Abstract: Background: Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the western world and shows a very heterogeneous clinical course. While the genetic landscape of CLL has been well characterized during recent years it can only partially explain the underlying biology of this heterogeneity. Proteogenomics could offer a valuable tool to fill this gap and improve the understanding of CLL biology. Methods: Here, we performed a large proteogenomic analysis (n=263) of three clinically annotated CLL cohorts: For the discovery cohort (Germany_1: n=68) we performed in-depth HiRIEF LC-MS based proteomics (more than 9000 proteins quantified) alongside genome-, transcriptome and ex-vivo drug response-profiling with 43 clinically established drugs. The proteome of two additional validation cohorts (Germany_2: n=44, Sweden_1: n=89), were characterized by data-independent acquisition (DIA) mass spectrometry. Results: To connect the CLL genotype with the molecular phenotype, we investigated associations between recurrent genetic alterations of CLL, mRNA expression and protein abundance. We found that trisomy 12, IGHV status and SF3B1 mutations had the greatest impact on protein abundances. CLL specific recurrent chromosomal deletions and gains (trisomy 12, del17p, del13q, del11q, gain8q24) consistently impacted on gene expression and protein abundance through gene dosage effects. We explored functional consequences of these gene dosage effects and found that the additional copy of chromosome 12 increased the abundance of central B-cell receptor (BCR) protein complexes through cis- and trans-effects, which could explain the increased response of trisomy 12 patient samples to BCR inhibition. Somatic mutations of TP53, ATM and XPO1 were associated with less, but specific and biologically-relevant protein abundance changes. p53 for instance, was the most upregulated protein in TP53 mutated samples, owing to the known stabilisation of mutant p53. This effect was not detectable on transcript level. ATM and XPO1 protein abundances were significantly lower in ATM and XPO1 mutated cases, indicating loss-of-function phenotypes of these mutations. To understand global similarities and differences between CLL patients on the proteomic level, we performed unsupervised clustering and identified clinically meaningful subgroups. Unsupervised clustering of the proteomics data identified six subgroups with contrasting clinical behaviour. TP53 mutations, IGHV status, trisomy 12 and their interactions explained five subgroups. These results show that quantitative mass spectrometry-based proteomics distinguished clinically relevant subgroups of CLL. Most importantly, we identified a previously unappreciated subgroup of CLL, comprising 20% of all cases, which could be uncovered by proteomic profiling and showed no association with frequent genetic or transcriptional alterations. This new CLL subgroup was characterized by accelerated disease progression, SF3B1 mutation-independent splicing alterations, metabolomic reprogramming and increased vulnerability to inhibitors of metabolic enzymes and the proteasome. Surprisingly, major BCR signaling proteins were downregulated in this subgroup, suggesting less dependence on BCR activity. In accordance with this observation, an unsupervised analysis revealed that low levels of many BCR signaling proteins (e.g. PLCG2 and PIK3CD) were associated with short time to next treatment. The existence of this subgroup could be confirmed in the validation cohorts. Finally, we performed an unsupervised multi-omics factor analysis (MOFA) across all omics data sets in parallel. This unsupervised analysis confirmed the existence of the above identified CLL subgroups and an important role of SF3B1 mutation-independent splicing alterations in CLL. Conclusion: Our integrative multi-omics analysis provides the first comprehensive overview of the interplay between genetic variants, the transcriptome, and the proteome, along with functional consequences for drug response and clinical outcome in CLL. Importantly, we identified a new subgroup with accelerated disease progression, a distinct proteomic signature and a clinically exploitable drug sensitivity profile. Figure Disclosures Mueller-Tidow: BiolineRx: Research Funding; Daiichi Sankyo: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMBF: Research Funding; Wilhelm-Sander-Stiftung: Research Funding; Jose-Carreras-Siftung: Research Funding; Bayer AG: Research Funding; Deutsche Krebshilfe: Research Funding; Deutsche Forschungsgemeinschaft: Research Funding; Janssen-Cilag Gmbh: Membership on an entity's Board of Directors or advisory committees. Dreger:Neovii: Research Funding; Roche: Consultancy, Speakers Bureau; Riemser: Consultancy, Research Funding, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Janssen: Consultancy; Gilead: Consultancy, Speakers Bureau; AstraZeneca: Consultancy; AbbVie: Consultancy, Speakers Bureau. Stilgenbauer:Pharmacyclics: Consultancy, Honoraria, Other, Research Funding; Novartis: Consultancy, Honoraria, Other, Research Funding; Mundipharma: Consultancy, Honoraria, Other, Research Funding; Janssen-Cilag: Consultancy, Honoraria, Other: travel support, Research Funding; GlaxoSmithKline: Consultancy, Honoraria, Other: travel support, Research Funding; Gilead: Consultancy, Honoraria, Other: travel support, Research Funding; Genzyme: Consultancy, Honoraria, Other: travel support, Research Funding; Genentech: Consultancy, Honoraria, Other: travel support, Research Funding; F. Hoffmann-LaRoche: Consultancy, Honoraria, Other: travel support, Research Funding; Celgene: Consultancy, Honoraria, Other: travel support, Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Other: travel support, Research Funding; Amgen: Consultancy, Honoraria, Other: travel support, Research Funding; AbbVie: Consultancy, Honoraria, Other: travel support, Research Funding. Tausch:Roche: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Janssen-Cilag: Consultancy, Honoraria, Research Funding. Dietrich:Roche: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; KITE: Membership on an entity's Board of Directors or advisory committees.

Abstract: A female, 48-year old patient was referred to an emergency ward with dyspnoea, haemoptysis and suspected respiratory infection that had been refractory to prior antimicrobial therapy. The onset of dyspnoea dated back to a febrile episode three months prior to presentation. The patient continued to work in spite of severe shortness of breath. Her history revealed a cervical cancer which had been treated by neoadjuvant radiochemotherapy and extended hysterectomy in 2005. Upon admission, extensive spontaneous haematomas were noted on the upper extremities. Following a diagnostic puncture of the radial artery, severe haemorrhage and compartmentalization developed, and the patient was transferred to our hospital for a surgical emergency intervention and haemostaseological counselling. A decreased factor VIII activity was noted, with a prolonged partial thromboplastin time, a normal von Willebrand Factor activity and concentration, a prolonged bleeding time and a low-titer inhibitor to factor VIII. Additionally, a profound hyponatremia (117 mmol/l) awaited explanation. Haemorrhage was treated with recombinant Factor VIIa, allowing for a successful surgical de-compartmentalization. With regard to the consistent feature of dyspnoea and partial respiratory insufficiency, bronchoscopic examination revealed only minor hemorrhagic suffusion of the bronchial epithelium, but no sign of infection or major pulmonary bleeding. Chest X-ray and laboratory findings showed right ventricular enlargement, bilateral infiltrates and an excessively high NT-pro-BNP concentration in plasma (13027 ng/l). Chest X-ray radiographs performed two months previously for suspected respiratory infection showed pulmonary infiltrates of comparable size, albeit no right ventricular enlargement. Computerized tomography of the chest showed a complete obstruction of the right pulmonary artery and of the lower branch of the left pulmonary artery. Pulmonary arterial pressure was measured at 70 mm Hg. Immunosuppressive therapy was initiated, with concomitant aPTT-adjusted anticoagulation. Upon clinical deterioration with right-ventricular failure, a catheter-based disruption of the emboli was attempted, albeit with no success. A cardio-surgical intervention with thrombendectomy was performed successfully; however, the patient succumbed to sepsis and multiorgan failure ten days later. The autopsy revealed multiple thrombi, a bilateral pulmonary haemorrhagic infarction and a disseminated lymphangiosis and hemangiosis carcinomatosa of the lungs. We conclude that the acquired inhibitor to factor VIII was of paraneoplastic origin and, although detected at a low titre, clinically relevant. Based on previous radiographic findings, a massive pulmonary embolism must be assumed to have occurred months prior to presentation. The anticoagulatory effect of the acquired inhibitor to Factor VIII may have averted the timely detection of the pulmonary embolism.

Abstract: Apoptosis-related proteins are important molecules for predicting chemotherapy response and prognosis in adult acute myeloid leukemia (AML). However, data on the expression and prognostic impact of these molecules in childhood AML are rare. Using flow cytometry and western blot analysis, we therefore investigated 45 leukemic cell samples of children with de novo AML enrolled and treated within the German AML-BFM93 study for the expression of apoptosis-regulating proteins (CD95, Bcl-2, Bax, Bcl-xL, Procaspase-3, XIAP, cIAP-1, Survivin). XIAP (p & lt;0.002) but no other apoptosis regulators showed maturation-dependent expression differences as determined by FAB morphology with the highest expression levels observed within the immature M0/1 subtypes. XIAP (p & lt;0.01) and Bcl-xL (p & lt;0.01) expression was lower in patients with favorable than intermediate/poor cytogenetics. After a mean follow-up of 34 months, a shorter overall survival was associated with high expression levels of XIAP {30 (n=10) vs. 41 months (n=34); p & lt;0.05} and Survivin {27 (n=10) vs. 41 months (n=34); p & lt;0.05}. We conclude that apoptosis-related molecules are associated with maturation stage, cytogenetic risk groups and therapy outcome in childhood de novo AML. The observed association of XIAP with immature FAB types, intermediate/poor cytogenetics and poor overall survival should be confirmed within prospective pediatric AML trials.

Abstract: Abstract 546 TP53 is commonly mutated across lymphoma entities. TP53 mutations have been found in 20–25 % of aggressive B-cell lymphomas and most studies suggest an impact on outcome. Few studies have addressed the impact of TP53 mutations in larger trial cohorts. In order to evaluate the contribution of TP53 mutation status to current risk models, we investigated TP53 gene mutations within the RICOVER-60 trial of the DSHNHL. Of 1222 elderly patients (aged 61–80 years) randomly assigned to six or eight cycles of CHOP-14 with or without Rituximab, 265 patients (representative of the whole cohort) were analyzed for TP53 mutations. Genomic DNA samples extracted from paraffin embedded tissue were used and mutations were studied by Sanger sequencing of exons 4–9. All patients had untreated CD20+ aggressive B-cell lymphoma according to the World Health Organization classification as confirmed by reference pathology. The primary endpoint of the trial was event-free survival. Analyses were done by intention to treat. The trial is registered on National Cancer Institute website (NCT00052936). TP53 mutations were demonstrated in 64 of 265 patients (24.1%). The incidence is in accordance to prior studies. TP53 mutation was associated with higher LDH (66% vs. 37%), higher IPI-Scores (IPI 4/5 27% vs. 12%), and B-symptoms (41% vs. 24%) (TP53 mutant (mut) and wild type (wt) groups resp.). Patients with TP53 mutation were less likely to obtain a CR/Cru 62.5% (mut.) vs. 79.6% (wt). The median observation time was 40.2 months and the presence of TP53 mutations was associated with decreased OS; EFS and PFS. The respective 3 year overall and event-free survival were 49% (vs. 77%) and 41% (vs. 60.5%) for the group with TP53 mutation (p 〈 0.0001; 〈 0.01). The analysis of the TP53 mutant subgroup (n=64) showed no impact of the treatment arm (R-CHOP vs. CHOP; 6 vs. 8 cycles) for OS, EFS or PFS in univariate analysis. In a Cox proportional hazard models including the IPI, ECOG, LDH, stage and treatment arms, TP53 mutation was shown to be an independent predictor of EFS (HR 1.5), PFS (HR 2.1) and most importantly OS (HR 2.4; p 〈 0.001). Additional factors associated with EFS were LDH, ECOG, stage and Rituximab arm. Our findings suggest that TP53 mutations are independent predictors of poor PFS, EFS and OS in untreated patients with aggressive CD20+ lymphoma. Based on the analysis within a homogenous trial cohort, strategies to improve outcome for the 20–25% of patients with mutant p53 should be developed. Recurrent genetic lesions should be integrated into risk models in DLBCL. Fig.: Overall survival and TP53 mutation in aggressive B-NHL Fig.:. Overall survival and TP53 mutation in aggressive B-NHL Disclosures: No relevant conflicts of interest to declare.

Abstract: We aimed at understanding the relapse-driving processes in pediatric T-ALL and performed an integrated longitudinal multi-level omics analysis of 13 T-ALL patients at initial diagnosis (INI) and relapse (REL). We compared the mutation (SNV/InDels) and copy number alteration (CNA) patterns as well as gene expression, methylation levels and chromatin accessibility by ATAC-Seq. Aberrant expression of T-ALL transcription factors (TAL1, TAL2,LMO2, TLX1, TLX3, NKX2.4 and NKX2.5) was preserved from initial presentation to relapse in all patients. These leukemia-driving events defined the expression patterns, methylation profiles and the chromatin accessibility landscapes. A global differential analysis of the RNA-Seq data (DESeq2, padj 〈 .05) revealed only 0.3% of the genes to be either up- or down-regulated at relapse when compared to the matched initial sample. Likewise, we detected methylation changes in only 3% of the promoters and differential chromatin accessibility in only 0.26% of the analyzed ATAC-peaks (DESeq2, padj 〈 .05). We then focused our analysis on the 2 types of relapse in pediatric T-ALL, which we have previously defined on the basis of subclonal mutation profiles (Kunz et al., 2015). These types of relapse are characterized by either clonal evolution of cells derived from the major clone at initial presentation (type 1) or emergence and evolution of a minor initial clone showing a molecular profile that is distinct from the predominant initial clone (type 2). When considering type 1 and type 2 relapses separately we identified a strong trend for type 2 relapses to acquire more mutations (p=0.0879, ttest) than type 1 relapses. Further to the known activating mutations in NT5C2 acquired at relapse by 8/13 patients no other mutations or CNAs were recurrently acquired in the relapses of this group of patients. However, mutations in proto-oncogenes or genes involved in DNA surveillance were acquired by 7/8 type 2 relapse patients in our series. Changes of CNAs also occurred more frequently in type 2 than in type 1 relapses (pval= 0.0267, ttest). This increased complexity on the genetic level was also apparent on the epigenetic level, with an increase of changes in the methylation pattern (mean difference in β value between INI and REL: type 1 - 0.00034; type2 - 0.002 (pval 〈 0.0001, chi2)), chromatin accessibility (number of differentially accessible ATAC-peaks: type 1 - 4 (0.006%) ; type 2 - 1018 (1.3%); (pval 〈 0.0001, chi2)) and on the expression level (number of differentially expressed genes: type 1 - 11; type 2 - 111, pval 〈 0.0001, chi2). When considering differences between leukemias at the time of initial diagnosis, which later develop either type 1 or type 2 relapses we found 1.016 genes to be differentially expressed (524: up; 492: down in type 1; DE-Seq2: padj 〈 0.05). Differential expression analysis revealed that genes involved in early T-cell differentiation were upregulated at initial diagnosis of type 1 in comparison to initial diagnosis of type 2, which was also reflected by more accessible chromatin surrounding their cis-regulatory regions as analyzed by ATAC-seq suggesting an early arrest of these samples during differentiation process. Altogether 1.4% of all ATAC-peaks were more accessible in type 1, whereas 0.7% of peaks were more accessible in type 2 leukemia (DE-Seq2: p 〈 0.05). Remarkably, the chromatin surrounding DNA-repair genes was more accessible in initial leukemias of type 1, which was reflected by up-regulated mRNA expression level of such genes. These data suggest that an increased propensity of DNA repair may represent an important mechanism of T-ALL to develop a type 1 relapse, an interpretation that is consistent with previous reports linking the epigenetic silencing of the methylguanine-DNA methyltransferase promoter with compromised DNA repair and longer survival in patients with glioblastoma receiving alkylating agents (Esteller et. al., 2000). In sum, the multilevel omic comparison of pediatric T-ALL that develop either a type 1 or a type 2 relapse show remarkably more complex changes of the genetic and epigenetic profiles during the transition from initial to relapsing disease. Notably, pediatric T-ALLs, who later develop a type 1 relapse display an epigenetic and transcriptomic landscape predicting an upregulation of DNA repair functions, which we suggest to potentially play a role in developing resistance to DNA damaging agents in this type of relapse. Disclosures Muckenthaler: Novartis: Research Funding. Bourquin:Amgen: Other: Travel Support. Kulozik:bluebird bio: Consultancy, Honoraria.

Abstract: Classic Hodgkin lymphoma (cHL) is the cancer type most susceptible to antibodies targeting programmed cell death protein 1 (PD1) and is characterized by scarce Hodgkin and Reed-Sternberg cells (HRSCs), perpetuating a unique tumor microenvironment (TME). Although anti-PD1 effects appear to be largely mediated by cytotoxic CD8+ T cells in solid tumors, HRSCs frequently lack major histocompatibility complex expression, and the mechanism of anti-PD1 efficacy in cHL is unclear. Rapid clinical responses and high interim complete response rates to anti-PD1 based first-line treatment were recently reported for patients with early-stage unfavorable cHL treated in the German Hodgkin Study Group phase 2 NIVAHL trial. To investigate the mechanisms underlying this very early response to anti-PD1 treatment, we analyzed paired biopsies and blood samples obtained from NIVAHL patients before and during the first days of nivolumab first-line cHL therapy. Mirroring the rapid clinical response, HRSCs had disappeared from the tissue within days after the first nivolumab application. The TME already shows a reduction in type 1 regulatory T cells and PD-L1+ tumor-associated macrophages at this early time point of treatment. Interestingly, a cytotoxic immune response and a clonal T-cell expansion were not observed in the tumors or peripheral blood. These early changes in the TME were distinct from alterations found in a separate set of cHL biopsies at relapse during anti-PD1 therapy. We identify a unique very early histologic response pattern to anti-PD1 therapy in cHL that is suggestive of withdrawal of prosurvival factors, rather than induction of an adaptive antitumor immune response, as the main mechanism of action.

Abstract: The class of the so-called long non-coding RNAs (lncRNAs) is defined by their lacking coding potential and their length of more than 200 nucleotides. LncRNAs are readily detected in transcriptome analysis but have so far not been characterized or catalogued exhaustively. Although lncRNAs make up a major portion of the mammalian non-coding transcriptome, it is challenging to predict their functions due to their diversity in terms of size, sequence and biogenesis. However, several lncRNAs have already been shown to be involved in crucial cellular processes such as imprinting, regulation of cell proliferation and cellular differentiation. Their mode of action generally involves epigenetic modifications as well as transcriptional and post-transcriptional regulation. Within the ICGC (International Cancer Genome Consortium)-MMML-Seq (Molecular Mechanisms in Malignant Lymphoma by Sequencing) Consortium, which aims at fully characterizing a total of 250 germinal center derived B-cell lymphomas, including Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL), we conducted a search for yet unidentified genes. We here report the identification of a novel transcript, which we have named BLUT (Burkitt Lymphoma Unknown Transcript). BLUT is highly expressed in BL, with only very low expression levels in FL and no detectable expression in DLBCL. It is around 1050 nucleotides long with splice evidence for three exons, whereby exons 1 and 3 are shared between all isoforms. BLUT represents a putative lncRNA since it lacks conserved protein domains and has a splice site conservation pattern typical of non-coding transcripts. Furthermore, it is predicted as non-coding by the software Coding Potential Calculator and there was not a single peptide match between a published peptide database (Human Proteome Map) and any potential BLUT peptide (all isoforms). Moreover, BLUT shares a high sequence identity with two (so far) uncharacterized non-coding RNAs (Pan Troglodyte, Chlorocebus sabeus). We tested a cell line panel (consisting of 16 different cell lines including non-lymphoma cell lines) for BLUT expression but could only detect it in the two BL cell lines Namalwa and Raji. Cellular fractionation experiments showed that BLUT is predominantly localized in the nucleus in both Namalwa and Raji. Overexpression of BLUT in SU-DHL-4 resulted in the identification of 222 differentially regulated genes (FDR 〈 0.05) with a logFC of ± 0.25, among which 49 genes had a logFC of ± 0.50. Several lymphoma relevant genes were part of this list including BIN1, CCL22, CCR7, EBI3, ID2, JAG1, NQO1, and RGS1. Given its nuclear localization, we currently perform chromatin isolation by RNA purification experiments to identify the genomic regions which BLUT binds to. In combination with our overexpression data this will allow further insights into the functions of BLUT. In summary, we have identified a novel long-noncoding RNA termed BLUT, which is differentially expressed between lymphoma subtypes with a high expression in Burkitt lymphoma. Overexpression of BLUT resulted in the deregulation of several genes with known roles in lymphomagenesis. Given its predominantly nuclear localization, we are currently investigating its functions on chromatin regulation. (Supported by BMBF through 01KU1002A-J and by the Duesseldorf School of Oncology (funded by the Comprehensive Cancer Center Duesseldorf/Deutsche Krebshilfe and the Medical Faculty HHU Düsseldorf)) Disclosures Truemper: Amgen, roche, Mundipharma: Research Funding; Sandoz, Celgene, AMGEN, Nordic Nanovector: Other: Advisory board.

Abstract: Introduction: Chronic myeloproliferative neoplasms (CMN) are characterized by expansion of myeloid cells, splenomegaly and thrombosis. Recent studies have demonstrated that chronic inflammation promotes the disease development as mutated hematopoietic cells are stimulated by pro-inflammatory cytokines. The most common driver mutation in BCR-ABL negative CMN is the activating point mutation JAK2-V617F. The constitutively activated JAK2 signaling leads to strongly elevated TNF levels in polycythemia vera, essential thrombocythemia and primary myelofibrosis (Fleischman et al, Blood, 2011). Recently, it has been reported that defective negative regulation of Toll-like receptor signals leads to excessive TNF in monocytes from CMN. Interestingly, this is not directly driven by JAK2-V617F in a cell-intrinsic manner (Lai et al., Blood, 2019). Thus, blockade of TNF signaling may represent a valuable anti-inflammatory pharmacological target in CMN. Therefore, in the Vav1-Cre x JAK2+/V617F (JAK2VF+) mouse model, we investigated the effects of TNFR1 or TNFR2 blockade using neutralizing antibodies (Ab) and we applied genetic disruption of the TNFRs. Methods: To study the genetic disruption of TNFR1 or TNFR2, TNFR1-/- or TNFR2-/- mice were crossed with the JAK2VF+ mouse model. For αTNFR1 Ab study, JAK2VF+ were crossed with a mouse (huTNFR1) expressing a chimeric TNFR1 consisting of the human extracellular domain and the murine transmembrane and intracellular domains, allowing treatment with an anti-human TNFR1 Ab. Over 3 weeks, JAK2VF+ mice received i.p. 3 x per week αTNFR1 (H398; 20 mg/kg) or 2 x per week αTNFR2 (TR75-54.7; 5 mg/kg) Abs or specific IgG controls. Blood was collected weekly for blood count measurements. After treatment, cell composition of blood, bone marrow and spleen was analyzed by flow cytometry. Serum cytokines were measured using a bead-based assay. HSCs and progenitor cells (MPP) were isolated from untreated huTNFR1 x JAK2VF+ mice and plated in MethoCult ± αTNFR1 or αTNFR2 (10µg/l). Colonies were analyzed after 7 days. Results: Blood count and spleen size of TNFR1-/- or TNFR2-/- x JAK2VF+ mice showed no differences as compared to TNFRWT x JAK2VF+ mice. In addition, the immune cell composition was similar to TNFRWT x JAK2VF+ mice. These results indicate that the disruption of a single cytokine pathway has no influence in this model, as other cytokines likely will compensate the signal during initiation of the disease.In contrary, αTNFR1 Ab treatment (n = 6) reduced the mean HCT after 3 weeks from 72.5 to 60.0%, which was not observed in the control group (n = 5). This was partly a result of a reduced MCV in αTNFR1 treated mice (34.9 versus 30.9 fl) as there was no reduction of the RBC. WBC number increased upon αTNFR1 treatment from 14.3 to 17.9 x 109 cells/l. Remarkably, αTNFR1 Ab treatment resulted in a major decrease of total cytokines as TNF, IL-1β, IL-10 and others when compared to the control group. Thus, αTNFR1 treatment is able to reduce chronic inflammation in the JAK2VF+ mouse but has minor impact on the HCT and WBC. Similar experiments were performed using αTNFR2 Ab (n = 4). This treatment showed only a minimal change on the complete blood counts of αTNFR2 treated mice in comparison to the IgG treated mice (n = 4). Thus, the HCT of αTNFR2 treated mice declined from 79.3 to 71.3 %, MCV and RBC remained stable during treatment phase. Of note, αTNFR2 treatment had no major impact on serum cytokine levels. Further, no differences in spleen size or immune cell composition were detected. In total, αTNFR2 therapy failed to reduce inflammation in the JAK2VF+model. To test whether αTNFR1/2 Ab treatment would impact on the stem cell compartment, a clonogenic assay was performed employing HSCs and MPPs ± αTNFR1 or αTNFR2. Almost no (αTNFR1) or minor (αTNFR2) reduction in colony number was measured; indicating that blockade of TNFRs has minor impact on the stem cell compartment in the JAK2VF+ mouse. Conclusions: Our study employing the JAK2VF+ mouse model revealed an involvement of TNF-TNFR1 in induction of chronic inflammation rather than TNF-TNFR2. αTNFR1 treatment strongly reduces cytokine levels but only slightly reduces HCT. Therefore targeting TNFR1 in CMN may have beneficial effects on chronic inflammation. In our view, a clinical trial investigating combination therapy with JAK1/2 inhibitors such as Ruxolitinib in patients with persisting symptoms of chronic inflammation is warranted. Disclosures Richter: Baliopharm GmbH: Other: F.R. is named inventor on patent applications covering Fc heterodimerization modules and monovalent inhibitors of TNFR1 interaction.. Pfizenmaier:Baliopharm GmbH: Other: K.P. is named inventor on patent applications covering Fc heterodimerization modules and monovalent inhibitors of TNFR1 interaction..