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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.

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.

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.

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.

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.