A gut microbiota rheostat forecasts responsiveness to PD-L1 and VEGF blockade in mesothelioma.

Malignant mesothelioma is a rare tumour caused by asbestos exposure that originates mainly from the pleural lining or the peritoneum. Treatment options are limited, and the prognosis is dismal. Although immune checkpoint blockade (ICB) can improve survival outcomes, the determinants of responsiveness remain elusive. Here, we report the outcomes of a multi-centre phase II clinical trial (MiST4, NCT03654833) evaluating atezolizumab and bevacizumab (AtzBev) in patients with relapsed mesothelioma. We also use tumour tissue and gut microbiome sequencing, as well as tumour spatial immunophenotyping to identify factors associated with treatment response. MIST4 met its primary endpoint with 50% 12-week disease control, and the treatment was tolerable. Aneuploidy, notably uniparental disomy (UPD), homologous recombination deficiency (HRD), epithelial-mesenchymal transition and inflammation with CD68+ monocytes were identified as tumour-intrinsic resistance factors. The log-ratio of gut-resident microbial genera positively correlated with radiological response to AtzBev and CD8+ T cell infiltration, but was inversely correlated with UPD, HRD and tumour infiltration by CD68+ monocytes. In summary, a model is proposed in which both intrinsic and extrinsic determinants in mesothelioma cooperate to modify the tumour microenvironment and confer clinical sensitivity to AtzBev. Gut microbiota represent a potentially modifiable factor with potential to improve immunotherapy outcomes for individuals with this cancer of unmet need.

Clinical efficacy and molecular mechanism of Chinese medicine in the treatment of autoimmune thyroiditis.

ETHNOPHARMACOLOGICAL RELEVANCE: Autoimmune Thyroiditis (AIT) is a common refractory autoimmune disease of the endocrine system that may eventually lead to complete loss of thyroid function, with subsequent severe effects on the metabolism. Because of the deficiency in current clinical management of AIT, the need for alternative therapies is highlighted. With its multi-component and multi-target characteristics, Chinese medicine has good potential as an alternative therapy for AIT. AIM OF THE STUDY: The aim of this study was to systematically summarize the clinical efficacy and safety evaluation of TCM and its active ingredients in the treatment and regulation of AIT. Additionally, we provide an in-depth discussion of the relevant mechanisms and molecular targets to understand the protective effects of traditional Chinese medicine on AIT and explore new ideas for clinical treatment. MATERIALS AND METHODS: The literature related to "Hashimoto", "autoimmune thyroiditis", "traditional Chinese medicine," and "Chinese herbal medicine" was systematically summarized and reviewed from Web of Science Core Collection, PubMed, CNKI, and other databases. Domestic and international literature were analyzed, compared, and reviewed. RESULTS: An increasing number of studies have demonstrated that herbal medicines can intervene in immunomodulation, with pharmacological effects such as antibody lowering, anti-inflammatory, anti-apoptotic thyroid follicular cells, regulation of intestinal flora, and regulation of estrogen and progesterone levels. The signaling pathways and molecular targets of the immunomodulatory effects of Chinese herbal medicine for AIT may include Fas/FasL, Caspase, BCL-2, and TLRs/MyD88/NF-κB et al. CONCLUSIONS: The use of Chinese herbs in the treatment and management of AIT is clinically experienced, satisfactory, and safe. Future studies may evaluate the influence of herbal medicines on the occurrence and development of AIT by modulating the interaction between immune factors and conventional signaling pathways.

Evaluating niraparib versus active symptom control in patients with previously treated mesothelioma (NERO): a study protocol for a multicentre, randomised, two-arm, open-label phase II trial in UK secondary care centres.

BACKGROUND: Malignant mesothelioma is a rapidly lethal cancer that has been increasing at an epidemic rate over the last three decades. Targeted therapies for mesothelioma have been lacking. A previous study called MiST1 (NCT03654833), evaluated the efficacy of Poly (ADP-ribose) polymerase (PARP) inhibition in mesothelioma. This study met its primary endpoint with 15% of patients having durable responses exceeding 1 year. Therefore, there is a need to evaluate PARP inhibitors in relapsed mesothelioma patients, where options are limited. Niraparib is the PARP inhibitor used in NERO. METHODS: NERO is a multicentre, two-arm, open-label UK randomised phase II trial designed to evaluate the efficacy of PARP inhibition in relapsed mesothelioma. 84 patients are being recruited. NERO is not restricted by line of therapy; however, eligible participants must have been treated with an approved platinum based systemic therapy. Participants will be randomised 2:1, stratified according to histology and response to prior platinum-based chemotherapy, to receive either active symptom control (ASC) and niraparib or ASC alone, for up to 24 weeks. Participants will be treated until disease progression, withdrawal, death or development of significant treatment limiting toxicity. Participants randomised to niraparib will receive 200 or 300 mg daily in a 3-weekly cycle. The primary endpoint is progression-free survival, where progression is determined by modified Response Evaluation Criteria in Solid Tumors (mRECIST) or RECIST 1.1; investigator reported progression; or death from any cause, whichever comes first. Secondary endpoints include overall survival, best overall response, 12-week and 24 week disease control, duration of response, treatment compliance and safety/tolerability. If NERO shows niraparib to be safe and biologically effective, it may lead to future late phase randomised controlled trials in relapsed mesothelioma. ETHICS AND DISSEMINATION: The study received ethical approval from London-Hampstead Research Ethics Committee on 06-May-2022 (22/LO/0281). Data from all centres will be analysed together and published as soon as possible. TRIAL REGISTRATION NUMBER: ISCRTN16171129; NCT05455424.

Transcriptional perturbation of protein arginine methyltransferase-5 exhibits MTAP-selective oncosuppression.

We hypothesized that small molecule transcriptional perturbation could be harnessed to target a cellular dependency involving protein arginine methyltransferase 5 (PRMT5) in the context of methylthioadenosine phosphorylase (MTAP) deletion, seen frequently in malignant pleural mesothelioma (MPM). Here we show, that MTAP deletion is negatively prognostic in MPM. In vitro, the off-patent antibiotic Quinacrine efficiently suppressed PRMT5 transcription, causing chromatin remodelling with reduced global histone H4 symmetrical demethylation. Quinacrine phenocopied PRMT5 RNA interference and small molecule PRMT5 inhibition, reducing clonogenicity in an MTAP-dependent manner. This activity required a functional PRMT5 methyltransferase as MTAP negative cells were rescued by exogenous wild type PRMT5, but not a PRMT5E444Q methyltransferase-dead mutant. We identified c-jun as an essential PRMT5 transcription factor and a probable target for Quinacrine. Our results therefore suggest that small molecule-based transcriptional perturbation of PRMT5 can leverage a mutation-selective vulnerability, that is therapeutically tractable, and has relevance to 9p21 deleted cancers including MPM.

Clonal architecture in mesothelioma is prognostic and shapes the tumour microenvironment.

Malignant Pleural Mesothelioma (MPM) is typically diagnosed 20-50 years after exposure to asbestos and evolves along an unknown evolutionary trajectory. To elucidate this path, we conducted multi-regional exome sequencing of 90 tumour samples from 22 MPMs acquired at surgery. Here we show that exomic intratumour heterogeneity varies widely across the cohort. Phylogenetic tree topology ranges from linear to highly branched, reflecting a steep gradient of genomic instability. Using transfer learning, we detect repeated evolution, resolving 5 clusters that are prognostic, with temporally ordered clonal drivers. BAP1/-3p21 and FBXW7/-chr4 events are always early clonal. In contrast, NF2/-22q events, leading to Hippo pathway inactivation are predominantly late clonal, positively selected, and when subclonal, exhibit parallel evolution indicating an evolutionary constraint. Very late somatic alteration of NF2/22q occurred in one patient 12 years after surgery. Clonal architecture and evolutionary clusters dictate MPM inflammation and immune evasion. These results reveal potentially drugable evolutionary bottlenecking in MPM, and an impact of clonal architecture on shaping the immune landscape, with potential to dictate the clinical response to immune checkpoint inhibition.

The Circulating Nucleic Acid Characteristics of Non-Metastatic Soft Tissue Sarcoma Patients.

Soft tissue sarcomas (STS) are rare, malignant tumours with a generally poor prognosis. Our aim was to explore the potential of cell free DNA (cfDNA) and circulating tumour DNA (ctDNA) analysis to track non-metastatic STS patients undergoing attempted curative treatment. The analysed cohort (n = 29) contained multiple STS subtypes including myxofibrosarcomas, undifferentiated pleomorphic sarcomas, leiomyosarcomas, and dedifferentiated liposarcomas amongst others. Perioperative cfDNA levels trended towards being elevated in patients (p = 0.07), although did not correlate with tumour size, grade, recurrence or subtype, suggesting a limited diagnostic or prognostic role. To characterise ctDNA, an amplicon panel covering three genes commonly mutated in STSs was first trialled on serial plasma collected from nine patients throughout follow-up. This approach only identified ctDNA in 2.5% (one in 40) of the analysed samples. Next custom-designed droplet digital PCR assays and Ion AmpliSeq™ panels were developed to track single nucleotide variants identified in patients' STSs by whole exome sequencing (1-6 per patient). These approaches identified ctDNA in 17% of patients. Although ctDNA was identified before radiologically detectable recurrence in two cases, the absence of demonstrable ctDNA in 83% of cases highlights the need for much work before circulating nucleic acids can become a useful means to track STS patients.

Utilizing network pharmacology to explore the underlying mechanism of Radix Salviae in diabetic retinopathy.

INTRODUCTION: Radix Salviae (Dan-shen in pinyin), a classic Chinese herb, has been extensively used to treat diabetic retinopathy in clinical practice in China for many years. However, the pharmacological mechanisms of Radix Salviae remain vague. The aim of this study was to decrypt the underlying mechanisms of Radix Salviae in the treatment of diabetic retinopathy using a systems pharmacology approach. METHODS: A network pharmacology-based strategy was proposed to elucidate the underlying multi-component, multi-target, and multi-pathway mode of action of Radix Salviae against diabetic retinopathy. First, we collected putative targets of Radix Salviae based on the Traditional Chinese Medicine System Pharmacology database and a network of the interactions among the putative targets of Radix Salviae and known therapeutic targets of diabetic retinopathy was built. Then, two topological parameters, "degree" and "closeness certainty" were calculated to identify the major targets in the network. Furthermore, the major hubs were imported to the Database for Annotation, Visualization and Integrated Discovery to perform a pathway enrichment analysis. RESULTS: A total of 130 nodes, including 18 putative targets of Radix Salviae, were observed to be major hubs in terms of topological importance. The results of pathway enrichment analysis indicated that putative targets of Radix Salviae mostly participated in various pathways associated with angiogenesis, protein metabolism, inflammatory response, apoptosis, and cell proliferation. The putative targets of Radix Salviae (vascular endothelial growth factor, matrix metalloproteinases, plasminogen, insulin-like growth factor-1, and cyclooxygenase-2) were recognized as active factors involved in the main biological functions of treatment, which implied that these were involved in the underlying mechanisms of Radix Salviae on diabetic retinopathy. CONCLUSIONS: Radix Salviae could alleviate diabetic retinopathy via the molecular mechanisms predicted by network pharmacology. This research demonstrates that the network pharmacology approach can be an effective tool to reveal the mechanisms of traditional Chinese medicine from a holistic perspective.

Sensitivity of Colorectal Cancer to Arginine Deprivation Therapy is Shaped by Differential Expression of Urea Cycle Enzymes.

Tumors deficient in the urea cycle enzymes argininosuccinate synthase-1 (ASS1) and ornithine transcarbamylase (OTC) are unable to synthesize arginine and can be targeted using arginine-deprivation therapy. Here, we show that colorectal cancers (CRCs) display negligible expression of OTC and, in subset of cases, ASS1 proteins. CRC cells fail to grow in arginine-free medium and dietary arginine deprivation slows growth of cancer cells implanted into immunocompromised mice. Moreover, we report that clinically-formulated arginine-degrading enzymes are effective anticancer drugs in CRC. Pegylated arginine deiminase (ADI-PEG20), which degrades arginine to citrulline and ammonia, affects growth of ASS1-negative cells, whereas recombinant human arginase-1 (rhArg1peg5000), which degrades arginine into urea and ornithine, is effective against a broad spectrum of OTC-negative CRC cell lines. This reflects the inability of CRC cells to recycle citrulline and ornithine into the urea cycle. Finally, we show that arginase antagonizes chemotherapeutic drugs oxaliplatin and 5-fluorouracil (5-FU), whereas ADI-PEG20 synergizes with oxaliplatin in ASS1-negative cell lines and appears to interact with 5-fluorouracil independently of ASS1 status. Overall, we conclude that CRC is amenable to arginine-deprivation therapy, but we warrant caution when combining arginine deprivation with standard chemotherapy.

Circulating tumour-derived DNA in metastatic soft tissue sarcoma.

Following treatment 40% of soft tissue sarcoma (STS) patients suffer disease recurrence. In certain cancers circulating cell free DNA (cfDNA) and circulating tumour-derived DNA (ctDNA) characteristics correlate closely with disease burden, making them exciting potential sources of biomarkers. Despite this, the circulating nucleic acid characteristics of only 2 STS patients have been reported to date. To address this we used an Ion AmpliSeq™ panel custom specifically designed for STS patients to conduct a genetic characterisation of plasma cfDNA, buffy coat (germline) DNA and where available Formalin-Fixed Paraffin-Embedded (FFPE) primary STS tissue DNA in a cohort of 11 metastatic STS patients. We found that total cfDNA levels were significantly elevated in the STS patients analysed, and weakly correlated with disease burden. Using our Ion AmpliSeq™ panel we also successfully detected ctDNA in 4/11 (36%) patients analysed with a wide variety of STS subtypes and disease burdens. This evidence included the presence of cancer associated TP53 / PIK3CA mutations in 2 patients' plasma and matched primary STS tumour tissue, and in the plasma alone for 2 patients. We also identified 2 potential examples of allelic loss of heterozygosity in an additional patient's STS DNA and cfDNA. This is the largest study performed characterising STS patient cfDNA/ctDNA and confirms that the field remains an attractive potential source of novel STS biomarkers. Further work is required to investigate the circulating nucleic acid characteristics of individual STS subtypes, and the potential prognostic or therapeutic roles that cfDNA/ctDNA may hold for patients with these complex tumours.

Posttranscriptional Upregulation of p53 by Reactive Oxygen Species in Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) cells multiply and become more resistant to immunochemotherapy in "proliferation centers" within tissues, whereas apoptosis occurs in the periphery. Various models recapitulate these microenvironments in vitro, such as stimulation with CD154 and IL4. Using this system, we observed a 30- to 40-fold induction of wild-type p53 protein in 50 distinct human CLL specimens tested, without the induction of either cell-cycle arrest or apoptosis. In contrast, the mRNA levels for p53 did not increase, indicating that its elevation occurred posttranscriptionally. Mechanistic investigations revealed that under the conditions studied, p53 was phosphorylated on residues associated with p53 activation and increased half-life. However, p53 protein induced in this manner could transcriptionally activate only a subset of target genes. The addition of a DNA-damaging agent further upregulated p53 protein levels, which led to apoptosis. p53 induction relied on the increase in intracellular reactive oxygen species observed after CD154 and IL4 stimulation. We propose that chronic oxidative stress is a characteristic of the microenvironment in B-cell "proliferation centers" in CLL that are capable of elevating the basal expression of p53, but to levels below the threshold needed to induce arrest or apoptosis. Our findings suggest that reactivation of the full transcriptional activities of p53 in proliferating CLL cells may offer a possible therapeutic strategy. Cancer Res; 76(21); 6311-9. ©2016 AACR.

Lysine-specific demethylase 1 regulates the embryonic transcriptome and CoREST stability.

Lysine-specific demethylase 1 (LSD1), which demethylates mono- and dimethylated histone H3-Lys4 as part of a complex including CoREST and histone deacetylases (HDACs), is essential for embryonic development in the mouse beyond embryonic day 6.5 (e6.5). To determine the role of LSD1 during this early period of embryogenesis, we have generated loss-of-function gene trap mice and conditional knockout embryonic stem (ES) cells. Analysis of postimplantation gene trap embryos revealed that LSD1 expression, and therefore function, is restricted to the epiblast. Conditional deletion of LSD1 in mouse ES cells, the in vitro counterpart of the epiblast, revealed a reduction in CoREST protein and associated HDAC activity, resulting in a global increase in histone H3-Lys56 acetylation, but not H3-Lys4 methylation. Despite this biochemical perturbation, ES cells with LSD1 deleted proliferate normally and retain stem cell characteristics. Loss of LSD1 causes the aberrant expression of 588 genes, including those coding for transcription factors with roles in anterior/posterior patterning and limb development, such as brachyury, Hoxb7, Hoxd8, and retinoic acid receptor γ (RARγ). The gene coding for brachyury, a key regulator of mesodermal differentiation, is a direct target gene of LSD1 and is overexpressed in e6.5 Lsd1 gene trap embryos. Thus, LSD1 regulates the expression and appropriate timing of key developmental regulators, as part of the LSD1/CoREST/HDAC complex, during early embryonic development.