Investigating the effects of arginine methylation inhibitors on microdissected brain tumour biopsies maintained in a miniaturised perfusion system.

Arginine methylation is a post-translational modification that consists of the transfer of one or two methyl (CH3) groups to arginine residues in proteins. Several types of arginine methylation occur, namely monomethylation, symmetric dimethylation and asymmetric dimethylation, which are catalysed by different protein arginine methyltransferases (PRMTs). Inhibitors of PRMTs have recently entered clinical trials to target several types of cancer, including gliomas (NCT04089449). People with glioblastoma (GBM), the most aggressive form of brain tumour, are among those with the poorest quality of life and likelihood of survival of anyone diagnosed with cancer. There is currently a lack of (pre)clinical research on the possible application of PRMT inhibitors to target brain tumours. Here, we set out to investigate the effects of clinically-relevant PRMT inhibitors on GBM biopsies. We present a new, low-cost, easy to fabricate perfusion device that can maintain GBM tissue in a viable condition for at least eight days post-surgical resection. The miniaturised perfusion device enables the treatment of GBM tissue with PRMT inhibitors ex vivo, and we observed a two-fold increase in apoptosis in treated samples compared to parallel control experiments. Mechanistically, we show thousands of differentially expressed genes after treatment, and changes in the type of arginine methylation of the RNA binding protein FUS that are consistent with hundreds of differential gene splicing events. This is the first time that cross-talk between different types of arginine methylation has been observed in clinical samples after treatment with PRMT inhibitors.

The Use of Tissue-on-Chip Technology to Focus the Search for Extracellular Vesicle miRNA Biomarkers in Thyroid Disease.

Small extracellular vesicles (sEVs) contain microRNAs (miRNAs) which have potential to act as disease-specific biomarkers. The current study uses an established method to maintain human thyroid tissue ex vivo on a tissue-on-chip device, allowing the collection, isolation and interrogation of the sEVs released directly from thyroid tissue. sEVs were analysed for differences in miRNA levels released from benign thyroid tissue, Graves' disease tissue and papillary thyroid cancer (PTC), using miRNA sequencing and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) to identify potential biomarkers of disease. Thyroid biopsies from patients with benign tissue (n = 5), Graves' disease (n = 5) and PTC (n = 5) were perfused with medium containing sEV-depleted serum for 6 days on the tissue-on-chip device. During incubation, the effluents were collected and ultracentrifuged to isolate sEVs; miRNA was extracted and sequenced (miRNASeq). Out of the 15 samples, 14 passed the quality control and miRNASeq analysis detected significantly higher expression of miR-375-3p, miR-7-5p, miR-382-5p and miR-127-3p in the sEVs isolated from Graves' tissue compared to those from benign tissue (false discovery rate; FDR p < 0.05). Similarly, miR-375-3p and miR-7-5p were also detected at a higher level in the Graves' tissue sEVs compared to the PTC tissue sEVs (FDR p < 0.05). No significant differences were observed between miRNA in sEVs from PTC vs. those from benign tissue. These results were supported by Quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR). The novel findings demonstrate that the tissue-on-chip technology is a robust method for isolating sEVs directly from the tissue of interest, which has permitted the identification of four miRNAs, with which further investigation could be used as biomarkers or therapeutic targets within thyroid disease.

The Epigenetic Regulatory Protein CBX2 Promotes mTORC1 Signalling and Inhibits DREAM Complex Activity to Drive Breast Cancer Cell Growth.

Chromobox 2 (CBX2) is a chromatin-binding component of polycomb repressive complex 1, which causes gene silencing. CBX2 expression is elevated in triple-negative breast cancer (TNBC), for which there are few therapeutic options. Here, we aimed to investigate the functional role of CBX2 in TNBC. CBX2 knockdown in TNBC models reduced cell numbers, which was rescued by ectopic expression of wild-type CBX2 but not a chromatin binding-deficient mutant. Blocking CBX2 chromatin interactions using the inhibitor SW2_152F also reduced cell growth, suggesting CBX2 chromatin binding is crucial for TNBC progression. RNA sequencing and gene set enrichment analysis of CBX2-depleted cells identified downregulation of oncogenic signalling pathways, including mTORC1 and E2F signalling. Subsequent analysis identified that CBX2 represses the expression of mTORC1 inhibitors and the tumour suppressor RBL2. RBL2 repression, in turn, inhibits DREAM complex activity. The DREAM complex inhibits E2F signalling, causing cell senescence; therefore, inhibition of the DREAM complex via CBX2 may be a key oncogenic driver. We observed similar effects in oestrogen receptor-positive breast cancer, and analysis of patient datasets suggested CBX2 inhibits RBL2 activity in other cancer types. Therapeutic inhibition of CBX2 could therefore repress mTORC1 activation and promote DREAM complex-mediated senescence in TNBC and could have similar effects in other cancer types.

The Histone Demethylase Enzymes KDM3A and KDM4B Co-Operatively Regulate Chromatin Transactions of the Estrogen Receptor in Breast Cancer.

Many estrogen receptor (ER)-positive breast cancers develop resistance to endocrine therapy but retain canonical receptor signalling in the presence of selective ER antagonists. Numerous co-regulatory proteins, including enzymes that modulate the chromatin environment, control the transcriptional activity of the ER. Targeting ER co-regulators has therefore been proposed as a novel therapeutic approach. By assessing DNA-binding dynamics in ER-positive breast cancer cells, we have identified that the histone H3 lysine 9 demethylase enzymes, KDM3A and KDM4B, co-operate to regulate ER activity via an auto-regulatory loop that facilitates the recruitment of each co-activating enzyme to chromatin. We also provide evidence that suggests that KDM3A primes chromatin for deposition of the ER pioneer factor FOXA1 and recruitment of the ER-transcriptional complex, all prior to ER recruitment, therefore establishing an important mechanism of chromatin regulation involving histone demethylases and pioneer factors, which controls ER functionality. Importantly, we show via global gene-expression analysis that a KDM3A/KDM4B/FOXA1 co-regulated gene signature is enriched for pro-proliferative and ER-target gene sets, suggesting that abrogation of this network could be an efficacious therapeutic strategy. Finally, we show that depletion of both KDM3A and KDM4B has a greater inhibitory effect on ER activity and cell growth than knockdown of each individual enzyme, suggesting that targeting both enzymes represents a potentially efficacious therapeutic option for ER-driven breast cancer.

Does radiation-induced c-MYC amplification initiate breast oncogenesis?

The MYC (v-myc avian myelocytomatosis viral oncogene homolog; c-MYC) locus on chromosome 8q is susceptible to high-level amplification following exposure of human breast cells to ionizing radiation, and c-MYC amplification is a common feature of both radiogenic adenocarcinoma and radiogenic angiosarcoma of the breast. Taken together, these observations suggest common breast-specific susceptibility factors that predispose cells to amplification of this critical proto-oncogene and the development of radiogenic cancer in multiple tissue types of this radiosensitive organ.

Common cancer-associated imbalances in the DNA damage response confer sensitivity to single agent ATR inhibition.

ATR is an attractive target in cancer therapy because it signals replication stress and DNA lesions for repair and to S/G2 checkpoints. Cancer-specific defects in the DNA damage response (DDR) may render cancer cells vulnerable to ATR inhibition alone. We determined the cytotoxicity of the ATR inhibitor VE-821 in isogenically matched cells with DDR imbalance. Cell cycle arrest, DNA damage accumulation and repair were determined following VE-821 exposure.Defects in homologous recombination repair (HRR: ATM, BRCA2 and XRCC3) and base excision repair (BER: XRCC1) conferred sensitivity to VE-821. Surprisingly, the loss of different components of the trimeric non-homologous end-joining (NHEJ) protein DNA-PK had opposing effects. Loss of the DNA-binding component, Ku80, caused hypersensitivity to VE-821, but loss of its partner catalytic subunit, DNA-PKcs, did not. Unexpectedly, VE-821 was particularly cytotoxic to human and hamster cells expressing high levels of DNA-PKcs. High DNA-PKcs was associated with replicative stress and activation of the DDR. VE-821 suppressed HRR, determined by RAD51 focus formation, to a greater extent in cells with high DNA-PKcs.Defects in HRR and BER and high DNA-PKcs expression, that are common in cancer, confer sensitivity to ATR inhibitor monotherapy and may be developed as predictive biomarkers for personalised medicine.

The histone demethylase enzyme KDM3A is a key estrogen receptor regulator in breast cancer.

Endocrine therapy has successfully been used to treat estrogen receptor (ER)-positive breast cancer, but this invariably fails with cancers becoming refractory to treatment. Emerging evidence has suggested that fluctuations in ER co-regulatory protein expression may facilitate resistance to therapy and be involved in breast cancer progression. To date, a small number of enzymes that control methylation status of histones have been identified as co-regulators of ER signalling. We have identified the histone H3 lysine 9 mono- and di-methyl demethylase enzyme KDM3A as a positive regulator of ER activity. Here, we demonstrate that depletion of KDM3A by RNAi abrogates the recruitment of the ER to cis-regulatory elements within target gene promoters, thereby inhibiting estrogen-induced gene expression changes. Global gene expression analysis of KDM3A-depleted cells identified gene clusters associated with cell growth. Consistent with this, we show that knockdown of KDM3A reduces ER-positive cell proliferation and demonstrate that KDM3A is required for growth in a model of endocrine therapy-resistant disease. Crucially, we show that KDM3A catalytic activity is required for both ER-target gene expression and cell growth, demonstrating that developing compounds which target demethylase enzymatic activity may be efficacious in treating both ER-positive and endocrine therapy-resistant disease.