IntroductionChronic myeloid leukemia (CML) is a myeloproliferative disease in which leukemic cells exhibit the reciprocal translocation t(9;22) and the bcr/abl oncogene. [1][2][3] The resulting oncoprotein, BCR/ABL, displays constitutive tyrosine kinase activity and activates a number of signaling molecules including RAS/RAF/MAP kinases, the phosphoinositide 3-kinase (PI3-kinase), and signal transducer and activator of transcription 5 (STAT5). [4][5][6][7][8] In addition, BCR/ABL converts cytokine-dependent cell lines to growth factor independence and acts oncogenic in mice. 9 Several different mechanisms have been implicated in BCR/ ABL-dependent growth and accumulation of leukemic cells in CML. [10][11][12] One important mechanism may be inhibition of apoptosis. 10,[13][14][15][16] Thus, a number of antiapoptotic molecules are expressed in CML cells and may contribute to enhanced survival of leukemic cells. [10][11][12][13][14][15][16][17] Likewise, it has been shown that CML cells express several members of the BCL-2 family including BCL-2, BCL-x L , or A1. [13][14][15][16][17] However, the relative contribution of each of these molecules to inhibition of apoptosis in CML cells has not been defined yet. In addition, some of these molecules may only be expressed in leukemic cells in a subgroup of patients. 17 MCL-1 is a well-characterized member of the BCL-2 family that is considered to act antiapoptotic in various neoplastic cells including several leukemia-derived cell lines. [18][19][20][21] Originally, MCL-1 was described as a survival-enhancing molecule that is expressed during 12-O-tetra decanoyl phorbol 13-acetate (TPA) induced differentiation of leukemic ML-1 cells. 18 The CML-derived cell line K562 has also been described to express the MCL-1 protein. 19,[21][22][23] However, little is known so far about expression of MCL-1 in primary CML cells, the regulation of expression of MCL-1 in these cells, and the role that this antiapoptotic molecule may play in survival and accumulation of leukemic cells in patients with CML.In the present study, we have investigated the role of BCR/ABL in expression of MCL-1 in leukemic cells and analyzed underlying signaling pathways. The results of our study show that primary CML cells express MCL-1 in a constitutive manner and that BCR/ABL promotes the expression of MCL-1 through activation of the RAS/RAF/MAP kinase pathway. Moreover, our data show that down-regulation of MCL-1 by antisense oligonucleotides (ASOs) counteracts growth Patients, materials, and methods ConstructsPlasmids used in this study were pDCR-ras-G12V 24,25 (kindly provided by Yoel Kloog, Hadassah Medical Center, Jerusalem, Israel), pMT-Ha-ras-N17 26 (kindly provided by Mark Ewen, Dana Farber Cancer Institute, Boston, MA), and pBSK-mcl-1 27,28 (kindly provided by Stanley J. Korsmeyer, Dana Farber Cancer Institute). cDNAs were cloned into pcDNA3.1 ϩ vector (Invitrogen, Carlsbad, CA). The mcl-1 reporter gene construct (mcl-1-Luc) 29 was a kind gift from Steven W. Edwards (School of Biological Sciences...

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Recently, betulinic acid was identified as a highly selective inhibitor of human melanoma growth and was reported to induce apoptosis in these cells. We have investigated the growth-inhibitory properties of this compound alone and in combination with ionizing radiation in a panel of established human melanoma cell lines as well as in normal human melanocytes. Betulinic acid strongly and consistently suppressed the growth and colony-forming ability of all human melanoma cell lines investigated. In combination with ionizing radiation the effect of betulinic acid on growth inhibition was additive in colony-forming assays. Betulinic acid also induced apoptosis in human melanoma cells as demonstrated by Annexin V binding and by the emergence of cells with apoptotic morphology. The growth-inhibitory action of betulinic acid was more pronounced in human melanoma cell lines than in normal human melanocytes. Notably, despite the induction of apoptosis, analysis of the expression of Bcl-2 family members in betulinic-acid-treated cells revealed that expression of the anti-apoptotic protein Mcl-1 was induced. Furthermore, the antiproliferative action of betulinic acid seemed to be independent of the p53 status. The properties of betulinic acid make it an interesting candidate, not only as a single agent but also in combination with radiotherapy. We conclude that the strictly additive mode of growth inhibition in combination with irradiation suggests that the two treatment modalities may function by inducing different cell death pathways or by affecting different target cell populations.

Purpose: MET, the receptor for hepatocyte growth factor (HGF), has been implicated in driving tumor proliferation and metastasis. High MET expression is correlated with poor prognosis in multiple cancers. Activation of MET can be induced either by HGF-independent mechanisms such as gene amplification, specific genetic mutations, and transcriptional upregulation or by HGF-dependent autocrine or paracrine mechanisms.Experimental Design/Results: Here, we report on LY2875358, a novel humanized bivalent anti-MET antibody that has high neutralization and internalization activities, resulting in inhibition of both HGFdependent and HGF-independent MET pathway activation and tumor growth. In contrast to other bivalent MET antibodies, LY2875358 exhibits no functional agonist activity and does not stimulate biologic activities such as cell proliferation, scattering, invasion, tubulogenesis, or apoptosis protection in various HGFresponsive cells and no evidence of inducing proliferation in vivo in a monkey toxicity study. LY2875358 blocks HGF binding to MET and HGF-induced MET phosphorylation and cell proliferation. In contrast to the humanized one-armed 5D5 anti-MET antibody, LY2875358 induces internalization and degradation of MET that inhibits cell proliferation and tumor growth in models where MET is constitutively activated. Moreover, LY2875358 has potent antitumor activity in both HGF-dependent and HGF-independent (METamplified) xenograft tumor models. Together, these findings indicate that the mechanism of action of LY2875358 is different from that of the one-armed MET antibody.Conclusions: LY2875358 may provide a promising therapeutic strategy for patients whose tumors are driven by both HGF-dependent and HGF-independent MET activation. LY2875358 is currently being investigated in multiple clinical studies. Clin Cancer Res; 20(23); 6059-70. Ó2014 AACR.

Leukaemic stem cells in patients with CD33(+) AML express CD33. This observation is in favour of novel treatment concepts employing CD33-targeting antibodies in AML.

The mTOR pathway is active in 40% of patients with HCC undergoing OLT, but has no influence of DFS or OS. No direct correlation was observed between up- and downstream proteins limiting the use of upstream proteins to predict mTOR activity. Prospective clinical trials are needed to test whether the activation status of the mTOR pathway in HCCs predicts the antitumor effect of rapamycin derivative in the posttransplantation course.

BACKGROUND. The nonstructural protein NS1 of influenza virus counteracts the interferon-mediated immune response of the host. By deleting the open reading frame of NS1, we have generated a novel type of influenza vaccine. We studied the safety and immunogenicity of an influenza strain lacking the NS1 gene (DeltaNS1-H1N1) in healthy volunteers. METHODS. Healthy seronegative adult volunteers were randomized to receive either a single intranasal dose of the DeltaNS1-H1N1 A/New Caledonia vaccine at 1 of 5 dose levels (6.4, 6.7, 7.0, 7.4, and 7.7 log(10) median tissue culture infective dose) (n = 36 recipients) or placebo (n = 12 recipients). RESULTS. Intranasal vaccination with the replication-deficient DeltaNS1-H1N1 vaccine was well tolerated. Rhinitis-like symptoms and headache were the most common adverse events identified during the 28-day observation period. Adverse events were similarly distributed between the treatment and placebo groups. Vaccine-specific local and serum antibodies were induced in a dose-dependent manner. In the highest dose group, vaccine-specific antibodies were detected in 10 of 12 volunteers. Importantly, the vaccine also induced neutralizing antibodies against heterologous drift variants. CONCLUSIONS. We show that vaccination with an influenza virus strain lacking the viral interferon antagonist NS1 induces statistically significant levels of strain-specific and cross-neutralizing antibodies despite the highly attenuated replication-deficient phenotype. Further studies are warranted to determine whether these results translate into protection from influenza virus infection. TRIAL REGISTRATION. ClinicalTrials.gov identifier: NCT00724997 .