Terry H. Landowski

Constitutive Activation of Stat3 Signaling Confers Resistance to Apoptosis in Human U266 Myeloma Cells

Interleukin 6 (IL-6) is the major survival factor for myeloma tumor cells and induces signaling through the STAT proteins. We report that one STAT family member, Stat3, is constitutively activated in bone marrow mononuclear cells from patients with multiple myeloma and in the IL-6-dependent human myeloma cell line U266. Moreover, U266 cells are inherently resistant to Fas-mediated apoptosis and express high levels of the antiapoptotic protein Bcl-xL. Blocking IL-6 receptor signaling from Janus kinases to the Stat3 protein inhibits Bcl-xL expression and induces apoptosis, demonstrating that Stat3 signaling is essential for the survival of myeloma tumor cells. These findings provide evidence that constitutively activated Stat3 signaling contributes to the pathogenesis of multiple myeloma by preventing apoptosis.

Bone marrow stromal-derived soluble factors and direct cell contact contribute to de novo drug resistance of myeloma cells by distinct mechanisms

The tumor microenvironment plays a critical role in determining the fate of tumor cells. We have previously reported that adhesion of human myeloma and leukemia cell lines to the extracellular matrix protein, fibronectin, confers a multidrug-resistant phenotype. Mechanisms associated with this cell adhesion-mediated drug resistance are drug-type specific. In the present study, we examined the influence of bone marrow stromal cells (BMSCs) on myeloma cell response to the topoisomerase II inhibitor, mitoxantrone. Apoptosis was inhibited by more than 50% when cells were adhered to BMSCs as compared to myeloma cells maintained in suspension. To investigate the mechanisms contributing to the resistance of myeloma cells in contact with BMSCs, we examined the protective effects of BMSCs under four separate conditions: (1) direct cell contact; (2) BMSCs conditioned medium; (3) medium conditioned by coculturing myeloma cells in direct contact with BMSCs; and (4) medium conditioned by coculturing myeloma cells and BMSCs without direct physical contact. Conditioned medium from BMSCs alone was not sufficient to protect myeloma cells from drug-induced apoptosis; however, soluble factors produced during the myeloma-BMSCs interaction decreased the sensitivity of myeloma cells to mitoxantrone, suggesting a dynamic interaction between myeloma cells and BMSCs. We also found that myeloma cells in direct contact with BMSCs underwent growth arrest, whereas soluble factors produced by myeloma cells-BMSCs coincubation stimulated the proliferation of myeloma cells. These data show that both cell–cell adhesion of BMSCs with myeloma cells and soluble factors induced by this cell–cell interaction are involved in the protection of myeloma cells from mitoxantrone-induced apoptosis; however, the mechanisms contributing to the drug resistance are different.

Mitochondrial-Mediated Disregulation of Ca2+ Is a Critical Determinant of Velcade (PS-341/Bortezomib) Cytotoxicity in Myeloma Cell Lines

The proteasome inhibitor bortezomib (also known as PS-341/Velcade) is a dipeptidyl boronic acid that has recently been approved for use in patients with multiple myeloma. Bortezomib inhibits the activity of the 26S proteasome and induces cell death in a variety of tumor cells; however, the mechanism of cytotoxicity is not well understood. In this report, oligonucleotide microarray analysis of the 8226 multiple myeloma cell line showed a predominant induction of gene products associated with the endoplasmic reticulum secretory pathway following short-term, high-dose exposure to bortezomib. Examination of mediators of endoplasmic reticulum stress-induced cell death showed specific activation of caspase 12, as well as of caspases 8, 9, 7, and 3, and cleavage of bid. Treatment of myeloma cells with bortezomib also showed disregulation of intracellular Ca2+ as a mechanism of caspase activation. Cotreatment with a panel of Ca2+-modulating agents identified the mitochondrial uniporter as a critical regulatory factor in bortezomib cytotoxicity. The uniporter inhibitors ruthenium red and Ru360 prevented caspase activation and bid cleavage, and almost entirely inhibited bortezomib-induced cell death, but had no effect on any other chemotherapeutic drug examined. Additional Ca2+-modulating agents, including 2-amino-ethoxydiphenylborate, 1,2-bis (o-aminophenoxy) ethane-tretraacetic acid (acetoxymethyl) ester, and dantrolene, did not alter bortezomib cytotoxicity. Analysis of intracellular Ca2+ showed that the ruthenium-containing compounds inhibited Ca2+ store loading and abrogated the desensitized capacitative calcium influx associated with bortezomib treatment. These data support the hypothesis that intracellular Ca2+ disregulation is a critical determinant of bortezomib cytotoxicity.

Role of the tumor microenvironment in mediating de novo resistance to drugs and physiological mediators of cell death

The emergence of clinical drug resistance continues to be an obstacle for the successful treatment of cancer. Our current understanding of mechanisms associated with drug resistance has been ascertained by investigating drug-resistant models created by exposing a parental population to increasing concentrations of a cytotoxic. These unicellular drug-resistant models have been critical in elucidating drug-resistant mechanism and in some cases have aided in the identification of drug targets. However, these models do not address resistance mechanisms that contribute to de novo drug resistance. We propose that specific niches within the tumor microenvironment may provide a sanctuary for subpopulations of tumors cells that affords a survival advantage following initial drug exposure and may facilitate the acquisition of acquired drug resistance. More specifically, we propose that the bone marrow microenvironment is a sanctuary for hema-topoietic cancers. This review will focus on the bone marrow microenvironment and its role in conferring resistance to cytotoxics and physiological mediators of cell death.

Cell adhesion-mediated drug resistance (CAM-DR) is associated with activation of NF-κB (RelB/p50) in myeloma cells

The microenvironment has been shown to influence tumor cell phenotype with respect to growth, metastasis, and response to chemotherapy. We have utilized oligonucleotide microarray analysis to identify signal transduction pathways and gene products altered by the interaction of myeloma tumor cells with the extracellular matrix component fibronectin that may contribute to the antiapoptotic phenotype conferred by the microenvironment. Genes with altered expression associated with fibronectin cell adhesion, either induced or repressed, were numerically ranked by fold change. FN adhesion repressed the expression of 469 gene products, while 53 genes with known coding sequences were induced by twofold or more. Of these 53 genes with two fold, or greater increase in expression, 11 have been reported to be regulated by the nuclear factor-kappa B (NF-κB) family of transcription factors. EMSA analysis demonstrated NF-κB binding activity significantly increased in cells adhered to fibronectin compared to cells in suspension. This DNA binding activity consisted primarily of RelB-p50 heterodimers, which was distinct from the NF-κB activation of TNFα. These data demonstrate the selectivity of signal transduction from the microenvironment that may contribute to tumor cell resistance to programmed cell death.

Inhibition of Bcr-Abl kinase activity by PD180970 blocks constitutive activation of Stat5 and growth of CML cells

Chronic myelogenous leukemia (CML) is a myeloproliferative disease characterized by the BCR–ABL genetic translocation and constitutive activation of the Abl tyrosine kinase. Among members of the Signal Transducers and Activators of Transcription (STAT) family of transcription factors, Stat5 is activated by the Bcr–Abl kinase and is implicated in the pathogenesis of CML. We recently identified PD180970 as a new and highly potent inhibitor of Bcr–Abl kinase. In this study, we show that blocking Bcr–Abl kinase activity using PD180970 in the human K562 CML cell line resulted in inhibition of Stat5 DNA-binding activity with an IC50 of 5 nM. Furthermore, abrogation of Abl kinase-mediated Stat5 activation suppressed cell proliferation and induced apoptosis in K562 cells, but not in the Bcr–Abl-negative myeloid cell lines, HEL 92.1.7 and HL-60. Dominant-negative Stat5 protein expressed from a vaccinia virus vector also induced apoptosis of K562 cells, consistent with earlier studies that demonstrated an essential role of Stat5 signaling in growth and survival of CML cells. RNA and protein analyses revealed several candidate target genes of Stat5, including Bcl-x, Mcl-1, c-Myc and cyclin D2, which were down-regulated after treatment with PD180970. In addition, PD180970 inhibited Stat5 DNA-binding activity in cultured primary leukemic cells derived from CML patients. To detect activated Stat5 in CML patient specimens, we developed an immunocytochemical assay that can be used as a molecular end-point assay to monitor inhibition of Bcr–Abl signaling. Moreover, PD180970 blocked Stat5 signaling and induced apoptosis of STI-571 (Gleevec, Imatinib)-resistant Bcr–Abl-positive cells. Together, these results suggest that the mechanism of action of PD180970 involves inhibition of Bcr–Abl-mediated Stat5 signaling and provide further evidence that compounds in this structural class may represent potential therapeutic agents for CML.

Adhesion-Mediated Intracellular Redistribution of c-Fas-Associated Death Domain-Like IL-1-Converting Enzyme-Like Inhibitory Protein-Long Confers Resistance to CD95-Induced Apoptosis in Hematopoietic Cancer Cell Lines

Evasion of immune surveillance is a key step in malignant progression. Interactions between transformed hematopoietic cells and their environment may initiate events that confer resistance to apoptosis and facilitate immune evasion. In this report, we demonstrate that β1 integrin-mediated adhesion to fibronectin inhibits CD95-induced caspase-8 activation and apoptosis in hematologic tumor cell lines. This adhesion-dependent inhibition of CD95-mediated apoptosis correlated with enhanced c-Fas-associated death domain-like IL-1-converting enzyme-like inhibitory protein-long (c-FLIPL) cytosolic solubility compared with nonadhered cells. Cytosolic c-FLIPL protein preferentially associated with cytosolic Fas-associated death domain protein (FADD) and localized to the death-inducing signal complex after CD95 ligation in adherent cells. The incorporation of c-FLIPL in the death-inducing signal complex prevented procaspase-8 processing and activation of the effector phase of apoptosis. Adhesion to fibronectin increased c-FLIPL cytosolic solubility and availability for FADD binding by redistributing c-FLIPL from a preexisting membrane-associated fraction. Increased cytosolic availability of c-FLIPL for FADD binding was not related to increased levels of RNA or protein synthesis. These data show that adhesion of anchorage-independent cells to fibronectin provides a novel mechanism of resistance to CD95-mediated programmed cell death by regulating the cellular localization and availability of c-FLIPL.

The tumor microenvironment as a determinant of cancer cell survival: A possible mechanism for de novo drug resistance

The influence of the microenvironment in the pathogenesis and progression of human cancer has traditionally been considered in the context of solid tumors. More recently, evidence has been accumulating to support the role of the bone marrow microenvironment in hematologic malignancies as well, particularly in multiple myeloma. This review focuses on myeloma as a model to demonstrate that the bone marrow microenvironment provides a sanctuary against programmed cell death and promotes tumor cell survival and progression. Additionally, the protective effects of the bone marrow milieu may confer a protection from cytotoxic drugs, allowing the emergence of drug-resistant tumors. These advances may assist in the design of novel therapeutic approaches to enhance the efficacy of standard chemotherapeutic drugs.

The farnesyl transferase inhibitor, FTI-277, inhibits growth and induces apoptosis in drug-resistant myeloma tumor cells

Mutations of the ras gene are among the most commonly identified transforming events in human cancers, including multiple myeloma. Farnesyltransferase inhibitors (FTI) were developed to prevent Ras processing and induce cancer cell death. Several FTIs are in phase II and one is in phase III clinical trials. Preclinically, most of the focus has been on solid tumors, and the effects of FTIs in multiple myeloma have not been investigated. In this study we examined the cytotoxic activity and inhibition of Ras processing in three myeloma cell lines with differing Ras mutation status. H929 cells with activated N-Ras were more sensitive to FTI-277 treatment than 8226 and U266 cells with activated K-Ras or wild-type Ras, respectively. A combination of FTI-277 and a geranylgeranyltransferase I inhibitor (GGTI)-2166 inhibited K-Ras processing and enhanced cell death in 8226 cells. U266 cells and Bcl-xL transfectants were equally sensitive to FTI-277 treatment. Similarly, 8226 cells selected for resistance to various chemotherapeutic agents, which resulted in either P-glycoprotein overexpression, altered topoisomerase II activity, or elevated glutathione levels, were equally sensitive to FTI-277. These preclinical studies suggest that prenylation inhibitors may represent new therapeutic agents for the treatment of refractory or drug-resistant multiple myeloma.

CD95 antigen mutations in hematopoietic malignancies

The CD95 receptor, also known as Fas/Apo-1, is a member of the Tumor Necrosis Factor receptor (TNF-R) family of death receptors. Apoptosis mediated by CD95 plays a central role in maintaining homeostasis of the immune system. Dysregulation of the CD95 apoptotic pathway has been proposed as a mechanism of oncogenesis by providing a survival advantage to potentially malignant cells. This extended lifespan could allow the accumulation of further mutations leading to malignant transformation. Several mechanisms of resistance to CD95 mediated apoptosis have been identified, including reduced surface expression of the receptor, overexpression of anti-apoptotic molecules, and loss of function mutations. This review will focus on the potential role of the CD95-CD95 ligand system in the pathogenesis of hematological malignancies, with particular emphasis on recent work from our laboratory examining the expression of CD95 in B cell lymphomas. We demonstrate that CD95 mutations occur at low frequency in NHL tumors, however, surface expression of the CD95 protein varies with the subtype of lymphoma. Loss of surface CD95 is more likely to occur in lymphomas of aggressive histology, and is unrelated to the detection of CD95 mutations.