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Dysregulation of the intrinsic BCL-2 pathway-mediated apoptosis cascade is a common feature of hematological malignancies including acute B-lymphoblastic leukemia (B-ALL). The KMT2A-rearranged high-risk cytogenetic subtype is characterized by high expression of antiapoptotic protein BCL-2, likely due to the direct activating binding of KMT2A fusion proteins to the BCL2 gene. The BCL-2 inhibitor venetoclax (VEN) has proven great clinical value in other blood cancers, however, data on B-ALL is sparse and past studies have not so far described the effects of VEN on gene and protein expression profiles. Using cell lines and patient-derived in vivo xenograft models, we show BCL-2 pathway-mediated apoptosis induction and decelerated tumor cell counts in KMT2A-rearranged B-ALL but not in other cytogenetic subtypes. VEN treatment of cell line- and patient-derived xenografts reduced blast frequencies in blood, bone marrow, and spleen, and tumor cell doubling times were increased. Growth rates are further correlated with VEN concentrations in blood. In vitro incubation with VEN resulted in BCL-2 dephosphorylation and targeted panel RNA sequencing revealed reduced gene expression of antiapoptotic pathway members BCL2, MCL1, and BCL2L1 (BCL-XL). Reinforced translocation of BAX proteins towards mitochondria induced caspase activation and cell death commitment. Prolonged VEN application led to upregulation of antiapoptotic proteins BCL-2, MCL-1, and BCL-XL. Interestingly, the extrinsic apoptosis pathway was strongly modulated in SEM cells in response to VEN. Gene expression of members of the tumor necrosis factor signaling cascade was increased, resulting in canonical NF-kB signaling. This possibly suggests a previously undescribed mechanism of BCL-2-independent and NF-kB-mediated upregulation of MCL-1 and BCL-XL. In summary, we herein prove that VEN is a potent option to suppress tumor cells in KMT2A-rearranged B-ALL in vitro and in vivo. Possible evasion mechanisms, however, must be considered in subsequent studies.

Denileukin Diftitox (ONTAK(®), DAB(389) IL-2) is a recombinant DNA-derived fusion protein depleting cells that express high-affinity IL-2 receptor. Important cell targets are CD4(+)CD25(+)Foxp3(+) regulatory T cells (T(reg)). Elimination of immunosuppressive T(reg) by Denileukin Diftitox may provide a way to modulate immune tolerance following stem cell transplantation. Here, we combined T(reg) depletion with a vaccination approach to induce donor-specific immune reactions. To investigate this approach we chose the mixed chimerism canine stem cell transplantation model which represents a high state of tolerance between two hematopoietic systems. The aim was therefore to induce a graft versus hematopoiesis effect thereby converting mixed to full donor chimerism. Dog leukocyte antigen identical siblings that had developed a stable mixed chimerism after non-myeloablative stem cell transplantation received a single dose of Denileukin Diftitox (18μg/kg, i.v.) followed by several cell-lysate vaccinations. Host peripheral blood mononuclear cell lysates combined with CpG-ODN, and Montanide(®) ISA 51 were locally applied. In vitro studies demonstrated that canine T(reg) are a target of Denileukin Diftitox. The suppression of T-cell proliferation by T(reg) was abolished by addition of Denileukin Diftitox (10nM). An increase of proliferation of median 300% (range: 200%-425%) was observed. No change in donor chimerism was observed after administration of Denileukin Diftitox and vaccination. This study highlights that application of Denileukin Diftitox resulted in a depletion of T(reg) followed by an increase of immune response in vitro. This effect could not be confirmed in vivo even if the immune system was stimulated by vaccinations.

During aging, mitochondrial DNA (mtDNA) can accumulate mutations leading to increasing levels of reactive oxygen species (ROS). Increased ROS were described to activate formerly quiescent hematopoietic stem cells (HSC). Mutations in mtDNA were shown to enhance the risk for myelodysplastic syndrome and leukemia. However, the complex relationship between mtDNA variations, ROS and aging of the hematopoietic system is not fully understood. Herein, three mouse strains with mtDNA polymorphisms in genes of respiratory chain complexes I (ND4), III (CYTB) and IV (COX3) were compared to a reference strain during aging. Analysis focused on ROS and ATP levels, bone marrow composition and blood counts. Additionally, hematopoietic restoration capacity following cytotoxic stress was tested. Mice with polymorphisms in ND4 and CYTB gene had significantly decreasing ROS levels in bone marrow cells during aging, without effecting ATP levels. In addition, the frequency of stem and progenitor cells increased during aging but the amount of lymphocytes in the peripheral blood decreased during aging. In summary, the presence of mtDNA polymorphisms affecting the respiratory chain complexes I, III and IV was associated with altered ROS levels as well as changes in BM and peripheral blood composition during aging.

// Wen Liu 1, 2, * , Julia Beck 3, * , Laura C. Schmidt 1, 2 , Catrin Roolf 1 , Anahit Pews-Davtyan 4 , Barbara C. Rutgen 5 , Sabine Hammer 6 , Saskia Willenbrock 2 , Anett Sekora 1 , Arndt Rolfs 7, 8 , Matthias Beller 4 , Bertram Brenig 9 , Ingo Nolte 2 , Christian Junghanss 1 , Ekkehard Schutz 3, 9, * , Hugo Murua Escobar 1, 2, * 1 Department of Medicine, Clinic III - Hematology/Oncology/Palliative Care, Rostock University Medical Center, Rostock, Germany 2 Small Animal Clinic, University of Veterinary Medicine Hannover, Hannover, Germany 3 Chronix Biomedical, Gottingen, Germany 4 Leibniz-Institute for Catalysis at the University of Rostock, Rostock, Germany 5 Clinical Pathology, Department of Pathobiology, University of Veterinary Medicine Vienna, Vienna, Austria 6 Institute of Immunology, Department of Pathobiology, University of Veterinary Medicine Vienna, Vienna, Austria 7 Albrecht-Kossel-Institute for Neuroregeneration (Akos), Center for Mental Health, University of Rostock, Rostock, Germany 8 Centogene AG, Rostock, Germany 9 Institute of Veterinary Medicine, Georg-August-University Gottingen, Gottingen, Germany * The authors contributed equally Correspondence to: Hugo Murua Escobar, email: hugo.murua.escobar@med.uni-rostock.de Keywords: PDA, arylindolylmaleimides, canine lymphoma, transcriptome sequencing Received: September 14, 2015 Accepted: April 02, 2016 Published: May 12, 2016 ABSTRACT Protein kinase inhibitors are widely used in chemotherapeutic cancer regimens. Maleimide derivatives such as SB-216763 act as GSK-3 inhibitor targeting cell proliferation, cell death and cell cycle progression. Herein, the two arylindolylmaleimide derivatives PDA-66 and PDA-377 were evaluated as potential chemotherapeutic agents on canine B-cell lymphoma cell lines. Canine lymphoma represents a naturally occurring model closely resembling the human high-grade non-Hodgkin’s lymphoma (NHL). PDA-66 showed more pronounced effects on both cell lines. Application of 2.5μM PDA-66 resulted in a significant induction of apoptosis (approx. 11 %), decrease of the metabolic activity (approx. 95 %), anti-proliferative effect (approx. 85 %) and cell death within 48h. Agent induced mode of action was characterized by whole transcriptome sequencing, 12 h and 24 h post-agent exposure. Key PDA-66-modulated pathways identified were cell cycle, DNA replication and p53 signaling. Expression analyses indicated that the drug acting mechanism is mediated through DNA replication and cycle arrest involving the spindle assembly checkpoint. In conclusion, both PDA derivatives displayed strong anti-proliferation activity in canine B-cell lymphoma cells. The cell and molecular PDA-induced effect characterization and the molecular characterization of the agent acting mechanism provides the basis for further evaluation of a potential drug for canine lymphoma serving as model for human NHL.

Introduction: Aberrant methylation of tumor suppressor gene promoters is frequently observed in acute lymphoblastic leukemia (ALL). Decitabine (DAC) and Azacitidine (AZA) are methyltransferase inhibitors (hypomethylating agents, HMA) which partially reverse aberrant DNA methylation. Recently, it was reported that DNA methylation patterns are regulated by casein kinase 2 (CK2) mediated phosphorylation of DNMT3a. CK2 is a key regulator of cell proliferation and survival and modulates hematopoiesis associated signaling cascades by phosphorylation of PTEN and AKT. Elevated CK2 expression has been demonstrated in hematological malignancies and plays a significant role in cell survival. However, it is not yet clear if CK2 inhibition is effective in ALL cells. Therefore, we examined the impact of conventional cytostatics, demethylation and CK2 inhibition on B- and T-ALL cells in single application and in combination. We hypothesized that demethylation and inhibition of CK2 act synergistically inducing greater impact on DNA hypomethylation and ALL cell proliferation. Methods: Several B- and T-ALL cell lines (SEM, RS4;11, Jurkat, CEM) as well as de novo ALL cells were treated with DAC and AZA in mono application. Further, DAC was combined with AraC, Doxorubicin (Doxo) or a CK2 inhibitor for up to 72 h. Cell proliferation and metabolism were determined (trypan blue staining & WST-1 assay). Methylation patterns of bisulfite converted DNA samples were examined using methylation specific qPCR on LINE-1 and the CDH13 gene. For in vivo studies, the SEM cell line was stably transfected with a dual firefly luciferase (ffluc) and GFP expression plasmid. NOD scid gamma (NSG) mice were intravenously injected with 2.5x106 SEM-ffluc-GFP cells. Starting on day (d) 7, mice were treated intraperitoneal. with a vehicle (saline: d7-d10), daily 0.5 mg/kg DAC (d7-d10), daily with 150 mg/kg AraC (d7, d8), or both. Leukemic engraftment and drug response were investigated weekly by flow cytometry (GFP) and bioluminescence imaging (ffluc) for up to 31 days, respectively. Additionally, for therapy monitoring 18F-FDG metabolism of spleen was evaluated using PET/CT on d21 and d28. Results : Mono application of HMA reduced metabolic activity and proliferation significantly (p Moreover, we evaluated the in vivo efficacy of DAC and DAC+AraC. Transplantation of SEM-ffluc-GFP into NSG mice resulted in stable engraftment of ALL cells. Using bioluminescence imaging, leukemic organ infiltration in bone marrow, spleen, lung, liver and brain increased in saline treated mice from d7: 3.0x107 ± 2.3x107 to d31: 6.2x109 ± 5.7x108 ph/s (n=9) continuously. In contrast, treatment with DAC alone or in combination with AraC significantly inhibited the engraftment of ALL cells in vivo. Notably, strongest anti-leukemic effects were induced with DAC alone and not in combination with AraC (d31: DAC: 3.1 x109 ± 1.7 x109 ph/s; n=11 vs. DAC+ AraC: 4.8 x109 ± 1.7 x109 ph/s, n=11). In addition, metabolic spleen volume determined by 18F-FDG-PET/CT on d28 was also markedly reduced in DAC-treated (DAC: 22.8 ± 15.5 mm3, DAC+AraC: 48.7 ± 15.4 mm3) compared to saline treated mice (81.1 ± 21.8 mm3). In vitro combination of DAC with an CK2 inhibitor (CX-4945) induced strong synergistic effects. Here, metabolic activity decreased significantly after 72 h (DAC+CX-4945: 32.2 ± 2.4 % compared to DAC: 72.6 ± 6.4 % and CX-4945: 68.6 ± 8.4 %; DMSO-control: 100 %). The efficacy of DAC and CK2 inhibition in an ALL-xenograft model is currently evaluated. In conclusion, we have shown that HMA significantly inhibit ALL cell proliferation (DAC more than AZA). Further, in vivo combination of DAC with AraC was less effective than DAC alone pointing at antagonistic effects. Of note, our in vitro data indicate that treatment with DAC and CK2 inhibition is synergistic and reveals significant anti-leukemic effects which have to be further elucidated. Disclosures No relevant conflicts of interest to declare.

Progress of age-related hematopoietic diseases such as myelodysplastic syndrome has previously been linked to enhanced levels of reactive oxygen species (ROS). Uncoupling protein 2 (UCP2) was found to reduce mitochondrial ROS production through uncoupling of the respiratory chain. The impact of UCP2 loss and elevated ROS on hematopoiesis during aging has not yet been investigated. In this study, UCP2 knockout mice were analyzed at aging stages of 3, 12, and 24 months with respect to oxidative and energy status of bone marrow cells. Further, the cellular bone marrow subpopulation composition was characterized, as were the differential blood counts at all time points. UCP2 knockout mice revealed enhanced levels of mitochondrial superoxide in elderly animals. Following oxidative stress, adenosine triphosphate (ATP) levels decreased more in the knockout mice than in the wild type. Investigation of bone marrow and blood counts of the knockout mice revealed an enhanced amount of monocytes and neutrophils, as well as a decreased amount of B cells and impaired erythropoiesis throughout aging. In summary, UCP2 induces protective effects on ROS and ATP levels during aging. Additionally, the results suggest an imbalance in hematopoiesis because of the lack of UCP2.

Regulatory T cells (T(reg)) are CD4(+) T lymphocytes with constitutive expression of CD25 and FOXP3, as well as the ability to modulate cellular immune responses. In this study, the phenotypic characteristics, function and feasibility of enrichment and expansion of canine T(reg) were examined. Canine peripheral blood mononuclear cells were isolated and enriched by labelling of CD25, and expansion of T(reg) was achieved by adding interleukin (IL)-2 for 1 week. Phenotypic and functional analyses of T(reg) were performed prior to and after expansion. Canine T(reg) could be phenotypically characterized by CD4, CD25, and FOXP3 expression. Isolation and enrichment of canine T(reg) is possible, but high purities are difficult to achieve without significant cell loss. Expansion of canine T(reg) was possible by adding IL-2 without other growth factors. Higher initial cell numbers seeded allow more substantial T(reg) expansion in vitro. Canine T(reg) have the potential to suppress proliferation of effector T cells (T(eff)). By adding expanded T(reg), a higher capability for suppressing T(eff) could be shown in comparison with freshly isolated T(reg). Enrichment and expansion of canine T(reg) is feasible, and canine T(reg) had similar characteristics to T(reg) from other species.

Introduction: Successful engraftment following hematopoietic stem cell transplantation (HSCT) depends on factors like immunosuppression, graft composition and number of infused HSC. Whereas the immunosuppression as well as the type and composition of the graft are influenceable low numbers of available HSCs i.e. “weak grafts” remain a clinical challenge. Weak grafts are accompanied by increased graft failure rates and longer cytopenias associated with increased morbidity. Intra bone marrow (IBM) infusion of HSC might be an approach to overcome these problems. Studies in rodents demonstrated faster engraftment with an IBM HSCT approach compared to intravenous (IV) HSCT following myeloablative conditioning. Studies of IBM HSCT following non-myeloablative or reduced intensity conditioning (RIC) are missing. Aims: Exploring the feasibility and efficiency of IBM allogeneic HSCT in comparison to IV HSCT in dog leukocyte antigen (DLA) identical canine littermates using a RIC regimen. Methods: DLA-identical siblings were used as donor/recipient pairs for HSCT. Recipient dogs were conditioned with 4.5 Gy total body irradiation before HSCT (d0) and received 15 mg/kg Cyclosporin A BID as pre- and postgrafting immunosuppression (d-1 to d+35). BM grafts were harvested at d0. In the control group (CON, n=7) unmodified BM was transplanted IV. In the IBM group (n=7) BM harvests were centrifuged and buffy coat of the BM was then transfused simultaneously into the recipient humeri and femura (50 ml, 10 min). 10 dogs are currently evaluable. Chimerism of the peripheral blood mononuclear cells (PBMC) and granulocytes (G) were tested weekly until week 8 and afterwards in larger intervals. Blood cell counts and clinical toxicities such as weight loss were monitored. Results: Infusion of BM directly into the bone was feasible. All animals engrafted. Median number of infused total nucleated cells was 4.0*108/kg (range 2.3-6.0*108/kg, IBM) and 3.3*108/kg (range 1.9-5.0*108/kg, CON, IBM vs CON: p=0.4). Median CD34+ numbers infused were 3.1*106/kg (range:1.2-10.0*106/kg, IBM) and 3.9*106/kg (range: 1.0-7.2*106/kg, CON; IBM vs CON: p= 0.8). Hematopoietic recovery in the IBM and CON groups were similar. Leukocytes recovery (>1.0*109/l) occurred at median d+11 (range: d+10 - d+16, IBM) and d+10 (range: d+9-d+12, CON; IBM vs CON: p=0.3). Median leukocytes nadirs amounted to 0.23*109/l (IBM) and 0.28*109/l (CON; IBM vs CON: p=0.3) and median duration of leukopenia ( Chimerism analyses showed an early and fast increase in donor chimerism in both groups. The PBMC donor chimerism at d+14, d+28 and d+56 were 46% (range: 30-53%), 57% (range: 40-73%), 64% (range: 60-83%) for IBM. Results in CON were 37% (range: 17-93%), 60% (range: 49-100%), 57% (range: 40-100%) (IBM vs CON, p=n.s. (all time points)). The G chimerism values at that specific points were 95% (range: 53-100%), 100% (range: 53-100%), 96% (range: 88-100%) for IBM and 100% (range: 93-100%), 99% (range: 92-100%), 98% (range: 93-100%) for CON (IBM vs CON, p=n.s. (all time points)). Primary goal of the study was the feasibility of the IBM approach. Ethics regulations did not allow to use weak grafts (≤2.0*106/kg) intentionally. However, 4 animals received weak grafts (CON n=2, 1.0 and 2.0*106/kg; IBM n=2, 1.2 and 1.3 *106/kg). Of interest, comparing data of these dogs showed that durations of leukopenia were similar (median 10 days, both groups), but duration of thrombocytopenia were different (median 8 days, IBM vs 22 days, CON). Additionally, long term donor chimerism was higher in the IBM (median 80% PBMC, 100% G) vs CON (median 61% PBMC, 42% G). Conclusion: First, IBM HSCT is a feasible and effective method to deliver HSC directly into the bone marrow following RIC in a canine HSCT model. Second, our preliminary data suggest that IBM HSCT reveals advantageous engraftment differences in regards to platelet recovery and donor chimerism kinetics compared to the IV HSCT when grafts with low HSC numbers were infused. Follow up data of this study and future studies will have to clarify these observations further. Disclosures No relevant conflicts of interest to declare.

Mitochondria are complex cell compartments characterized by a nuclear genome independent own genome referred to as mitochondrial genome (mtDNA). MtDNA encodes 13 proteins which are part of the five enzyme complexes of the mitochondrial respiratory chain. The respiratory chain is responsible for ATP synthesis and is the main source of reactive oxygen species (ROS) in the cell. During cellular aging, mutations in mtDNA accumulate leading potentially to respiratory chain deficiency. Due to the lack of DNA repair mechanisms within the mitochondrion itself, the mtDNA is especially vulnerable towards ROS. Until now it is not fully understood whether intracellular ROS levels increase with aging in all cell types, and whether this process is due to a potential impaired function of the respiratory chain. Thus, the influence of mtDNA mutations on oxidative stress and aging of hematopoietic cells remains to be investigated. Herein, we compared two conplastic mouse strains differing in one point mutation in the mtDNA affecting the respiratory chain. The C5BL/6Ntac-mtFVB/NJ (mtFVB) strain carries a point mutation at nt7778G/T leading to a D>Y replacement in ATP8 protein, while the control stain C5BL/6Ntac-mtAKR (mtAKR) does not. The targeted protein is part of the F0sub unit of complex V in the respiratory chain and we hypothesize that the mutation affects the transport of protons within the complex V. In our study, we analyzed bone marrow cells of both strains at four different aging stages (3 to 24 months) for ROS and ATP levels by DCFH-fluorescence and luminescence staining, respectively. Additionally, the subpopulations of bone marrow cells were analyzed by flow cytometric immunophenotyping. Further, blood counts of the mice treated with a single dose of 5 Fluorouracil (5-FU, 150mg/kg BW, i.p.) were performed every 3 days during a total time span of 21 days. The mtAKR strain showed increasing levels of ROS ranging from 7.1 x 103 to 12 x 103 relative fluorescence units (RFU) and decreasing levels of ATP (from 0.99 to 0.36 µM) during the measurement time points 3 to 24 months. In comparison, the mtFVB strain showed a significant decrease in ROS levels ranging from 5.5 x 103 to 2.4 x 103 RFU and a significant increase of ATP levels (from 0.23 to 0.64 µM) during the same period. Hematopoietic cells of aged mtFVB mice (24 months) contained significantly more ATP than cells of mtAKR mice. Analysis of bone marrow cell composition of both strains showed an increase of hematopoietic stem cells (LSK cells: lineage-, sca-1+, c-kit+) during experimental time span from 3 to 24 months (mtAKR: from 2.13 to 2.81 % of lineage- cells, mtFVB: from 2.13 to 3.52 %). However, only in the mtFVB strain this increase was significant. Interestingly, the amount of LSK cells was significant higher in mtFVB compared to the mtAKR strain (mtFVB: 2.36 %, mtAKR: 1.23 %) at 6 months. Furthermore, erythroide cells (Ter119+) in mtAKR increased with aging (from 25.22 % to 32.69 %), while the mtFVB strain showed a decreased rate (from 25.88 % to 18.9 %). Blood counts of 3 months old mtFVB mice treated with 5-FU were similar to those of the mtAKR strain in the myelosuppression phase after application, but demonstrated higher regeneration peaks at day 15 in white blood cells (mtAKR: 20.24 x 103/µl, mtFVB: 25.72 x 103/µl), platelets (mtAKR: 2016 x 103/µl, mtFVB: 2848 x 103/µl), neutrophilic (mtAKR: 0.8 x 103/µl, mtFVB: 2.36 x 103/µl) and eosinophilic granulocytes (mtAKR: 0.28 x 103/µl, mtFVB: 0.76 x 103/µl) and lymphocytes (mtAKR: 17.12 x 103/µl, mtFVB: 21.84 x 103/µl). The mtDNA polymorphism in complex V of the respiratory chain in mtFVB strain influences the development of ROS and ATP levels in hematopoietic cells during aging, and seems to have a protective impact concerning ROS and ATP levels in aged mice. Furthermore, the capability of LSK cells to expand with age seems to be enhanced in mtFVB strain. Moreover, the regeneration capacity after 5-FU myelosuppression seems also to be increased in 3 months old mice. Disclosures: No relevant conflicts of interest to declare.

The PI3K/Akt pathway is dysregulated is some acute lymphoblastic leukemias (ALL) and might therefore serve as therapeutic target. Indolylmaleimides exhibit inhibitory potencies against different protein kinases -like glycogen synthase kinase 3 (GSK3β) or protein kinase c- influencing thereby several cellular processes. Recently, it was demonstrated that PDA-66, a newly synthesized indolylmalemide based on the well known GSK3β inhibitor SB-216763, hinders microtubule polymerization in human neuronal progenitor and neuroblastoma cells. GSK3β is a downstream substrate of the PI3K/Akt and Wnt pathways and is often deregulated in tumor tissues. Herein, we investigated the effects of PDA-66 and its derivates (PDA-66E und PDA-377) on B and T-lymphoblastic leukemia cells. Methods B- and T-ALL cell lines (SEM, RS4;11, REH, Jurkat, MOLT-4 and CEM) were incubated for 72 h with increasing concentrations (0.1 µM-5.0 µM) of PDA-66, PDA-66E, PDA-377 and comparatively analyzed to SB-216763. To evaluate the effect of each substance WST-1 assay, cell proliferation, cell cycle analyses as well as apoptosis rates were determined. Activities of indolylmalemides were analyzed by GSK3β kinase assay. Detection of key molecules of Wnt and PI3K/Akt signaling pathway was performed using Western blot. Results PDA-66 and derivates inhibited proliferation and metabolism of ALL cells significantly in a dose dependent manner. Interestingly, all PDA derivates showed a stronger inhibitory effect on proliferation than SB-216763 but the inhibitory effect on GSK3β kinase was lower than SB-216763. Antiproliferative effects of PDA-66 were studied in more detail. The incubation of 1 µM PDA-66 led to condensation of chromatin in the nucleus, karyorrhexis and an increasing amount of vacuoles after 48 h of treatment. PDA-66 influenced the cell cycle distribution of ALL cell lines differently. While RS4;11 and MOLT-4 cells were arrested in G2 phase, SEM cells remained increasingly in G0/1 phase. After 48 h all PDA-66 treated cell lines showed a significant increase in apoptosis compared to control cells (SEM: 2.1 ± 0.9 % to 10.5 ± 1.3 %; RS4;11: 2.5 ± 0.7 % to 7.4 ± 1.1 %; Jurkat: 3.8 ± 0.6 % to 8.3 ± 1.9 %; MOLT-4: 3.7 ± 1.2 % to 16.3 ± 5.1 %). Apoptosis of ALL cells was initiated by cleavage of caspase 3 and 7 and Poly (ADP-ribose) polymerase (PARP). Furthermore, with increasing concentration of PDA-66 a decrease of pGSK3βSer9 was observed after 4 h in SEM cells. However, no influence on the total form of β-catenin was detectable. Nevertheless, there was an influence of PDA-66 on the expression of 4EBP-1 and p4EBP-1Ser65. SEM, RS4;11 and Jurkat cells showed a decrease of the phosphorylated as well as the total form of 4EBP-1 after an incubation of 4 and 24 h. In conclusion, our results demonstrate that the newly synthesized indolymalemides have pronounced antiproliferative effects in ALL cells. In particular, the indolymalemide PDA-66 should be further investigated concerning it’s clinical efficiency as well as well as it's intracellular ways of action. Disclosures: No relevant conflicts of interest to declare.

Abstract 3711 Introduction: Successful engraftment following transplantation of hematopoietic stem cells (HSCT) depends mainly on pre- and posttransplant immunosuppression, graft type and composition as well as on the HSC numbers infused. Whereas some of the aforementioned parameters can be influenced in the clinical setting, the latter one is more difficult to address. HSCTs of grafts with limited HSC numbers are accompanied by increased graft failure rates, longer cytopenias and increased morbidity. Current concepts to overcome low HSC numbers include the combination of two unrelated grafts, expansion techniques, modification of the graft composition or the site of graft infusion. In preliminary rodent studies intra-bone marrow (IBM) compared to intravenous (IV) HSCT led to faster engraftment which might be explained by closer location of the HSC to the stem cell niches. Aims: To investigate the feasibility and efficiency of IBM-HSCT following a non-myeloablative conditioning regimen in a dog-leukocyte antigen (DLA) identical canine HSCT model. Method: DLA-identical siblings were used as donor/recipient pairs for HSCTs. Recipients received a single dose of 2 Gy total body irradiation before HSCT (day 0). Pre- and postgrafting immunosuppression consisted of CSA (d-1 to d+35) and MMF (d0 to d+27). Two IBM-HSCT cohorts were investigated and data compared to IV controls (CON). BM-grafts of the respective donors were infused unmodified IV (CON, n=9) or IBM after HSC enrichment using a buffy coat followed by ficoll density centrifugation (IBM-I, n=6; 5ml total volume) or IBM after HSC enrichment using buffy coat centrifugation only (IBM-II, n=6; 25 ml total volume). In the CON group the graft was infused in the cubital vein. In the IBM-groups the grafts were infused through a BM aspiration needle into the BM of the left humerus and femur over a period of 5 minutes. In 4 IBM animals graft migration analyses were performed using technecium99 marking. Chimerism and BM cellularity were determined at injection and opposite sides. Analyses of chimerism were performed via polymorphic nucleotide repeat analyses weekly. BM cellularity was determined biweekly. Complete blood count was performed daily. Result: Infusion of grafts directly into the BM was feasible: both volumes (5ml, 25ml) could be infused without any leakage at the injection sites. Tc99-marked BM cells stayed predominately at the injection site for the first 24 hours. All animals engrafted. Mean TNC numbers infused were 2.6 ×108/kg (range: 1.6–11.4; CON), 1.6 ×108/kg (range: 1–2.4; IBM-I), 3.7 ×108/kg (range: 2.1–5.8; IBM-II) (IBM-I vs CON: p=0.08, IBM-II vs CON: p=0.9, IBM-I vs II: p Conclusion: Infusion of HSC grafts up to volumes of 25ml directly into the BM is feasible and allows successful donor engraftment following non-myeloablative conditioning. Duration of cytopenias following IBM-HSCT is still significant, perhaps due to the loss of precursor cells during graft preparation. Further studies are warranted to determine optimal graft preparation and IBM application techniques. Disclosures: No relevant conflicts of interest to declare.

Abstract 4115 Introduction Inhibition of specific signal transduction pathways in neoplastic cells has been successfully used to regulate aberrantly activated cellular processes such as proliferation. Specific target agents such as tyrosine kinase inhibitors have been successfully introduced into the treatment of several cancers. The novel dual PI3K and mTOR inhibitor, NVP-BEZ235, has shown antitumor activity in vitro and is being investigated in phase I clinical trials in solid tumors. It is currently unclear whether PI3K/Akt and mTOR inhibition might also be usefull in acute leukemia cells, and if so, what would be the best modalities for combination approaches. Here, we examined the antileukemic activity of NVP-BEZ234 in acute lymphoblastic (ALL) and myeloid leukemia (AML) cells. Methods ALL (SEM, RS4;11, Jurkat and MOLT-4) and AML (MV4;11, MONO-MAC-1, HL-60 and NB-4) cell lines with different cytogenetics and PI3K/Akt activity were investigated. Cells were treated for 96h with NVP-BEZ435 (1 nM, 10 nM and 100 nM) alone or in combination with cytarabine, doxorubicin or dexamethasone. Cytotoxic drugs were added simultaneously, 24h before or 24h after treatment with NVP-BEZ235. Cell count, apoptosis rates, necrosis rates, cell cycle distrubution, protein phosphorylation and metabolic activity were determined at 0.5h, 4h, 24h, 48h, 72h, and 96h by microscopy, flow cytometry, western blot and WST-1 testing, respectively. Results NVP-BEZ235 potently inhibited the proliferation of SEM, Jurkat, RS4;11, MV4;11, MONO-MAC-1 and NB-4 significantly. Most pronounced effects were seen after 72h. In HL-60, which has a low PI3K/Akt activation, inhibition of proliferation to a lesser degree was observed. Inhibition of PI3K and mTOR activity and G1/G0 arrest was detected at 10 nM and 100 nM in B- and T-ALL cell lines, respectively. Metabolic activity decreased significantly as early as 48h after exposure. Changes in apoptosis and necrosis rates were not observed in B-ALL and AML cells. However, in Jurkat and MOLT-4 (both T-ALL) highest doses of NVP-BEZ seemed to slightly increase. In a time and concentration dependent manner the phosphorylation status of Akt (Ser473 and Thr308), FoxO3A (Thr32) and p70S6 kinase (Thr389) decreased early (0.5-4h) after treatment. Of interest, subsequently an increase of phosphorylation of the same proteins was observed (24-48h), followed by another dephosphorylation episode. Concomittant treatment with NVP-BEZ235 and cytarabine, doxorubicin or dexamethasone enhanced the cytotoxicity in B-cells compared to single drug treatment. The most significant effects on proliferation inhibition and metabolic activity were observed for the simultaneous exposure to both drugs or pretreatment of cells with NVP-BEZ235 followed by dexamethasone, doxorubicin and cytarabine addition after 24h. No additive effect was observed when cytostatic drugs were added before inhibition of PI3K and mTOR by NVP-BEZ235 was induced. Conclusions NVP-BEZ235 reduces metabolic activity and induces cell cycle arrest in acute lymphoblastic and myeloid leukemia cells. Treatment of ALL cells with NVP-BEZ combined with glucocorticoids or conventional chemotherapy drugs enhances the cytotoxicity of the dual PI3K and mTOR inhibitor when NVP-BEZ235 is given upfront or simultaneously. In contrast, pretreatment with conventional chemotherapy drugs followed by NVP-BEZ235 exposure does not induce additive cytotoxicity. Our data indicate that NVP-BEZ might be a usefull drug in ALL as well AML treatment. Furthermore, simultaneous treatment with chemotherapy seems more advisable compared to NVP-BEZ interruption. Disclosures: No relevant conflicts of interest to declare.