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Locus HLA-DRB3 codes for the serologically defined supertypic specificity DRw52 in HLA-DR3, -5 and -w6 haplotypes. Three specificities of DRw52 (DRw52a, -b and -c) can further be distinguished by cellular techniques or by DNA typing with allele-specific oligonucleotide probes. These specificities were recently reported to have significant importance in antigen presentation. To avoid a time-consuming hybridization procedure, we have developed a simple typing system using PCR and subsequent digestion by allele-specific restriction endonucleases. A system was established with locus-specific amplification of HLA-DRB3 and digestion by the enzymes KpnI, Seal and HinfI which recognize unique restriction sites within the amplified region. This allowed HLA-DRB3 typing on agarose gel by determining whether the amplification product has been digested or not. This typing system was compared to conventional oligotyping by analyzing 145 RFLP-typed individuals for their DRw52 specificity using both methods. Agarose typing correlated well with oligotyping and was shown to be more simple and practical even in heterozvaous individuals.

Danon disease is an X-linked disorder resulting from mutations in the lysosome-associated membrane protein-2 (LAMP2) gene. We report a male patient with skeletal myopathy, mental retardation, and massive hypertrophic obstructive cardiomyopathy necessitating heart transplantation. Immunohistochemistry of skeletal muscle and leukocytes, western blot analysis of leukocytes and cardiac muscle, flow cytometry, and DNA sequencing were performed. Muscle biopsy revealed autophagic vacuolar myopathy and lack of immunohistochemically detectable LAMP-2. Diagnosis of Danon disease was confirmed by western blot analysis of myocardial tissue and peripheral blood sample of the patient showing deficiency of LAMP-2 in myocardium and leukocytes. Moreover, absence of LAMP-2 in lymphocytes, monocytes and granulocytes was shown by flow cytometric analysis. Genetic analysis of the LAMP2 gene revealed a novel 1-bp deletion at position 179 (c.179delC) at the 3′ end of exon 2, resulting in a frameshift with a premature stop codon.

More than 95% of mugwort pollen-allergic individuals are sensitized to Art v 1, the major allergen in mugwort pollen. Interestingly, the CD4 T cell response to Art v 1 involves only one single immunodominant peptide, Art v 125–36 (KCIEWEKAQHGA), and is highly associated with the expression of HLA-DR1. Therefore, we investigated the molecular basis of this unusual immunodominance among allergens. Using artificial APC expressing exclusively HLA-DRB1*0101 and HLA-DRA*0101, we formally showed that DR1 acts as restriction element for Art v 125–36-specific T cell responses. Further assessment of binding of Art v 125–36 to artificial HLA-DR molecules revealed that its affinity was high for HLA-DR1. Amino acid I27 was identified as anchor residue interacting with DR molecules in pocket P1. Additionally, Art v 125–36 bound with high affinity to HLA-DRB1*0301 and *0401, moderately to HLA-DRB1*1301 and HLA-DRB5*0101, and weakly to HLA-DRB1*1101 and *1501. T cell activation was also inducible by Art v 125–36-loaded, APC-expressing HLA molecules other than DR1, indicating degeneracy of peptide binding and promiscuity of TCR recognition. Specific binding of HLA-DRB1*0101 tetramers containing Art v 119–36 allowed the identification of Art v 125–36-specific T cells by flow cytometry. In summary, the immunodominance of Art v 125–36 relies on its affinity to DR1, but is not dictated by it. Future investigations at the molecular HLA/peptide/TCR and cellular level using mugwort pollen allergy as a disease model may allow new insights into tolerance and pathomechanisms operative in type I allergy, which may instigate new, T cell-directed strategies in specific immunotherapy.

Objective Advanced mast cell (MC) neoplasms are usually resistant to conventional therapy. Therefore, current research focuses on new targets in neoplastic MC and development of respective targeted drugs. Mastocytomas in dogs often behave as aggressive tumors. We report that heat-shock protein 32 (Hsp32), also known as heme oxygenase-1, is a survival-enhancing molecule and new target in canine mastocytoma cells. Materials and Methods As assessed by reverse transcriptase polymerase chain reaction, Northern blotting, immunocytochemistry, and Western blotting, primary neoplastic dog MC, and the canine mastocytoma-derived cell line C2 expressed Hsp32 mRNA and the Hsp32 protein in a constitutive manner. Results The KIT-targeting drug midostaurin inhibited expression of Hsp32, as well as survival in C2 cells. Confirming the functional role of Hsp32, the inhibitory effect of midostaurin on C2 cells was markedly reduced by the Hsp32-inductor hemin. Two pharmacologic Hsp32-inhibitors, styrene maleic-acid micelle-encapsulated ZnPP (SMA-ZnPP) and pegylated zinc-protoporphyrin (PEG-ZnPP) were applied. Both drugs were found to inhibit proliferation of C2 cells as well as growth of primary neoplastic canine MC. The growth-inhibitory effects of SMA-ZnPP and PEG-ZnPP were dose- and time-dependent (IC 50 : 1–10 μM) and found to be associated with induction of apoptosis. Conclusions Hsp32 is an important survival factor and interesting new target in neoplastic canine MC. Trials with Hsp32-targeted drugs are now warranted to define the clinical efficacy of these drugs.

Background and Objectives In a majority of all patients with systemic mastocytosis (SM) including those with mast cell leukemia (MCL), neoplastic mast cells (MC) display the D816V-mutated variant of KIT . The respective oncoprotein, KIT D816V, exhibits constitutive tyrosine kinase (TK) activity and has been implicated in malignant cell growth. Therefore, several attempts have been made to identify KIT D816V-targeting drugs. Design and Methods We examined the effects of the novel TK-inhibitor dasatinib alone and in combination with other targeted drugs on growth of neoplastic MC. Results Confirming previous studies, dasatinib was found to inhibit the TK activity of wild type (wt) KIT and KIT -D816V as well as growth and survival of neoplastic MC and of the MCL cell line, HMC-1. The growth-inhibitory effects of dasatinib in HMC-1 cells were found to be associated with a decrease in expression of CD2 and CD63. In addition, we found that dasatinib blocks KIT D816V-induced cluster-formation and viability in Ba/F3 cells. In drug combination experiments, dasatinib was found to co-operate with PKC412, AMN107, imatinib, and 2CdA in producing growth-inhibition and apoptosis in neoplastic MC. In HMC-1.1 cells lacking KIT D816V, all drug interactions were found to be synergistic in nature. By contrast, in HMC-1.2 cells exhibiting KIT D816V, only the combinations dasatinib+PKC412 and dasatinib+2CdA were found to produce synergistic effects. Interpretation and Conclusions Combinations of targeted drugs may represent an interesting pharmacologic approach for the treatment of aggressive SM or MCL.

The PI3Kδ isoform is a candidate drug target in leukemia. Here, we explored its role in Abelson-induced leukemia. Frank leukemia emerges if the tumor cells have managed to outwit the immune system. The absence of PI3Kδ affected both the tumor cells and the NK cells. Abelsontransformed PI3Kδ-/cells induced leukemia in RAG2-/animals with a significantly increased latency, implicating PI3Kδ in tumor progression. NK cell function, however, was also contingent on PI3Kδ. PI3Kδ-/NK cells failed to lyse target cells. Capacitance measurements revealed the underlying defect: in PI3Kδ-/NK cells lytic granules did not fuse with the cell membrane. Accordingly, transplanted leukemic cells killed PI3Kδ-/animals more rapidly, both in syngeneic (PI3Kδ-/-) or immunocompromised (RAG2-/PI3Kδ-/-) animals. Our observations define a dual function of PI3Kδ in leukemia and document that the action of PI3Kδ in the NK compartment is as relevant to the survival of the mice as the delayed tumor progression. from 13th Scientific Symposium of the Austrian Pharmacological Society (APHAR). Joint Meeting with the Austrian Society of Toxicology (ASTOX) and the Hungarian Society for Experimental and Clinical Pharmacology (MFT) Vienna, Austria. 22–24 November 2007

Chronic graft-versus-host disease (cGVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (HSCT) and a leading cause of non-relapse mortality (NRM). Currently, biology-based markers are lacking both for diagnosis and for monitoring the activity of cGVHD. Seventy patients who received HSCT were enrolled in a pilot study, including 21 without cGVHD and 49 with active or resolved cGVHD. Evaluations were comprised of clinical parameters including cGVHD severity and infections. Peripheral blood cells were analyzed by multi-parameter flow cytometry. The CD19+ B cell compartment was further subdivided by staining for surface IgD, CD21 and CD27. No significant differences in absolute B, T, and natural killer (NK) cell numbers were observed between the groups with and without cGVHD. However, elevated numbers (>15% of B lymphocytes) of immature/transitional CD19+/CD21- B cells were associated with the occurrence of severe infections (P = .003). Most significantly, all patients with active cGVHD and elevated numbers of CD19+/CD21- B lymphocytes experienced severe infections (P = .00016). The numbers of both non-class-switched and class-switched memory B cells were significantly lower in patients with active cGVHD when compared to patients who never experienced cGVHD (P = .002 and P = .001). Perturbation of circulating B lymphocyte compartments may serve as a novel biomarker for monitoring cGVHD activity and its impact on the immune system. A prospective study on unselected patients assessed serially for B cell reconstitution after HSCT is warranted.

B-cell-derived non-Hodgkin's lymphoma (NHL) of childhood and adolescence can be subdivided into mature B-cell neoplasms and B-cell precursor (BCP) lymphoblastic lymphomas (LBL). The former comprise Burkitt's lymphoma and leukemia, diffuse large B-cell lymphoma (DLBCL) and primary mediastinal large B-cell lymphoma. With adequate therapy, about 80–90% of the patients with mature B-cell malignancies can now become long-term, disease-free survivors.1 However, the prognosis of children who suffer from a relapse is still very poor. In most therapy studies, risk stratification for mature B-cell NHL is largely based on stage of disease and the initial serum lactate dehydrogenase level.1 Moreover, to date, evaluation of minimal disseminated disease (MDD) at the time of diagnosis, that is in the bone marrow (BM) or peripheral blood, and of the kinetics of minimal residual disease (MRD) have not yet been implemented into the risk assignment of modern protocols. Nevertheless, as Burkitt's lymphoma is genetically characterized by the presence of a chromosomal translocation involving the C-MYC oncogene on chromosome 8 and one of the loci of the immunoglobulin (Ig) heavy- or light-chain genes on chromosomes 14, 22 or 2, the molecular fusion products have been explored in a few polymerase chain reaction (PCR)-based studies to assess both minimal disseminated and residual diseases.2, 3 However, considering the long-time experience of different techniques on MRD analysis in childhood acute lymphoblastic leukemia (ALL), it seems very likely that besides PCR-based examinations of disease-specific fusion genes or clone-specific Ig gene rearrangements, flow cytometry could also allow MDD and MRD detection in Burkitt's malignancies.4, 5

MCL-1 is a Bcl-2 family member that has been described as antiapoptotic in various myeloid neoplasms. Therefore, MCL-1 has been suggested as a potential new therapeutic target. Systemic mastocytosis (SM) is a myeloid neoplasm involving mast cells (MCs) and their progenitors. In the present study, we examined the expression and functional role of MCL-1 in neoplastic MCs and sought to determine whether MCL-1 could serve as a target in SM. As assessed by RT-PCR and immunohistochemical examination, primary neoplastic MCs expressed MCL-1 mRNA and the MCL-1 protein in all SM patients examined. Moreover, MCL-1 was detectable in both subclones of the MC line HMC-1—HMC-1.1 cells, which lack the SM-related KIT mutation D816V, and HMC-1.2 cells, which carry KIT D816V. Exposure of HMC-1.1 cells or HMC-1.2 cells to MCL-1–specific antisense oligonucleotides (ASOs) or MCL-1–specific siRNA resulted in reduced survival and increased apoptosis compared with untreated cells. Moreover, MCL-1 ASOs were found to cooperate with various tyrosine kinase inhibitors in producing growth inhibition in neoplastic MCs, with synergistic effects observed with PKC412, AMN107, and imatinib in HMC-1.1 cells and with PKC412 in HMC-1.2 cells. Together, these data show that MCL-1 is a novel survival factor and an attractive target in neoplastic MCs.

Many infectious viruses coevolved with the vertebrate immune system. During the assembly of enveloped viruses, lipid ordered domains of the host cell plasma membrane, called lipid rafts, frequently function as a natural meeting point for viral proteins. The role of lipid rafts in the organization of complex combinations of immune receptors during antigen presentation and T cell signaling is widely recognized. In our studies, we determined whether lipid rafts, virus budding, and molecular interactions during T cell activation could be brought into a novel context to create artificial antigen-presenting particles. We show here that cell-free virus-like particles (VLP) expressing a surrogate TCR/CD3 ligand (OKT3scFv) and the costimulator CD80 polyclonally activate human T cells independently of accessory cells. VLP expressing the glycoprotein epitope 33–41 of the lymphocytic choriomeningitis virus in the context of H-2D b activate and expand naïve, antigen-specific CD8 + T lymphocytes and differentiate them into cytotoxic effector cells. Efficient targeting of T cell ligands to lipid rafts and ultimately to VLP is achieved by C-terminal introduction of glycosyl phosphatidyl inositol acceptor sequences, replacing transmembrane and intracellular domains. In this work, basic functions of immunostimulatory molecules meet virus biology and translate into a reductionist antigen-specific T lymphocyte-stimulating vehicle, which we refer to as immunosomes. A large variety of agonistic and antagonistic accessory molecules on genuine antigen-presenting cells may complicate the predictable manipulation of T cells as well as the analysis of selected receptor combinations, making immunosomes potentially useful reagents for such purposes in the future.

Basophil numbers are typically elevated in chronic myeloid leukemia (CML) and increase during disease progression. Histamine is an essential mediator and marker of basophils and is highly up-regulated in CML. We examined the biochemical basis of histamine synthesis in CML cells. The CML-specific oncoprotein BCR/ABL was found to promote expression of histidine decarboxylase (HDC) and synthesis of histamine in Ba/F3 cells. Moreover, the BCR/ABL tyrosine kinase inhibitors imatinib (STI571) and nilotinib (AMN107) decreased histamine levels and HDC mRNA expression in BCR/ABL-transformed Ba/F3 cells, in the CML-derived basophil cell line KU812, and in primary CML cells. Synthesis of histamine was found to be restricted to the basophil compartment of the CML clone and to depend on signaling through the PI3-kinase pathway. CML cells also expressed histamine receptors (HRs), including HR-1, HR-2, HR-4, and histamine-binding CYP450 isoenzymes which also serve as targets of HR antagonists. The HR-1 antagonists loratadine and terfenadine, which bind to CYP450, were found to counteract proliferation of CML cells, whereas no growth inhibition was observed with the HR-1 antagonist fexofenadine which is not targeted or metabolized by CYP450. Moreover, DPPE, an inhibitor of histamine-binding CYP450 isoenzymes, produced growth inhibition in CML cells. Together, these data show that BCR/ABL promotes histamine production in CML cells and that certain HR-targeting drugs exert antileukemic effects on CML cells.

Vascular endothelial growth factor (VEGF) is a major regulator of angiogenesis and a potential autocrine growth factor for neoplastic cells in various malignancies. In the present study, we have investigated expression of VEGF and VEGF receptors in canine mastocytomas and the canine mastocytoma cell line C2. As assessed by immunostaining of tissue sections and cytospin slides, primary neoplastic mast cells (MC) and C2 cells were found to express the VEGF protein. In Northern blot and RT-PCR experiments, C2 cells expressed VEGF mRNA in a constitutive manner. VEGF mRNA expression in C2 cells was counteracted by LY294002 and rapamycin, suggesting involvement of the PI3-kinase/mTOR pathway. Moreover, C2 cells were found to express VEGF receptor-1 (Flt-1) and VEGF receptor-2 (KDR). However, recombinant VEGF failed to promote 3 H-thymidine uptake in C2 cells, and a neutralizing anti-VEGF antibody (bevacizumab) failed to downregulate spontaneous proliferation in these cells. In addition, rapamycin decreased the expression of VEGF in C2 cells at the mRNA and protein level without suppressing their proliferation. Together, canine mastocytoma cells express VEGF as well as VEGF receptors. However, despite co-expression of VEGF and its receptors, VEGF is not utilized as an autocrine growth regulator by canine mastocytoma cells.

A number of autocrine and paracrine growth regulators are considered to be involved in the survival and proliferation of blast cells in acute myeloid leukemia (AML). We have recently shown that blast cells in a group of patients with AML produce and secrete the mitogenic enzyme tryptase. In the present study, we examined functional effects of tryptase in the context of AML. As assessed by 3H-thymidine uptake experiments, tryptase-containing serum from patients with AML as well as heparin-complexed recombinant tryptase were found to promote the proliferation of cultured bone marrow- and lung fibroblasts in a dose-dependent manner. A neutralizing antibody against human beta-tryptase was found to diminish these growth-stimulatory effects of serum-tryptase in all patients examined. Tryptase also induced the expression of mRNA for GM-CSF and SCF, two cytokines known to promote growth of AML cells, in cultured bone marrow fibroblasts. Neither recombinant tryptase nor tryptase-rich serum of AML patients, showed an effect on the growth of leukemic blast cells irrespective of the FAB category or expression of protease-activated receptor (PAR)-2, a putative molecular target of tryptase. Together, tryptase is secreted from AML blasts as a biologically active molecule that may exhibit paracrine rather than autocrine effects in AML.

Seizure prophylaxis with phenytoin is a common measure in oncologic patients with brain metastases. In these patients, generalized severe adverse drug reactions such as erythema multiforme (EEM) may occur. However, in a subgroup of patients with brain radiation therapy, EEM-like lesions develop particularly in the radiation field. Most recently, the acronym EMPACT (Erythema Multiforme associated with Phenytoin And Cranial radiation Therapy) was proposed to specifically describe this syndrome.Here, we report on EMPACT syndrome in a 46-year-old woman. Therapeutic measures included seizure prophylaxis with phenytoin and total brain radiation therapy of brain metastases from bronchial carcinoma. Three weeks after introduction of phenytoin, the patient presented with EEM-like skin lesions restricted to the original radiation field and facial mucocutaneous involvement. After a few days, the rash spread to the upper part of the body. She was also in poor general condition.The immediate cessation of phenytoin therapy, combined with administration of systemic corticosteroids and high dose immunoglobulins along with intensive local treatment and pain medications, resulted in complete resolution of the skin eruption. Patch testing to phenytoin was positive after 72 hours.EMPACT should be classified as an specific entity among the EEM-like drug reactions as it only appears after radiotherapy and seizure prophylaxis with the anticonvulsant phenytoin. We propose including specific type IV-sensitization to phenytoin into the definition of EMPACT.

Chronic myeloid leukemia (CML) is a myeloproliferative disease in which BCR/ABL enhances survival of leukemic cells through modulation of proapoptotic and antiapoptotic molecules. Recent data suggest that proapoptotic Bcl-2–interacting mediator (Bim) plays a role as a tumor suppressor in myeloid cells, and that leukemic cells express only low amounts of this cell death activator. We here show that primary CML cells express significantly lower amounts of bim mRNA and Bim protein compared with normal cells. The BCR/ABL inhibitors imatinib and AMN107 were found to promote expression of Bim in CML cells. To provide direct evidence for the role of BCR/ABL in Bim modulation, we employed Ba/F3 cells with doxycycline-inducible expression of BCR/ABL and found that BCR/ABL decreases expression of bim mRNA and Bim protein in these cells. The BCR/ABL-induced decrease in expression of Bim was found to be a posttranscriptional event that depended on signaling through the mitogen-activated protein kinase pathway and was abrogated by the proteasome inhibitor MG132. Interestingly, MG132 up-regulated the expression of bim mRNA and Bim protein and suppressed the growth of Ba/F3 cells containing wild-type BCR/ABL or imatinib-resistant mutants of BCR/ABL. To show functional significance of “Bim reexpression,” a Bim-specific small interfering RNA was applied and found to rescue BCR/ABL-transformed leukemic cells from imatinib-induced cell death. In summary, our data identify BCR/ABL as a Bim suppressor in CML cells and suggest that reexpression of Bim by novel tyrosine kinase inhibitors, proteasome inhibition, or by targeting signaling pathways downstream of BCR/ABL may be an attractive therapeutic approach in imatinib-resistant CML.

The mammalian target of rapamycin (mTOR) has recently been described to be constitutively activated in Bcr-Abl-transformed cells and to mediate rapamycin-induced inhibition of growth in respective cell lines. We have recently shown that rapamycin down-regulates expression of vascular endothelial growth factor (VEGF), a mediator of leukemia-associated angiogenesis, in primary CML cells. In the present study, we analyzed growth-inhibitory in vitro and in vivo effects of rapamycin on primary CML cells and asked whether rapamycin-induced suppression of VEGF in leukemic cells is related to growth inhibition. Rapamycin dose dependently inhibited growth of primary CML cells obtained from patients with imatinib-responsive or imatinib-resistant disease as well as growth of Bcr-Abl-transformed imatinib-resistant cell lines. Moreover, we observed potent cytoreductive effects of rapamycin in a patient with imatinib-resistant Bcr-Abl+ leukemia. The growth-inhibitory effects of rapamycin on CML cells were found to be associated with G1 cell cycle arrest and with induction of apoptosis. In all cell types tested, rapamycin was found to down-regulate expression of VEGF. However, exogenously added VEGF did not counteract the rapamycin-induced decrease in proliferation. In conclusion, rapamycin inhibits growth of CML cells in vitro and in vivo and, in addition, down-regulates expression of VEGF. Both effects may contribute to the antileukemic activity of the drug in CML.

Antiapoptotic members of the bcl-2 family have recently been implicated in the pathogenesis of chronic myeloid leukemia (CML), a hematopoietic neoplasm associated with the BCR/ABL oncogene. We have examined expression of MCL-1 in primary CML cells and BCR/ABL-transformed cell lines. Independent of the phase of disease, isolated primary CML cells expressed myeloid cell leukemia-1 (mcl-1) mRNA and the MCL-1 protein in a constitutive manner. The BCR/ABL inhibitor imatinib (=STI571) decreased the expression of MCL-1 in these cells. Correspondingly, BCR/ABL enhanced mcl-1 promoter activity, mcl-1 mRNA expression, and the MCL-1 protein in Ba/F3 cells. BCR/ABL-dependent expression of MCL-1 in Ba/F3 cells was counteracted by the mitogen-activated protein-kinase/extracellular signal-regulated kinase (MEK) inhibitor, PD98059, but not by the phosphoinositide 3-kinase inhibitor, LY294002. Identical results were obtained for constitutive expression of MCL-1 in primary CML cells and the CML-derived cell lines K562 and KU812. To investigate the role of MCL-1 as a survival-related target in CML cells, mcl-1 siRNA and mcl-1 antisense oligonucleotides (ASOs) were applied. The resulting down-regulation of MCL-1 was found to be associated with a substantial decrease in viability of K562 cells. Moreover, the mcl-1 ASO was found to synergize with imatinib in producing growth inhibition in these cells. Together, our data identify MCL-1 as a BCR/ABL-dependent survival factor and interesting target in CML. (Blood. 2005;105:3303-3311)

In an effort to characterize molecules with immunoregulatory potential, we raised mAbs to human dendritic cells. We selected an Ab that recognizes a molecule that is induced on monocytes differentiated in vitro toward dendritic cells. Retroviral expression cloning identified this molecule as B7-H3, a member of the B7 family described recently. In contrast to an earlier report, in which B7-H3 was described as a molecule consisting of two Ig-like domains, our cDNA encoded a type I membrane protein with four Ig-like domains, and the molecule identified by us was therefore named 4Ig-B7-H3. mRNA analysis as well as Western blotting experiments performed by us did not reveal evidence for a small B7-H3. B7-H3 is not expressed on peripheral blood lymphocytes, monocytes, or granulocytes. Upon in vitro stimulation, the expression of B7-H3 is induced on T cells, B cells, and NK cells. A number of different approaches were used to investigate the function of human B7-H3. In contrast to an earlier report, our data do not support a costimulatory role of B7-H3 in anti-CD3-mediated activation of the TCR-complex resulting in T cell proliferation and IFN-γ production.

Dendritic cells (DC) play a pivotal role in the function of the immune system, for they are the primary antigen-presenting cells (APC) in the activation of naive T-lymphocyte responses (1). Recent studies have uncovered complexity in the DC lineage with several subsets, functions, and maturational stages. Although it is generally accepted that human DC derive from hematopoietic progenitor cells (2-9), it is not clear at present whether DC cells and their precursors represent a separate hematopoietic lineage or whether DC should be seen as specialized macrophages with particular morphological, molecular, and functional features. Several lines of evidence point to DC and monocytes/ macrophages being offspring of the same CD34(+) hematopoietic progenitor cell (3-5,12-14, and reviewed in ([10,11].) DC committed precursor cells have also been identified in peripheral blood (15-18).

We have shown previously that the oligomeric endostatin domain of collagen XVIII (NC1) functioned as a motility-inducing factor regulating the extracellular matrix-dependent morphogenesis of endothelial cells. This motogenic activity gave rise to structures resembling filipodia and lamellipodia and was dependent on Rac, Cdc42, and mitogen-activated protein kinase. Here, we demonstrate that these properties of endostatin are primarily mediated by laminin in the basement membrane and heparan sulfates on the cell surface. The sites of interaction between laminin and oligomeric endostain include the N-terminal regions of all three laminin chains (amino acids 204-1243 of the alpha chain, 932-1161 of the beta chain, and 150-965 of the gamma chain). A monoclonal antibody that blocks the interactions between endostatin and laminin was utilized to inhibit the motogenic activity of endostatin. In parallel, we have engineered selective point mutations and produced recombinant forms that lack binding to heparan sulfates on the cell surface. Our data are consistent with a model of endostatin with two binding sites: one mainly to laminin in the basement membrane and the other to heparan sulfates on the cell surface. The two binding domains on endostatin appear to be separate with the possibility of some overlap between the two sites.

Specific interactions between envelope and core proteins govern the membrane assembly of most enveloped viruses. Despite this, mixed infections lead to pseudotyping, the association of the viral cores of one virus with the envelopes of another. How does this occur? We show here that the detergent-insoluble lipid rafts of the plasma membrane function as a natural meeting point for the transmembrane and core components of a phylogenetically diverse collection of enveloped viruses. As a result, viral particles preferentially incorporate both the envelope components of other viruses as well as the extra- and intracellular constituents of host cell lipid rafts, including gangliosides, glycosyl phosphatidylinositol-anchored surface proteins, and intracellular signal transduction molecules. Pharmacological disruption of lipid rafts interferes with virus production.

During paternity testing, we encountered the following constellation in the Jk system: the mother's phenotype was Jk(a-b+), while the son was typed as Jk(a+b-). The deduced genotype of the mother would have been Jkb Jkb, and each offspring should then express the Jk(b) antigen. Consequently, non-maternity would be deduced. Since no material was available for extended family studies or HLA typing, except for the DNA of the propositi, only RFLP analysis could bring clarification in this case. The application of four highly polymorphic single locus probes proved the maternity and hence the existence of a Jk-Null allele. We conclude that direct testing at the DNA level may help resolving cases where, by conventional parentage testing, conclusive results are unachievable because of putative ‘Null’ alleles.

Using a recently described serum-free culture system of purified human CD34+ progenitor cells, we show here a critical cooperation of flt3 ligand (FL) with transforming growth factor-beta1 (TGF-beta1) in the induction of in vitro dendritic cell/Langerhans cell (DC/LC) development. The addition of FL to serum-free cultures of CD34+ cells supplemented with TGF-beta1, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor alpha, and stem cell factor strongly increases both percentages (mean, 36% +/- 5% v 64% +/- 4%; P = .001) and total numbers (4.4- +/- 0.8-fold) of CD1a+ dendritic cells. These in vitro-generated CD1a+ cells molecularly closely resemble a particular type of DC known as an epidermal Langerhans cell. Generation of DC under serum-free conditions was found to strictly require supplementation of culture medium with TGF-beta1. Upon omission of TGF-beta1, percentages of CD1a+ DC decreased (to mean, 10% +/- 8%; P = .001) and, in turn, percentages of granulomonocytic cells (CD1a- cells that are lysozyme [LZ+]; myeloperoxidase [MPO+]; CD14+) increased approximately threefold (P.05). Furthermore, in the absence of TGF-beta1, FL consistently promotes generation of LZ+, MPO+, and CD14+ cells, but not of CD1a+ cells. Serum-free single-cell cultures set up under identical TGF-beta1- and FL-supplemented culture conditions showed that high percentages of CD34+ cells (mean, 18% +/- 2%; n = 4) give rise to day-10 DC colony formation. The majority of cells in these DC-containing colonies expressed the Langerhans cell/Birbeck granule specific marker molecule Lag. Without TGF-beta1 supplementation, Lag+ colony formation is minimal and formation of monocyte/macrophage-containing colonies predominates. Total cloning efficiency in the absence and presence of TGF-beta1 is virtually identical (mean, 41% +/- 6% v 41% +/- 4%). Thus, FL has the potential to strongly stimulate DC/LC generation, but has a strict requirement for TGF-beta1 to show this costimulatory effect.

Several studies have shown that substantial numbers of functional dendritic cells (DC) can be generated in vitro from human CD34+ progenitor cells upon culture with the two cytokines GM-CSF and TNFα.1,2,3 More recent data suggest that at least two in vitro differentiation pathways for the development of DC seem to exist.4,5,6 One pathway gives rise to Langerhans (LC) type DC. Phenotypically, this pathway follows the route CD34+ to CD1a+ to CD1a+/Lag+ and does not involve a CD14+ monocytoid intermediate cell stage.5

Background Pyogenic sterile arthritis, pyoderma gangrenosum, and acne (PAPA) syndrome (OMIM 604416) is a rare autosomal dominant inherited autoinflammatory syndrome characterized by pyogenic sterile arthritis and less frequently accompanied by pyoderma gangrenosum and acne. It is associated with dominant missense mutations in the proline-serine-threonine phosphatase–interacting protein 1 gene (PSTPIP1) located on chromosome 15. The patient was diagnosed as having features of a PAPA-like syndrome in which cutaneous manifestations, such as pyoderma gangrenosum and acne fulminans, predominated. Observations Sequencing of the PSTPIP1 gene was performed in the patient and his extended family. The patient's DNA analysis revealed a homozygous nucleotide exchange c.773G>C in the PSTPIP1 gene, leading to the substitution of glycine 258 by alanine (p.Gly258Ala), a previously reported heterozygous polymorphism. Heterozygous changes were identified in both of the patient's parents and in 7 other family members, all of whom were asymptomatic. The patient was treated with canakinumab, a human anti–interleukin 1β monoclonal antibody, which led to rapid remission of the symptoms. Conclusions To our knowledge, this is the first reported case of the resolution of dermatological symptoms associated with a PAPA-like syndrome using canakinumab treatment. Further study of the p.Gly258Ala variant is warranted to determine whether this mutation has a role in causing an apparently recessive cutaneous syndrome resembling PAPA syndrome.

Dendritic cells (DC) are the most potent APCs within the immune system. We show here that highly purified CD14(bright) peripheral blood monocytes supplemented with granulocyte-monocyte (GM)-CSF plus IL-4 develop with high efficacy (>95% of input cells) into DC. They neo-expressed CD1a, CD1b, CD1c, CD80, and CD5; they massively up-regulated CD40 (109-fold) and HLA-DQ and DP (125- and 87-fold); and significantly (>5-fold) up-regulated HLA-DR, CD4, CD11b, CD11c, CD43, CD45, CD45R0, CD54, CD58, and CD59. CD14, CD15s, CD64, and CDw65 molecules were down-regulated to background levels, and no major changes were observed for HLA class I, CD11a, CD32, CD33, CD48, CD50, CD86, CDw92, CD93, or CD97. Monocytes cultured in parallel with GM-CSF plus TNF-alpha were more heterogeneous in expression densities but otherwise similar in their surface molecule repertoire. They clearly differed, however, in their accessory cell capacity. Only GM-CSF plus IL-4-cultured cells were found to be potent stimulators in allogeneic and autologous MLR and they presented tetanus toxoid 100- to 1000-fold more efficiently than other cell populations tested. Furthermore, only cytokine-treated monocytes formed clusters with resting T cells. At variance from all these similarities between in vitro-generated monocyte-derived DC and in vivo-developing DC, the DC populations generated by us contained significant amounts of myeloperoxidase and also expressed lysozyme. At least in this respect they, thus, differ from "classical" DC types.

Monoclonal antibodies recognizing CD27, a member of the nerve growth factor receptor family, have been observed to enhance or inhibit PHA- or CD3 mAb-stimulated T cell proliferation. In this study, we investigate the effects of CD27 stimulation on the proliferation and cytokine production of T cells stimulated simultaneously through CD3, CD2, or CD28. Here we report that simultaneous crosslinking of CD27 plus CD28 results in T cell proliferation and in the production of IL-2, IL-4, IL-10, and IFN-γ. CD27 plus CD28 stimulation thus introduces a novel Ag-independent pathway of T cell activation. We further show that all major T cell subsets (CD4 + , CD8 + , CD4 + CD45RA + , or CD4 + CD45RO + T cells) respond to CD27 plus CD28-mediated costimulation. Synergistic effects on T cell stimulation are also found when CD27 is crosslinked on cells stimulated simultaneously through CD3 or CD2. In further experiments we show that crosslinking of CD27 elevates cytoplasmic free Ca 2+ levels. Finally, both the CD3 plus CD27 and the CD28 plus CD27-stimulated T cell proliferation is sensitive to inhibition by CsA. Taken together, these data emphasize the importance of CD27 stimulation in the context of simultaneously received signals through CD3, CD2 or, most importantly, through CD28. Furthermore, signaling through CD27 appears to involve Ca 2+ -dependent mechanisms sensitive to CsA.

Background Currently, T cell receptor (TCR) transgenic (tg) mice with a murine TCR specific for chicken ovalbumin in the context of a murine restriction element (I-Ad) are frequently used in allergy research to investigate T helper cell differentiation and allergy treatment in vivo. Methods We here aimed to generate double tg mice expressing a human TCR specific for the immuno-dominant epitope of the major mugwort (Artemisia vulgaris) pollen allergen Art v 1 in the context of the human restriction element HLA-DR1 to provide a valid model for studying allergy development and treatment in vivo. To obtain high expression levels the allergen-specific human TCR variable sequences were chimerized with murine TCR constant sequences. Resulting transgenes were cloned into the pTcass vector system and thus put under the transcriptional control of the natural TCR alpha and beta promotor/enhancer elements. Allergen-specific TCR tg founder mice were cross-bread with HLA-DR1+ B10.M-DR1dlAb1-Ea mice. Results Immunophenotyping of double tg TCR/HLA-DR1 mice revealed clear-cut expression of the Art v 1-specific TRBV18 chain on peripheral blood CD3+ T lymphocytes and HLA-DR1 expression on CD14+ monocytes and B220+ B lymphocytes. In vitro, splenocytes from TCR/HLA-DR1 double tg mice but not of HLA-DR1 single tg mice or wt mice specifically proliferated upon incubation with the human-relevant immuno-dominant Art v 125 to 36 peptide or whole Art v 1 protein. No proliferation was observed upon incubation with control peptides or proteins. Allergen-specific cellular proliferation is accompanied by the production of a balanced cytokine milieu including IFN-gamma, IL-2, IL-4, IL-6, IL-13 and IL-17 (>50 pg/mL per 2 × 105 splenocytes). No cytokine secretion was evident upon incubation of splenocytes with a control peptide or medium alone. Importantly, double tg mice are proficient to mount both IgG2a and IgG1, IgE responses when i.p. immunized with antigen plus alum. Conclusions A fully humanized allergy model, in which all components of the allergen-specific synapse are well-defined enables to analyze the relevant T-cell dependent (and independent) pathways by which allergic diseases can be influenced in vivo and will provide important insights into the pathophysiology of allergic diseases and their possible cure in the future.

CCLE, a disease entity at the benign end of the lupus spectrum, is characterized by marked photosensitivity and skin lesions in sun-exposed areas. The histopathology of lesions resembles hypersensitivity type IV reactions. We have asked whether an association between class II alleles and CCLE exists. RFLP analysis of HLA-DQA genes revealed a Taq I HLA-DQA1 allelic restriction fragment overrepresented in a group consisting of 26 patients as compared to healthy control individuals. This result was corroborated by typing with oligonucleotide probes. The presence of the DQA1*0102 allele in the patients' group led to a relative risk of 4.57, with a statistical significance of p0.05 after correction for 36 comparisons. Although not statistically significant, it is interesting that all patients possess in at least one of their HLA-DQA1 alleles a nucleotide sequence coding for the amino acid glutamine at position 34 of the DQ alpha molecule. The expected frequency of these alleles in the control population amounts to 82%. The HLA-DRB1*16 allele, which is found in linkage disequilibrium with the HLA-DQA1*0102 allele, is also observed at an increased frequency in the patient's group, though the association was not significant after correction for the number of comparisons. However, no associations of CCLE with alleles at the HLA-DPB1 locus was found. The association of CCLE with certain HLA class II alleles points to an involvement of HLA-DQ and/or -DR molecules in the pathogenesis of the disease. Alternatively, genetic loci in linkage disequilibrium may code for elements which contribute to the development of CCLE.(ABSTRACT TRUNCATED AT 250 WORDS)

The present study investigates the effect of transforming growth factor (TGF)-beta on the production of IL-4 and IFN-gamma by the leukemia Th0 type cell line HUT78, by freshly isolated human T cells, and by antigen specific human T cell clones. We found that IL-4 and IFN-gamma, but not IL-2, production by stimulated HUT78 cells was inhibited by TGF-beta 1. TGF-beta 1 also reduced the accumulation of IL-4 and IFN-gamma specific mRNA in stimulated HUT78 cells. However, IL-2 and IL-7 co-stimulated IL-4 and IFN-gamma production, whereas IL-1, IL-3, IL-5, IL-6, IL-8, tumor necrosis factor-alpha or granulocyte macrophage colony stimulating factor had no effect. Because IL-2 is an important helper cytokine for the production of IL-4 and IFN-gamma, we investigated whether signal transduction through the IL-2 receptor is impaired by TGF-beta 1. We found that tyrosine phosphorylation in response to IL-2 in HUT78 cells was strongly inhibited by a short preincubation with TGF-beta 1. Evidence for an antagonistic role for TGF-beta 1 and IL-2 comes from the finding that high doses of IL-2 could partially overcome TGF-beta 1 mediated inhibition of IL-4 and IFN-gamma production. Similar to its effect on HUT78 cells, TGF-beta 1 also inhibited IL-4 and IFN-gamma production by freshly isolated T cells as well as by human T cell clones. Taken together, our experiments show that the IL-2 dependent cytokines IL-4 and IFN-gamma are both negatively controlled by TGF-beta under conditions where IL-2 production is unaffected by a mechanism which partially involves an inhibition of IL-2/IL-2R signal transduction. These data identify TGF-beta and IL-2 as mutual antagonists in the regulation of IL-4 and IFN-gamma production.

The transmembrane glycoprotein CD34 shows a highly restricted expression on a crucial subset of hematopoietic cells. We show here that engagement of particular determinants of CD34 can lead to signal transduction and to enhanced adhesiveness of CD34+ hematopoietic cells. Monoclonal antibodies (MoAbs) directed against O-sialoglycoprotease- sensitive epitopes of CD34 (QBEND10, ICH3, BI.3C5, MY10) but not MoAbs against O-sialoglycoprotease-resistant epitopes (9F2, 8G12) induce actin polymerization in KG-1a and KG-1 cells and strongly enhanced cytoadhesiveness. The capacity to induce adhesion requires cellular energy, divalent cations, and intact cytoskeleton but not de novo protein synthesis. The observed cytoadhesion seems at least in part to be caused by a concomitant activation of the beta 2 integrin cytoadhesion pathway. It can be significantly inhibited with lymphocyte function-associated antigen-1 and intercelluar adhesion molecule-1 antibodies. Protein kinase inhibition analyses suggest that the pathways initiated by engagement of the CD34 molecule with certain CD34 MoAbs involves protein tyrosine kinases but that protein kinase C is not critically involved.

Abstract 4414 Resistance against one or more tyrosine kinase inhibitors (TKI) prevents eradication of Ph+ chronic myeloid leukemia (CML). In many patients BCR/ABL1 mutations are detectable. We have recently identified two targeted drugs that exert major growth-inhibitory effects on drug-resistant CML cells, the triterpenoid CDDO-Me (Bardoxolone-methyl, REATA Pharma) that blocks several signalling molecules including mTOR, Akt, and STAT3, and upregulates expression of heat shock protein 32 (Hsp32 = heme oxygenase 1, HO-1), and styrene-maleic acid-copolymer micelle-encapsulated ZnPP (SMA-ZnPP), a water-soluble inhibitor of Hsp32/HO-1. In the current project, we asked whether CDDO-Me exerts inhibitory effects on growth of TKI-resistant CML cells and whether the combination of CDDO-Me and SMA-ZnPP would produce synergistic effects in drug-resistant CML cells. As determined by 3H-thymidine incorporation, CDDO-Me was found to inhibit the proliferation of imatinib-responsive and imatinib-resistant K562, imatinib-resistant KCL-22, KU812, and Ba/F3 cells transfected with various TKI-resistant mutants of BCR/ABL1 (T315I, E255K, Y253F, H396P). In each case, IC50 values Disclosures: Valent: Novartis: Consultancy, Honoraria, Research Funding.

Background Regulatory T (Treg) cells establish and maintain tolerance to self-antigens and many foreign antigens, such as allergens, by suppressing effector T-cell proliferation and function. We have previously shown that human T-cell receptor (TCR) αβ-chains specific for allergen-derived epitopes confer allergen specificity on peripheral blood T cells of individuals with and without allergy. Objective To study the feasibility of generating allergen-specific human Treg cells by retroviral transduction of a transcription unit encoding forkhead box protein 3 (FOXP3) and allergen-specific TCR αβ- chains. Methods cDNAs encoding the α and β −chains of a Bet v 1 142-153 −specific TCR (TCR alpha variable region 6/TCR beta variable region 20) and human FOXP3 were linked via picornaviral 2A sequences and expressed as single translational unit from an internal ribosomal entry site—green fluorescence protein−containing retroviral vector. Retrovirally transduced peripheral blood T cells were tested for expression of transgenes, Treg phenotype, and regulatory capacity toward allergen-specific effector T cells. Results Transduced T cells displayed a Treg phenotype with clear-cut upregulation of CD25, CD39, and cytotoxic T-lymphocyte antigen 4. The transduced cells were hyporesponsive in cytokine production and secretion and, like naturally occurring Treg cells, did not proliferate after antigen-specific or antigen-mimetic stimulation. However, proliferation was inducible upon exposure to exogenous IL-2. In coculture experiments, TRAV6 + TRBV20 + FOXP3 + transgenic T cells, unlike FOXP3 + single transgenic T cells or naturally occurring Treg cells, highly significantly suppressed T cell cytokine production and proliferation of corresponding allergen-specific effector T cells in an allergen-specific, dose-dependent manner. Conclusion We demonstrate a transgenic approach to engineer human allergen-specific Treg cells that exert their regulatory function in an activation-dependent manner. Customized Treg cells might become useful for tolerance induction therapies in individuals with allergic and other immune-mediated diseases.

Monoclonal antibodies (moAbs) that recognize common or variable determinants of the gamma delta T-cell receptor (TcR) were used to assess gamma delta T-cell distribution on biopsy specimens and/or peripheral blood leukocytes (PBL) from 30 patients suffering from chronic cutaneous lupus erythematosus (CCLE). CD3+/gamma delta TcR + T cells were evaluated in 15 biopsies from patients with CCLE lesions, their numbers varying from 0.5 to 15.0% of all intralesional CD3 +T cells present. In all specimens from lesional skin gamma delta TCR+T cells were BB3 + and/or Ti gamma A +, indicating predominant use of the V gamma 2/V delta 2 phenotype. In the CCLE lesions the intraepidermal V gamma 2/V delta +T cells were observed in close vicinity to the damaged basal keratinocyte (KC) layer, and also randomly scattered among the densely packed inflammatory infiltrate in the dermis. In contrast to the immunohistologic findings, no numerical increase of gamma delta TcR+T cells could be observed among PBL from 28 of 30 CCLE patients. Only one CCLE patient being treated with hydroxychloroquine for two months had 15% CD3 +/gamma delta TcR+T cells among the PBL. Based on the immunohistologic findings one may infer that in CCLE, a skin-restricted form of LE, V gamma 2/V delta 2 +T cells expand extrathymically to an as yet unknown stimulus. One may also propose that these gamma delta T cells--based on their cytotoxic capacity--may contribute to the epidermal damage. It remains to be determined whether the extrathymic expansion of V gamma 2/V delta 2 +Tells occurs within lesional skin or in the periphery within subsequent recruitment into skin lesions. The results obtained by fluorescence-activated cell sorter analysis favor the first possibility.

Abstract 3972 Systemic mastocytosis (SM) is a myeloid neoplasm defined by abnormal growth and accumulation of neoplastic mast cells (MC) in one or more internal organs. In most patients, the D816V-mutated variant of KIT is detectable. This mutant supposedly confers resistance against several tyrosine kinase inhibitors including imatinib and masitinib. In aggressive SM (ASM) or mast cell leukemia (MCL) the response to conventional drugs is poor and the prognosis is grave. In these patients, additional KIT-independent signalling pathways and molecules, such as BTK and LYN may play an important role in disease evolution and MC proliferation. R763/AS703569 is a multikinase inhibitor that blocks the kinase activity of KIT, BTK, LYN, Aurora-Kinase-A, Aurora-Kinase-B, ABL, AKT, and FLT3. We analyzed the effects of R763/AS703569 on growth and survival of the human mast cell leukemia cell line HMC-1 and the canine mastocytoma cell line C2. Two subclones of HMC-1 were used, one expressing KIT D816V (HMC-1.2) and one lacking KIT D816V (HMC-1.1). Both HMC-1 subclones were found to express Aurora-Kinase-A mRNA and Aurora-Kinase-B mRNA in RT-PCR experiments. As assessed by 3H-thymidine uptake, R763/AS703569 was found to inhibit proliferation of HMC-1 cells in a dose-dependent manner, with lower IC50 values obtained in HMC-1.2 cells (1-5 nM) compared to HMC-1.1 cells (10-10-50 nM). Moreover, R763/AS703569 produced growth inhibition in C2 cells (IC50: 1–5 nM). As assessed by light microscopy and Tunel assay, the growth-inhibitory effects of R763/AS703569 were found to be accompanied by apoptosis in all three cell lines. Correspondingly, R763/AS703569 was found to induce cleavage of caspase-3, caspase-8, and caspase-9 in HMC-1 cells. Moreover, R763/AS703569 was found to induce a G2/M cell cycle arrest in HMC-1 cells and C2 cells after 24 hours. In order to define the target spectrum for R763/AS703569 in HMC-1 cells, Western blot experiments were performed. In these experiments, R763/AS703569 was found to inhibit the phosphorylation of KIT, Aurora-Kinase-A, and BTK in HMC-1.1 cells, whereas no effects of R763/AS703569 on phosphorylation of LYN were seen. We then combined R763/AS703569 with dasatinib, a drug known to block LYN activation in HMC-1 cells. In these experiments, we were able to show that both drugs cooperate with each other in inducing apoptosis in HMC-1.1 cells and HMC-1.2 cells. In summary, our data suggest that R763/AS703569 is a novel promising drug that should be tested for its anti-neoplastic effects in patients with ASM and MCL in clinical trials. Complete inhibition of growth of neoplastic MC may require drug combinations employing R763/AS703569 and other targeted or cytotoxic drugs. Disclosures: Sarno: Merck-Serono: Employment. Valent:Novartis: Research Funding; Bristol-Myers Squibb: Research Funding; Merck-Serono: Research Funding.

Abstract 2280 In most patients with chronic myeloid leukemia (CML), complete cytogenetic remission can be achieved with the BCR/ABL tyrosine kinase inhibitor (TKI) imatinib. However, not all patients are long-term responders. A major cause of acquired resistance against imatinib is the development of BCR/ABL mutations in subclones. In most of these patients, a second generation TKI is prescribed. However, the T315I mutant of BCR/ABL introduces resistance against most TKI, including nilotinib and dasatinib. One approach to overcome drug resistance in BCR/ABL T315I+ CML cells may be to apply drug combinations. Recent data suggest that the mechanisms through which dasatinib and nilotinib act on BCR/ABL differ from each other and that both drugs act on multiple additional targets in CML cells. Here, we show that dasatinib and nilotinib cooperate with each other in producing growth inhibition in imatinib-sensitive and imatinib-resistant CML cells, including subclones bearing BCR/ABL T315I. The drug combination was tested on leukemic cells obtained from 9 patients with chronic phase (CP) CML and 3 with blast phase (PB) of CML. Samples were assessed from 4 patients at the time of diagnosis, and against cells from 8 patients (CP, n=5; BP, n=3) who had developed resistance against one or more BCR/ABL TKI. In all 3 patients in PB, the T315I mutant was detectable. As expected, nilotinib and dasatinib failed to inhibit proliferation of cells harbouring BCR/ABL T315I when applied as single agents. However, the combination xnilotinib+dasatinibx produced synergistic effects in most samples, including primary CML cells and Ba/F3 cells harbouring BCR/ABL T315I. Interestingly, in all 3 patients with BP (BCR/ABL T315I+), strong cooperative or even synergistic growth-inhibitory effects were observed in primary CML cells, resulting in substantial anti-leukemic effects seen at reasonable (pharmacologic) drug concentrations (< 1 μ M) (figure). Based on these results, we treated one patient with TKI-resistant CML in hematologic relapse in whom 2 BCR/ABL mutant-bearing subclones, one clinically resistant against nilotinib (F359V) and one clinically resistant against dasatinib (F317L) had been detected, with a combination of nilotinb (800 mg p.o. daily) and dasatinib (50 mg/day p.o., days 1–5 every third week). A transient hematologic response was obtained in this patient, and except for mild bone pain, no side effects were recorded. Moreover, we were able to show that during treatment with xnilotinib+dasatinibx, the number of CD34+/CD38-/CD33+ CML stem cells decreased from clearly measurable levels (0.005%) to nearly undetectable levels (0.0002%). Finally, ex vivo analyses of leukemic blood cells confirmed, that the combination xnilotinib+dasatinibx produced strong cooperative growth-inhibitory effects in both disease-components, i.e. the F359V-bearing subclone and the F317L-bearing subclone. In summary, our data show that the combination of dasatinib and nilotinib can override acquired TKI resistance in CML, and can suppress growth of various imatinib-resistant subclones including cells that bear BCR/ABL T315I or other BCR/ABL mutants. Whether this combination can suppress imatinib-resistant subclones in CML for prolonged time periods or even can eradicate neoplastic stem cells remains in CML patients to be determined. Synergistic effects of nilotinib and dasatinib on primary leukemic cells obtained from a patient with a BCR/ABL T315I+ blast phase of CML Disclosures: Valent: Novartis: Research Funding; Bristol-Myers Squibb: Research Funding.

Abstract 4936 Advanced mast cell (MC) disorders are characterized by uncontrolled growth of neoplastic MC in various organ systems, resistance to conventional cytoreductive drugs, and a poor prognosis. In most patients, transforming mutations in the KIT proto-oncogene are detectable and are considered to contribute to resistance. MC lines are an important model for analyzing drug resistance in neoplastic MC. We have established a novel canine mastocytoma cell line, NI-1 from a canine patient suffering from mast cell leukemia. NI-1 cells were found to harbour several homozygous KIT mutations including two single nucleotide mutations, one at nucleotide 107 (C to T, leading to a missense mutation, P36L) and one at nucleotide 1187 (A to G, leading to a missense mutation, Q396R), a 12 bp duplication at nucleotide 1263, and a 12 bp deletion at nucleotide 1550, the latter reflecting a transcriptional variant. NI-1 cells contained histamine and formed tumors in NOD-SCID IL-2Rgammanull (NSG) mice. As assessed by 3H-thymidine uptake, a number of targeted drugs were found to inhibit the proliferation of NI-1 cells at pharmacologically relevant concentrations. Among these drugs were the KIT kinase blockers midostaurin (IC50 2 μM). However, the HDAC inhibitor vorinostat was found to block growth of NI-1 cells (0.1-0.5 μM). Drug response profiling also revealed that NI-1 cells differ markedly from canine C2 mastocytoma cells harbouring an exon 11 KIT mutation, and the human mast cell lines HMC-1.1 (exon 11 mutation in KIT) and HMC-1.2 (exon 11 and exon 17 KIT mutations). In contrast to C2 cells, NI-1 cells were found to be largely resistant against the growth-inhibitory effects of masatinib, sorafenib, and sunitinib. By contrast, NI-1 cells were found to be even more sensitive against the growth-inhibitory effects of the mTOR blocker RAD001 and the PI3-kinase/mTOR inhibitor NVP-BEZ235. Together, our data suggest that certain KIT mutations may be associated with relative resistance of neoplastic MC against KIT-targeting drugs, a phenomenon that has also been described for human MC and the KIT mutant D816V that mediates resistance against masatinib and imatinib. The novel MC line NI-1 may serve as a novel tool to investigate the mechanisms of resistance against TKI in neoplastic MC. Disclosures: Valent: Novartis: Research Funding; Bristol-Myers Squibb: Research Funding.

Abstract 900 Introduction: Bronchiolitis obliterans syndrome (BOS) characterized by the new development of fixed airflow obstruction after allogeneic hematopoietic cell transplantation (HCT) is an increasingly recognized manifestation of chronic graft-versus-host disease (cGVHD) affecting up to 30% of patients. BOS is associated with increased non-relapse mortality and poor patient outcome after HCT. Its cause is currently unknown but allorecognition of lung antigens is the suspected etiology since BOS has been observed in states of alloimmunity such as cGVHD and after lung transplantation. Currently, the diagnosis of BOS is based on radiologic findings in high resolution expiratory chest CTs (HR-CT) and pulmonary function tests (PFTs). For early diagnosis of BOS biomarkers would be highly desirable allowing efficient treatment of early disease before progression to irreversibility is observed. B-cells are of importance in cGVHD and excess of B-cell activation factor (BAFF), increased CD19+CD21- immature transitional B-cells and dysgammaglobulinemia characterize cGVHD. Objective: We investigated circulating B-cell subpopulations in cGVHD patients to define novel cellular biomarkers for diagnosis of BOS and scoring its severity. Patients and methods: One hundred and thirty-five patients (74 males, 61 females) with a median age of 46 (range, 20–65) years, given myeloablative (n=76) or reduced-intensity (n=59) conditioning for related (n=108) or unrelated donor (n=27) HCT were prospectively evaluated for presence of cGVHD from day 100 after HCT and thereafter serially every 3 months for 2 years. At the same time points PFTs, multicolor flow cytometric analyses of peripheral blood (PB) cells stained for CD19, CD21, CD27, CD10, CD38, IgM, IgD, CD3, CD4, CD8, and CD56, measurements of serum immunoglobulin levels and BAFF were performed. BOS and its severity were defined according to the NIH consensus criteria. All patients with BOS except 2 had HR-CTs of the lung, bronchoalveolar lavage and transbronchial biopsies performed. Results: After a mean follow-up of 250 (range, 100–640) days after HCT 35 of 135 (26%) patients developed BOS. Other manifestations of cGVHD included skin in 72%, eyes in 58%, oral mucosa in 50%, and liver in 65% of patients. Severity of BOS consisted of NIH score I (FEV1=60-79%) in 14 (40%), NIH score II (FEV1=40-59%) in 16 (46%), and NIH score III (FEV Conclusion: Disturbance of B-cell homeostasis is more pronounced in cGVHD of the lung compared to other main organ manifestations. B-cell subpopulation numbers and excess of BAFF can serve as novel cellular biomarkers for BOS. Disclosures: No relevant conflicts of interest to declare.

Abstract 3394 Resistance to imatinib is a major clinical problem and challenge in advanced chronic myeloid leukemia (CML). In most patients, drug-resistant mutants of BCR/ABL are detectable. Although most of these mutants still are responsive to second generation BCR/ABL kinase inhibitors (KI) such as nilotinib or dasatinib, drug responses are often short-lived. The BCR/ABL mutant T315I confers resistance against all available BCR/ABL KI, including nilotinib and dasatinib. More recent data suggest that several Aurora kinase (AuK) inhibitors block the kinase activity of BCR/ABL T315I. We have examined the growth-inhibitory effects of the AuK/ABL inhibitor R763/AS703569 (Merck-Serono, Darmstadt, Germany) on primary CML cells (chronic phase, n=12), the CML cell line K562, and Ba/F3 cells transfected with various imatinib-resistant mutants of BCR/ABL. As assessed by 3H-thymidine-uptake, R763/AS703569 was found to inhibit proliferation in imatinib-sensitive and imatinib-resistant primary CML cells in all donors tested, in imatinib-resistant and imatinib-responsive K562 cells, and in Ba/F3 cells harbouring various mutants of BCR/ABL (E255K, Y253F, H396P, T315I). The effects of R763/AS703569 on BCR/ABL-transformed cells were dose-dependent with IC50 values ranging between 0.001–0.1 μ M in K562 cells, Synergistic growth-inhibitory effects of R763/AS703569 and nilotinib in BCR/ABL T315I+ Ba/F3 cells (left), and R763/AS703569 and dasatinib in K562 cells (right). Disclosures: Valent: Novartis: Research Funding; Bristol-Myers Squibb: Research Funding; Merck-Serono: Research Funding.

The restriction fragment length polymorphism (RFLP) of HLA-DR beta, -DQ alpha and -DQ beta genes was analyzed in 637 unrelated individuals from the Austrian population. The restriction enzyme was Taq I, and three exon-specific probes were applied. The gene frequencies, the Hardy-Weinberg equilibrium in DR beta and DQ loci, linkage disequilibria between the loci and haplotype frequencies are calculated. Rare associations between DR and DQ loci are described. Two RFLP patterns are demonstrated which were unique in the overall 1,000 individuals tested so far.

Abstract 2226 Poster Board II-203 Chronic graft-versus-host disease (cGVHD) is a major cause of mortality after allogeneic hematopoietic stem cell transplantation (HCT) and deleteriously affects the quality of life in surviving patients who otherwise have been cured of their underlying disease. Corticosteroids are the mainstay of therapy for cGVHD. Patients with steroid-refractory or steroid-intolerant cGVHD have received numerous immunosuppressive agents with a wide range of responses. Due to possible side effects of long-term immunosuppression including infectious complications and severe organ toxicities, biomarkers for prediction of response would be highly desirable. Recently, we reported that proportions of immature CD19+CD21- B-lymphocytes predict the response to extracorporeal photopheresis (ECP) in patients with cGVHD (Blood 114: 744-746, 2009). Whether these findings were ECP-specific had to remain speculative since no other treatment cohorts had been analyzed and the mechanisms of action of ECP are still being elucidated. Therefore, we investigated B cell subpopulations in peripheral blood (PB) of 74 patients (median age 40 years, range 20-59 years) given different therapies including cyclosporine A (CSA n=24), tacrolimus (n=13), sirolimus (n=18) and ECP (n=19) for moderate (n=29) or severe (n=45) cGVHD according to the NIH Consensus. Duration of cGVHD before assessment of B cell subpopulations was a median of 14 (range, 0.5-120) months. Organ involvement at study entry consisted of skin in 45 patients (61%), oral mucosa in 50 (68%), eyes in 28 (38%), liver in 22 (30%) and lungs in 23 (31%), respectively . Seventy-three percent of patients had more than 2 organs affected by cGVHD. PB leukocytes were analyzed by multiparameter flow cytometry after staining for CD19, CD27, CD21 and surface Ig. Patients were assessed for cGVHD activity and response to therapy according to the NIH Consensus Development Project criteria prior to start of immunosuppressive therapy and every 3 months thereafter. B cell subpopulations of responders and non-responders after 6 months of therapy were compared. Overall, 45 patients (61%) responded to therapy including 20/24 (83%) given CSA, 6/13 (46%) on tacrolimus, 7/18 (39%) on sirolimus, and 12/19 (63%) given ECP, respectively. Among responders 16 (9 on CSA, 7 on ECP) achieved a complete resolution of cGVHD after 6 months of therapy. Prior to start of immunosuppressive therapy non-responders had significantly (p=0.03) higher proportions of immature CD19+CD21- B-lymphocytes with a mean of 19.4% (range, 4.17-45) compared with a mean of 13.3% (range, 1.3-54) in responders to 6 months of therapy. Highest immature CD19+CD21- B-lymphocyte numbers were observed in patients not responding to 6 months of therapy with sirolimus or ECP. Comparisons revealed significantly higher proportions of immature CD19+CD21- B-lymphocytes in non-responders compared to responders to either sirolimus (mean of 23% vs 9.3%, p=0.02), tacrolimus (mean of 17.9% vs 8.6%,p=0.02), or ECP (mean of 22% vs 13.7%, p=0.02), respectively. In the CSA cohort no significant difference in immature CD19+CD21- B-lymphocyte numbers between responders and non-responders was observed. In addition, no significant difference in memory CD27+ B-lymphocytes was observed prior to start of immunosuppressive therapy in any patient cohort between responders and non-responders. After 6 months of immunosuppressive therapy all responding patients had a significant (p=0.01) decrease of percentages of immature CD19+CD21- B-lymphocytes from a mean of 13.3% (range, 1.3-54) prior to therapy to a mean of 8.2% (range, 1.1-30) 6 months later. In addition, in complete responders the immature CD19+CD21- B-lymphocytes significantly (p=0.04) decreased from a mean of 14.06% (range, 1.3-54.6) to a mean of 7% (range, 1.1-36) after 6 months. In all non-responder percentages of immature CD19+CD21- B-lymphocytes either increased or remained unchanged after 6 months of therapy. In conclusion, relative amounts of immature CD19+CD21- B-lymphocytes assessed prior to start of cGVHD therapy may predict response to ECP, tacrolimus, and sirolimus. Increased proportions of CD21- B-lymphocytes could be part of the autoimmune pathogenesis resulting in autoreactive B-cells in cGVHD. The correlation of numbers of B cell subsets and activity of cGVHD as observed in our study supports this hypothesis. Therefore, this novel cellular biomarker should be investigated in larger treatment cohorts of cGVHD patients. Disclosures: Off Label Use: Sirolimus and ECP for therapy of chronic GVHD. Greinix:Therakos: Consultancy, Speakers Bureau. Knobler:Therakos: Consultancy, Speakers Bureau.

Determination of ethanol concentration in a blood sample drawn from a person who caused a serious car crash showed a level which was markedly above the upper limit tolerated legally i.e. 0.08%. At the court hearing the accused car driver challenged the drunken driving charge and claimed that there might have been a mix up of the blood samples, whereby his was replaced by another blood sample, since the tube containing his blood was not marked with his name. The blood sample had been stored without anticoagulants for about 6 months at -20 degrees C. Due to haemolysis it was impossible to determine conventional haemogenetic marker systems. We therefore tried to extract DNA from the blood sample and to determine the restriction fragment length polymorphism (RFLP) by means of five DNA probes recognizing highly polymorphic single-locus systems as described by Jeffreys et al. We analyzed the RFLP's of both the old blood sample and of fresh blood drawn from the accused car driver and we were able to identify the blood sample as certainly having been taken from the accused.

Chronic graft-versus-host disease (cGVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (HCT) requiring prolong immunosuppressive therapy and increasing non-relapse mortality. Immune mechanisms underlying cGVHD have remained elusive. Recently, we reported a severe disturbance in B cell homeostasis seen in a significant expansion of immature/transitional B lymphocytes (CD21−) and a significant decrease of non-class switched (CD19+/IgD+/CD27+) and class-switched memory B cells (CD19+/IgD−/CD27+) in patients with active chronic GVHD. In long-lasting cGVHD irreversible tissue damage cannot be distinguished from active cGVHD with certainty since no biomarkers for monitoring of cGVHD activity as well as for assessment of therapeutic response are available. We investigated serially every 3 to 6 months B cell subpopulations in the peripheral blood (PB) of 47 patients (median age 40 years, range 18–62 years) given extracorporeal photopheresis (ECP) as first line (n=13) or salvage therapy (n=34). Twenty-nine patients (64%) had more than 2 organs affected by cGVHD and in 21 patients (45%) severity of organ involvement was grade 3 according to the NIH Consensus. Duration of cGVHD before ECP was a median of 2.5 years. ECP was performed initially every two weeks on 2 consecutive days and monthly thereafter until improvement. The median duration of ECP was 30 (range, 8–50) cycles during a median of 13 (range, 3–36) months and ECP is still ongoing in 14 patients. A total of 257 samples with a median of 6 analyses per patient (range, 3 to 10) was assessed. PB leukocytes were analyzed by multiparameter flow cytometry after staining for CD19, CD27, CD21 and surface Ig. Patients were scored for cGVHD activity and response evaluation according to the NIH Consensus Development Project criteria at every sampling event. Thirty-five patients (75%) responded to ECP, including 22 with complete resolution, whereas 12 (25%) were nonresponders. Retrospective correlation of clinical response to ECP with B cell subpopulation numbers revealed that patients not responding to ECP had a significantly higher number of immature/transitional B cells (CD19+/CD21−) (mean 16.6%, range 0.8–57%) in PB samples taken prior to start of ECP compared to responders (mean 13%, range 1–54%, p 0.0001). The numbers of both non-class-switched and class-switched memory B cells were significantly higher in ECP-responders, mean 5.1% (range 1–21%) compared to a mean of 4.2% (range 0.3–14%) in nonresponders (p 0.041) The CD21−/CD27+ ratio was significantly higher in nonresponders with a mean of 8.6 (range 0.5–50) compared to a mean of 6.6 (range 0.3–89) in the responding group(p 0.0005). Three months after start of ECP patients responding to therapy had a decrease in their CD21−/CD27+ ratio (mean 3, range, 0.3–18, p=0.05) whereas in nonresponders B cell subpopulations were unchanged. After 6 months a significant decrease in immature/transitional B cell numbers (CD19+/CD21−) compared to baseline (p=0.03) values was observed in ECP responders whereas ECP nonresponders presented a further increase in immature/transitional B cell (CD19+/CD21−) numbers. One year after start of ECP 22 patients with durable complete responses had a further decrease of the CD21−/CD27+ ratio (mean 1.6, range 0.2–5.4) comparable to patients with resolved cGVHD as previously reported (Greinix et al, BBMT14:208–219, 2008). In contrast, nonresponders to ECP with persistent active cGVHD had a significantly higher CD21−/CD27+ ratio (mean 3.3, range, 0.1–11, p=0.08) in PB samples. In conclusion, determining the degree of disturbance of B cell homeostasis as seen in elevated numbers of immature/transitional B cells and decreased memory B cell subsets during active cGVHD could be used for predicting therapeutic response to ECP. Moreover, 3 to 6 months after start of ECP distribution of B cell subpopulations differed significantly between ECP responders and nonresponders. Thus, B cell subsets serially assessed could serve as possible biomarkers of response to ECP in cGVHD. Our preliminary findings should be confirmed in a larger prospective patient cohort receiving ECP.

Chronic myeloid leukemia (CML) is a stem cell disease characterized by the BCR/ABL oncoprotein. The ABL kinase inhibitor imatinib is effective in most patients and considered standard first line therapy. However, not all patients show a long-lasting response. Treatment failure is usually associated with the occurrence of imatinib-resistant mutants of BCR/ABL. For these patients, novel multi-kinase inhibitors such as dasatinib represent alternative treatment options. Still, however, not all patients respond to these drugs, especially when leukemic cells bear the BCR/ABL mutant T315I that confers resistance against most kinase-blockers. Bosutinib is a novel multi-kinase inhibitor that has been described to act growth-inhibitory in ABL-transformed leukemias. In the current study, we examined the effects of bosutinib alone and in combination with dasatinib on growth and survival of CML cells. Bosutinib was found to inhibit 3H-thymidine uptake and thus proliferation in imatinib-sensitive and imatinib-resistant K562 cells in a dose-dependent manner, with identical IC50 values (10–100 nM). Moreover, bosutinib was found to inhibit the growth of primary CML cells and Ba/F3 cells bearing various imatinibresistant mutants of BCR/ABL, except the T315I mutant (IC50>1 μM). The growth-inhibitory effects of bosutinib were found to be associated with signs of apoptosis. Dasatinib showed similar effects on CML cells, and again did not block the growth of subclones bearing BCR/ABL T315I. Unexpectedly, however, we found that bosutinib and dasatinib synergize with each other in producing growth inhibition in primary CML cells exhibiting BCR/ABL T315I at pharmacologic concentrations (0.01–1 μM). Clear synergistic effects were also observed in imatinib-sensitive and imatinib-resistant K562 cells as well as in Ba/F3 cells bearing BCR/ABL T315I. In parallel, we performed multiplexed kinase assays as well as chemical proteomics analysis and mass spectrometry using K562 cells and primary CML cells and coupleable dasatinib and bosutinib analogues. In these experiments, dasatinib and bosutinib were found to express an overlapping, but non-identical profile of target kinases. As expected, both drugs were found to bind to wt ABL, SRC kinases, and TEC-family kinases including BTK. Specific targets preferentially bound and inhibited by bosutinib were STE20s, the FES/FER family, CAMKIIG, PYK2 and TBK1. We were also able to confirm that the dasatinib-targets KIT and PDGFRA are not recognized by bosutinib. Interestingly, whereas wt ABL (IC50

Mcl-1 is a Bcl-2 family-member that has been described to act anti-apoptotic in various myeloid neoplasms. We and others have recently shown that neoplastic mast cells (MC) in patients with systemic mastocytosis (SM) display Mcl-1, Bcl-2, and Bcl-xL. In the present study, we examined the effects of the Mcl-1/Bcl-2-targeting drug GX015-070 (obatoclax®; GeminX, Montréal, Quebéc, Canada) on growth and viability of primary neoplastic MC obtained from patients with SM (n=3), the human MC leukemia cell line HMC-1, and the canine mastocytoma cell line C2. Two HMC-1 subclones, one lacking KIT D816V (HMC- 1.1) and one expressing KIT D816V (HMC-1.2) were examined. As assessed by RT-PCR and immunostaining, primary neoplastic MC as well as HMC-1 cells (both subclones) were found to express Mcl-1 mRNA and the Mcl-1 protein in a constitutive manner, but did not express significant amounts of proapoptotic Bim. Transfection of HMC-1 cells with Mcl-1-specific siRNA resulted in reduced proliferation and increased apoptosis compared to cells transfected with a control siRNA. GX015-070 was found to inhibit 3H-thymidine uptake and thus proliferation in HMC-1 cells in a dose-dependent manner, with higher IC50 values obtained in HMC-1.2 cells (0.5 μM) compared to HMC-1.1 cells (0.05 μM). GX015-070 also inhibited the growth and survival in the canine mastocytoma cell line C2 (IC50: 0.5-1 μM). Moreover, GX015-070 was found to inhibit the proliferation of primary human neoplastic MC in all SM patients tested (IC50: 0.05-0.1 μM). We next attempted to combine obatoclax with a modulator of Mcl-1/Bim expression in MC, in order to enhance drug effects. Since Bim is degraded via the proteasome, we applied the proteasome inhibitor bortezomib. Whereas GX015-07 did not modulate the production/expression of Mcl-1 or Bim in HMC-1 cells, bortezomib was found to promote the expression of Bim in our Western blot experiments. In addition, bortezomib was found to suppress 3H-thymidine uptake in both HMC-1 subclones. Finally, bortezomib was found to cooperate with GX015-070 in producing apoptosis in HMC-1.1 cells, HMC-1.2 cells, and C2 cells. Together, our data show that the Mcl-1/Bcl-2-targeting drug GX015-070 is a potent inhibitor of in vitro growth and survival of canine and human neoplastic MC. Targeting of Mcl-1 in neoplastic MC alone or in combination with a Bim-regulator may be an interesting pharmacologic approach in advanced SM.

In cases of disputed parentage usually more than 20 polymorphic systems (red and white cell antigens, serum markers and enzyme markers) have to be analyzed using a battery of different techniques. A more recent method involving analysis of restriction fragment length polymorphism in highly polymorphic genes allows assignment of offspring to their parents. To test the latter method, 28 paternity cases were studied in parallel using conventional systems and detection of RFLPs of probes MS 1, MS 31, MS 43, and g 3, developed by Jeffreys et al. Furthermore, the cogency of exclusion of parentage, the average power of exclusion and the probability of parentage is calculated using published mutation rates and gene frequencies of the four probes. In conclusion, use of the four gene probes has both theoretically and practically turned out to be a powerful method for parentage testing.

Systemic mastocytosis (SM) is a myeloid neoplasm characterized by increased growth and survival of neoplastic mast cells (MC). Aggressive SM (ASM) and MC leukemia (MCL) are advanced disease variants that usually are drug-resistant and have an unfavorable prognosis. In most patients, the D816V-mutated ′oncogenic′ variant of KIT is detectable. However, the mutant is also detectable in patients with indolent SM exhibiting a normal life-expectancy, and therefore is not considered to represent a fully transforming oncoprotein. This assumption is also supported by studies in Ba/F3 cells, and whether KIT D816V-targeting drugs are able to induce long-term remission in ASM or MCL, remains to be seen. Therefore, it has been hypothesized that in addition to KIT, other pro-oncogenic molecules and signaling pathways play a role in malignant transformation/progression in SM. We here describe a novel KIT D816V-independent oncogenic pathway in neoplastic MC that involves Lyn and Bruton’s tyrosine kinase (Btk). Western blotting and immunostaining revealed that neoplastic MC display the Btk- and Lyn protein. Both molecules were found to be constitutively phosphorylated in primary neoplastic MC and in the MC leukemia cell line HMC-1. Lyn/Btk-activation was not only detectable in KIT D816V-positive HMC-1.2 cells, but also in the KIT D816V-negative HMC-1.1 subclone. In studies employing Ba/F3 cells with doxycycline-inducible expression of KIT, we were able to show that KIT D816V induces activation of STAT5 and Akt, but does not induce activation of Btk. Correspondingly, pharmacologic deactivation/dephosphorylation of KIT in HMC-1 cells by midostaurin (PKC412) (Novartis, Basel, Switzerland) was not accompanied by a decrease in phosphorylation of Lyn or Btk. The functional significance of Btk expression/activation in neoplastic MC could be demonstrated by a Btk-specific siRNA that reduced the proliferation and survival in HMC-1 cells, and was found to cooperate with midostaurin in producing growth inhibition. In consecutive experiments, we identified the Src/Abl kinase-targeting drug dasatinib (BMS, Princeton, NJ) as a potent inhibitor of Lyn/Btk activation in neoplastic MC. In particular, dasatinib (1 μM) was found to block Lyn and Btk activity in HMC-1.1 cells as well as in HMC-1.2 cells, and corresponding results were obtained with primary neoplastic MC. Finally, as assessed by a chemical proteomics approach, we were able to show that dasatinib directly binds to Btk and Lyn in neoplastic MC. In summary, our data show that a KIT-independent Lyn/Btk-driven signaling pathway contributes to growth and survival of neoplastic MC, and possibly to disease progression in SM. Our study also identifies dasatinib as a potent inhibitor of the Lyn/Btk pathway, which may have clinical implications and may explain some of the synergistic effects obtained with combinations of dasatinib and other KIT-targeting TK inhibitors in neoplastic MC.

Chronic eosinophilic leukemia (CEL) is a myeloproliferative disorder characterized by molecular and/or cytogenetic evidence of monoclonality of eosinophils, sustained marked eosinophilia, and consecutive organ damage. In a majority of patients with CEL with or without associated mastocytosis, the transforming mutation FIP1L1-PDGFRα and the related CHIC2 deletion is found. The respective oncoprotein, FIP1L1-PDGFRα, is considered to play a major role in malignant cell growth in CEL. The tyrosine kinase (TK) inhibitor imatinib (STI571) has been described to counteract the TK activity of FIP1L1-PDGFRα in most patients, and has been introduced as a novel effective therapy in CEL. However, not all patients with CEL show a response to imatinib. Therefore, several attempts have been made to identify other TK inhibitors that counteract growth of neoplastic eosinophils in CEL. We provide evidence that dasatinib, a multi-targeted kinase inhibitor, blocks the growth and survival of EOL-1, an eosinophil leukemia cell line carrying FIP1L1-PDGFRα. The effects of dasatinib on proliferation of EOL-1 cells were dose-dependent, with an IC50 of 0.5–1 nM, that was found to be in the same range compared to IC50 values produced by imatinib. Dasatinib was also found to induce apoptosis in EOL-1 cells in a dose-dependent manner (IC-50: 1–10 nM). The apoptosis-inducing effects of dasatinib on EOL-1 cells were demonstrable by light microscopy, flow cytometry, and by a Tunel assay. To further examine the mechanism of growth inhibition induced by dasatinib in neoplastic eosinophils, Western blot experiments were performed using antibodies directed against phosphorylated or total PDGFRα. In these experiments, we were able to show that dasatinib at 1 μM completely blocks the phosphorylation of FIP1L1-PDGFRα in EOL-1 cells. In summary, our data show that dasatinib inhibits the growth of leukemic eosinophils through targeting of the TK activity of the disease-related oncoprotein FIP1L1-PDGFRα. Based on this observation, dasatinib may be considered as a new interesting treatment option for patients with CEL. As dasatinib is also known to block various KIT mutants as well as wild type KIT, such therapy may also be of interest for patients who have systemic mastocytosis (SM) with an associated CEL (SM-CEL).