Incorporation of CENP-A/CID into centromeres during early Drosophila embryogenesis does not require RNA polymerase II–mediated transcription

In many species, centromere identity is specified epigenetically by special nucleosomes containing a centromere-specific histone H3 variant, designated as CENP-A in humans and CID in Drosophila melanogaster. After partitioning of centromere-specific nucleosomes onto newly replicated sister centromeres, loading of additional CENP-A/CID into centromeric chromatin is required for centromere maintenance in proliferating cells. Analyses with cultured cells have indicated that transcription of centromeric DNA by RNA polymerase II is required for deposition of new CID into centromere chromatin. However, a dependence of centromeric CID loading on transcription is difficult to reconcile with the notion that the initial embryonic stages appear to proceed in the absence of transcription in Drosophila, as also in many other animal species. To address the role of RNA polymerase II–mediated transcription for CID loading in early Drosophila embryos, we have quantified the effects of alpha-amanitin and triptolide on centromeric CID-EGFP levels. Our analyses demonstrate that microinjection of these two potent inhibitors of RNA polymerase II–mediated transcription has at most a marginal effect on centromeric CID deposition during progression through the early embryonic cleavage cycles. Thus, we conclude that at least during early Drosophila embryogenesis, incorporation of CID into centromeres does not depend on RNA polymerase II–mediated transcription.

Anti-CD37 Alpha-Amanitin Conjugated Antibodies As Therapeutic Weapons for Richter's Syndrome

The leukocyte surface antigen CD37 (TSPAN26), a member of the tetraspanin superfamily, is widely expressed on most malignant B cells, making it an actionable target for treatment of patients with chronic lymphocytic leukemia (CLL) and other B-cell non-Hodgkin lymphoma (NHL) indications. Accordingly, αCD37 antibodies have shown promising results in phase 1/2 clinical trials for CLL and NHL. Richter's syndrome (RS) is the transformation of CLL into an aggressive and rapidly fatal lymphoma, typically a diffuse large B cell lymphoma (DLBCL). RS is a challenging disease since very few effective treatment options exist for these patients and the available regimens, mainly based on R-CHOP scheme, show limited efficacy. We recently established patient-derived xenograft (PDX) models from RS patients and have shown that they can be used to test the efficacy of novel drugs and drug combinations 1,2. All available RS-PDX models were characterized by high-levels of CD37 expression, when assessed by RNA sequencing, reverse-transcriptase-polymerase chain reaction (RT-PCR), flow cytometry (FACS), western blot (WB) and immunohistochemistry (IHC). More precisely, two models (RS1316 and IP867/17) showed slightly higher CD37 levels compared to the others (RS9737 and RS1050). These models were used to test three different αCD37-ATACs®, ADCs which comprise amanitin-derivatives as payload. Amanitin (the main poison in the green deathcap mushroom) belongs to the well-known amatoxin family. Amanitin is taken up by OATP1B3 transporter, solely expressed on hepatocytes. Upon mushroom intoxication, it can lead to severe liver toxicity by inhibiting the RNA polymerase II. Upon conjugation to target-specific antibodies, the maximal tolerated dose is significantly increased by reducing the non-specific liver uptake. By binding to its antigen, ATACs deliver amanitin only into target-positive cancer cells while target negative cells show no off-target toxicity. Consistent with CD37 expression on the cell surface, ex-vivo treatment of freshly purified cells from RS-PDX tumor masses to αCD37-ATACs® resulted in increased apoptosis after 72 hours of treatment, with only minor differences among the 3 ATACs® and the models used. Since alpha-amanitin is a deadly toxin known to target human RNA polymerase II and, at high doses, also RNA polymerase III, we checked messenger RNA levels in basal conditions and after CD37-ATAC® treatment by looking at different housekeeping genes, and confirmed a reduction in global mRNA levels. αCD37-ATAC® efficacy was then assessed in vivo in systemic RS-PDX models where RS cells are intra-venously (i.v.) injected in the tail vein and cells distribute to different tissues (blood, spleen and bone marrow), resembling the human disease. Cells from RS1316, RS1050 and RS9737 models were injected into the tail vein and left to engraft 14 days, before mice were randomly assigned to vehicle or ATAC® groups. A single i.v. treatment for each αCD37-ATAC® was administered, testing two different doses for each compound, and mice were then monitored for survival. Overall, the single administration of all three ATACs® caused highly significant disease regression. In the RS1316 model, independently of the dose or tested ATAC®s, all treated mice, except one, were alive and disease-free until the end of the experiment, 140 days post cells injection, while survival of vehicle-treated mice was 65 days. FACS analysis to trace neoplastic cells in parenchymatous organs and bone marrow confirmed the absence of neoplastic cells. In the other 2 models, RS9737 and RS1050, even though ATAC® treatment did not result in complete disease eradication, a single administration of αCD37-ATAC® resulted in a dramatically increased survival (approximately 35-60 days, depending on the model and ATAC® used). Finally, CD37 expression was confirmed by RNA sequencing on a cohort of 15 primary RS samples, even though with variable levels. Compared to CLL cells, RS samples showed CD37 expression levels comparable to those reported for DLBCL cells. Overall, these data indicate CD37 as a potential target to treat RS patients with selective targeting αCD37-ATACs®. ATACs® is a registered trade mark of Heidelberg Pharma Research GmbH, Germany References Vaisitti T et al. Blood. 2021;137(24):3365-3377. Iannello A, et al. Blood. 2021;137(24):3378-3389. Disclosures Orlik: Heidelberg Pharma: Current Employment. Kulke: Heidelberg Pharma: Current Employment. Pahl: Heidelberg Pharma: Current Employment. Deaglio: Heidelberg Pharma: Research Funding; Astra Zeneca: Research Funding.

Defining a Role of Amanita phalloides Toxins in Cancer: Research and Therapy

Despite the progress of diagnostic and therapy, the cancer burden is still rising worldwide. The new chemotherapeutical toxicity to somatic cells and its tolerance to tumor cells illustrates the immediate demand through recent pharmaceutical products with less harmful impacts. The use of natural anticancer products, like alpha-amanitin toxins have reached the cancer field therapy since the separation of Amanita phalloides fungi was performed. Application of Amanita phalloides affects tumor cell activity. It is thought that Amanita phalloides dilutions are recommended for a patient suffering from various cancer types and have no severe side effects resulting from amanita therapy. This review aims to explain the use of the therapeutic potential of -amanitin toxin against different cancer types.

Identification of Decrease in TRiC Proteins as Novel Targets of Alpha-Amanitin-Derived Hepatotoxicity by Comparative Proteomic Analysis In Vitro

Alpha-amanitin (α-AMA) is a cyclic peptide and one of the most lethal mushroom amatoxins found in Amanita phalloides. α-AMA is known to cause hepatotoxicity through RNA polymerase II inhibition, which acts in RNA and DNA translocation. To investigate the toxic signature of α-AMA beyond known mechanisms, we used quantitative nanoflow liquid chromatography–tandem mass spectrometry analysis coupled with tandem mass tag labeling to examine proteome dynamics in Huh-7 human hepatoma cells treated with toxic concentrations of α-AMA. Among the 1828 proteins identified, we quantified 1563 proteins, which revealed that four subunits in the T-complex protein 1-ring complex protein decreased depending on the α-AMA concentration. We conducted bioinformatics analyses of the quantified proteins to characterize the toxic signature of α-AMA in hepatoma cells. This is the first report of global changes in proteome abundance with variations in α-AMA concentration, and our findings suggest a novel molecular regulation mechanism for hepatotoxicity.

Chlorophyllum Molybdites “Delicious Poisoning Snack” in Patients Diagnosed with Acute Gastroenteritis

Introduction: Since time immemorial, mushrooms have been used as a part of human diet, some of them are very well known for their nutritive and medicinal properties and some are known to cause poisoning to the human body. A number of post ingestion fatalities due to poisonous mushrooms has been reported worldwide. These poisonous mushrooms are often misidentified as edible ones, which accounts for accidental poisoning.Objective: The main objective of this report was to describe the clinical manifestations of mushroom poisoning cases presented at the Emergency Department (ED), Taiping Hospital.Case Presentation: There were two cases presented, who suffered from moderate dehydration due to acute gastroenteritis after taking 'delicious mushrooms', also known as Chlorophyllum Molybdites. This study found that both cases had complaints of abdominal cramping, diarrhoea and vomiting more than twenty times a day. There was no history of numbness or weakness noted, and no chest pain or shortness of breath. On arrival, both cases presented signs of moderate dehydration with coated tongue and normal blood pressure, with slightly increased in temperature (37.30C). Abdomen was soft but discomfort upon palpation and described as bloated. Both cases were resuscitated with 20ml/kg normal saline. Charcoal, antiemetic, proton pump inhibitor and ceftriaxone antibiotic were given at the ED. Both survived and were treated as infectious acute gastroenteritis. Nausea and vomiting were the most common early symptoms of intoxication and should be considered as a medical emergency. Alpha Amanitin levels should be checked where possible if amanita poisoning is suspected. An early diagnosis and immediate treatment are required for a successful outcome.Conclusion: All patients with the history of mushroom ingestion should be admitted. If laboratory detection of toxin is not available, history of mushroom ingestion, clinical manifestation and their trends could define mushroom poisoning.International Journal of Human and Health Sciences Supplementary Issue: 2021 Page: S17