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Background: Bidirectional interactions between the tumor microenvironment (TME) and AML cells lead to disease progression through induction of angiogenesis, migration, cancer stemness and local immunosuppression. Leukemia-associated macrophages (LAM) constitute an important cell population within the TME, but little is known about the phenotype, function, and plasticity of these cells. In the present study we provide an extensive characterization of the macrophage population in patients with AML. Methods: The phenotype and expression of co-regulatory receptors was assessed on different bone marrow-derived CD68 +CD14 + LAM populations, in comparison to corresponding CD3 + T-cells and CD117 +CD34 + AML cells (n=35), as well as peripheral blood monocytes from healthy donors (HD, n=16) using multi-parameter flow cytometry. The expression of surface markers and the distribution of LAM subpopulations was correlated with clinical parameters. The effect of a blocking anti-TIGIT antibody on the in vitro plasticity on primary LAMs and monocyte-derived macrophages from healthy donors was investigated. Furthermore, we analyzed if the treatment with blocking anti-TIGIT and anti-CD47 antibodies could increase the anti-leukemic phagocytosis of AML cell lines and in vitro polarized monocyte-derived M2 macrophages. Results: Phenotypic analysis of M1 and M2 macrophages in AML and HD revealed that the predominant macrophage population in patients with AML is made up of immunosuppressive alternatively activated M2 LAMs defined by expression of CD163 and CD86 (M1 AML vs. HD p Importantly, in vitro blockade of TIGIT in primary LAMs of AML patients or differentiated PB-derived M2 macrophages of HDs resulted in a change in polarization from the M2 towards the M1 phenotype after 24 hours (AML: anti-TIGIT vs. IgG2a p Moreover, the additional blockade of TIGIT on PB-derived M2 macrophages augmented the anti-CD47-mediated phagocytosis of the AML cell lines MOLM-13 and MV4-11 after 4 hours (MOLM-13: anti-CD47 vs. IgG1a 31% vs. 10.9%, p=0.04; anti-CD47 vs. combined anti-CD47 + anti-TIGIT 31% vs. 46.4%, p Next, we correlated the phenotypic data with clinical parameters. AML patients of the intermediate risk group according to ELN criteria exhibited a significantly higher frequency of M2 LAMs co-expressing TIGIT and LAG-3 than those in the favorable group (p=0.04 and p=0.01). Moreover, the frequency of TIM-3 + M2 LAMs was significantly increased in patients with adverse and intermediate risk in comparison to those with a favorable risk (p=0.01, p=0.0053). Furthermore, TIGIT + M2 LAMs were significantly more frequent in patients with the FLT3 ITD mutation in comparison with the wilde type (p=0.03). Conclusions: Our findings suggest that the proven clinical effect of monoclonal antibodies against TIGIT and TIM-3 in cancer may be due in part to their action on macrophages and depend on macrophage polarization. Our study identifies TIGIT + M2 LAMs co-expressing TIM-3 and LAG-3 as a promising effector population in AML. Further experiments should be conducted to investigate macrophage-mediated cytotoxicity in AML. Disclosures Brauneck: Daiichi Sankyo: Consultancy, Honoraria, Other: meeting attendance; Servier: Consultancy, Honoraria, Other: meeting attendance; Jazz Pharmaceuticals: Other: meeting attendance; Novartis: Other: meeting attendance. Bokemeyer: BMS: Honoraria, Other: Travel accomodation, Research Funding; Sanofi: Consultancy, Honoraria, Other: Travel accomodation; Merck Serono: Consultancy, Other: Travel accomodation ; Bayer Schering Pharma: Consultancy; GSO: Consultancy; AOK Health insurance: Consultancy; Abbvie: Research Funding; ADC Therapeutics: Research Funding; Agile Therapeutics: Research Funding; Alexion Pharmaceuticals: Research Funding; Amgen: Research Funding; Apellis Pharmaceuticals: Research Funding; Astellas: Research Funding; BerGenBio: Research Funding; Blueprint Medicine: Research Funding; Boehringer Ingelheim: Research Funding; Celgene: Research Funding; Daiichi Sankyo: Research Funding; Eisai: Research Funding; Gilead Sciences: Research Funding; Gylcotope GmbH: Research Funding; GlaxoSmithKline: Research Funding; Inside: Research Funding; IO Biotech: Research Funding; Isofol Medical: Research Funding; Janssen-Cilag: Research Funding; Karyopharm Therapeutics: Research Funding; Lilly: Research Funding; Millenium: Research Funding; MSD: Research Funding; Merck KGaA: Honoraria; Bayer: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Merck Sharp Dohme: Consultancy, Honoraria; AstraZeneca: Honoraria, Research Funding; Lilly/ImClone: Consultancy; Nektar: Research Funding; Rafael Pharmaceuticals: Research Funding; Springworks Therapeutics: Research Funding; Taiho Pharmaceutical: Research Funding; Pfizer: Other. Fiedler: Celgene: Consultancy; Servier: Consultancy, Other: support for meeting attendance; Abbvie: Consultancy, Honoraria; Morphosys: Consultancy; Pfizer: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Other: support for meeting attendance; Jazz Pharmaceuticals: Consultancy, Other: support for meeting attendance; Stemline: Consultancy; Novartis: Consultancy; ARIAD/Incyte: Consultancy; Amgen: Consultancy, Other: support for meeting attendance, Patents & Royalties, Research Funding.

Immune checkpoint therapy has revolutionized the treatment of patients with cancer. Checkpoint receptors and their ligands play an important role in T cell activation and exhaustion and are currently the focus in understanding the antitumoral immune responses. In patients with acute myeloid leukemia (AML) limited data have been published that comprehensively describe the expression of checkpoint receptors on different T cell subsets. We performed multicolor flow cytometry on peripheral blood mononuclear cells (PB, PBMCs) from patients with newly diagnosed AML (n=20) and PBMCs from age matched healthy donors (HDs; n=12), focusing on differentiation, the clinically actionable exhaustion receptor T cell immunoglobulin and ITIM domain (TIGIT), the two ectoenzymes ectonucleoside triphosphate diphosphohydrolase-1 (CD39) and ecto-5′-nucleotidase (CD73). Our studies included also analysis of interleukin-7 receptor-α (CD127) and the intracellular expression of the transcription factor T cell factor 1 (TCF-1). Both markers are known to be expressed in long living memory CD8+ T cells and harboring the ability for self-renewal. The thymocyte selection-associated high mobility group box protein (TOX) was also analyzed, since this molecule has recently been described as regulator of CD8+ T cell exhaustion. Comparison of PB from patients with newly diagnosed AML vs. HDs revealed that the frequency of CD8+, CD4con (CD4+CD127+CD25-) and CD4reg (CD4+CD127-CD25+) T cells was similar among both groups. However, the frequency of CD8+ EMRA T cells (CCR7- CD45RO- CD8+ CD3+) was increased in PB from patients with AML compared to PB from HDs (39,05 ± 4,38 vs. 14,29 ± 3,83; p TIGIT, CD39 and CD73 emerged as checkpoints of interest on CD8+ T cells. The frequency of TIGIT+ CD8+ T cells and CD39+ CD8+ T cells in PBs from patients with newly diagnosed AML was increased compared with that in HDs (42,60 ± 4,67 vs. 20,36 ± 3,68; p=0,00 and 6,54 ± 2,05 vs. 1,31 ± 0,32; p=0,05). Whereas reduced frequency of CD73+ CD8+ T cells occurred in PB from patients with AML vs. HD (41,35 ± 4,75 vs. 66,18 ± 7,28; p=0,01). Analysis between TIGIT and CD73 expression showed inverse correlation between both targets in AML (r=-0,53; p=0,01). The frequency of the TIGIT+ CD73- CD8+ T cell population was increased in AML (36,85 ± 5,17, vs. 16,23 ± 5,09 ;p=0,01). This increased frequency of TIGIT+ CD73- cells in AML was related to EM (CCR7- CD45RO+ CD8+ CD3+)T cells (42,89 ± 4,78 vs. 25,27 ± 3,39; p=0,01) and EMRA CD8+ T cells (56,01 ± 5,68 vs. 36,54 ± 4,85; p=0,03). Moreover, CD39 was aberrantly expressed on this population: we observed an increased frequency of CD39+ TIGIT+ CD73- CD8+ T cells in PB from patients with newly diagnosed AML compared to PB from HDs (13,47 ± 4,03 vs. 3,03 ± 1,21; p=0,02). Next we focused on CD127 and TCF-1 which are involved in creating long living antigen independent memory CD8+ T cells and ability to self-renewal while producing differentiated effector cells. Comparing expression of CD127 and TCF-1 on CD39+ TIGIT+ CD73- CD8+ T cells showed a significantly decreased frequency of CD127 (17,73 ± 2,16, vs. 30,72 ± 7,12; p=0,04) and TCF-1 (14,67 ± 2,90 vs. 39,74 ± 9,32; p=0,03) in PB from patients with newly diagnosed AML compared to HDs. Expression of TIGIT and TCF-1 inversely correlated in AML (r =-0,87; p To further evaluate the exhaustion status of TIGIT+ CD73- CD8+ T cells we examined the expression of TOX, recently described as one of the key regulators governing CD8+ T cell exhaustion, the frequency of TOX+ cells was increased in AML (50,57 ± 8,21 vs. 22,14 ± 4,86; p=0,01). Analysis of co-expression showed that the TOX+ CD39+ TIGIT+ CD73- CD8+ population was significantly increased in PB from patients with newly diagnosed AML compared to their counterparts in PB from HDs (21,81 ± 3,14 vs. 3,84 ± 1,10; p=0,04). In summary, we could show that in PB from patients with newly diagnosed AML an aberrant cell population of CD39+ TIGIT+ CD73- CD8+ T cells is prevalent in contrast to the PB from HDs. In this cell population we observed elevated expression of TOX which has recently described as one of the key regulators governing CD8+ T cell exhaustion. In contrast downregulation of CD127 and TCF-1 was found in these cells. These data might contribute to CD8+ T cell exhaustion in AML and support further functional analysis to investigate the relevance of combinatorial inhibition of TIGIT and CD39. Disclosures Brauneck: Daiichi Sankyo: Consultancy, Honoraria, Other: support for meeting attendance; Novartis: Other: support for meeting attendance; Jazz Pharmaceuticals: Other: support for meeting attendance. Bokemeyer:Merck KGaA: Honoraria; Janssen-Cilag: Research Funding; Roche: Honoraria, Research Funding; Bayer: Honoraria, Research Funding; Taiho Pharmaceutical: Research Funding; Pfizer: Other; Karyopharm Therapeutics: Research Funding; Millenium: Research Funding; MSD: Research Funding; Nektar: Research Funding; Novartis: Research Funding; Rafael Pharmaceuticals: Research Funding; Springworks Therapeutics: Research Funding; Sanofi: Consultancy, Honoraria, Other: travel accomodations; Bristol-Myers Squibb: Honoraria, Other: travel accomodations, Research Funding; AstraZeneca: Honoraria, Research Funding; Merck Sharp & Dohme: Consultancy, Honoraria; Lilly/ImClone: Consultancy, Research Funding; Merck Serono: Consultancy, Other: travel accomodations; Bayer Schering Pharma: Consultancy; GSO: Consultancy; AOK Health Insurance: Consultancy; Abbvie: Research Funding; ADC Therapeutics: Research Funding; Agile Therapeutics: Research Funding; Alexion Pharmaceuticals: Research Funding; Amgen: Research Funding; Apellis Pharmaceuticals: Research Funding; Astellas: Research Funding; BerGenBio: Research Funding; Blueprint Medicines: Research Funding; Boehringer Ingelheim: Research Funding; Celgene: Research Funding; Daiichi Sankyo: Research Funding; Eisai: Research Funding; Gilead Sciences: Research Funding; Glycotope GmbH: Research Funding; GSK: Research Funding; Incyte: Research Funding; IO Biotech: Research Funding; Isofol Medical: Research Funding. Fiedler:Amgen: Consultancy, Honoraria, Other: support for meeting attendance, Patents & Royalties, Research Funding; ARIAD/Incyte: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Morphosys: Consultancy, Honoraria; Abbvie: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Honoraria, Other: support for meeting attendance; Gilead: Other: support for meeting attendance; Daiichi Sankyo: Other: support for meeting attendance.

Aberrant activation of hedgehog signaling (HH) is associated with a wide variety of neoplasms, including acute myeloid leukemia (AML). The GLI transcription factors are the main downstream effectors of the HH signaling cascade and play a fundamental role in cancer development, progression and maintenance of leukemic stem cells, which are responsible for therapy failure and tumor relapse due to their resistance to chemotherapy. Moreover, the GLI transcription factors represent central hubs in oncogenic signaling and thus are an attractive therapeutic target for anti-cancer therapy. However non-canonical GLI activation by multiple oncogenic signaling pathways limit the use of SMO inhibitors, while treatment strategies directly targeting the GLI transcription factors are limited. Mebendazole (MBZ) is a commonly used anthelmintic drug with a favorable toxicity profile. MBZ has been shown to exhibit strong anti-tumor effects in different cancer entities, including AML. We treated AML cell lines and primary AML samples with different MBZ concentrations and could show a dose-dependent effect on the proliferation, colony formation and apoptosis in AML cells. In addition, therapeutic synergy between cytarabine and MBZ was demonstrated in OCI-AML3 cells with a combination index To further evaluate if MBZ is a suitable GLI inhibitor in clinical practice, we transferred these findings into the clinical setting by treating two patients with refractory AML with MBZ monotherapy in an off-label setting. We demonstrated an effective MBZ concentration in the plasma using a modified plasma inhibitory assay (PIA) by incubating an indicator cell lines carrying the GLI luciferase promoter transgene with the patient's plasma. In one patient, a clear continuous decrease in leukemic blasts in peripheral blood was noted. In this patient a reduction in the luciferase activity in the PIA assay and a fast reduction in GLI2 levels in peripheral leukemic blood were detected. Moreover, a healthy volunteer ingested MBZ at a dose of 50 mg/kg divided in two doses at time 0h and 12h. Blood was drawn at 4h and at 24h. PIA results indicated a biological active plasma concentration. Taken together, our results demonstrate that MBZ functions as an effective GLI inhibitor with strong anti-leukemic activity in a clinical setting and encourage for further studies to translate the scientific findings into clinical practice. A clinical study of mebendazole plus low dose cytarabine in patients with refractory AML is planned. Figure Disclosures Stamm: AstraZeneca: Employment. Modemann:Servier, Incyte, Gilead, Jazz Pharmaceuticals, Novartis, Teva, Pfizer, Amgen: Other: Support for meeting attendance ; Servier: Honoraria; Daiichi Sankyo: Research Funding. Fiedler:Amgen, Pfizer, Abbvie: Other: Support in medical writing; Amgen: Research Funding; Amgen, Pfizer, Novartis, Jazz Pharmaceuticals, Ariad/Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen, Jazz Pharmaceuticals, Daiichi Sanchyo Oncology, Servier: Other: Support for meeting attendance. OffLabel Disclosure: Mebendazole is an approved oral broad-spectrum anthelmintic drug for the treatment of worm infections. Data from preclinical studies demonstrated strong anti-tumor effects. We used it as an antineoplastic drug in a compassionate and off-label setting for treatment of two AML patients.

The relevance of the Hedgehog signaling pathway in the pathophysiology of acute myeloid leukemia (AML) has been demonstrated by us and others. Inhibition of the downstream Hedgehog transcription factors GLI1 and GLI2 results in strong anti-leukemic effects. Therefore, Hedgehog pathway inhibitors represent a promising therapeutic approach in AML. Mebendazole is an anthelmintic drug commonly used for the treatment of various parasitic worm infections. Recently, mebendazole has been shown to exhibit strong anti-tumor effects in different cancer entities including AML. In the work presented here, we investigated the effect of mebendazole on expression and activity of GLI transcription factors and its anti-leukemic activity. To determine the effect of mebendazole on GLI transcription factors, we treated the AML cell lines MV4-11, MOLM-13, THP-1 and OCI-AML3 with different concentrations of mebendazole and analyzed its impact on GLI1 and GLI2 protein- and mRNA levels. Furthermore, GLI reporter assays (Cignal GLI Reporter (luc) Kit, Qiagen) were performed to determine the effect of mebendazole on the GLI1 and -2 transcriptional activity. Mebendazole strongly inhibited GLI1 and GLI2 signaling activity in a dose-dependent manner. Exemplarily, treatment with 500 nM mebendazole reduced the GLI1 and -2 transcriptional activity in all cell lines tested by 54.8 % (± 9.6) after 24h and 73.2 % (± 11.6) after 48h. We could demonstrate by Western Blotting that GLI1 and -2 protein levels were clearly reduced 24h and 48h after mebendazole exposure, whereas GLI1 and -2 mRNA levels did not decrease. These data suggest that mebendazole may increase degradation of GLI proteins via the proteasome pathway. Therefore, we evaluated the influence of the 26s proteasome inhibitor bortezomib on GLI levels after mebendazole treatment. Inhibiting the 26s proteasome with 2 nM, 5 nM and 10 nM of bortezomib increased GLI signaling activity by 13.6 % (± 8.0), 84.6 % (± 39.2) and 137.1 % (± 37.9), respectively. Furthermore, 10 nM bortezomib abolished the effect of mebendazole on GLI protein levels. Taken together, mebendazole increased the proteasomal degradation of GLI1 and GLI2. These observations were extended to samples from AML patients. After mebendazole treatment for 24h or 48h all analyzed patients had reductions of GLI1 protein levels as confirmed by Western blotting (n=4), whereas GLI1 and GLI2 mRNA levels were not changed (n=7), indicating that proteasomal degradation was operational in primary blasts as well. Evaluating the anti-leukemic effects of mebendazole, we also investigated its combination with the small molecule GLI inhibitor GANT61. We treated the AML cell lines MV4-11, MOLM-13, THP-1 and OCI-AML3 with combinations of mebendazole and GANT61 and analyzed cell proliferation, apoptosis and colony formation. Mebendazole treatment alone already resulted in decreased proliferation and colony forming capacity as well as increased apoptosis rates in a dose-dependent manner. The combination of mebendazole with the GLI inhibitor GANT61 synergistically increased the anti-proliferative effects of mebendazole on all 4 AML cell lines tested. Additionally, GANT61 further increased the effect of mebendazole on colony formation significantly. Incubation with 100 nM, 200 nM and 500 nM mebendazole inhibited the proliferation of primary blasts from AML patients by 15.1 % (± 7.5), 31.6 % (± 16.8) and 66.0 % (± 17.4), respectively (n=8). Moreover, the combination with GANT61 significantly increased these anti-proliferative effects. This work indicates that mebendazole exerts profound anti-leukemic effects by decreasing GLI1 and GLI2 intracellular levels by promoting its proteasomal degradation. Combining mebendazole with GLI1 and GLI2 inhibitors such as GANT61 enhances this effect considerably. These observations may lead to the introduction of novel treatment strategies in AML. Disclosures Stamm: Amgen Research (Munich) GmbH / Amgen Inc.: Patents & Royalties; Astellas GmbH: Other: Travel Grant. Wellbrock:Amgen Research (Munich) GmbH: Patents & Royalties. Fiedler:GSO: Other: support for meeting attendance; Gilead: Other: support for meeting attendance; Amgen: Other: support for meetíng attendance; Pfizer: Research Funding; Amgen: Research Funding; Amgen: Patents & Royalties; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; ARIAD/Incyte: Membership on an entity's Board of Directors or advisory committees, support for meeting attendance; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Other: support for meeting attendance; JAZZ Pharmaceuticals: Other: support for meeting attendance; Teva: Other: support for meeting attendance.

Leukemia-initiating cells reside within the bone marrow (BM) in specialized niches where they undergo complex interactions with their surrounding stromal cells. In order to identify genes being implicated in the interaction of acute myeloid leukemia (AML) cells and stromal cells, we performed co-cultures of primary AML cells with primary endothelial cells and osteoblasts. The gene expression of co-cultured AML blasts was compared to AML cells grown without adherent cells using microarray analysis. Amongst those genes being dysregulated upon co-culture was the actin binding protein plastin-3 (PLS3). Further RT-qPCR analysis revealed an endogenous PLS3 expression in about 50% of BM samples from AML patients (n=25). In contrast, expression of PLS3 was only detected in 2 of 12 analyzed AML cell lines with Kasumi-1 showing strong and THP-1 showing only weak expression. Therefore, functional analysis of PLS3 in AML was studied using shRNA knockdown and overexpression of PLS3 in Kasumi-1 cells. We could show that PLS3 has an impact on the colony formation capacity of AML cells in vitro as the knockdown resulted in significantly reduced colony numbers while increased colony growth was observed in the Kasumi-1 cells overexpressing PLS3 (p Finally, we investigated whether the expression of PLS3 was associated with AML patients' outcome using published microarray-based gene expression data (Verhaak et al, Haematologica 2009;94). Clinical data of 290 AML patients were available. Based on the mean gene expression value, the patient cohort was divided into high vs low PLS3 expressors. The overall survival was analyzed in a multivariate Cox proportional hazards model including PLS3 gene expression and the baseline parameters age, karyotype and FLT3 mutational status. After a stepwise removal of insignificant terms, the patient's age and a high PLS3 expression remained as independent prognostic survival markers (for PLS3: HR 1.58 (CI 1.05 - 2.37) and for age: HR 1.01 (CI 1.00 - 1.03)). In conclusion, our results identify the actin binding protein PLS3 as potential novel therapeutic target in AML. Disclosures Stamm: Astellas: Other: Travel, Accommodation, Expenses. Heuser:BerGenBio: Research Funding; Tetralogic: Research Funding; Novartis: Consultancy, Research Funding; Celgene: Honoraria; Bayer Pharma AG: Research Funding; Pfizer: Research Funding; Karyopharm Therapeutics Inc: Research Funding. Fiedler:Kolltan: Research Funding; Ariad/Incyte: Consultancy; Novartis: Consultancy; Gilead: Other: Travel; Teva: Other: Travel; GSO: Other: Travel; Pfizer: Research Funding; Amgen: Consultancy, Other: Travel, Patents & Royalties, Research Funding. Wellbrock:Astellas: Other: Travel, Accommodation, Expenses.

In this issue of Blood , Jiang et al identify the cell adhesion molecule CD146 as novel co-receptor for vascular endothelial growth factor receptor 2 (VEGFR2).[1][1] The cell adhesion molecule CD146 was first described in 1987 because of its expression on malignant melanocytes and was correlated

Background: T-cell activity is regulated by immune checkpoints to maintain the sensitive balance of co-stimulatory and inhibitory immune signals. Therapeutic blockade of checkpoint molecules on tumor or T cells such as CTLA-4 or PD-L1 has shown clinical success in several tumor types including Hodgkin´s disease. Furthermore, Blinatumomab, a bispecific T-cell engager (BiTE antibody construct) directing cytotoxic T cells to CD19 positive leukemic cells has been approved for treatment of acute lymphoblastic leukemia. The CD33 specific BiTE antibody construct AMG 330 has been developed for the therapy of acute myeloid leukemia (AML) and will be evaluated in phase I studies shortly. In our current study, we investigated the therapeutic utility of blockade of the novel checkpoint proteins PVR (poliovirus receptor) and PVRL2 (poliovirus receptor-related 2) alone and in combination with AMG 330 in AML. Methods and results: Samples from 140 treatment naive patients with newly diagnosed AML (AMLSG 07-04, NCT00151242) were analyzed by RT-qPCR for expression of the immune checkpoint molecules PVR, PVRL2 and Galectin-9 (Gal-9). Expression was correlated with patient demographics (age, karyotype, FLT3 mutation status) and clinical survival data by multivariate cox regression. The majority of patients showed mRNA expression of PVR (94%), PVRL2 (95%) and Gal-9 (92%). In a multivariate stepwise cox regression for overall survival, an unfavorable karyotype, high PVR and high Gal-9 expression were identified as independent prognostic markers (p In a second, independent patient cohort containing microarray-based gene expression and clinical data of 291 AML patients (Verhaak et.al., Haematologica 2009;94) a high PVR and PVRL2 expression in contrast to expression of CD80, CD86 or PD-L1 was associated with poor overall survival (log-rank test p=0.003 and p=0.032, respectively). In in vitro killing assays the therapeutic effect of PVR and PVRL2 blockade was studied by FACS using 7-AAD staining. AML cell lines MV4-11, Kasumi-1 and Molm-13 were preincubated with blocking antibodies against PVR, PVRL2 or both and co-cultured for 24h with peripheral blood mononuclear cells (PBMCs) of healthy donors in the presence or absence of AMG 330. In the absence of AMG 330, the cell kill of MV4-11 increased from 12.6±4.7% (control) to 33.0±8.8% (PVR), to 40.4±10.4% (PVRL2) and to 56.0±12.0% (both PVR + PVRL2). In the presence of suboptimal concentration of AMG 330 (0.1 ng/ml) MV4-11 cell lysis was 29.4±9.0% (AMG 330 alone), 49.7±12.6% (AMG 330 + PVR), 57.9±11.3% (AMG 330 + PVRL2) and 70.0±9.8% (AMG 330 + PVR + PVRL2; n=4, p Conclusion: The expression of immune checkpoint ligands PVR and PVRL2 confers a negative prognosis to AML patients possibly due to immune evasion. We could further show that the killing of AML cells by PBMCs could be augmented by blockade of these novel checkpoint inhibitors. Furthermore, addition of PVR and/or PVRL2 blocking antibodies to AMG 330 could enhance cytotoxicity. Therefore, blockade of PVR and PVRL2 represents a promising target for the treatment of AML. Disclosures Kischel: Amgen Research (Munich) GmbH: Employment. Stienen:Amgen Research (Munich) GmbH: Employment. Friedrich:Amgen Research (Munich) GmbH: Employment. Lutteropp:Amgen Research (Munich) GmbH: Employment. Nagorsen:Amgen: Employment, Equity Ownership, Patents & Royalties: Inventor on blinatumomab-related patent.

Background Leukemic stem cells depend on signals provided by bone marrow (BM) niche cells. Due to its function in stem cell biology, we investigated expression and prognostic impact of the Hedgehog pathway in acute myeloid leukemia (AML). Methods and results Pre-treatment samples from 104 patients with newly diagnosed AML (AMLSG 07-04 trial) were analyzed by quantitative PCR. Expression of receptors Smoothened and Patched and downstream transcription factors Gli1, Gli2 and Gli3 was found in 69%, 41%, 73%, 20% and 26% of cases, respectively. However, no expression of Hedgehog ligands was observed. Gli2 expression had a significant influence on event-free, relapse-free and overall survival (p=0.037, p=0.026 and p=0.013, respectively) and was furthermore correlated to FLT3 mutational status (p To verify our findings in a second, independent patient cohort, microarray-based gene expression data of patients published by Valk et.al.(N Engl J Med. 2004 Apr 15;350(16):1617-28) was used. Clinical data of 291 AML patients from this cohort were available and the negative prognostic impact of Gli2 expression on overall and event-free survival could be confirmed (p=0.003 for overall and p=0.007 for event-free survival, respectively; no data for relapse-free survival available). Additionally, the correlation of Gli2 expression to FLT3 mutational status was also observed in this patient cohort (p=0.003). Although Hedgehog ligands were not expressed in AML bone marrow aspirates, Desert Hedgehog (DHH) plasma levels were significantly increased in AML patients compared to healthy subjects (p=0.002). Provision of Desert Hedgehog could be ascribed to BM niche cells including endothelial cells and osteoblasts, since DHH expression was found in outgrowth endothelial cells (OECs) and osteoblasts in vitro and on immunohistochemistry of leukemic bone marrow samples in patients. Next, we investigated the effect of Gli inhibition in vitro. AML cell lines KG-1, OCI-AML5 and UKE-1 as well as freshly isolated primary AML cells were treated with the specific Gli inhibitor GANT61. GANT61 dose-dependently induced apoptosis in all three cell lines investigated. Furthermore, GANT61 treatment resulted in significantly increased apoptosis in primary AML cells upon treatment with 10 µM, 30 µM and 90 µM GANT61 (n=10 pts). Additionally, a pronounced growth inhibitory effect on primary AML cells was observed with a significant reduction in cell count to 82 ± 13%, to 54 ± 11% and to 47 ± 13% upon treatment with 10 µM, 30 µM and 90 µM of GANT61, respectively (n=9 pts). Incubation of KG-1, UKE-1 and OCI-AML5 cells for 7 days with 30 µM GANT61 resulted in reductions in cell numbers compared to the control to 14 ± 2%, to 44 ± 6% and to 31 ± 0%, respectively. Moreover, GANT61 mediated a strong and significant inhibitory effect on the colony formation capacity of AML cell lines and primary AML cells. The average number of colonies was reduced to 44 ± 16% and 4 ± 4% upon treatment with 10 µM and 30 µM GANT61, respectively, while no colony formation was observed with a concentration of 90 µM GANT61 in primary AML cells (n=4 pts). Conclusion In conclusion, Gli2 expression is a negative prognostic factor in AML that mirrors activated Hedgehog signaling induced by paracrine stimulation through BM niche cells. Inhibition of Hedgehog signaling by blocking Gli activity might represent a promising therapeutic approach for AML treatment with special regard to patients carrying a mutated FLT3. Disclosures: Fiedler: Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding.

Abstract 5189 Background: Pulmonary arterial hypertension (PAH) is a severe and life-threatening disease. It is characterized by excessive growth of pulmonary artery endothelial and smooth muscle cells leading to a profound pulmonary artery remodeling and consequently increased pulmonary artery pressure and vascular resistance. Most patients with the heritable form of PAH harbor a mutation in the bone morphogenic protein (BMP) receptor 2 (BMPR2) resulting in dysregulated BMP signaling. In addition, aberrant BMP signaling was also observed in the idiopathic form of PAH although the underlying molecular mechanisms have not been elucidated. Recently, it was shown that BMP antagonist Gremlin-1 was elevated in pulmonary vessels of mice during development of hypoxic pulmonary hypertension (Cahill et al, Circulation. 2012;125(7):920–30). Methods and Results: The aim of this prospective study was to investigate the plasma levels of Gremlin-1 in PAH patients (Dana point classification group I) and to correlate Gremlin-1 levels to clinical and hemodynamic parameters. Thirty subjects were included in the study (19 patients with PAH treated at the PH clinics of the University Medical Center Hamburg-Eppendorf, Germany and 11 healthy volunteers) after giving informed consent. The mean Gremlin-1 plasma level was 2. 6-fold increased with 333 ± 160 ng/ml, in patients with pulmonary arterial hypertension compared to those of healthy control subjects with a mean Gremlin-1 plasma level of 118 ± 115 ng/ml (p=0. 001 in t-test). Gremlin-1 plasma levels of PAH patients were correlated to demographic, clinical and hemodynamic parameters including age, sex, 6-minute walk distance, systemic and pulmonary blood pressure & vascular resistance, lung function testing, NT-proBNP (N terminal pro-brain natriuretic peptide) and NYHA/WHO functional classification. A positive correlation between Gremlin-1 plasma levels and NT-proBNP plasma levels was observed (Spearman Rho 0. 809 with p Conclusion: The plasma levels of BMP antagonist Gremlin-1 are significantly elevated in patients with pulmonary arterial hypertension and may serve as new serological marker. Gremlin-1 might mirror the state of BMP dysregulation and represent a potential follow up marker under a future targeted therapy. Furthermore, since Gremlin-1 was shown to induce proliferative effects on both endothelial as well as smooth muscle cells, it might also contribute directly to the aberrant vessel growth observed in PAH. Gremlin-1 plasma levels of patients with pulmonary hypertension (n=19) were analyzed in an enzyme-linked immunosorbent assay. Compared to healthy subjects (n=11), mean plasma levels of Gremlin-1 were 2. 6-fold increased in PH patients (t-test p=0. 001). Box plots show the median (center horizontal line), the 25th to the 75th percentile (box) and the range (whiskers).** indicates p Disclosures: Hennigs: Bayer: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Actelion: Research Funding; GlaxoSmithKline: Honoraria; Novartis: Honoraria. Fiedler:Pfizer Inc. : Consultancy, Research Funding; Novartis: Consultancy, Research Funding.

Abstract 3269 The Loeys-Dietz syndrome (LDS) is an inherited connective tissue disorder with symptoms similar to those of Marfan syndrome and the vascular type of Ehlers-Danlos syndrome. Most patients with LDS develop severe aortic aneurysms resulting in early need of surgical intervention. Patients with LDS harbour a mutation in the transforming growth factor β (TGF-β) receptors TGFBR1 (also named ALK-5) or TGFBR2. Since the TGF-β pathway plays a crucial role in many cellular processes including angiogenesis, we focussed our analyses on endothelial cell dysfunction in patients with Loeys-Dietz syndrome. We isolated circulating outgrowth endothelial cells (OEC) from the peripheral blood of two LDS patients (one female, 54 years; one male, 26 years old) both harbouring a mutation in the TGFBR2 gene. Gene expression profiles of OEC clones were performed using Affymetrix Human Genome U133 Plus 2.0 Arrays and confirmed by quantitative PCR analysis for genes of interest. OEC clones isolated from age- and sex-matched healthy controls served as reference subjects. We demonstrate that several genes belonging to the TGF-β pathway had altered expression in OECs isolated from LDS patients compared to those from healthy controls. For example, mRNA levels of bone morphogenic proteins (BMP) 2 and 4 were decreased in both LDS OEC clones (mean decrease 4 and 6 fold, respectively) whereas gene expression of inhibitory downstream molecule SMAD-6 was increased 2-fold. In both analysed OEC clones from LDS patients, gene expression of BMP antagonist Gremlin-1 (also known as Drm) showed the most prominent dysregulation with a 1136-fold and 164-fold higher expression in LDS OECs compared to healthy controls, respectively. Interestingly, in OECs isolated from healthy donors, Gremlin-1 expression was significantly down-regulated after incubation with SB431542 (5 μM), a small molecule inhibitor of the TGF-β receptor complex (mean decrease 4 fold; t-test: p = 0.002; n = 6). In contrast, the stimulation of OEC clones with TGF-β1 (1 ng/ml) resulted in significant up-regulation of Gremlin-1 mRNA levels (mean increase 7 fold; t-test: p = 0.014; n = 6). Apparently, the up-regulation of Gremlin-1 in LDS OECs seems to mirror an activated TGF-β signalling cascade in outgrowth endothelial cells. These findings are in line with other studies published on LDS where hyperactivity of the TGF-β downstream signalling was demonstrated by higher phosphorylation levels of SMAD-2 in the aortic media of LDS patients (Loeys et al., Nat Genet. 2005 Mar;37(3):275–81). Gremlin-1 might represent a second gene supporting the concept of increased TGF-β signalling in Loeys-Dietz syndrome. Gremlin-1 itself displays opposing effects on angiogenesis. First, it is known as a pro-angiogenic factor and was recently shown to stimulate angiogenesis via direct binding to the VEGF receptor 2 (Mitola et al., Blood. 2010 Nov 4;116(18):3677–80). On the other hand, as antagonist of bone morphogenic proteins, Gremlin-1 possesses anti-angiogenic properties by suppressing pro-angiogenic effects of BMP-2 and BMP-4. In summary, we believe that due to its drastic up-regulation in OECs of LDS patients, Gremlin-1 represents a crucial effector of dysregulated TGF-β signalling in endothelial cells inducing vascular pathology in Loeys-Dietz syndrome. Disclosures: Fiedler: Pfizer: Research Funding.