Single-cell RNA sequencing uncovers the nuclear decoy lincRNA PIRAT as a regulator of systemic monocyte immunity during COVID-19

The systemic immune response to viral infection is shaped by master transcription fac-tors, such as NF-κB, STAT1, or PU.1. Although long noncoding RNAs (lncRNAs)have been suggested as important regulators of transcription factor activity, their contri-butions to the systemic immunopathologies observed during SARS-CoV-2 infectionhave remained unknown. Here, we employed a targeted single-cell RNA sequencingapproach to reveal lncRNAs differentially expressed in blood leukocytes during severeCOVID-19. Our results uncover the lncRNA PIRAT (PU.1-induced regulator of alar-min transcription) as a major PU.1 feedback-regulator in monocytes, governing the pro-duction of the alarmins S100A8/A9, key drivers of COVID-19 pathogenesis. Knockoutand transgene expression, combined with chromatin-occupancy profiling, characterizedPIRATasanucleardecoyRNA,keepingPU.1frombindingtoalarminpromotersandpromoting its binding to pseudogenes in naïve monocytes. NF-κB–dependent PIRATdown-regulation during COVID-19 consequently releases a transcriptional brake, fuelingalarmin production. Alarmin expression is additionally enhanced by the up-regulation ofthe lncRNA LUCAT1, which promotes NF-κB–dependentgeneexpressionattheexpenseof targets of the JAK-STAT pathway. Our results suggest a major role of nuclear noncod-ing RNA networks in systemic antiviral responses to SARS-CoV-2 in humans

Glucocorticoid-induziertes Wachstum von Tumorzellen: Systematische Quantifizierung, Signalmechanismen und Inhibition.

Glucocorticoide (GC) wie Dexamethason (Dex) werden in der Tumortherapie häufig eingesetzt, zum einen als Zytostatika zur Behandlung hämatopoetischer Tumoren und zum anderen als Adjuvantien zur Reduktion von Nebenwirkungen in der Behandlung solider Tumoren. In den letzten Jahren wurde jedoch gezeigt, dass GC die Wirkung von Zytostatika auf Zellen solider Tumore abschwächen können. Durch Vorarbeiten in unserer Arbeitsgruppe kam der Verdacht auf, dass GC in der Lage sind, Proliferation von Tumorzellen zu induzieren. In der vorliegenden Arbeit wurde erstmals die GC-induzierte Proliferation von Tumorzellen systematisch untersucht. Das beschleunigte Tumorzellwachstum wurde mittels repetitiver Mikroskopie, Impedanzanalyse, Bestimmung der DNASyntheserate, der enzymatischen Aktivität sowie der absoluten Zellzahl validiert. Eine Quantifizierung ergab, dass 6 von 10 Zelllinien verschiedenster solider Tumoren mit Proliferation auf GC reagierten. Darüber hinaus konnten die in vitro erhobenen pro-proliferativen Effekte der GC durch einen Tierversuch mit einer Lungenkarzinomzelllinie ebenfalls in vivo bestätigt werden. Des Weiteren wurden 139 primäre Proben von Kindern mit akuter Leukämie getestet und bei 15% der Proben ein Überlebensvorteil der Tumorzellen durch GC gemessen; eine Probe zeigte sogar GC-induzierte Proliferation. Demzufolge konnte der anti-apoptotische und pro-proliferative Effekt von GC nicht nur auf etablierten Zelllinien solider Tumore, sondern auch auf primären hämatopoetischen Tumorzellen nachgewiesen werden. Knockdown-Studien in Zellen solider Tumoren zeigten eine wichtige Rolle des Glucocorticoidrezeptors für die GC-induzierte Proliferation, welche des Weiteren durch die beiden Proteinkinasen Akt und p38-MAPK vermittelt wurde. GC-induzierte Proliferation konnte durch Apoptoseinduktion verhindert werden, die einerseits durch klinisch einsetzbare Substanzen, wie beispielsweise Vincristin herbeigeführt wurde, andererseits durch induzierbare Expression des pro-apoptotischen Moleküls Caspase-3. Zusammenfassend charakterisiert die vorliegende Arbeit GC-induzierte Proliferation von Tumorzellen als neue, Tumorzell-gerichtete Nebenwirkung von GC. Die Daten sprechen für einen zurückhaltenden Einsatz von GC während der Tumortherapie sowie für die Durchführung weiterführender präklinischer und klinischer Studien, die einen effektiveren und sichereren Einsatz von GC während der Tumortherapie aufzeigen

Glucocorticoids augment survival and proliferation of tumor cells.

BACKGROUND: Glucocorticoids are widely used for cancer patients, although they can reduce the efficacy of anticancer treatment. MATERIALS AND METHODS: We characterized non-apoptotic actions of glucocorticoids on tumor cell lines, primary tumor cells and an in vivo model, together with molecular signaling studies. RESULTS: Glucocorticoids enhanced cell proliferation in 9/17 cell lines and significantly promoted tumor cell proliferation in a pre-clinical mouse model of lung carcinoma. 65/139 primary acute childhood leukemia samples were glucocorticoid-resistant. Both dexamethasone and prednisolone increased in vitro survival in 21/65 samples from glucocorticoid-resistant primary leukemias, revealing a completely new action of glucocorticoids. Dexamethasone-induced proliferation was mediated by glucocorticoid receptor and activated the proliferation signaling pathways of protein kinase B/AKT and p38 mitogen-activated protein kinase. CONCLUSION: Our data suggest that restriction of the use of glucocorticoids during anticancer treatment might improve the outcome of patients with solid tumors

The adaptor protein FADD and the initiator caspase-8 mediate activation of NF-κB by TRAIL.

Besides inducing apoptosis, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) activates NF-κB. The apoptosis signaling pathway of TRAIL is well characterized involving TRAIL receptors, Fas-associated protein with death domain (FADD) and caspase-8. In contrast, the molecular mechanism of TRAIL signaling to NF-κB remains controversial. Here, we characterized the receptor-proximal mediators of NF-κB activation by TRAIL. Deletion of the DD of TRAIL receptors 1 and 2 revealed that it is essential in NF-κB signaling. Because FADD interacts with the TRAIL receptor DD, FADD was tested. RNAi-mediated knockdown of FADD or FADD deficiency in JURKAT T-cell leukemia cells decreased or disabled NF-κB signaling by TRAIL. In contrast, TRAIL-induced activation of NF-κB was maintained upon loss of receptor interacting protein 1 (RIP1) or knockdown of FLICE-like inhibitory protein (FLIP). Exogenous expression of FADD rescued TRAIL-induced NF-κB signaling. Loss-of-function mutations of FADD within the RHDLL motif of the death effector domain, which is required for TRAIL-induced apoptosis, abrogated FADD's ability to recruit caspase-8 and mediate NF-κB activation. Accordingly, deficiency of caspase-8 inhibited TRAIL-induced activation of NF-κB, which was rescued by wild-type caspase-8, but not by a catalytically inactive caspase-8 mutant. These data establish the mechanism of TRAIL-induced NF-κB activation involving the TRAIL receptor DD, FADD and caspase-8, but not RIP1 or FLIP. Our results show that signaling of TRAIL-induced apoptosis and NF-κB bifurcates downstream of caspase-8

Leukemia-initiating cells of patient-derived acute lymphoblastic leukemia xenografts are sensitive toward TRAIL.

Cancer stem cells represent the most important target cells for antitumor therapy. TRAIL (TNF-related apoptosis inducing ligand) is a potential anticancer agent that induces apoptosis in a wide variety of tumor cells, but its ability to target cancer stem cells is currently unknown. Here we investigated whether TRAIL targets leukemia-initiating cells. Limiting dilution transplantation assays were performed on xenografts from pediatric patients with precursor B-cell acute lymphoblastic leukemia (pre-B ALL) in NSG mice. In vitro treatment of xenograft cells with TRAIL significantly reduced and delayed their engraftment and procrastinated animal death from leukemia. Systemic TRAIL treatment of mice injected with patient-derived pre-B ALL xenograft cells abrogated leukemia in 3 of 5 mice in 1 sample. In conclusion, our data suggest that TRAIL targets leukemia-initiating cells derived from pre-B ALL xenografts in vitro and in vivo, and hence constitutes an attractive candidate drug for treatment of ALL