ALBERT

Description: Resistance toward imatinib and other BCR/ABL tyrosine kinase inhibitors remains an increasing clinical problem in the treatment of advanced stages of chronic myeloid leukemia (CML). We recently have identified the heat shock protein 32 (Hsp32)/heme oxygenase-1 (HO-1) as a BCR/ABL-dependent survival molecule in CML cells. We here show that silencing Hsp32/HO-1 in CML cells by an siRNA approach results in induction of apoptosis. Moreover, targeting Hsp32/HO-1 by either pegylated zinc protoporphyrine (PEG-ZnPP) or styrene maleic acid-micelle–encapsulated ZnPP (SMA-ZnPP) resulted in growth inhibition of BCR/ABL-transformed cells. The effects of PEG-ZnPP and SMA-ZnPP were demonstrable in Ba/F3 cells carrying various imatinib-resistant mutants of BCR/ABL, including the T315I mutant, which exhibits resistance against all clinically available BCR/ABL tyrosine kinase inhibitors. Growth-inhibitory effects of PEG-ZnPP and SMA-ZnPP also were observed in the CML-derived human cell lines K562 and KU812 as well as in primary leukemic cells obtained from patients with freshly diagnosed CML or imatinib-resistant CML. Finally, Hsp32/HO-1–targeting compounds were found to synergize with either imatinib or nilotinib in producing growth inhibition in imatinib-resistant K562 cells and in Ba/F3 cells harboring the T315I mutant of BCR/ABL. In summary, these data show that HO-1 is a promising novel target in imatinib-resistant CML.

Description: Systemic mastocytosis (SM) is a myeloid neoplasm characterized by abnormal growth and accumulation of mast cells (MC) in various internal organs. In most patients, the D816V-mutated variant of c-KIT, which mediates resistance against several tyrosine kinase (TK) inhibitors like imatinib, is found. In advanced SM, the response of neoplastic MC to conventional drugs is poor and the prognosis is grave. Therefore current research is attempting to identify novel targets in neoplastic MC. Polo-like kinase 1 (Plk-1) is a serine/threonine kinase that plays an essential role in mitosis and has recently been introduced as a new target in myeloid leukemias. In the present study, we analyzed expression and function of Plk-1 in neoplastic human MC, and asked whether Plk-1 can serve as a target of therapy in SM. As determined by immunohistochemistry, primary neoplastic MC were found to display activated/phosphorylated Plk-1 in all patients examined (n=5). The human MC leukemia cell line HMC-1 was also found to exhibit activated Plk-1. In addition, we found that primary neoplastic MC as well as HMC-1 cells express Plk-1 mRNA in RT-PCR experiments. As assessed by 3H-thymidine-uptake experiments, the Plk-1-targeting drug BI 2536 (Boehringer Ingelheim GmbH, Germany) was found to inhibit the proliferation of HMC-1 cells in a dose-dependent manner (IC50 5–15 nM). The effect of BI 2536 was seen in both subclones of HMC-1, i.e. in HMC-1.1 cells displaying KIT G560V (but not KIT D816V), and HMC-1.2 cells exhibiting both KIT G560V and KIT D816V, with comparable IC50 values. Moreover, BI 2536 was found to inhibit the proliferation of primary neoplastic cells, with IC50 values ranging between 5 and 50 nM. The growth-inhibitory effects of BI 2536 on HMC-1 cells were found to be associated with mitotic arrest and G2-M cell cycle arrest as well as consecutive apoptosis. In normal bone marrow or peripheral blood mononuclear cells, neither mitotic cell arrest nor apoptosis were observed after treatment with BI 2536. In a consecutive phase of the study, we asked whether combined targeting of KIT D816V and Plk-1 would lead to synergistic drug-interactions. For this purpose, HMC-1 cells and primary neoplastic MC were coincubated with BI 2536 and midostaurin (PKC412), a multitargeted kinase inhibitor that blocks KIT D816V TK activity. In these experiments, BI 2536 was found to synergize with midostaurin in counteracting the proliferation of HMC-1 cells and primary neoplastic MC. In conclusion, our data show that activated Plk-1 is detectable in MC neoplasms and plays a role in cell cycle progression and viability of neoplastic MC. Targeting of Plk-1 with BI 2536 leads to growth inhibition and apoptosis in neoplastic MC. Furthermore, BI 2536 synergizes with midostaurin in counteracting growth of neoplastic MC. Targeting of Plk-1 may be an attractive new pharmacologic concept in advanced SM.

Description: Abstract 213 Systemic mastocytosis (SM) is a neoplastic disease of mast cells (MC) and their bone marrow-derived progenitors. The clinical picture in SM is variable ranging from an indolent course to highly aggressive variants with short survival time. The pathologic hallmark in SM is the multifocal dense infiltrate of MC in the bone marrow. Other typical features of SM include alterations of the bone marrow microenvironment such as increased angiogenesis and fibrosis. In a majority of patients, MC display the KIT mutation D816V which affects the activation loop at the entrance to the enzymatic pocket of the KIT kinase. As a consequence, KIT D816V exhibits constitutive tyrosine kinase activity and promotes cytokine-independent differentiation of MC. However, so far, little is known about KIT D816V-dependent expression of pathogenetically relevant molecules in neoplastic MC. Oncostatin M (OSM) is a pleiotropic cytokine of the interleukin-6 family which is produced mainly by activated T cells and monocytes. OSM has been shown to inhibit cell growth in cell lines derived from solid tumors but to stimulate proliferation of fibroblasts and endothelial cells. Recently, it has been reported that OSM produced by activated MC promotes growth of human dermal fibroblasts. Moreover, it has been suggested that OSM stimulates growth of murine bone marrow-derived mast cells in a mast cell/fibroblast coculture. However, expression of OSM in neoplastic MC or a potential pathogenetic role of OSM in SM have not been examined so far. The aim of the present study was to analyze expression of OSM in neoplastic human MC and to determine the role of KIT D816V in OSM expression. As assessed by immunohistochemistry performed on bone marrow sections of patients with SM, typical spindle-shaped neoplastic MC were found to express OSM. Serial section-staining confirmed that tryptase-positive MC co-express OSM. Expression of OSM was found in neoplastic MC in all patients investigated (n=15) and in all variants of SM (indolent SM as well as aggressive variants) with comparable staining intensities. Preincubation of anti-OSM antibody with a specific blocking peptide resulted in a negative stain. In Ba/F3 cells, doxycycline-inducible expression of KIT D816V led to a substantial upregulation of OSM mRNA and OSM protein, whereas expression of wild type KIT did not affect expression of OSM. In addition, the KIT D816V-positive HMC-1.2 mast cell line was found to express OSM at high levels, whereas the KIT D816V-negative HMC-1.1 subclone expressed only baseline levels of OSM. Correspondingly, the KIT D816V-targeting drug midostaurine (PKC412) decreased the expression of OSM in HMC-1.2 cells as well as in KIT D816V-expressing Ba/F3 cells in a dose-dependent manner. To investigate signaling pathways involved in KIT D816V-dependent expression of OSM, we applied pharmacologic inhibitors and dominant negative-acting signaling molecules. We found that KIT D816V-dependent expression of OSM is inhibited by the mitogen-activated protein-kinase/extracellular signal-regulated kinase (MEK) inhibitor, PD98059, but not by the phosphoinositide 3-kinase inhibitor, LY294002. Expression of dominant negative mutants of signal transducer and activator of transcription 5 (STAT5) did not affect expression of OSM in KIT D816V-expressing cells. In summary, our data identify OSM as a novel cytokine expressed in neoplastic MC in patients with SM and show that KIT D816V directly promotes expression of OSM through activation of the mitogen-activated protein-kinase pathway. OSM may be an important KIT D816V-dependent effector promoting angiogenesis and fibrogenesis/sclerosis in patients with SM. Disclosures: No relevant conflicts of interest to declare.

Description: Heme oxygenase 1 (HO-1), also known as heat shock protein 32 (Hsp32), has recently been identified as a stress-related survival molecule that acts anti-apoptotic and cytoprotective in inflammatory reactions. Recent data suggest that HO-1/Hsp32 is also expressed in neoplastic cells in various malignancies. In the present study, we provide evidence that HO-1 is constitutively expressed in primary leukemic cells in patients with acute myeloid leukemia (AML, n=17) and in various AML cell lines such as HL60, KG1, KG1a, and U937. Expression of HO-1 mRNA was demonstrable by RT-PCR, and the HO-1 protein by immunocytochemistry and Western blotting. In addition, we were able to demonstrate expression of HO-1 mRNA and of HO-1 protein in the CD34+/CD38− progenitor/stem cell fraction in the leukemic clone in patients with AML. The HO-1 inductor hemin (10 μM) was found to promote expression of HO-1 in AML cells. Incubation with the HO-1-targeting drugs pegylated zink protoporphyrin (PEG-ZnPP) or styrene maleic acid-conjugated ZnPP (SMA-ZnPP), resulted in a dose-dependent inhibition of growth of leukemic cells at pharmacologic concentrations (IC50: 5–20 μM for cell lines and primary AML cells). The SMA-ZnPP-induced growth-inhibition of AML cells were found to be associated with induction of apoptosis as evidenced by light microscopy, electron microscopy, and by a Tunel assay. In consecutive experiments, combination experiments were performed using SMA-ZnPP and AML cell lines. In these experiments, SMA-ZnPP was found to synergize with cytarabine in producing growth inhibition in all AML cell lines tested. In summary, these data show that HO-1/Hsp32 is a novel survival factor and interesting target in AML. The clinical significance of this observation remains to be determined in forthcoming trials.

Description: The signal transducer and activator of transcription 5 (STAT5) has recently been implicated as essential pro-oncogenic factor in the pathogenesis of myeloid leukemias in mice (Cancer Cell2005;7:87–99). More recently, STAT5 activation has also been described to occur in human leukemias. However, so far, little is known about the expression of activated/tyrosine phosphorylated STAT5 (pSTAT5) in various myeloid neoplasms and about the distribution of pSTAT5 in the cellular compartments of the normal and leukemic bone marrow (bm). We have examined the expression of pSTAT5 in the bm in patients with acute myeloid leukemia (AML, FAB M0, n=3, M1, n=6, M2, n=4, M3, n=5, M4, n=5, M5, n=4, M6, n=5, M7, n=4), chronic myeloid leukemia (CML, chronic phase, n=4, accelerated phase, n=5, blast phase, n=5), and systemic mastocytosis (SM, n=30), as well as in the normal bm (n=5). Expression of pSTAT5 was determined on paraffin-embedded bm sections by immunohistochemistry using the pSTAT5-specific antibody AX1. In the normal bm, the antibody AX1 was found to react with megakaryocytes and immature myeloid progenitor cells, whereas erythroid cells and mature granulocytic cells did not stain positive for AX1. In patients with AML and CML, the distribution of pSTAT5 showed a similar pattern. In fact, pSTAT5 was found to be expressed in leukemic blast cells without differences among FAB types as well as megakaryocytic cells, but not in erythroid cells. In patients with SM, neoplastic mast cells were found to be immunoreactive for pSTAT5. Interestingly, in all patients and all cells examined, pSTAT5 was found to be localized in the cytoplasm rather than in the nucleus. The cytoplasmic distribution of pSTAT5 in neoplastic cells was confirmed by immunocytochemical staining experiments performed on primary isolated neoplastic cells (AML, CML, mastocytosis) and respective cell lines (U937, KG1, K562, KU812, HMC-1). In each case, the reactivity of neoplastic cells with the AX1 antibody was abrogated by preincubation of the antibody with a pSTAT5-specific blocking peptide. Moreover, the expression of cytoplasmic pSTAT5 in the leukemic cell lines was demonstrable by flow cytometry. To study the molecular mechanisms underlying STAT5-activation in neoplastic cells, Ba/F3 cells with doxycycline-inducible expression of disease-specific oncoproteins, namely BCR/ABL (CML) and KIT-D816V (SM) were employed. Induction of these oncoproteins in Ba/F3 cells resulted in massive activation of pSTAT5 and DNA binding activity as shown by EMSA and supershift assays. In summary, our data show that neoplastic cells in AML, CML, and SM express cytoplasmic pSTAT5, and that disease-related oncoproteins contribute to STAT5-activation. The particular cytoplasmic localization of pSTAT5 in neoplastic cells suggests that apart from its function as a transcription factor, pSTAT5 may have an additional role as a cytoplasmic regulator in these malignancies.

Description: In most patients with systemic mastocytosis (SM), including aggressive SM and mast cell leukemia (MCL), neoplastic cells express the oncogenic KIT mutation D816V. KIT D816V is associated with constitutive tyrosine kinase (TK) activity and thus represents an attractive drug target. However, imatinib and most other TK inhibitors fail to block the TK activity of KIT D816V. We show that the novel TK-targeting drugs PKC412 and AMN107 counteract TK activity of D816V KIT and inhibit the growth of Ba/F3 cells with doxycycline-inducible expression of KIT D816V as well as the growth of primary neoplastic mast cells and HMC-1 cells harboring this KIT mutation. PKC412 was a superior agent with median inhibitory concentration (IC50) values of 50 to 250 nM without differences seen between HMC-1 cells exhibiting or lacking KIT D816V. By contrast, AMN107 exhibited more potent effects in KIT D816V- HMC-1 cells. Corresponding results were obtained with Ba/F3 cells exhibiting wild-type or D816V-mutated KIT. The growth-inhibitory effects of PKC412 and AMN107 on HMC-1 cells were associated with induction of apoptosis and down-regulation of CD2 and CD63. PKC412 was found to cooperate with AMN107, imatinib, and cladribine (2CdA) in producing growth inhibition in HMC-1, but synergistic drug interactions were observed only in cells lacking KIT D816V. Together, PKC412 and AMN107 represent promising novel agents for targeted therapy of SM. (Blood. 2006;107: 752-759)

Description: 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)

Description: Chronic myeloid leukemia (CML) is a hematopoietic stem cell disease in which BCR/ABL enhances growth and survival of leukemic cells. In most patients, the disease can be kept under control by the BCR/ABL tyrosine kinase inhibitor imatinib (STI571; Novartis Basel, Switzerland). However, resistance or intolerance against imatinib may occur during therapy. Therefore, current research is focusing on novel targets and targeted drugs in CML. Polo-like kinase 1 (Plk-1) is a serine/threonine kinase that plays an essential role in mitosis and is expressed in activated/phosphorylated form in various malignancies including acute myeloid leukemia (AML). BI 2536 (Boehringer Ingelheim GmbH, Germany) is a novel selective inhibitor of Plk-1, that is currently tested in AML-trials. In this study, we have evaluated expression and the potential role of Plk-1 as a novel target in CML cells. As assessed by PCR, Plk-1 mRNA was found to be expressed abundantly in primary CML cells and in the CML cell line K562, whereas normal peripheral blood cells did not express detectable levels of Plk-1 mRNA. The Plk-1 protein was detected in primary CML cells and K562 cells by immunocytochemistry. In consecutive experiments, we were able to show that CML cells display phosphorylated Plk-1. As assessed by 3H-thymidine-uptake experiments, BI 2536 was found to inhibit the proliferation of K562 cells in a dose-dependent manner (IC50 5–15 nM). Moreover, BI 2536 was found to inhibit the proliferation of both imatinib-naive (n=6) and imatinib-resistant (n=3) primary CML cells (IC50: 1–15 nM). The growth-inhibitory effect of BI 2536 on CML cells was found to be associated with mitotic arrest, a G2-M cell cycle arrest, and consecutive apoptosis. In normal bone marrow or peripheral blood mononuclear cells, neither mitotic cell arrest nor apoptosis were observed after exposure to BI 2536. In further experiments, primary CML cells were coincubated with BI 2536 plus imatinib or with BI 2536 plus nilotinib (AMN107; Novartis) at fixed ratio of drug concentrations. In these experiments, BI 2536 was found to synergize with both tyrosine kinase inhibitors in counteracting the proliferation of CML cells. In conclusion, our data show that Plk-1 is expressed in activated form in CML cells and plays a role in cell cycle progression and cell viability. Targeting Plk-1 with BI 2536 leads to mitotic arrest, growth inhibition, and apoptosis in imatinib-naive and imatinib-resistant leukemic cells. Moreover, BI 2536 synergizes with imatinib and nilotinib in counteracting the growth of neoplastic cells in CML. Targeting of Plk-1 may be a novel interesting pharmacologic approach to counteract growth of CML cells.

Description: In a majority of all patients with systemic mastocytosis (SM) including aggressive SM and mast cell leukemia (MCL), neoplastic cells 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. We found that the TK-inhibitor dasatinib (BMS-354825) inhibits TK activity of wild type (wt) KIT and KIT-D816V in Ba/F3 cells with doxycycline-inducible KIT-expression. In addition, dasatinib was found to inhibit KIT D816V-induced cluster formation and viability in Ba/F3 cells as well as growth of HMC-1.1 cells (KIT-D816V-negative) and HMC-1.2 cells (KIT-D816V-positive). The effects of dasatinib on growth of HMC-1 cells were dose-dependent, with 100–1,000-fold higher IC50-values in cells harbouring KIT-D816V compared to cells lacking KIT-D816V. Furthermore, dasatinib was found to inhibit the growth of primary neoplastic mast cells in SM in all patients examined. The inhibitory effects of dasatinib in HMC-1 cells were found to be associated with apoptosis and a decrease in expression of CD2 and CD63 as determined by flow cytometry. In addition, dasatinib was found to cooperate with the tyrosine kinase inhibitors PKC412 (midostaurin), AMN107 (nilotinib), and STI571 (imatinib), as well as with 2CdA (cladribine) in producing growth-inhibition in neoplastic mast cells. In HMC-1.1 cells, all drug-interactions applied were found to be synergistic. By contrast, in HMC-1.2 cells, only the combinations “dasatinib+PKC412” and “dasatinib+2CdA” were found to produce synergistic effects. These drug-combinations may thus represent an interesting pharmacologic approach for the treatment of patients with aggressive systemic mastocytosis or mast cell leukemia.

Description: Heat shock proteins (Hsp) are increasingly employed as therapeutic targets in various solid tumors and leukemias. We have recently shown that Hsp32 is expressed in leukemic cells and serves as a survival-factor and molecular target in Ph+ chronic myeloid leukemia. In the present study, we examined the expression and functional role of Hsp32 in acute lymphoblastic leukemia (ALL). Leukemic cells were obtained from patients with Ph+ ALL (n=5) and Ph− ALL (n=5). In addition, Ph+ ALL cell lines (Z-119, BV-173, TOM-1, NALM-1) and Ph− ALL cell lines (RAJI, RAMOS, REH, BL-41) were used. As assessed by immunocytochemistry and qRT-PCR, leukemic cells were found to express the Hsp32 protein as well as Hsp32 mRNA in all patients and in all cell lines examined. The Hsp32-inductor hemin was found to promote the expression of Hsp32 in leukemic cells. To determine the functional role of Hsp32 in lymphoblasts, an siRNA against Hsp32 was applied. The siRNA-induced knock down of Hsp32 in RAJI cells was found to be associated with reduced growth and with an increase in apoptotic cells compared to a control siRNA against luciferase (p