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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.

In acute lymphoblastic leukemia (ALL), conventional cell lines do not recapitulate the clonal diversity and microenvironment. Orthotopic patient-derived xenograft models (PDX) overcome these limitations and mimic the clinical situation, but molecular stability and engraftment patterns have not yet been thoroughly assessed. We herein describe and characterize the PDX generation in NSG mice. In vivo tumor cell proliferation, engraftment and location were monitored by flow cytometry and bioluminescence imaging. Leukemic cells were retransplanted for up to four passages, and comparative analyses of engraftment pattern, cellular morphology and genomic hotspot mutations were conducted. Ninety-four percent of all samples were successfully engrafted, and the xenograft velocity was dependent on the molecular subtype, outcome of the patient and transplantation passage. While BCR::ABL1 blasts were located in the spleen, KMT2A-positive cases had higher frequencies in the bone marrow. Molecular changes appeared in most model systems, with low allele frequency variants lost during primary engraftment. After the initial xenografting, however, the PDX models demonstrated high molecular stability. This protocol for reliable ALL engraftment demonstrates variability in the location and molecular signatures during serial transplantation. Thorough characterization of experimentally used PDX systems is indispensable for the correct analysis and valid data interpretation of preclinical PDX studies.

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.

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.

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.

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.