IL-4 downregulates expression of the target receptor CD30 in neoplastic canine mast cells.

CD30 is a novel therapeutic target in human mast cell (MC) neoplasms. In this 'comparative oncology' study, we examined CD30 expression and regulation in neoplastic canine MC using a panel of immunomodulatory cytokines [interleukin-2 (IL-2), IL-4, IL-5, IL-6, IL-13 and stem cell factor (SCF)] and the canine mastocytoma cell lines NI-1 and C2. Of all cytokines tested IL-4 was found to downregulate expression of CD30 in NI-1 and C2 cells. We also found that the CD30-targeting antibody-conjugate brentuximab vedotin induces growth inhibition and apoptosis in both MC lines. Next, we asked whether IL-4-induced downregulation of CD30 interferes with brentuximab vedotin-effects. Indeed, pre-incubation of NI-1 cells with IL-4 decreased responsiveness towards brentuximab vedotin. To overcome IL-4-mediated resistance, we applied drug combinations and found that brentuximab vedotin synergizes with the Kit-targeting drugs masitinib and PKC412 in inhibiting growth of NI-1 and C2 cells. In summary, CD30 is a new marker and IL-4-regulated target in neoplastic canine MC.

Next-generation sequencing identifies major DNA methylation changes during progression of Ph+ chronic myeloid leukemia.

Little is known about the impact of DNA methylation on the evolution/progression of Ph+ chronic myeloid leukemia (CML). We investigated the methylome of CML patients in chronic phase (CP-CML), accelerated phase (AP-CML) and blast crisis (BC-CML) as well as in controls by reduced representation bisulfite sequencing. Although only ~600 differentially methylated CpG sites were identified in samples obtained from CP-CML patients compared with controls, ~6500 differentially methylated CpG sites were found in samples from BC-CML patients. In the majority of affected CpG sites, methylation was increased. In CP-CML patients who progressed to AP-CML/BC-CML, we identified up to 897 genes that were methylated at the time of progression but not at the time of diagnosis. Using RNA-sequencing, we observed downregulated expression of many of these genes in BC-CML compared with CP-CML samples. Several of them are well-known tumor-suppressor genes or regulators of cell proliferation, and gene re-expression was observed by the use of epigenetic active drugs. Together, our results demonstrate that CpG site methylation clearly increases during CML progression and that it may provide a useful basis for revealing new targets of therapy in advanced CML.

NOX4-driven ROS formation mediates PTP inactivation and cell transformation in FLT3ITD-positive AML cells.

Activating mutations of FMS-like tyrosine kinase 3 (FLT3), notably internal tandem duplications (ITDs), are associated with a grave prognosis in acute myeloid leukemia (AML). Transforming FLT3ITD signal transduction causes formation of reactive oxygen species (ROS) and inactivation of the protein-tyrosine phosphatase (PTP) DEP-1/PTPRJ, a negative regulator of FLT3 signaling. Here we addressed the underlying mechanisms and biological consequences. NADPH oxidase 4 (NOX4) messenger RNA and protein expression was found to be elevated in FLT3ITD-positive cells and to depend on FLT3ITD signaling and STAT5-mediated activation of the NOX4 promoter. NOX4 knockdown reduced ROS levels, restored DEP-1 PTP activity and attenuated FLT3ITD-driven transformation. Moreover, Nox4 knockout (Nox4(-/-)) murine hematopoietic progenitor cells were refractory to FLT3ITD-mediated transformation in vitro. Development of a myeloproliferative-like disease (MPD) caused by FLT3ITD-transformed 32D cells in C3H/HeJ mice, and of a leukemia-like disease in mice transplanted with MLL-AF9/ FLT3ITD-transformed murine hematopoietic stem cells were strongly attenuated by NOX4 downregulation. NOX4-targeting compounds were found to counteract proliferation of FLT3ITD-positive AML blasts and MPD development in mice. These findings reveal a previously unrecognized mechanism of oncoprotein-driven PTP oxidation, and suggest that interference with FLT3ITD-STAT5-NOX4-mediated overproduction of ROS and PTP inactivation may have therapeutic potential in a subset of AML.

Identification of bromodomain-containing protein-4 as a novel marker and epigenetic target in mast cell leukemia.

Advanced systemic mastocytosis (SM) is a life-threatening neoplasm characterized by uncontrolled growth and accumulation of neoplastic mast cells (MCs) in various organs and a poor survival. So far, no curative treatment concept has been developed for these patients. We identified the epigenetic reader bromodomain-containing protein-4 (BRD4) as novel drug target in aggressive SM (ASM) and MC leukemia (MCL). As assessed by immunohistochemistry and PCR, neoplastic MCs expressed substantial amounts of BRD4 in ASM and MCL. The human MCL lines HMC-1 and ROSA also expressed BRD4, and their proliferation was blocked by a BRD4-specific short hairpin RNA. Correspondingly, the BRD4-targeting drug JQ1 induced dose-dependent growth inhibition and apoptosis in HMC-1 and ROSA cells, regardless of the presence or absence of KIT D816V. In addition, JQ1 suppressed the proliferation of primary neoplastic MCs obtained from patients with ASM or MCL (IC50: 100-500 nm). In drug combination experiments, midostaurin (PKC412) and all-trans retinoic acid were found to cooperate with JQ1 in producing synergistic effects on survival in HMC-1 and ROSA cells. Taken together, we have identified BRD4 as a promising drug target in advanced SM. Whether JQ1 or other BET-bromodomain inhibitors are effective in vivo in patients with advanced SM remains to be elucidated.

Oncostatin M is a FIP1L1/PDGFRA-dependent mediator of cytokine production in chronic eosinophilic leukemia.

BACKGROUND: Chronic eosinophilic leukemia (CEL) is a myeloproliferative neoplasm characterized by expansion of neoplastic eosinophils, tissue infiltration, and organ damage. In a subset of these patients, the FIP1L1/PDGFRA (F/P) oncoprotein is detectable. F/P exhibits constitutive tyrosine kinase activity and activates a number of signaling pathways. So far, however, little is known about the role of F/P-dependent proteins in the pathogenesis of CEL. METHODS: A screen for F/P-dependent cytokines was performed in growth factor-dependent human cell lines lentivirally transduced with F/P. Signal transduction pathways were characterized in Ba/F3 cells with doxycycline-inducible expression of F/P and in EOL-1 cells. Cytokine expression was confirmed in patients' material by immunohistochemistry, immunofluorescence, and confocal microscopy. Gene expression analysis, proliferation assays, and chemotaxis assays were used to elucidate paracrine interactions between neoplastic eosinophils and stromal cells. RESULTS: We show that F/P upregulates expression of oncostatin M (OSM) in various cell line models in a STAT5-dependent manner. Correspondingly, neoplastic eosinophils in the bone marrow were found to overexpress OSM. OSM derived from F/P + cells stimulated proliferation of stromal cells. Moreover, OSM-containing supernatants from F/P + cells were found to upregulate production of stromal cell-derived factor-1 (SDF-1)/CXCL12 in human fibroblasts. SDF-1, in turn, induced migration of EOL-1 cells in a dose-dependent manner. CONCLUSIONS: We have identified a F/P-driven paracrine interaction between neoplastic eosinophils and stromal cells that may contribute to tissue fibrosis and accumulation of neoplastic eosinophils in CEL.

NI-1: a novel canine mastocytoma model for studying drug resistance and IgER-dependent mast cell activation.

BACKGROUND: Advanced mast cell (MC) disorders are characterized by uncontrolled growth of neoplastic MC in various organs, mediator-related symptoms, and a poor prognosis. Kit mutations supposedly contribute to abnormal growth and drug resistance in these patients. METHODS: We established a novel canine mastocytoma cell line, NI-1, from a patient suffering from MC leukemia. RESULTS: NI-1 cells were found to form mastocytoma lesions in NOD/SCID IL-2Rgamma(null) mice and to harbor several homozygous Kit mutations, including missense mutations at nucleotides 107(C→T) and 1187(A→G), a 12-bp duplication (nucleotide 1263), and a 12-bp deletion (nucleotide 1550). NI-1 cells expressed several MC differentiation antigens, including tryptase, Kit, and a functional IgE receptor. Compared to the C2 mastocytoma cell line harboring a Kit exon 11 mutation, NI-1 cells were found to be less responsive against the Kit tyrosine kinase inhibitors (TKI) masitinib and imatinib, but were even more sensitive against proliferation-inhibitory effects of the mammalian target of rapamycin (mTOR) blocker RAD001 and PI3-kinase/mTOR blocker NVP-BEZ235. The Kit-targeting multikinase inhibitors PKC412 and dasatinib were also found to override TKI resistance in NI-1 cells, and produced growth inhibition with reasonable IC(50) values (<0.1 µM). CONCLUSION: NI-1 may serve as a useful tool to investigate IgE-dependent reactions and mechanisms of abnormal growth and drug resistance in neoplastic MC in advanced mastocytosis.

The Hsp32 inhibitors SMA-ZnPP and PEG-ZnPP exert major growth-inhibitory effects on D34+/CD38+ and CD34+/CD38- AML progenitor cells.

Heat shock protein 32 (Hsp32), also known as heme oxygenase 1 (HO-1), has recently been identified as a potential target in various hematologic malignancies. We provide evidence that Hsp32 is constitutively expressed in primary leukemic cells in patients with acute myeloid leukemia (AML) and in various AML cell lines (HL60, U937, KG1). Expression of Hsp32 mRNA was demonstrable by qPCR, and expression of the Hsp32 protein by immunocytochemistry and Western blotting. The stem cell-enriched CD34+/CD38+ and CD34+/CD38- fractions of AML cells were found to express Hsp32 mRNA in excess over normal CD34+ progenitor cells. Two Hsp32-targeting drugs, pegylated zinc-protoporphyrin (PEG-ZnPP) and styrene-maleic-acid-copolymer-micelle-encapsulated ZnPP (SMAZnPP), were found to inhibit cytokine-dependent and spontaneous proliferation in all 3 AML cell lines as well as in primary AML cells. Growth inhibitory effects of SMA-ZnPP and PEG-ZnPP were dose-dependent with IC50 values ranging between 1 and 20 µM, and were accompanied by apoptosis as evidenced by light- and electron microscopy, Tunel assay, and caspase-3 activation. Finally, we were able to demonstrate that SMA-ZnPP inhibits cytokine-dependent proliferation of CD34+/CD38+ and CD34+/CD38- AML progenitor cells in vitro in all patients as well as leukemiainitiation of AML stem cells in NOD-SCID IL-2Rγ(-/-) (NSG) mice in vivo. Together, our data suggest that Hsp32 plays an important role as a survival factor in leukemic stem cells and as a potential new target in AML.

Targeting of Hsp32 in solid tumors and leukemias: a novel approach to optimize anticancer therapy.

Heat shock protein 32 (Hsp32), also known as heme oxygenase-1 (HO-1), is a stress-related anti-apoptotic molecule, that has been implicated in enhanced survival of neoplastic cells and in drug-resistance. We here show that Hsp32 is expressed in most solid tumors and hematopoietic neoplasms and may be employed as a new therapeutic target as evidenced by experiments using specific siRNA and a Hsp32-targeting pharmacologic inhibitor. This Hsp-32 targeting drug, SMA-ZnPP, was found to inhibit the proliferation of neoplastic cells with IC(50) values ranging between 1 and 50 microM. In addition, SMA-ZnPP induced apoptosis in all neoplastic cells examined. Furthermore, SMA-ZnPP was found to synergize with other targeted and conventional drugs in producing growth-inhibition. Resulting synergistic effects were observed in all tumor and leukemia cells examined. Interestingly, several of the drug partners, when applied as single agents, induced the expression of Hsp32 in neoplastic cells, suggesting that synergistic effects resulted from SMA-ZnPP-induced ablation of a Hsp32-mediated survival-pathway that is otherwise used by tumor cells to escape drug-induced apoptosis. Together, Hsp32 is an important survival factor and target in solid tumors and hematopoietic neoplasms, and may be used to optimize anticancer therapy by combining conventional or targeted drugs with Hsp32-inhibitors. Based on these data, it seems desirable to explore the value of Hsp32-targeting drugs as anti-cancer agents in clinical trials.

Evidence that the sheep associated form of malignant catarrhal fever is caused by a herpes virus.

Out of a cow, which was infected with the sheep form of malignant catarrhal fever (MCF), blood and spleen samples were inoculated into rabbits. From the spleen cells of an infected rabbit, which were cocultivated with bovine embryonic gingiva cells, a herpesvirus could be isolated. The isolate showed crossreactions with reference sera against the strain WC 11 (wildebeest form) in the SNT. An immunosuppressed heifer, which was infected with the isolate, contracted typical clinical symptoms of MCF. The isolate was named No. 732.

[Seroepidemiologic studies of the agent of respiratory diseases in cattle]. / Seroepidemiologische Untersuchungen auf Erreger respiratorischer Erkrankungen beim Rind.

In a period of three years epidemiological studies were carried out in Styria concerning the occurrence of antibodies against BRSV (Bovine Respiratory Syncytial Virus), Adeno I Species 3-Virus, Adeno II Species 8-Virus, PI-III (Parainfluenza 3-Virus), IBR/IPV-Virus and Chlamydia. A total of 1376 serum samples were examined and showed different titers of specific antibodies against respiratory pathogens. 449 blood samples were submitted to serological examinations in the year 1984, 525 in 1985 and 402 in 1986. Antibodies against BRSV could be found in 17.4% of the sera or in 55.9% of the herds. Specific antibodies against Adeno I Species 3 occurred in 33.9% of the sera resp. in 74.2% of the units. 35.2% of the cows or 80.1% of the herds showed positive SN-titres against Adeno II Species 8. IBR/IPV-specific antibodies were detected in 2.3% of the sera resp. 3.8% of the herds. Antibodies against Chlamydia could be found in 8.8% of the cows or in 22.6% of the units. In the years mentioned above significant differences of antibodies against BRSV, Adeno I Species 3, Adeno II Species 8 and Chlamydia could be observed, but not against PI-III and IBR/IPV as well. By means of the Chi-Square-Test significant differences between Simmentaler, Austrian Brown Cattle and Holstein Friesian regarding antibodies against IBR/IPV and Adeno II Species 8 could be found. No significant differences between the serological results of herds, which contained on of the three breeds could be observed.