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Systemic steroids are the standard first-line treatment for acute graft-vs-host disease (aGVHD), but approximately 50% of patients become steroid refractory or dependent (SR/D). Ruxolitinib is the only FDA and EMA approved therapy for patients with SR/D aGVHD. In the phase 3 REACH2 trial (NCT02913261), ruxolitinib demonstrated superior efficacy to best available therapy (BAT) in treating SR/D aGVHD, with significantly higher overall response rate (ORR) at day 28, durable ORR at day 56, and longer median overall survival. Identifying biomarkers and clinical characteristics associated with increased probability of response could guide treatment decisions. In this exploratory analysis of the REACH2 study, we developed baseline (pretreatment) and Day 14 models to identify patient characteristics and biomarkers (12 aGVHD-associated cytokines/chemokines, 6 immune cell types, and 3 inflammatory proteins) before and during treatment that affected the probability of response at day 28. Treatment with ruxolitinib versus BAT, type of conditioning, skin involvement, and age were strongly associated with increased likelihood of response in ≥1 model. Lower levels of most aGVHD and immune cell markers at baseline were associated with increased probability of response. In the Day 14 model, levels of aGVHD markers at day 14, rather than change from baseline, impacted probability of response. For both models, bias-corrected area under receiver operating characteristic (AUROC) values (baseline, 0.73; Day 14, 0.80) indicated high level of correspondence between fitted and actual outcomes. This first biomarker study in context of a randomized phase 3 aGVHD trial, highlights the potential prognostic value of select biomarkers and patient characteristics.

Peripheral T-cell lymphomas (PTCL) - especially angioimmunoblastic (AITL) and follicular TCL - have a dismal prognosis due to lack of efficient therapies, and patients` symptoms are often dominated by an inflammatory phenotype including fever, night sweats, weight loss and skin rash. In this study, we investigated the role of inflammatory granulocytes and activated cytokine signaling on PTCL-TFH (T-follicular helper type) disease progression and symptoms. We show, that ITK-SYK driven murine PTCLs and primary human PTCL-TFH xenografts both induce inflammation in mice including murine neutrophil expansion and massive cytokine release. Granulocyte/lymphoma interactions were mediated by positive autoregulatory cytokine loops involving INF-γ (CD4+malignant T-cells) and IL-6 (activated granulocytes), ultimately inducing broad JAK kinase activation (Jak1/2/3, Tyk2) in both cell types. Depletion of inflammatory granulocytes via antibodies (Ly6G), genetic granulocyte depletion (LyzM-Cre/MCL1flox/flox) or the deletion of IL-6 within microenvironmental cells blocked inflammatory symptoms, reduced lymphoma infiltration and enhanced mouse survival. Furthermore, unselective JAK kinase inhibitors (ruxolitinib) inhibited both, TCL progression and granulocyte activation in various PTCL mouse models. Our results support the important role of granulocyte-driven inflammation, cytokine-induced granulocyte/CD4+ TCL interactions and the requirement of an intact JAK/STAT signaling pathway for PTCL-TFH development, and support broad JAK kinase inhibition as an effective treatment strategy in early disease stages.

Acute graft-versus-host disease (GVHD) is a major life-threatening complication after allogeneic hematopoietic cell transplantation (allo-HCT). The classical target organs of acute GVHD include the intestines, liver, and skin. The damage of these organs is relatively easy to detect for the clinician as diarrhea, increased bilirubin, and rash. However, there is increasing evidence that other organs, where the acute damage is less apparent or more difficult to distinguish from drug toxicity, such as the central nervous system, lungs, ovaries and testis, thymus, bone marrow, and kidney, can be target organs of acute GVHD. Here, we review current evidence for nonclassical manifestations of acute GVHD in rodent models and in patients and discuss them in the context of novel emerging therapies for GVHD. A better understanding of the involvement of nonclassical GVHD target organs may help to improve patient outcomes after allo-HCT.

High-dose chemotherapy, followed by autologous hematopoietic stem cell transplantation (auto-HSCT), is an established therapy for patients with hematological malignancies. The age of patients undergoing auto-HSCT and, therefore, the comorbidities, has increased over the last decades. However, the assessment of organ dysfunction prior to auto-HSCT has not been well studied. Therefore, we retrospectively analyzed the association of clinical factors and lung and cardiac function with outcome and complications after conditioning with BEAM (BCNU/carmustine, etoposide, cytarabine, melphalan) or high-dose melphalan in patients undergoing auto-HSCT. This study included 629 patients treated at our institution between 2007 and 2017; 334 and 295 were conditioned with BEAM or high-dose melphalan, respectively. The median follow-up was 52 months (range, 0.2-152) and 50 months (range, 0.5-149), respectively. In the multivariate analysis, we identified that progressive disease, CO-diffusion capacity corrected for hemoglobin (DLCOcSB) ≤ 60% of predicted, Karnofsky Performance Status (KPS) ≤ 80%, Hematopoietic Cell Transplantation Comorbidity Index (HCT-CI) score ≥ 4, and age > 70 years were associated with decreased overall survival (OS) in patients treated with BEAM. Similarly, DLCOcSB ≤ 60% of predicted, HCT-CI score ≥ 4, and age > 60 years were identified in patients treated with high-dose melphalan. Abnormalities in DLCOcSB ≤ 60% of predicted were associated with chemotherapy with lung-toxic substances, mediastinal radiotherapy, KPS ≤ 80%, current/previous smoking, and treatment in the intensive care unit. More often, patients with DLCOcSB ≤ 60% of predicted experienced nonrelapse mortality, including pulmonary causes of death. In summary, we identified DLCOcSB ≤ 60% of predicted as an independent risk factor for decreased OS in patients conditioned with BEAM or high-dose melphalan prior to auto-HSCT.

The nuclear receptor (NR) subclass, retinoid X receptors (RXRs), exert immunomodulatory functions that control inflammation and metabolism via homodimers and heterodimers, with several other NRs, including retinoic acid receptors. IRX4204 is a novel, highly specific RXR agonist in clinical trials that potently and selectively activates RXR homodimers, but not heterodimers. In this study, in vivo IRX4204 compared favorably with FK506 in abrogating acute graft-versus-host disease (GVHD), which was associated with inhibiting allogeneic donor T-cell proliferation, reducing T-helper 1 differentiation, and promoting regulatory T-cell (Treg) generation. Recipient IRX4204 treatment reduced intestinal injury and decreased IFN-γ and TNF-α serum levels. Transcriptional analysis of donor T cells isolated from intestines of GVHD mice treated with IRX4204 revealed significant decreases in transcripts regulating proinflammatory pathways. In vitro, inducible Treg differentiation from naive CD4 T cells was enhanced by IRX4204. In vivo, IRX4204 increased the conversion of donor Foxp3 T cells into peripheral Foxp3 Tregs in GVHD mice. Using Foxp3 lineage-tracer mice in which both the origin and current FoxP3 expression of Tregs can be tracked, we demonstrated that IRX4204 supports Treg stability. Despite favoring Tregs and reducing Th1 differentiation, IRX4204-treated recipients maintained graft-versus-leukemia responses against both leukemia and lymphoma cells. Notably, IRX4204 reduced in vitro human T-cell proliferation and enhanced Treg generation in mixed lymphocyte reaction cultures. Collectively, these beneficial effects indicate that targeting RXRs with IRX4204 could be a novel approach to preventing acute GVHD in the clinic. Key Points: • A novel RXR agonist attenuates acute GVHD, while retaining graft-versus-leukemia responses. • The RXR agonist enhances Treg generation and stabilizes Foxp3, even in a highly inflammatory environment.

Edited by Associate Editor Robert Zeiser, this How I Treat series focuses on post–allogeneic transplant management of high-risk patients. These patients are at risk for relapse of malignancy and severe graft-versus-host disease (GVHD). Two articles in the series discuss adoptive cellular immunotherapy and maintenance therapy to preempt relapse of acute myeloid leukemia. The third article addresses the state of the art for preventing GVHD in this subset of stem cell transplant recipients.

BACKGROUND Standard first-line treatment of cGVHD includes systemic corticosteroids; however, about 50% of patients (pts) are steroid refractory or dependent (SR/D) and require additional treatment. The best second-line therapy option has not yet been defined. RUX, an oral JAK1/2 inhibitor, was superior to BAT in SR acute GVHD (aGVHD) in a phase 3 study (REACH2). Here we present the primary analysis of the REACH3 study (NCT03112603), a phase 3, open-label, randomized study evaluating RUX vs BAT in pts with SR/D cGVHD. METHODS Eligible pts were ≥ 12 years old, had received allogeneic hematopoietic cell transplant, and had developed moderate or severe SR/D cGVHD. Pts transitioning from aGVHD to cGVHD without tapering steroids were excluded. Pts treated with JAK inhibitors for aGVHD were allowed if they achieved complete response (CR) or partial response (PR) and had been off JAK inhibitor treatment for ≥ 8 weeks prior to cycle 1 day 1. Those treated with ≥ 2 prior lines of systemic therapy for cGVHD in addition to corticosteroids ± calcineurin inhibitors (CNI) were ineligible. Pts were randomized (1:1) to RUX 10 mg bid or investigator-selected BAT (10 options) and were treated for 6 cycles (cycle = 28 days). Pts continued receiving their regimen of corticosteroids ± CNI. Viral prophylaxis and antibiotics were allowed as needed for infection prevention and treatment. Addition or initiation of a new BAT was allowed only after lack of response, intolerable toxicity, or cGVHD flare and was considered treatment failure. Crossover from BAT to RUX was allowed on or after cycle 7 day 1 (C7D1) in pts who did not achieve or maintain CR/PR, developed toxicity to BAT, or had a cGVHD flare. The primary endpoint was overall response rate (ORR) at C7D1. ORR was defined as the proportion of pts achieving CR or PR, per NIH consensus criteria. Key secondary endpoints were failure-free survival (FFS; defined as time to the earliest of recurrence of underlying disease, start of new systemic treatment for cGVHD, or death) and improvement in symptoms based on change in the modified Lee symptom score (mLSS; 0 [no symptoms] to 100 [worst symptoms]) at C7D1. An mLSS responder was defined as having achieved a ≥ 7-point reduction from baseline in the total symptom score. The primary and key secondary endpoints were α-controlled via an overall hierarchical testing procedure. RESULTS A total of 329 pts were randomized to RUX (n = 165) or BAT (n = 164). Baseline characteristics were balanced in the 2 arms; 61% were male, and median age was 49 years (range, 12-76 years). Twelve pts were < 18 years old. Overall, 48% and 52% of pts had moderate and severe cGVHD, respectively. At data cutoff (May 8, 2020), 125 pts (38%) were on randomized treatment (RUX, 50%; BAT, 26%). Eighty-two (50%) and 122 (74%) pts discontinued RUX and BAT, respectively; 61 (37%) had crossed over to RUX. Reasons for discontinuation included lack of efficacy (15% RUX vs 43% BAT), adverse events (AEs; 17% vs 5%), and relapse (5% vs 4%). The study met its primary endpoint with the efficacy boundary crossed at interim analysis (ORR, P = 0.0003). At C7D1, ORR was significantly higher in the RUX arm vs BAT (50% vs 26%; odds ratio, 2.99; P < 0.0001a); the CR rate was higher with RUX (7% vs 3%; Table). Both key secondary endpoints showed superiority of RUX vs BAT. FFS was significantly longer for RUX-treated pts (median FFS, not reached vs 5.7 months; HR, 0.370 [95% CI, 0.268-0.510]; P < 0.0001; Figure), and the mLSS responder rate was higher (24% vs 10%; odds ratio, 3.00; P = 0.0003). Overall, 31 RUX (19%) and 27 BAT (16%) pts died; the main cause of death was cGVHD (RUX, n = 22 [13%]; BAT, n = 13 [8%], including 2 deaths after crossover to RUX). Rates of AEs up to C7D1 were comparable in the 2 arms (RUX, 98% [grade ≥ 3, 57%]; BAT, 92% [grade ≥ 3, 58%]. The most common AEs (≥ 15%) in the RUX vs BAT arms were anemia (29% vs 13%), hypertension (16% vs 13%), pyrexia (16% vs 9%), and ALT increase (15% vs 4%). Infections of any type occurred in 64% of RUX and 56% of BAT pts (19% vs 18% grade 3, grading based on Cordonnier et al 2006), and included fungal (12% vs 6%), viral (34% vs 29%), and bacterial (28% vs 26%) infections. CONCLUSIONS This is the first successful randomized phase 3 trial of RUX in pts with SR/D cGVHD. RUX demonstrated superior efficacy vs BAT, measured by a higher ORR, longer FFS, and greater symptom improvement. RUX was effective for moderate or severe SR/D cGVHD and its safety profile is consistent with that expected for this drug and this population. Disclosures Zeiser: Malinckrodt: Honoraria; Novartis: Honoraria; Incyte: Honoraria. Polverelli:Novartis: Consultancy. Chakraverty:Mallinckrodt Pharmaceuticals (Therakos (UK) Ltd): Honoraria; Neovii: Honoraria. Flowers:Novartis: Current Employment. Dawkins:Incyte: Current Employment. Hollaender:Novartis Pharma AG: Current Employment. Gowda:Novartis Pharmaceuticals Corporation: Current Employment. Stefanelli:Novartis AG: Current Employment, Current equity holder in publicly-traded company. Lee:Takeda: Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Research Funding; Amgen: Research Funding; Kadmon: Research Funding; Incyte: Consultancy, Research Funding; Syndax: Research Funding; AstraZeneca: Research Funding. Teshima:Sharp & Dohme Corp: Consultancy, Honoraria; Astellas Pharma Inc.: Research Funding; Bristol-Myers Squibb: Honoraria; Japan Society for the Promotion of Science KAKENHI (17H04206): Other; The Center of Innovation Program from Japan Science and Technology Agency: Other; Kyowa Kirin Co., Ltd.: Honoraria, Research Funding; Merck: Consultancy, Honoraria; Janssen Pharmaceutical K.K.: Other; NIPPON SHINYAKU CO., LTD.: Honoraria; Fuji Pharma Co., Ltd.: Honoraria; TEIJIN PHARMA LIMITED: Honoraria; Sanofi K.K.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Novartis Pharma K.K.: Consultancy, Other: Manuscript preparation, Research Funding; Pfizer Japan Inc.: Honoraria; Takeda Pharmaceutical Company Limited: Consultancy, Honoraria. Locatelli:Jazz Pharmaceeutical: Speakers Bureau; Medac: Speakers Bureau; Miltenyi: Speakers Bureau; Bellicum Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. OffLabel Disclosure: Ruxolitinib for the treatment of chronic GVHD.

Allogeneic hematopoietic cell transplantation is a potentially curative treatment of different hematological malignancies. A major life-threatening complication is acute graft-versus-host disease (GVHD), in particular when the disease becomes steroid refractory. Based on the detection of pathogenic cytokines, chemokines, and T-cell subsets in individuals developing GVHD or experimental GVHD models, different therapeutic strategies have been developed. A potential cause why targeting individual receptors can lack efficacy could be that multiple cytokines, danger signals, and chemokine that have redundant functions are released during GVHD. To overcome this redundancy, novel strategies that do not target individual surface molecules like chemokine receptors, integrins, and cytokine receptors, but instead inhibit signaling pathways downstream of these molecules, have been tested in preclinical GVHD models and are currently being tested in clinical GVHD trials. Another important development is tissue regenerative approaches that promote healing of GVHD-related tissue damage as well as strategies that rely on microbiota modifications. These approaches are promising because they act very differently from conventional immunosuppression, instead aiming at reinstalling tissue homeostasis and microbiome diversity. This review discusses major novel developments in GVHD therapy that are based on a better understanding of GVHD biology, the repurposing of novel kinase inhibitors, microbiome modification strategies, and tissue-regenerative approaches.

Conditioning-induced damage of the intestinal tract plays a critical role during the onset of acute graft-versus-host disease (GVHD). Therapeutic interference with these early events of GVHD is difficult, and currently used immunosuppressive drugs mainly target donor T cells. However, not donor T cells but neutrophils reach the sites of tissue injury first, and therefore could be a potential target for GVHD prevention. A detailed analysis of neutrophil fate during acute GVHD and the effect on T cells is difficult because of the short lifespan of this cell type. By using a novel photoconverter reporter system, we show that neutrophils that had been photoconverted in the ileum postconditioning later migrated to mesenteric lymph nodes (mLN). This neutrophil migration was dependent on the intestinal microflora. In the mLN, neutrophils colocalized with T cells and presented antigen on major histocompatibility complex (MHC)-II, thereby affecting T cell expansion. Pharmacological JAK1/JAK2 inhibition reduced neutrophil influx into the mLN and MHC-II expression, thereby interfering with an early event in acute GVHD pathogenesis. In agreement with this finding, neutrophil depletion reduced acute GVHD. We conclude that neutrophils are attracted to the ileum, where the intestinal barrier is disrupted, and then migrate to the mLN, where they participate in alloantigen presentation. JAK1/JAK2-inhibition can interfere with this process, which provides a potential therapeutic strategy to prevent early events of tissue damage-related innate immune cell activation and, ultimately, GVHD.

BACKGROUND Approximately 50% of patients (pts) who receive initial cGVHD therapy are steroid refractory or dependent (SR/D); standard second-line therapy has yet to be established. Quality of life (QOL) in these pts is poor and worsens with disease severity. In the phase 3 REACH3 study in SR/D cGVHD (NCT03112603), RUX demonstrated superior efficacy compared with BAT, with a higher overall response rate at wk 24 (primary endpoint), longer failure-free survival, and a higher proportion of pts with a significant improvement in symptoms on the cGVHD-specific modified Lee Symptom Scale (mLSS; 24.2% vs 11.0% at wk 24; P=0.001) (Zeiser NEJM 2021). Given the significant impact of symptoms on QOL, the collection of PROs is recommended by NIH consensus criteria, and assessing QOL via PRO measures specifically designed to capture cGVHD symptoms (ie, the mLSS) is important in evaluating treatment. We present an analysis of PROs in REACH3 and compare mLSS subscale results with objective organ responses (data cutoff: May 8, 2020). METHODS Pts ≥12 years old with moderate or severe SR/D cGVHD were randomized (1:1) to RUX 10 mg twice daily or investigator-selected BAT (prespecified from 10 options). Randomized treatment was administered for ≥6 cycles (28 d/cycle) along with corticosteroids ± a calcineurin inhibitor. Addition or initiation of a new systemic agent for cGVHD was considered treatment failure. PROs were collected at baseline and at 4-wk intervals through wk 24 or until treatment failure or discontinuation from the main study period. The rate of responders per improvement of ≥7 points from baseline in mLSS summary score (0 [no symptoms] to 100 [worst symptoms]) at wk 24 was a key secondary endpoint (alpha controlled). Additional endpoints were individual organ response and changes in other PROs (Functional Assessment of Cancer Therapy-Bone Marrow Transplantation [FACT-BMT], EQ-5D-5L, Patient Global Impression of Severity [PGIS], Patient Global Impression of Change [PGIC]). mLSS data at wk 24 were available for 55.8% of RUX pts (92/165) and 53.0% of BAT pts (87/164) (see Table for pts evaluable for other PROs) . RESULTS A total of 329 pts received RUX (n=165) or BAT (n=164). Baseline characteristics, including symptom burden, were balanced between arms; 48% and 52% of pts had moderate or severe cGVHD, respectively. The baseline median summary mLSS score was 18.7 (RUX) vs 18.5 (BAT), indicating a moderate symptom burden. More pts receiving RUX had an mLSS response at wk 24, at any time up to wk 24, and for ≥2 consecutive visits than those receiving BAT (Table). RUX was associated with a rapid and continued reduction in mean summary mLSS symptom score, whereas only an initial reduction at wk 4 was seen with BAT (Figure 1). In subset analysis, mLSS response was similar in RUX pts with moderate and severe cGVHD. Among pts achieving a complete or partial cGVHD response, those treated with RUX were more likely to have an mLSS response (RUX, 40.2%; BAT, 28.6%). Response rates in mLSS subscales were higher with RUX vs BAT (odds ratio, >1.7; P More pts receiving RUX than BAT reported "no symptoms" (28.6% vs 17.3%) according to PGIS and were "very much better" or "moderately better" (63.1% vs 39.5%) based on PGIC. RUX treatment also had a positive effect on non-cGVHD-specific PRO measures (EQ-5D-5L and FACT-BMT) at wk 24. CONCLUSIONS Compared with BAT, RUX resulted in more substantial improvement in PROs, with rapid, continued symptom improvement and benefits across mLSS subscales, indicating that RUX is superior to BAT not only in physician-assessed responses but also in terms of treating patients' cGVHD symptoms. EQ-5D-5L and FACT-BMT scores were numerically higher with RUX than BAT, but the smaller changes suggest that these measures are too generic to fully capture the impact of cGVHD on QOL while confirming that multidimensional QOL was not reduced with RUX or BAT. Importantly, the pt experience of organ-specific symptom improvements was consistent with physician-assessed objective organ responses and both were greater with RUX. Figure 1 Figure 1. Disclosures Lee: Amgen: Research Funding; AstraZeneca: Research Funding; Incyte: Other: Membership on Steering Committee, Research Funding; Kadmon: Research Funding; Novartis: Research Funding; Pfizer: Consultancy, Research Funding; Syndax: Research Funding; Takeda: Research Funding; 4SC: Consultancy; EMD Serono: Consultancy, Research Funding; Genzyme: Consultancy; Merck Sharpe Dohme: Consultancy; Regeneron: Consultancy; Sanofi: Consultancy; BMS: Research Funding; Wolters Kluwer: Research Funding. Locatelli: Miltenyi: Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Medac: Speakers Bureau; Jazz Pharamceutical: Speakers Bureau; Takeda: Speakers Bureau. Ayuk: Gilead: Honoraria; Mallinckrodt/Therakos: Honoraria, Research Funding; Takeda: Honoraria; Janssen: Honoraria; Celgene/BMS: Honoraria; Miltenyi Biomedicine: Honoraria; Novartis: Honoraria. Zuckerman: Gilead Sciences: Honoraria, Speakers Bureau; Novartis: Honoraria; AbbVie: Honoraria; BioSight Ltd: Honoraria; Orgenesis Inc.: Honoraria; Janssen: Honoraria; Cellect Biotechnology: Honoraria. Pidala: Pharmacyclics: Other: Clinical trial support, Research Funding; BMS: Other: Clinical trial support, Research Funding; Novartis: Other: Clinical trail support; Takeda: Other: Clinical trail support; Jannssen: Other: Clinical trial support; Johnson and Johnson: Other; AbbVie: Other; BMS: Other; Incyte: Consultancy; Regeneron: Consultancy; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Clinical trial support; CTI Biopharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Clinical trial support; Syndax: Consultancy, Membership on an entity's Board of Directors or advisory committees. Hamad: Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Zeiser: Incyte, Mallinckrodt, Novartis: Honoraria, Speakers Bureau. Bhatt: Incyte: Current Employment. Gowda: Novartis: Current Employment. Han: Novartis: Current Employment. Stefanelli: Novartis: Current Employment, Current equity holder in publicly-traded company. Zuurman: Novartis: Current Employment. Teshima: Pfizer Inc.: Honoraria; Astellas Pharma Inc.: Research Funding; TEIJIN PHARMA Limited: Research Funding; Merck Sharp & Dohme: Membership on an entity's Board of Directors or advisory committees; Nippon Shinyaku Co., Ltd.: Research Funding; Bristol Myers Squibb: Honoraria; Gentium/Jazz Pharmaceuticals: Consultancy; Takeda Pharmaceutical Company: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis International AG: Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; Kyowa Kirin Co.,Ltd.: Honoraria, Research Funding; Janssen Pharmaceutical K.K.: Other; Fuji pharma CO.,Ltd: Research Funding; Sanofi S.A.: Research Funding.

BACKGROUND aGVHD, a common complication of allogeneic stem cell transplant (alloSCT), is driven by proinflammatory cytokines and chemokines that activate the immune system, resulting in end-organ damage. Steroids are first-line treatment but up to 50% of pts are steroid refractory (SR), resulting in high mortality and morbidity. RUX, a JAK1/JAK2 inhibitor, inhibits cytokine-dependent activation of the JAK-STAT pathway and cell proliferation and differentiation, which prevents worsening of aGVHD and allows recovery. JAK pathway inhibition by RUX may also lead to modulation of proinflammatory cytokines and prognostic GVHD biomarkers. The phase 3 randomized REACH2 trial (NCT02913261) in SR aGVHD demonstrated superiority of RUX vs BAT, with a significantly higher overall response rate (ORR; complete [CR] + partial response [PR]) at D28 (62% vs 39%; P < .001) and higher durable ORR at D56 (40% vs 22%; P < .001) (Zeiser R, et al. N Engl J Med. 2020). In this exploratory analysis of REACH2, we assessed whether baseline (BL) levels of proinflammatory cytokines and GVHD markers were prognostic for response and how this changed over time related to treatment. METHODS Pts (N = 309; ≥ 12 y old with grade II-IV SR aGVHD after alloSCT) were randomized 1:1 to RUX (starting at 10 mg bid) or investigator-selected BAT and stratified by aGVHD grade; 295 pts had valid biomarker levels. Serum samples were collected at BL, D14, and D28 and biomarkers, including inflammatory cytokines, soluble receptors of cytokines, chemokines, and tissue-specific markers for gastrointestinal (GI), liver, and skin GVHD, were assessed (Table). Biomarker levels at BL were stratified by response (CR, PR, none [NR]) at D28 for each treatment arm; those that differed by response were analyzed by logistic regression (LR) to assess the association (CR+PR vs NR), adjusting for treatment. The analysis was repeated, adjusting for key covariates that had significant impact on the biomarker-response relationship. Change in biomarkers over time (BL to D28) was assessed via geometric mean values at each visit, along with fold change from BL for each treatment arm. RESULTS Of 22 GVHD biomarkers assessed, 17 (77%) were evaluable (Table). Higher median BL levels of proinflammatory cytokines (IL-6, IL-8, TNF-α), soluble cytokine receptors (IL2RA, TNFRSF1A, ST2) and tissue-specific GVHD markers (REG3A, HGF) were generally observed in NR vs CR pts (Table) and were further analyzed. Higher BL levels of these markers were significantly associated with a lower probability of OR (P[OR]) in the LR analysis (Figure). An interaction term was investigated to assess whether there was a differing biomarker effect within treatment groups; however, this was insignificant and not retained. The addition of skin involvement to the LR for all biomarkers had an impact on P(OR), with increased P(OR) for pts with skin involvement at BL. Liver involvement at BL was added to the LR for HGF, IL6, REG3A, TNFα, and TNFRSF1A. The LR models showed a significant decrease in P(OR) for pts with liver involvement at BL. GI involvement did not have a significant impact for any biomarker. Geometric mean levels of TNFα, TNFRSF1A, IL2RA, REG3A, and ST2 decreased from BL to D14 in CR pts in the RUX arm but increased with BAT. NR pts had increases in these biomarkers regardless of treatment; however, longer follow-up is needed. Treatment had an underlying impact on P(OR) but no further impact on the effect of biomarkers on P(OR). Maximum change from BL was observed at D14; a slight return to BL levels was observed at D28 in CR pts. Surprisingly, IL6 levels increased with RUX regardless of response. IL6, like TNFα, is directly modulated by the JAK-STAT pathway and was therefore expected to decrease with RUX. CONCLUSIONS This is the first biomarker study in a phase 3 trial in GVHD. BL levels of IL6, IL8, TNFα, IL2RA, TNFRSF1A, ST2, REG3A, and HGF tended to be higher in NR vs CR pts; higher levels were associated with lower P(OR) when adjusted for treatment arm. Skin and liver involvement also had an impact on response. Skin involvement was associated with higher P(OR), regardless of biomarker BL levels, possibly because no skin involvement implied other organ involvement and tended to be associated with a higher overall severity grade at BL. CR pts in the RUX group had lower BL levels of TNFα and showed trends toward decreases from BL to D14 in the soluble cytokine receptors ST2, TNFRSF1A, and IL2RA and tissue-specific marker REG3A. Disclosures Socié: Elsalys: Honoraria; Alexion: Consultancy, Honoraria, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte: Speakers Bureau. Niederwieser:Amgen: Speakers Bureau; Daiichi: Research Funding; Novartis: Speakers Bureau; Cellectis: Membership on an entity's Board of Directors or advisory committees. Von Bubnoff:Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Clinical biomarker research, steering committee, Patents & Royalties: Research support, Research Funding; Astra Zeneca: Honoraria, Other: Lectures, Patents & Royalties: Astra Zeneca. Mohty:BMS: Consultancy, Honoraria, Research Funding, Speakers Bureau; Sanofi: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; GSK: Consultancy, Honoraria, Research Funding, Speakers Bureau; Stemline: Consultancy, Honoraria, Research Funding, Speakers Bureau; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Takeda: Consultancy, Honoraria, Research Funding, Speakers Bureau. Szer:Alexion Pharmaceuticals Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; Apellis: Consultancy; Pfizer: Honoraria, Speakers Bureau; Takeda: Honoraria, Speakers Bureau; Prevail Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees. Or:Hadassah Hebrew University Medical Center: Consultancy, Current Employment. Wagner:Novartis: Membership on an entity's Board of Directors or advisory committees; Kite/Gilead: Membership on an entity's Board of Directors or advisory committees; MSD: Membership on an entity's Board of Directors or advisory committees; Medac: Other: Travel grand; Shire: Other: Travel grand. Forcade:JAZZ: Other: Travel grant for congress; NEOVII: Other: Travel grant for congress; Gilead: Speakers Bureau; Sanofi: Other: Travel grant for congress; Novartis: Other: Travel grant for congress. Civriz Bozdag:Novartis: Research Funding. Chaturvedi:Novartis: Current Employment. Wilke:Novartis Pharma AG: Current Employment, Other: Stock owner. Zeiser:Novartis: Honoraria; Incyte: Honoraria; Malinckrodt: Honoraria.

Relapse after allogeneic hematopoietic stem cell transplantation (HSCT) remains an unmet medical need. The ETCTN 9204 study evaluated in 71 study subjects if immune checkpoint blockade with anti-CTLA-4 (Ipilimumab (Ipi), n = 43) or anti-PD-1 (Nivolumab (Nivo), n = 28) antibodies could stimulate graft-versus-leukemia (GvL) responses in this setting. We focused mainly on patients (pts) with relapsed AML/MDS, which constituted the majority of pts (n = 38; 54%). Ipi induced both long-term complete remissions (CR; n = 3) and transient CRs (TR; n = 3), while Nivo did not generate any CRs, but 4 patients demonstrated partial remissions (PR). To define the molecular features associated with response to Ipi, we performed bulk transcriptomic analyses of formalin-fixed paraffin-embedded biopsies of sites of AML/MDS involvement (n = 35) collected before and after Ipi treatment from 13 pts. Our analysis of matched pre- and post-Ipi samples of patients with CRs identified 50 differentially expressed genes. By gene ontology analysis, these were enriched for signatures of 'lymphocyte activation' and 'antigen-receptor signaling'. Principal component analysis using these genes separated post-Ipi CR samples as a distinct cluster, transcriptionally apart from pre-Ipi CR samples and from non-responder (NR, n = 15) pre and post-Ipi samples. Of note, post-Ipi CR samples were similar to both pre- and post-Ipi TR samples as well as samples from GvHD/toxicity biopsies (n = 9). Consistent with these findings, we detected increased CD8+ T cell abundance post-Ipi in CR but not NR samples with CIBERSORTx (pre vs post, median score 0.043 vs. 0.56, p < 0.01). Likewise, reconstruction of T cell receptor (TCR) CDR3 sequences using the tool TRUST showed an increase in TCR clonotypes per million reads post-Ipi in CR samples (pre vs post, median 0.33 vs. 1.65, p < 0.05). In sum, response to CTLA-4 blockade is characterized by transcriptional evidence of T cell infiltration and activation within the tumor microenvironment, with similar gene programs observed in the setting of GvHD. To determine if the changes within tissue sites following Ipi are also observed in peripheral blood (PB), we analyzed the immunophenotype and TCR repertoire of T cells from serially collected PB samples from pts with AML/MDS (n = 14) or non-myeloid malignancy (n = 6). In responders and non-responders, exposure to Ipi was associated with decreased naïve, increased effector memory, and increased expression of HLA-DR on central memory T cells (p < 0.05), consistent with T cell differentiation and activation. From 9 of 14 AML/MDS pts (CR = 4, NR = 5), bulk TCR sequencing before and after 1 cycle of Ipi yielded 572,017 PB TCR sequences. Only 776 clonotypes demonstrated significant change in frequency, and these TCRs were detected in greater proportion among responders (613/776 versus NR 163/776, p < 0.01). Thus, Ipi alters the differentiation states of circulating T cells irrespective of response and leads to higher TCR repertoire turnover in CR patients. Of the AML patients achieving PR following Nivo, we also observed transcriptional evidence of CD8+ T cell infiltration in one patient. This signature, however, was not detected in a second such patient. In a separate instance, we had the opportunity to dissect the molecular features of a partial response in a patient with JAK2V617F+ secondary AML following Nivo through single-cell RNA and ATAC-sequencing (scRNA-seq and scATAC-seq, 10x Genomics) of PB mononuclear cells collected serially across 6 timepoints. In total, we analyzed the transcriptomes and chromatin accessibility data from of 27,777 and 28,713 individual cells, respectively. At time of response, non-exhausted CD4+ T cells expanded while both exhausted CD8+ T cells and a malignant subpopulation with increased expression of PD-L1 and features of megakaryocytic differentiation preferentially contracted. The subsequent expansion of a PD-L1- malignant population at progression suggests that decreased leukemic PD-L1 expression was associated with relapse. Altogether, these data highlight the molecular and cellular features of successful reinvigoration of GvL using CTLA-4 blockade, from increased local T cell infiltration and activation in the leukemic microenvironment to peripheral T cell turnover. In addition, the selection of therapy-resistant subclones after PD-1 blockade underscores the need for further high-resolution studies of GvL responses. Disclosures Zeiser: Novartis: Honoraria; Incyte: Honoraria; Malinckrodt: Honoraria. Ritz:Rheos Medicines: Consultancy; LifeVault Bio: Consultancy; Infinity Pharmaceuticals: Consultancy; Falcon Therapeutics: Consultancy; Avrobio: Consultancy; Kite Pharma: Research Funding; Talaris Therapeutics: Consultancy; Equillium: Research Funding; Amgen: Research Funding; TScan Therapeutics: Consultancy. Neuberg:Madrigak Pharmaceuticals: Current equity holder in publicly-traded company; Celgene: Research Funding; Pharmacyclics: Research Funding. Soiffer:alexion: Consultancy; Gilead: Consultancy; Be the Match/ National Marrow Donor Program: Membership on an entity's Board of Directors or advisory committees; Rheos Therapeutics: Consultancy; Juno: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Mana Therapeutics: Consultancy; Precision Bioscience: Consultancy; VOR Biopharma: Consultancy; Kiadis: Membership on an entity's Board of Directors or advisory committees; Cugene: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees. Liu:GV20 Oncotherapy: Consultancy, Membership on an entity's Board of Directors or advisory committees; 3DMed Care: Consultancy; Sanofi: Research Funding; Takeda: Research Funding. Davids:AbbVie: Consultancy; Gilead Sciences: Consultancy; Sunesis: Consultancy; BeiGene: Consultancy; AstraZeneca: Consultancy, Research Funding; Syros Pharmaceuticals: Consultancy; Research to Practice: Honoraria; Pharmacyclics: Consultancy, Research Funding; TG Therapeutics: Consultancy, Research Funding; Verastem: Consultancy, Research Funding; Adaptive Biotechnologies: Consultancy; Janssen: Consultancy; Surface Oncology: Research Funding; Novartis: Consultancy, Research Funding; MEI Pharma: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Eli Lilly: Consultancy; Zentalis: Consultancy; Celgene: Consultancy; Bristol Myers Squibb: Research Funding; Merck: Consultancy; Ascentage Pharma: Consultancy, Research Funding. Wu:Pharmacyclics: Research Funding; BionTech: Current equity holder in publicly-traded company. OffLabel Disclosure: Ipilimumab and Nivolumab were tested in the setting of relapsed hematological malignancies after allogeneic stem cell transplantation.

Edited by Associate Editor Robert Zeiser, this Review Series presents the current understanding of the pathophysiology of and therapeutic approaches to acute graft-versus-host disease (aGVHD). The articles discuss the role of the microbiome in contributing to aGVHD and review how novel cellular therapies, biologicals, and small-molecule inhibitors may advance its treatment.

Activating mutations in FMS-like tyrosine kinase receptor-3 (FLT3) and Nucleophosmin-1 (NPM1) are most frequent alterations in acute myeloid leukemia (AML), and are often coincidental. The mutational status of NPM1 has strong prognostic relevance to patients with point mutations of the FLT3 tyrosine kinase domain (TKD), but the biological mechanism underlying this effect remains unclear. In the present study, we investigated the effect of the coincidence of NPM1c and FLT3-TKD. Although expression of FLT3-TKD is not sufficient to induce a disease in mice, coexpression with NPM1c rapidly leads to an aggressive myeloproliferative disease in mice with a latency of 31.5 days. Mechanistically, we could show that FLT3-TKD is able to activate the downstream effector molecule signal transducer and activator of transcription 5 (STAT5) exclusively in the presence of mutated NPM1c. Moreover, NPM1c alters the cellular localization of FLT3-TKD from the cell surface to the endoplasmic reticulum, which might thereby lead to the aberrant STAT5 activation. Importantly, aberrant STAT5 activation occurs not only in primary murine cells but also in patients with AML with combined FLT3-TKD and NPM1c mutations. Thus, our data indicate a new mechanism, how NPM1c mislocalizes FLT3-TKD and changes its signal transduction ability.

Relapse of the original disease is a major cause of death after allogeneic hematopoietic cell transplantation for acute leukemias. There is growing evidence that relapses may be explained not only by resistance to chemotherapy but also by the escape of tumor cells from the control of the allogeneic immune response. Mechanisms of immune evasion can involve abrogation of leukemia cell recognition due to loss of HLA genes, immunosuppression by immune-checkpoint ligand expression, production of anti-inflammatory factors, release of metabolically active enzymes, loss of proinflammatory cytokine production, and acquisition of novel driver mutations that promote leukemia outgrowth. These mechanisms, and therapeutic targeting of immune escape, will be discussed. We divide the evidence in support of immune-escape mechanisms into animal studies, human laboratory studies, and human clinical experience. A better understanding of the molecular pathways connected to immune escape and relapse may help to improve our therapeutic armamentarium against acute myeloid leukemia relapse.

Myeloid-derived suppressor cells (MDSCs) can subdue inflammation. In mice with acute graft-versus-host disease (GVHD), donor MDSC infusion enhances survival that is only partial and transient because of MDSC inflammasome activation early posttransfer, resulting in differentiation and loss of suppressor function. Here we demonstrate that conditioning regimen-induced adenosine triphosphate (ATP) release is a primary driver of MDSC dysfunction through ATP receptor (P2x7R) engagement and NLR pyrin family domain 3 (NLRP3) inflammasome activation. P2x7R or NLRP3 knockout (KO) donor MDSCs provided significantly higher survival than wild-type (WT) MDSCs. Although in vivo pharmacologic targeting of NLRP3 or P2x7R promoted recipient survival, indicating in vivo biologic effects, no synergistic survival advantage was seen when combined with MDSCs. Because activated inflammasomes release mature interleukin-1�� (IL-1��), we expected that IL-1�� KO donor MDSCs would be superior in subverting GVHD, but such MDSCs proved inferior relative to WT. IL-1�� release and IL-1 receptor expression was required for optimal MDSC function, and exogenous IL-1�� added to suppression assays that included MDSCs increased suppressor potency. These data indicate that prolonged systemic NLRP3 inflammasome inhibition and decreased IL-1�� could diminish survival in GVHD. However, loss of inflammasome activation and IL-1�� release restricted to MDSCs rather than systemic inhibition allowed non-MDSC IL-1�� signaling, improving survival. Extracellular ATP catalysis with peritransplant apyrase administered into the peritoneum, the ATP release site, synergized with WT MDSCs, as did regulatory T-cell infusion, which we showed reduced but did not eliminate MDSC inflammasome activation, as assessed with a novel inflammasome reporter strain. These findings will inform future clinical using MDSCs to decrease alloresponses in inflammatory environments.

Despite major advances in recent years, graft-versus-host disease (GVHD) remains a major life-threatening complication of allogeneic hematopoietic cell transplantation (allo-HCT). To improve our therapeutic armory against GVHD, preclinical evidence is most frequently generated in mouse and large animal models of GVHD. However, because every model has shortcomings, it is important to understand how predictive the different models are and why certain findings in these models could not be translated into the clinic. Weaknesses of the animal GVHD models include the irradiation only-based conditioning regimen, the homogenous donor/recipient genetics in mice, canine or non-human primates (NHP), anatomic site of T cells used for transfer in mice, the homogenous microbial environment in mice housed under specific pathogen-free conditions, and the lack of pharmacologic GVHD prevention in control groups. Despite these major differences toward clinical allo-HCT, findings generated in animal models of GVHD have led to the current gold standards for GVHD prophylaxis and therapy. The homogenous nature of the preclinical models allows for reproducibility, which is key for the characterization of the role of a new cytokine, chemokine, transcription factor, microRNA, kinase, or immune cell population in the context of GVHD. Therefore, when carefully balancing reasons to apply small and large animal models, it becomes evident that they are valuable tools to generate preclinical hypotheses, which then have to be rigorously evaluated in the clinical setting. In this study, we discuss several clinical approaches that were motivated by preclinical evidence, novel NHP models and their advantages, and highlight the recent advances in understanding the pathophysiology of GVHD.

The activating mutation of the BRAF serine/threonine protein kinase (BRAF V600E) is the key driver mutation in hairy cell leukemia (HCL), suggesting opportunities for therapeutic targeting. We analyzed the course of 21 HCL patients treated with vemurafenib outside of trials with individual dosing regimens (240-1920 mg/d; median treatment duration, 90 days). Vemurafenib treatment improved blood counts in all patients, with platelets, neutrophils, and hemoglobin recovering within 28, 43, and 55 days (median), respectively. Complete remission was achieved in 40% (6/15 of evaluable patients) and median event-free survival was 17 months. Response rate and kinetics of response were independent of vemurafenib dosing. Retreatment with vemurafenib led to similar response patterns (n = 6). Pharmacodynamic analysis of BRAF V600E downstream targets showed that vemurafenib (480 mg/d) completely abrogated extracellular signal-regulated kinase phosphorylation of hairy cells in vivo. Typical side effects also occurred at low dosing regimens. We observed the development of acute myeloid lymphoma (AML) subtype M6 in 1 patient, and the course suggested disease acceleration triggered by vemurafenib. The phosphatidylinositol 3-kinase hotspot mutation (E545K) was identified in the AML clone, providing a potential novel mechanism for paradoxical BRAF activation. These data provide proof of dependence of HCL on active BRAF signaling. We provide evidence that antitumor and side effects are observed with 480 mg vemurafenib, suggesting that dosing regimens in BRAF-driven cancers could warrant reassessment in trials with implications for cost of cancer care.

Over the last decade, our understanding of the pathophysiology of chronic graft-versus-host disease (cGVHD) has improved considerably. In this spotlight, we discuss emerging insights into the pathophysiology of cGVHD with a focus on B cells. First, we summarize supporting evidence derived from mouse and human studies. Next, novel cGVHD therapy approaches that target B cells will be covered to provide treating physicians with an overview of the rationale behind the emerging armamentarium against cGVHD.

Myeloid-derived suppressor cells (MDSCs) are a naturally occurring immune regulatory population associated with inhibition of ongoing inflammatory responses. In vitro generation of MDSCs from bone marrow has been shown to enhance survival in an acute model of lethal graft-versus-host disease (GVHD). However, donor MDSC infusion only partially ameliorates GVHD lethality. In order to improve the potential therapeutic benefit and ultimately survival outcomes, we set out to investigate the fate of MDSCs after transfer in the setting of acute GVHD (aGVHD). MDSCs transferred to lethally irradiated recipients of allogeneic donor hematopoietic grafts are exposed to an intense inflammatory environment associated with aGVHD, which we now show directly undermines their suppressive capacity. Under a conditioning regimen and GVHD inflammatory settings, MDSCs rapidly lose suppressor function and their potential to inhibit GVHD lethality, which is associated with their induced conversion toward a mature inflammasome-activated state. We find even brief in vitro exposure to inflammasome-activating mediators negates the suppressive potential of cultured murine and human-derived MDSCs. Consistent with a role for the inflammasome, donor MDSCs deficient in the adaptor ASC (apoptosis-associated speck-like protein containing a CARD), which assembles inflammasome complexes, conferred improved survival of mice developing GVHD compared with wild-type donor MDSCs. These data suggest the use of MDSCs as a therapeutic approach for preventing GVHD and other systemic inflammatory conditions will be more effective when combined with approaches limiting in vivo MDSC inflammasome activation, empowering MDSCs to maintain their suppressive potential.

The successful treatment of acute leukemias with allogeneic hematopoietic cell transplantation (allo-HCT) is limited by acute graft-versus-host disease (GVHD). Because microRNA-155 (miR-155) regulates activation of the innate immune system, we aimed to determine its function in dendritic cells (DCs) during GVHD in an experimental model. We observed that miR-155 deficiency of the recipient led to improved survival, reduced serum levels of proinflammatory cytokines, and lower GVHD histopathology scores. In addition, miR-155(-/-) bone marrow chimeric mice receiving allo-HCT and miR-155(-/-) DCs showed that miR-155 deficiency in the DC compartment was responsible for protection from GVHD. Activated miR-155(-/-) DCs displayed lower expression of various purinergic receptors and impaired migration toward adenosine triphosphate (ATP). Microarray analysis of lipopolysaccharide/ATP-stimulated miR-155(-/-) DCs revealed mitogen-activated protein kinase pathway dysregulation and reduced inflammasome-associated gene expression. Consistent with this gene expression data, we observed reduced ERK activation, caspase-1 cleavage, and IL-1β production in miR-155(-/-) DCs. The connection between miR-155 and inflammasome activation was supported by the fact that Nlrp3/miR-155 double-knockout allo-HCT recipient mice had no increased protection from GVHD compared with Nlrp3(-/-) recipients. This study indicates that during GVHD, miR-155 promotes DC migration toward sites of ATP release accompanied by inflammasome activation. Inhibiting proinflammatory miR-155 by antagomir treatment could help reduce this complication of allo-HCT.

Members of the B7 family have been shown to be important for regulating immune responses by providing either positive or negative costimulatory signals. The function of B7-H3 has been controversial. We show that B7-H3 is upregulated in graft-versus-host disease (GVHD) target organs, including the colon, liver, and lung. Infusion of allogeneic donor T cells into B7-H3(-/-) vs wild-type (WT) recipients resulted in increased GVHD lethality associated with increased T-cell proliferation, colonic inflammatory cytokines, and destruction of epithelial barriers. Allogeneic B7-H3(-/-) vs WT donor T cells also had increased T-cell proliferation and GVHD lethality associated with increased proliferation and cytokine secretion in the spleen, intraepithelial lymphocyte inflammatory cytokines, and intestinal permeability. Both resting and activated regulatory T cells (Tregs) lack B7-H3 messenger RNA. Consistent with these data, GVHD was augmented in recipients of B7-H3(-/-) Treg-depleted grafts. In two delayed lymphocyte infusion (DLI) models, T cells lacking B7-H3 are capable of providing graft-versus-leukemia (GVL) effects. We conclude that B7-H3 is responsible for providing a negative costimulatory signal. Our studies provide support for developing and testing new therapies directed toward the B7-H3 pathway, including approaches to augment host B7-H3 early after bone marrow transplantation to prevent GVHD and to develop potent antagonistic antibodies later after transplant to facilitate DLI-mediated GVL without GVHD complications.

Acute graft-versus-host disease (GVHD) limits the success of allogeneic hematopoietic cell transplantation (allo-HCT); therefore, a better understanding of its biology may improve therapeutic options. We observed miR-146a up-regulation in T cells of mice developing acute GVHD compared with untreated mice. Transplanting miR-146a(-/-) T cells caused increased GVHD severity, elevated tumor necrosis factor (TNF) serum levels, and reduced survival. TNF receptor-associated factor 6 (TRAF6), a verified target of miR-146a, was up-regulated in miR-146a(-/-) T cells following alloantigen stimulation. Higher TRAF6 levels translated into increased nuclear factor-κB activity and TNF production in miR-146a(-/-) T cells. Conversely, the detrimental effect of miR-146a deficiency in T cells was antagonized by TNF blockade, whereas phytochemical induction of miR-146a or its overexpression using a miR-146a mimic reduced GVHD severity. In humans, the minor genotype of the single nucleotide polymorphism rs2910164 in HCT donors, which reduces expression of miR-146a, was associated with severe acute GVHD (grade III/IV). We show that miR-146a functions as a negative regulator of donor T cells in GVHD by targeting TRAF6, leading to reduced TNF transcription. Because miR-146a expression can be exogenously enhanced, our results provide a novel targeted molecular approach to mitigate GVHD.

Graft-versus-host-disease (GVHD) is a severe complication of allogeneic hematopoietic cell transplantation (allo-HCT) characterized by the production of high levels of proinflammatory cytokines. Activated Janus kinases (JAKs) are required for T-effector cell responses in different inflammatory diseases, and their blockade could potently reduce acute GVHD. We observed that inhibition of JAK1/2 signaling resulted in reduced proliferation of effector T cells and suppression of proinflammatory cytokine production in response to alloantigen in mice. In vivo JAK 1/2 inhibition improved survival of mice developing acute GVHD and reduced histopathological GVHD grading, serum levels of proinflammatory cytokines, and expansion of alloreactive luc-transgenic T cells. Mechanistically, we could show that ruxolitinib impaired differentiation of CD4(+) T cells into IFN-γ- and IL17A-producing cells, and that both T-cell phenotypes are linked to GVHD. Conversely, ruxolitinib treatment in allo-HCT recipients increased FoxP3(+) regulatory T cells, which are linked to immunologic tolerance. Based on these results, we treated 6 patients with steroid-refractory GVHD with ruxolitinib. All patients responded with respect to clinical GVHD symptoms and serum levels of proinflammatory cytokines. In summary, ruxolitinib represents a novel targeted approach in GVHD by suppression of proinflammatory signaling that mediates tissue damage and by promotion of tolerogenic Treg cells.

Inherited bone marrow failure syndromes (IBMFS) are a heterogeneous group of disorders characterized by impaired stem cell function resulting in pancytopenia. Diagnosis of IBMFS presents a major challenge due to limited diagnostic tests and overlapping phenotypes. For that reason, novel and clinical relevant biomarkers and possible targets are urgently needed. Our study defines NIPA as an IBMFS gene, which is significantly downregulated in a distinct subset of MDS-type refractory cytopenia of childhood patients. Mechanistically, NIPA binds FANCD2 and regulates its nuclear abundance. The stabilization of both non- and monoubiquitinated FANCD2 identifies NIPA as an essential player in the Fanconi Anemia (FA) pathway. NIPA thereby prevents MMC hypersensitivity visualized by increased numbers of chromosome radials and reduced cell survival after induction of interstrand crosslinks. To provide proof of principle, re-expression of FANCD2 in Nipa deficient cells restores MMC sensitivity. In a knockout mouse model, Nipa deficiency leads to major cell intrinsic long-term repopulation defects of hematopoietic stem cells (HSCs), with impaired self-renewal in serial transplantations and a bias towards myeloid differentiation. Unresolved DNA damage in Nipa deficient HSCs causes increased sensitivity to cell death and leads to progressive, age-related loss of the HSC pool. Induction of replication stress triggers the phenotypic reduction and functional decline of murine HSCs, resulting in complete bone marrow failure and death of the mice thereby mimicing Fanconi Anemia. Taken together, our study adds NIPA to the short list of FA-associated proteins being essential for a functional DNA repair/FA/BRCA axis and thereby emphasizing its impact as potential diagnostic marker and/or possible target in bone marrow failure syndromes. Disclosures Niemeyer: Celgene: Consultancy.

Chimeric antigen receptor T-cells (CAR Tc) have yielded impressive remission rates in treatment-refractory B-cell malignancies (B-ALL and B-lymphomas) by targeting CD19, resulting in the first FDA approved CAR Tc therapies, Kymriah and Yescarta. However, the translation of these results for other cancer entities remains a challenge. Pre-clinical studies using second-generation CAR Tc against the interleukin-3 receptor alpha chain (CD123) engendered strong anti-leukemic activity. CD123 CAR Tc clinical studies resulted in transient responses, or complete remission but at the expense of on-target off-tumor toxicities. Our studies employing third-generation anti-CD123 CAR Tc demonstrate strong anti-leukemic activity with no adverse effects in vivo. However, the leukemia was not completely eradicated. Combining anti-CD123 CAR Tc with DNA hypomethylating (HMA) agents may enhance the anti-leukemic effect and survival. HMAs such as azacytidine (Aza) activate key epigenetically silenced pathways in AML cells, inhibiting cell proliferation while enhancing cell immunogenicity. We hypothesized that Aza will increase the expression of CD123 on AML cells resulting in long-term disease eradication by anti-CD123 CAR Tc. The anti-leukemic efficacy, survival advantage, safety and feasibility of the combination treatment with Aza and anti-CD123 CAR Tc were evaluated in vivo. HL-60 (CD123med), MLL-2 (CD123lo), MOLM-13 (CD123hi), primary de novo and relapsed/refractory (r/r) AML cells were cultured for 0-8 days in the presence of Aza (0µM-5µM) and analysed for their CD123 expression by flow cytometry, quantitative western blot and RNAseq. The anti-CD123 CAR was constructed with the humanized CSL362-based ScFv and the CD28-OX40-CD3ζ signaling domain, encoded in a third-generation lentiviral vector and expressed in CD3+ Tc from healthy donors. Rag2γc-/- mice (n=12-16/ group) were engrafted with 1x105 MOLM13/ffLuc AML cells and treated with PBS, 5x106 Non-transduced (NTD) Tc orCAR Tc, 4x 2.5mg/kg Aza, or 5x106 CAR Tc following 4x Aza (2.5mg/kg). Leukemic burden was assessed weekly by bioluminescence imaging. Tc activity and immunophenotyping was performed using flow cytometry at day 35 post engraftment, and survival was monitored. HL-60, MLL-2 and MOLM-13 cells showed significant increases in HLA-DR, PD-L1, STAT1 and IRF7 expression, as well as CD123 when exposed to Aza (Fig 1A,B). Interestingly, the increased effect was seen from day one regardless of concentration. This was similarly reflected in AML patient cells. Aza treatment also arrested cell proliferation and decreased viability in both cell lines and patient cells suggesting Aza can aid in the anti-leukemic effect. Rag2γc-/- mice engrafted with MOLM-13 and treated with Aza and CD123 CAR Tc demonstrated suppressed growth, and eradication of MOLM-13 cells compared to mice treated with CD123 CAR Tc or Aza alone. Additionally, a significant decrease in residual CD123+ cells in the bone marrow (BM) of dual treated mice was seen (Fig 1C). A higher frequency of residual CD8+ T-cells in the BM, and CD4+ Tc in the peripheral blood (PB) and BM of dual treated mice was observed compared to CAR Tc only treated mice. Most prominently, we found a significantly higher mean number of stem cell-like and central memory CD8+ Tc in the BM of dual treated mice (232 cells/µl and 208cells/µl, respectively) compared to the CAR Tc only group (55 cells/µl and 23 cells/µl, respectively). Assessment of immune checkpoint markers on residual CAR Tc of dual treated mice revealed significantly decreased levels of CTLA-4, PD-1 and TIM-3 in the BM, and CTLA-4 in the PB compared to the CAR Tc only group. While CAR Tc treatment alone demonstrated a survival advantage compared to PBS, NTD or Aza treated mice, Aza and CAR Tc treatment had a significantly higher survival rate compared to the CAR Tc only group (92% vs. 46% at day 50, p Our findings indicate that Aza increases immunogenicity and augments the cell surface expression of CD123 on AML cells, allowing enhanced recognition and elimination of malignant cells by CD123 CAR Tc. This is the first demonstration that HMAs and CAR Tc immunotherapy can be used synergistically to treat AML. Considering HMAs are currently under clinical investigation in AML, our data encourage further clinical evaluation of this dual treatment in r/r AML, including high-risk patients that are chemotherapy or allogeneic transplantation ineligible. Disclosures Hughes: Novartis, Bristol-Myers Squibb, Celgene: Research Funding; Novartis, Bristol-Myers Squibb: Consultancy, Other: Travel. White:BMS: Honoraria, Research Funding; AMGEN: Honoraria, Speakers Bureau. Yong:Novartis: Honoraria, Research Funding; Celgene: Research Funding; BMS: Honoraria, Research Funding.

Allogeneic hematopoietic cell transplantation (allo-HCT) is a curative therapy for different malignant and non-malignant hematologic diseases. Major life-threatening complications after allo-HCT are acute graft-versus-host disease (aGVHD) and severe infections. The incidence of GVHD after allo-HCT remains high, despite prophylactic immunosuppressive medication. According to the CIBMTR database, 60% of patients undergoing allo-HCT develop at least grade II-IV aGVHD. Microbial invasion into the intestinal mucosa after allo-HCT triggers the activation of neutrophil granulocytes (neutrophils) and requires antibiotic treatment to prevent sepsis. However, antibiotics lead to a loss of microbial diversity, which is connected to a higher incidence of aGVHD. Anti-microbial therapies which eliminate invading bacteria and diminish neutrophil-mediated damage without reducing the diversity of the microbiota are therefore highly desirable. A potential solution for this could be the use of anti-microbial antibodies that target and mark invading pathogens resulting in their elimination by innate immune cells. Therefore we investigated the potency of both active and passive immunization against the conserved microbial surface polysaccharide Poly-N-acetylglucosamine (PNAG) which is expressed on various pathogens in a murine aGVHD model. We could detect reduced aGVHD related mortality in mice which underwent treatment with an antibody against PNAG (anti-PNAG) or were actively immunized against PNAG. Sequencing analysis of the microbiota before and after allo-HCT showed that anti-PNAG treatment did not have any effect luminal microbial diversity. Mechanistically, we could show that anti-PNAG antibody treatment caused more abundant neutrophil recruitment to the intestinal submucosa. This supports the concept that neutrophils are able to effectively eliminate any opsonized bacteria in this tissue, leading to reduced local inflammation. In agreement with this we observed lower intestinal inflammation and reduced myeloperoxidase activity as well as proliferation of neutrophils in the small intestine of mice treated with anti-PNAG antibody. We demonstrate the potency of targeting PNAG as a novel antimicrobial treatment option in aGVHD by reducing uncontrolled neutrophil activation and thereby interfering with aGVHD without affecting commensal intestinal microbial diversity. Anti-PNAG-antibodies enhance anti-microbial immunity, while reducing the activation of neutrophil-mediated downstream immunopathology which is a novel approach to reduce the severity of GVHD. This could be translated into a clinical application given the modest toxicity profile of vaccination and the availability of fully human anti-PNAG antibodies, both of which have been tested in phase 1 trials in humans (ClinicalTrials.gov Identifier: NCT02853617). Disclosures Cywes-Bentley: OneBiopharma, Inc.: Honoraria. Koenecke:Novartis: Other: none. Pier:OneBiopharma, Inc.: Equity Ownership, Honoraria; Alopexx Vaccine, LLC: Equity Ownership, Honoraria.

Acute lymphoblastic leukemia (ALL) is a heterogeneous disease and treatment guidelines are still evolving. Allogeneic stem cell transplantation (allo-SCT) offers a potentially curative option and is recommended in first relapse and for high-risk patients in first complete remission (CR). Survival after allo-SCT could be substantially improved due to better risk stratification, patient selection and adapted treatment protocols leading to reduced non-relapse mortality (NRM). Prognostic factors for survival after allo-SCT still need to be defined: pheno- and genotype, patients´ age, conditioning regimens and remission status prior to allo-SCT are under discussion. We analyzed the outcome of 180 consecutive ALL patients who received allo-SCT at the Freiburg University Medical Center between 1995 and 2018 with regard to treatment response, survival, adverse reactions, and performed subgroup analyses to identify prognostic factors. The median age in our cohort was 37 years (ys), 19% were older than 55 ys. 27% were diagnosed with Philadelphia (Ph)-positive ALL, 24% with T-ALL. 36% were treated with relapsed/refractory disease. 48% of allo-SCTs were conducted with a HLA-matched, 19% with a HLA-mismatched unrelated and 33% with a related donor. In 61% the conditioning regimen included total body irradiation (TBI). In 48% no minimal residual disease (MRD) was detected prior to allo-SCT, 20% were transplanted in MRD-positive CR. The overall response rate was 86%, with MRD-negativity in 78%. With a median follow up of 10 ys, we observed a median overall survival (OS) of 23 months and a median progression free survival (PFS) of 11 months. The 10ys-OS was 33%, the 10ys-PFS 31%. The cumulative incidence of relapse was 68% at 10 ys, the cumulative incidence of NRM 12%. Acute graft-versus-host disease (GvHD) III-IV° occurred in 17%, severe chronic GvHD in 9%. Survival was significantly better in patients reaching MRD-negative CR before allo-SCT (10ys-OS 48% vs. 19%, p With a very long follow-up and high rate of MRD-assessment, we observed a high response rate and a low rate of severe GvHD. Our data confirm that allo-SCT enables long-term survival in high-risk ALL, suggest that, in certain subgroups, survival may be best in patients transplanted in CR and receiving TBI for conditioning, including the relevant observation that allo-SCT can be performed in carefully selected elderly patients. Disclosures Finke: Medac: Honoraria, Other: research support, Speakers Bureau; Neovii: Honoraria, Other: research support, Speakers Bureau; Riemser: Honoraria, Other: research support, Speakers Bureau. Wäsch:Pfizer: Consultancy; Sanofi: Other: Travel, Research Funding; Celgene: Other: travel, Research Funding; Jazz: Other: travel, Research Funding; Amgen: Consultancy; Gilead: Consultancy; Sanofi: Consultancy; Novartis: Consultancy; Takeda: Consultancy; Amgen: Other: travel, Research Funding; Gilead: Other: travel, Research Funding.

Anaplastic large cell lymphoma (ALCL) represents a heterogeneous group of T-cell non-Hodgkin lymphoma (NHL) mainly affecting children and young adults. About 70% of systemic ALCLs are associated with a characteristic chromosomal translocation, t(2;5)(p23;35) which fuses the anaplastic lymphoma kinase (ALK) gene on chromosome 2 with the nucleophosmin (NPM) gene on chromosome 5, resulting in the NPM-ALK fusion gene, its over-expression and constitutive kinase activity. Immunophenotypic characterization of human ALCL revealed highly CD30-positive cells of T- or Null-Cell-origin and resulted in promising clinical trials with CD30-coupled antibodies. However, the impact of CD30 on disease development remains unclear and the relationship between NPM-ALK and CD30 has been investigated mostly using cell line models. Syngeneic mouse models of cancer can serve as useful models since the tumors develop in situ where the contribution made by the immune system and the extracellular matrix can be investigated. Here, we focus on the involvement of CD30 in a retroviral murine bone marrow transplantation model of ALCL. In this model, the BM of Lck-Cre-transgenic mice is infected with a MSCV-Stop-NPM-ALK-IRES-EGFP vector leading to expression of NPM-ALK in early T-cells. With a latency of 3-4 months, mice develop lymphomas and die from neoplastic T-cell-infiltration of BM and lymphatic organs. To investigate the impact of abrogation of CD30 signals on the development of NPM-ALK+ ALCL in our model, CD30 knockout mice were crossed with Lck-Cre mice. Both Lck-Cre NPM-ALK CD30 wt and Lck-Cre NPM-ALK CD30 ko recipients develop a human ALCL-like lymphoma with a pure T-cell phenotype characterized by Thy1.2+ cells infiltrating the thymus, lymph nodes, spleen and BM. First results from Lck-Cre NPM-ALK CD30 ko transplanted mice showed impaired disease induction and prolonged survival compared to CD30 wt animals. Moreover, secondary transplantation of NPM-ALK thymic lymphomas led to distinct deceleration of disease development upon CD30 deletion. Microarray analyses have shed some light on the mechanisms underlying the delayed lymphoma progression of CD30 deleted tumors with an upregulation of inflammatory pathways and proteins that are master players in inflammation and immune responses. Further characterization of the role of CD30-mediated immune response in disease progression using this mouse model and immunocompromised mice is ongoing. An improved understanding of how the immune system affects tumor progression will extend the rationale in translational strategies to use immunotherapies for patients with T-NHLs. Disclosures No relevant conflicts of interest to declare.

Multiple myeloma (MM) is closely dependent on cross-talk between malignant plasma cells and cellular components of the inflammatory bone marrow milieu, which promotes disease progression, bone destruction and immune-impairment. However, the molecular mechanisms initiating this inflammatory microenvironment remain poorly defined. Beta-2-microglobulin (β2M) accumulates during MM and represents an established prognostic biomarker, but little is known regarding its immunoregulatory potential. Here, we found that β2m triggers an enhanced caspase-1 dependent release of mature IL-1β (15.8 ± 4.5 ng/ml) and IL-18 (5.8 ± 2.1 ng/ml) by macrophages in a NLRP3 dependent way. Activation of the NLRP3 inflammasome was initiated, after phagocytosis of β2m and subsequent lysosomal rupture due to amyloid fibril formation in lysosomes, in a process which partially involved lysosomal protease cathepsin B. β2m mediated NLRP3 inflammasome activation also occurs in tumor-associated macrophages (TAMs) of MM patients leading to the release of IL-1β and IL-18 towards MM cells. Moreover, TAMs in a murine MM model (5TMM) displayed an active NLRP3 inflammasome as well. Treatment of 5TMM mice with MCC950, a selective NLRP3 inhibitor, reduced tumor progression and thus attenuated the severity of MM. Our findings shed new light on β2m as macrophagic NLRP3 inflammasome activator which contributes to MM cell progression, and open a potential novel avenue for therapies of this severe malignancy. Figure Disclosures Bruns: Morphosys AG: Research Funding.

Acute graft-versus-host disease (GvHD) is a complex process involving endothelial damage and neovascularization. Better understanding of the pathophysiology of neovascularization during GvHD could help to target this process while leaving T-cell function intact. Under ischemic conditions, neovascularization is regulated by different micro RNAs (miRs), which potentially play a role in inflamed hypoxic GvHD target organs. We observed strong neovascularization in the murine inflamed intestinal tract (IT) during GvHD. Positron emission tomography imaging demonstrated abundant αvβ3 integrin expression within intestinal neovascularization areas. To interfere with neovascularization, we targeted αv integrin-expressing endothelial cells, which blocked their accumulation in the IT and reduced GvHD severity independent of immune reconstitution and graft-versus-tumor effects. Additionally, enhanced neovascularization and αv integrin expression correlated with GvHD severity in humans. Expression analysis of miRs in the inflamed IT of mice developing GvHD identified miR-100 as significantly downregulated. Inactivation of miR-100 enhanced GvHD indicating a protective role for miR-100 via blocking inflammatory neovascularization. Our data from the mouse model and patients indicate that inflammatory neovascularization is a central event during intestinal GvHD that can be inhibited by targeting αv integrin. We identify negative regulation of GvHD-related neovascularization by miR-100, which indicates common pathomechanistic features of GvHD and ischemia.

Despite major advances in recent years, graft-versus-host disease (GVHD) remains a major life-threatening complication of allogeneic hematopoietic cell transplantation (allo-HCT). To improve our therapeutic armory against GVHD, preclinical evidence is most frequently generated in mouse and large animal models of GVHD. However, because every model has shortcomings, it is important to understand how predictive the different models are and why certain findings in these models could not be translated into the clinic. Weaknesses of the animal GVHD models include the irradiation only-based conditioning regimen, the homogenous donor/recipient genetics in mice, canine or non-human primates (NHP), anatomic site of T cells used for transfer in mice, the homogenous microbial environment in mice housed under specific pathogen-free conditions, and the lack of pharmacologic GVHD prevention in control groups. Despite these major differences toward clinical allo-HCT, findings generated in animal models of GVHD have led to the current gold standards for GVHD prophylaxis and therapy. The homogenous nature of the preclinical models allows for reproducibility, which is key for the characterization of the role of a new cytokine, chemokine, transcription factor, microRNA, kinase, or immune cell population in the context of GVHD. Therefore, when carefully balancing reasons to apply small and large animal models, it becomes evident that they are valuable tools to generate preclinical hypotheses, which then have to be rigorously evaluated in the clinical setting. In this study, we discuss several clinical approaches that were motivated by preclinical evidence, novel NHP models and their advantages, and highlight the recent advances in understanding the pathophysiology of GVHD.

Chronic graft-versus-host disease (cGVHD) remains a major complication after allogeneic hematopoietic cell transplantation. Over the last decade, clinical success in patients with cGVHD has been hampered by the lack of insight into the complex pathobiological mechanisms of the disease, the paucity

CD4+ natural killer T (NKT) cells, along with CD4+CD25+ regulatory T cells (Tregs), are capable of controlling aberrant immune reactions. We explored the adoptive transfer of highly purified (> 95%) CD4+NKT cells in a murine model of allogeneic hematopoietic cell transplantation (HCT). NKT cells follow a migration and proliferation pattern similar to that of conventional T cells (Tcons), migrating initially to secondary lymphoid organs followed by infiltration of graft-versus-host disease (GVHD) target tissues. NKT cells persist for more than 100 days and do not cause significant morbidity or mortality. Doses of NKT cells as low as 1.0 × 104 cells suppress GVHD caused by 5.0 × 105 Tcons in an interleukin-4 (IL-4)–dependent mechanism. Protective doses of NKT cells minimally affect Tcon proliferation, but cause significant reductions in interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) production by donor Tcons and in skin, spleen, and gastrointestinal pathology. In addition, NKT cells do not impact the graft-versus-tumor (GVT) effect of Tcons against B-cell lymphoma-1 (BCL-1) tumors. These studies elucidate the biologic function of donor-type CD4+NKT cells in suppressing GVHD in an allogeneic transplantation setting, demonstrating clinical potential in reducing GVHD in HCT.

Allogeneic hematopoietic cell transplantation (allo-HCT) is the treatment strategy of choice for the majority of patients with acute myeloid leukemia (AML). However, about 40% of patients relapse, which is associated with a poor prognosis. Donor lymphocyte infusions (DLIs) induce remission only in a minority of relapsed AML patients. In order to understand the impact of AML cells on T cell bioenergetics, we investigated the metabolic phenotype and immunological profile of CD8+ T cells isolated from AML patients at primary diagnosis, during remission and relapse after allo-HCT. At diagnosis, T cells exhibited robust metabolic activity and strong immune activation. In contrast, during relapse, T cells demonstrated reduced glycolytic and respiratory activity compared to their status at remission. The metabolic phenotype was accompanied by reduced cytotoxic ability. Metabolomics revealed lactate accumulation and substantial arginine depletion in the serum of these patients. The results observed in the human setting could be reproduced in a murine graft versus leukemia (GVL) model where T cells isolated from AML bearing mice exhibited lower metabolic activity compared to control animals. In vitro co-culture experiments enabled tightly controlled conditions for mechanistic studies. The metabolic impairment induced by AML cells was mediated by soluble factors, mirroring the major changes in serum metabolites of patients, and interfered with the crucial metabolic reprogramming in the priming phase of T cells. Well-known mechanisms such as nutrient deprivation could be excluded. Extensive transcriptomic and metabolomic analyses have been undertaken in order to identify the molecules responsible for the diminished metabolic activation. Influencing mitochondrial architecture thereby boosting respiratory activity prior to infusion led to enhanced anti-leukemic activity of T cells in vivo. The pre-treatment favored an immunological memory phenotype essential for a potent GVL effect. Using metabolic stimuli as pre-treatment of DLIs could promote T cell resistance against tumor evasion and increase anti-tumor immunity. This could greatly improve the success of DLIs and other adoptive T cell transfer strategies in the treatment of AML relapse. Disclosures Finke: Neovii: Consultancy, Honoraria, Other: travel grants, Research Funding; Medac: Consultancy, Honoraria, Other: travel grants, Research Funding; Novartis: Consultancy, Honoraria, Other: travel grants, Research Funding; Riemser: Consultancy, Honoraria, Research Funding.

Oncogenic Ras mutations occur frequently in myelodysplastic (MDS) and myeloproliferative syndromes (MPN). It was unclear if besides the direct transforming effect via constant RAS/MEK/ERK signaling, an inflammation related effect of KRAS contributes to the development of the disease. To address this question we performed a microarray based analysis of bone marrow derived from Rosa26CreERT2; LSL-KrasG12D mice after induction of KrasG12D with tamoxifen. We found that the NLRP3 inflammasome and related genes were upregulated. In agreement with increased NLRP3 sensitivity, KrasG12D bone marrow derived dendritic cells (BMDCs) showed increased inflammasome activation upon stimulation with LPS and ATP as quantified by intracellular levels of IL-1β and caspase-1 p20 subunit. To validate the functional role of the NLRP3 inflammasome for the MDS phenotype in vivo we generated Rosa26CreERT2; LSL-KrasG12D; NLRP3-/- mice. We transferred BM (CD45.2) from WT mice, Rosa26CreERT2; LSL-KrasG12D or Rosa26CreERT2; LSL-KrasG12D; NLRP3-/- mice into C57BL/6 WT recipients (CD45.1) after myeloablative conditioning and induced KrasG12D by tamoxifen treatment. Rosa26CreERT2; LSL-KrasG12D BM recipient mice developed MPN stigmata including anemia, leukopenia and thrombocytopenia. The mice also presented weight loss, splenomegaly, and an increase in myeloid cells in peripheral blood, spleen and BM, as well as increased CD11b+ckit+ immature myeloid progenitors. In contrast, Rosa26CreERT2; LSL-KrasG12D; NLRP3-/- recipient mice developed no disease phenotype. BMDCs isolated from Rosa26CreERT2; LSL-KrasG12D recipient mice exhibited strong NLRP3 inflammasome activation and high IL-1β levels which was not found in BMDCs of Rosa26CreERT2; LSL-KrasG12D; NLRP3-/- mice. CD11b+ cells of a Juvenile myelomonocytic leukemia (JMML) patient with an activating KRAS mutation showed increased inflammasome activation as compared to a non-mutant patient, which is in agreement with our observations in mouse models. Our findings support the concept that oncogenic KrasG12D does not only act via its oncogenic driver function, but also enhances activation of the NLRP3/IL-1β axis. This could lead to novel therapeutic approaches combining inhibition of oncogenic signaling and immune modulation via IL-1β blockade. Disclosures No relevant conflicts of interest to declare.

Mutations in the colony-stimulating factor 3 receptor (CSF3R) have been identified in 90% of chronic neutrophilic leukemia (CNL) and lower frequency in atypical chronic myeloid leukemia (aCML). It was shown recently, that signaling of the different types of CSF3R mutations varies, but a central role of the JAK/STAT pathway activation by oncogenic CSF3R membrane proximal point mutations has been anticipated. However, the exact downstream signaling pathways mediated by CSF3R mutations are still not completely understood. Recent studies in JAK mutated myeloproliferative diseases uncovered the vital importance of JAK/STAT signaling in PD-L1 mediated immune escape, thereby suggesting a possible role and mechanistic link for PD-1/PD-L1 immune checkpoint regulation in CNL and aCML. To investigate the dependence of the oncogenic CSF3R signaling cascade on an activated JAK/STAT axis, we performed Ruxolitinib treatment in MTT assays with BaF3 cells retrovirally infected with pMIG-R1 vectors harboring either CSF3R point or truncation mutations. Our data indicate, that both, CSF3R point and truncation mutations induced cell growth could be inhibited by Ruxolitinib treatment. To narrow down the involvement of the different JAK family kinases, we included CSF3R mutation transduced JAK1 deficient U4C, JAK2 deficient y2A and TYK2 deficient U1A cell lines in combination with different JAK inhibitors. Interestingly, here we were able to show that CSF3R point mutations require functional JAK1/TYK2-STAT3 signaling for oncogenic potential, whereas truncation mutations depend on all JAK kinases through STAT3/5 for activation. To further study potential therapeutic strategies, we examined checkpoint inhibition in CSF3R mutated disease. In this regard we demonstrate a significant induction of PD-L1 expression in murine cell lines upon expression of CSF3R point mutations T615A or T618I. To investigate the impact of PD-L1 upregulation in vivo, we used a retroviral murine bone marrow (BM) transplantation model where we infected BM of Balb/c mice with a vector carrying CSF3R T618I point mutation and transplanted it into lethally irradiated recipients. Transplanted mice developed a CSF3R driven leukemia and died by leukocytosis and organ infiltration, whereas mice transplanted with BM harboring CSF3RT618I and additional ADA mutation failed to induce leukemia. To test checkpoint inhibition as a potential therapeutic strategy in vivo, CSF3RT618I transplanted mice were treated regularly with anti-PD-L1-antibody or isotype control. Interestingly, checkpoint inhibition led to reduced EGFP burden and prolonged survival of responder mice suggesting a treatment response of immunooncologic agents in these rare diseases. In immunophenotypic analyses of primary patient material, we were able to show increased PD-L1 expression in primary diseased myeloid cells isolated from peripheral blood of CNL and aCML patients, thereby defining this structure as a possible new therapeutic target. To summarize, our data expand the knowledge of oncogenic CSF3R signaling pathways and implicate an immunotherapeutic strategy by checkpoint inhibition in CSF3R driven CNL and atypical CML. Disclosures Lubbert: Teva: Other: Study drug; Celgene: Other: Travel Grant; Janssen: Honoraria, Research Funding.

Introduction: MM is characterized by the accumulation of aberrant BM plasma cells (aPCs). The use of novel agents for anti-MM treatment has enabled the achievement of deeper responses and prolonged progression free survival (PFS) and overall survival (OS). These advances have created the need for sensitive means to detect residual PCs. MFC allows aPC detection with high sensitivity and is used with NGS in clinical trials (CTs) (Paiva, 2015-2018). It allows the detection of residual cells after anti-MM therapies and is a risk denominator of pt subgroups and post-therapeutic outcome. The incorporation of cost-effective, readily available and standardized MRD tools into CTs and clinical practice seems a key requisite. It may allow the improvement of clinical decisions: 1) timing of stem cell transplantation (SCT), 2) need for consolidation, 3) duration of maintenance, 4) therapy modulations and 5) early relapse detection. Since MRD gains importance for novel agents' potency to achieve MRD negativity, including FDA/EMA-approvals, we assessed MFC for detection of aPCs, their change to treatment and over time. Methods: We systematically validated a 6-, 8- and 10-color panel using CD38, CD138, CD19, CD45, CD27, CD56, CD28, CD81, CD117, CD200 and kappa/lambda (k/l) in 9 different MM cell lines (MMCLs) and 213 BM, PB, leukapheresis (LA), extramedullary (EM) samples of 122 different MM pts and 14 healthy individuals (HI). These were immediately analyzed after sampling, using a bulk-lysis protocol. To reach a high sensitivity, >3x106 viable nucleated cells were analyzed. MFC validation included fluorescence-minus-one-(FMO), isotype- and spike-in-controls to evaluate the reliability and sensitivity of our panels. Spike-in controls were performed using a dilution assay to recover defined MMCL cells in the PB of HI. Data analysis was accomplished using the BeckmanCoulter software Kaluza®. We compared MFC using BM and PB samples of HI, MM pts at initial diagnosis (ID) or with progression (PD), after standard therapy (ST) and SCT. Additionally, BM samples at ID were sorted into each 4 aPC- and normal PC- (nPC) subpopulations for DNA and RNA sequencing and follow-up. The study was performed with written consent of all pts and HIs and approval of the ethics committee. Results: The antigen expression levels in 9 MMCLs using our 6-, 8- and 10-color panel could be confirmed as reported. The expression levels of antigens included in all panels were similar in all MMCLs (RPMI 8226; U266; IM-9; MM1.S; MM1.R; L363; Karpas620; NCI-H929; OPM-2). For all panels, we established easy to adapt gating strategies, using commercially available software to identify aPCs vs. nPCs in all analyzed samples. Using the 6-color panel, differentiation into up to 4 phenotypic subpopulations of aPCs and nPCs in MM pts and up to 4 subpopulations of nPCs in HI was achieved, depending on the heterogeneous phenotype the individual was expressing. Moreover, both DNA and RNA of obtained subpopulations were collected and analyzed via sequencing. Since in 1% of our samples, aPCs remained undetected via 6-color panel using surface antigens exclusively, we added k/l and did detect aPCs in all pts. The 10-color panel identified aPC- and nPC-subpopulations in even more detail. We determined a MRD sensitivity of 10-5, confirmed via independent dilution assays. In 48% of BM and PB samples, aPCs were determined at ID and PD; 52% were assessed for MRD after ST and SCT. Of the latter, 55% were BM samples, of which 5% showed MRD negativity of Conclusion: Our 6-, 8-, and 10-color panels allow highly sensitive detection of aPCs vs. nPCs in MM samples. These panels have been validated using various controls, dilution assays, 9 MMCLs, MM pt samples and HI and can be implemented into routine diagnostics. The analysis of DNA and RNA expression patterns in distinct subpopulations will generate relevant insight, which subclones contribute to response vs. resistance mechanisms. The EuroFlow Consortium have established a two 8-color tubes test for identification of aPCs, which - albeit used worldwide - may not be applicable for every center and every pt outside CTs. Therefore, meticulously validated alternatives as shown here are of interest. Our 8-color panel has been included into our CT portfolio. Disclosures Azab: Cellatrix LLC: Equity Ownership, Other: Founder and owner; Targeted Therapeutics LLC: Equity Ownership, Other: Founder and owner; Ach Oncology: Research Funding; Glycomimetics: Research Funding. Zeiser:Jazz Pharmaceuticals: Research Funding. Wäsch:Pfizer: Honoraria.

Robert Zeiser, MD, and Michel Sadelain, MD, PhD, share some highlights of this review series. Allogeneic hematopoietic cell transplantation (allo-HCT) is the only curative approach for a large group of hematological malignancies, and it relies heavily on graft-versus-leukemia (GVL) effects. Relapse

CD4+CD25+ regulatory T cells (Tregs) suppress immune responses to alloantigens. The in vivo circulation and tissue localization of Tregs during an adaptive immune response remain unclear. We noninvasively tracked luciferase-expressing Tregs over time in an allogeneic bone marrow transplant model and demonstrated colocalization with effector T cells and initial expansion in secondary lymphoid organs before migration into inflamed tissues. Inflammation induced by irradiation and the allogeneic setting provided crucial stimuli for early Treg expansion and migration, leading to parallel reduction of effector T-cell proliferation in lymphoid organs and peripheral tissues. Treg transplants conferred long-term protection from systemic inflammatory challenge consistent with Treg in vivo survival. Suppression occurred during multiple phases of inflammation, but is optimal in the initial phase, providing protection from graft-versus-host disease while maintaining the graft-versus-tumor effect even at physiologic doses of Tregs due to their in vivo expansion, hence overcoming a major barrier to potential clinical applications of Tregs given their rarity.

Background: Treatment with the DNA hypomethylating agents (HMAs) 5-azacytidine (AZA) or DAC has become a well-established option for elderly MDS and AML patients (pts). Regarding their mechanism of action, HMAs not only reactivate tumor suppressor genes, but also lead to an immune activation response, by expression of Cancer/testis antigens (Almstedt et al., Leuk. Res. 2010; Goodyear et al., Blood 2010) and endogenous retroviruses (Chiappinelli et al., Cell 2015; Roulois et al., Cell 2015). Hypothesizing that HMA-mediated immune activation may sensitize blasts to subsequent DLIs, we and others showed that AZA combined with DLIs can induce long-term remissions in pts with relapsed MDS/AML after allogeneic transplantation, particularly with a low leukemic burden, and after failure to respond to DLIs alone (Steinmann et al., BMT 2015, Schroeder et al., BBMT 2015). As DAC is considered useful also in pts with higher WBC and higher blast counts, we now treated 18 pts (most of them with more aggressive hematologic relapse) with DAC+DLIs. Patients and Methods: Between 10/06 and 03/16, 18 pts (median age 63 years, range 29-76, 10 males) with hematological relapse after allogeneic transplantation were treated with DAC and (where feasible) DLIs. Thirteen pts had AML (genetic risk by ELN: 3x int-1, 4x int-2, 6x adverse (4 MK+), 3 had MDS, 1 had polycythemia vera, 1 had atypical CML. Sixteen and 2 pts had received reduced-toxicity conditioning (mostly FBM) and myeloablative conditioning, respectively. Donors: matched unrelated in 8 pts, matched sibling in 4, mismatched in 6. DLIs at relapse after 1st/2nd transplant in 15/3 pts (1st/2nd/3rd/4th relapse in 11/5/1/1 pts). Median duration from transplant to hematologic relapse was 306 days (range, 56-4943). Median % blasts before DAC was 4% in peripheral blood (pb, range, 0-40%), 30 % in bone marrow (range, 7-60%), the median WBC was 4,770/µl (range, 430-30,800). DAC (20 mg/m2 per day) was administered either as a standard 5-day (15 pts) or a 10-day course (3 pts). DLIs were to be given 7 days after the last HMA dose. Prior AZA as relapse treatment was allowed. Results: Of 18 pts with myeloid neoplasias (mostly AML) with hematologic relapse after allografting, 15 received DAC+DLIs (for 8 of them after previous DLI-only, for 3 after previous AZA+DLI), 3 pts only received DAC (median number of DAC cycles: 2, range 1-8, median number of DLIs: 2, range 1-10). Two AML pts (aged 64 and 76 yr, no previous AZA, 7 and 4% pb blasts, respectively) attained a complete hematologic and molecular remission after 5-day DAC+DLI, with a duration of 250+ and 391 days, respectively. One of them is still in CR with no signs of GvHD, the other died in CR because of liver GvHD and hemosiderosis. A third AML pt attained complete donor chimerism following DAC+DLI but died at day 35 because of a fungal pneumonia (concurrent Sorafenib). Eleven additional pts achieved a temporary disease control (median duration 93 days, range 34-212), 5 of them were subsequently re-transplanted. Four pts showed no response to DAC+DLI, 3 developed GvHD. Seven pts died of PD, 6 pts of infections, 1 pt of intracranial hemorrhage, 1 pt of GvHD. Conclusions: DAC in combination with DLIs showed good feasibility in pts with relapsed AML/MDS after allogeneic transplantation. Despite overall higher median WBC and blast expansion than a large previous cohort treated with AZA+DLI, clinical activity was comparable, and in 2/18 pts (11%) a complete hematologic and molecular remission was achieved (despite the presence of peripheral blood blasts). This approach is investigated prospectively within the RELDAC substudy of the EORTC trial AML21 of older AML patients (inDACtion vs. induction, NCT02172872). Disclosures Claus: Janssen-Cilag: Consultancy, Honoraria, Other: Travel Funding; Gilead: Consultancy, Honoraria; Roche: Consultancy, Honoraria. Marks:Pfizer: Honoraria. Lübbert:Celgene: Other: Travel Funding; Ratiopharm: Other: Study drug valproic acid; Janssen-Cilag: Other: Travel Funding, Research Funding.

We have previously reported on the efficacy of the JAK1/2 inhibitor ruxolitinib in corticosteroid-refractory (SR) acute (a) and chronic (c) graft-versus-host disease (GVHD) in 95 patients (pts) (Leukemia 2015;29(10):2062-8). To assess long-term follow-up results, we collected data from the same pts treated in 19 centers in Europe and the US. Pts were classified as SR-aGVHD (n=54, all grade III or IV) or SR-cGvHD (n=41, all moderate or severe). Median numbers of pre-ruxolitinib GVHD treatment lines were 3 (1-7) and 3 (1-10) for SR-aGVHD and SR-cGvHD, respectively. The median follow-up was 19 and 24 months for aGVHD and cGVHD, respectively. The 1-year overall survival (OS) from was 62.4% (CI: 49.4%-75.4%) and 92.7% (CI: 84.7%-100%) for SR-aGVHD and SR-cGvHD, respectively. The estimated median OS (50% death) was 18 months for aGVHD and not reached for cGVHD patients. The median duration of ruxolitinib treatment was 5 and 10 months for patients with SR-aGVHD and SR-cGVHD, respectively reflecting the different biology of the diseases. At follow-up, 22/54 (41%) of SR-aGVHD patients and 10/41 (24%) of SR-cGVHD patients have an ongoing response and are free of any immunosuppression. GVHD relapse or progression after achieved PR/CR was observed in 14/45 (31%) and 13/36 (36%) patients with SR-aGVHD and SR-cGVHD, respectively. Response to re-treatment with Ruxolitinib or any immunosupressive therapy was seen in 11/14 (78%) and 11/13 (86%) patients with SR-aGVHD and SR-cGVHD, respectively. Cytopenia (any grade) and CMV-reactivation were observed during ruxolitinib-treatment in both SR-aGVHD (30/54, 55.6% and 18/54, 33.3%) and SR-cGVHD (7/41, 17.1% and 6/41, 14.6%) patients. These findings extend our previous report by showing that patients with SR-aGVHD and SR-cGVHD may benefit long-term from ruxolitinib treatment with an OS that is relatively high for steroid-refractory GVHD. GVHD-relapse or GVHD-progression rates were moderate and more than 75% of the relapse/progression patients responded to re-treatment with ruxolitinib or other immunosuppression. Disclosures Meyer: Stanford University: Patents & Royalties. Marks:Pfizer: Honoraria. Lübbert:Ratiopharm: Other: Study drug valproic acid; Celgene: Other: Travel Funding; Janssen-Cilag: Other: Travel Funding, Research Funding. Scheid:Novartis: Other: funding outside this work; Celgene: Other: funding outside this work; Janssen: Other: funding outside this work. Kobbe:Celgene: Honoraria, Other: travel support, Research Funding; Jansen: Honoraria, Other: travel support. Negrin:Stanford University: Patents & Royalties. Brune:Meda Pharma: Consultancy. Mielke:JAZZ Pharma: Speakers Bureau; Novartis: Consultancy; MSD: Consultancy, Other: Travel grants; Gilead: Other: Travel grants; Celgene: Other: Travel grants, Speakers Bureau. Kuball:Gadeta B.V,: Membership on an entity's Board of Directors or advisory committees. Kröger:Sanofi: Honoraria, Research Funding; Neovii: Honoraria, Research Funding; Riemser: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Peschel:MophoSys: Honoraria. von Bubnoff:BMS: Honoraria; Amgen: Honoraria; Novartis: Honoraria, Research Funding.

Based on their ability to control T-cell homeostasis, Foxp3+CD4+CD25+ regulatory T cells (Tregs) are being considered for treatment of autoimmune disorders and acute graft-versus-host disease (aGVHD). When combining Tregs with the immunosuppressant rapamycin (RAPA), we observed reduced alloreactive conventional T-cell (Tconv) expansion and aGVHD lethality compared with each treatment alone. This synergistic in vivo protection was paralleled by intact expansion of polyclonal Tregs with conserved high FoxP3 expression. In contrast to Tconv, activation of Tregs with alloantigen and interleukin-2 preferentially led to signal transducer and activator of transcription 5 (STAT5) phosphorylation and not phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway activity. Expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a negative regulator of the PI3K/Akt/mTOR pathway, remained high in Tregs but not Tconv during stimulation. Conversely, targeted deletion of PTEN increased susceptibility of Tregs to mTOR inhibition by RAPA. Differential impact of RAPA as a result of reduced usage of the mTOR pathway in Tregs compared with conventional T cells explains the synergistic effect of RAPA and Tregs in aGVHD protection, which has important implications for clinical trials using Tregs.

Murine CD4+CD25+ regulatory T cells (Treg cells) reduce acute graft-versus-host disease (aGvHD). However, surface molecules critical for suppression are unclear. Deficiency of CD30 (CD30−/−) leads to impaired thymic negative selection and augmented T-cell autoreactivity. Therefore, we investigated the role of CD30 signaling in Treg-cell function during aGvHD. Treg cells derived from CD30−/− animals were significantly less effective in preventing aGvHD lethality. Early blockade of the CD30/CD153 pathway with a neutralizing anti-CD153 mAb reduced Treg-mediated protection from proinflammatory cytokine accumulation and donor-type T-cell apoptosis. In vivo bioluminescence imaging demonstrated intact homing but reduced expansion of luciferase-expressing Treg cells when CD153 was blocked during the early phase after adoptive transfer. CD30 surface expression on Treg cells increased with alloantigen exposure, and CD153 expression on recipient-type dendritic cells increased in the presence of a proinflammatory environment. These data demonstrate that early CD30 signaling is critical for Treg-mediated aGvHD protection after major MHC-mismatch bone marrow transplantation.

JAK2 V617F can be identified in the majority of polycythemia vera cases and in 50% of essential thrombocythemia and idiopathic myelofibrosis patients. JAK2 inhibitors including ruxolitinib, fedratinib and lestaurtinib display clinical activity in trials for PV, ET and IMF, and ruxolitinib has recently been approved for the treatment of primary and secondary myelofibrosis. In other malignancies it has been demonstrated that acquired resistance to kinase inhibitors emerge due to secondary resistance mutations in the targeted kinase. To identify JAK2 point mutations mediating ruxolitinib resistance and predict alternatively treatment strategies, we exposed JAK2 V617F expressing Ba/F3 cells to ruxolitinib. Surprisingly, sublines resistant to ruxolitinib at 1000nM, 2000nM and even 4000nM did not harbor point mutations neither in the kinase nor in the pseudokinase domain of JAK2. However, western blot analysis of sublines resistant to ruxolitinib revealed a 45-kDa JAK2 variant together with full length JAK2 V617F protein in 87% of the cases. Sequencing of the short form in drug resistant clones revealed a novel JAK2 variant missing amino acids 76 to 820 resulting in the N-terminal FERM domain directly fused to the kinase domain of JAK2 (FERM-JAK2). FERM-JAK2 was highly resistant to the ATP-competitive JAK2 inhibitors ruxolitinib and fedratinib. Phospho-deficient mutant studies provided evidence that FERM-JAK2 preferentially exist in an inactive state. This would impede drug binding and explain the resistant phenotype of FERM-JAK2. However, in this study we could not identify other genetic TKI resistant JAK2 variants than FERM-JAK2. This is in remarkable contrast to BCR-ABL mediated CML, where more than 90 residues have been described as TKI resistant variants and might indicate that in case of JAK2, the residues which mediate the drug resistance might comprise the JAK2 kinase function. Therefore, we treated JAK2 V617F expressing Ba/F3 cells with a chemical mutagen (ENU) to predict point mutations in JAK2 that might cause resistance towards the ruxolitinib. Surprisingly, after ENU pretreatment the frequency of drug resistant clones increased and most of the drug resistant clones displayed point mutations in the JAK2 kinase domain. In this screen, we identified L902Q, Y931C, L983F as the most frequent mutations and L902Q+R938E, L902Q+N947Q, L902Q+E1028K and L983F+Q959H as minor mutations. All these mutations confer cross-resistance across a panel of different JAK2 kinase inhibitors except L983FJAK2. L983FJAK2 reduces the sensitivity of JAK2-dependent cells to ruxolitinib and JAK inhibitor-1 but does not reduce the sensitivity of cell proliferation against fedratinib, thereby indicated that we identified a compound specific resistant mutations which is not an ATP-competitor specific mutation. Structure based modeling studies suggest that ruxolitinib is held by numerous hydrophobic interactions with residues Leu 855, Val 863, Ala 880, Val 911, Met 929, Leu 932 and Leu 983 that line the binding pocket. The aromatic ring of Tyr 931 is close enough to the pyrimidine ring to have p-p interaction. Surprisingly, this important interaction does not seem to be critical for the orientation of the inhibitor in the binding pocket as the Y931C mutation does not lead to a different binding pose. Leu 902 does not directly interact with ruxolitinib, however, it is close to the binding pocket and its mutation to Gln with a polar side chain significantly disturbs the inhibitor binding, thus explaining the high resistance of this mutation to ruxolitinib. Mutation of Leu 983 to Phe disrupts important hydrophobic interactions (e.g. Ala 880, Val 911, Met 929) with the pyrrolopyrimidine moiety and induces aromatic-aromatic interaction between the new phenyl ring and the pyrrol and pyrazol rings. Finally, our study also provided evidence that HSP90 inhibitors 17-AAG and geldanamycin are highly potent against TKI resistant FERM-JAK2, L902Q, Y931C and L983FJAK2 through JAK2 degradation and inhibition of the JAK2-STAT5 signaling axis. Taken together, our study highlight HSP90 inhibitors as a promising therapeutic agent in drug resistant mutations against tyrosine kinase inhibitors in MPN. Disclosures No relevant conflicts of interest to declare.

Background: Allogeneic hematopoietic cell transplantation is a potentially curative therapy for patients with hematological malignancies. However a fraction of patients will develop corticosteroid-refractory (SR) acute (a) and chronic (c) graft-versus-host disease (GVHD) which both cause a high mortality and impaired quality of life. Pre-clinical evidence indicates the potent anti-inflammatory properties of the JAK1/2 inhibitor ruxolitinib by modification of T cells and dendritic cells. Methods: In this retrospective analysis, 19 stem cell transplant centers in Europe and the United States reported clinical outcome data from 95 patients who had received ruxolitinib as salvage-therapy for SR-GVHD. Patients were classified as having SR-aGVHD (n=54, all grade III or IV) or SR-cGvHD (n=41, all moderate or severe). The median number of previous GVHD-therapies was 3 for both SR-aGVHD (1-7) and SR-cGvHD (1-10). The median follow-up times were 26.5 (3-106) for SR-aGVHD and 22.4 (3-135) weeks for SR-cGVHD-patients. Results: The ORR was 81.5% (44/54) in SR-aGVHD including 25 CRs (46.3%), while for SR-cGVHD the ORR was 85.4% (35/41). The median time to response was 1.5 (1-11) and 3 (1-25) weeks after initiation of ruxolitinib treatment in SR-aGVHD and SR-cGVHD, respectively. Of those patients responding to ruxolitinib, the rate of GVHD-relapse was 6.8% (3/44) and 5.7% (2/35) for SR-aGVHD and SR-cGVHD, respectively. The 6-month-survival was 79% (67.3%-90.7%,95% CI) and 97.4% (92.3%-100%,95% CI) for SR-aGVHD and SR-cGVHD, respectively. Cytopenia and CMV reactivation were observed during ruxolitinib-treatment in both SR-aGVHD (30/54, 55.6% and 18/54, 33.3%) and SR-cGVHD (7/41, 17.1% and 6/41, 14.6%) patients. Relapse of the underlying malignancy occurred in 9.3% (5/54) and 2.4% (1/41) of the patients with SR-aGVHD or SR-cGVHD, respectively. Conclusion: Ruxolitinib constitutes a promising new treatment option for SR-aGVHD and SR-cGVHD. Its activity in SR-aGVHD and SR-cGVHD should be validated in a prospective trials in both, SR-aGvHD and cGvHD. Disclosures Bertz: GILEAD Sciences: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Scheid:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees. Bug:TEVA Oncology, Astellas: Other: Travel Grant; NordMedica, Boehringer Ingelheim, Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene, Novartis: Research Funding.

The constitutively activated chimeric Tyrosine kinase BCR-ABL is critical for initiation, progression and maintenance of chronic myelogenous leukemia (CML). Imatinib and second generation BCR-ABL tyrosine kinase inhibitors (TKIs) serve now as standard therapies for Ph+-patients. However, disease persistence occurs frequently and insensitivity of CML stem cells to TKI treatment is discussed as one major reason for this. Recent evidence accumulates, that autophagy, a genetically-regulated process of adaptation to metabolic stress, is involved in TKI-induced cell death. It is hypothesized, that TKI-induced autophagy could allow CML stem cells to become metabolically dormant enabling their survival under conditions that may mimic growth factor deprivation and thereby "antagonize" TKI-induced cell death. However, the molecular mechanism of BCR-ABL and TKI induced autophagy as well as its role as tumor suppressor or promoter is poorly understood. In our study, we aim to identify the precise role of autophagy and its´ effector molecules in a murine CML model. To test whether BCR-ABL regulates autophagy, we measured LC3 as a marker for autophagy in BCR-ABL+-K562 cell. Interestingly, inhibition of BCR-ABL activity by nilotinib led to increased LC3-II expression and punctual LC3 accumulation, indicating, that BCR-ABL activity can suppress autophagy. Consistent with this, Ba/F3 cells expressing BCR-ABL WT induce autophagy, whereas Ba/F3 cell expressing BCR-ABL-T315I fail to induce autophagy by nilotinib treatment, pointing to a BCR-ABL specific autophagy induction than an unspecific effect of TKI treatment. Next, we investigated the proteins involved in BCR-ABL mediated autophagosome formation. Recruitment of VPS34 and ATG14 to Beclin1 was increased in case of nilotinib treatment and could thereby positively regulate autophagosome formation, whereas Rubicon, a negative regulator was less recruited to the Beclin1-complex. To further identify the impact of Beclin1 as a key regulator of autophagy in BCR-ABL-driven leukemia, we used a targeted genetic approach in a CML mouse model. Interestingly, mice transplanted with Belin1 knockdown, BCR-ABL expressing bone marrow showed a less aggressive disease with significantly lower WBC-count, leukemic burden and prolonged overall survival of the mice. In contrast, deletion of ATG5, another central regulator of autophagy, was not able to change disease onset or progression in the CML model. To further clarify the function of Beclin1, we performed biochemical binding analyses and were able to show, that Beclin1 binds to BCR-ABL independent of BCR-ABL kinase activity and Beclin1 is phosphorylated by BCR-ABL. Interestingly, Beclin1 is an exclusive target of BCR-ABL and can not be phosphorylated by other aberrantly activated tyrosine kinases like Flt3-ITD, NPM-ALK and PDGFRA-D842V. In vitro kinase assay with active ABL-kinase confirm Beclin1 as a specific substrate of BCR-ABL. GST pulldown experiments mapped the N-terminal region of Beclin1 to interact with BCR-ABL. Cloning of different phospho-deficient mutants identified tyrosine residues Y233 and Y352 of Beclin1 as the crucial sites for specific BCR-ABL phosphorylation. To test the impact of BCR-ABL mediated Beclin1-phosphorylation on autophagy induction, we generated Beclin1 phospho-mimic (Y233E/Y352E) and phospho-deficient (Y233F/Y352F) mutations. Interestingly, nilotinib treatment fails to induce autophagy in cells expressing the Beclin1 phospho-mimic mutations, thereby highlighting the necessity of Beclin1 in BCR-ABL-mediated autophagy. Expression of Beclin1 mutations in Beclin1 knockout MEFs and K562 cells show decreased binding of UVRAG, ATG14 and VPS34 to Beclin1 Y233E/Y352E, suggesting an important role of Beclin1 phosphorylation for complex stabilization and autophagy suppression. Taken together our findings identify Beclin1 as a specific substrate of BCR-ABL. Downregulation of Beclin1 is associated with a prolonged overall survival of BCR-ABL transplanted animals; direct phosphorylation of Beclin1 on Tyrosine residues Y233 and Y352 lead to LC3 inhibition and suppression of autophagy. Our results thereby highlight the importance of Beclin1 in BCR-ABL-mediated leukemogenesis and show, that autophagy induction in CML cells may be rather a specific Beclin1-BCR-ABL interaction effect than a general microenvironmental stress phenomenon. Disclosures No relevant conflicts of interest to declare.