Ferroptosis Biology.

Ferroptosis is a regulated cell death modality triggered by iron-dependent lipid peroxidation. Ferroptosis plays a causal role in the pathophysiology of various diseases, making it a promising therapeutic target. Unlike all other cell death modalities dependent on distinct signaling cues, ferroptosis occurs when cellular antioxidative defense mechanisms fail to suppress the oxidative destruction of cellular membranes, eventually leading to cell membrane rupture. Physiologically, only two such surveillance systems are known to efficiently prevent the lipid peroxidation chain reaction by reducing (phospho)lipid hydroperoxides to their corresponding alcohols or by reducing radicals in phospholipid bilayers, thus maintaining the integrity of lipid membranes. Mechanistically, these two systems are linked to the reducing capacity of glutathione peroxidase 4 (GPX4) by consuming glutathione (GSH) on the one and ferroptosis suppressor protein 1 (FSP1, formerly AIFM2) on the other hand. Notably, the importance of ferroptosis suppression in physiological contexts has been linked to a particular vulnerability of renal tissue. In fact, early work has shown that mice genetically lacking Gpx4 rapidly succumb to acute renal failure with pathohistological features of acute tubular necrosis. Promising research attempting to implicate ferroptosis in various renal disease entities, particularly those with proximal tubular involvement, has generated a wealth of knowledge with widespread potential for clinical translation. Here, we provide a brief overview of the involvement of ferroptosis in nephrology. Our goal is to introduce this expanding field for clinically versed nephrologists in the hope of spurring future efforts to prevent ferroptosis in the pathophysiological processes of the kidney.

A tangible method to assess native ferroptosis suppressor activity.

Ferroptosis, a regulated cell death hallmarked by unrestrained lipid peroxidation, plays a pivotal role in the pathophysiology of various diseases, making it a promising therapeutic target. Glutathione peroxidase 4 (GPX4) prevents ferroptosis by reducing (phospho)lipid hydroperoxides, yet evaluation of its actual activity has remained arduous. Here, we present a tangible method using affinity-purified GPX4 to capture a snapshot of its native activity. Next to measuring GPX4 activity, this improved method allows for the investigation of mutational GPX4 activity, exemplified by the GPX4U46C mutant lacking selenocysteine at its active site, as well as the evaluation of GPX4 inhibitors, such as RSL3, as a showcase. Furthermore, we apply this method to the second ferroptosis guardian, ferroptosis suppressor protein 1, to validate the newly identified ferroptosis inhibitor WIN62577. Together, these methods open up opportunities for evaluating alternative ferroptosis suppression mechanisms.

High RIPK3 expression is associated with a higher risk of early kidney transplant failure.

Renal ischemia-reperfusion injury (IRI) is associated with reduced allograft survival, and each additional hour of cold ischemia time increases the risk of graft failure and mortality following renal transplantation. Receptor-interacting protein kinase 3 (RIPK3) is a key effector of necroptosis, a regulated form of cell death. Here, we evaluate the first-in-human RIPK3 expression dataset following IRI in kidney transplantation. The primary analysis included 374 baseline biopsy samples obtained from renal allografts 10 minutes after onset of reperfusion. RIPK3 was primarily detected in proximal tubular cells and distal tubular cells, both of which are affected by IRI. Time-to-event analysis revealed that high RIPK3 expression is associated with a significantly higher risk of one-year transplant failure and prognostic for one-year (death-censored) transplant failure independent of donor and recipient associated risk factors in multivariable analyses. The RIPK3 score also correlated with deceased donation, cold ischemia time and the extent of tubular injury.

Diverse biological functions of vitamin K: from coagulation to ferroptosis.

Vitamin K is essential for several physiological processes, such as blood coagulation, in which it serves as a cofactor for the conversion of peptide-bound glutamate to γ-carboxyglutamate in vitamin K-dependent proteins. This process is driven by the vitamin K cycle facilitated by γ-carboxyglutamyl carboxylase, vitamin K epoxide reductase and ferroptosis suppressor protein-1, the latter of which was recently identified as the long-sought-after warfarin-resistant vitamin K reductase. In addition, vitamin K has carboxylation-independent functions. Akin to ubiquinone, vitamin K acts as an electron carrier for ATP production in some organisms and prevents ferroptosis, a type of cell death hallmarked by lipid peroxidation. In this Perspective, we provide an overview of the diverse functions of vitamin K in physiology and metabolism and, at the same time, offer a perspective on its role in ferroptosis together with ferroptosis suppressor protein-1. A comparison between vitamin K and ubiquinone, from an evolutionary perspective, may offer further insights into the manifold roles of vitamin K in biology.

Tumor-Agnostic Precision Medicine from the AACR GENIE Database: Clinical Implications.

Biomarker-driven cancer therapy has revolutionized precision oncology. With a better understanding of tumor biology, tissue-agnostic targets have been characterized and explored, which ultimately led to therapeutics with pan-cancer efficacy. To date, five molecular biomarkers have obtained FDA tissue-agnostic approval for targeted therapies and immunotherapies. Those include BRAFV600E mutations, RET fusions, NTRK fusions, high tumor mutation burden (TMB), and deficient mismatch repair/high microsatellite instability (dMMR/MSI-High). Herein, we have used data from AACR project GENIE to explore the clinico-genomic landscape of these alterations. AACR GENIE is a publicly accessible registry of genomic data from multiple collaborating cancer centers. Current database (version 13.0) includes sequencing data of 168,423 samples collected from patients with different cancers. We were able to identify BRAFV600E, RET fusions, NTRK fusions, and high TMB in 2.9%, 1.6%, 1.5%, and 15.2% of pan-cancer samples, respectively. In this article, we describe the distribution of those tissue-agnostic targets among different cancer types. In addition, we summarize the current prospect on the biology of these alterations and evidence on approved drugs, including pembrolizumab, dostarilmab, larotrectinib, entrectinib, selpercatinib, and dabrafenib/trametinib combination.

N-of-1 Trials in Cancer Drug Development.

SUMMARY: The current approaches for cancer drug development lag behind an accelerated need in the field for a fast and efficient method for evaluating drugs in the personalized medicine era. In that regard, N-of-1 studies emerge as a potential addition to the drug development arsenal, although there are several considerations before its broad application becomes feasible. In essence, N-of-1 trials are a departure from the traditional "drug-centric" model to a "patient-centric" model. Herein, we review the concept of N-of-1 trials and provide real-world examples of their use in the developmental therapeutics field. N-of-1 trials offer an exceptional opportunity for fast-tracking of cancer drug development in the precision oncology era.

Ferroptosis: Under pressure!

Ferroptosis is a disease-relevant and pervasive form of cell death triggered by iron-dependent lipid peroxidation and resulting in membrane rupture. A new study addresses how tension-sensing channels can balance and modulate membrane tension in the context of ferroptotic cell death.

Platelet-instructed SPP1+ macrophages drive myofibroblast activation in fibrosis in a CXCL4-dependent manner.

Fibrosis represents the common end stage of chronic organ injury independent of the initial insult, destroying tissue architecture and driving organ failure. Here we discover a population of profibrotic macrophages marked by expression of Spp1, Fn1, and Arg1 (termed Spp1 macrophages), which expands after organ injury. Using an unbiased approach, we identify the chemokine (C-X-C motif) ligand 4 (CXCL4) to be among the top upregulated genes during profibrotic Spp1 macrophage differentiation. In vitro and in vivo studies show that loss of Cxcl4 abrogates profibrotic Spp1 macrophage differentiation and ameliorates fibrosis after both heart and kidney injury. Moreover, we find that platelets, the most abundant source of CXCL4 in vivo, drive profibrotic Spp1 macrophage differentiation. Single nuclear RNA sequencing with ligand-receptor interaction analysis reveals that macrophages orchestrate fibroblast activation via Spp1, Fn1, and Sema3 crosstalk. Finally, we confirm that Spp1 macrophages expand in both human chronic kidney disease and heart failure.

The coming decade in precision oncology: six riddles.

High-throughput methods to investigate tumour omic landscapes have quickly catapulted cancer specialists into the precision oncology era. The singular lesson of precision oncology might be that, for it to be precise, treatment must be personalized, as each cancer's complex molecular and immune landscape differs from patient to patient. Transformative therapies include those that are targeted at the sequelae of molecular abnormalities or at immune mechanisms, and, increasingly, pathways previously thought to be undruggable have become druggable. Critical to applying precision medicine is the concept that the right combination of drugs must be chosen for each patient and used at the right stage of the disease. Multiple puzzles remain that complicate therapy choice, including evidence that deleterious mutations are common in normal tissues and non-malignant conditions. The host's role is also likely to be key in determining treatment response, especially for immunotherapy. Indeed, maximizing the impact of immunotherapy will require omic analyses to match the right immune-targeted drugs to the individualized patient and tumour setting. In this Perspective, we discuss six key riddles that must be solved to optimize the application of precision oncology to otherwise lethal malignancies.

Cancer, cancer treatment and aneurysmatic ascending aorta growth within a retrospective single center study.

Background: Multi-morbidity poses a substantial challenge for health care in an aging population. Recent studies did not provide evidence for general side effects of anti-cancer therapy regarding the growth rate of coincident abdominal aortic aneurysms, although it was suggested that specific therapeutic substances might accelerate growth. Aneurysm pathology, however, differs with respect to localization. Hence, we present the first ever analysis on the association of cancer and cancer therapy with growth alteration of aneurysms of the ascending aorta (AscAA). Patients and methods: A retrospective single-center identification of AscAA+cancer patients was performed in the institutional picture archiving and communication system (PACS). Included were all patients with ≥2 CT angiograms over ≥6 months and additional malignancy. Clinical data and aneurysm diameters were retrieved and analyzed for an association of cancer (stratified by tumor entity) or cancer therapy (stratified by several classes of chemotherapeutic agents and radiation therapy) with annual growth rate, respectively. Statistics included t-test, Wilcoxon test, and a linear regression model accounting for initial AscAA diameter and type of treatment. Results: From 2003 to 2021, 151 patients (median age 70 years; 85% male) with AscAA and coincident 163 malignancies were identified. Prostate (37%) and hematologic cancer (17%) were most frequent. One-hundred-eleven patients (74%) received chemotherapy and 75 patients (50%) had radiation. After exclusion of six patients with an initial AscAA diameter >55 mm, the average annual AscAA growth rate was 0.18±0.64 mm/year, with only 12 patients experiencing a growth rate >1mm/year. Neither tumor entity nor radiation or chemotherapy - alone or in combination - were significantly associated with an alteration of the annual AscAA growth rate. Likewise, a subanalysis for singular chemotherapeutic agents did not reveal a specific association with AscAA growth alteration. Conclusions: Growth rates of AscAA are low in this cohort with coincident malignancy. Cancer and/or chemotherapy or radiation are not associated with an alteration of the annual growth rate. Additional control examinations seem unnecessary.

Molecular landscape of immune pressure and escape in aplastic anemia.

Idiopathic aplastic anemia (IAA) pathophysiology is dominated by autoreactivity of human leukocyte antigen (HLA)-restricted T-cells against antigens presented by hematopoietic stem and progenitor cells (HSPCs). Expansion of PIGA and HLA class I mutant HSPCs have been linked to immune evasion from T-cell mediated pressures. We hypothesized that in analogy with antitumor immunity, the pathophysiological cascade of immune escape in IAA is initiated by immunoediting pressures and culminates with mechanisms of clonal evolution characterized by hits in immune recognition and response genes. To that end, we studied the genetic and transcriptomic make-up of the antigen presentation complexes in a large cohort of patients with IAA and paroxysmal nocturnal hemoglobinuria (PNH) by using single-cell RNA, high throughput DNA sequencing and single nucleotide polymorphism (SNP)-array platforms. At disease onset, HSPCs displayed activation of selected HLA class I and II-restricted mechanisms, without extensive inhibition of immune checkpoint apparatus. Using a newly implemented bioinformatic framework we found that not only class I but also class II genes were often impaired by acquisition of genetic aberrations. We also demonstrated the presence of novel somatic alterations in immune genes possibly contributing to the evasion from the autoimmune T-cells. In contrast, these hits were absent in myeloid neoplasia. These aberrations were not mutually exclusive with PNH and did not correlate with the accumulation of myeloid-driver hits. Our findings shed light on the mechanisms of immune activation and escape in IAA and define alternative modes of clonal hematopoiesis.

A non-canonical vitamin K cycle is a potent ferroptosis suppressor.

Ferroptosis, a non-apoptotic form of cell death marked by iron-dependent lipid peroxidation1, has a key role in organ injury, degenerative disease and vulnerability of therapy-resistant cancers2. Although substantial progress has been made in understanding the molecular processes relevant to ferroptosis, additional cell-extrinsic and cell-intrinsic processes that determine cell sensitivity toward ferroptosis remain unknown. Here we show that the fully reduced forms of vitamin K-a group of naphthoquinones that includes menaquinone and phylloquinone3-confer a strong anti-ferroptotic function, in addition to the conventional function linked to blood clotting by acting as a cofactor for γ-glutamyl carboxylase. Ferroptosis suppressor protein 1 (FSP1), a NAD(P)H-ubiquinone reductase and the second mainstay of ferroptosis control after glutathione peroxidase-44,5, was found to efficiently reduce vitamin K to its hydroquinone, a potent radical-trapping antioxidant and inhibitor of (phospho)lipid peroxidation. The FSP1-mediated reduction of vitamin K was also responsible for the antidotal effect of vitamin K against warfarin poisoning. It follows that FSP1 is the enzyme mediating warfarin-resistant vitamin K reduction in the canonical vitamin K cycle6. The FSP1-dependent non-canonical vitamin K cycle can act to protect cells against detrimental lipid peroxidation and ferroptosis.

Executioner caspases 3 and 7 are dispensable for intestinal epithelium turnover and homeostasis at steady state.

Apoptosis is widely believed to be crucial for epithelial cell death and shedding in the intestine, thereby shaping the overall architecture of the gastrointestinal tract, but also regulating tolerance induction, pinpointing a role of apoptosis intestinal epithelial cell (IEC) turnover and maintenance of barrier function, and in maintaining immune homeostasis. To experimentally address this concept, we generated IEC-specific knockout mice that lack both executioner caspase-3 and caspase-7 (Casp3/7ΔIEC), which are the converging point of the extrinsic and intrinsic apoptotic pathway. Surprisingly, the overall architecture, cellular landscape, and proliferation rate remained unchanged in these mice. However, nonapoptotic cell extrusion was increased in Casp3/7ΔIEC mice, compensating apoptosis deficiency, maintaining the same physiological level of IEC shedding. Microbiome richness and composition stayed unaffected, bearing no sign of dysbiosis. Transcriptome and single-cell RNA sequencing analyses of IECs and immune cells revealed no differences in signaling pathways of differentiation and inflammation. These findings demonstrate that during homeostasis, apoptosis per se is dispensable for IEC turnover at the top of intestinal villi intestinal tissue dynamics, microbiome, and immune cell composition.

A study of Telomerase Reverse Transcriptase rare variants in myeloid neoplasia.

Telomere dysfunctions are associated with several hematopoietic stem cell (HSC) malignancies. Recent findings have indicated that the occurrence of rare variants of unknown significance (VUS) in the Telomerase Reverse Transcriptase (TERT) gene influences the outcomes of patients with myelodysplastic syndromes undergoing allogeneic HSC transplantation. However, the role of TERT variants has been historically controversial as initially considered pathogenic variants (H412Y, A202T) presenting functional consequences, were found very frequent in general population questioning their pathogenicity and risk allele significance. Herein, we show that overall TERT VUS are non-recurrent in myeloid disorders and cannot be considered risk alleles individually nor can their biological impact.

Concomitantly discovered visceral artery aneurysms do rarely grow during cancer therapy.

Visceral artery aneurysms (VAA) are a rare entity of arterial aneurysms with the imminent threat of rupture. The impact of cancer and chemotherapy on the growth of VAAs is unknown. A retrospective dual center cohort study of patients with concomitant VAA and different types of cancer was conducted and the impact of various chemotherapeutic agents on VAA growth was studied by sequential CT analysis. For comparison, a non-cancer all comer cohort with VAAs and no cancer was studied to compare different growth rates. The primary endpoint was aneurysm progress or regression >1.75 mm. Chi-square test, Fisher's exact test and Mann-Whitney test was used for statistical comparison. In the 17-year-period from January 2003 to March 2020, 59 patients with 30 splenic artery aneurysms, 14 celiac trunk aneurysms, 11 renal artery aneurysms and 4 other VAA and additional malignancy were identified. 20% of patients suffered from prostate cancer, the rest were heterogeneous. The most prevalent chemotherapies were alkylating agents (23%), antimetabolites (14%) and mitose inhibitors (10%). Eight patients had relevant growth of their VAA and one patient showed diameter regression (average growth rate 0.1 ± 0.5 mm/year). Twenty-nine patients with 14 splenic, 11 RAAs (seven right) and 4 celiac trunk aneurysms were available in the non-cancer comparison cohort (average growth rate 0.5 ± 0.9 mm/year, p = 0.058). However, the growth rate of patients receiving operative treatment for relevant VAA growth was significantly higher (p = 0.004). VAAs grow rarely, and rather slow. Cancer and/or chemotherapy do not significantly influence the annual growth rate. Additional control examinations seem unnecessary.

XIAP restrains TNF-driven intestinal inflammation and dysbiosis by promoting innate immune responses of Paneth and dendritic cells.

Deficiency in X-linked inhibitor of apoptosis protein (XIAP) is the cause for X-linked lymphoproliferative syndrome 2 (XLP2). About one-third of these patients suffer from severe and therapy-refractory inflammatory bowel disease (IBD), but the exact cause of this pathogenesis remains undefined. Here, we used XIAP-deficient mice to characterize the mechanisms underlying intestinal inflammation. In Xiap−/− mice, we observed spontaneous terminal ileitis and microbial dysbiosis characterized by a reduction of Clostridia species. We showed that in inflamed mice, both TNF receptor 1 and 2 (TNFR1/2) cooperated in promoting ileitis by targeting TLR5-expressing Paneth cells (PCs) or dendritic cells (DCs). Using intestinal organoids and in vivo modeling, we demonstrated that TLR5 signaling triggered TNF production, which induced PC dysfunction mediated by TNFR1. TNFR2 acted upon lamina propria immune cells. scRNA-seq identified a DC population expressing TLR5, in which Tnfr2 expression was also elevated. Thus, the combined activity of TLR5 and TNFR2 signaling may be responsible for DC loss in lamina propria of Xiap−/− mice. Consequently, both Tnfr1−/−Xiap−/− and Tnfr2−/−Xiap−/− mice were rescued from dysbiosis and intestinal inflammation. Furthermore, RNA-seq of ileal crypts revealed that in inflamed Xiap−/− mice, TLR5 signaling was abrogated, linking aberrant TNF responses with the development of a dysbiosis. Evidence for TNFR2 signaling driving intestinal inflammation was detected in XLP2 patient samples. Together, these data point toward a key role of XIAP in mediating resilience of TLR5-expressing PCs and intestinal DCs, allowing them to maintain tissue integrity and microbiota homeostasis.

Rethinking phage-bacteria-eukaryotic relationships and their influence on human health.

There is a rapidly growing body of research demonstrating the unique and often surprising mechanisms by which bacteriophages, specialized viruses of bacteria, can influence human health and disease states. This can occur directly by shaping their bacterial host's ecology through top-down pressure or via more indirect routes, including influencing the human body's metabolism or immune system. These microbial interactions can affect health and disease states in both the local environment or by influencing the body's distal organs or systems. Here we provide an update on the current understanding of bacteriophages' influence on human health within the context of tripartite symbioses with their bacterial and human hosts.

ABO subgroup incompatibility with severe hemolysis after consecutive allogeneic stem cell transplantations.

Allogeneic hematopoietic stem cell transplantations (HSCTs) represent a curative strategy for treating hematologic malignancies yet bear dangerous and frequently life-threatening complications including the development of graft-versus-host disease. Here, we present a case of a patient that suffered from relapsed/refractory multiple myeloma, a hematologic neoplasm characterized by clonal proliferation of malignant plasma cells in the bone marrow. During the course of his disease, the patient underwent consecutive allogeneic HSCTs, during which he developed a clinical meaningful and hitherto unreported ABO subgroup incompatibility, leading to persistent hemolysis. Testing for ABO subgroups during donor selection, especially after consecutive allogeneic HSCTs, may therefore aid to prevent these complications.