Aberrant epigenetic regulation is a hallmark of diffuse midline glioma (DMG), an incurable pediatric brain tumor. The H3K27M driver histone mutation leads to transcriptional dysregulation, indicating that targeting the epigenome and transcription may be key therapeutic strategies against this highly aggressive cancer. One such target is the facilitates chromatin transcription (FACT) histone chaperone. We found FACT to be enriched at developmental gene promoters, coinciding with open chromatin and binding motifs of core DMG regulatory transcription factors. Furthermore, FACT co-occurred with the bromodomain and extraterminal domain (BET) protein BRD4 at promoters and enhancers, suggesting functional cooperation between FACT and BRD4 in DMG. In vitro, a combinatorial therapeutic approach using the FACT inhibitor CBL0137, coupled with BET inhibition, revealed potent and synergistic cytotoxicity across a range of DMG cultures. These results were recapitulated in vivo, extending survival in three independent orthotopic patient-derived xenograft models of DMG. Mechanistically, we show that CBL0137 treatment decreased chromatin accessibility and combined with BET inhibition to cause broad transcriptional collapse; silencing of several key oncogenes including MYC, PDGFRA, MDM4, and SOX2; and alterations to the splicing landscape. This combination also elicited immune-related effects, including activation of the interferon response and antigen presentation mechanisms in DMG cells and induction of an activated state in macrophages and T cells, as demonstrated in an immunocompetent setting with spatial transcriptomics. Together, our data highlight the therapeutic promise of simultaneously targeting FACT and BET proteins in DMG, offering a dual tumor-intrinsic and immune-mediated strategy for combating this devastating pediatric brain tumor.

Background Diffuse midline glioma, characterized by H3K27 alteration (DMG), is the predominant high-grade glioma in children. It commonly originates in the brainstem, yet effective treatments for these patients remain elusive.Methods To identify novel therapies for DMG, we conducted high-throughput drug screens (HTS) using biologically active, clinically approved compounds against DMG neurospheres. Multiple primary DMG cultures were utilized in conjunction with in vitro cytotoxicity and clonogenic assays to validate the efficacy of top compounds. Molecularly diverse patient-derived and transgenic DMG orthotopic models were employed to assess therapeutic efficacy alongside pharmacokinetic and immunohistochemical analyses. Mechanistic studies, including RNA sequencing, western blotting, and flow cytometry, were conducted to elucidate the antitumor efficacy of the most promising compound, fenretinide, in DMG cells.Results Through HTS, 6 compounds were identified and validated for their potent cytotoxic activity. However, most of these compounds failed to improve survival in an orthotopic Diffuse Midline Glioma (DMG) model due to limited blood-brain barrier (BBB) penetration. In contrast, fenretinide exhibited effective BBB penetration, significantly enhancing the survival of tumor-bearing animals. Mechanistic studies revealed that fenretinide increased reactive oxygen species (ROS) generation and induced apoptosis by inhibiting PDGFR alpha. RNA-sequencing further elucidated that fenretinide upregulates the Unfolded Protein Response (UPR) and endoplasmic reticulum (ER) stress pathways while downregulating neurogenesis. The in vivo antitumor efficacy of 2 fenretinide formulations was demonstrated in PDGFR alpha-amplified and transgenic DMG models.Conclusion This comprehensive study has identified new DMG therapeutic vulnerabilities and highlights fenretinide as a brain-penetrant, anti-DMG agent.

Abstract BACKGROUND Diffuse Midline Gliomas (DMGs), characterised by prevalent H3K27M mutations, exhibit epigenetic modifications that drive a proliferative state resistant to conventional therapies. The interplay of the cell cycle with PI3K and RAS/MYC growth signalling pathways, pivotal in the context of the H3K27M mutation, is yet to be fully understood. The aim of this study was to assess the efficacy of the inhibition of Polo-like kinase 1 (PLK1), a critical regulator of mitosis and cell cycle progression, identify mechanisms of action, and to develop effective combination strategies against DMG. METHODS AND RESULTS PLK1 inhibition demonstrated significant anti-tumour activities both in vitro and within aggressive in vivo orthotopic DMG models, notably doubling the median survival of mice bearing tumours. High-throughput RNA-seq analysis of DMG samples particularly sensitive to PLK1 inhibition, revealed a distinct pattern of negative enrichment in ribosomal genes, indicating a marked reduction in ribosomal function-associated gene expression. This alteration suggests a potential for innovative drug combination strategies targeting the PI3K/mTOR and RAS/MYC pathways, crucial for ribosomal and RNA processing. Subsequently, our in vitro drug sensitivity assays demonstrated that PLK1 inhibition in combination with PI3K/mTOR and MEK inhibitors, exhibit strong drug synergies, evidenced by high Bliss synergy scores. CONCLUSIONS Our findings underscore the efficacy of simultaneous targeting of PI3K/mTOR and RAS/MYC pathways with PLK1 inhibition as an attractive strategy to mitigate DMG tumorigenesis. Furthermore this research not only sheds light on the crucial interplay between cell cycle and signalling pathways in DMG but also facilitates novel intervention strategies for a range of other paediatric CNS malignancies with similar characteristic molecular drivers.

Abstract Aberrant epigenetic regulation is a hallmark of Diffuse Midline Glioma (DMG), an incurable tumor that primarily arises in the brainstem of young children. The H3K27M driver histone mutation and resulting permissive chromatin indicates epigenetic targeting as a key therapeutic strategy against this aggressive cancer. One such epigenetic target is the Facilitates Chromatin Transcription (FACT) histone chaperone. FACT is targeted by the curaxin compound CBL0137 which is currently in phase I/II clinical trial for pediatric cancer patients (NCT04870944). This study aims to decipher the mechanism of CBL0137 to find effective combination therapies for the next round of clinical implementation. To this end, we applied a multi-omics approach by integrating epigenetic profiling with transcriptomic data sets (CUT&RUN, ATAC-seq and RNA-seq) to interrogate the therapeutic mechanism of CBL0137 in DMG. We then employed in vitro cytotoxicity assays and in vivo orthotopic patient-derived xenograft (PDX) pre-clinical models to test a novel, mechanistic-anchored epigenetic combination therapy. We found FACT to be enriched at the promoters of developmental genes, coinciding with regions of open chromatin and binding motifs of the core DMG regulatory transcription factors TCF12 and OLIG2. Furthermore, FACT interacted and co-localized with the Bromodomain and Extra-Terminal Domain (BET) protein BRD4, suggesting cooperation between FACT and BRD4 in DMG. Given that BRD4 is an established therapeutic target in DMG and other cancers, we investigated the combination of the FACT-inhibitor CBL0137 with the BET-inhibitor JQ1. Combination treatment was cytotoxic against DMG in vitro, with enhanced cytotoxicity observed against H3K27M-mutant cells. We demonstrated a significant survival extension in three independent orthotopic PDX mouse models treated with CBL0137 and JQ1 in combination. Mechanistically, CBL0137 and JQ1 decreased chromatin accessibility and transcription of genes involved in RNA regulatory processes, such as RNA methylation and splicing. Consistently, combination treatment led to increased intron retention, highlighting a possible link between chromatin disruption and the splicing machinery in DMG. Future work will investigate whether treatment-induced intron retention acts as a source of neoantigens, with the potential to enhance DMG immunogenicity. In sum, this study has identified a promising new epigenetic combination therapy – FACT + BET inhibition – while uncovering intriguing new insights into DMG epigenetics and pathobiology. Citation Format: Holly Holliday, Aaminah Khan, Nisitha Jayatilleke, Chelsea Mayoh, Samuel E. Ross, Yolanda Colino Sanguino, Anjana Gopalakrishnan, Anahid Ehteda, Benjamin Rayner, Maria Tsoli, David S. Ziegler. Targeting the epigenome through combined Facilitates Chromatin Transcription (FACT) and Bromodomain and Extra-Terminal Domain (BET) inhibition in Diffuse Midline Glioma (DMG) [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr B016.

Protein post-translational modifications (PTMs) alter the properties of protein through proteolytic cleavage, or the addition of modifying groups to one or more amino acids. Histones are major targets of PTMs, including through SUMOylation of lysine residues and citrullination of arginine residues, with both processes able to regulate gene transcription. We aimed to delineate the potential role of SUMOylation and citrullination in the epigenetic dysregulation that drives Diffuse Midline Glioma (DMG) tumorigenicity. DMG tumours demonstrated an upregulation of SUMOylation machinery pathway components and increased expression of peptidylarginine deiminases (PADs), the enzymes responsible for citrullination. Nanomolar doses of the SUMOylation inhibitor TAK-981 blocks DMG cell cycle G2/M transition and results in potent decreases in cell viability and clonogenic potential, triggering ‘viral mimicry’ in DMG cells, resulting in increased expression of interferon stimulated genes and inflammatory mediators including IL-1β, IL-6 and TNFα, whilst also causing a pro-inflammatory M1 macrophage response. These data suggest that SUMOylation inhibition may lead to a double-punch effect against DMG by inhibiting tumorgenicity and stimulating an immune response. In further studies, we have shown that pharmacological inhibition of PAD activity in DMG decreases histone citrullination and restores histone methylation – considered the Achilles heel of DMG. This was accompanied by induction of reactive oxygen species and an unfolded protein response, resulting in DMG cell death in a dose dependent manner. In addition to fully characterising the epigenetic and global changes within DMG initiated through SUMOylation or PAD inhibition, we are currently performing preliminary drug efficacy studies in orthotopic DMG models, providing the necessary pre-clinical research pipeline towards an effective treatment for DMG. Protein SUMOylation and citrullination are previously unrecognised therapeutic targets in DMG and potentially play key roles in tumour initiation and pathogenesis. Our data suggests a novel therapeutic approach for children with DMG.

Objective: Myocardial infarction (MI) is the primary cause of ischaemic heart disease. ApoA-I, the main apolipoprotein constituent of high-density lipoproteins (HDLs) has anti-inflammatory properties and protects against post-MI ischemia/reperfusion (I/R) injury. However, HDLs from MI survivors do not protect against I/R injury. This study asks whether a newly developed peptide that has anti-inflammatory properties can mimic the ability of apoA-I to reduce I/R injury in a rat model of MI, and protect myocardial fibroblasts from hypoxic damage. Methods: The left anterior descending (LAD) coronary artery of Wistar rats (n≥6/group) was temporarily ligated for 1 h to induce I/R injury. Rats that underwent the procedure were randomly allocated to no treatment or treatment with PBS or peptide. The procedure was also performed on rats without LAD ligation (sham control). The rats were treated immediately after the procedure with peptide (10 mg/kg, ip) or PBS, then re-treated every second day for 7 days. H9C2 myocardial fibroblasts were incubated for 24 h under hypoxic (0.4 % oxygen) conditions ± peptide (0.05-1 mg/ml). Results: Peptide treatment improved the ejection fraction of rats that underwent LAD ligation by 16.4±7% (p<0.05) compared to control. It also reduced fibrosis in the left ventricle by 12.6±2.6% (p<0.0001). These results were recapitulated in vitro in myocardial fibroblasts incubated under hypoxic conditions, with peptide treatment (1 mg/ml) decreasing cell death by 19.8±4.0% (p<0.05). Conclusions: The peptide protects myocardial cell viability and reduces myocardial death and is therefore of potential therapeutic benefit for MI survivors with I/R injury.

Abstract The BRAFV600E mutation has recently been discovered as a driver mutation in a subset of pediatric high-grade gliomas (pHGG), with tumors driven by this mutation responding to targeted BRAF and MEK inhibitors. Unfortunately, resistance inevitability develops, resulting in disease progression. There is currently limited understanding of the mechanisms underlying drug resistance in BRAFV600E pHGG, and no effective counter-therapies exist. This study aimed to identify driver pathways of resistance in BRAFV600E pHGG and subsequently novel therapeutic targets. BRAF inhibitor-resistant, MEK inhibitor-resistant and BRAF+MEK inhibitor-resistant BRAFV600E pHGG cultures were derived through chronic exposure of a BRAFV600E patient-derived culture to vemurafenib (BRAF inhibitor), trametinib (MEK inhibitor) or a combination of both drugs, respectively. Cytotoxicity assays confirmed resistance. All resistant cell lines spontaneously changed from a spheroid phenotype to adherent growth, indicating changes in tumor characteristics. RNAseq identified > 1500 genes of interest, with subsequent Gene Set Enrichment Analysis identifying key pathways as novel drivers of resistance, the top-ranked gene sets being neural development and plasma membrane/cell adhesion. Approximately 45% of gene sets were enriched across all drug-resistant lines, indicating overlapping resistance mechanisms. Interrogation of the receptor tyrosine kinase (RTK) gene sets identified potent upregulation of several RTKs. Notably, overexpression of the RTK, EGFR, was confirmed through RNAseq and western blot as a potential key mediator of the acquired resistance. Combined treatment with vemurafenib and the EGFR inhibitor, dacomitinib, resulted in synergistic activity against both vemurafenib-resistant BRAFV600E pHGG cells and matched parental cells, providing a viable therapeutic option for drug-resistant BRAFV600E pHGG. Overall, novel drivers of resistance have been identified in BRAFV600E pHGG. EGFR overexpression was pinpointed as a key mediator and dual BRAF/EGFR inhibition displayed promising efficacy as a therapeutic option. Future studies will continue to elucidate the mechanisms of resistance using multi-omic approaches and the in vivo efficacy of dual BRAF/EGFR inhibition.

Heart failure (HF) is the leading cause of hospitalisations worldwide, with only 35% of patients surviving the first 5 years after diagnosis. The pathogenesis of HF with preserved ejection fraction (HFpEF) is still unclear, impeding the implementation of effective treatments. FK506-binding protein like (FKBPL) and its therapeutic peptide mimetic, AD-01, are critical mediators of angiogenesis and inflammation. Thus, in this study, we investigated—for the first time—FKBPL’s role in the pathogenesis and as a biomarker of HFpEF. In vitro models of cardiac hypertrophy following exposure to a hypertensive stimulus, angiotensin-II (Ang-II, 100 nM), and/or AD-01 (100 nM), for 24 and 48 h were employed as well as human plasma samples from people with different forms of HFpEF and controls. Whilst the FKBPL peptide mimetic, AD-01, induced cardiomyocyte hypertrophy in a similar manner to Ang-II (p < 0.0001), when AD-01 and Ang-II were combined together, this process was abrogated (p < 0.01–0.0001). This mechanism appears to involve a negative feedback loop related to FKBPL (p < 0.05). In human plasma samples, FKBPL concentration was increased in HFpEF compared to controls (p < 0.01); however, similar to NT-proBNP and Gal-3, it was unable to stratify between different forms of HFpEF: acute HFpEF, chronic HFpEF and hypertrophic cardiomyopathy (HCM). FKBPL may be explored for its biomarker and therapeutic target potential in HFpEF.

Diffuse Midline Gliomas (DMGs) are devastating and incurable pediatric brain tumours. DMGs are characterised by epigenetic aberrations in the form of histone modifications, coupled with mitotic abnormalities, which together drive a stem-like transcriptional cell state that is critically dependent on the cell cycle. Polo-like kinase 1 (PLK1) is a pivotal regulator of mitosis and cell cycle progression. To determine an effective therapeutic strategy against DMG, we characterised the anti-tumour potential of PLK1 inhibition, either alone or in combination with radiotherapy. We show that targeting PLK1 with small molecule inhibitors BI2536 and BI6727 (volasertib) reduced DMG colony formation and cell proliferation, and induced G2/M checkpoint arrest and apoptosis in vitro at physiologically achievable, clinically relevant concentrations. When combined with radiotherapy, PLK1 inhibitors acted as potent radiosensitisers with synergistic suppression of DMG survival. We further show that PLK1 inhibitors exert potent anti-tumour effects in highly aggressive orthotopic DMG models, effectively extending the median survival of tumour harbouring mice both as a single agent and enhanced in combination with radiotherapy. We further demonstrate a potential novel role for PLK1 in defining an oncogenic transcriptional state in DMG by acting as a conduit between cell cycle progression and the DMG epigenome through modulation of polycomb repressive complex 2 (PRC2) activity and its resultant histone methyltransferase function. Together, these data indicate that targeting PLK1 is a highly promising therapeutic strategy to arrest DMG tumorgenicity. This data has formed the basis for a planned international Phase 1/2 trial of the PLK1 inhibitor volasertib in combination with radiotherapy in paediatric patients with DMG. Citation Format: Elisha Hayden, Laura Franshaw, Dannielle Upton, Jie Liu, Hieu Nguyen, Swapna Joshi, Maria Tsoli, Emmy Dolman, Benjamin Rayner, David Ziegler. Preclinical examination of PLK1 inhibitors for the treatment of diffuse midline gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1561.

The BRAFV600E mutation has recently been discovered as a driver mutation in a subset of pediatric high-grade gliomas (pHGG), with tumors driven by this mutation responding to targeted BRAF and MEK inhibitors. Unfortunately, resistance inevitability develops, resulting in disease progression. There is currently limited understanding of the mechanisms underlying drug resistance in BRAFV600E pHGG, and no effective counter-therapies exist. This study aimed to identify driver pathways of resistance in BRAFV600E pHGG and subsequently novel therapeutic targets. BRAF inhibitor-resistant, MEK inhibitor-resistant and BRAF+MEK inhibitor-resistant BRAFV600E pHGG cultures were derived through chronic exposure of a BRAFV600E patient-derived culture to vemurafenib (BRAF inhibitor), trametinib (MEK inhibitor) or a combination of both drugs, respectively. Cytotoxicity assays confirmed resistance. All resistant cell lines spontaneously changed from a spheroid phenotype to adherent growth, indicating changes in tumor characteristics. RNAseq identified > 1500 genes of interest, with subsequent Gene Set Enrichment Analysis identifying key pathways as novel drivers of resistance, the top-ranked gene sets being neural development and plasma membrane/cell adhesion. Approximately 45% of gene sets were enriched across all drug-resistant lines, indicating overlapping resistance mechanisms. Interrogation of the receptor tyrosine kinase (RTK) gene sets identified potent upregulation of several RTKs. Notably, overexpression of the RTK, EGFR, was confirmed through RNAseq and western blot as a potential key mediator of the acquired resistance. Combined treatment with vemurafenib and the EGFR inhibitor, dacomitinib, resulted in synergistic activity against both vemurafenib-resistant BRAFV600E pHGG cells and matched parental cells, providing a viable therapeutic option for drug-resistant BRAFV600E pHGG. Overall, novel drivers of resistance have been identified in BRAFV600E pHGG. EGFR overexpression was pinpointed as a key mediator and dual BRAF/EGFR inhibition displayed promising efficacy as a therapeutic option. Future studies will continue to elucidate the mechanisms of resistance using multi-omic approaches and the in vivo efficacy of dual BRAF/EGFR inhibition.

Diffuse Intrinsic Pontine Gliomas (DIPG) are a subset of Diffuse Midline Gliomas (DMG) and are the most devastating of all brain tumours. There are currently no effective treatments. ACT001 is a novel anti-cancer agent in clinical development that is blood-brain-barrier permeable. Previous studies undertaken in adult glioblastoma suggest that ACT001 exerts an anti-tumour effect via induction of oxidative stress and inhibition of NF-kB and STAT3 pathways. In DIPG, we have found that ACT001 demonstrated potent cytotoxic activity against a panel of DIPG neurospheres and also inhibited colony formation. Flow cytometric analysis confirmed the induction of apoptosis in vitro. Interestingly, we also observed increased expression of some markers of the ROS-dependant NRF2 endogenous antioxidant pathway within DIPG neurospheres suggesting an alternative mechanism of inhibition of DIPG cell proliferation involving ROS generation. In vivo testing of ACT001 in a highly aggressive DIPG-orthotopic model showed that ACT001 was well tolerated and significantly improved survival. ACT001 treatment of HSJD-DIPG007 tumour-bearing animals extended median survival from 59 to 78 days (p=0.0005). ACT001 treated mice had significantly decreased proliferating DIPG cells, analysed by Ki67 staining (p=0.01), and significantly increased H3K27me3 staining (p=0.0479). Given the significant effects on H3K27me3 we evaluated two potential drug combinations with known, clinically available epigenetic modifers. Combination of ACT001 with the FACT inhibitor CBL0137 or the HDAC inhibitor SAHA each significantly and synergistically decreased colony formation. These combinations are currently being evaluated in vivo in orthotopic models of DIPG. ACT001 is currently in a Phase 1 paediatric trial for children with DIPG/DMG. Clinical activity has been demonstrated in DIPG/DMG patients, including a reduction in tumour burden and clinical response. These combined preclinical and clinical results suggest that ACT001 is a viable therapy for patients with DIPG/DMG and preclinical combination therapy testing may guide further clinical trials.

Objective: Particulate matter (PM) with a diameter of 2.5 μm or less (PM2.5) can cross the blood-placental barrier causing adverse foetal outcomes. However, the impact of maternal exposure to low-levels of PM2.5 on liver health and the metabolic profile is unclear. This study aimed to investigate hepatic responses to long-term gestational low-dose PM2.5 exposure, and whether the removal of PM after conception can prevent such effects. Method: Female Balb/c mice (8 weeks) were exposed to PM2.5 (5 μg/day) for 6 weeks prior to mating, during gestation and lactation to model living in a polluted environment (PM group). In a sub-group, PM2.5 exposure was stopped post-conception to model mothers moving to areas with clean air (pre-gestation, Pre) group. Livers were studied in 13-week old offspring. Results: Female offspring in both PM and Pre groups had increased liver triglyceride and glycogen levels, glucose intolerance, but reduced serum insulin and insulin resistance. Male offspring from only the Pre group had increased liver and serum triglycerides, increased liver glycogen, glucose intolerance and higher fasting glucose level. Markers of oxidative stress and inflammation were increased in females from PM and Pre groups. There was also a significant sex difference in the hepatic response to PM2.5 with differential changes in several metabolic markers identified by proteomic analysis. Conclusions: Maternal PM exposure exerted sex-dependent effects on liver health with more severe impacts on females. The removal of PM2.5 during gestation provided limited protection in the offspring’s metabolism regardless of sex.

AIM:Imaging mass cytometry (IMC) affords simultaneous immune-labelling/imaging of multiple antigens in the same tissue. Methods utilizing multiplex data beyond co-registration are lacking. This study developed and applied an innovative spatial analysis workflow for multiplex imaging data to IMC data determined from cardiac tissues and revealed the mechanism(s) of neutrophil-mediated post-myocardial-infarction damage.METHODS:IMC produced multiplex images with various redox/inflammatory markers. The cardiac peri-infarct zone (PIZ) was determined to be up to 240 µm from the infarct border based on the presence of neutrophils. The tissue region beyond the infarct was defined as the remote area (RA). ImageJ was used to quantify the immunoreactivity. Functional assessments included infarct size, cell necro/apoptosis, total thiol assay and echocardiogram.RESULTS:Expression of damage markers decreased in order from the infarct area to PIZ and then RA, reflecting the neutrophil density in the regions. Concentrically spaced "shoreline contour analysis" around the cardiac infarct extending into the PIZ showed that immunoreactivity for damage markers decreased linearly with increasing distance from the infarct, concomitant with a decreasing neutrophil-myeloperoxidase (MPO) gradient from the infarct to the PIZ. Stratifying by concentric bands around individual MPO+ -signal identified that the immunoreactivity of haem-oxygenase-1 (HO-1) and phosphorylated-p38 mitogen-activated protein kinase (pP38) peaked near neutrophils. Furthermore, spatial dependence between neutrophils and markers of cardiac cellular damage was confirmed by nearest-neighbour distance analysis. Post-infarction tissue exhibited declined functional parameters that were associated with neutrophil migration from the infarct to PIZ.CONCLUSION:This image-based quantitative protocol revealed the spatial association and provided potential molecular pathways responsible for neutrophil-mediated damage post-infarction.

Systemic sclerosis (SSc) is characterised by progressive multiple organ fibrosis leading to morbidity and mortality. Lysyl oxidases play a vital role in the cross-linking of collagens and subsequent build-up of fibrosis in the extracellular matrix. As such, their inhibition provides a novel treatment paradigm for SSc. A novel small molecule pan-lysyl oxidase inhibitor, PXS-5505, currently in clinical development for myelofibrosis treatment was evaluated using in vivo rodent models resembling the fibrotic conditions in SSc. Both lysyl oxidase and lysyl oxidase-like 2 (LOXL2) expression were elevated in the skin and lung of SSc patients. The oral application of PXS-5505 inhibited lysyl oxidase activity in the skin and LOXL2 activity in the lung. PXS-5505 exhibited anti-fibrotic effects in the SSc skin mouse model, reducing dermal thickness and α-smooth muscle actin. Similarly, in the bleomycin-induced mouse lung model, PXS-5505 reduced pulmonary fibrosis toward normal levels, mediated by its ability to normalise collagen/elastin crosslink formation. PXS-5505 also reduced fibrotic extent in models of the ischaemia-reperfusion heart, the unilateral ureteral obstruction kidney, and the CCl4-induced fibrotic liver. PXS-5505 consistently demonstrates potent anti-fibrotic efficacy in multiple models of organ fibrosis relevant to the pathogenesis of SSc, suggesting that it may be efficacious as a novel approach for treating SSc.

The release of myeloperoxidase (MPO) produced through the activation and infiltration of immune cells within sites of inflammation and damage to the vasculature is mechanistically involved in atherosclerosis. This effect of active MPO and its derived products on the endothelial lining of the blood vessels perturbs vascular function. Mechanisms involved in this perturbation include disruption to nitric oxide signaling and mediation of the endothelial cell inflammatory response, through the oxidant-induced upregulation of cell adhesion molecules. These processes result in the ongoing chronic exacerbation of immune cell recruitment and inflammation that defines this disease. Additionally, at the endothelial cell level, the exposure to MPO-derived oxidants causes loss of intracellular glutathione and protein-bound thiols, activates signaling pathways involving intracellular calcium mishandling, resulting in mitochondrial dysfunction and endothelial cell death. Together, these events directly contribute to endothelial dysfunction that precedes atherosclerotic lesion formation, highlighting MPO as one of the most attractive targets of therapeutic intervention for this debilitating condition, the underlying cause of cardiac ischemia and heart failure.

Diffuse midline gliomas (DMGs) are invariably fatal pediatric brain tumours that are inherently resistant to conventional therapy. In recent years our understanding of the underlying molecular mechanisms of DMG tumorigenicity has resulted in the identification of novel targets and the development of a range of potential therapies, with multiple agents now being progressed to clinical translation to test their therapeutic efficacy. Here, we provide an overview of the current therapies aimed at epigenetic and mutational drivers, cellular pathway aberrations and tumor microenvironment mechanisms in DMGs in order to aid therapy development and facilitate a holistic approach to patient treatment.

Augmentation of endogenous nitric oxide (NO) synthesis, either by the classical L-arginine-NO synthase pathway, or the recently discovered entero-salivary nitrate-nitrite-NO system, may slow the progression of autosomal dominant polycystic kidney disease (ADPKD). To test this hypothesis, the expression of NO in human ADPKD cell lines (WT 9-7, WT 9-12), and the effect of L-arginine on an in vitro model of three-dimensional cyst growth using MDCK cells, was examined. In addition, groups of homozygous Pkd1RC/RC mice (a hypomorphic genetic ortholog of ADPKD) received either low, moderate or high dose sodium nitrate (0.1, 1 or 10 mmol/kg/day), or sodium chloride (vehicle; 10 mmol/kg/day), supplemented drinking water from postnatal month 1 to 9 (n = 12 per group). In vitro, intracellular NO, as assessed by DAF-2/DA fluorescence, was reduced by >70% in human ADPKD cell lines, and L-arginine and the NO donor, sodium nitroprusside, both attenuated in vitro cyst growth by up to 18%. In contrast, in Pkd1RC/RC mice, sodium nitrate supplementation increased serum nitrate/nitrite levels by ~25-fold in the high dose group (P<0.001), but kidney enlargement and percentage cyst area was not altered, regardless of dose. In conclusion, L-arginine has mild direct efficacy on reducing renal cyst growth in vitro, whereas long-term sodium nitrate supplementation was ineffective in vivo. These data suggest that the bioconversion of dietary nitrate to NO by the entero-salivary pathway may not be sufficient to influence the progression of renal cyst growth in ADPKD.

Background and Aims: Neutrophil extracellular trap (NET) release in response to inflammatory stimuli is increasingly implicated in many chronic disease settings, including driving lesion development and thrombosis in atherosclerosis. Macrophages also release extracellular traps or “METs”, but the pathways responsible for triggering MET release in vivo, and the role of these structures in pathological processes are poorly defined, though these cells are dominant in lesions. In this study, we examined MET release from human macrophages using different inflammatory stimuli.