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Background: Myelofibrosis (MF) is a myeloproliferative neoplasm (MPN) characterized by progressive bone marrow fibrosis, ineffective erythropoiesis, dysplastic megakaryocyte hyperplasia, and extramedullary hematopoiesis. MF includes primary MF (PMF), post-polycythemia vera MF (post-PV-MF), and post-essential thrombocythemia MF (post-ET-MF). Clinical presentation is heterogeneous, marked by splenomegaly, progressive anemia, and constitutional symptoms. The median survival in patients with high-risk disease is approximately 2 years. Hematopoietic Stem Cell Transplant (HSCT) is a potentially curative therapy, however due to considerable morbidity and mortality rates, HSCT is not appropriate for most patients, including elderly patients with intermediate-II and high-risk disease. The Janus kinase (JAK) inhibitor ruxolitinib is approved in the US and EU for the treatment of patients with intermediate or high-risk MF, including PMF, post-PV-MF, and post-ET-MF. In clinical studies, treatment with ruxolitinib has been shown to reduce spleen volume by International Working Group (IWG) criteria in approximately 28% to 42% of patients and improve constitutional symptoms of MF in approximately 46% of patients (Verstovsek, J Hematol Oncol. 2017). Ruxolitinib provides symptomatic improvement, however, does not target the malignant clone or appreciably reduce the degree of fibrosis; some patients experience disease progression and leukemic transformation while on therapy (Versotvsek, NEJM. 2010; Harrison, NEJM. 2012; Kremyanskaya, Br J Hem. 2014). Moreover, ruxolitinib is associated with AEs including anemia and thrombocytopenia, which can lead to discontinuation. Approximately 50% of patients treated with ruxolitinib discontinued treatment within 3 years and 73% at 5 years (Verstovsek, Haematologica. 2015; Verstovsek, J Hematol Oncol. 2017; Cervantes, Blood. 2013; Harrison, Leukemia. 2016). Median overall survival in patients who discontinue ruxolitinib is 14-16 months, highlighting the need for novel therapies targeting alternative pathways in the setting of failure or intolerance of JAK inhibitor therapy (Newberry, Blood. 2017). The tumor suppressor protein p53 is the master regulator of cell-cycle arrest and apoptosis in response to cellular stress or DNA damage. Murine double minute 2 (MDM2) is a key regulator of p53, inhibiting its activity via ubiquitination, nuclear export, and direct inhibition of transcriptional activity. Increased MDM2 protein expression has been observed in MF CD34+ cells, suggesting that MF might be sensitive to MDM2 inhibition (Lu M, Blood. 2017). KRT-232 is a potent and selective, oral, small molecule drug that targets MDM2 and prevents MDM2-mediated p53 inhibition, allowing p53 to mediate tumor cell-cycle arrest and apoptosis. In MF, TP53 is observed to be wild-type in 96% of MF patients, suggesting MDM2 inhibition could be a successful therapeutic strategy in this disease (Raza, Am J Hematol. 2012). KRT-232 has been investigated as monotherapy and in combination with trametinib or dabrafenib in phase I studies of AML and melanoma; the most common treatment-related adverse events (TRAEs) observed were nausea, diarrhea, vomiting, decreased appetite, anemia, leukopenia, thrombocytopenia, and fatigue. The majority of TRAEs were grade 1 or 2. Methods: KRT-232 is being evaluated in an open-label phase 2 study in patients with MF who relapsed on or are refractory to JAK inhibitors (Figure). Up to 247 patients ≥ 18 years of age, with ECOG performance status ≤ 2, with high-, intermediate-2, or intermediate-1 risk disease by Dynamic International Prognostic System (DIPSS), and failure of prior treatment with JAK inhibitors will be enrolled. The study will be conducted in 2 parts. Part A will identify the recommended dose and schedule by testing varying doses and schedules across 7 treatment cohorts. Part B will evaluate safety and efficacy using the recommended dose and schedule from Part A. The primary endpoint of the study is to determine spleen response at week 24; secondary endpoints include improvement in MPN-SAF Total Symptom Score (weeks 24 and 48), red blood cell (RBC) transfusion independence, and rates of complete remission and partial remission (IWG-ERT and ELN) at week 24. This trial is enrolling at multiple sites in the United States and Europe (NCT03662126, EudraCT: 2018-001671-21). Disclosures Garcia-Delgado: Hospital Virgen De La Victoria Malaga: Employment; Novartis: Consultancy, Speakers Bureau; Celgene: Speakers Bureau. McLornan:Jazz Pharmaceuticals: Honoraria, Speakers Bureau; Novartis: Honoraria. Jourdan:Novartis: Honoraria; Astellas: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Al-Ali:Celgene: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; CTI: Honoraria. Pluta:Freelight Poland: Honoraria; Sandoz: Honoraria; Servier: Honoraria; Jansen-Cilag: Honoraria; Novartis: Honoraria; Takeda: Honoraria; Roche: Honoraria; Specialistic Hospital in Brzozow,Dept of Haematooncology Ks.Bielawskiego 18 36-200 Brzozow, Poland: Employment; Teva: Honoraria; Roche Poland: Membership on an entity's Board of Directors or advisory committees; Jansen Cilag Poland: Membership on an entity's Board of Directors or advisory committees. Ewing:Novartis: Honoraria, Other: Meeting attendance sponsorship ; Bristol Myers-Squibb: Other: Meeting attendance sponsorship . Khan:Amgen: Consultancy; Celgene: Consultancy; Incyte: Honoraria; Pfizer: Consultancy; Takeda: Research Funding. Jost:Novartis: Research Funding; Celgene: Other: Travel Support; Pfizer: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Abbvie: Consultancy, Patents & Royalties: Royalty payments for the drug compound ABT-199, Research Funding; Bohringer: Consultancy, Research Funding; BMS: Consultancy, Speakers Bureau. Rothbaum:Kartos Therapeutics: Employment, Patents & Royalties: Pending; Quogue Bioventures LLC: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. McGreivy:Kartos Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Verstovsek:Incyte: Research Funding; Roche: Research Funding; NS Pharma: Research Funding; Celgene: Consultancy, Research Funding; Gilead: Research Funding; Promedior: Research Funding; CTI BioPharma Corp: Research Funding; Genetech: Research Funding; Blueprint Medicines Corp: Research Funding; Novartis: Consultancy, Research Funding; Sierra Oncology: Research Funding; Pharma Essentia: Research Funding; Astrazeneca: Research Funding; Ital Pharma: Research Funding; Protaganist Therapeutics: Research Funding; Constellation: Consultancy; Pragmatist: Consultancy. OffLabel Disclosure: Yes, KRT-232 is an investigational small molecule MDM2 inhibitor. This trial-in-progress abstract describes a registered clinical trial that will evaluate the safety and efficacy of KRT-232 for patients with myelofibrosis.

Patients with X-linked lymphoproliferative syndrome type 2 (XLP-2) (BIRC4 deficiency) suffer from hyperinflammation often observed during the conditioning regimen prior to allogeneic bone marrow transplant. This article shows that in mice hematopoietic recipient cells contribute to graft-versus-host disease by the secretion of elevated levels of proinflammatory cytokines during engraftment when BIRC4 is absent.

Purpose Rapidly evolving genomics technologies, in particular comprehensive next-generation sequencing (NGS), have led to exponential growth in the understanding of cancer biology, shifting oncology toward personalized treatment strategies. However, comprehensive NGS approaches, such as whole-exome sequencing, have limitations that are related to the technology itself as well as to the input source. Hence, clinical implementation of comprehensive NGS in a quality-controlled diagnostic workflow requires both the standardization of sequencing procedures and continuous validation of sequencing results by orthogonal methods in an ongoing program to enable the determination of key test parameters and continuous improvement of NGS and bioinformatics pipelines. Patients and Methods We present validation data on 220 patients who were enrolled between 2013 and 2016 in a multi-institutional, genomics-guided precision oncology program (Molecularly Aided Stratification for Tumor Eradication Research) of the National Center for Tumor Diseases Heidelberg and the German Cancer Consortium. Results More than 90% of clinically actionable genomic alterations identified by combined whole-exome sequencing and transcriptome sequencing were successfully validated, with varying frequencies of discordant results across different types of alterations (fusions, 3.7%; single-nucleotide variants, 2.6%; amplifications, 1.1%; overexpression, 0.9%; deletions, 0.6%). The implementation of new computational methods for NGS data analysis led to a substantial improvement of gene fusion calling over time. Conclusion Collectively, these data demonstrate the value of a rigorous validation program that partners with comprehensive NGS to successfully implement and continuously improve cancer precision medicine in a clinical setting.

Background Monoclonal gammopathy causes several kinds of renal pathology. A rare and special form is monoclonal gammopathy-induced C3 glomerulopathy (MG-C3G). Like idiopathic C3G, MG-C3G frequently leads to end-stage renal disease. MG-C3G frequently recurs after renal transplantation, leading to graft failure in most of the patients. While there is some evidence for successful treatment of recurrent idiopathic C3 glomerulopathy with eculizumab after renal transplantation, nothing is known about its efficacy in the setting of recurrent MG-C3G. Case presentation We report a patient with recurrent MG-C3G in a renal allograft that was successfully treated with eculizumab in addition to standard immunosuppression. He had early recurrence of MG-C3G 2 months after transplantation. His graft function successively declined despite high dose steroids and plasmapheresis. Only after therapy with three cycles of bortezomib and continuous therapy with eculizumab, his graft function stabilized. He was still in clinical remission after 28 months of follow-up without having experienced major infectious complications. Conclusions Eculizumab may be a safe and effective treatment of recurrent MG-C3G. Because of the high and early recurrence risk, renal transplantation should be reviewed carefully for every individual patient. Subsequent hematopoietic stem cell transplantation may ameliorate long-term renal allograft survival. Eculizumab might serve as a bridging therapy until stem cell transplantation.

Deregulated apoptosis is an identifying feature of myelodysplastic syndromes (MDS). Whereas apoptosis is increased in the bone marrow (BM) of low-risk MDS patients, progression to high-risk MDS correlates with an acquired resistance to apoptosis and an aberrant expression of BCL-2 proteins. To overcome the acquired apoptotic resistance in high-risk MDS, we investigated the induction of apoptosis by inhibition of pro-survival BCL-2 proteins using the BCL-2/-XL/-W inhibitor ABT-737 or the BCL-2-selective inhibitor ABT-199. We characterized a cohort of 124 primary human BM samples from MDS/secondary acute myeloid leukemia (sAML) patients and 57 healthy, age-matched controls. Inhibition of anti-apoptotic BCL-2 proteins was specifically toxic for BM cells from high-risk MDS and sAML patients, whereas low-risk MDS or healthy controls remained unaffected. Notably, ABT-737 or ABT-199 treatment was capable of targeting the MDS stem/progenitor compartment in high-risk MDS/sAML samples as shown by the reduction in CD34(+) cells and the decreased colony-forming capacity. Elevated expression of MCL-1 conveyed resistance against both compounds. Protection by stromal cells only partially inhibited induction of apoptosis. Collectively, our data show that the apoptotic resistance observed in high-risk MDS/sAML cells can be overcome by the ABT-737 or ABT-199 treatment and implies that BH3 mimetics might delay disease progression in higher-risk MDS or sAML patients.

Cells exposed to extreme physicochemical or mechanical stimuli die in an uncontrollable manner, as a result of their immediate structural breakdown. Such an unavoidable variant of cellular demise is generally referred to as ?accidental cell death' (ACD). In most settings, however, cell death is initiated by a genetically encoded apparatus, correlating with the fact that its course can be altered by pharmacologic or genetic interventions. "Regulated cell death" (RCD) can occur as part of physiologic programs or can be activated once adaptive responses to perturbations of the extracellular or intracellular microenvironment fail. The biochemical phenomena that accompany RCD may be harnessed to classify it into a few subtypes, which often (but not always) exhibit stereotyped morphologic features. Nonetheless, efficiently inhibiting the processes that are commonly thought to cause RCD, such as the activation of executioner caspases in the course of apoptosis, does not exert true cytoprotective effects in the mammalian system, but simply alters the kinetics of cellular demise as it shifts its morphologic and biochemical correlates. Conversely, bona fide cytoprotection can be achieved by inhibiting the transduction of lethal signals in the early phases of the process, when adaptive responses are still operational. Thus, the mechanisms that truly execute RCD may be less understood, less inhibitable and perhaps more homogeneous than previously thought. Here, the Nomenclature Committee on Cell Death formulates a set of recommendations to help scientists and researchers to discriminate between essential and accessory aspects of cell death Fil: Rabinovich, Gabriel Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina Fil: Nomenclature Committee on Cell Death. Equipe 11 Apoptose, Cancer et Immunité. Centre de Recherche des Cordeliers; Francia

The concept of 'personalised medicine' aims at allocating patients to different treatment options based on individual characteristics to optimise treatment benefit and side effects. In oncology, personalised treatments coupled to biomarkers have led to the approval of targeted agents with high anti-tumour activity. However, these therapies are often limited to narrow, molecularly defined subsets of patients with a specific morphomolecular tumour profile. Recently, it became obvious that the same molecular alteration might drive oncogenesis in many different tumours, and it might be beneficial to target the alteration in a histology informed but entity-overarching way. Consequently, Universal Genomic Testing (UGT) of tumours encompassing panel sequencing to whole-exome and transcriptome sequencing is propagated to revolutionise oncology. This article will describe the developments leading to identification and application of potential biomarkers using UGT. On this basis, it will review the clinical evidence of this approach and summarise recommendations for the ongoing evaluation of UGT as the next step in oncological decision-making.

Acute myeloid leukemia (AML) is a heterogeneous group of haematopoietic neoplasms driven partly by the loss of differentiation and the blockade of cell death. AML is sustained by leukemia-initiatin...

Precision oncology implies the ability to predict which patients will likely respond to specific cancer therapies based on high-resolution molecular diagnostics. The value of comprehensive molecular profiling based on whole-exome/genome sequencing (WES/WGS) and global RNA sequencing to guide therapeutic decisions in individual patients remains to be established. We report the results of MASTER (Molecularly Aided Stratification for Tumor Eradication Research), a multicenter registry trial for prospective, biology-driven stratification of younger adults with advanced-stage cancer across all histologies and patients with rare tumors conducted under the auspices of NCT Heidelberg/Dresden and the German Cancer Consortium (DKTK). Based on a standardized workflow for selection and consenting of patients, sample processing, WES/WGS and RNA sequencing, bioinformatic analysis, and technical validation of potentially actionable findings, we have analyzed more than 1300 poor-prognosis (median overall survival, 12 months) patients representing a broad spectrum of entities. Evaluation of the data by a cross-institutional molecular tumor board has allowed categorization into 7 different intervention baskets and formulation of evidence-based recommendations for clinical management in more than 80% of patients, which were implemented in approximately one third of cases. Overall response and disease control rates on molecularly guided treatment were significantly improved compared to prior systemic therapies, which translated into a progression-free survival (PFS) ratio of greater than 1.3 in more than 40% of cases. In 5% of patients, comprehensive genomic profiling allowed to refine the clinical diagnosis, as exemplified by several soft-tissue sarcomas not otherwise specified and carcinomas of unknown primary site that could be categorized based on their genotypes and subsequent histopathologic re-evaluation. Finally, systematic analysis of germline alterations revealed that 11% of patients had pathogenic (ACMG Class 4 or 5) variants in known tumor predisposition genes, and that 4% were carriers for autosomal recessive disorders. This prospective trial demonstrates that molecular profiling based on WES/WGS and RNA sequencing in a multi-institutional clinical setting is feasible, complements and advances routine molecular diagnostics, and creates clinically meaningful therapeutic opportunities in a significant proportion of patients. To improve clinical translation, the MASTER platform is now linked to a growing portfolio of cross-institutional basket trials. In the intermediate term, genomic profiling within MASTER will be integrated with additional layers of patient characterization and extended to additional treatment modalities (e.g. radiotherapy and surgical interventions). Citation Format: Peter Horak, Christoph Heining, Simon Kreutzfeldt, Christoph E. Heilig, Lino Möhrmann, Laura Gieldon, Martina Fröhlich, Sebastian Uhrig, Daniel Hübschmann, Katja Beck, Daniela Richter, Stephan Wolf, Katrin Pfütze, Christina Geörg, Bettina Meißburger, Frederick Klauschen, Sebastian Ochsenreither, Gunnar Folprecht, Jens Siveke, Sebastian Bauer, Thomas Kindler, Christian Brandts, Melanie Börries, Nikolas von Bubnoff, Karsten Spiekermann, Philipp J. Jost, Klaus Schulze-Osthoff, Michael Bitzer, Peter Schirmacher, Christof von Kalle, Richard Schlenk, Barbara Klink, Barbara Hutter, Wilko Weichert, Albrecht Stenzinger, Evelin Schröck, Benedikt Brors, Hanno Glimm, Stefan Fröhling. Clinical relevance of comprehensive genomic analysis in advanced-stage cancers and rare malignancies: Results from the MASTER trial of the German Cancer Consortium [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 468.

Purpose: In gastrointestinal stromal tumor (GIST), there is no biomarker available that indicates success or failure of therapy. We hypothesized that tumor-specific v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (CKIT)- or platelet-derived growth factor receptor-α (PDGFRA)–mutant DNA fragments can be detected and quantified in plasma samples of patients with GIST. Experimental Design: We prospectively collected 291 plasma samples from 38 subjects with GIST harboring activating mutations of CKIT or PDGFRA detected in tumor tissue, irrespective of current disease status or treatment. We used allele-specific ligation PCR to detect mutant free circulating DNA (fcDNA). Results: We were able to detect fcDNA harboring the tumor mutation in 15 of 38 patients. Patients with active disease displayed significantly higher amounts of mutant fcDNA compared with patients in complete remission (CR). The amount of mutant fcDNA correlated with disease course. We observed repeated positive test results or an increase of mutant fcDNA in five patients with progressive disease or relapse. A decline of tumor fcDNA or conversion from positive to negative was seen in five patients responding to treatment. A negative to positive conversion was seen in two patients with relapse and one patient with progression. In two cases, we aimed to identify additional mutations and found four additional exchanges, including mutations not known from sequentially conducted tumor biopsies. Conclusions: Our results indicate that fcDNA harboring tumor-specific mutations in the plasma of patients with GIST can be used as tumor-specific biomarker. The detection of resistance mutations in plasma samples might allow earlier treatment changes and obviates the need for repeated tumor biopsies. Clin Cancer Res; 19(17); 4854–67. ©2013 AACR.

Local cooling is very common after bone and joint surgery. Therefore the knowledge of bone blood flow during local cooling is of substantial interest. Previous studies revealed that hypothermia leads to vasoconstriction followed by decreased blood flow levels. The aim of this study was to characterize if local cooling is capable of inducing reduced blood flow in bone tissue using a stepwise-reduced temperature protocol in experimental rabbits. To examine bone blood flow we utilized the fluorescent microsphere (FM) method. In New Zealand white rabbits one randomly chosen hind limb was cooled stepwise from 32 to 2°C, whereas the contra lateral hind limb served as control. Injection of microspheres was performed after stabilization of bone and muscle temperature at each temperature level. Bones were removed, dissected and fluorescence intensity was determined to calculate blood flow values. We found that blood flow of all cooled regions decreased relative to the applied external temperature. At maximum cooling blood flow was almost completely disrupted, indicating local cooling as powerful regulatory mechanism for regional bone blood flow (RBBF). Postoperative cooling therefore may lead to strongly decreased bone blood flow values. As a result external cooling has capacity to both diminish bone healing and reduce bleeding complications.

Acute myeloid leukemia (AML) is sustained by leukemia-initiating cells (LICs) with blocked myeloid differentiation and increased self-renewal capacities. These cells arise from pre-leukemic hematopoietic stem and progenitor cells (HSPCs) that carry genetic alterations being selected for during leukemogenesis [1]. The resistance of LICs to standard chemotherapies presents a major clinical challenge as they eventually cause disease relapse and death [2]. Understanding the mechanisms of LIC resistance to undergoing cell death is therefore critical for a curative therapy of AML. The ability of LICs and pre-leukemic HSCs to evade cell death has been attributed to several pathways that are distinct from normal HSPCs. LICs for example have constitutive NF-κB activity while normal HSPCs do not. In addition, analysis of patient-derived cells showed that in some cases the apoptotic cell death pathway in LICs is skewed. However, work in mature myeloid cells has shown that these cells are exquisitely sensitive to undergoing cell death driven by receptor-interacting serine-threonine kinase 3 (RIPK3) [3]. In addition to inducing apoptosis, RIPK3 can also induce regulated necrosis. In contrast to apoptosis, regulated necrosis in myeloid cells is accompanied by inflammation due to the release of cytokines and DAMPs from dying cells. We found that in a similar fashion LICs are also sensitive to RIPK3dependent cell death and that loss of RIPK3 expression underlies the developmental block and oncogenic process in many subtypes of AML [4]. In our study, we focused on one of the recurring genetic alterations found in AML that gives rise to internal tandem duplications of the tyrosine kinase receptor FLT3 (FLT3-ITD). In human patients FLT3-ITD is associated with poor prognosis and lower overall survival. Using a model of adoptive transfer of bone marrow (BM) cells transduced with FLT3-ITD we demonstrated that in the absence of Ripk3, FLT3-ITD-induced leukemia was accelerated and significantly more aggressive. The enhanced disease was due to the expansion of common myeloid progenitors (CMPs) and short-term hematopoietic stem cells (ST-HSCs), being the cellular compartment to contain LICs. RIPK3 functioned to induce cell death of these cells and acted as a tumor suppressor. We further showed that RIPK1 and the TNF receptors 1 and 2 (TNFR1/2) functioned upstream of RIPK3 to regulate cell death. However, Mlkl deficiency did not abrogate cell death in vitro, and disease progression in vivo was also not significantly different, suggesting that RIPK3 may also induce apoptosis of LICs, a function that is consistent with recently published results [5]. Importantly, WT FLT3-ITD-transformed cells produced substantial amounts of the inflammatory cytokine IL-1β which was severely blunted by Ripk3 or Mlkl deficiency. The role of IL-1β in promoting LIC differentiation and thus restricting leukemogenesis was confirmed with Pycard-/and Il1r1-/HSPCs. Hence, RIPK3 signals through two complementary pathways, cell death and IL-1β production, to restrict LICs and to promote myeloid cell differentiation, respectively (Figure 1). Using several human patient sample cohorts, we observed a reduced expression of both RIPK3 and Commentary: Autophagy and Cell Death

The mitotic spindle assembly checkpoint (SAC) maintains genome stability and marks an important target for antineoplastic therapies. However, it has remained unclear how cells execute cell fate decisions under conditions of SAC-induced mitotic arrest. Here, we identify USP9X as the mitotic deubiquitinase of the X-linked inhibitor of apoptosis protein (XIAP) and demonstrate that deubiquitylation and stabilization of XIAP by USP9X lead to increased resistance toward mitotic spindle poisons. We find that primary human aggressive B-cell lymphoma samples exhibit high USP9X expression that correlate with XIAP overexpression. We show that high USP9X/XIAP expression is associated with shorter event-free survival in patients treated with spindle poison-containing chemotherapy. Accordingly, aggressive B-cell lymphoma lines with USP9X and associated XIAP overexpression exhibit increased chemoresistance, reversed by specific inhibition of either USP9X or XIAP. Moreover, knockdown of USP9X or XIAP significantly delays lymphoma development and increases sensitivity to spindle poisons in a murine El-Myc lymphoma model. Together, we specify the USP9X–XIAP axis as a regulator of the mitotic cell fate decision and propose that USP9X and XIAP are potential prognostic biomarkers and therapeutic targets in aggressive B-cell lymphoma. peerReviewed

Nucleotide-binding and oligomerization domain (NOD)-like receptors constitute a first line of defense against invading bacteria. X-linked Inhibitor of Apoptosis (XIAP) is implicated in the control of bacterial infections, and mutations in XIAP are causally linked to immunodeficiency in X-linked lymphoproliferative syndrome type-2 (XLP-2). Here, we demonstrate that the RING domain of XIAP is essential for NOD2 signaling and that XIAP contributes to exacerbation of inflammation-induced hepatitis in experimental mice. We find that XIAP ubiquitylates RIPK2 and recruits the linear ubiquitin chain assembly complex (LUBAC) to NOD2. We further show that LUBAC activity is required for efficient NF-κB activation and secretion of proinflammatory cytokines after NOD2 stimulation. Remarkably, XLP-2-derived XIAP variants have impaired ubiquitin ligase activity, fail to ubiquitylate RIPK2, and cannot facilitate NOD2 signaling. We conclude that XIAP and LUBAC constitute essential ubiquitin ligases in NOD2-mediated inflammatory signaling and propose that deregulation of NOD2 signaling contributes to XLP-2 pathogenesis.

Fas (also called CD95 or APO-1), a member of a subgroup of the tumour necrosis factor receptor superfamily that contain an intracellular death domain, can initiate apoptosis signalling and has a critical role in the regulation of the immune system. Fas-induced apoptosis requires recruitment and activation of the initiator caspase, caspase-8 (in humans also caspase-10), within the death-inducing signalling complex. In so-called type 1 cells, proteolytic activation of effector caspases (-3 and -7) by caspase-8 suffices for efficient apoptosis induction. In so-called type 2 cells, however, killing requires amplification of the caspase cascade. This can be achieved through caspase-8-mediated proteolytic activation of the pro-apoptotic Bcl-2 homology domain (BH)3-only protein BH3-interacting domain death agonist (Bid), which then causes mitochondrial outer membrane permeabilisation. This in turn leads to mitochondrial release of apoptogenic proteins, such as cytochrome c and, pertinent for Fas death receptor (DR)-induced apoptosis, Smac/DIABLO (second mitochondria-derived activator of caspase/direct IAP binding protein with low Pi), an antagonist of X-linked inhibitor of apoptosis (XIAP), which imposes a brake on effector caspases. In this review, written in honour of Juerg Tschopp who contributed so much to research on cell death and immunology, we discuss the functions of Bid and XIAP in the control of Fas DR-induced apoptosis signalling, and we speculate on how this knowledge could be exploited to develop novel regimes for treatment of cancer.

Apoptotic death of hepatocytes, a contributor to many chronic and acute liver diseases, can be a consequence of overactivation of the immune system and is often mediated by TNFalpha. Injection with lipopolysaccharide (LPS) plus the transcriptional inhibitor D(+)-galactosamine (GalN) or mitogenic T cell activation causes fatal hepatocyte apoptosis in mice, which is mediated by TNFalpha, but the effector mechanisms remain unclear. Our analysis of gene-targeted mice showed that caspase-8 is essential for hepatocyte killing in both settings. Loss of Bid, the proapoptotic BH3-only protein activated by caspase-8 and essential for Fas ligand-induced hepatocyte killing, resulted only in a minor reduction of liver damage. However, combined loss of Bid and another BH3-only protein, Bim, activated by c-Jun N-terminal kinase (JNK), protected mice from LPS+GalN-induced hepatitis. These observations identify caspase-8 and the BH3-only proteins Bid and Bim as potential therapeutic targets for treatment of inflammatory liver diseases.

FAS (also called APO-1 and CD95) and its physiological ligand, FASL, regulate apoptosis of unwanted or dangerous cells, functioning as a guardian against autoimmunity and cancer development. Distinct cell types differ in the mechanisms by which the 'death receptor' FAS triggers their apoptosis. In type I cells, such as lymphocytes, activation of 'effector caspases' by FAS-induced activation of caspase-8 suffices for cell killing, whereas in type II cells, including hepatocytes and pancreatic beta-cells, caspase cascade amplification through caspase-8-mediated activation of the pro-apoptotic BCL-2 family member BID (BH3 interacting domain death agonist) is essential. Here we show that loss of XIAP (X-chromosome linked inhibitor of apoptosis protein) function by gene targeting or treatment with a second mitochondria-derived activator of caspases (SMAC, also called DIABLO; direct IAP-binding protein with low pI) mimetic drug in mice rendered hepatocytes and beta-cells independent of BID for FAS-induced apoptosis. These results show that XIAP is the critical discriminator between type I and type II apoptosis signalling and suggest that IAP inhibitors should be used with caution in cancer patients with underlying liver conditions.

During T cell development in the thymus, high-affinity/avidity TCR engagement induces negative selection by apoptosis, while lower affinity/avidity TCR interactions lead to positive selection and survival of thymocytes. Yet, the mechanisms that discriminate between positive and negative selection are not fully understood. One major regulator of survival and apoptosis in lymphoid cells is the transcription factor NF-κB. Several reports have indicated key roles for NF-κB in positive and negative selection. In peripheral T cells, TCR ligation activates NF-κB through a selective pathway that involves protein kinase Cθ, Bcl10, and Malt1. While protein kinase Cθ is dispensable for thymic TCR signaling, the molecular roles of Bcl10 and Malt1 in thymocytes have not been investigated. In the present study, we show that both Bcl10 and Malt1 are essential for TCR signaling in thymocytes as a genetic disruption of either molecule blocks TCR-induced NF-κB activation in these cells. To investigate the function of this pathway in thymic selection, we introduced the Bcl10 or Malt1 mutations into three well-established TCR transgenic mouse models. Surprisingly, using several in vivo or in vitro assays, we were unable to demonstrate a role for TCR-induced NF-κB activation in either positive or negative selection. Thus, while TCR signaling to NF-κB controls the activation of mature T cells, we suggest that this pathway is not involved in the positive or negative selection of thymocytes.

T lymphocyte non-Hodgkin's lymphoma (T-NHL) represents an aggressive and largely therapy-resistant subtype of lymphoid malignancies. As deregulated apoptosis is a frequent hallmark of lymphomagenesis, we analyzed gene expression profiles and protein levels of primary human T-NHL samples for various apoptotic regulators. We identified the apoptotic regulator MCL-1 as the only pro-survival BCL-2 family member to be highly expressed throughout all human T-NHL subtypes. Functional validation of pro-survival protein members of the BCL-2 family in two independent T-NHL mouse models identified that the partial loss of Mcl-1 significantly delayed T-NHL development in vivo. Moreover, the inducible reduction of MCL-1 protein levels in lymphoma-burdened mice severely impaired the continued survival of T-NHL cells, increased their susceptibility to chemotherapeutics and delayed lymphoma progression. Lymphoma viability remained unaffected by the genetic deletion or pharmacological inhibition of all alternative BCL-2 family members. Consistent with a therapeutic window for MCL-1 treatment within the context of the whole organism, we observed an only minimal toxicity after systemic heterozygous loss of Mcl-1 in vivo. We conclude that re-activation of mitochondrial apoptosis by blockade of MCL-1 represents a promising therapeutic strategy to treat T-cell lymphoma.

The MASTER (Molecularly Aided Stratification for Tumor Eradication Research) Program of the NCT (National Center for Tumor Diseases) Heidelberg and the DKTK (German Cancer Consortium) is situated at the interface of cancer genomics and clinical oncology to provide whole exome/genome and transcriptome sequencing to selected patients with unmet medical need, and to evaluate the utility of such an approach regarding molecular stratification and individualized, biology-guided treatment. The program has enabled implementation of a shared, DKTK-wide workflow for rapid-turnaround clinical sequencing, comprising all steps from sample processing to reporting of results by a dedicated molecular tumor board. In clinical cancer genome sequencing programs, bioinformatics pipelines normally exclude germline variants detected in matched normal tissues to select for somatic mutations. However, in younger cancer patients (less than 51 years of age) and/or patients with rare cancer entities, which are eligible for analysis within MASTER, we anticipated an enrichment of patients with a possible hereditary background. If germline variants would be excluded in such cases a priori, relevant pathogenic mutations responsible for cancer development might be missed. Importantly, such mutations could also be therapeutically relevant. This applies in particular to tumors with mutations in genes involved in DNA damage response signaling, e.g. BRCA1/2, PALB2, ATM, and others, which respond to treatment with PARP inhibitors and platinum-based chemotherapy. Furthermore, germline mutations in these genes are responsible for hereditary breast and ovarian cancer and more than 120 tumor predisposition syndromes (TPS) known to date. Therefore analysis of germline variants of all known hereditary cancer genes was included as part of the NCT/DKTK MASTER workflow since 2015. A board-certified clinical geneticists experienced in evaluating rare private germline variants performed data interpretation. Recommendations were provided to the clinical oncologists for referral of patients to a TPS center for genetic counseling, further diagnostics, surveillance, and tumor prevention measures. We here present results from 321 NCT/DKTK MASTER patients analyzed thus far. Previously unknown pathogenic germline variants in 22 different tumor susceptibility genes, such as BRCA1/2, PALB2, ATM, NF1, MEN1, RB1, APC, SDHB, CDH1, and others, were detected in 36 patients (11%) with various cancers. We thus demonstrate the importance of evaluating germline variants obtained by “omics”-based molecular diagnostic approaches under clinical conditions. Furthermore, our results not only had implications for further surveillance of patients and their families, but also contributed to clinically “actionable” treatment recommendations. Citation Format: Evelin Schrock, Barbara Hutter, Martina Fröhlich, Falk Zakrzewski, Sebastian Uhrig, Andreas Rump, Karl Hackmann, Joseph Porrmann, Laura Gieldon, Daniela Richter, Albrecht Stenzinger, Thomas Kindler, Wilko Weichert, Philipp J. Jost, Christian Brandts, Klaus Schulze-Osthoff, Johanna Falkenhorst, Sebastian Bauer, Frederick Klauschen, Konrad Klinghammer, Gunnar Folprecht, Martin Wermke, Karsten Spiekermann, Benedikt Brors, Stefan Gröschel, Christoph Heining, Peter Horak, Hanno Glimm, Stefan Fröhling, Barbara Klink. Identification of patients at risk for tumor predisposition syndromes based on the evaluation of sporadic cancer exome sequencing data: experiences from the NCT/DKTK MASTER program [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-287. doi:10.1158/1538-7445.AM2017-LB-287

There is currently immense interest in understanding the biological consequences of aberrant necroptosis. The recently uncovered role for X-linked inhibitor of apoptosis protein (XIAP) in blocking tumor necrosis factor-dependent necroptosis explains, at least in part, the systemic hyperinflammatory syndrome XLP-2. However, it also points to rather unexpected differences between XIAP and the related proteins baculoviral IAP repeat containing 2 and 3 (cIAP1/2).

X-linked inhibitor of apoptosis protein (XIAP) has been identified as a potent regulator of innate immune responses, and loss-of-function mutations inXIAP cause the development of the X-linked lymphoproliferative syndrome type 2 (XLP-2) in humans. Using gene-targeted mice, we show that loss of XIAPor deletion of its RING domain lead to excessive cell death and IL-1β secretion from dendritic cells triggered by diverse Toll-like receptor stimuli. Aberrant IL-1β secretion is TNF dependent and requires RIP3 but is independent of cIAP1/cIAP2. The observed cell death also requires TNF and RIP3 but proceeds independently of caspase-1/caspase-11 or caspase-8 function. Loss of XIAP results in aberrantly elevated ubiquitylation of RIP1 outside of TNFR complex I. Virally infected Xiap(-/-) mice present with symptoms reminiscent of XLP-2. Our data show that XIAP controls RIP3-dependent cell death and IL-1β secretion in response to TNF, which might contribute to hyperinflammation in patients with XLP-2.

The routine study of human malaria liver-stage biology in vitro is hampered by low infection efficiency of human hepatocellular carcinoma (HCC) lines (

Acute myeloid leukemia (AML) is a heterogeneous group of hematopoietic neoplasms driven partly by the loss of differentiation and theblockade of cell death. AML is sustained by leukemia-initiating cells (LICs) that arise from pre-leukemic hematopoietic stem and progenitor cells (HSPCs) that carry genetic alterations being selected for during leukemogenesis. The resistance of LICs to standard chemotherapies presents a major clinical challenge as they eventually cause disease relapse and death. Understanding the mechanisms of LIC resistance to undergoing cell death is therefore critical for a curative therapy of AML. While the regulatory factors that maintain HSPC proliferation and differentiation under normal conditions are well understood, significantly less is known about how LIC fate is regulated. As many hematopoietic disorders are characterized by the overproduction of pro-inflammatory cytokines, we hypothesized that necroptosis controlled cytokine secretion and inflammatory cell death might influence AML development. We therefore addressed the role of MLKL and XIAP in AML and tested whether deletion of Mlkl or Xiap would affect disease progression. Here we show that MLKL limits oncogene-mediated leukemogenesis by promoting the inflammatory cell death of common myeloid progenitors (CMPs) and short-term hematopoietic stem cells (HSCs) in experimental mice. Upon oncogenic stress MLKL-dependent necroptosis and subsequent inflammasome activation were triggered, promoting the production of IL-1β, a potent stimulator of HSPC differentiation and maturation, thus, suppressing the emergence of LICs and limiting leukemogenesis. In a murine bone marrow transplantation model of AML the absence of MLKL accelerated AML development significantly. The enhanced disease was due to the expansion of common myeloid progenitors (CMPs) and short-term hematopoietic stem cells (ST-HSCs), being the cellular compartments to contain LICs. The survival advantage of Mlkl-/- HSPCs became apparent in colony-forming assays and liquid cultures specifically within the CMP and ST-HSC compartments. Sorted ST-HSCs from Mlkl-/- produced more GEMM colonies than WT, the colony type harboring the multipotential myeloid progenitor cells, and both ST-HSCs and CMPs retained significantly more lineage-negative cells in liquid culture. In addition, Mlkl-/- colonies showed a reduction in propidium iodide (PI)-positive dead cells compared with WT colonies. Importantly, WT cells showed caspase activation and produced substantial amounts of the inflammatory cytokine IL-1β which was severely blunted by Mlkl deficiency. We also observed reduced expression of MLKL in leukemic cells on both mRNA and protein level, implying that suppression of cell death was beneficial for the survival of LICs. In contrast, deletion of Xiap did not alter survival or differentiation of leukemic cells when compared with WT cells. Furthermore, XIAP was not differentially expressed on mRNA or protein level compared with WT, indicating that XIAP does not play a critical role in leukemogenesis. In agreement with the murine data, gene expression analysis from primary leukemia cells from two large patient cohorts newly diagnosed with AML showed significantly lower expression of MLKL, but not XIAP, in a variety of AML subtypes compared to healthy controls. Overall, our data demonstrate a key role for MLKL-mediated cell death and activation of the inflammasome in AML and represents a novel tumor-suppressive mechanism. Disclosures Peschel: MophoSys: Honoraria.

Introduction: Myelodysplastic syndromes (MDS) are one of the most common haematological disorders of the older patient. Allogeneic stem cell stransplantation (ASCT) is the sole curative treatment. The number of eligible patients is limited due to age and comorbidities and only approximately one-third of these patients are cured by ASCT. Further therapeutic strategies such as hypomethylating agents (HMA) have often a short response duration. We recently found that ABT-199 effectively induces apoptosis in the leukemic progenitor compartment of higher-risk MDS and sAML patients, whereas healthy controls and low-risk patients remain unaffected (Jilg et al., 2016). These data suggest that pro-apoptotic ABT-199 might harbor potential as a novel treatment modality in higher-risk MDS patients. However, it remains unclear whether ABT-199 is a powerful option in patients after HMA failure. Synergistic effects of ABT-199 and 5-azacytidine (5-AZA) were also not analyzed in primary material of MDS/sAML patients yet. Here we investigate the effect of ABT-199 as single treatment and in combination with 5-azacytdine in bone marrow samples of 28 high-risk MDS/sAML patients under HMA therapy. We measured induction of apoptosis after ABT-199/5-azacytidine treatment and analyzed the effect on colony forming capacity. Methods: Purified bone marrow mononuclear cells (BMMNC) were treated with 1μM ABT-199 or a soluble control (DMSO) and/or 5-AZA (from 0.25µM to 10µM) for 72h in vitro. Apoptosis was analyzed by flow cytometry after staining for 7-AAD, Annexin V, and CD34 as a progenitor marker. The long-term survival was investigated by colony formation assay. Control samples were obtained from human femoral heads discarded after implantation of total endoprosthesis of the hip joint from hematologically healthy age-matched donors. Combination indices were calculated using ComboSyn. Results: 28 primary samples of patients under HMA treatment (complete response n=5; stable disease n=10 and primary failure n=13) were treated with either ABT-199 single and/or combination therapy with ABT-199 and 5-AZA. Combination therapy showed a clear synergistic effect with the most favorable combination index (CI=0.1446) at ABT-199 (1µM) and 5-AZA (1µM). As expected ABT-199 only showed reduced activity in patients with complete remission. However BCL-2 inhibition effectively decreased the number of viable CD34+ cells in patients with stable disease under HMA treatment. Patients with HMA have a very poor prognosis. As expected 5-AZA (1µM) had only slight effects on MDS stem/progenitor cells of these patients in the ex vivo setting. ABT-199 monotherapy was still able to effectively induce apoptosis in a 72h read-out. Combination therapy (ABT-199 and 5-AZA at a concentration of 1µM each) efficiently induced apoptosis in the CD34+stem/progenitor cells as well as in the bulk of BMMNCs. When compared with single treatment, we found that induction of apoptosis was significantly increased in the stem/progenitor population from patients with HMA failure after combination treatment (ABT-199 mono vs combination p= 0.0089 and 5-AZA mono vs. combination p=0.0006). Conclusion: To identify novel treatment options in higher-risk MDS/sAML patients, we analyzed the apoptotic effect of ABT-199 single and ABT-199/5-azacytidine in combination in primary samples of patients under 5-azacytidine treatment. ABT-199 and 5-azacytidine showed strong synergistic effects with the most favorable combination index with 5-azacytidine (1µM) and ABT-199 (1µM). Monotherapy with ABT-199 was able to induce cell death ex vivo in high-risk MDS/sAML evenafter HMA failure. Combination therapy induced apoptosis very effectively and significantly reduced colony forming capacity. Age-matched healthy controls were only marginally effected. We therefore conclude that combination of low-dose 5-azacytidine with ABT-199 is more effective than single treatment in this pre-treated cohort of high-risk MDS/sAML patients. Since patients with HMA failure have a very poor prognosis with limited treatment options combination therapy of ABT-199 and 5-azacytidine seems to be a promising therapy option. Disclosures No relevant conflicts of interest to declare.

The ETV6-ABL1 fusion gene, as consequence of a t(9;12)(q34;p13), is a rare but recurrent genetic aberration found in chronic or blast phase of eosinophilia-associated myeloproliferative neoplasms (MPN-eo) and de novo acute B-cell lymphoblastic leukemias (B-ALL) or lymphoblastic T-cell lymphomas (T-LBL). Here, we sought to evaluate a) relevant clinical characteristics, b) treatment options and c) survival in 7 ETV6-ABL1 positive patients (male, n=4; median age 46 years; range 20-61). Cytogenetic analyses revealed a conventional reciprocal translocation t(9;12)(q34;p13) in 3 cases, an ins(12;9)(p13;q34q22) and a normal karyotype in one patient each, and a complex karyotype in 2 patients. In all cases, ETV6-ABL1 was confirmed by FISH analysis and/or RT-PCR. Histopathological diagnoses of the hypercellular bone marrow (BM) included atypical chronic myeloid leukemia (n=3), MPN-eo (n=3, concomitant T-LBL in one patient) or chronic myelomonocytic leukemia (n=1). In peripheral blood, all patients presented with left shifted leukocytosis (median 84 x 109/l, range 21-143) and significant (>1.5 x 109/l) eosinophilia (median 6.1 x 109/l, range 2.0-7.1) but without increased blast cells. Splenomegaly was present in 60% of patients. After a median of 3 months (range 0-6) from diagnosis, all patients were treated with a TKI (imatinib, n=5; dasatinib, n=1; nilotinib, n=1) at standard doses. On dasatinib (#2) or nilotinib (#3), 2 patients achieved a complete hematologic remission (CHR) within 3 months and complete cytogenetic remission (CCR) after 5 months (#3) or complete molecular remission (CMR) after 18 months (#2), respectively (Figure). On imatinib (n=5), 2 of 5 patients (#4 and #5) achieved a CHR within 3 months with loss of CHR after 9 (#4) and 5 months (#5), respectively. Patient #4 developed a myeloid sarcoma. After local radiation, he received an allogeneic stem cell transplant (SCT) but died 8 months later due to GvHD while in CMR. Patient #5 switched to nilotinib and achieved a CCR and CMR after 3 and 10 months, respectively. Patient #1 has not achieved a significant response after 4 months on imatinib. Patient #6 showed progressive disease within 2 months on imatinib, but achieved a rapid CHR and durable CCR and CMR on dasatinib after 13 and 14 months, respectively. Patient #7 presented with a classical myeloid/lymphoid (T-LBL) neoplasm with eosinophilia (MLN-eo) according to the WHO classification. On imatinib, a regression of lymphadenopathy, but persistence of leukocytosis and eosinophilia was observed. With increasing leukocytosis and reappearance of lymphadenopathy, the patient was switched to dasatinib (9 months), followed by nilotinib (2 months). The responses were only partial and transient and the patient died 23 months after diagnosis with myeloid blast phase (secondary acute myeloid leukemia). Overall, after a median treatment time of 22 months (range, 3-58), 4 patients are in CCR (n=1, patient #3) or CMR (n=3; patients #2, #5, #6) while on a second generation TKI and two patients (#4, #7) have died. On imatinib, none of 5 patients achieved a CCR or CMR. We conclude that a) cytogenetic analysis is an important tool for the identification of potential TK fusion genes such as ETV6-ABL1, b) ETV6-ABL1 is a candidate for incorporation into the WHO-defined subcategory ´MLN-eo´, c) patients with MPN-eo can achieve durable CHR, CCR or CMR on TKI with second generation TKI being more effective than imatinib d) close monitoring by cytogenetics, FISH and RT-PCR is recommended for early identification of inadequate response or resistance. Figure (A) responses and (B) overall survival in 7 patients with ETV6-ABL1 positive MPN-eo treated with various tyrosine kinase inhibitors.Abbreviation, CHR:complete hematologic response; CCR: complete cytogenetic response; CMR: complete molecular response; CP: chronic phase; BP: blast phase; allo SCT: allogeneic stem cell transplantation. Figure. (A) responses and (B) overall survival in 7 patients with ETV6-ABL1 positive MPN-eo treated with various tyrosine kinase inhibitors.Abbreviation, CHR:complete hematologic response; CCR: complete cytogenetic response; CMR: complete molecular response; CP: chronic phase; BP: blast phase; allo SCT: allogeneic stem cell transplantation. Disclosures Somervaille: Novartis: Consultancy, Honoraria; Imago Biosciences: Consultancy. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

We have investigated the usefulness of two small synthetic peptides comprising either a linear or a cyclic PLAEIDGIEL domain and a DNA-binding moiety of 16 lysine residues to mediate gene transfer selectively into alpha9beta1-integrin-displaying cells. Such specific gene delivery could only be achieved with the peptide containing the cyclic PLAEIDGIEL domain. However, inclusion of the cationic liposome Lipofect-AMINE into the peptide/DNA complexes resulted for both peptides in efficient gene transfer with significant targeting specificity. Naturally, the integrin alpha9beta1 is present only in a few highly specialised tissues and abundant throughout the human airway epithelia in vivo. Targeting gene vectors to this integrin therefore appears a useful approach to gene therapy of lung diseases such as cystic fibrosis. As the integrin alpha9beta1 is associated with tissue differentiation during foetal development and may cause resurgence of the foetal phenotype in colon cancers, such vectors may also be applicable for prenatal and cancer gene therapy.

X-linked Inhibitor of Apoptosis (XIAP) is an essential ubiquitin ligase for pro-inflammatory signalling downstream of the nucleotide-binding oligomerization domain containing (NOD)-1 and -2 pattern recognition receptors. Mutations in XIAP cause X-linked lymphoproliferative syndrome type-2 (XLP2), an immunodeficiency associated with a potentially fatal deregulation of the immune system, whose aetiology is not well understood. Here, we identify the XIAP baculovirus IAP repeat (BIR)2 domain as a hotspot for missense mutations in XLP2. We demonstrate that XLP2-BIR2 mutations severely impair NOD1/2-dependent immune signalling in primary cells from XLP2 patients and in reconstituted XIAP-deficient cell lines. XLP2-BIR2 mutations abolish the XIAP-RIPK2 interaction resulting in impaired ubiquitylation of RIPK2 and recruitment of linear ubiquitin chain assembly complex (LUBAC) to the NOD2-complex. We show that the RIPK2 binding site in XIAP overlaps with the BIR2 IBM-binding pocket and find that a bivalent Smac mimetic compound (SMC) potently antagonises XIAP function downstream of NOD2 to limit signalling. These findings suggest that impaired immune signalling in response to NOD1/2 stimulation is a general defect in XLP2 and demonstrate that the XIAP BIR2-RIPK2 interaction may be targeted pharmacologically to modulate inflammatory signalling. The X-linked lymphoproliferative syndrome type-2 is an immunodeficiency disease caused by mutations in the XIAP gene. BIR2 domain mutations in patients impair RIPK2 binding and NOD2-dependent innate immune signaling, explaining some of the pathology.

Local cooling is very common after bone and joint surgery. Therefore the knowledge of bone blood flow during local cooling is of substantial interest. Previous studies revealed that hypothermia leads to vasoconstriction followed by decreased blood flow levels. The aim of this study was to characterize if local cooling is capable of inducing reduced blood flow in bone tissue using a stepwise-reduced temperature protocol in experimental rabbits. To examine bone blood flow we utilized the fluorescent microsphere (FM) method. In New Zealand white rabbits one randomly chosen hind limb was cooled stepwise from 32 to 2°C, whereas the contra lateral hind limb served as control. Injection of microspheres was performed after stabilization of bone and muscle temperature at each temperature level. Bones were removed, dissected and fluorescence intensity was determined to calculate blood flow values. We found that blood flow of all cooled regions decreased relative to the applied external temperature. At maximum cooling blood flow was almost completely disrupted, indicating local cooling as powerful regulatory mechanism for regional bone blood flow (RBBF). Postoperative cooling therefore may lead to strongly decreased bone blood flow values. As a result external cooling has capacity to both diminish bone healing and reduce bleeding complications.

Neutrophils are essential players in the first-line defense against invading bacteria and fungi. Besides its antiapoptotic role, the inhibitor of apoptosis protein (IAP) family member X-linked IAP (XIAP) has been shown to regulate innate immune signaling. Whereas the role of XIAP in innate signaling pathways is derived mostly from work in macrophages and dendritic cells, it is not known if and how XIAP contributes to these pathways in neutrophils. Here we show that in response to bacterial lipopolysaccharides (LPS), mouse neutrophils secreted considerable amounts of tumor necrosis factor-α (TNFα) and interleukin-1β (IL-1β) and, in accordance with earlier reports, XIAP prevented LPS-induced hypersecretion of IL-1β also in neutrophils. Interestingly, and in contrast to macrophages or dendritic cells, Xiap-deficient neutrophils were insensitive to LPS-induced cell death. However, combined loss of function of XIAP and cIAP1/-2 resulted in rapid neutrophil cell death in response to LPS. This cell death occurred by classical apoptosis initiated by a TNFα- and RIPK1-dependent, but RIPK3- and MLKL-independent, pathway. Inhibition of caspases under the same experimental conditions caused a shift to RIPK3-dependent cell death. Accordingly, we demonstrate that treatment of neutrophils with high concentrations of TNFα induced apoptotic cell death, which was fully blockable by pancaspase inhibition in wild-type neutrophils. However, in the absence of XIAP, caspase inhibition resulted in a shift from apoptosis to RIPK3- and MLKL-dependent necroptosis. Loss of XIAP further sensitized granulocyte–macrophage colony-stimulating factor (GM-CSF)-primed neutrophils to TNFα-induced killing. These data suggest that XIAP antagonizes the switch from TNFα-induced apoptosis to necroptosis in mouse neutrophils. Moreover, our data may implicate an important role of neutrophils in the development of hyperinflammation and disease progression of patients diagnosed with X-linked lymphoproliferative syndrome type 2, which are deficient in XIAP.

Blood flow in various organs is determined by an autoregulatory mechanism that guarantees constant organ perfusion over a wide range of arterial blood pressure changes. This physiological principle has been proven for the kidney, brain and intestinal tract, but so far not for bone. This study was carried out to determine whether there is an autoregulatory mechanism of bone or not. The fluorescent microsphere reference sample method was used to determine blood flow within the bone and kidneys. Eight anesthetized female New Zealand rabbits received left ventricular injections of fluorescent microspheres over a wide range of arterial pressure levels prior to removal of kidney, femur and tibia. Blood flow values were calculated by measurement of fluorescence intensity in kidney and bone and correlated to fluorescence intensity in the peripheral blood (reference sample). Despite a reduction of mean arterial pressure from 100 to 80 mmHg bone blood flow remained constant. Further reduction of mean arterial pressure results in a linear decrease in bone blood flow. The correlation between arterial pressure and organ perfusion in the bone is similar to blood flow within the kidney, indicating the presence of an autoregulated blood flow mechanism within the bone tissue.

Endothelial injury is perhaps the inciting factor leading to the microangiopathic process that initiates thrombotic thrombocytopenic purpura-hemolytic uremic syndrome (TTP-HUS). TTP-HUS after postendoscopic retrograde cholangiopancreatography (ERCP) pancreatitis is extremely rare, but potentially is life threatening. Here, we describe a case of a 23-year-old man with a history of choledocholithiasis, who developed TTP-HUS, 2 days after the onset of post-ERCP pancreatitis. It is important that physicians recognize TTP-HUS as one of the potential causes of acute kidney injury in cases of acute pancreatitis and post-ERCP pancreatitis for adult patients, especially when there is concomitant thrombocytopenia and hemolytic anemia. The early initiation of plasma exchange has a major impact on the survival and preservation of renal function. Exchange transfusion of fresh frozen plasma remains the cornerstone treatment of TTP-HUS.

Germinal Center Survival The humoral immune response, which comprises antibodies secreted by B lymphocytes, is critical for protection against pathogens. In response to infection, B lymphocytes proliferate and differentiate into antibody-producing effector cells. After an infection clears, a small number of cells persist as memory B cells; however, the survival signals that regulate effector and memory B lymphocyte generation are not well understood. To probe this question, Vikstrom et al. (p. 1095 , published online 7 October) deleted prosurvival genes in activated, antigen-specific B cells during a T lymphocyte–dependent immune response in mice. They found that a specific programmed cell death inhibitor, known as Mcl1, was required for the formation of germinal-center B cells (an effector cell population) and memory B cells but not for their maintenance. Dysregulation of the B cell responses mediated by Mcl1 may be a trigger for lymphomagenesis.

The transcription factor NF-κB is a tightly regulated positive mediator of T- and B-cell development, proliferation, and survival. The controlled activity of NF-κB is required for the coordination of physiologic immune responses. However, constitutive NF-κB activation can promote continuous lymphocyte proliferation and survival and has recently been recognized as a critical pathogenetic factor in lymphoma. Various molecular events lead to deregulation of NF-κB signaling in Hodgkin disease and a variety of T- and B-cell non-Hodgkin lymphomas either upstream or downstream of the central IκB kinase. These alterations are prerequisites for lymphoma cell cycling and blockage of apoptosis. This review provides an overview of the NF-κB pathway and discusses the mechanisms of NF-κB deregulation in distinct lymphoma entities with defined aberrant pathways: Hodgkin lymphoma (HL), diffuse large B-cell lymphoma (DLBCL), mucosa-associated lymphoid tissue (MALT) lymphoma, primary effusion lymphoma (PEL), and adult T-cell lymphoma/leukemia (ATL). In addition, we summarize recent data that validates the NF-κB signaling pathway as an attractive therapeutic target in T- and B-cell malignancies.

A 29-year-old woman presented to the emergency department with exhaustion, fatigue, and abdominal pain. She reported having received a diagnosis of bulimia nervosa 10 years before. On examination, she had a marked pallor and was severely malnourished. Laboratory analysis revealed a dramatically low hemoglobin level of 1.7 g/dL (ref: 11.5-15.8 g/dL). Serum iron was quantified as 1.4 micromol/L (ref: 7-26 micromol/L), ferritin as 5 ng/mL (ref: 10-120 ng/mL), and the level of serum transferrin as 212 mg/dL (ref: 200-360 mg/dL). A duodenal biopsy revealed villous atrophy in the mucosal layer indicative for celiac disease. This diagnosis was confirmed by serum levels of endomysial antibodies, tissue transglutaminase antibody, and antigliadin antibodies. The newly diagnosed gluten-sensitive enteropathy is likely to be in part responsible for the severe symptoms reported. The extent of hemoglobin decline in combination with an astonishing lack of critical symptoms seen in this patient is a rarity. We conclude that anorectic patients with severe anemia and malnutrition should be evaluated for the presence of additional somatic conditions.

The heterogeneous subgroup of T-lymphocyte non-Hodgkin lymphoma (T-NHL) exhibits an aggressive growth pattern and is relatively insensitive to chemotherapy. The molecular mechanisms of survival and resistance to therapy remain largely undefined. We hypothesized that BCL-2 proteins with anti-apoptotic properties protect T-NHL cells from cell death, cause resistance to therapy and therefore represent an attractive target for therapy. MCL-1 potently protects physiologically healthy T-lymphocytes from cell death, but its function in transformed T-cells is incompletely understood. We therefore dissected the role of MCL-1 using in silico meta-analyses on available gene expression data on human primary T-NHL samples. We identified MCL-1 as the primary BCL-2 family protein to be highly expressed across most major T-NHL subsets. Expression of MCL-1 was restricted to its anti-apoptotic full-length splice variant as opposed to its pro-apoptotic short isoform. To functionally characterize the requirement for MCL-1, we utilized a T-NHL mouse model, which is based on four consecutive low-dose whole-body γ-irradiations in 4 week-old mice resulting in the development of T-NHL mimicking human peripheral T-cell lymphoma. We utilized mice harbouring loxP-flanked Mcl-1 or loxP-flanked Bcl-x(L) in combination with inducible Cre recombinase to conditionally delete the gene of interest after lymphoma induction. Interestingly, conditional deletion of only one allele of Mcl-1 in fully established primary T-NHL cells ex vivo led to a significant and specific loss of viability, whereas survival remained unaffected by full deletion of Bcl-x(L). Reduced MCL-1 levels resulted in substantially elevated sensitivity to standard chemotherapeutics such as anthracyclins, cyclophosphamide and etoposide. In addition, mono-allelic deletion of Mcl-1 in vivo prolonged survival of lymphoma-bearing mice. Together, these data argue that anti-apoptotic MCL-1 is the single most important BCL-2 family member involved in the sustained survival of T-NHL. Disclosures: Strasser: Genentech Inc: Consultancy.

10508 Background: In gastrointestinal stromal tumor (GIST), there is no biomarker available that indicates success or failure of therapy. We hypothesized that tumor specific CKIT or PDGFRA mutant DNA fragments can be detected and quantified in plasma samples of GIST patients. Methods: We prospectively collected 291 plasma samples from 38 subjects with GIST harbouring activating mutations of CKIT or PDGFRA detected in tumor tissue, irrespective of current disease status or treatment. We used allele-specific Ligation PCR to detect mutant free circulating (fc)DNA. Results: We were able to detect fcDNA harbouring the tumor mutation in 15 out of 38 patients. Patients with active disease displayed significantly higher amounts of mutant fcDNA compared to patients in CR. The amount of mutant fcDNA correlated with disease course. We observed repeated positive test results or an increase of mutant fcDNA in five patients with progressive disease or relapse. A decline of tumor fcDNA or conversion from positive to negative was seen in five patients responding to treatment. A negative to positive conversion was seen in two patients with relapse and one patient with progression. In two cases, we aimed to identify additional mutations, and found four additional exchanges, including mutations not known from sequentially performed tumor biopsies. Conclusions: Our results indicate that free circulating DNA harbouring tumor specific mutations in the plasma of patients with GIST can be used as tumor-specific biomarker. The detection of resistance mutations in plasma samples might allow earlier treatment changes and obviates the need for repeated tumor biopsies. Clinical trial information: NCT01462994.

The FasL/Fas system is critical for deletion of autoreactive and antigen-activated T and B cells. Accordingly, mutations in these proteins result in lymphadenopathy and autoimmunity in gld and lpr mutant mice, which lack functional FasL or Fas, respectively. Upon antigenic stimulation of T cells, FasL is sythesised, directed to and stored in secretory lysosomes followed by extrusion at the immunological synapse where it is rapidly downregulated by a metalloprotease, shedding the extracellular portion (sFasL) to prevent non-specific killing. It is unclear whether the pathology observed in gld mutant mice is due to the loss of the membrane-bound or the secreted form of FasL or both. We have produced a panel of mutant FasL knock-in mice to address this question. In the first mutant strain the cytoplasmic and trans-membrane domains of FasL were replaced with the signal peptide from G-CSF. Activated T cells from these mutant mice can produce cytoplasmic but no membrane bound FasL and, interestingly, they are defective in FasL-mediated cytotoxic function and undergo significantly less activation-induced cell death upon re-stimulation with anti-CD3 antibodies than wt T cells. The extent of these defects is similar to that seen in FasL mutant gld T cells. With age these FasL mutant knock-in mice develop lymphadenopathy and splenomegaly and CD3+B220+CD4-CD8- T cells accumulate, similarly to what has been observed in gld and lpr mutant mice. In contrast to gld mice, the FasL mutant knock-in mice on the C57BL/6 background develop haemopoietic tumours and reticular cell sarcomas, suggesting that while membrane-bound FasL is the guardian against autoimmunity, secreted FasL may play a critical role in tissue damage and tumour suppression.

Development of immature T cells in the thymus requires signals through the clonotypic T cell receptor (TCR). Thymocytes expressing a functionally inactive or autoreactive TCR are deleted via apoptosis (negative selection). Thymocytes expressing a functionally active but not autoreactive TCR are selected through inhibition of cell death (positive selection). Deregulation of this process is likely to result in autoimmunity or lymphomagenesis of T cells. The intracellular mechanisms by which the balance between TCR-dependent survival and apoptosis are regulated are largely unknown. A central regulator of survival and apoptosis in the immune system is the transcription factor NF-κB. Activation of NF-κB in mature T-cells requires the adaptor proteins Bcl10 and Malt1. Using gene-targeted mice deficient for Bcl10 or Malt1, we show that Bcl10 and Malt1 are also required for TCR-induced NF-κB activation in immature T cells. Furthermore, to elucidate the process of T cell selection within the thymus, we have crossed Bcl10 or Malt1 deficient mice into mice with genetic backgrounds expressing defined TCR transgenes. Using specific peptide agonists of these TCR transgenes, we show that neither in vivo nor in vitro development into single positive (SP) CD4 or CD8 positive T cells is altered in Bcl10 or Malt1 deficient mice. Absolute numbers and ratio of SP T cells found within the thymus or in peripheral lymphnodes of transgenic animals are normal. These findings indicate that Bcl10 and Malt1 activate NF-κB in thymocytes but are dispensable for maturation of immature T cells in this model system.

Targeting gene vectors to human airway epithelial cells may help to overcome the current inefficiency of gene transfer as the major problem confronting cystic fibrosis gene therapy. To elucidate novel ligands targeting abundant, apically located receptors on airway epithelial cells, a phage display library was screened for peptides binding with high affinity to such cells. This screening yielded a selectively enriched amino acid sequence, Thr-His-Ala-Leu-Trp-His-Thr (THALWHT). Subsequent binding studies confirmed that THALWHT-displaying phages bound much stronger than phages displaying control peptides to human airway epithelial cells. In contrast, no significant binding differences were observed on a variety of non-airway-derived human cell lines suggesting selective binding of the THALWHT motif to airway epithelia. Confocal microscopy of such cells after exposure to labelled synthetic THALWHT peptide indicated that its binding is followed by specific internalisation via endocytosis. A synthetic peptide comprising a cyclic CTHALWHTC domain and a DNA binding moiety enabled efficient targeted gene delivery into human airway epithelial cells. Competition assays with free THALWHT peptide confirmed the specificity of gene delivery. Thus, the THALWHT motif may prove a useful targeting moiety for both non-viral and viral gene therapy vectors.

FAS (also known as APO-1 or CD95) belongs to the subgroup of the tumor necrosis factor receptor (TNF-R) family that contain an intra-cellular ‘death domain’ and can trigger apoptosis. Its physiological ligand, FASL (CD95L), is a member of the corresponding TNF cytokine family. Studies with spontaneous mutant mice, gene-targeted mice and cells from human patients have shown that FAS and FASL play critical roles in the immune system, in particular in the killing of pathogen infected target cells and the death of no longer needed, potentially deleterious as well as autoreactive lymphocytes. This ligand-receptor pair thereby functions as a guardian against autoimmunity and tumor development. FASL-FAS signaling triggers apoptosis through FADD (Fas-associated protein with death domain, also called MORT1) adaptor protein-mediated recruitment and activation of the aspartate-specific cysteine protease, caspase-8. In certain cells such as hepatocytes, albeit not in lymphocytes, FAS-induced apoptosis signaling requires amplification through proteolytic activation of the pro-apoptotic BCL-2 family member BID. Curiously, several components of the FAS signaling machinery have been implicated in non-apoptotic processes, including cellular activation, differentiation and proliferation. Here we describe current knowledge of the roles of FASL and FAS in the immune system, discuss important unresolved issues and propose experimental approaches to address them.

The development of lymphomas and leukemias is frequently caused by chromosomal translocations that deregulate cellular pathways of differentiation, proliferation or survival. The molecules that are involved in these aberrations provide rational targets for selective drug therapies. Recently, several disease specific translocations have been identified in human MALT lymphoma. These aberrations either upregulate the expression of BCL10 or MALT1 or induce the formation of API2-MALT1 fusion proteins. Genetic and biochemical experiments identified BCL10 and MALT1 as central components of an oligomerization-ubiquitinylation-phosphorylation cascade that activates the transcription factor NF-kappaB in response to antigen receptor ligation. Deregulation of the signaling cascade is directly associated with antigen independent MALT lymphoma growth. Here we provide an overview of the physiological and pathological functions of BCL10/MALT1 signal transduction and discuss the potential of this pathway as a drug target.

Predicting minimal residual disease (MRD) levels in tyrosine kinase inhibitor (TKI)-treated chronic myeloid leukemia (CML) patients is of major clinical relevance. The reason is that residual leukemic (stem) cells are the source for both, potential relapses of the leukemicclone but also for its clonal evolution and, therefore, for the occurrence of resistance. The state-of-the art method for monitoring MRD in TKI-treated CML is the quantification of BCR-ABL levels in the peripheral blood (PB) by PCR. However, the question is whether BCR-ABL levels in the PB can be used as a reliable estimate for residual leukemic cells at the level of hematopoietic stem cells in the bone marrow (BM). Moreover, once the BCR-ABL levels have been reduced to undetectable levels, information on treatment kinetics is censored by the PCR detection limit. Clearly, BCR-ABL negativity in the PB suggests very low levels of residual disease also in the BM, but whether the MRD level remains at a constant level or decreases further cannot be read from the BCR-ABL negativity itself. Thus, also the prediction of a suitable time point for treatment cessation based on residual disease levels cannot be obtained from PCR monitoring in the PB and currently remains a heuristic decision. To overcome the current lack of a suitable biomarker for residual disease levels in the BM, we propose the application of a computational approach to quantitatively describe and predict long-term BCR-ABL levels. The underlying mathematical model has previously been validated by the comparison to more than 500 long-term BCR-ABL kinetics in the PB from different clinical trials under continuous TKI-treatment [1,2,3]. Here, we present results that show how this computational approach can be used to estimate MRD levels in the BM based on the measurements in the PB. Our results demonstrate that the mathematical model can quantitatively reproduce the cumulative incidence of the loss of deep and major molecular response in a population of patients, as published by Mahon et al. [4] and Rousselot et al. [5]. Furthermore, to demonstrate how the model can be used to predict the BCR-ABL levels and to estimate the molecular relapse probability of individual patients, we compare simulation results with more than 70 individual BCR-ABL-kinetics. For this analysis we use patient data from different clinical studies (e.g. EURO-SKI: NCT01596114, STIM(s): NCT00478985, NCT01343173) where TKI-treatment had been stopped after prolonged deep molecular response periods. Specifically, we propose to combine statistical (non-linear regression) and mechanistic (agent-based) modelling techniques, which allows us to quantify the reliability of model predictions by confidence regions based on the quality (i.e. number and variance) of the clinical measurements and on the particular kinetic response characteristics of individual patients. The proposed approach has the potential to support clinical decision making because it provides quantitative, patient-specific predictions of the treatment response together with a confidence measure, which allows to judge the amount of information that is provided by the theoretical prediction. References [1] Roeder et al. (2006) Dynamic modeling of imatinib-treated chronic myeloid leukemia: functional insights and clinical implications, Nat Med 12(10):1181-4 [2] Horn et al. (2013) Model-based decision rules reduce the risk of molecular relapse after cessation of tyrosine kinase inhibitor therapy in chronic myeloid leukemia, Blood 121(2):378-84. [3] Glauche et al. (2014) Model-Based Characterization of the Molecular Response Dynamics of Tyrosine Kinase Inhibitor (TKI)-Treated CML Patients a Comparison of Imatinib and Dasatinib First-Line Therapy, Blood 124:4562 [4] Mahon et al. (2010) Discontinuation of imatinib in patients with chronic myeloid leukaemia who have maintained complete molecular remission for at least 2 years: the prospective, multicentre Stop Imatinib (STIM) trial. Lancet Oncol 11(11):1029-35 [5] Rousselot et al. (2014) Loss of major molecular response as a trigger for restarting TKI therapy in patients with CP- CML who have stopped Imatinib after durable undetectable disease, JCO 32(5):424-431 Disclosures Glauche: Bristol Meyer Squib: Research Funding. von Bubnoff:Amgen: Honoraria; Novartis: Honoraria, Research Funding; BMS: Honoraria. Saussele:ARIAD: Honoraria; Novartis: Honoraria, Other: Travel grants, Research Funding; Pfizer: Honoraria, Other: Travel grants; BMS: Honoraria, Other: Travel grants, Research Funding. Mustjoki:Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Ariad: Research Funding; Novartis: Honoraria, Research Funding. Guilhot:CELEGENE: Consultancy. Mahon:NOVARTIS PHARMA: Honoraria, Research Funding; BMS: Honoraria; PFIZER: Honoraria; ARIAD: Honoraria. Roeder:Bristol-Myers Squibb: Honoraria, Research Funding.