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Immune checkpoint blockade trials have yet to produce a robust anti-cancer response in prostate cancer patients as a monotherapy due to the immunosuppressed prostate cancer tumour immune microenvironment. ST3Gal1 and other sialyltransferases are implicated in cancer and immune suppression by synthesizing sialoglycans, which act as ligands for Siglec receptors. These checkpoints are important for the immune response. However, it’s unclear how the synthesis of Siglec ligands is regulated, and little is known about the role of sialoglycan-Siglec-axis in prostate cancer’s evasion of anti-tumour immunity. We report that ST3Gal1 levels negatively correlate with androgen signalling in prostate tumours. Utilising syngeneic mouse models, we demonstrate that ST3Gal1 plays an important role in modulating tumour immune evasion. Using mouse models, patient samples andin vitromodels we show that ST3Gal1 synthesises sialoglycans with the capacity to engage the Siglec-7 and Siglec-9 immunoreceptors preventing immune clearance of cancer cells. For the first time we provide evidence of the expression of Siglec-7/9 ligands and their respective immunoreceptors in prostate tumours. Importantly, we show that these interactions can be modulated by enzalutamide and may maintain immune suppression in enzalutamide treated tumours. We conclude that the activity of ST3Gal1 is critical to prostate cancer anti-tumour immunity and provide rationale for the use of glyco-immune checkpoint targeting therapies in advanced prostate cancer.

Mutations in pre-mRNA processing factor 31 (PRPF31), a core protein of the spliceosomal tri-snRNP complex, cause autosomal-dominant retinitis pigmentosa (adRP). It has remained an enigma why mutations in ubiquitously expressed tri-snRNP proteins result in retina-specific disorders, and so far, the underlying mechanism of splicing factors-related RP is poorly understood. Here, we used iPSC technology to generate retinal organoids and RPE models from three patients with severe and very severe PRPF31-adRP, normal individuals and a CRISPR/Cas9-corrected isogenic control. To fully assess the impacts of PRPF31 mutations, quantitative proteomics analyses of retinal organoids and RPE cells was carried out showing RNA splicing, autophagy and lysosome, unfolded protein response (UPR) and visual cycle-related pathways to be significantly affected. Strikingly, the patient-derived RPE and retinal cells were characterised by the presence of large amounts of cytoplasmic aggregates containing the mutant PRPF31 and misfolded, ubiquitin-conjugated proteins including key visual cycle proteins, which accumulated progressively with time. Mutant PRPF31 variant was not incorporated into splicing complexes, but reduction of PRPF31 wildtype levels led to tri-snRNP assembly defects in Cajal bodies of PRPF31 patient retinal cells with reduced U4/U6 snRNPs and accumulation of U5, smaller nuclear speckles and reduced formation of active spliceosomes giving rise to global splicing dysregulation. Moreover, the impaired waste disposal mechanisms further exacerbated aggregate formation, and targeting these by activating the autophagy pathway using Rapamycin resulted in reduction of cytoplasmic aggregates and improved cell survival. Our data demonstrate that it is the progressive aggregate accumulation that overburdens the waste disposal machinery rather than direct PRPF31-initiated mis-splicing, and thus relieving the RPE cells from insoluble cytoplasmic aggregates presents a novel therapeutic strategy that can be combined with gene therapy studies to fully restore RPE and retinal cell function in PRPF31-adRP patients.HighlightsPRPF31 RP mutations lead to formation of insoluble aggregates containing the mutant PRPF31 and misfolded, ubiquitin conjugated proteins including key visual cycle proteins (e.g. RLBP1) in RPE cells, which accumulate progressively with time and affect tight junctions and cell survival.Mutant PRPF31 is predominantly localised in cytoplasmic aggregates of patient specific RPE and retinal cells and is not able to be incorporated into splicing complexes to cause direct mis-splicing.High-throughput quantitative proteomics identifies significantly altered RNA splicing, visual perception, retinoid metabolism, waste disposal and unfolded protein response pathways in patient RPE cells, and autophagy and lysosome, unfolded protein response (UPR) and visual cycle-related pathways in photoreceptor cells.Accumulation of PRPF31 mutant variant as cytoplasmic aggregates reduces wildtype PRPF31 in the nucleus leading to tri-snRNP assembly defects, characterised by accumulation of U5 and reduction of U4/U6 snRNPs in Cajal bodies, altered morphology of nuclear speckles and consequently downregulation of active spliceosomes (Bact and C complexes) in PRPF31 patient RPE and retinal cells.Proteomic study of insoluble aggregates identifies other RP-linked splicing factors and multiple key retinal-specific proteins, whose variants are linked to retinitis pigmentosa, within the aggregates of patient RPE cells.PRPF31 patient RPE cells have impaired waste disposal and proteasome mediated degradation, which together with the impaired autophagy pathway, further exacerbate aggregate formation.Phagocytosis of photoreceptor outer segment fragments (POS) shed daily by RPE cells accelerates aggregation of key proteins indicating enhanced cytoplasmic aggregate formation under physiological conditions in patient RPE cells.Activation of autophagy via administration of rapamycin results in reduction of cytoplasmic aggregates in RPE cells, correct localisation of mislocated and misfolded proteins to the nucleus, thereby improving cell survival.

Two cell lines were generated by CRISPR/Cas9 mediated knockout of MKK7 (MAP2K7) by removal of exon 1 or exons 4 through 7. These knockouts were confirmed at the transcript and protein levels. These hESCs are pluripotent and maintain tri-lineage differentiation capacity. These cell lines are a useful resource for studying MKK7 function in humans.

Primary culture of human prostate organoids and patient‐derived xenografts is inefficient and has limited access to clinical tissues. This hampers their use for translational study to identify new treatments. To overcome this, we established a complementary approach where rapidly proliferating and easily handled induced pluripotent stem cells enabled the generation of human prostate tissue in vivo and in vitro. By using a coculture technique with inductive urogenital sinus mesenchyme, we comprehensively recapitulated in situ 3D prostate histology, and overcame limitations in the primary culture of human prostate stem, luminal and neuroendocrine cells, as well as the stromal microenvironment. This model now unlocks new opportunities to undertake translational studies of benign and malignant prostate disease., We demonstrate for the first time that induced pluripotent stem cells (iPSCs) can generate human prostate tissue both in vivo and in vitro, overcoming some significant limitations with patient‐derived xenografts and primary culture to provide a complimentary approach. Furthermore, the in vitro model provides a new platform to create a living biobank of prostate cancer organoids derived from gene editing of iPSCs for preclinical drug testing.

Retinitis pigmentosa (RP) is the most common form of inherited vision loss and is characterized by degeneration of retinal photoreceptor cells and the retinal pigment epithelium (RPE). Mutations in pre-mRNA processing factor 31 (PRPF31) cause dominant RP via haploinsufficiency with incomplete penetrance. There is good evidence that the diverse severity of this disease is a result of differing levels of expression of the wild type allele among patients. Thus, we hypothesize that PRPF31-related RP will be amenable to treatment by adeno-associated virus (AAV)-mediated gene augmentation therapy. To test this hypothesis, we used induced pluripotent stem cells (iPSC) with mutations in PRPF31 and differentiated them into RPE cells. The mutant PRPF31 iPSC-RPE cells recapitulate the cellular phenotype associated with the PRPF31 pathology, including defective cell structure, diminished phagocytic function, defects in ciliogenesis, and compromised barrier function. Treatment of the mutant PRPF31 iPSC-RPE cells with AAV-PRPF31 restored normal phagocytosis and cilia formation, and partially restored structure and barrier function. These results provide proof-of concept that AAV-based gene therapy can be used to treat patients with PRPF31-related RP.

One of the key issues hampering the development of effective treatments for prostate cancer is the lack of suitable, tractable, and patient-specific in vitro models that accurately recapitulate this disease. In this review, we address the challenges of using primary cultures and patient-derived xenografts to study prostate cancer. We describe emerging approaches using primary prostate epithelial cells and prostate organoids and their genetic manipulation for disease modelling. Furthermore, the use of human prostate-derived induced pluripotent stem cells (iPSCs) is highlighted as a promising complimentary approach. Finally, we discuss the manipulation of iPSCs to generate ‘avatars’ for drug disease testing. Specifically, we describe how a conceptual advance through the creation of living biobanks of “genetically engineered cancers” that contain patient-specific driver mutations hold promise for personalised medicine.

Mutations in pre-mRNA processing factors (PRPFs) cause 40% of autosomal dominant retinitis pigmentosa (RP), but it is unclear why mutations in ubiquitously expressed PRPFs cause retinal disease. To understand the molecular basis of this phenotype, we have generated RP type 11 (PRPF31-mutated) patient-specific retinal organoids and retinal pigment epithelium (RPE) from induced pluripotent stem cells (iPSC). Impaired alternative splicing of genes encoding pre-mRNA splicing proteins occurred in patient-specific retinal cells and Prpf31 /- mouse retinae, but not fibroblasts and iPSCs, providing mechanistic insights into retinal-specific phenotypes of PRPFs. RPE was the most affected, characterised by loss of apical-basal polarity, reduced trans-epithelial resistance, phagocytic capacity, microvilli, and cilia length and incidence. Disrupted cilia morphology was observed in patient-derived-photoreceptors that displayed progressive features associated with degeneration and cell stress. In situ gene-editing of a pathogenic mutation rescued key structural and functional phenotypes in RPE and photoreceptors, providing proof-of-concept for future therapeutic strategies.

SummaryMutations in pre-mRNA processing factors (PRPFs) cause 40% of autosomal dominant retinitis pigmentosa (RP), but it is unclear why mutations in ubiquitously expressed PRPFs cause retinal disease. To understand the molecular basis of this phenotype, we have generated RP type 11 (PRPF31-mutated) patient-specific retinal organoids and retinal pigment epithelium (RPE) from induced pluripotent stem cells (iPSC). Impaired alternative splicing of genes encoding pre-mRNA splicing proteins occurred in patient-specific retinal cells and Prpf31+/− mouse retinae, but not fibroblasts and iPSCs, providing mechanistic insights into retinal-specific phenotypes of PRPFs. RPE was the most affected, characterised by loss of apical-basal polarity, reduced trans-epithelial resistance, phagocytic capacity, microvilli, and cilia length and incidence. Disrupted cilia morphology was observed in patient-derived-photoreceptors that displayed progressive features associated with degeneration and cell stress. In situ gene-editing of a pathogenic mutation rescued key structural and functional phenotypes in RPE and photoreceptors, providing proof-of-concept for future therapeutic strategies.eTOCPRPF31 is a ubiquitously expressed pre-mRNA processing factor that when mutated causes autosomal dominant RP. Using a patient-specific iPSC approach, Buskin and Zhu et al. show that retinal-specific defects result from altered splicing of genes involved in the splicing process itself, leading to impaired splicing, loss of RPE polarity and diminished phagocytic ability as well as reduced cilia incidence and length in both photoreceptors and RPE.HighlightsSuccessful generation of iPSC-derived RPE and photoreceptors from four RP type 11 patientsRPE cells express the mutant PRPF31 protein and show the lowest expression of wildtype proteinPRPF31 mutations result in altered splicing of genes involved in pre-mRNA splicing in RPE and retinal organoidsPrpf31 haploinsufficiency results in altered splicing of genes involved in pre-mRNA splicing in mouse retinaRPE cells display loss of polarity, reduced barrier function and phagocytosisPhotoreceptors display shorter and fewer cilia and degenerative featuresRPE cells display most abnormalities suggesting they might be the primary site of pathogenesisIn situ gene editing corrects the mutation and rescues key phenotypes

The m.3243A > G mitochondrial DNA mutation was originally described in patients with mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes. The phenotypic spectrum of the m.3243A > G mutation has since expanded to include a spectrum of neuromuscular and ocular manifestations, including reduced vision with retinal degeneration, the underlying mechanism of which remains unclear. We used dermal fibroblasts, from patients with retinal pathology secondary to the m.3243A > G mutation to generate heteroplasmic induced pluripotent stem cell (hiPSC) clones. RPE cells differentiated from these hiPSCs contained morphologically abnormal mitochondria and melanosomes, and exhibited marked functional defects in phagocytosis of photoreceptor outer segments. These findings have striking similarities to the pathological abnormalities reported in RPE cells studied from post-mortem tissues of affected m.3243A > G mutation carriers. Overall, our results indicate that RPE cells carrying the m.3243A > G mutation have a reduced ability to perform the critical physiological function of phagocytosis. Aberrant melanosomal morphology may potentially have consequences on the ability of the cells to perform another important protective function, namely absorption of stray light. Our in vitro cell model could prove a powerful tool to further dissect the complex pathophysiological mechanisms that underlie the tissue specificity of the m.3243A > G mutation, and importantly, allow the future testing of novel therapeutic agents.

Age-related macular degeneration (AMD) is the most common cause of blindness, accounting for 8.7% of all blindness globally. Vision loss is caused ultimately by apoptosis of the retinal pigment epithelium (RPE) and overlying photoreceptors. Treatments are evolving for the wet form of the disease; however, these do not exist for the dry form. Complement factor H polymorphism in exon 9 (Y402H) has shown a strong association with susceptibility to AMD resulting in complement activation, recruitment of phagocytes, RPE damage, and visual decline. We have derived and characterized induced pluripotent stem cell (iPSC) lines from two subjects without AMD and low-risk genotype and two patients with advanced AMD and high-risk genotype and generated RPE cells that show local secretion of several proteins involved in the complement pathway including factor H, factor I, and factor H-like protein 1. The iPSC RPE cells derived from high-risk patients mimic several key features of AMD including increased inflammation and cellular stress, accumulation of lipid droplets, impaired autophagy, and deposition of "drüsen"-like deposits. The low- and high-risk RPE cells respond differently to intermittent exposure to UV light, which leads to an improvement in cellular and functional phenotype only in the high-risk AMD-RPE cells. Taken together, our data indicate that the patient specific iPSC model provides a robust platform for understanding the role of complement activation in AMD, evaluating new therapies based on complement modulation and drug testing. Stem Cells 2017;35:2305-2320.

Background: Sapacitabine is an oral nucleoside analogue that is metabolised in-vivo to CNDAC (2′-C-cyano-2′-deoxy-1-β-D-arabino-pentofuranosylcytosine). It has a novel mechanism of action by causing single-strand breaks and replication-associated double-strand breaks (DSBs) after its incorporation into DNA. DSB, if unrepaired, are cytotoxic and are predominantly repaired by homologous recombination (HR). Over 50% of high-grade serous ovarian cancers are estimated to be defective in HR, only a small proportion of which are accounted for by germline BRCA mutations. HR-defective ovarian cancers have been shown to be sensitive to PARP inhibitors ex-vivo and platinum in-vivo and we therefore hypothesised that HR defective ovarian cancers would be sensitive to CNDAC and aimed to explore the potential use of CNDAC in ovarian cancer stratified for HR function. Methods: Primary cultures (PCO) were generated from ascitic fluid collected from patients undergoing ovarian cancer surgery, characterised and confirmed to be ovarian and epithelial in origin based upon morphology, expression of immunofluorescent-labelled markers and cross-referencing with formal examination of FFPE tissue and cytology of ascites. The functional status of HR was determined for each cell line and PCO culture by quantification of nuclear Rad51 focus formation following induction of DNA DSB. SRB assays were used to calculate the GI50 for cisplatin and CNDAC, (Cyclacel, UK), in cell lines nad PCO cultures. Results: As hypothesised, HR defective cell lines were highly sensitive to CNDAC (VC8 BRCA2 mutant: GI50=366 nM) in comparison to their matched HR competent cell lines (VC8-BRCA2 reconstructed: GI50=18 μM; VC8-PARPi-resistant BRCA revertant: GI50=3 μM). Sensitivity to CNDAC in patient PCO cultures (n=39) was heterogeneous with a mean GI50 of 297 nM (95% CI 136-646 nM). Stratifying PCO cultures according to their HR status demonstrated greater sensitivity in HR defective cultures with a mean GI50 of 135 nM (46-394; n=12) in defective cultures, compared to 477 nM in HR competent cultures (164-1386; n=27). Of the 34 cisplatin resistant PCO cultures (GI50 >5 μM), 20 (59%) remained sensitive to CNDAC with a mean GI50 of 426 nM (148-1223; n=13) in HR competent cultures and a GI50 of 230 nM (70-759; n=7) in HR defective cultures. CNDAC sensitivity was evaluated in base excision repair (BER) defective EM9 cell line (GI50=374 nM) compared with parental wild type AA8 cells (GI50=6.9 μM), p Conclusions: Cell lines defective in HR or BER are highly sensitive to CNDAC suggesting a possible use of HR or BER status as biomarkers for the stratified use of CNDAC in the treatment of ovarian cancer. When applied to ex-vivo studies HR status as a single marker cannot reliably predict sensitivity and a panel of pathways may therefore be needed to enable reliable treatment stratification. Nevertheless, stratification based on these markers is likely to permit enrichment of patients more likely to respond to CNDAC. Additionally CNDAC may have a role in the treatment of platinum resistant disease as approximately 60% of patients with resistant disease may benefit. Further work is required to explore the possibility of CNDAC as a chemosensitiser in platinum sensitive disease and as a second line agent in platinum resistant disease. Citation Format: Rachel L. O'Donnell, James CC Murray, Adriana GB Buskin, Sheelagh Frame, David G. Blake, Michelle Dixon, Aiste McCormick, Angelika Kaufmann, E Ruth Plummer, Richard J. Edmondson, Nicola J. Curtin. Therapeutic potential of sapacitabine in ovarian cancers defective in homologous recombination. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A33.