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The occurrence of repetitive genomic changes that provide a selective growth advantage in pluripotent stem cells is of concern for their clinical application. However, the effect of different culture conditions on the underlying mutation rate is unknown. Here we show that the mutation rate in two human embryonic stem cell lines derived and banked for clinical application is low and not substantially affected by culture with Rho Kinase inhibitor, commonly used in their routine maintenance. However, the mutation rate is reduced by >50% in cells cultured under 5% oxygen, when we also found alterations in imprint methylation and reversible DNA hypomethylation. Mutations are evenly distributed across the chromosomes, except for a slight increase on the X-chromosome, and an elevation in intergenic regions suggesting that chromatin structure may affect mutation rate. Overall the results suggest that pluripotent stem cells are not subject to unusually high rates of genetic or epigenetic alterations. Mutations in human pluripotent stem cells (PSC) and whether any form during culture prior to use in a human clinical context are a concern. Here, the authors use hPSCs derived to cGMP standards and show they have low mutation rates after culture, noting this decreases on culturing in low (5%) oxygen conditions.

The Ataxia telangiectasia and Rad3-related (ATR) inhibitor ceralasertib in combination with the PD-L1 antibody durvalumab demonstrated encouraging clinical benefit in melanoma and lung cancer patients who progressed on immunotherapy. Here we show that modelling of intermittent ceralasertib treatment in mouse tumor models reveals CD8 + T-cell dependent antitumor activity, which is separate from the effects on tumor cells. Ceralasertib suppresses proliferating CD8 + T-cells on treatment which is rapidly reversed off-treatment. Ceralasertib causes up-regulation of type I interferon (IFNI) pathway in cancer patients and in tumor-bearing mice. IFNI is experimentally found to be a major mediator of antitumor activity of ceralasertib in combination with PD-L1 antibody. Improvement of T-cell function after ceralasertib treatment is linked to changes in myeloid cells in the tumor microenvironment. IFNI also promotes anti-proliferative effects of ceralasertib on tumor cells. Here, we report that broad immunomodulatory changes following intermittent ATR inhibition underpins the clinical therapeutic benefit and indicates its wider impact on antitumor immunity. The ATR inhibitor ceralasertib has shown clinical activity in combination with immune-checkpoint inhibitors in several cancer types. Here the authors report the anti-tumor activity and the immunomodulatory changes, dependent on up-regulation of type I interferon pathway, following intermittent ATR inhibition in preclinical cancer models.

Background Loss of motor neurons in amyotrophic lateral sclerosis (ALS) leads to progressive paralysis and death. Dysregulation of thousands of RNA molecules with roles in multiple cellular pathways hinders the identification of ALS-causing alterations over downstream changes secondary to the neurodegenerative process. How many and which of these pathological gene expression changes require therapeutic normalisation remains a fundamental question. Methods Here, we investigated genome-wide RNA changes in C9ORF72-ALS patient-derived neurons and Drosophila , as well as upon neuroprotection taking advantage of our gene therapy approach which specifically inhibits the SRSF1-dependent nuclear export of pathological C9ORF72 -repeat transcripts. This is a critical study to evaluate (i) the overall safety and efficacy of the partial depletion of SRSF1, a member of a protein family involved itself in gene expression, and (ii) a unique opportunity to identify neuroprotective RNA changes. Results Our study shows that manipulation of 362 transcripts out of 2257 pathological changes, in addition to inhibiting the nuclear export of repeat transcripts, is sufficient to confer neuroprotection in C9ORF72-ALS patient-derived neurons. In particular, expression of 90 disease-altered transcripts is fully reverted upon neuroprotection leading to the characterisation of a human C9ORF72-ALS disease-modifying gene expression signature. These findings were further investigated in vivo in diseased and neuroprotected Drosophila transcriptomes, highlighting a list of 21 neuroprotective changes conserved with 16 human orthologues in patient-derived neurons. We also functionally validated the high neuroprotective potential of one of these disease-modifying transcripts, demonstrating that inhibition of ALS-upregulated human KCNN1–3 ( Drosophila SK) voltage-gated potassium channel orthologs mitigates degeneration of human motor neurons and Drosophila motor deficits. Conclusions Strikingly, the partial depletion of SRSF1 leads to expression changes in only a small proportion of disease-altered transcripts, indicating that not all RNA alterations need normalization and that the gene therapeutic approach is safe in the above preclinical models as it does not disrupt globally gene expression. The efficacy of this intervention is also validated at genome-wide level with transcripts modulated in the vast majority of biological processes affected in C9ORF72-ALS. Finally, the identification of a characteristic signature with key RNA changes modified in both the disease state and upon neuroprotection also provides potential new therapeutic targets and biomarkers.

Of familial amyotrophic lateral sclerosis (fALS) cases, 20% are caused by mutations in the gene encoding human cytosolic Cu/Zn superoxide dismutase (hSOD1). Efficient translation of the therapeutic potential of RNAi for the treatment of SOD1-ALS patients requires the development of vectors that are free of significant off-target effects and with reliable biomarkers to discern sufficient target engagement and correct dosing. Using adeno-associated virus serotype 9 to deliver RNAi against hSOD1 in the SOD1G93A mouse model, we found that intrathecal injection of the therapeutic vector via the cisterna magna delayed onset of disease, decreased motor neuron death at end stage by up to 88%, and prolonged the median survival of SOD1G93A mice by up to 42%. To our knowledge, this is the first report to demonstrate no significant off-target effects linked to hSOD1 silencing, providing further confidence in the specificity of this approach. We also report the measurement of cerebrospinal fluid (CSF) hSOD1 protein levels as a biomarker of effective dosing and efficacy of hSOD1 knockdown. Together, these data provide further confidence in the safety of the clinical therapeutic vector. The CSF biomarker will be a useful measure of biological activity for translation into human clinical trials. Development of off-target effect-free AAV9-shRNA-mediated SOD1 silencing in an ALS mouse model, with reliable biomarkers to discern sufficient target engagement and correct dosing.

Insulin-like growth factor-I (IGF-I) provides pivotal cell survival and differentiation signals during inner ear development throughout evolution. Homozygous mutations of human IGF1 cause syndromic sensorineural deafness, decreased intrauterine and postnatal growth rates, and mental retardation. In the mouse, deficits in IGF-I result in profound hearing loss associated with reduced survival, differentiation and maturation of auditory neurons. Nevertheless, little is known about the molecular basis of IGF-I activity in hearing and deafness. A combination of quantitative RT-PCR, subcellular fractionation and Western blotting, along with in situ hybridization studies show IGF-I and its high affinity receptor to be strongly expressed in the embryonic and postnatal mouse cochlea. The expression of both proteins decreases after birth and in the cochlea of E18.5 embryonic Igf1(-/-) null mice, the balance of the main IGF related signalling pathways is altered, with lower activation of Akt and ERK1/2 and stronger activation of p38 kinase. By comparing the Igf1(-/-) and Igf1(+/+) transcriptomes in E18.5 mouse cochleae using RNA microchips and validating their results, we demonstrate the up-regulation of the FoxM1 transcription factor and the misexpression of the neural progenitor transcription factors Six6 and Mash1 associated with the loss of IGF-I. Parallel, in silico promoter analysis of the genes modulated in conjunction with the loss of IGF-I revealed the possible involvement of MEF2 in cochlear development. E18.5 Igf1(+/+) mouse auditory ganglion neurons showed intense MEF2A and MEF2D nuclear staining and MEF2A was also evident in the organ of Corti. At P15, MEF2A and MEF2D expression were shown in neurons and sensory cells. In the absence of IGF-I, nuclear levels of MEF2 were diminished, indicating less transcriptional MEF2 activity. By contrast, there was an increase in the nuclear accumulation of FoxM1 and a corresponding decrease in the nuclear cyclin-dependent kinase inhibitor p27(Kip1). We have defined the spatiotemporal expression of elements involved in IGF signalling during inner ear development and reveal novel regulatory mechanisms that are modulated by IGF-I in promoting sensory cell and neural survival and differentiation. These data will help us to understand the molecular bases of human sensorineural deafness associated to deficits in IGF-I.

Background Most analyses of microarray data are based on point estimates of expression levels and ignore the uncertainty of such estimates. By determining uncertainties from Affymetrix GeneChip data and propagating these uncertainties to downstream analyses it has been shown that we can improve results of differential expression detection, principal component analysis and clustering. Previously, implementations of these uncertainty propagation methods have only been available as separate packages, written in different languages. Previous implementations have also suffered from being very costly to compute, and in the case of differential expression detection, have been limited in the experimental designs to which they can be applied. Results puma is a Bioconductor package incorporating a suite of analysis methods for use on Affymetrix GeneChip data. puma extends the differential expression detection methods of previous work from the 2-class case to the multi-factorial case. puma can be used to automatically create design and contrast matrices for typical experimental designs, which can be used both within the package itself but also in other Bioconductor packages. The implementation of differential expression detection methods has been parallelised leading to significant decreases in processing time on a range of computer architectures. puma incorporates the first R implementation of an uncertainty propagation version of principal component analysis, and an implementation of a clustering method based on uncertainty propagation. All of these techniques are brought together in a single, easy-to-use package with clear, task-based documentation. Conclusion For the first time, the puma package makes a suite of uncertainty propagation methods available to a general audience. These methods can be used to improve results from more traditional analyses of microarray data. puma also offers improvements in terms of scope and speed of execution over previously available methods. puma is recommended for anyone working with the Affymetrix GeneChip platform for gene expression analysis and can also be applied more generally.

Neutrophils are rapidly recruited to sites of tissue injury or infection, where they protect against invading pathogens. Neutrophil functions are limited by a process of neutrophil senescence, which renders the cells unable to respond to chemoattractants, carry out respiratory burst, or degranulate. In parallel, aged neutrophils also undergo spontaneous apoptosis, which can be delayed by factors such as GMCSF. This is then followed by their subsequent removal by phagocytic cells such as macrophages, thereby preventing unwanted inflammation and tissue damage. Neutrophils translate mRNA to make new proteins that are important in maintaining functional longevity. We therefore hypothesised that neutrophil functions and lifespan might be regulated by microRNAs expressed within human neutrophils. Total RNA from highly purified neutrophils was prepared and subjected to microarray analysis using the Agilent human miRNA microarray V3. We found human neutrophils expressed a selected repertoire of 148 microRNAs and that 6 of these were significantly upregulated after a period of 4 hours in culture, at a time when the contribution of apoptosis is negligible. A list of predicted targets for these 6 microRNAs was generated from http://mirecords.biolead.org and compared to mRNA species downregulated over time, revealing 83 genes targeted by at least 2 out of the 6 regulated microRNAs. Pathway analysis of genes containing binding sites for these microRNAs identified the following pathways: chemokine and cytokine signalling, Ras pathway, and regulation of the actin cytoskeleton. Our data suggest that microRNAs may play a role in the regulation of neutrophil senescence and further suggest that manipulation of microRNAs might represent an area of future therapeutic interest for the treatment of inflammatory disease.

BackgroundCeralasertib is a specific ATR inhibitor (ATRi) that hinders the DNA damage response in tumor cells, making them more susceptible to death in situations of high replication stress. Ceralasertib in combination with the PD-L1 antibody durvalumab demonstrated encouraging clinical benefit in melanoma and lung cancer patients who progressed on immunotherapy. The mechanism of this effect remained unclear.MethodsIn this study, we employed different mouse tumor models treated with ceralasertib to investigate the effect of the ATRi on tumor microenvironment.ResultsAntitumor effect of ceralasertib was dependent on the presence of CD8 cells, since in vivo depletion of CD8 cells abrogated therapeutic effect of the ATRi. Analysis of the gene expression profile using RNAseq demonstrated significant up-regulation of type I interferon (IFNI) pathway in tumors of mice treated with ceralasertib. Neutralizing anti IFNI receptor (IFNAR1) antibody abrogated antitumor effect of ceralasertib. Antitumor effect of ceralasertib in combination with anti-PD-L1 was eliminated in mice reconstituted with bone marrow from IFNAR1-KO mice. Reconstitution with bone marrow from mice with constitutively active IFNAR1 markedly enhanced antitumor effect of ceralasertib. Treatment of tumor-bearing mice with ceralasertib caused accumulation of DCs with activated phenotype (up-regulation of CD40, CD86, MHC class II). DCs isolated from tumor of ceralasertib-treated mice demonstrated an enhanced ability to stimulate T cells in a mixed lymphocyte reaction assay. Notably, this effect was reversed when DCs were isolated from IFNAR1 KO mice. Moreover, in vitro-generated DCs treated with ceralasertib exhibited activation similar to that induced by lipopolysaccharide (LPS), but this effect was not observed in DCs generated from IFNAR1 KO mice. Treatment with ceralasertib led to the depletion in tumor of M-MDSC and TAMs, but not PMN-MDSC. However, PMN-MDSCs suppressive activity was abrogated after the treatment. This was associated with increased IFNI signature observed in tumor PMN-MDSC. Treatment with ceralasertib enhanced tumor antigen-specific response of T cells.ConclusionsOur findings demonstrate IFNI mediated modulation of TME by ceralasertib resulting in enhanced antitumor activity of T cells and potentiated effect of PD-L1 antibody.

BackgroundRecent clinical trials have shown the potential therapeutic benefit of combining durvalumab (anti-PD-L1) with the ataxia telangiectasia and rad3-related (ATR) protein inhibitor ceralasertib (AZD6738).1 2 Here, we characterized the effect of ceralasertib on T cells, tumor cells, and cancer-associated fibroblasts (CAFs) in vitro to understand how ceralasertib may synergize with anti-PD-L1 in modulating various aspects of tumor immune biology.MethodsTo determine the effect of ceralasertib on CD8 T-cells, melanoma dissociated tumor cells (DTCs) or CD3/CD28-stimulated healthy donor (HD) CD8 T-cells, were cultured in the presence or absence of ceralasertib at 6 uM (concentration determined by pRAD50 EC50 (H-Score)). Changes to the phenotype of CD8 T-cells were analyzed by multi-parameter flow cytometry, and polyfunctionality was assessed by single-cell intracellular proteomics (Isoplexis technology). Changes in markers of immunogenic cell death (ICD) and senescence were assessed to determine the effect of ceralasertib on tumor cells and CAFs using melanoma (SKMEL-2) and fibroblast (IMR-90) cell lines. Statistical analysis was done using paired student t-test.ResultsTreatment of HD CD3/CD28-activated CD8 T-cells with 6 uM ceralasertib for 3 days led to a reduction (87.98% ± 2.34 of DMSO control; n=5) of T central memory (TCM) CD8 T-cells (CD3+CD8+CD45RA-CCR7+). Similarly, the frequency of TCM CD8 T-cells from DTCs was reduced when compared to DMSO (p=0.0085; n=7). Ceralasertib treatment reduced proliferation of HD TCM CD8 T-cells (CD3+CD8+KI67+CD45RA-CCR7+) (19.18% ± 2.67 of DMSO control; n=5) and increased the frequency of CD8 T-cells expressing gH2AX, consistent with ceralasertib’s mechanism of action on DNA damage. HD ceralasertib-treated CD3/CD28-activated CD8 T-cells demonstrated a higher Polyfunctional Strength Index (PSI), defined here as a greater number of cells secreting 2 or more of the following cytokines: GM-CSF, Granzyme B, IFN-g, MIP-1a, MIP-1b and TNF-a versus control-treated cells. Treatment of the melanoma cell line SKMEL-2 with ceralasertib for 3 days induced ICD (higher ATP, HMGB1, calreticulin and cleaved gasdermin D levels). Treatment for only 2 days and culture for an additional 5 days induced senescence (higher b-galactosidase activity, higher calreticulin and HLA Class I membrane surface expression). Interestingly, ceralasertib treatment of IMR-90 fibroblasts induced only senescence regardless of treatment length (2–7 days).ConclusionsCeralasertib combines with anti-PD-L1 to enhance anti-tumor activity in clinical studies. In vitro experiments demonstrate that ceralasertib has pro-immunogenic effects on multiple cell types of the tumor microenvironment, which may contribute to the therapeutic benefit of ceralasertib when combined with durvalumab in patients.ReferencesKwon M, Kim G, Kim R, Kim KT, Kim ST, Smith S, Mortimer PGS, Hong JY, Loembé AB, Irurzun-Arana I, Koulai L, Kim KM, Kang WK, Dean E, Park WY, Lee J. Phase II study of ceralasertib (AZD6738) in combination with durvalumab in patients with advanced gastric cancer. J Immunother Cancer. 2022 Jul;10(7)Kim R, Kwon M, An M, Kim ST, Smith SA, Loembé AB, Mortimer PGS, Armenia J, Lukashchuk N, Shah N, Dean E, Park WY, Lee. Phase II study of ceralasertib (AZD6738) in combination with durvalumab in patients with advanced/metastatic melanoma who have failed prior anti-PD-1 therapy. J.Ann Oncol. 2022 Feb;33(2):193–203.Ethics ApprovalAll suppliers met AstraZeneca’s HBS standards and provided informed consent.

BackgroundThe combination of an ATR inhibitor ceralasertib and anti-PD-L1 antibody durvalumab is being tested in Phase III clinical trials in patients who have progressed on prior immunotherapy. Preclinical experiments were performed to build a greater understanding of the potential immune driven mechanisms-of-action by which ceralasertib enhances antitumor efficacy in combination with anti-PD-L1 in the context of the clinical dose and schedule.MethodsTo assess the antitumor efficacy and associated pharmacodynamic effects ceralasertib ceralasertib was administered to CT26 tumor-bearing BALB/c immunocompetent mice twice daily with continuously and intermittently 7 day-on/7 day-off schedules alone or in combination with PD-L1 blockade. Flow cytometry and immunohistochemistry was used to quantify CD8+ T-cells when on and off ceralasertib treatment. Immunophenotyping was performed using single-cell CyTOF protein expression mass cytometry and bulk tumor or single-cell RNA sequencing (scRNA-seq) transcriptomics analysis to evaluate intratumoral T-cell populations.ResultsModelling of intermittent ceralasertib dosing regimen in mouse tumor models revealed CD8+ T-cell and type I interferon (IFNI) dependent antitumor activity, which was enhanced in combination with anti-PD-L1 immune checkpoint blockade. Ceralasertib suppressed highly proliferating CD8+ T-cells when on-treatment which was rapidly reversed when off-treatment. This was linked to significant relative reductions in T-cells which displayed markers commonly associated with an exhausted phenotype and a concomitant increase in cells with naïve non-activated phenotypes with improved T-cell function in the tumor microenvironment. Continuous daily administration of ceralasertib led to the sustained suppression of CD8+ T-cells and impaired antitumor activity compared to intermittent dosing. In addition, ceralasertib caused up-regulation of type I interferon (IFNI) which may promote an inflammatory environment and drive direct anti-proliferative effects on tumor cells.ConclusionsBroad immunostimulatory and immunomodulatory changes following intermittent ATR treatment in combination with immune checkpoint blockade may underpin the durable clinical therapeutic benefit observed in patients and indicates its potential wider impact on antitumor immunity.