Transcription factor NFE2L1 decreases in glomerulonephropathies after podocyte damage

Funding: This work was supported by NHS Lothian. This project received funding from the European Union’s Horizon 2020 research and innovation programme under Grant Agreement No. 101017453 as part of the KATY project, and from NuCana plc.Podocyte cellular injury and detachment from glomerular capillaries constitute a critical factor contributing to kidney disease. Notably, transcription factors are instrumental in maintaining podocyte differentiation and homeostasis. This study explores the hitherto uninvestigated expression of Nuclear Factor Erythroid 2-related Factor 1 (NFE2L1) in podocytes. We evaluated the podocyte expression of NFE2L1, Nuclear Factor Erythroid 2-related Factor 2 (NFE2L2), and NAD(P)H:quinone Oxidoreductase (NQO1) in 127 human glomerular disease biopsies using multiplexed immunofluorescence and image analysis. We found that both NFE2L1 and NQO1 expressions were significantly diminished across all observed renal diseases. Furthermore, we exposed human immortalized podocytes and ex vivo kidney slices to Puromycin Aminonucleoside (PAN) and characterized the NFE2L1 protein isoform expression. PAN treatment led to a reduction in the nuclear expression of NFE2L1 in ex vivo kidney slices and podocytes.Publisher PDFPeer reviewe

Mechanisms of action of 3’-deoxyadenosine in treating clear cell renal cell carcinoma

Although treatment strategies for advanced and metastatic clear cell renal cell carcinoma (ccRCC) have markedly evolved with the recent use of immune-checkpoint inhibitor (ICI)-based combinations, most patients eventually develop resistance to these therapies. Therefore, there is still an urgent need for the development of effective treatment options. NUC-7738, a novel ProTide transformation of the nucleoside analogue 3ﹶ-deoxyadenosine, releases 3ﹶ-deoxyadenosine monophosphate (3’-dAMP) in cells which is then phosphorylated to the di- (3’-dADP) and tri-phosphate forms (3’-dATP). 3’-dAMP might have the ability to activate AMP-activated protein kinase (AMPK), a key cellular energy sensor, and thus disrupt metabolic homeostasis in cancer cells. Furthermore, 3’-dATP might interfere with RNA synthesis affecting protein expression and survival of cancer cells. The ability of NUC-7738 to activate AMPK through phosphorylation of Th172 was tested in ccRCC cell lines and ex vivo tissue slices of ccRCC from patients. The effect of NUC-7738 on mRNA synthesis and polyadenylation was investigated in two ccRCC cell lines, 786-O and 769-P. AMPK activation by NUC-7738 showed inter-replicate variability and inter-patient variability in ccRCC cell lines and ex vivo tissue slices, respectively, indicating the complexity of the regulation of AMPK phosphorylation. Moreover, mass spectrometry analysis showed that 3’-dATP is the main active metabolite of NUC-7738. Transcriptome data analysis showed mitochondrial gene transcripts of electron transport chain (ETC) complexes were the most significantly altered in both 786-O and 769-P cell lines, with lower expression levels in response to NUC-7738 treatment. This was accompanied by downregulation of the protein expression of ETC complexes subunits. NUC-7738 induced the intrinsic pathway of apoptosis in these cells through the release of cytochrome c from mitochondria and the subsequent activation of caspases -9 and -7. These data suggest that NUC-7738 might inhibit tumour cells growth and proliferation through the inhibition of mitochondrial respiration and the subsequent induction of apoptosis

Follicular fluid Vascular Endothelial Growth Factor (VEGF) could be a predictor for pregnancy outcome in normo-responders and polycystic ovary syndrome women undergoing IVF/ICSI treatment cycles

Objective: The aim of this study was to evaluate the serum and follicular fluid levels of Vascular Endothelial Growth Factor (VEGF) as possible predictive markers for IVF outcome in PCOS patients. Design: Prospective cross-sectional study. Materials and Methods: Forty patients with PCOS and 40 age and weight matched control women were included in this study. All of them were enrolled in the IVF program. Blood and follicular fluid (FF) samples were collected from all subjects on the day of oocyte retrieval. VEGF levels were measured in serum and follicular fluid (FF) using enzyme linked immunosorbent assay (ELISA). The correlation between VEGF levels and number of retrieved oocyte, number of mature oocyte, number of fertilized oocyte, number of embryos, percent of mature oocytes, fertilization rate and pregnancy was assessed. Results: PCOS patients showed higher serum VEGF levels than controls (297.46 ± 159.45 pg/ml vs. 134.08 ± 113.06 pg/ml, p value = 0.0001) and higher FF VEGF levels compared to controls (1358.92 ± 820.86 pg/ml vs. 987.58 ± 236.62 pg/ml, p value = 0.007). Serum VEGF levels were not significantly correlated with the number of retrieved oocyte in both PCOS (r = 0.37, p value = 0.19) and control women (r = −0.03, p value = 0.83), and FF VEGF levels were not significantly correlated with the number of retrieved oocyte in both PCOS (r = −0.06, p value = 0.7) and control women (r = −0.13, p value = 0.43). Both serum and FF VEGF levels were not significantly correlated with the number of mature oocyte, number of fertilized oocyte, number of embryos, percent of mature oocytes or fertilization rate. Serum VEGF levels were not significantly different between pregnant and non-pregnant women in both PCOS (256.69 ± 139.11 pg/ml vs. 300.20 ± 114.60 pg/ml, p value = 0.416) and control women (107.98 ± 115.51 pg/ml vs. 154.39 ± 111.09 pg/ml, p value = 0.271), whereas FF VEGF levels were significantly lower in FFs from the group of pregnancy than those from the group of no-pregnancy in both PCOS (812.04 ± 308.16 pg/ml vs. 1936.78 ± 771.79 pg/ml, p value = 0.0001) and control women (822.45 ± 199.45 pg/ml vs. 1171.98 ± 139.60 pg/ml, p value = 0.0001). FF VEGF levels had a significant predictive value of pregnancy in both PCOS (p value = 0.0001) and control women (p value = 0.0001). Conclusion: FF VEGF levels may serve as a reliable predictive marker for pregnancy in both normal women and women with polycystic ovary syndrome undergoing IVF

The novel nucleoside analogue ProTide NUC-7738 overcomes cancer resistance mechanisms in vitro and in a first-in-human Phase 1 clinical trial

We thank the Oxford Genomics Centre at the Wellcome Centre for Human Genetics (funded by Wellcome Trust grant reference 203141/Z/16/Z) for the generation and initial processing of the sequencing data. This work was funded in part by NuCana plc. The Oxford Early Phase Clinical Trials Unit, the Edinburgh and Newcastle Trials units are grateful for support from CRUK and Experimental Cancer Medicine Centre (ECMC) funding.Purpose: Nucleoside analogues form the backbone of many therapeutic regimens in oncology and require the presence of intracellular enzymes for their activation. A ProTide is comprised of a nucleoside fused to a protective phosphoramidate cap. ProTides are easily incorporated into cells whereupon the cap is cleaved and a pre-activated nucleoside released. 3'-deoxyadenosine (3'-dA) is a naturally-occurring adenosine analogue with established anti-cancer activity in vitro but limited bioavailability due to its rapid in vivo deamination by the circulating enzyme adenosine deaminase, poor uptake into cells and reliance on adenosine kinase for its activation. In order to overcome these limitations, 3'-dA was chemically modified to create the novel ProTide NUC-7738. Experimental Design: We describe the synthesis of NUC-7738. We determine the IC50 of NUC-7738 using pharmacokinetics (PK) and conduct genome-wide analyses to identify its mechanism of action using different cancer model systems. We validate these findings in cancer patients. Results: We show that NUC-7738 overcomes the cancer resistance mechanisms that limit the activity of 3'-dA and that its activation is dependent on ProTide cleavage by the enzyme histidine triad nucleotide binding protein 1. PK and tumour samples obtained from the ongoing first-in-human Phase 1 clinical trial of NUC-7738 further validate our in vitro findings and show NUC-7738 is an effective pro-apoptotic agent in cancer cells with effects on the NF-kappaB pathway. Conclusions: Our study provides proof that NUC-7738 overcomes cellular resistance mechanisms and support its further clinical evaluation as a novel cancer treatment within the growing pantheon of anti-cancer ProTides.Publisher PDFPeer reviewe