u-0126 and Multiple-Myeloma

CD200 is a relatively ubiquitously expressed molecule that plays a role in cancer immune evasion through interaction with its receptors. High expression levels of CD200 have been described in different human malignancies. For example, CD200 has been shown to be targeted after RAS/RAF/MEK/ERK activation in melanoma. Here we present the analysis of CD200 expression in human Multiple Myeloma (MM) samples. We found that CD200-positive cells express ERK and p-ERK. Moreover, UO126, a MEK inhibitor, reduces CD200 expression. Furthermore, we observe that CD200-positive cells show reduced immunogenicity compared to normal lymphocytes and that such immunogenicity increases when UO126 is used. We therefore hypothesize that CD200 expression in MM could suppress antitumor response and that anti-CD200 treatment might be therapeutically beneficial in CD200-expressing tumors.

Topics: Antigens, CD; Antigens, Surface; Biomarkers, Tumor; Butadienes; CD4-Positive T-Lymphocytes; Cells, Cultured; Enzyme Inhibitors; Humans; Lymphocyte Activation; Lymphocyte Culture Test, Mixed; MAP Kinase Signaling System; Multiple Myeloma; Nitriles; Orexin Receptors; Receptors, Cell Surface; Tumor Escape

When mTOR inhibitor rapalogs prevent cap-dependent translation of cell-cycle proteins like c-myc, continuing tumor cell growth depends on cap-independent translation, which is mediated by internal ribosome entry sites (IRESes) located in the 5'-UTR (untranslated region) of transcripts. To investigate if rapalog-induced activation of MNK kinases had a role in such IRES activity, we studied multiple myeloma (MM) cells. Rapamycin (RAP)-activated MNK1 kinase activity in MM cell lines and primary specimens by a mitogen-activated protein kinase-dependent mechanism. Pharmacological inhibition of MNK activity or genetic silencing of MNK1 prevented a rapalog-induced upregulation of c-myc IRES activity. Although RAP, used alone, had little effect on myc protein expression, when combined with a MNK inhibitor, myc protein expression was abrogated. In contrast, there was no inhibition of myc RNA, consistent with an effect on myc translation. In a RAP-resistant MM cell lines as well as a resistant primary MM specimen, co-exposure to a MNK inhibitor or MNK1 knockdown significantly sensitized cells for RAP-induced cytoreduction. Studies in MNK-null murine embryonic fibroblasts additionally supported a role for MNK kinases in RAP-induced myc IRES stimulation. These results indicate that MNK kinase activity has a critical role in the fail-safe mechanism of IRES-dependent translation when mTOR is inhibited. As kinase activity also regulated sensitivity to RAP, the data also provide a rationale for therapeutically targeting MNK kinases for combined treatment with mTOR inhibitors.

Topics: 5' Untranslated Regions; Aniline Compounds; Animals; Butadienes; Cell Line, Tumor; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Fibroblasts; Genes, myc; Humans; Imidazoles; Intracellular Signaling Peptides and Proteins; Mice; Multiple Myeloma; Nitriles; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Biosynthesis; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-myc; Purines; Pyridines; RNA Interference; RNA, Messenger; RNA, Small Interfering; Sirolimus; TOR Serine-Threonine Kinases; Up-Regulation

The PI3K/Akt/mTOR signal transduction pathway plays a central role in multiple myeloma (MM) disease progression and development of therapeutic resistance. mTORC1 inhibitors have shown limited efficacy in the clinic, largely attributed to the reactivation of Akt due to rapamycin induced mTORC2 activity. Here, we present promising anti-myeloma activity of MK-2206, a novel allosteric pan-Akt inhibitor, in MM cell lines and patient cells. MK-2206 was able to induce cytotoxicity and inhibit proliferation in all MM cell lines tested, albeit with significant heterogeneity that was highly dependent on basal pAkt levels. MK-2206 was able to inhibit proliferation of MM cells even when cultured with marrow stromal cells or tumor promoting cytokines. The induction of cytotoxicity was due to apoptosis, which at least partially was mediated by caspases. MK-2206 inhibited pAkt and its down-stream targets and up-regulated pErk in MM cells. Using MK-2206 in combination with rapamycin (mTORC1 inhibitor), LY294002 (PI3K inhibitor), or U0126 (MEK1/2 inhibitor), we show that Erk- mediated downstream activation of PI3K/Akt pathway results in resistance to Akt inhibition. These provide the basis for clinical evaluation of MK-2206 alone or in combination in MM and potential use of baseline pAkt and pErk as biomarkers for patient selection.

Topics: Apoptosis; Butadienes; Cell Line, Tumor; Cell Proliferation; Chromones; Gene Expression Regulation, Neoplastic; Heterocyclic Compounds, 3-Ring; Humans; MAP Kinase Signaling System; Mechanistic Target of Rapamycin Complex 1; Morpholines; Multiple Myeloma; Multiprotein Complexes; Nitriles; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transcriptional Activation

Constitutive activation of the transcription factor STAT3 contributes to the pathogenesis of many cancers, including multiple myeloma (MM). Since STAT3 is dispensable in most normal tissue, targeted inhibition of STAT3 is an attractive therapy for patients with these cancers. To identify STAT3 inhibitors, we developed a transcriptionally based assay and screened a library of compounds known to be safe in humans. We found the drug nifuroxazide to be an effective inhibitor of STAT3 function. Nifuroxazide inhibits the constitutive phosphorylation of STAT3 in MM cells by reducing Jak kinase autophosphorylation, and leads to down-regulation of the STAT3 target gene Mcl-1. Nifuroxazide causes a decrease in viability of primary myeloma cells and myeloma cell lines containing STAT3 activation, but not normal peripheral blood mononuclear cells. Although bone marrow stromal cells provide survival signals to myeloma cells, nifuroxazide can overcome this survival advantage. Reflecting the interaction of STAT3 with other cellular pathways, nifuroxazide shows enhanced cytotoxicity when combined with either the histone deacetylase inhibitor depsipeptide or the MEK inhibitor UO126. Therefore, using a mechanistic-based screen, we identified the clinically relevant drug nifuroxazide as a potent inhibitor of STAT signaling that shows cytotoxicity against myeloma cells that depend on STAT3 for survival.

Topics: Butadienes; Cell Line, Tumor; Cell Survival; Depsipeptides; Humans; Hydroxybenzoates; Janus Kinases; Multiple Myeloma; Myeloid Cell Leukemia Sequence 1 Protein; Nitriles; Nitrofurans; Phosphorylation; Proto-Oncogene Proteins c-bcl-2; STAT3 Transcription Factor

Macrophage inflammatory protein 1 alpha (MIP-1 alpha) is detected at high concentrations in patients with multiple myeloma, and it is thought to play an important role in the etiology of multiple myeloma and osteolysis. Thus, we investigated whether or not YM529/ONO-5920, a new bisphosphonate, inhibited MIP-1 alpha mRNA expression in, and MIP-1 alpha secretion from, mouse myeloma cells. When the cells were stimulated by lipopolysaccharide, increased MIP-1 alpha mRNA expression and MIP-1 alpha secretion were observed. YM529/ONO-5920 inhibited MIP-1 alpha mRNA expression and MIP-1 alpha secretion in a concentration-dependent manner. A transient increase in the phosphorylation of extracellular-regulated kinase 1/2 (ERK1/2) and Akt was observed after lipopolysaccharide stimulation. After YM529/ONO-5920 was given, there was no transient increase in the phosphorylation of ERK1/2 or Akt. These results indicated that YM529/ONO-5920 inhibited the expression and secretion of MIP-1 alpha through blocking the signaling pathway of the Ras/mitogen-activated protein kinase kinase/ERK and Ras/phosphatidylinositol-3 kinase/Akt. Accordingly, YM529/ONO-5920 appears to have promise for use in effective future therapy for osteolysis and myeloma cell growth that depends on MIP-1 alpha.

Topics: Animals; Butadienes; Cell Growth Processes; Cell Line, Tumor; Chemokine CCL3; Chromones; Diphosphonates; I-kappa B Proteins; Imidazoles; Lipopolysaccharides; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Morpholines; Multiple Myeloma; Nitriles; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Polyisoprenyl Phosphates; Proto-Oncogene Proteins c-akt; RNA, Messenger; Sesquiterpenes

We recently reported that internal deletion of PTEN tumor suppressor gene in OPM2 and Delta47 myeloma lines led to high Akt activation. Re-expression of PTEN induced strong apoptosis and growth inhibition. To understand the biologic importance of the phosphatidylinositol 3 kinase (PI3K)/Akt activation affected by PTEN deletion, we analysed apoptosis and growth inhibition by applying PI3K inhibitors to myeloma lines and by expressing Akt constructs. The PI3K inhibitors preferentially suppressed PTEN-null myeloma growth to those expressing PTEN, indicating that PI3K activation is more critical for growth and survival of those lines with PTEN mutations than others expressing a functional PTEN gene. Since PTEN-null myeloma lines exhibited much stronger Akt activation than PTEN-expressing cells in response to insulin-like growth factor I stimulation, we determined whether Akt could be responsible for PI3K-mediated cell survival and growth of PTEN-null myeloma lines. Expression of an active Akt, but not its kinase dead mutant, reversed wortmannin- and dexamethasone-induced apoptosis and growth inhibition in PTEN-null myeloma lines, suggesting that Akt lies downstream of PI3K for PTEN-null myeloma survival and dexamethasone resistance. In summary, we have provided evidence that PTEN-null myeloma cells are stringently dependent on the PI3K/Akt activation for cell survival. These results may provide a basis to treat myeloma patients with PI3K and Akt inhibitors.

Topics: Androstadienes; Antineoplastic Agents; Apoptosis; Butadienes; Cell Division; Cell Survival; Chromones; Dexamethasone; Enzyme Activation; Enzyme Inhibitors; Gene Deletion; Genes, Tumor Suppressor; Humans; Insulin-Like Growth Factor I; Interleukin-6; Morpholines; Multiple Myeloma; Nitriles; Phosphoinositide-3 Kinase Inhibitors; Phosphoric Monoester Hydrolases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Signal Transduction; Tumor Cells, Cultured; Tumor Suppressor Proteins; Wortmannin

The evolutionarily conserved Ras/Raf/MEK/ERK pathway is thought to be essential for proliferation of eukaryotic cells. The human multiple myeloma (MM) cell line 8226 encodes an activated K-ras allele and proliferates without requirement for the main MM growth and survival factor IL-6. Surprisingly, the addition of the MEK1/2 inhibitors PD98059 or U0126 to 8226 cultures at doses that block virtually all ERK1/2 activity had minimal effects on the rapid proliferation of this cell line. In contrast, proliferation of the IL-6-dependent MM cell line, ANBL-6 was blocked by PD98059. Levels of activated forms of the other classical MAP kinases (JNK and p38) were very low during MM cell proliferation and, therefore, do not substitute for the mitogenic activities normally regulated by ERK kinases. These data demonstrate that proliferation of 8226 cells does not require ERK1/2 activity, and suggest that IL-6-independent growth of MM may correlate with independence from a requirement for ERK activity. Other signal transduction pathways that appear to regulate cell cycle progression in these cells were examined.

Topics: Antibiotics, Antineoplastic; Blotting, Western; Butadienes; Cell Cycle; Cell Division; Chromones; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Flavonoids; Genes, ras; Humans; Interleukin-6; JNK Mitogen-Activated Protein Kinases; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Morpholines; Multiple Myeloma; Nitriles; p38 Mitogen-Activated Protein Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Ribosomal Protein S6 Kinases; Signal Transduction; Sirolimus; Tumor Cells, Cultured