High-dose chemotherapy and autologous haematopoietic stem-cell transplantation in older, fit patients with primary diffuse large B-cell CNS lymphoma (MARTA): a single-arm, phase 2 trial.

BACKGROUND: Available treatments for older patients with primary diffuse large B-cell CNS lymphoma (PCNSL) offer progression-free survival of up to 16 months. We aimed to investigate an intensified treatment of high-dose chemotherapy and autologous haematopoietic stem-cell transplantation (HSCT) in older patients with PCNSL. METHODS: MARTA was a prospective, single-arm, phase 2 study done at 15 research hospitals in Germany. Patients aged 65 years or older with newly diagnosed, untreated PCNSL were enrolled if they had an Eastern Cooperative Oncology Group performance status of 0-2 and were fit for high-dose chemotherapy and autologous HSCT. Induction treatment consisted of two 21-day cycles of high-dose intravenous methotrexate 3·5 g/m2 (day 1), intravenous cytarabine 2 g/m2 twice daily (days 2 and 3), and intravenous rituximab 375 mg/m2 (days 0 and 4) followed by high-dose chemotherapy with intravenous rituximab 375 mg/m2 (day -8), intravenous busulfan 3·2 mg/kg (days -7 and -6), and intravenous thiotepa 5 mg/kg (days -5 and -4) plus autologous HSCT. The primary endpoint was progression-free survival at 12 months in all patients who met eligibility criteria and started treatment. The study was registered with the German clinical trial registry, DRKS00011932, and recruitment is complete. FINDINGS: Between Nov 28, 2017, and Sept 16, 2020, 54 patients started induction treatment and 51 were included in the full analysis set. Median age was 71 years (IQR 68-75); 27 (53%) patients were female and 24 (47%) were male. At a median follow-up of 23·0 months (IQR 16·8-37·4), 23 (45%) of 51 patients progressed, relapsed, or died. 12-month progression-free survival was 58·8% (80% CI 48·9-68·2; 95% CI 44·1-70·9). During induction treatment, the most common grade 3-5 toxicities were thrombocytopenia and leukopenia (each in 52 [96%] of 54 patients). During high-dose chemotherapy and autologous HSCT, the most common grade 3-5 toxicity was leukopenia (37 [100%] of 37 patients). Treatment-related deaths were reported in three (6%) of 54 patients, all due to infectious complications. INTERPRETATION: Although the primary efficacy threshold was not met, short induction followed by high-dose chemotherapy and autologous HSCT is active in selected older patients with PCNSL and could serve as a benchmark for comparative trials. FUNDING: Else Kröner-Fresenius Foundation, Riemser Pharma, and Medical Center-University of Freiburg.

Entirely noninvasive outcome prediction in central nervous system lymphomas using circulating tumor DNA.

ABSTRACT: State-of-the-art response assessment of central nervous system lymphoma (CNSL) by magnetic resonance imaging is challenging and an insufficient predictor of treatment outcomes. Accordingly, the development of novel risk stratification strategies in CNSL is a high unmet medical need. We applied ultrasensitive circulating tumor DNA (ctDNA) sequencing to 146 plasma and cerebrospinal fluid (CSF) samples from 67 patients, aiming to develop an entirely noninvasive dynamic risk model considering clinical and molecular features of CNSL. Our ultrasensitive method allowed for the detection of CNSL-derived mutations in plasma ctDNA with high concordance to CSF and tumor tissue. Undetectable plasma ctDNA at baseline was associated with favorable outcomes. We tracked tumor-specific mutations in plasma-derived ctDNA over time and developed a novel CNSL biomarker based on this information: peripheral residual disease (PRD). Persistence of PRD after treatment was highly predictive of relapse. Integrating established baseline clinical risk factors with assessment of radiographic response and PRD during treatment resulted in the development and independent validation of a novel tool for risk stratification: molecular prognostic index for CNSL (MOP-C). MOP-C proved to be highly predictive of outcomes in patients with CNSL (failure-free survival hazard ratio per risk group of 6.60; 95% confidence interval, 3.12-13.97; P < .0001) and is publicly available at www.mop-c.com. Our results highlight the role of ctDNA sequencing in CNSL. MOP-C has the potential to improve the current standard of clinical risk stratification and radiographic response assessment in patients with CNSL, ultimately paving the way toward individualized treatment.

Diagnostic Accuracy of MR Spectroscopic Imaging and 18F-FET PET for Identifying Glioma: A Biopsy-Controlled Hybrid PET/MRI Study.

Contrast-enhanced MRI is the method of choice for brain tumor diagnostics, despite its low specificity for tumor tissue. This study compared the contribution of MR spectroscopic imaging (MRSI) and amino acid PET to improve the detection of tumor tissue. Methods: In 30 untreated patients with suspected glioma, O-(2-[18F]fluoroethyl)-l-tyrosine (18F-FET) PET; 3-T MRSI with a short echo time; and fluid-attenuated inversion recovery, T2-weighted, and contrast-enhanced T1-weighted MRI were performed for stereotactic biopsy planning. Serial samples were taken along the needle trajectory, and their masks were projected to the preoperative imaging data. Each sample was individually evaluated neuropathologically. 18F-FET uptake and the MRSI signals choline (Cho), N-acetyl-aspartate (NAA), creatine, myoinositol, and derived ratios were evaluated for each sample and classified using logistic regression. The diagnostic accuracy was evaluated by receiver operating characteristic analysis. Results: On the basis of the neuropathologic evaluation of tissue from 88 stereotactic biopsies, supplemented with 18F-FET PET and MRSI metrics from 20 areas on the healthy-appearing contralateral hemisphere to balance the glioma/nonglioma groups, 18F-FET PET identified glioma with the highest accuracy (area under the receiver operating characteristic curve, 0.89; 95% CI, 0.81-0.93; threshold, 1.4 × background uptake). Among the MR spectroscopic metabolites, Cho/NAA normalized to normal brain tissue showed the highest diagnostic accuracy (area under the receiver operating characteristic curve, 0.81; 95% CI, 0.71-0.88; threshold, 2.2). The combination of 18F-FET PET and normalized Cho/NAA did not improve the diagnostic performance. Conclusion: MRI-based delineation of gliomas should preferably be supplemented by 18F-FET PET.

Morphological and molecular comparison of HIV-associated and sporadic inclusion body myositis.

OBJECTIVE: The molecular characteristics of sporadic inclusion body myositis (sIBM) have been intensively studied, and specific patterns on the cellular, protein and RNA level have emerged. However, these characteristics have not been studied in the context of HIV-associated IBM (HIV-IBM). In this study, we compared clinical, histopathological, and transcriptomic patterns of sIBM and HIV-IBM. METHODS: In this cross-sectional study, we compared patients with HIV-IBM and sIBM based on clinical and morphological features as well as gene expression levels of specific T-cell markers in skeletal muscle biopsy samples. Non-disease individuals served as controls (NDC). Cell counts for immunohistochemistry and gene expression profiles for quantitative PCR were used as primary outcomes. RESULTS: 14 muscle biopsy samples (7 HIV-IBM, 7 sIBM) of patients and 6 biopsy samples from NDC were included. Clinically, HIV-IBM patients showed a significantly lower age of onset and a shorter period between symptom onset and muscle biopsy. Histomorphologically, HIV-IBM patients showed no KLRG1+ or CD57+ cells, while the number of PD1+ cells did not differ significantly between the two groups. All markers were shown to be significantly upregulated at gene expression level with no significant difference between the IBM subgroups. CONCLUSION: Despite HIV-IBM and sIBM sharing important clinical, histopathological, and transcriptomic signatures, the presence of KLRG1+ cells discriminated sIBM from HIV-IBM. This may be explained by longer disease duration and subsequent T-cell stimulation in sIBM. Thus, the presence of TEMRA cells is characteristic for sIBM, but not a prerequisite for the development of IBM in HIV+ patients.

AMD3100-Mediated CXCR4 Inhibition Impairs Development of Primary Lymphoma of the Central Nervous System.

A hallmark of primary lymphoma of the central nervous system (CNS; PCNSL) is the strong CXCR4 expression of the tumor cells, the function of which is still unknown. In vitro treatment of BAL17CNS lymphoma cells by AMD3100, which inhibits CXCR4-CXCL12 interactions, resulted in the significantly differential expression of 273 genes encoding proteins involved in cell motility, cell-cell signaling and interaction, hematological system development and function, and immunologic disease. Among the genes down-regulated was the one encoding CD200, a regulator of CNS immunologic activity. These data directly translated into the in vivo situation; BAL17CNS CD200 expression was down-regulated by 89% (3% versus 28% CD200+ lymphoma cells) in AMD3100-treated versus untreated mice with BAL17CNS-induced PCNSL. Reduced lymphoma cell CD200 expression may contribute to the markedly increased microglial activation in AMD3100-treated mice. AMD3100 also maintained the structural integrity of blood-brain barrier tight junctions and the outer basal lamina of cerebral blood vessels. Subsequently, lymphoma cell invasion of the brain parenchyma was impaired, and maximal parenchymal tumor size was significantly reduced by 82% in the induction phase. Thus, AMD3100 qualified as a potentially attractive candidate to be included into the therapeutic concept of PCNSL. Beyond therapy, CXCR4-induced suppression of microglial activity is of general neuroimmunologic interest. This study identified CD200 expressed by the lymphoma cells as a novel mechanism of immune escape in PCNSL.

Glioneuronal tumor with ATRX alteration, kinase fusion and anaplastic features (GTAKA): a molecularly distinct brain tumor type with recurrent NTRK gene fusions.

Glioneuronal tumors are a heterogenous group of CNS neoplasms that can be challenging to accurately diagnose. Molecular methods are highly useful in classifying these tumors-distinguishing precise classes from their histological mimics and identifying previously unrecognized types of tumors. Using an unsupervised visualization approach of DNA methylation data, we identified a novel group of tumors (n = 20) that formed a cluster separate from all established CNS tumor types. Molecular analyses revealed ATRX alterations (in 16/16 cases by DNA sequencing and/or immunohistochemistry) as well as potentially targetable gene fusions involving receptor tyrosine-kinases (RTK; mostly NTRK1-3) in all of these tumors (16/16; 100%). In addition, copy number profiling showed homozygous deletions of CDKN2A/B in 55% of cases. Histological and immunohistochemical investigations revealed glioneuronal tumors with isomorphic, round and often condensed nuclei, perinuclear clearing, high mitotic activity and microvascular proliferation. Tumors were mainly located supratentorially (84%) and occurred in patients with a median age of 19 years. Survival data were limited (n = 18) but point towards a more aggressive biology as compared to other glioneuronal tumors (median progression-free survival 12.5 months). Given their molecular characteristics in addition to anaplastic features, we suggest the term glioneuronal tumor with ATRX alteration, kinase fusion and anaplastic features (GTAKA) to describe these tumors. In summary, our findings highlight a novel type of glioneuronal tumor driven by different RTK fusions accompanied by recurrent alterations in ATRX and homozygous deletions of CDKN2A/B. Targeted approaches such as NTRK inhibition might represent a therapeutic option for patients suffering from these tumors.

European Association of Neuro-Oncology (EANO) guidelines for treatment of primary central nervous system lymphoma (PCNSL).

The management of primary central nervous system (PCNSL) is one of the most controversial topics in neuro-oncology because of the complexity of the disease and the limited number of controlled studies available. In 2021, given recent advances and the publication of practice-changing randomized trials, the European Association of Neuro-Oncology (EANO) created a multidisciplinary task force to update the previously published evidence-based guidelines for immunocompetent adult patients with PCNSL and added a section on immunosuppressed patients. The guideline provides consensus considerations and recommendations for the treatment of PCNSL, including intraocular manifestations and specific management of the elderly. The main changes from the previous guideline include strengthened evidence for the consolidation with ASCT in first-line treatment, prospectively assessed chemotherapy combinations for both young and elderly patients, clarification of the role of rituximab even though the data remain inconclusive, of the role of new agents, and the incorporation of immunosuppressed patients and primary ocular lymphoma. The guideline should aid the clinicians in everyday practice and decision making and serve as a basis for future research in the field.

Colloid cyst with hyphal-like structures: A rarity that mimics actinomycosis of athe third ventricle.

Colloid cysts are histologically well defined and consist of three main components, a capsule, with an underlying epithelial layer, and a mucinous heart. In our case, we present a 35-year-old female with acute deterioration of level of consciousness. An emergent CT scan showed a cystic lesion occluding the intraventricular foramen. The lesion was endoscopically excised through a transfrontal approach. Microscopic examination of the resected specimen revealed hyphal-like structures (HLS). This rare finding was first described by Dodds and Powers in 1977 and, in its microscopic nature, it mimics actinomyces of the third ventricle.

Expression of Cas9 in a Syngeneic Model of Primary Central Nervous System Lymphoma Induces Intracerebral NK and CD8 T Cell-Mediated Lymphoma Cell Lysis Via Perforin.

The development of clustered regulatory interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR-Cas9)-mediated gene modification has opened an exciting avenue of targeting genes to study the pathogenesis of diseases and to develop novel therapeutic concepts. However, as the effector protein Cas9 is of bacterial origin, unwanted side effects due to a host immune response against Cas9 need to be considered. Here, we used the syngeneic model of BAL17CNS-induced primary lymphoma of the central nervous system (PCNSL, CNS) in BALB/c mice to address this issue. Surprisingly, stable expression of Cas9 in BAL17CNS (BAL17CNS/Cas9) cells rendered them unable to establish PCNSL on intracerebral transplantation. Instead, they induced a prominent intracerebral immune response mediated by CD8 T cells, which lysed BAL17CNS/Cas9 cells via perforin. In addition, B cells contributed to the immune response as evidenced by serum anti-Cas9 antibodies in BALB/c mice as early as day 8 after transplantation of BAL17CNS/Cas9 cells. In athymic BALB/cnu/nu mice, NK cells mounted a vigorous intracerebral immune response with perforin-mediated destruction of BAL17CNS/Cas9 cells. Thus, in the CNS, perforin produced by NK and CD8 T cells was identified as a mediator of cytotoxicity against BAL17CNS/Cas9 cells. These observations should be taken into account when considering therapeutic CRISPR-Cas9-mediated tumor cell manipulation for PCNSL.

Efficacy and tolerability of regorafenib in pretreated patients with progressive CNS grade 3 or 4 gliomas.

BACKGROUND: The phase 2 REGOMA trial suggested an encouraging overall survival benefit in glioblastoma patients at first relapse treated with the multikinase inhibitor regorafenib. Here, we evaluated the efficacy and side effects of regorafenib in a real-life setting. METHODS: From 2018 to 2021, 30 patients with progressive WHO CNS grade 3 or 4 gliomas treated with regorafenib (160 mg/day; first 3 weeks of each 4-week cycle) with individual dose adjustment depending on toxicity were retrospectively identified. Side effects were evaluated according to the Common Terminology Criteria for Adverse Events (version 5.0). MRI was obtained at baseline and after every second cycle. Tumor progression was assessed according to RANO criteria. After regorafenib initiation, the median PFS and OS were calculated. RESULTS: The median number of treatment lines before regorafenib was 2 (range 1-4). Most patients (73%) had two or more pretreatment lines. At first relapse, 27% of patients received regorafenib. A total of 94 regorafenib cycles were administered (median 2 cycles; range 1-9 cycles). Grade 3 and 4 side effects were observed in 47% and 7% of patients, respectively, and were not significantly increased in patients with two or more pretreatments (P > 0.05). The most frequent grade 3 or 4 side effects were laboratory abnormalities (62%). PFS was 2.6 months (range 0.8-8.2 months), and the OS was 6.2 months (range 0.9-24 months). CONCLUSIONS: In patients with progressive WHO grade 3 or 4 gliomas, predominantly with two pretreatment lines or more, regorafenib seems to be effective despite considerable grade 3 or 4 side effects.

Long-term efficacy, safety and neurotolerability of MATRix regimen followed by autologous transplant in primary CNS lymphoma: 7-year results of the IELSG32 randomized trial.

219 HIV-negative adults ≤70 years with primary CNS lymphoma (PCNSL) were enrolled in the randomized IELSG32 trial. Enrolled patients were randomly assigned to receive methotrexate-cytarabine (arm A), or methotrexate-cytarabine-rituximab (B), or methotrexate-cytarabine-thiotepa-rituximab (MATRix; arm C). A second randomization allocated patients with responsive/stable disease to whole-brain irradiation (WBRT) or carmustine-thiotepa-conditioned autologous transplantation (ASCT). First results, after a median follow-up of 30 months, showed that MATRix significantly improves outcome, with both WBRT and ASCT being similarly effective. However, sound assessment of overall survival (OS), efficacy of salvage therapy, late complications, secondary tumors, and cognitive impairment requires longer follow-up. Herein, we report the results of this trial at a median follow-up of 88 months. As main findings, MATRix was associated with excellent long-lasting outcome, with a 7-year OS of 21%, 37%, and 56% respectively for arms A, B, and C. Notably, patients treated with MATRix and consolidation had a 7-year OS of 70%. The superiority of arm B on arm A suggests a benefit from the addition of rituximab. Comparable efficacy of WBRT and ASCT was confirmed. Salvage therapy was ineffective; benefit was recorded only in patients with late relapse re-treated with methotrexate. Eight (4%) patients developed a second cancer. Importantly, MATRix and ASCT did not result in higher non-relapse mortality or second tumors incidence. Patients who received WBRT experienced impairment in attentiveness and executive functions, whereas patients undergoing ASCT experienced improvement in these functions as well as in memory and quality of life.

Radiomics for the noninvasive prediction of the BRAF mutation status in patients with melanoma brain metastases.

BACKGROUND: The BRAF V600E mutation is present in approximately 50% of patients with melanoma brain metastases and an important prerequisite for response to targeted therapies, particularly BRAF inhibitors. As heterogeneity in terms of BRAF mutation status may occur in melanoma patients, a wild-type extracranial primary tumor does not necessarily rule out a targetable mutation in brain metastases using BRAF inhibitors. We evaluated the potential of MRI radiomics for a noninvasive prediction of the intracranial BRAF mutation status. METHODS: Fifty-nine patients with melanoma brain metastases from two university brain tumor centers (group 1, 45 patients; group 2, 14 patients) underwent tumor resection with subsequent genetic analysis of the intracranial BRAF mutation status. Preoperative contrast-enhanced MRI was manually segmented and analyzed. Group 1 was used for model training and validation, group 2 for model testing. After radiomics feature extraction, a test-retest analysis was performed to identify robust features prior to feature selection. Finally, the best performing radiomics model was applied to the test data. Diagnostic performances were evaluated using receiver operating characteristic (ROC) analyses. RESULTS: Twenty-two of 45 patients (49%) in group 1, and 8 of 14 patients (57%) in group 2 had an intracranial BRAF V600E mutation. A linear support vector machine classifier using a six-parameter radiomics signature yielded an area under the ROC curve of 0.92 (sensitivity, 83%; specificity, 88%) in the test data. CONCLUSIONS: The developed radiomics classifier allows a noninvasive prediction of the intracranial BRAF V600E mutation status in patients with melanoma brain metastases with high diagnostic performance.

Impact of a Faulty Germinal Center Reaction on the Pathogenesis of Primary Diffuse Large B Cell Lymphoma of the Central Nervous System.

Primary lymphoma of the central nervous system (PCNSL, CNS) is a specific diffuse large B cell lymphoma (DLBCL) entity confined to the CNS. Key to its pathogenesis is a failure of B cell differentiation and a lack of appropriate control at differentiation stages before entrance and within the germinal center (GC). Self-/polyreactive B cells rescued from apoptosis by MYD88 and/or CD79B mutations accumulate a high load of somatic mutations in their rearranged immunoglobulin (IG) genes, with ongoing somatic hypermutation (SHM). Furthermore, the targeting of oncogenes by aberrant SHM (e.g., PIM1, PAX5, RHOH, MYC, BTG2, KLHL14, SUSD2), translocations of the IG and BCL6 genes, and genomic instability (e.g., gains of 18q21; losses of 9p21, 8q12, 6q21) occur in these cells in the course of their malignant transformation. Activated Toll-like receptor, B cell receptor (BCR), and NF-κB signaling pathways foster lymphoma cell proliferation. Hence, tumor cells are arrested in a late B cell differentiation stage, corresponding to late GC exit B cells, which are genetically related to IgM+ memory cells. Paradoxically, the GC reaction increases self-/polyreactivity, yielding increased tumor BCR reactivity for multiple CNS proteins, which likely contributes to CNS tropism of the lymphoma. The loss of MHC class I antigen expression supports tumor cell immune escape. Thus, specific and unique interactions of the tumor cells with resident CNS cells determine the hallmarks of PCNSL.

Clinical Characteristics and Magnetic Resonance Imaging-Based Prediction of the KLF4K409Q Mutation in Meningioma.

BACKGROUND: Meningioma is the most common primary brain tumor in adults. In recent years, several non-neurofibromin 2 mutations, i.e., AKT1, SMO, TRAF7, and KLF4 mutations, specific for meningioma have been identified. This study aims to analyze the clinical impact and imaging characteristics of the KLF4K409Q mutation in meningioma. METHODS: Clinical, neuropathologic, and imaging data of 170 patients who underwent meningioma resection between 2013 and 2018 were retrospectively collected and tumors were analyzed for the presence of the KLF4K409Q mutation. We collected imaging characteristics, performed volumetric analysis of tumor size and peritumoral edema (PTBE), and calculated the edema index (EI, i.e., ratio of PTBE to tumor volume). Receiver operating characteristic curve analysis was performed to identify cut-off EI values to predict the mutational status of KLF4. RESULTS: Eighteen (10.6%) of the meningiomas carried the KLF4K409Q mutation; these were significantly associated with a secretory subtype (P < 0.001) and sphenoid wing location (P = 0.029). Smaller tumor size (P = 0.007), an increased PTBE (P = 0.012), and an increased EI (P = 0.001) proved to be significantly associated with the KLF4K409Q mutation. In receiver operating characteristic curve analysis, EI predicted the KLF4K409Q mutation with an area under the curve of 0.728 (P = 0.0016). CONCLUSIONS: The KLF4K409Q mutation is associated with a distinct small tumor subtype, prone to substantial PTBE. EI is a reliable parameter to predict the KLF4K409Q mutation in meningioma, thus providing a tool for improvement of pre- and perioperative medical management.

Treatment patterns and disease course of previously untreated Primary Central Nervous System Lymphoma: Feasibility of MTX-based regimens in clinical routine.

BACKGROUND: Primary central nervous system lymphoma (PCNSL) is a rare type of aggressive lymphoma of the central nervous system. Treatment strategies improved significantly over the past decades differ regionally but mainly consist of rituximab and high-dosed methotrexate (MTX)-based therapies. METHODS: We assessed clinical outcomes of 100 patients with newly diagnosed PCNSL between 2010-2020 at the University Hospital of Cologne, Germany. RESULTS: Patients were 23-88 years of age and either treated with MTX-based regimens (PRIMAIN, MARTA, MATRix), individual regimens, or best supportive care, respectively. Overall response rates were generally high (66,7-83,8%), but different organ toxicities required dose adjustments in most groups. Two-year overall survival rates were 57,9% (PRIMAIN), 63,6% (MARTA), 65,4% (MATRix), and 37,5% (Other), respectively. Out of 9 patients suffering from relapse >12 months from primary diagnosis, 7 patients (77,8%) received methotrexate-based salvage therapy with 2-year overall survival of 4/6 patients (66,7%). CONCLUSION: Although a relevant proportion of patients are not eligible for clinical trials due to age, performance status, or comorbidities, these results prove feasibility of different MTX-based treatment strategies in clinical routine. Even elderly patients displayed surprisingly favorable outcomes. However, with compromising organ toxicities, reduction of intensity should be part of strategies in future clinical trials.

OTUB1 prevents lethal hepatocyte necroptosis through stabilization of c-IAP1 during murine liver inflammation.

In bacterial and sterile inflammation of the liver, hepatocyte apoptosis is, in contrast to necroptosis, a common feature. The molecular mechanisms preventing hepatocyte necroptosis and the potential consequences of hepatocyte necroptosis are largely unknown. Apoptosis and necroptosis are critically regulated by the ubiquitination of signaling molecules but especially the regulatory function of deubiquitinating enzymes (DUBs) is imperfectly defined. Here, we addressed the role of the DUB OTU domain aldehyde binding-1 (OTUB1) in hepatocyte cell death upon both infection with the hepatocyte-infecting bacterium Listeria monocytogenes (Lm) and D-Galactosamine (DGal)/Tumor necrosis factor (TNF)-induced sterile inflammation. Combined in vivo and in vitro experiments comprising mice lacking OTUB1 specifically in liver parenchymal cells (OTUB1LPC-KO) and human OTUB1-deficient HepG2 cells revealed that OTUB1 prevented hepatocyte necroptosis but not apoptosis upon infection with Lm and DGal/TNF challenge. Lm-induced necroptosis in OTUB1LPC-KO mice resulted in increased alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) release and rapid lethality. Treatment with the receptor-interacting serine/threonine-protein kinase (RIPK) 1 inhibitor necrostatin-1s and deletion of the pseudokinase mixed lineage kinase domain-like protein (MLKL) prevented liver damage and death of infected OTUB1LPC-KO mice. Mechanistically, OTUB1 reduced K48-linked polyubiquitination of the cellular inhibitor of apoptosis 1 (c-IAP1), thereby diminishing its degradation. In the absence of OTUB1, c-IAP1 degradation resulted in reduced K63-linked polyubiquitination and increased phosphorylation of RIPK1, RIPK1/RIPK3 necrosome formation, MLKL-phosphorylation and hepatocyte death. Additionally, OTUB1-deficiency induced RIPK1-dependent extracellular-signal-regulated kinase (ERK) activation and TNF production in Lm-infected hepatocytes. Collectively, these findings identify OTUB1 as a novel regulator of hepatocyte-intrinsic necroptosis and a critical factor for survival of bacterial hepatitis and TNF challenge.

CD8 T cell-Derived Perforin and TNF-α Are Crucial Mediators of Neuronal Destruction in Experimental Autoimmune Enteric Ganglionitis.

In neuron-specific ovalbumin-transgenic CKTAC mice, antigen-specific OT-I CD8 T cells home to the enteric nervous system, where they attack and destroy neurons of the myenteric and submucosal plexus. Clinically, experimental autoimmune enteric ganglionitis (EAEG) manifests with gastrointestinal dysmotility and rapidly progresses to lethal ileus. Although interferon-γ has been identified as capable of damaging neurons in EAEG, the role of perforin, Fas/FasL, and tumor necrosis factor-α (TNF-α) in this disease is still a matter of debate. Thus, CKTAC mice were adoptively transferred with either perforin-/- or wild-type OT-I CD8 T cells. In addition, CKTAC mice that had received wild-type OT-I CD8 T cells were treated by either anti-TNF-α or anti-FasL. Furthermore, wild-type OT-I CD8 T cells were adoptively transferred into CKTAC mice with neuron-specific deletion of Fas. Although neither inactivation of enteric neuronal Fas nor anti-FasL treatment improved the disease, the absence of perforin from OT-I CD8 T cells and anti-TNF-α treatment significantly ameliorated EAEG and prevented lethal ileus by rescue of enteric neurons. Thus, these experiments identify perforin and TNF-α as important in the pathogenesis of EAEG.

An Autochthonous Mouse Model of Myd88- and BCL2-Driven Diffuse Large B-cell Lymphoma Reveals Actionable Molecular Vulnerabilities.

Based on gene expression profiles, diffuse large B cell lymphoma (DLBCL) is sub-divided into germinal center B cell-like (GCB) and activated B cell-like (ABC) DLBCL. Two of the most common genomic aberrations in ABC-DLBCL are mutations in MYD88, as well as BCL2 copy number gains. Here, we employ immune phenotyping, RNA-Seq and whole exome sequencing to characterize a Myd88 and Bcl2-driven mouse model of ABC-DLBCL. We show that this model resembles features of human ABC-DLBCL. We further demonstrate an actionable dependence of our murine ABC-DLBCL model on BCL2. This BCL2 dependence was also detectable in human ABC-DLBCL cell lines. Moreover, human ABC-DLBCLs displayed increased PD-L1 expression, compared to GCB-DLBCL. In vivo experiments in our ABC-DLBCL model showed that combined venetoclax and RMP1-14 significantly increased the overall survival of lymphoma bearing animals, indicating that this combination may be a viable option for selected human ABC-DLBCL cases harboring MYD88 and BCL2 aberrations.

The deubiquitinase OTUB1 augments NF-κB-dependent immune responses in dendritic cells in infection and inflammation by stabilizing UBC13.

Dendritic cells (DCs) are indispensable for defense against pathogens but may also contribute to immunopathology. Activation of DCs upon the sensing of pathogens by Toll-like receptors (TLRs) is largely mediated by pattern recognition receptor/nuclear factor-κB (NF-κB) signaling and depends on the appropriate ubiquitination of the respective signaling molecules. However, the ubiquitinating and deubiquitinating enzymes involved and their interactions are only incompletely understood. Here, we reveal that the deubiquitinase OTU domain, ubiquitin aldehyde binding 1 (OTUB1) is upregulated in DCs upon murine Toxoplasma gondii infection and lipopolysaccharide challenge. Stimulation of DCs with the TLR11/12 ligand T. gondii profilin and the TLR4 ligand lipopolysaccharide induced an increase in NF-κB activation in OTUB1-competent cells, resulting in elevated interleukin-6 (IL-6), IL-12, and tumor necrosis factor (TNF) production, which was also observed upon the specific stimulation of TLR2, TLR3, TLR7, and TLR9. Mechanistically, OTUB1 promoted NF-κB activity in DCs by K48-linked deubiquitination and stabilization of the E2-conjugating enzyme UBC13, resulting in increased K63-linked ubiquitination of IRAK1 (IL-1 receptor-associated kinase 1) and TRAF6 (TNF receptor-associated factor 6). Consequently, DC-specific deletion of OTUB1 impaired the production of cytokines, in particular IL-12, by DCs over the first 2 days of T. gondii infection, resulting in the diminished production of protective interferon-γ (IFN-γ) by natural killer cells, impaired control of parasite replication, and, finally, death from chronic T. encephalitis, all of which could be prevented by low-dose IL-12 treatment in the first 3 days of infection. In contrast, impaired OTUB1-deficient DC activation and cytokine production by OTUB1-deficient DCs protected mice from lipopolysaccharide-induced immunopathology. Collectively, these findings identify OTUB1 as a potent novel regulator of DCs during infectious and inflammatory diseases.