus antibodies. Dexamethasone reduced the number of long-term survivors and decreased the median survival (50 days for Delta-24-RGD + dexamethasone vs undetermined for Delta-24-RGD alone). In summary, we have developed the first adenovirus-permissive, immunocompetent hamster glioma model, addressing a critical need for a model in which to study the role of direct oncolysis in driving immune mediated viral clearance versus driving an antiglioma immune response. Understanding these mechanisms is critical to optimizing the success of oncolytic adenoviral therapy in the clinic.

Investigating Radiotherapy Response in a Novel Syngeneic Model of Prostate Cancer

The prostate cancer (PCa) field lacks clinically relevant, syngeneic mouse models which retain the tumour microenvironment observed in PCa patients. This study establishes a cell line from prostate tumour tissue derived from the Pten−/−/trp53−/− mouse, termed DVL3 which when subcutaneously implanted in immunocompetent C57BL/6 mice, forms tumours with distinct glandular morphology, strong cytokeratin 8 and androgen receptor expression, recapitulating high-risk localised human PCa. Compared to the commonly used TRAMP C1 model, generated with SV40 large T-antigen, DVL3 tumours are immunologically cold, with a lower proportion of CD8+ T-cells, and high proportion of immunosuppressive myeloid derived suppressor cells (MDSCs), thus resembling high-risk PCa. Furthermore, DVL3 tumours are responsive to fractionated RT, a standard treatment for localised and metastatic PCa, compared to the TRAMP C1 model. RNA-sequencing of irradiated DVL3 tumours identified upregulation of type-1 interferon and STING pathways, as well as transcripts associated with MDSCs. Upregulation of STING expression in tumour epithelium and the recruitment of MDSCs following irradiation was confirmed by immunohistochemistry. The DVL3 syngeneic model represents substantial progress in preclinical PCa modelling, displaying pathological, micro-environmental and treatment responses observed in molecular high-risk disease. Our study supports using this model for development and validation of treatments targeting PCa, especially novel immune therapeutic agents.

BK Virus RNA in Renal Allograft Biopsies

BK polyomavirus–associated nephropathy (BKpyVAN) remains a cause of graft loss in kidney transplant recipients on immunosuppressive therapy. Its diagnosis relies on the identification of BK virus (BKV) in the renal allograft biopsy by positive immunohistochemical (IHC) stain for the viral SV40 large T antigen, although in situ hybridization (ISH) for viral DNA is used in some centers. We examined tissue detection of BKV RNA by RNAscope, a novel, automated ISH test, in 61 allograft biopsies from 56 patients with BKpyVAN. We found good correlation between the estimate of BKV tissue load by RNAscope ISH and SV40 IHC ( R2 = 0.65, p<0.0001). RNAscope ISH showed 88% sensitivity and 79% specificity and, as an alternative test, could confirm the presence of BKV tissue in presumed BKpyVAN and rule out BKV as the causative agent in JC virus nephropathy. We also used tissue BK viral load estimates by both RNAscope ISH and SV40 IHC to examine the relation between tissue and plasma BK levels and found significant correlation only between BK viremia and tissue BK measured by RNAscope ISH. Our findings suggest that the RNAscope ISH assay could be a reliable test for BKV detection in allograft biopsies.