Michaela Wagner

Histone Deacetylase Inhibitors Induce a Very Broad, Pleiotropic Anticancer Drug Resistance Phenotype in Acute Myeloid Leukemia Cells by Modulation of Multiple ABC Transporter Genes

Purpose: Histone deacetylase inhibitors (HDACi) are being studied in clinical trials with the aim to induce cellular differentiation, growth arrest, and apoptosis of tumor cells. Recent reports suggest that the multidrug resistance-1 (MDR1) gene is regulated by epigenetic mechanisms. To investigate whether additional drug transporters are regulated by HDACi and how this affects cytotoxicity, acute myeloid leukemia (AML) cells were examined. Experimental Design: AML cells were cultured in the presence of phenylbutyrate, valproate, suberoylanilide hydroxamic acid, or trichostatin A and analyzed for drug transporter expression and function as well as sensitivity to anticancer drugs. Results:MDR1, breast cancer resistance protein (BCRP), and multidrug resistance-associated proteins (MRP) 7 and 8 were induced in a dose- and time-dependent manner as shown by semiquantitative PCR. The pattern of gene induction was cell line specific. Phenylbutyrate induced P-glycoprotein and BCRP expression and the efflux of drugs as determined with labeled substrates. KG-1a cells treated with phenylbutyrate developed resistance to daunorubicin, mitoxantrone, etoposide, vinblastine, paclitaxel, topotecan, gemcitabine, and 5-fluorouracil; as a result drug-induced apoptosis was impaired. Chromatin immunoprecipitation revealed the hyperacetylation of histone proteins in the promoter regions of MDR1, BCRP, and MRP8 on valproate treatment. Furthermore, an alternative MRP8 promoter was induced by HDACi treatment. Conclusions: Exposure of AML cells to HDACi induces a drug resistance phenotype broader than the “classic multidrug resistance,” which might negatively affect treatment effectiveness.

Protein Kinase C-β-Dependent Activation of NF-κB in Stromal Cells Is Indispensable for the Survival of Chronic Lymphocytic Leukemia B Cells In Vivo

Summary Tumor cell survival critically depends on heterotypic communication with benign cells in the microenvironment. Here, we describe a survival signaling pathway activated in stromal cells by contact to B cells from patients with chronic lymphocytic leukemia (CLL). The expression of protein kinase C (PKC)-βII and the subsequent activation of NF-κB in bone marrow stromal cells are prerequisites to support the survival of malignant B cells. PKC-β knockout mice are insusceptible to CLL transplantations, underscoring the in vivo significance of the PKC-βII-NF-κB signaling pathway in the tumor microenvironment. Upregulated stromal PKC-βII in biopsies from patients with CLL, acute lymphoblastic leukemia, and mantle cell lymphoma suggests that this pathway may commonly be activated in a variety of hematological malignancies.

Sorafenib induces cell death in chronic lymphocytic leukemia by translational downregulation of Mcl-1.

Chronic lymphocytic leukemia (CLL) has a high prevalence in western countries and remains incurable to date. Here, we provide evidence that the multikinase inhibitor sorafenib induces apoptosis in primary CLL cells. This strong pro-apoptotic effect is not restricted to any subgroup of patients, based on Binet stage and the expression of ZAP70 or CD38. Mechanistically, sorafenib-induced cell death is preceded by a rapid downregulation of Mcl-1 through the inhibition of protein translation. Subsequently, the cell intrinsic apoptotic pathway is activated, indicated by destabilization of the mitochondrial membrane potential and activation of caspase-3 and -9. In contrast to sorafenib, the monoclonal vascular epidermal growth factor (VEGF)-antibody bevacizumab failed to induce apoptosis in CLL cells, suggesting that sorafenib induces cell death irrespectively of VEGF signalling. Notably, although sorafenib inhibits phosphorylation of the Scr-kinase Lck, knock-down of Lck did not induce apoptosis in CLL cells. Of note, the pro-apoptotic effect of sorafenib is not restricted to cell-cycle arrested cells, but is also maintained in proliferating CLL cells. In addition, we provide evidence that sorafenib can overcome drug resistance in CLL cells protected by microenvironmental signals from stromal cells. Conclusively, sorafenib is highly active in CLL and may compose a new therapeutic option for patients who relapse after immunochemotherapy.

Recruitment of PKC-βII to lipid rafts mediates apoptosis-resistance in chronic lymphocytic leukemia expressing ZAP-70

ZAP-70 is a key signaling molecule in T cells. It couples the antigen-activated T-cell receptor to downstream signaling pathways. Its expression in leukemic B-cells derived from a subgroup of patients with chronic lymphocytic leukemia (CLL) is associated with an aggressive course of the disease. However, its implication for the pathogenesis of aggressive CLL is still unclear. In this study, we show that the expression of ZAP-70 enhances the signals associated with the B-cell receptor, recruiting protein kinase C-βII (PKC-βII) into lipid raft domains. Subsequently, PKC-βII is activated and shuttles from the plasma membrane to the mitochondria. We unravel that the antiapoptotic protein Bcl-2 and its antagonistic BH3-protein BimEL are putative substrates for PKC-βII. PKC-βII-mediated phosphorylation of Bcl-2 augments its antiapoptotic function by increasing its ability to sequester more pro-apoptotic BimEL. In addition, the phosphorylation of BimEL by PKC-βII leads to its proteasomal degradation. These changes confer leukemic cells to a more antiapoptotic state with aggressiveness of the disease. Most importantly, these molecular changes can be therapeutically targeted with the small molecule inhibitor Enzastaurin. We provide evidence that this compound is highly active in leukemic cells and augments the cytotoxic effects of standard chemotherapeutic drugs.

Immunotoxin BL22 induces apoptosis in mantle cell lymphoma (MCL) cells dependent on Bcl-2 expression

Mantle cell lymphoma (MCL) is an incurable mature B cell proliferation, combining the unfavourable clinical features of aggressive and indolent lymphomas. The blastic variant of MCL has an even worse prognosis and new treatment options are clearly needed. We analysed the effects of BL22, an immunotoxin composed of the Fv portion of an anti‐ CD22 antibody fused to a 38‐kDa Pseudomonas exotoxin‐A fragment on four MCL cell lines as well as on primary cells of four MCL patients. Apoptosis induction by BL22 was much more pronounced in MCL cell lines with low Bcl‐2 expression (NCEB‐1, JeKo‐1 and JVM‐2) compared to Granta‐519 cells with high Bcl‐2 expression. While the expression of the antiapoptotic protein Mcl‐1 declined (NCEB‐1, Granta‐519), Bcl‐2 levels remained unchanged in Granta‐519 cells. However transfection of BCL2 cDNA into NCEB‐1, JeKo‐1 and JVM‐2 cells significantly reduced BL22‐mediated toxicity. Accordingly we examined the effects of Bcl‐2 inactivation in Granta‐519 cells using siRNA. Indeed, apoptosis induction was strongly enhanced in Granta‐519 cells with silenced Bcl‐2. Our results were confirmed in freshly isolated MCL‐cells from patients with leukaemic MCL. We conclude that Bcl‐2 expression is important for mediating resistance against the immunotoxin BL22 in MCL cells.

Hypoxia regulates proliferation of acute myeloid leukemia and sensitivity against chemotherapy.

Reduced oxygen partial pressure (pO2, hypoxia) is an important component of the bone marrow microenvironment and the hematopoietic stem cell niche. It is unclear whether this applies to the leukemic stem cell as well and if differences in pO2 between the normal hematopoetic and the leukemic stem cell niche exits. Here, we demonstrate that while there is no detectable difference in the hypoxic level of bone marrow infiltrated by acute myeloid leukemia (AML) and healthy bone marrow, physiological hypoxia of 1% O2 itself leads to cell cycle arrest of AML blasts (both cell lines and primary AML samples) in the G0/G1 phase with upregulation of p27 and consecutive decrease of cells in the S phase. Hence, susceptibility of AML blasts toward cytarabine as S phase dependent drug is significantly decreased as shown by decreased cytotoxicity in vitro. In addition, cells exposed to hypoxia activate PI3K/Akt and increase expression of anti-apoptotic XIAP. Inhibition of PI3K can restore cytarabine sensitivity of AML blasts at hypoxic conditions. In conclusion, hypoxia mediated effects encountered in the bone marrow might contribute to chemoresistance of AML blasts.

Integration of innate into adaptive immune responses in ZAP-70–positive chronic lymphocytic leukemia

The crucial dependence of chronic lymphocytic leukemia (CLL) cells on signals derived from the B cell receptor (BCR) has encouraged the development of new inhibitors, which interfere with BCR signaling and demonstrate clinical benefits in nearly all patients. In addition, signaling through Toll-like receptor (TLR) 9 of the innate immune system has been shown to further contribute to the activation of CLL cells. However, responses to TLR9 engagement are not uniform, but diametrically opposed with cell death in some patients and cell proliferation in others. We now provide evidence that heterogeneous responses to TLR agonists are related to differences in the ability of CLL cells to activate the BCR-associated kinase Syk. Notably, expression of ZAP-70 appears to be of crucial importance for TLR9-mediated activation of Syk. We show that the activation of Syk provides an antiapoptotic signal, which is independent of Mcl-1, Bcl-2, and Bcl-XL, but related to the degradation of the proapoptotic Bim. Mechanistically, TLR9-mediated antiapoptotic signals in ZAP-70-positive CLL trigger secretion of immunoglobulin M, which then serves as (auto-) antigen for a prosurvival BCR signal. Thus, our data show that single activation of the innate immune receptor TLR9 is sufficient to fully engage BCR signaling in ZAP-70-positive CLL, protecting malignant cells from apoptosis. We conclude that the integration of TLR signaling into an adaptive immune response can further promote survival of CLL cells and may contribute to the unfavorable prognosis of ZAP-70-positive CLL.

IL-8 as mediator in the microenvironment-leukaemia network in acute myeloid leukaemia.

The bone marrow microenvironment is physiologically hypoxic with areas being as low as 1% O2, e.g. the stem cell niche. Acute myeloid leukaemia (AML) blasts misuse these bone marrow niches for protection by the local microenvironment, but also might create their own microenvironment. Here we identify IL-8 as a hypoxia-regulated cytokine in both AML cell lines and primary AML samples that is induced within 48 hours of severe hypoxia (1% O2). IL-8 lacked effects on AML cells but induced migration in mesenchymal stromal cells (MSC), an integral part of the bone marrow. Accordingly, MSC were significantly increased in AML bone marrow as compared to healthy bone marrow. Interestingly, mononuclear cells obtained from healthy bone marrow displayed both significantly lower endogenous and hypoxia-induced production of IL-8. IL-8 mRNA expression in AML blasts from 533 patients differed between genetic subgroups with significantly lower expression of IL-8 in acute promyelocytic leukaemia (APL), while in non APL-AML patients with FLT ITD had the highest IL-8 expression. In this subgroup, high IL-8 expression was also prognostically unfavourable. In conclusion, hypoxia as encountered in the bone marrow specifically increases IL-8 expression of AML, which in turn impacts niche formation. High IL-8 expression might be correlated with poor prognosis in certain AML subsets.

The bone marrow microenvironment is a critical player in the NK cell response against acute myeloid leukaemia in vitro

Immune therapy for acute myeloid leukaemia (AML) has been largely disappointing. One possible explanation might lie in the microenvironment of the bone marrow, comprising cellular (e.g. mesenchymal stromal cells, MSC) and non-cellular components (e.g. hypoxia). The purpose of this study was to investigate the effects of these components in the immune response against AML in vitro. In vitro exposure of lymphocytes to hypoxia resulted in an increased expression of CD69 as an activation marker in NK cells only, with subsequently enhanced cell lysis of K-562 cell line by NK cells but not in lysis of primary blast. However, co-culture of AML cells with MSC significantly protected leukemic blasts from NK cell mediated lysis, mainly in a specific manner requiring cell-to-cell contact with supportive MSC. These data imply a relevant but unequivocal role of hypoxia and MSC the immune response against AML blasts.

Microenvironmental Hypoxia regulates FLT3 expression and biology in AML

Fms-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase constitutively expressed by acute myeloid leukaemia (AML) blasts. In addition, 25% of AML patients harbour a FLT3-ITD mutation, associated with inferior outcome due to increased relapse rate. Relapse might be propagated by interactions between AML blasts and the bone marrow microenvironment. Besides cellular elements of the microenvironment (e.g. mesenchymal stromal cells), bone marrow hypoxia has emerged as an additional crucial component. Hence, effects of hypoxia on FLT3 expression and biology could provide novel insight into AML biology. Here we show that 25% of AML patients down-regulate FLT3 expression on blasts in response to in vitro hypoxia (1% O2), which was independent of its mutational state. While virtually no AML cell lines regulate FLT3 in response to hypoxia, the down-regulation could be observed in Ba/F3 cells stably transfected with different FLT3 mutants. Hypoxia-mediated down-regulation was specific for FLT3, reversible and proteasome-dependent; with FLT3 half-life being significantly shorter at hypoxia. Also, PI-3K inhibition could partially abrogate down-regulation of FLT3. Hypoxia-mediated down-regulation of FLT3 conferred resistance against cytarabine in vitro. In conclusion, FLT3 expression in AML is dependent on the oxygen partial pressure, but response to hypoxia differs.