Search

Background: DNA-hypomethylating agents (HMAs) show an encouraging but not yet well-understood activity in AML/MDS patients (pts) with adverse cytogenetics, such as -7/del(7q) embedded in a complex, monosomal karyotype (MK). We have noted that MK pts with multiple monosomies responded better to DAC treatment than pts with only one monosomal autosome (Blum, Greve and Lübbert, Curr Opin Hematol. 2017). This important characteristic of HMAs differentiates them from cytarabine (AraC, lacking DNMT-inhibitory activity) as shown in randomized studies with DAC (Wierzbowska et al., Am. J. Hematology 2018) and AZA (Döhner et al., Leukemia 2018). The mechanism of action is currently under investigation and may involve preferential gene reactivation on monosomic chromosomes by DAC (Greve et al., ASH Annual Meeting 2017, #2612). In order to address this important difference between DAC and AraC, we compared their antileukemic activity in PDX models representing cytogenetically normal (CN) AML and adverse cytogenetics AML (complex, monosomal karyotypes including del(7q) and 1 or more monosomies). Materials and Methods: PDX models were generated for 5 CN and 3 MK AML pts with a median age of 68 years (range 55-80), a median WBC of 3.0 x 103/µl (range 1.4-139.7) and a median of 42% bone marrow (BM) blasts (range 5-97). 7/8 pts were sampled at diagnosis, i.e. prior to first-line treatment (3 pts received DAC, 3 pts induction and 1 pt upfront allografting); 1/8 pt had had multiple relapses. T-cell depleted peripheral blood or bone marrow cells (3x106 cells/mouse) were injected into NSG (NOD/Shi-scid/IL-2Rγnull) mice. Leukemic cell engraftment was determined by flow cytometry (FC) in BM, peripheral blood (PB) and spleen during the course of the experiment and at the end of a study. Leukemic cells were transplanted serially at least 4-5 times to propagate and ensure full leukemic potential of the cells. Mice were treated with low-toxic concentrations of DAC (1 mg/kg/day given intraperitoneally), AraC (15 mg/kg/day given intravenously) or vehicle (PBS) for 5 consecutive days (one cycle). Drug doses were titrated to be equitoxic and as potent as possible without causing side effects. Treatment commenced upon disease onset, as evaluated by FC positivity for human CD33, increased WBC in PB and weight loss. Overall survival served as the read-out. The study was carried out in accordance with the recommendations by the Society of Laboratory Animal Science in an AAALCA accredited animal facility. The animal experiments were approved by the regional council (Regierungspräsidium Freiburg, ref. 35, permit no. G-12/86). Results: We successfully established well-characterized PDX from pts with CN (n = 5), and with MK (n = 3) with a median of 2 autosomal monosomies (range 1-6; of chromosomes 4, 6, 7 [2x], 15, 17 [2x], 18, 21). TP53 (mutational status available for 6/8 pts) was wildtype in 4 CN pts and 1 MK pt, and mutated in 1 MK pt. All models were treated with either DAC, AraC or vehicle. Across both cytogenetic cohorts, DAC treatment significantly prolonged median survival by 36 days when compared to vehicle administration (p = 0.0002, see Fig 1A). Specifically, DAC increased median survival of CN PDX by 33 days (p = 0.0104, range 61-105 days with DAC, compared to 43-65 days with vehicle, Fig 1B), and in MK PDX by 40 days (p = 0.0197; range 72-105 days with DAC, compared to 39-50 days with vehicle, Fig 1C). Notably, disease progression in two DAC-treated PDX (one CN, one MK with 6 monosomies) was delayed to 105 days (Fig 1, marked with ⋆). AraC treatment also resulted in longer median survival across both cohorts compared to vehicle, albeit not as pronounced as with DAC, by an average of 20 days (p = 0.0066). In more detail, AraC increased median survival of CN PDX by 19 days (p = 0.0025, range 53-82 days), and, though not significantly, of MK PDX by 15 days (p = 0.2048, range 46-92 days). Conclusion: In this PDX study recapitulating two different cytogenetic risk groups of AML, DAC showed encouraging activity also in models with complex, monosomal karyotype, even in the presence of 6 autosomal monosomies, and was superior to AraC in extending survival. These data lend further support to the treatment of such adverse genetics AML patients with HMAs. Disclosures No relevant conflicts of interest to declare.

The receptor tyrosine kinase (RTK) insulin-like growth factor-1 receptor (IGF1R) is implicated in various tumor entities including chronic lymphocytic leukemia (CLL), but its functional significance in this disease remains poorly characterized. Here, we show that the IGF1R protein is overexpressed in various CLL subsets, suggesting a contribution to CLL pathology. Indeed, we show that IGF1R knockdown in primary human CLL cells compromised their viability. Likewise, IGF1R inhibition with 3 structurally distinct compounds induced apoptosis, even in the presence of protective stroma components. Furthermore, IGF1R inhibition effectively limited CLL development in Eμ-TCL1 transgenic mice and of primary human CLL xenografts. In agreement with its prosurvival function, IGF1R inhibition affected the phosphorylation and/or expression of multiple signaling proteins. The multikinase inhibitor sorafenib yielded similar effects on these signaling elements as IGF1R inhibitors. Indeed, IGF1R appears to be a direct sorafenib target because sorafenib decreased IGF1R expression and phosphorylation, counteracted insulin-like growth factor-1 (IGF-1) binding to CLL cells, and lowered the in vitro kinase activity of recombinant, purified IGF1R. Thus, we demonstrate that blockade of IGF1R-mediated signaling represents a novel mechanism of action for sorafenib in CLL. Importantly, IGF1R inhibitors compromise CLL viability in their microenvironment context, implicating this RTK as a promising therapeutic target.

Background: Translocations involving the MLL gene located on chromosome 11q23 are usually found in de novo acute myeloid leukemia (AML) and generally confer a dismal prognosis unless the AF9 gene is involved (Döhner et al., Blood 2010;115:453-74). MLL can be fused to multiple different genes, resulting in the large and growing "MLL recombinome" (Meyer et al., Leukemia 2013;27:2165-76). Thus far, only two genes encoding proteins that are part of the mRNA decapping protein complex (i.e., DCP1A, DCPS) have been described as rarely being fused to MLL. Here, we describe an AML with an indolent disease course arising from myelodysplastic syndrome (MDS) that disclosed a unique MLL fusion with another component of the mRNA decapping complex, i.e., EDC4. Patient and Methods: Briefly, a 55 year-old female patient presented with an MDS [timepoint (t) -1] that within 10 months progressed to an AML with 2% blood and 40% bone marrow myeloblasts (t0). The patient refused treatment beyond supportive care. Six months later, a marked blast expansion of 80% was detectable in the blood (t1). The patient received 5 cycles of decitabine (t2, cycle 5), followed by 3 months of hydroxyurea (t3). Samples were depleted from CD3+ cells via MACS; CD3+ cells served as germline control. RNA sequencing libraries were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs), and the SureSelect Human All Exon v5 kit (Agilent Technologies) was used for exome capturing from gDNA. Next generation sequencing was performed on an Illumina Hiseq 2500. The analyses were performed as previously described (Kataoka et al. Nature Genetics 2015;47:1304-15, Becker et al. Blood 2014;123:1883-6). NOD scid gamma mice were used as hosts for patient derived tumor xenografts (PDX). Results: Standard metaphase cytogenetics at the diagnosis of AML (t0) revealed a previously undescribed translocation involving the MLL gene, i.e., t(11;16)(q23;q22), as the sole cytogenetic abnormality. The unknown fusion partner on chromosome 16 was identified by RNA-sequencing as the EDC4 gene (a.k.a. Ge-1), which encodes a key scaffold protein of the mRNA decapping complex; the fusion was confirmed by PCR on cDNA. The translocation led to the in-frame fusion of MLL exon 13 to EDC4 exon 6 which was linked by 19 nucleotides from EDC4 intron 5. The predicted amino acid sequence of the linker was ALNTLLR. Further analyses including exome sequencing on the samples collected over the disease course demonstrated STAG2 as a potential founder mutation that was already present during the MDS (t-1) and persisted throughout the disease course at variant allele frequencies (VAFs) of approximately 45-50%. At the time of transformation to AML (t0), the MLL-EDC4 and two RAS mutations (KRAS p.G13D, NRAS p.G12C) were detectable. Towards the terminal phase (t3), the RAS mutations disappeared and a clone that acquired a mutation in the FLT3 tyrosine kinase domain (TKD; p.D835V; VAF 43%) expanded. Primary blasts from the patient engrafted in NOD scid gamma mice and established a stable PDX serial transplantation mouse model used for drug testing. Conclusions: This report provides the first demonstration of an MLL-EDC4 in-frame fusion, with potential cooperativity with a founder mutation in the STAG2 splicing factor gene during the transformation of MDS to AML and the additional acquisition of a FLT3-TKD mutation during disease progression. RNA sequencing proved to be a very feasible approach to identify novel fusion partners of known oncogenes such as MLL. Disclosures Becker: BMS: Honoraria; Novartis: Honoraria. Kataoka:Yakult: Honoraria; Boehringer Ingelheim: Honoraria; Kyowa Hakko Kirin: Honoraria. Schüler:Oncotest GmbH: Employment. Ogawa:Kan research institute: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding. Lübbert:Janssen-Cilag: Other: Travel Funding, Research Funding; Celgene: Other: Travel Funding; Ratiopharm: Other: Study drug valproic acid.

Introduction: Novel substances such as the next generation IMiD pomalidomide or the recently approved next generation proteasome inhibitor carfilzomib (Cfz) have considerably expanded our treatment options in MM, both of them influencing multiple myeloma (MM) interaction with bone marrow stroma cells (BMSCs), that provides an interesting target for anti MM therapy. More compounds directed at this disease critical crosstalk are currently under investigation, however the development of novel drugs remains inefficient, displayed by a substantial drop out rate of the 376 preclinical single agents tested since 1961 (Rongvaux, Annu Rev Immunol. 2013; Schüler, Expert Opin Biol Ther. 2013; Kortüm, CLML 2014). Our focus in the projected presented here was to develop a novel bone-derived in vitro 3D co-culture model specifically adapted to mimic the BM niche to more closely study the role of bone and BM bystander cells and to perform more reliable ex vivo compound screening in MM. Methods: Previous 2D models were compared to a novel 3D co-culture model (agarose matrix interlayer, 100 microwells/cm², 1.5mm in depth, permeable for oxygen+cytokines, but not for BMSCs utilizing U266, RPMI-8226, OPM-2 and primary BM patient (pt) cells, with and without HS-5 vs. M210B4 stroma support (Fig. A + Fig. B.a. for pt characteristics). Analyses covered Trypan Blue, Annexin/PI, MTT, FACS, cell cycle analyses and H2B-mCherry/cytochrome c-GFP assays (Udi, Br J Hematol. 2013). In a next step, primary bone-derived stroma cells were acquired from bones of C57BL/6 J mice. Bones were flushed, digested and FACS sorted in order to acquire single BM and bone bystander cell subtypes (MSPCs [mesenchymal stem and progenitor cells], endothelial cells, osteoblasts, PAS [PDFGRalphaSca1] and CaRs [CXCL12-abundant reticular cells]) which were then compared to HS-5 and M210B4 with regard to growth support, cytokine secretion and protection from anti-MM substances. Results: MMCLs and pt specimens were cultured at different concentrations (10 vs. 100 cells per microwell) with and without M210B4 demonstrating a growth advantage with vs. without M210B4 (Fig. B.b). Liquid overlay technique allowed cluster formation of pt specimens leading to more reliable propagation of pt material for up to 20d of culture. Apoptotic changes were assessed by confocal microscopy of RPMI8226 co-expressing fluorescently labelled histone 2B-mCherry (red) and cytochrome c-GFP (green) as indicators of late and early apoptosis. Comparing BMSCLs with regard to their MM growth support capacities, human HS-5 proved even more beneficent than M210B4 stroma (Fig. B.b). Phenotypic analyses of pt specimens showed decreased CXCR4 expression with vs. without BMSCs suggesting a dynamic regulation of homing molecules. The model was then used as an ex-vivo platform allowing both cytotoxicity and cell cycle analyses for the combination of bortezomib (Btz) vs. Cfz with ARRY-520 (kinesin spindle protein inhibitor). Btz (10nM) and Cfz (20nM) proved significantly cytotoxic compared to the control (U266 and pt specimens, respectively) after 48h of single agent treatment Fig.B.c). Compared to Btz (10nM, B10) and Cfz (20nM, C20) as single agents, the additional combination with ARRY-520 showed stronger additive cytotoxicity for Btz (A5+B10, median: 37.5% vs. 13.1%) than for Cfz (A5+C20, 38.6% vs. 34.1%). To note, the model could also be utilized for more profound analyses as depicted for G2/M cell cycle studies in Fig.B.d. 5nM ARRY-520 (A5) led to significant accumulation of OPM-2 cells in G2/M arrest after 48h of treatment confirming prior analyses (Hernández-Garcia Blood Suppl 4710,2014; Fig. B.d). Co-culture studies with different subsets of BM and bone-derived bystander cells are currently ongoing and will be presented at the meeting (Fig. B.e). Conclusions: More complex, 3D bone-derived high-throughput in vitro models are urgently needed to better predict the potency of preclinically tested agents and to better estimate the likelihood of their later clinical adoption into phase I-II trials. With this work, we provide an innovative model which reflects the BM microenvironment as a crucial predictor for in-vivo sensitivity as shown for ARRY-5200. This ex-vivo approach helps to better incorporate MM growth support by bone and BM-derived bystander cells and thus depicts a valid tool to better characterize the role of the BM niche in myeloma. Figure 1. Figure 1. Disclosures Engelhardt: Deutsch Krebshilfe: Other: grant. Wäsch:German Cancer Aid: Research Funding; Comprehensiv Cancer Center Freiburg: Research Funding; Janssen-Cilag: Research Funding; MSD: Research Funding.

Introduction: Hematopoietic stem and progenitor cells (HSPCs) represent the lifelong source of all blood cells and continuously renew the hematopoietic system by differentiation into mature blood cells. The process of differentiation is predominantly initiated in G1 phase of the cell cycle when stem cells leave their quiescent state. During G1 the anaphase-promoting complex or cyclosome (APC/C) associated with the coactivator Cdh1 is highly active and marks proteins for proteasomal degradation to regulate proliferation. In addition, Cdh1 has been shown to control terminal differentiation in neurons, muscle cells or osteoblasts. Here we show that Cdh1 is also a critical regulator of human HSPC differentiation and self-renewal. Methods: Human CD34+ cells were collected from peripheral blood (PB) of G-CSF mobilized donors and cultured in the presence of different cytokine combinations. To analyze cell division and self-renewal versus differentiation, CFSE staining was used in combination with flow cytometric detection of CD34 expression. The knockdown and overexpression of Cdh1 was achieved by lentiviral delivery of suitable vectors into target cells. After cell sorting transduced (GFP+) CD34+ cells were used for in vitro differentiation in liquid culture or CFU assay. For in vivo experiments purified cells were transplanted into NSG mice. Results: G-CSF mobilized CD34+ cells showed effective differentiation into granulocytes (SCF, G-CSF), erythrocytes (SCF, EPO) or extended self-renewal (SCF, TPO, Flt3-L) when stimulated in vitro. The differentiation was characterized by a fast downregulation of Cdh1 on protein level, while Cdh1 remained expressed under self-renewal conditions. A detailed analysis of different subsets, both in vitro and in vivo, showed high Cdh1 level in CD34+ cells and low expression in myeloid cells. Analysis of proliferation revealed lowest division rates during self-renewal, accompanied by higher frequency of CD34+ cells. The fastest proliferation was found after induction of erythropoiesis. These experiments also showed a more rapid decrease of HSPCs' colony-forming ability and of CD34+ cells during granulopoiesis after 2-3 cell divisions in contrast to a moderate decline under self-renewal conditions. The depletion of Cdh1 (Cdh1-kd) had no effect on total cell numbers or proliferation detected by CFSE during differentiation and self-renewal, but showed an increase in S phase cells. These results were confirmed at the single cell level by measuring the cell cycle length of individual cells. Independent of cell cycle regulation, Cdh1-kd cells showed a significant maintenance of CD34+ cells under self-renewal conditions and during erythropoiesis with lower frequency of Glycophorin A+ cells. In CFU assays, the Cdh1-kd resulted in less primary colony formation, notably CFU-GM and BFU-E, but significantly more secondary colonies compared to control cells. These results suggest that the majority of cells reside in a more undifferentiated state due to Cdh1-kd. The overexpression of Cdh1 showed reversed results with less S phase cells and tendency to increased differentiation in liquid culture and CFU assays. To further validate our results in vivo, we have established a NSG xenotransplant mouse model. Human CD34+ cells depleted of Cdh1 engrafted to a much higher degree in the murine BM 8 and 12 weeks after injection as shown by higher frequencies of human CD45+ cells. Moreover, we also found an increased frequency of human CD19+ B cells after transplantation of CD34+ Cdh1-kd cells. These results suggest an enhanced in vivo repopulation capacity of human CD34+ HSCs in NSG mice when Cdh1 is depleted. Preliminary data in murine hematopoiesis support our hypothesis showing enhanced PB chimerism upon Cdh1-kd. Looking for a mediator of these effects, we found the Cdh1 target protein TRRAP, a cofactor of many HAT complexes, increased upon Cdh1-kd under self-renewal conditions. We use currently RT-qPCR to determine, if this is caused by a transcriptional or post-translational mechanism. Conclusions: Loss of the APC/C coactivator Cdh1 supports self-renewal of CD34+ cells, represses erythropoiesis in vitro and facilitates engraftment capacity and B cell development of human HSPCs in vivo. This work was supported by Josè Carreras Leukemia Foundation grant DCJLS R10/14 (to ME+RW) Disclosures Ewerth: Josè Carreras Leukemia Foundation: Research Funding. Wäsch:German Cancer Aid: Research Funding; Comprehensiv Cancer Center Freiburg: Research Funding; Janssen-Cilag: Research Funding; MSD: Research Funding.

Introduction: In the past decade, substantial progress has been made in the understanding of multiple myeloma (MM) cell biology and its interaction with the bone marrow microenvironment (BMM). Binding of MM cells to BM stroma cells (BMSCs) alters the expression of SDF-1α and its receptor CXCR4, leading to the secretion of anti-apoptotic cytokines, promoting tumor growth, drug resistance and migration. MM cancer stem cells migrate to endosteal BM niches, where they escape therapies in a quiescent state causing relapse in the course of the disease. The development of novel agents that aim to target the MM and BMM interaction includes drugs as promising as 2nd and 3rdgeneration IMIDs or proteasome inhibitors. Despite these profound advances, the failure rate of preclinically proven cytotoxic single substances is sizeable, as preclinical models often lack the biological, genetic, etiological and immunological properties of the disease (Schüler, Expert. Opin. Biol. Ther. 2013; Kortüm. CLML 2014; Rongvaux. Annu Rev Immunol 2013). Methods & Results: We have previously demonstrated that BM interaction and homing to niches, mediated by the adhesion molecules CXCR4, CD49d and CD44, protect MM cell lines (MMCL) and primary plasma cells (PC) from the cytotoxic effect of anti-MM agents, such as bortezomib (Bor), vorinostat (Vor) and pomalidomide (Pom). Our in vitro and in vivo observed cytotoxic effects from Bor, Vor and Pom confirmed their potent cytotoxicity, whereas cocultivation with M2-10B4 substantially reduced apoptosis and induced tumor protective effects. Additional treatment with the CXCR4 inhibitor AMD3100 blocked CXCR4 in coculture, but left CD49d, CD44 and CD11a widely unchanged. Toxic or therapeutic effects from AMD3100 monotherapy were excluded for the doses used. Comparison of the CXCR4 antibody (ab)-clones 12G5, 44717 and 4G10 revealed that AMD3100 treatment of U266 cells reduced CXCR4 expression with use of 12G5 and 44717, whereas binding of both FITC- and PE-coupled 4G10 was not influenced, making the latter the most reliable for CXCR4 analysis. Use of image cytometry (IC) allowed accurate visualization of co-localisation of CXCR4 expression both on the cell surface and within the cytoplasm of MM cells. IC correlated with flow cytometry-determined CXCR4 expression and allowed the detailed assessment of treatment studies with and without anti-MM agents and AMD3100. Of note, AMD3100 resensitized MM cells to Bor, Vor and Pom (Waldschmidt. Blood 2012:2450), whereas carfilzomib (Cfz) reduced CXCR4 expression in MMCL and could not be antagonized by stroma coculture. Cfz sensitivity was not increased by adding AMD3100 (Simon. Blood 2013:3851). These preclinical studies need additional adaptation to the clinical setting in order to surpass prior drug failure rates, and there is a need to develop more broadly available and better predictive preclinical systems. Therefore, we are currently assessing a 3D co-culture MM model composed of agarose matrix interlayers, based on a novel liquid overlay technique. This model has been specifically adapted to MM cell and BM component interactions as described (Udi. BJH 2013; Zlei. Exp Hematol 2007; Schüler. EOBT 2013). MM cells are cultivated in conical microwells of a non-adherent agarose matrix after BMSCs were plated on the bottom of each plate, allowing the diffusion of soluble cytokines but no direct contact between BMSC and MMCL. Therein, we are presently testing novel anti-MM substances in comparison to our standard-coculture system. Conclusion: Targeting microenvironmental mediators, like SDF-1α and CXCR4, is a promising approach to expand the choice of antimyeloma agents and amplify the effects of established antimyeloma drugs, as previously shown by us and others for the combination of AMD3100 and Bor or Pom. However, as our knowledge on MM and its BMM has dramatically increased a great effort has been made in the preclinical testing of promising new anti-MM agents, and more complex high-throughput in vitro models are urgently needed to better predict the potency of these substances in order to reduce dropouts in clinical trials. We hereby provide a novel approach which better reflects the spatial growth of human MM samples in BMSC coculture, and more closely mimics the growth and proliferation of human MM clones in vivo. Disclosures No relevant conflicts of interest to declare.

Introduction Hematopoietic stem cells (HSCs) and multipotent progenitor cells continuously maintain hematopoiesis by self-renewal and differentiation. The stem cell fate is tightly connected with the cell cycle, where the major regulator anaphase-promoting complex or cyclosome (APC/C) with its co-activators Cdc20 and Cdh1 marks cell cycle regulatory proteins, such as cyclin A and B, for proteasomal degradation and thus controls their activity. Known targets of Cdh1 are involved in regulation of self-renewal and granulopoiesis. This raises the hypothesis that Cdh1 may be a critical mediator of HSC proliferation, self-renewal and differentiation. Methods CD34+ cells were collected from peripheral blood (PB) of G-CSF mobilized donors and cultured in the presence of different cytokines. To analyze cell division and self-renewal versus differentiation, CFSE staining was used in combination with CD34 detection. The knockdown (kd) of Cdh1 was achieved by lentiviral delivery of specific shRNA into target cells. Results In vitro cultivation of CD34+ cells under conditions resulting in either self-renewal (SCF, TPO, Flt3-l) or differentiation/granulopoiesis (SCF, G-CSF) showed impressive downregulation of Cdh1 during culture. A high Cdh1 expression in CD34+ cells and lower expression in myeloid cells (CD41a+, CD15+, Glycophorin A+) reflects the situation we found in vivo in bone marrow (BM) subsets. Western blotting also revealed inactivation of Cdh1 by its specific inhibitor Emi1 which stabilized the ubiquitin ligase Skp2 and promoted cell cycle entry and proliferation by degrading the Cdk inhibitor p27. In addition, the APC/CCdh1 target cyclin B was upregulated. These data indicate that initial Cdh1 downregulation is required to promote cell cycle entry and proliferation of CD34+ HSCs under conditions mediating both self-renewal as well as differentiation. When cultured under self-renewal conditions, CD34+ cells showed diminished proliferation with cells residing in lower generations, whereas during granulopoiesis, cells accumulated within higher generations. These experiments also revealed a more rapid decrease of CD34+ cells in granulopoiesis after three cell divisions in contrast to a moderate decline under self-renewal conditions. We also found a decreased colony-forming ability in cells divided more than twice during granulopoiesis, which correlates with their lower CD34 expression. This is consistent with more symmetric divisions into CD34+ daughter cells under self-renewal conditions and gradual commitment during granulopoiesis. Our current experiments extent these analyses to immunofluorescence of Numb distribution in individual cells to elucidate the impact of Cdh1 on symmetric/asymmetric cell division. We could already show that Cdh1-kd led to expansion of CD34+ HSCs in vitro. To further validate our results in vivo, we have established a NOD/SCID/IL-2Rγ chain-/- (NSG) xenotransplant mouse model. Human CD34+ cells depleted of Cdh1 engrafted to a much higher degree in the murine BM 8 and 12 weeks after injection as shown by higher frequencies of engrafted human CD45+ cells. Moreover, we also found an increased frequency of human CD19+ B cells after transplantation of CD34+ Cdh1-kd cells. Further analyses of the contributing subsets to the pool of CD45+ human cells are ongoing. These results suggest an enhanced in vivo repopulation capacity of human CD34+ HSCs in NSG mice when Cdh1 is depleted. Conclusions APC/CCdh1 mediates cell cycle entry and proliferation during self-renewal and differentiation in CD34+ HSCs in vitro and improves engraftment capacity in vivo. Disclosures: No relevant conflicts of interest to declare.

Abstract 3511 Introduction: In order to allow a better understanding of acute myeloid leukemia (AML) and develop more promising therapeutic strategies the establishment of functional and reproducible in vivo models is widely pursued. Of available model systems, xenografts in immunodeficient mice reproduce the clinical situation best. Here, we performed extensive analysis of AML engraftment in NOD/SCID-IL2-receptor-gamma-chain−/− (NSG) mice comparing tail vein versus intratibial injection and growth behavior of AML patient-derived bone marrow versus peripheral blood cells. Furthermore, tumor growth characteristics in the murine host were correlated with the disease stage and the molecular risk factor profile of the individual donors. Methods: Bone marrow and peripheral blood cells from 17 AML patients were injected intratibially into NSG mice (n=4–8/patient, 82 mice in total). As controls, 14 mice received bone marrow from three different donors and 5 mice were mock-injected. Tumor growth was monitored via a) determination of overall survival, b) fluorescence-based in vivo imaging (IVI, Kodak FX, Alexa750 labeled anti-human CD45 or CD33 and c) confirmation of IVI data by histological and immunohistochemical examination of bone marrow and spleen. When highly positive IVI signals and/or the overall condition of individual mice indicated enlarged tumor burden, the respective animals were sacrificed and the human AML cells transferred to another animal. In parallel the engraftment pattern of AML cells 2–4 weeks posttransplant was correlated with clinical disease activity, application route and origin of the particular tumor cells. Results: Patients included in the present study represent multiple different French-American-British (FAB) subtypes, various karyotypes and molecular features in terms of the mutational status of NPM1 and FLT3. All patient-derived specimens were capable of recapitulating the disease in NSG mice at 4–6 weeks after transplantation. Over a period of 13 months 12 out of 17 xenografts could be passaged once and 9 at least twice. Up to six passages were performed for an individual AML xenograft. In contrast, engraftment of healthy donor bone marrow cells could be determined merely until day 56 after implantation. The human bone marrow cells of the healthy donors did not engraft in serial passages. Mean survival time of AML bearing animals ranged between 21 and 82 days for a respective xenograft. No differences could be determined between engraftment capacities of peripheral blood or bone marrow cells of one patient. Neither karyotype, FAB classification nor leucocyte count or the percentage of monomorphic blasts in the bone marrow seemed to have an impact on engraftment capacity in the murine organism. However, mice bearing AML xenografts with mutations in FLT3 as well as in NPM1 showed particular short overall survival times and high tumor cell engraftment determined by IVI. This phenomenon became more obvious along the different passages. The intratibial approach proved to be superior in comparison to the intravenous application as cells of an individual patient engrafted faster when injected directly into the bone marrow microenvironment. Determination or tumor load via IVI permits to closely monitor not only the growth behavior but also the homing characteristics of the human cells over time. The positive IVI signals in bone marrow and spleen could be confirmed by histological examination as well as by immunohistochemistry specific for human CD45 and CD33. Conclusions: Our xenografts show a close resemblance to the AML-disease regarding the level of dissemination and organ involvement. Collection of whole-body IVI data proved to be a time- and animal-saving analysis that allows to closely monitor AML growth. As AML is characterized by an increasing number of molecular subtypes with completely different therapeutic options it seems to be extremely worthwhile to develop patient derived xenograft models representing as many AML subtypes as possible. Our results suggest that this model reflects the heterogeneity and important clinical characteristics of the disease, and thus may serve as a tool for preclinical drug testing and investigation of the pathophysiology of AML. Disclosures: No relevant conflicts of interest to declare.

Abstract 1236 Hematopoietic stem cells (HSCs) and multipotent progenitor cells continuously maintain hematopoiesis by self-renewal and differentiation to all types of blood lineages. These unique processes are regulated by intrinsic and extrinsic signals (e.g. cytokines, cell-cell contacts) and strongly connects stem cell fate with the cell cycle. The ubiquitin-proteasome system regulates spatial and temporal abundance of proteins in the cell. During cell cycle, the anaphase-promoting complex or cyclosome (APC/C) with its co-activators Cdc20 and Cdh1 marks proteins for proteasomal degradation and thus controls their activity. Known targets of Cdh1, namely Skp2 and Id2, are involved in regulation of self-renewal and granulopoiesis (Wang et al., Blood 2011; Buitenhuis et al., Blood 2005). This raises the hypothesis that Cdh1 may be a critical upstream regulator of HSC differentiation. The analysis of human bone marrow cell subsets (CD34+, lymphoid and myeloid cells) revealed highest protein level of Cdh1 in CD34+ cells, lower expression in more mature lymphoid subsets (CD3+, CD19+) and only marginal expression in mature myeloid cells (CD41a+, CD11b+). These data suggest that Cdh1 is important to induce differentiation, but dispensable for maintaining the differentiated state. In vitro cultivation of G-CSF mobilized peripheral blood CD34+ cells under conditions resulting in either self-renewal (SCF, TPO, Flt3-l) or differentiation/granulopoiesis (SCF, G-CSF) showed downregulation of Cdh1 during culture compared to d0. Western blots did not only reveal decreasing levels of Cdh1, but also its inactivation by its specific inhibitor Emi1 which stabilized the ubiquitin ligase Skp2 and promoted cell cycle entry and proliferation by degrading the cyclin-dependent-kinase inhibitor p27. In addition, the APC/CCdh1 target cyclin B was upregulated. These data indicate that initial Cdh1 downregulation is required to promote cell cycle entry and proliferation of CD34+ HSCs under conditions mediating both self-renewal as well as differentiation. To analyze cell division/proliferation and self-renewal versus differentiation more closely, we used the fluorescent dye CFSE as an indicator of cell division in combination with CD34 to indicate the differentiation status. When cultured under self-renewal conditions using SCF, TPO and Flt3-l, CD34+cells showed enhanced proliferation with increased cells in higher generations, whereas using SCF and G-CSF to induce granulopoiesis, cells within lower generations were more prominent. These experiments also revealed a rapid decrease of CD34 expression in granulopoiesis after 3 cell divisions in contrast to a moderate decline under self-renewal conditions. This is consistent with more symmetric divisions into CD34+ daughter cells under self-renewal conditions and gradual cell cycle exit and differentiation under conditions that induce granulopoiesis. To further elucidate the role of Cdh1 for stem/progenitor cell fate, we used a lentiviral knockdown of Cdh1 in CD34+ cells. After 4 days of transduction and cell sorting, the cells were cultivated for 1 week in medium containing SCF, TPO and Flt3-l. Cdh1 depleted cells showed enhanced proliferation compared to the empty vector control and a higher expression of CD34. In colony forming unit (CFU) assays, we observed that CD34+ cells with Cdh1-knockdown were less efficient to differentiate to CFU-G, CFU-M and BFU-E. A higher potential to self-renew was validated by replating of these colonies, where the number with Cdh1-knockdown increased during serial replating. To validate our results in vivo, we have established a NOD/SCID/IL-2Rγ chain−/− (NSG) xenotransplant mouse model. The evaluation of engraftment capacity and differentiation potential of human Cdh1 depleted CD34+ cells in this model is ongoing. Our data establish the central cell cycle regulator APC/CCdh1 as a novel regulator of self-renewal and differentiation in CD34+ HSCs. Disclosures: No relevant conflicts of interest to declare.

Abstract 4995 Introduction: Leukemia and lymphoma account for a notable proportion of cancers worldwide. The heterogeneity and biological characteristics of hematological malignancies induce unique therapeutic challenges. It is well known that pluripotent as compared to differentiated cells possess the potential for anchorage independent growth in semisolid medium. This can be monitored via clonogenic or colony formation assays, in which cells grow in vitro in a three-dimensional (3D) manner without adherence to plastic culture material support. These assays can be utilized to evaluate growth and drug sensitivity of tumor stem and progenitor cells (Fiebig HH et al. Eur J Cancer 40:802, 2004). In addition, these 3D cell culture assays often mimic the in vivo scenario better than 2D cell culture assays with adherent tumor cells. Material and methods: For our ex vivo anti-tumor efficacy profiling using clonogenic assays, we established a panel of 20 hematological cell lines comprising different entities like acute lymphoblastic leukemia (ALL, 4 cell lines), acute myeloid leukemia (AML, 6 cell lines), chronic myeloid leukemia (CML, 5 cell lines), Hodgkin- (1 cell line) and non-Hodgkin-lymphoma (NHL, 3 cell lines), as well as multiple myeloma (MM, 3 cell lines). Tumor cells were injected into the flanks of NOD/SCID mice in order to obtain subcutaneous tumor xenografts, which were kept at low passages (n Results: Twenty-four standard of care agents were tested in terms of their ex vivo chemosensitivity (e.g. cytarabine, cyclophosphamide, dexamethasone, doxorubicin, etoposide, melphalan, prednisolone, vincristine), including selected targeted drugs also (e.g. bortezomib, imatinib, nilotinib, sorafenib). The drugs showed diverse patterns of selectivity and potency: vincristine, doxorubicin and cytarabine, but also the proteasome inhibitor bortezomib exhibited pronounced activity with IC50 values in the nanomolar range (mean IC50 = 1 – 100nM), not only in their respective clinical application, but also in various other tumor entities, such as in ALL and AML with use of bortezomib. Differential activity was determined e.g. for prednisolone and dexamethasone, which were active in a micromolar range (mean IC50 = 22 – 58μM) in the ALL cell lines CCRF-CEM and MOLT-4, AML cell lines NOMO-1, NHL DAUDI and U-937, as well as the MM cell line IM-9. All-trans-retinoic acid (mean IC50 = 1.3μM) as well as interferon-gamma-1b (mean IC50 = 0.43 μM) showed specific activity patterns with pronounced growth inhibition in AML (3/6 tested AML cell lines: KG-1, NOMO-1, OCI-AML2), but also in CML (1/5 tested CML cell lines: EM-2) and MM (1/3 tested MM cell lines: L-363). The strong correlation of both tyrosine kinase inhibitors imatinib and nilotinib (spearman coefficient: 0.73, p Conclusions: The presented panel screen using clonogenic assays is of great value for time and cost effective profiling of traditional cytotoxic as well as new targeted anti-cancer agents which can be confirmed in tumor models of hematological malignancies and can thereby guide to more effectively designed in vivo experiments. Diverse activity and resistance patterns ex vivo and in vivo also contribute to create clinical development strategies of standard and novel compounds. Disclosures: No relevant conflicts of interest to declare.

Abstract 3924 Targeted therapies in terms of monoclonal antibodies have become standard in the treatment of various lymphomas. Albeit being more specific than conventional therapy, the used antibodies target surface receptors both present on polyclonal and monoclonal hematopoietic cells. Due to its specificity for the malignant B-cell clone the B-cell receptor (BCR) is an ideal therapeutic target in lymphoma therapy. Moreover, using peptides has several advantages over whole antibodies: reduced immunogenicity, better epitope mimicry and tissue penetration, easier synthesis and more favourable pharmacokinetics (no uptake into the reticulo-endothelial system). Peptides mimicking the epitope recognized by lymphoma BCRs have therefore been praised as promising therapeutic tools for years (Lam, West J Med., 1993) but a proof-of-concept has only been published recently in mice bearing subcutaneous A20 lymphoma (Palmieri et al., Blood, 2010). In the current study, we have established a human cell line-derived disseminated Burkitt′s lymphoma model (SUP-B8) in NOD/SCID mice by intravenous injection. Our active principle was the tetramerized BCR binding peptide YSFEDLYRRGGK-biotin (termed T-peptide, Renschler et al., PNAS, 1994) which was applied intravenously on day (d) 12, 14, 16 and 19 after injection of the tumor cells, respectively. The therapeutic efficacy was evaluated in comparison to several control groups (tetramerized control peptide (termed C-peptide, RDYSYERLFGGK-biotin), vehicle (0.8% ACN in water, 200μl/d) and untreated animals). Tumor cell engraftment was monitored via daily surveillance of disease symptoms, FACS (anti-human lambda, CD19, anti-murine CD45) and fluorescence-based in vivo imaging system (FI, Kodak FX, Alexa750 labeled anti-human CD45) on days 12 and 21. SUP-B8 engrafted predominantly in the bone marrow (BM, take rate = 100%) and marrow infiltration increased in untreated mice between start and end of therapy from 1 ± 0.4% (d 12) to 39.8 ± 9.4% (d 21). Other sites of engraftment were subcutis (38%) and spleen (8%). The examined compounds were well tolerated in tumor-bearing mice, no acute toxicity could be observed and maximal body weight loss was below 15%. Treatment of xenograft mice with the tetramerized BCR-binding peptide significantly reduced bone marrow infiltration compared to controls (T-peptide 8.1 ± 4.6%, C-peptide: 32.8 ± 8%, p=0.037, vehicle: 30.5 ± 7.9%, p=0.029). Considering the short half-life of uncoupled peptides and the injection schedule every second day, this is a remarkable reduction. For further optimization of this promising therapeutic approach we plan to couple peptides to effector molecules via acid labile linkers; this is based on the evidence that confocal imaging of Burkitt lymphoma cell lines showed the processing of specific BCR binding peptides in acidic organelles of the cell. In summary, we conclude that BCR targeted peptide-based therapy is a feasible method with remarkable therapeutic results in vivo and future studies will focus on coupling specific peptides to appropriate effector molecules or combinational therapeutic approaches using conventional chemotherapeutics. Disclosures: No relevant conflicts of interest to declare.

Abstract 2141 Acute myeloid leukemia (AML), one of the most common leukemia in adults, is characterized by the accumulation of abnormal white blood cells in the bone marrow. Even with modern advances in therapeutics and molecular diagnostics, the majority of AML patients die from their disease. Angiogenesis contributes to the development of hematologic malignancies, although its role has not been as clearly defined in hematologic malignancies as in solid tumors. In the present study we have determined antitumoral activity of two well-known inhibitors of angiogenesis. Sorafenib, a small molecule inhibitor affecting inter alia the VEGF pathway (100mg/kg/day (d); applied 12 consecutive days) and Cetuximab, a chimeric antibody against human EGFR (30 mg/kg/d; applied once weekly for 3 weeks) were evaluated in comparison to Etoposide, a topoisomerase II inhibitor (24mg/kg/day; applied 3 consecutive days) as a well established compound in AML treatment regimens in comparison with a control group. Equal parts of HL-60 cells were injected intravenously and into the peritoneal cavity of NOD/SCID mice and respective therapies were started 14 days after implantation. Tumor growth was monitored by a) daily monitoring of AML symptoms, and b) weekly fluorescence-based in vivo imaging (FI) using a Alexa750-labeled anti-human CD45 antibody and c) verification of the FI data by histological examination of bone marrow and spleen at the end of the study. Tumor inhibition was calculated as the proportional reduction of mean AML cell infiltration at the respective compartment of the test- compared to the control-group (in %). HL-60 cells engrafted predominantly in bone marrow (BM; take rate = 100%), but were as well detectable in the spleen (30%). At the respective doses and schedules the examined compounds were well tolerated in tumor-bearing mice. No acute toxicity could be observed and maximal body weight loss was below 15%. Tumor development was clearly reduced by Cetuximab (reduction of 53% vs control), albeit to a lesser extend then Sorafenib (reduction of 99% vs control), which induced a complete remission within the treatment period. Treatment of Etoposide induced no markable tumor growth inhibition (reduction of 10% vs control). Thus, HL-60 cells engrafted in NOD/SCID mice representing a valuable in vivo model for AML which exhibits high reproducibility and take-rates in relevant compartments closely mimicking the clinical situation. Collection of whole-body FI data proved to be a time- and animal-saving analysis that allows to closely monitor AML growth. With regard to the demographic development, AML will be more and more a disease of the elderly. Thus, development of new therapeutic options compared to high-dose chemotherapy will be highly required. As the VEGF and EGFR pathways are closely related, further investigations will include the evaluation of potentially synergistic effects in combination of Sorafenib with Cetuximab in human disseminated AML xenograft models. Disclosures: No relevant conflicts of interest to declare.

Abstract 3010 Introduction: In order to allow a better understanding of multiple myeloma (MM), the establishment of functional and reproducible in vivo models is widely pursued. Of available model systems, xenografts in immunodeficient mice reproduce the clinical situation advantageously. Here, the engraftment capacity of MM patient-derived bone marrow cells implanted into NOD/SCID-IL2-receptor-gamma-chain-/- (NSG) mice was meticulously investigated. Material and Methods: Bone marrow cells from 7 MM patients were injected intratibialy into NSG mice (n=5/patient). As controls, 5 mice received healthy donor bone marrow and 5 mice were mock-injected. Tumor growth was monitored via a) daily MM-symptom acquisition, such as hind limb paresis, apathy and consistent foot dragging, b) FACS (human HLA-A,B,C; CD138; CD45; CD38) and c) fluorescence-based in vivo imaging (FI, Kodak FX, Alexa750 labeled anti-human CD138, CD38, CD45 and HLA-ABC) in bone marrow, peripheral blood, spleen and lymph node sites of the respective animals. Results: There were significant differences in engraftment capacity, persistence of human cells and expression of selected markers between bone marrow of MM patients and healthy donors: 1.) infiltration of the spleen and lymph nodes was exclusively detected in NSG-mice bearing patient-derived MM cells, whereas cells of healthy donors were - if detected - exclusively found within the murine bone marrow; 2.) mean FI-areas in the bone marrow of MM-patient-derived injected mice were significantly increased as compared to mice bearing bone marrow cells of healthy donors (p=0.006); 3.) patient-derived MM cells expressed CD138, CD38 and HLA-ABC. In contrast, bone marrow cells of healthy donors expressed exclusively CD45 and CD138. The CD138 cell population determined by FACS in patients' bone marrow cells (before NSG-injection) decreased from a median of 11.3% to 0.8% 56 days after implantation (in NSG mice), either due to preferably CD138-negative plasma cell engraftment or the CD138 loss within the murine environment as previously described. Fifty-six days after implantation, patient-derived MM cells could be detected in all animals via FACS-analysis. Follow-up analyses by FI confirmed, that bone marrow engraftment was prominent and observed in all (35/35) NSG mice, albeit also in others organs. Patient-derived MM cells within the bone marrow could be detected in parallel via FACS- and FI-analyses in 10 NSG mice and within the peripheral blood in 12 NSG mice (total of 35 mice being examined). Maximal bone marrow-, peripheral blood- and spleen-engraftment numbers in NSG mice were as high as 4%, 25% and 52%, respectively, suggesting that in peripheral blood- and spleen-sites, MM-cell engraftment could even surmount that of bone marrow-sites. Spleen and other organ involvement observed in our xenografts have been confirmed in previous murine MM-models (Murillo et al. Clin Cancer Res, 2008), postulating that similarly to spleen-colony-forming-cells in hematopoiesis, spleen and other sites serve as fertile tumor engraftment locations.Differences in engraftment capacity and expression pattern between respective patient-derived MM specimen were evident, but did not strikingly correlate with MM-patients' characteristics, such as MM-subtypes, disease stage or expression pattern of the primary material; this observation also well correlating with previous reports (e.g. (Pilarski et al. Blood, 2000). Conclusions: Murine MM-models have shown to be exceedingly challenging in their ability to induce valid and trustworthy MM-patient-derived cell engraftment; here our NSG model suggest to harbor MM-cells. Our data demonstrates that intratibially-injected NSG mice mimic the clinical MM disease with respect to the disseminated nature of the disease and the indispensable engraftment of clonogenic plasma cells into the bone marrow. Collection of whole-body FI data proved to be a time- and animal-saving analysis that allows to closely monitor MM growth. Disclosures: No relevant conflicts of interest to declare.

Abstract 4912 Since novel treatment options are needed in multiple myeloma (MM), novel anti-MM agents and combinations are eagerly pursued to further improve the prognosis for MM patients. For potentially novel therapeutic agents, functional in vivo models are highly valuable. We have established a cell line-based, disseminated MM model in NOD/SCID-IL2-receptor-gamma-chain−/− (NSG) mice. In our current analysis, the multikinase inhibitor sorafenib was validated alone and in combination with the well-established anti-MM agent bortezomib in 6 independent experiments. Optimized dose and schedule were determined as follows: 1. sorafenib (100mg/kg/d; d0-11) alone, 2. bortezomib (0.7mg/kg/day (d); d0,4,11) alone, 3. both in combination with the respective doses and schedules compared to 4. a control group. L363 cells were injected intratibialy into NSG mice and respective therapies were started 7 days after L363-injection (d0). Tumor growth was monitored with daily monitoring of MM-symptoms, flow-cytometry (FACS) and fluorescence-based in vivo imaging (FI). Tumor inhibition was calculated as the proportional reduction of mean MM-cell-infiltration at the respective compartment of the test- compared to the control-group (optimal T/C in %). Furthermore, hollow bones of the injected mice were retrieved when mice were sacrificed, cells flushed out and MM cells purified by MACS microbeads. Total RNA was isolated from these cells and gene expression profiles analyzed using the HG-U133 Plus 2.0 array (Affymetrix) and the Expressionist software (Genedata AG, Basel). L363 engrafted reliably (take rate=100%) at the injection site and in distant organs, such as bone marrow (BM; 100%), spleen (38%) and rarely liver (8%); in the latter organs as previously reported. Control mice developed MM symptoms, such as hind limb pareses, weight loss and osteolyses. At the respective doses and schedules, the examined compounds were well tolerated in tumor-bearing mice. No acute toxicity could be observed and maximal body weight loss was 4% with mono- and 11% with combined therapy. Primary tumor development was markedly reduced by sorafenib (optimal T/C of 11% on d11), as well as with bortezomib, albeit to a lesser extend (optimal T/C: 22% on d5). BM metastases were also significantly reduced by sorafenib with an optimal T/C value of 21% on d11. Bortezomib reduced BM infiltration to an optimal T/C value of 46% on d5 as compared to the control. Combined therapy of sorafenib and bortezomib showed most pronounced anti-tumor and anti-metastatic effects, inducing T/C values of 17% (primary tumor) and 7% (BM) on day 11, respectively. Table 1. Antitumor effect of Sorafenib and Bortezomib in mono- and combined therapy in the L363-xenograft model Compound Side effects Primary tumor Bone marrow Dose Mortality Max. median bwc1 FI2 tumor inhibition FI2 tumor inhibition [mg/kg/d] [n] [%] [%] [%] Sorafenib 100 0 / 5 96 11 21 Bortezomib 0.7 0 / 5 97 22 46 Soraf. / Bortez. 100 / 0.7 0 / 5 89 17 7 1 bwc=body weight changes 2 Tumor inhibition was calculated as the median % of MM cells determined by FI at respective compartments of the test vs. control group multiplied by 100 (optimal test/control (T/C) in %) L363 engraftment into NSG is a valuable in vivo MM model which exhibits high reproducibility, take- and metastases-rates and closely mimics the clinical situation. Collection of whole-body FI data proved to be a time- and animal-saving analysis that allows to closely monitor MM growth. Sorafenib showed promising results in our MM model, in particular in combination with bortezomib. Amongst others, a detailed characterization of the anti-tumor activity of both compounds will be provided by gene expression analysis of L363 cells isolated from untreated vs. treated mice. Further investigations to validate other innovative anti-MM agents as well as their combinations are currently also pursued. Disclosures No relevant conflicts of interest to declare.

Abstract 2378 Poster Board II-355 B cell chronic lymphocytic leukemia (B-CLL), one of the most common leukemia in adults, is characterized by the accumulation of mature B cells expressing CD19 and CD5. Improved understanding of CLL has lead to new prognostic tools and therapeutic options. The protein kinase Syk as a key mediator of proximal B-cell receptor (BCR) signalling is constitutively phosphorylated in CLL B cells. For these novel therapeutics, functional in vivo models are highly valuable. We have established a cell line-based, disseminated B-CLL model in NOD/SCID-IL2-receptor-gamma-chain-/- (NSG) mice. In the current study, SYK inhibitor R788 (60mg/kg/day (d); applied daily) was evaluated in mono- and in combined therapy with Fludarabin (8mg/kg/day; applied 5 consecutive days in two cycles) as a well established compound in B-CLL treatment regimens in comparison with a control group. Equal parts of MEC1 cells were injected intravenously and into the peritoneal cavity of NSG mice and respective therapies were started 7 days after implantation. Tumor growth was monitored with flow-cytometry (FACS), daily monitoring of B-CLL symptoms and fluorescence-based in vivo imaging (FI). Tumor inhibition was calculated as the proportional reduction of mean B-CLL cell infiltration at the respective compartment of the test- compared to the control-group (in %). MEC1 cells engrafted predominantly in bone marrow (BM; take rate = 100%), but were as well detectable in spleen (33%) and peripheral blood (PB; 8%). At the respective doses and schedules the examined compounds were well tolerated in tumor-bearing mice. No acute toxicity could be observed and maximal body weight loss was below 15% in mono- and combined therapy. Tumor development was clearly reduced by R788 (optimal T/C of 48% on day 18), albeit to a lesser extend then standard anticancer agent Fludarabin (optimal T/C of 19% on day 18). Combined therapy induced no synergistic effects showing an optimal T/C value of 42% on day 19. Thus, MEC1 engraftment into NSG mice is a valuable in vivo model for B-CLL which exhibits high reproducibility and take-rates in relevant compartments closely mimicking the clinical situation. Collection of whole-body FI data proved to be a time- and animal-saving analysis that allows to closely monitor B-CLL growth. Further investigations will optimize the very promising antitumor activity of R788 and evaluate the potentially synergistic effect of R788 with additional well-established B-CLL therapeutics. Disclosures: No relevant conflicts of interest to declare.

Abstract 3032 Poster Board II-1008 Gamma delta T cells (GDTCs), a small subset of T-lymphocytes ( (Dhamko H was the recipient of an ASH Summer Trainee Research Award). Disclosures No relevant conflicts of interest to declare.

Abstract 4922 Introduction Thalidomide and lenalidomide possess antiangiogenic, antiproliferative, proapoptotic and immunomodulatory effects. The proteasome inhibitor bortezomib induces cell death in MMCLs and has demonstrated synergism on various tumor cell lines, when combined with the multikinase inhibitor sorafenib. Sorafenib targets various kinases involved in tumor growth and angiogenesis, which plays a governing role in numerous cancers, including MM. EGCG, the most active catechin in green tea, has been described to induce anti-MM- and anti-amyloid-, but recently also to prevent bortezomib-induced-effects. We therefore tested these compounds individually and in combinations on 3 MMCLs in order to assess their cytotoxicity, cell growth inhibition and phenotype changes. Material and Methods RPMI8226, U266 and L363 were cultured at 1×105 cells/ml, with RPMI1640, 10% FCS and 0.2% pen/strep. On day (d) 0, increasing concentrations of bortezomib, sorafenib, thalidomide, lenalidomide and EGCG were added. On d3 and d6, we determined cytotoxicity and cell viability via trypan blue dye exclusion assay and propidium iodide (PI) staining by flow-cytometry (FACS). Additionally, we analyzed phenotype changes by means of CD138-expression (FACS). To evaluate CD138-expression as well as morphologic changes after sorafenib treatment, we also performed confocal microscopy analyses. Results 100nM bortezomib showed pronounced cytotoxicity on all 3 MMCLs: mean PI-positivity in L363 was 83.9% on d3, remained stable on d6 and was significantly increased as compared to control-L363-cells (p With 10 and 100μM sorafenib in L363, we observed mean PI+ cells on d3 as high as 61.6% and 94.3% and on d6 of 80.8% and 91.8%, respectively (p Conclusions Bortezomib and sorafenib showed impressive cytotoxic effects as single agents and ongoing experiments suggest additive effects between both compounds, which is currently being investigated, both in vitro and in our in vivo NOD/SCID-IL2-receptor-gamma-chain−/− (NSG)-mouse-model. Further investigations will also validate the recently suggested inhibitory effects of EGCG on bortezomib-induced cytotoxicity. Thalidomide and lenalidomide moderately reduced viable cell numbers, confirming that other mechanisms, such as anti-angiogenesis and immunomodulation are of greater relevance on MM cells. In line with earlier work, EGCG induced a pronounced cytotoxic effect and inhibition of proliferation. Our results demonstrate that our in vivo model is valuable for the thorough analysis and discovery of innovative targeted anti-MM-agents. Disclosures No relevant conflicts of interest to declare.

For a better understanding of myeloma disease and biology, the establishment of reproducible in vivo models is pursued worldwide. We have established a cell linebased, disseminated myeloma model in NOD/SCID-IL2-receptor-gamma-chain−/− (IL2−/−) mice. In the current study, this model was validated in various treatment groups, using 1. bortezomib (0.7mg/kg/day (d); d0, d4, d11), 2. sorafenib (200mg/kg/d; d0–11), 3. dexamethasone (3mg/kg/d, d0–4 + 7–11), in comparison with 4. a control group. L363 cells were injected intratibialy (it) into IL2−/− mice and respective therapies were started 7 days after L363-it-injection (d0). Tumor growth was monitored with daily monitoring of MM symptoms, fluorescence-based in vivo imaging (FI) performed every 2. week and flow-cytometry (FACS; detection of human HLA−A, B, C + CD138) performed once weekly by sacrificing 2 mice per group and analyzing bone marrow (BM), spleen, peripheral blood (PB) and liver. Based on the FACS data, tumor inhibition was calculated as the median percentage of MM cells at respective compartments of the test- vs. control-group multiplied by 100 (optimal test/control (T/C) in %). Furthermore, hollow bones of the injected mice were retrieved when mice were sacrificed for FACS analyses, cells flushed out and MM cells purified by MACS microbeads. Total RNA was isolated from these cells and gene expression profiles will be analyzed using the HG-U133 Plus 2.0 array (Affymetrix) and the Expressionist software (Genedata AG, Basel). L363 engrafted reliably (take rate=100%) at the injection site and in distant organs, such as BM (100%), spleen (38%) and rarely liver (8%). Control mice developed MM symptoms, such as hind limb pareses, weight loss and osteolyses. L363 cells were detected by FACS and FI, not only at injection sites, but also in the BM, hollow bones and spleen. Primary tumor development was markedly reduced by sorafenib (optimal T/C of 23% on d14), as well as with dexamethasone and bortezomib, albeit to a much lesser extend (optimal T/C: 81% + 62% on d14, respectively). BM metastases were also significantly reduced by sorafenib with an optimal T/C value of 67% on d28. Dexamethasone and bortezomib, the latter possibly due to subclinical doses (determined after titration and toxicity experiments), had no relevant influence on BM metastases suppression (97% + 100% optimal T/C on d28, respectively). Thus, L363 engraftment into IL2−/− is a valuable in vivo model for MM which exhibits high reproducibility, take- and metastases-rates and closely mimics the clinical situation. Collection of whole-body FI data proved to be a time- and animal-saving analysis that allows to closely monitor MM growth. Further investigations will validate the very promising antitumor activity of sorafenib and evaluate the potentially synergistic effect of bortezomib and sorafenib. Amongst others, a detailed characterization of the antitumor activity of both compounds will be provided by the gene expression profile of L363 cells isolated from untreated as well as treated mice. The evaluation of new therapeutic approaches in comparison to standard agents was thus successfully conducted, suggesting that our model serves as a valuable tool in the development of new anticancer strategies.

Since MM expands preferentially in the bone marrow (BM) and the close myeloma and stroma interaction is well recognized, we analyzed whether various soluble factors and different stroma can enhance in vitro MM growth. The expansion and protective function of various growth factors (A. 10ng/ml of each IL-6, VEGF, IGF, +/−super (s) IL-6; B. combination A. plus 10ng/ml of each VEGF, HGF, and IL13, and C. 10ng/ml IL-6, IGF, SDF1, Galectin and IL-1) and different stroma (BM stroma from healthy donors [BMSCs], a mouse stroma line [M210B4], and osteoclasts [OC]) were used for MM cell lines (CL: L363, U266, RPMI 8226). Cell number, viability, percentage and number of CD138+ cells were assessed at day 3 and 6 after culture. The most beneficial culture settings were used to establish their relevance for CD138+ enriched BM cells from MM patients (pts), propagated for up to 14 days in culture. MM cell growth proved to be induced through a cell contact mediated mechanism, this being cytokine and stroma dependent. The most beneficial cytokine combination for CL survival and proliferation was combination B, which could substitute the stroma support. Compared to the control, consisting of culture medium only (RPMI 1640+10%FCS), we detected a median 3.1- (combination B) vs. 1.9-fold increase (BMSCs) at day 3, and 1.24- vs. 1.2-fold increase in cell numbers at day 6. BMSCs proved to be most supportive, especially when used together with combination B (1.8- vs. 1.2-fold increase with BMSCs used alone on day 6 of culture), suggesting that BMSCs provide growth factors acting synergistically with IL-6+VEGF+IGF+sIL-6. The cell viability, albeit not cell expansion potential, was best preserved by OC. With proliferation of myeloma CL, a decrease in the percentage of CD138+ cells was observed, consistent with the reported phenotypic shift of plasma cells (PC) from CD138+ to CD138− during cytokine stimulation. Especially OC favoured the expansion of CD138- cells, suggesting that MM clones require defined conditions for their growth and survival. Culture conditions B and C, with or without BMSCs, could sustain primary BM cells from MM pts for up to 14 days. With the inherent variation of different MM pt samples, the combination of 5 factors (C) was more beneficial for these cells. An additional support for cell viability was provided by BMSCs, especially with longer culture periods (beyond 5 days). The different growth and survival requirements for CL seemed to also apply for CD138+ cells from MM pts, which demonstrated to be remarkably heterogeneous as determined by FACS analysis. We identified at least four different PC populations which coexisted before culture in the same specimen. The phenotype of these cells differed in terms of CD45, CD138, CD38, CD56, CD126, CD221, CD19 and CD28, both in their presence on the cell surface and intensity of expression. Taken together, our data indicate that various culture conditions show substantial differences in their ability to preserve myeloma cells, aimed to reproduce the BM microenvironment where the malignancy develops unconstrained. The availability of in vitro systems will allow testing of novel anti-MM agents, alongside with the development of human MM models in mice.