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Background: Both bromodomain and extra-terminal domain (BET) proteins and spleen tyrosine kinase (SYK) represent promising targets in diffuse large B-cell (DLBCL) and Burkitt’s lymphoma (BL). We evaluated the anti-lymphoma activity of the isoform-specific bivalent BET inhibitor AZD5153 (AZD) and the pan-BET inhibitor I-BET151 (I-BET) as single agents and in combination with SYK inhibitor Entospletinib (Ento) in vitro. Methods: The effect of the single agents on cell proliferation and metabolic activity was evaluated in two DLBCL and two BL cell lines. Proliferation, metabolic activity, apoptosis, cell cycle and morphology were further investigated after a combined treatment of AZD or I-BET and Ento. RNAseq profiling of combined AZD+Ento treatment was performed in SU-DHL-4 cells. Results: Both BET inhibitors reduced cell proliferation and metabolic activity in a dose- and time-dependent manner. Combined BET and SYK inhibition enhanced the anti-proliferative effect and induced a G0/G1 cell cycle arrest. SU-DHL-4 demonstrated a pronounced modulation of gene expression by AZD, which was markedly increased by additional SYK inhibition. Functional enrichment analyses identified combination-specific GO terms related to DNA replication and cell division. Genes such as ADGRA2, MYB, TNFRSF11A, S100A10, PLEKHH3, DHRS2 and FOXP1-AS1 were identified as possible key regulators. Conclusion: Simultaneous inhibition of BET and SYK enhanced the anti-proliferative effects, and induced a combination-specific gene expression signature.

Kirsten rat sarcoma virus (KRAS) mutations are widespread in pancreatic ductal adenocarcinoma (PDAC) and contribute significantly to tumor initiation, progression, tumor relapse/resistance, and prognosis of patients. Although inhibitors against KRAS mutations have been developed, this therapeutic approach is not routinely used in PDAC patients. We investigated the anti-tumor efficacy of two KRAS inhibitors BI-3406 (KRAS::SOS1 inhibitor) and sotorasib (KRAS G12C inhibitor) alone or in combination with MEK1/2 inhibitor trametinib and/or PI3K inhibitor buparlisib in seven PDAC cell lines. Whole transcriptomic analysis of combined inhibition and control groups were comparatively analyzed to explore the corresponding mechanisms of inhibitor combination. Both KRAS inhibitors and corresponding combinations exhibited cytotoxicity against specific PDAC cell lines. BI-3406 enhance the efficacy of trametinib and buparlisib in BXPC-3, ASPC-1 and MIA PACA-2, but not in CAPAN-1, while sotorasib enhances the efficacy of trametinib and buparlisib only in MIA PACA-2. The whole transcriptomic analysis demonstrates that the two triple-inhibitor combinations exert antitumor effects by affecting related cell functions, such as affecting the immune system, cell adhesion, cell migration, and cytokine binding. As well as directly involved in RAF/MEK/ERK pathway and PI3K/AKT pathway affect cell survival. Our current study confirmed inhibition of KRAS and its downstream pathways as a potential novel therapy for PDAC and provides fundamental data for in vivo evaluations.

Background/aim Canine B-cell lymphoma represents a useful in vivo model for human diffuse large B-cell lymphoma (DLBCL). Pan-Bromodomain and extra-terminal (BET) inhibition targeting BRD2/3/4 and selective inhibition of BRD4, as well as spleen tyrosine kinase (SYK) inhibition, are currently evaluated as haematologic cancer therapy. Herein, we characterized the differences in the biologic response of isoform-specific or pan-BET inhibition alone or in combination with SYK inhibition. Materials and methods I-BET151 (pan-inhibitor) and AZD5153 (BRD4 inhibitor) were combined with Entospletinib (SYK inhibitor) and comparatively analysed in the canine DLBCL cell line CLBL-1. Dose- and time-dependent cellular responses were analysed by cell number, metabolic activity, apoptosis/necrosis, and cell morphology. The synergistic potential was evaluated through the Bliss independence model. Results I-BET151 and AZD5153 showed significant dose- and time-dependent inhibitory effects. Adding Entospletinib to I-BET151 or AZD5153 had no additional synergistic effects. Conclusion Entospletinib did not enhance the inhibitory effects of the pan- or isoform-specific BET.

Spirooxindole-1,3-oxazines are a small and structurally unique class of spirooxindole alkaloids. To date, only four of these compounds have been isolated from natural sources, and their biological properties remained unknown thus far. Dioxyreserpine is a synthetic spirooxindole-1,3-oxazine, that can readily be prepared from the Rauvolfia alkaloid (-)-reserpine by catalytic photooxygenation. While dioxyreserpine itself was now identified as a moderately effective antitumoral agent, structurally modified analogs of it emerged as a new class of highly potent and selective growth inhibitors of various human cancers, including pancreatic cancers. Systematic structural optimization ultimately led to an inhibitor displaying low-micromolar IC

The aberrant activation of the phosphoinositide 3-kinase (PI3K)/ protein kinase B (AKT) pathway is common in pancreatic ductal adenocarcinomas (PDAC). The application of inhibitors against PI3K and AKT has been considered as a therapeutic option. We investigated PDAC cell lines exposed to increasing concentrations of MK-2206 (an AKT1/2/3 inhibitor) and Buparlisib (a pan-PI3K inhibitor). Cell proliferation, metabolic activity, biomass, and apoptosis/necrosis were evaluated. Further, whole-exome sequencing (WES) and RNA sequencing (RNA-seq) were performed to analyze the recurrent aberrations and expression profiles of the inhibitor target genes and the genes frequently mutated in PDAC (Kirsten rat sarcoma virus (KRAS), Tumor protein p53 (TP53)). MK-2206 and Buparlisib demonstrated pronounced cytotoxic effects and limited cell-line-specific effects in cell death induction. WES revealed two sequence variants within the direct target genes (PIK3CA c.1143C > G in Colo357 and PIK3CD c.2480C > G in Capan-1), but a direct link to the Buparlisib response was not observed. RNA-seq demonstrated that the expression level of the inhibitor target genes did not affect the efficacy of the corresponding inhibitors. Moreover, increased resistance to MK-2206 was observed in the analyzed cell lines carrying a KRAS variant. Further, increased resistance to both inhibitors was observed in SU.86.86 carrying two TP53 missense variants. Additionally, the presence of the PIK3CA c.1143C > G in KRAS-variant-carrying cell lines was observed to correlate with increased sensitivity to Buparlisib. In conclusion, the present study reveals the distinct antitumor effects of PI3K/AKT pathway inhibitors against PDAC cell lines. Aberrations in specific target genes, as well as KRAS and TP53, individually or together, affect the efficacy of the two PI3K/AKT pathway inhibitors.

Casein kinase II (CK2) and cyclin-dependent kinases (CDKs) frequently interact within multiple pathways in pancreatic ductal adenocarcinoma (PDAC). Application of CK2- and CDK-inhibitors have been considered as a therapeutic option, but are currently not part of routine chemotherapy regimens. We investigated ten PDAC cell lines exposed to increasing concentrations of silmitasertib and dinaciclib. Cell proliferation, metabolic activity, biomass, and apoptosis/necrosis were evaluated, and bioinformatic clustering was used to classify cell lines into sensitive groups based on their response to inhibitors. Furthermore, whole exome sequencing (WES) and RNA sequencing (RNA-Seq) was conducted to assess recurrent mutations and the expression profile of inhibitor targets and genes frequently mutated in PDAC, respectively. Dinaciclib and silmitasertib demonstrated pronounced and limited cell line specific effects in cell death induction, respectively. WES revealed no genomic variants causing changes in the primary structure of the corresponding inhibitor target proteins. RNA-Seq demonstrated that the expression of all inhibitor target genes was higher in the PDAC cell lines compared to non-neoplastic pancreatic tissue. The observed differences in PDAC cell line sensitivity to silmitasertib or dinaciclib did not depend on target gene expression or the identified gene variants. For the PDAC hotspot genes kirsten rat sarcoma virus (KRAS) and tumor protein p53 (TP53), three and eight variants were identified, respectively. In conclusion, both inhibitors demonstrated in vitro efficacy on the PDAC cell lines. However, aberrations and expression of inhibitor target genes did not appear to affect the efficacy of the corresponding inhibitors. In addition, specific aberrations in TP53 and KRAS affected the efficacy of both inhibitors.

Bruton’s tyrosine kinase (BTK) and phosphoinositide 3-kinase (PI3K) in the B-cell receptor (BCR) signaling pathway are considered potential therapeutic targets for the treatment of B-cell lymphomas, among which, diffuse large B-cell lymphoma (DLBCL) is the most common type. Herein, we comparatively evaluated the single and combined application of the BTK inhibitor ibrutinib and the selective PI3Kγ inhibitor AS-605240 in the canine DLBCL cell line CLBL-1. For further comparison, key findings were additionally analyzed in canine B-cell leukemia GL-1 and human DLBCL cell line SU-DHL-4. While ibrutinib alone induced significant anti-proliferative effects on all cell lines in a dose-dependent manner, AS-605240 only induced anti-proliferative effects at high concentrations. Interestingly, ibrutinib and AS-605240 acted synergistically, reducing cell proliferation and increasing apoptosis/necrosis in all cell lines and inducing morphological changes in CLBL-1. Moreover, the combined application of ibrutinib and AS-605240 reduced relative phosphorylation and, in some instances, the levels of the BTK, AKT, GSK3β, and ERK proteins. Comparative variant analysis of RNA-seq data among canine B- and T-lymphoid cell lines and primary B-cell lymphoma samples revealed potentially high-impact somatic variants in the genes that encode PI3K, which may explain why AS-605240 does not singly inhibit the proliferation of cell lines. The combination of ibrutinib and AS-605240 represents a promising approach that warrants further in vivo evaluation in dogs, potentially bearing significant value for the treatment of human DLBCL.

Background Casein kinase II (CK2) is involved in multiple tumor-relevant signaling pathways affecting proliferation and apoptosis. CK2 is frequently upregulated in acute B-lymphoblastic leukemia (B-ALL) and can be targeted by the ATP-competitive CK2 inhibitor CX-4945. While reduced proliferation of tumor entities including B-ALL after CX-4945 incubation has been shown in vitro and in vivo, the detailed way of action is unknown. Here, we investigated the influence on the PI3K/AKT and apoptosis cascades in vivo and in vitro for further clarification. Methods A B-ALL xenograft model in NSG mice was used to perform in vivo longitudinal bioluminescence imaging during six day CX-4945 treatment. CX-4945 serum levels were determined at various time points. Flow cytometry of bone marrow and spleen cells was performed to analyze CX-4945-induced effects on tumor cell proliferation and distribution in B-ALL engrafted mice. ALL cells were enriched and characterized by targeted RNA sequencing. In vitro, B-ALL cell lines SEM, RS4;11 and NALM-6 were incubated with CX-4945 and gene expression of apoptosis regulators BCL6 and BACH2 was determined. Results In B-ALL-engrafted mice, overall tumor cell proliferation and distribution was not significantly influenced by CK2 inhibition. CX-4945 was detectable in serum during therapy and serum levels declined rapidly after cessation of CX-4945. While overall proliferation was not affected, early bone marrow and spleen blast frequencies seemed reduced after CK2 inhibition. Gene expression analyses revealed reduced expression of anti-apoptotic oncogene BCL6 in bone marrow blasts of CX-4945-treated animals. Further, BCL6 protein expression decreased in B-ALL cell lines exposed to CX-4945 in vitro. Surprisingly, levels of BCL6 opponent and tumor suppressor BACH2 also declined after prolonged incubation. Simultaneously, increased phosphorylation of direct CK2 target and tumor initiator AKT was detected at respective time points, even in initially pAKT-negative cell line NALM-6. Conclusions The CK2 inhibitor CX-4945 has limited clinical effects in an in vivo B-ALL xenograft model when applied as a single drug over a six day period. However, gene expression in B-ALL cells was altered and suggested effects on apoptosis via downregulation of BCL6. Unexpectedly, the BCL6 opponent BACH2 was also reduced. Interactions and regulation loops have to be further evaluated.

Constitutively activating internal tandem duplication (ITD) alterations of the receptor tyrosine kinase FLT3 (Fms-like tyrosine kinase 3) are common in acute myeloid leukemia (AML) and classifies FLT3 as an attractive therapeutic target. So far, applications of FLT3 small molecule inhibitors have been investigated primarily in FLT3-ITD+ patients. Only recently, a prolonged event-free survival has been observed in AML patients who were treated with the multikinase inhibitor sorafenib in addition to standard therapy. Here, we studied the sorafenib effect on proliferation in a panel of 13 FLT3-ITD- and FLT3-ITD+ AML cell lines. Sorafenib IC50 values ranged from 0.001 to 5.6 mu M, whereas FLT3-ITD-cells (MOLM-13, MV4-11) were found to be more sensitive to sorafenib than FLT3-ITD- cells. However, we identified two FLT3-ITD+ cell lines (MONO-MAC- 1 and OCI-AML-2) which were also sorafenib sensitive. Phosphoproteome analyses revealed that the affected pathways differed in sorafenib sensitive FLT3ITD(-) and FLT3-ITD+ cells. In MV4-11 cells sorafenib suppressed mTOR signaling by direct inhibition of FLT3. In MONO-MAC-1 cells sorafenib inhibited the MEK/ERK pathway. These data suggest that the FLT3 status in AML patients might not be the only factor predicting response to treatment with sorafenib.

Background Canine prostate cancer represents a unique model for human prostate cancer. In vitro systems offer various possibilities but Xenograft in vivo imaging allows studying complex tasks as tumor progression and drug intervention longitudinal. Herein, we established three canine prostate carcinoma cell lines stably expressing fluorescent proteins allowing deep tissue in vivo imaging. Methods Three canine prostate carcinoma (cPC) cell lines were stably transfected with fluorescent proteins in red, far-red and near infra-red spectrum, followed by G418 selection. Fluorescent protein expression was demonstrated by microscopy, flow cytometry and a NightOWL LB 983 in vivo imaging system. Cellular and molecular characteristics of the generated cell lines were compared to the parental cell line CT1258. Cell proliferation, metabolic activity and sphere formation capacity were analyzed. Stem cell marker expression was examined by qPCR and genomic copy number variation by genomic DNA whole genome sequencing. Results Three stably fluorescent protein transfected cPC cell lines were established and characterized. Compared to the parental cell line, no significant difference in cell proliferation and metabolic activity were detected. Genomic copy number variation analyses and stem cell marker gene expression revealed in general no significant changes. However, the generated cell line CT1258-mKate2C showed uniquely no distal CFA16 deletion and an elevated metabolic activity. The introduced fluorescencent proteins allowed highly sensitive detection in an in vivo imaging system starting at cell numbers of 0.156 × 106. Furthermore, we demonstrated a similar sphere formation capacity in the fluorescent cell lines. Interestingly, the clone selected CT1258-mKate2C, showed increased sphere formation ability. Discussion Starting from a well characterized cPC cell line three novel fluorescent cell lines were established showing high cellular and molecular similarity to the parental cell line. The introduction of the fluorescent proteins did not alter the established cell lines significantly. The red fluorescence allows deep tissue imaging, which conventional GFP labeling is not able to realize. Conclusion As no significant differences were detected between the established cell lines and the very well characterized parental CT1258 the new fluorescent cell lines allow deep tissue in vivo imaging for perspective in vivo evaluation of novel therapeutic regimens.

Aberrant PI3K/AKT signaling is a hallmark of acute B-lymphoblastic leukemia (B‑ALL) resulting in increased tumor cell proliferation and apoptosis deficiency. While previous AKT inhibitors struggled with selectivity, MK‑2206 promises meticulous pan‑AKT targeting with proven anti-tumor activity. We herein, characterize the effect of MK‑2206 on B‑ALL cell lines and primary samples and investigate potential synergistic effects with BCL‑2 inhibitor venetoclax to overcome limitations in apoptosis induction. MK‑2206 incubation reduced AKT phosphorylation and influenced downstream signaling activity. Interestingly, after MK‑2206 mono application tumor cell proliferation and metabolic activity were diminished significantly independently of basal AKT phosphorylation. Morphological changes but no induction of apoptosis was detected in the observed cell lines. In contrast, primary samples cultivated in a protective microenvironment showed a decrease in vital cells. Combined MK‑2206 and venetoclax incubation resulted in partially synergistic anti-proliferative effects independently of application sequence in SEM and RS4, 11 cell lines. Venetoclax-mediated apoptosis was not intensified by addition of MK‑2206. Functional assessment of BCL‑2 inhibition via Bax translocation assay revealed slightly increased pro-apoptotic signaling after combined MK‑2206 and venetoclax incubation. In summary, we demonstrate that the pan‑AKT inhibitor MK‑2206 potently blocks B‑ALL cell proliferation and for the first time characterize the synergistic effect of combined MK‑2206 and venetoclax treatment in B‑ALL.

BACKGROUND Mitochondrial DNA (mtDNA) encodes for the respiratory chain proteins. Genetic alterations in mtDNA have been described during aging and linked to impaired hematopoiesis. MATERIALS AND METHODS We investigated two novel conplastic mouse strains harboring a mitochondrial nt7778 G/T polymorphism leading to an amino acid exchange in respiratory chain complex V. Effects on reactive oxygen species (ROS) and adenosine triphosphate (ATP) levels, as well as bone marrow composition and peripheral blood counts, were investigated during aging (up to 24 month). RESULTS The polymorphism correlated with significantly decreased ROS levels in aged mice. Effects on hematopoiesis were marginal and not statistically significant: numbers of erythroid cells in bone marrow, as well as mean corpuscular hemoglobin, tended to decrease over time. CONCLUSION The investigated polymorphism is associated with decreased ROS levels in aged hematopoietic cells but does not significantly influence hematopoiesis itself.

Nitrogen-containing heterocycles such as quinoline, quinazolinones and indole are scaffolds of natural products and have broad biological effects. During the last years those structures have been intensively synthesized and modified to yield new synthetic molecules that can specifically inhibit the activity of dysregulated protein kinases in cancer cells. Herein, a series of newly synthesized isoquinolinamine (FX-1 to 8) and isoindoloquinazolinone (FX-9, FX-42, FX-43) compounds were evaluated in regards to their anti-leukemic potential on human B- and T- acute lymphoblastic leukemia (ALL) cells. Several biological effects were observed. B-ALL cells (SEM, RS4;11) were more sensitive against isoquinolinamine compounds than T-ALL cells (Jurkat, CEM). In SEM cells, metabolic activity decreased with 10 μM up to 26.7% (FX-3), 25.2% (FX-7) and 14.5% (FX-8). The 3-(p-Tolyl) isoquinolin-1-amine FX-9 was the most effective agent against B- and T-ALL cells with IC50 values ranging from 0.54 to 1.94 μM. None of the tested compounds displayed hemolysis on erythrocytes or cytotoxicity against healthy leukocytes. Anti-proliferative effect of FX-9 was associated with changes in cell morphology and apoptosis induction. Further, influence of FX-9 on PI3K/AKT, MAPK and JAK/STAT signaling was detected but was heterogeneous. Functional inhibition testing of 58 kinases revealed no specific inhibitory activity among cancer related kinases. In conclusion, FX-9 displays significant antileukemic activity in B- and T-ALL cells and should be further evaluated in regards to the mechanisms of action. Further compounds of the current series might serve as templates for the design of new compounds and as basic structures for modification approaches.

Background Promotor hypermethylation of CpG islands is common in B cell precursor acute lymphoblastic leukemia (BCP-ALL) with mixed lineage leukemia (MLL) gene rearrangements. Hypomethylating agents (HMA) such as azacitidine (AZA) and decitabine (DEC) reduce DNA hypermethylation by incorporation into DNA and were successfully introduced into the clinic for the treatment of myeloid neoplasias. Methods Here, we investigated whether HMA induce comparable biological effects in MLL-positive BCP-ALL. Further, efficacy of HMA and concomitant application of cytostatic drugs (cytarabine and doxorubicin) were evaluated on established SEM and RS4;11 cell lines. In addition, promising approaches were studied on BCP-ALL cell line- and patient-derived xenograft models. Results In general, DEC effects were stronger compared to AZA on MLL-positive BCP-ALL cells. DEC significantly reduced proliferation by induction of cell cycle arrest in G0/G1 phase and apoptosis. Most sensitive to HMA were SEM cells which are characterized by a fast cell doubling time. The combination of low-dose HMA and conventional cytostatic agents revealed a heterogeneous response pattern. The strongest antiproliferative effects were observed when ALL cells were simultaneously exposed to HMA and cytostatic drugs. Most potent synergistic effects of HMA were induced with cytarabine. Finally, the therapeutic potential of DEC was evaluated on BCP-ALL xenograft models. DEC significantly delayed leukemic proliferation in xenograft models as demonstrated longitudinally by non-invasive bioluminescence as well as 18F-FDG-PET/CT imaging. Unexpectedly, in vivo concomitant application of DEC and cytarabine did not enhance the antiproliferative effect compared to DEC monotherapy. Conclusions Our data reveal that DEC is active in MLL-positive BCP-ALL and warrant clinical evaluation. Electronic supplementary material The online version of this article (10.1186/s13045-018-0607-3) contains supplementary material, which is available to authorized users.

Graft-versus-host disease (GvHD) is an adverse effect following hematopoietic stem cell transplantation (HSCT) in humans. Dogs represent a key model organism for the development of treatment protocols for HSCT. However, detailed descriptions of canine GvHD and its treatment are rare. Herein we describe the development of canine GvHD and therapeutic intervention.A female Beagle received an allogeneic HSCT from a dog leukocyte antigen-identical littermate (conditioning with 4.5 Gy total body irradiation; immunosuppression with cyclosporine A).GvHD developed at day +52 and was treated with methylprednisolone, cyclosporine A, antibiotics, antiviral medication and analgesics. The dog initially responded to the treatment but GvHD relapsed twice. Within one week after discontinuation of glucocorticoid, GvHD recurred resulting in inevitable euthanasia of the animal.GvHD represents a life-threatening disease after HSCT in canines. Immediate therapeutic treatment is indicated and even a successful initial treatment response does not necessarily prevent GvHD recurrence.

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.

Dendritic cells (DCs) are important immune mediators following allogeneic hematopoietic stem cell transplantation (HSCT). We screened for DC frequency in the cornea and oral mucous membranes after HSCT by confocal laser scanning microscopy (CLSM) in a canine model.In vivo CLSM images of the epithelia were taken the day before and on days 28, 56 and 112 following HSCT. Peripheral blood counts and chimerism were determined.An increase of DCs after HSCT was detected in each animal in both investigated tissue types. Highest DC numbers in the flew and the gingiva were detected on day 28 and in the corneal epithelium on day 56 after HSCT, respectively.Changes of DCs in ocular and non-ocular mucous membranes can be monitored by CLSM in vivo. The DC frequency in the cornea and mucosa changes following HSCT. DC recovery is rapid and their numbers correlate with the development of chimerism of peripheral blood mononuclear cells.

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.

Background Prognosis of adult patients suffering from acute lymphoblastic leukemia (ALL) is still unsatisfactory. Targeted therapy via inhibition of deregulated signaling pathways appears to be a promising therapeutic option for the treatment of ALL. Herein, we evaluated the influence of a novel arylindolylmaleimide (PDA-66), a potential GSK3β inhibitor, on several ALL cell lines. Methods ALL cell lines (SEM, RS4;11, Jurkat and MOLT4) were exposed to different concentrations of PDA-66. Subsequently, proliferation, metabolic activity, apoptosis and necrosis, cell cycle distribution and protein expression of Wnt and PI3K/Akt signaling pathways were analyzed at different time points. Results PDA-66 inhibited the proliferation of ALL cells significantly by reduction of metabolic activity. The 72 h IC50 values ranged between 0.41 to 1.28 μM PDA-66. Additionally, caspase activated induction of apoptosis could be detected in the analyzed cell lines. PDA-66 influenced the cell cycle distribution of ALL cell lines differently. While RS4;11 and MOLT4 cells were found to be arrested in G2 phase, SEM cells showed an increased cell cycle in G0/1 phase. Conclusion PDA-66 displays significant antileukemic activity in ALL cells and classifies as candidate for further evaluation as a potential drug in targeted therapy of ALL.