nutlin-3a and Lymphoma--Mantle-Cell

PPM1D is a serine/threonine phosphatase that negatively regulates key DNA damage response proteins, such as p53, p38 MAPK, histone H2A.X, and ATM. We investigated the pathophysiological significance of PPM1D and its therapeutic targeting by the novel PPM1D inhibitor GSK2830371 in mantle cell lymphoma (MCL). Oncomine-based analyses indicated increased PPM1D mRNA levels in MCL cells compared with their normal counterpart cells. Higher PPM1D expression was associated with higher expression of the proliferation gene signature and poorer prognosis in patients. Eight MCL (three p53 wild-type and five mutant) cell lines were exposed to GSK2830371. GSK2830371 inhibited the cell growth, being prominent in p53 wild-type cells. GSK2830371 induced apoptosis in sensitive cells, as evidenced by induction of phosphatidylserine externalization and loss of mitochondrial membrane potential. p53 knockdown de-sensitized cell sensitivity. GSK2830371 increased the levels of total and Ser15-phosphorylated p53, and p53 targets p21 and PUMA. GSK2830371 and the MDM2 inhibitor Nutlin-3a acted synergistically in p53 wild-type cells. Interestingly, GSK2830371 sensitized MCL cells to bortezomib and doxorubicin in p53 wild-type and mutant cells; p38 signaling appeared to be involved in the GSK2830371/bortezomib lethality. PPM1D inhibition may represent a novel therapeutic strategy for MCL, which can be exploited in combination therapeutic strategies for MCL.

Topics: Aminopyridines; Apoptosis; Bortezomib; Cell Line, Tumor; Dipeptides; Doxorubicin; Genes, p53; Humans; Imidazoles; Lymphoma, Mantle-Cell; Phosphorylation; Piperazines; Prognosis; Protein Phosphatase 2C; Signal Transduction

This study demonstrated a pronounced synergistic growth-inhibitory effect of an MDM2 inhibitor Nutlin-3 and a proteasome inhibitor bortezomib in mantle cell lymphoma (MCL) cells regardless of TP53 mutant status and innate bortezomib sensitivity. In the mutant TP53 MCL cells which are intrinsically resistant to bortezomib, the combination of Nutlin-3/bortezomib synergistically induced cytotoxicity through the mitochondrial apoptotic pathway mediated by transcription-independent upregulation of NOXA, sequestration of MCL-1, activation of BAX, BAK, caspase-9 and -3. In the bortezomib sensitive wild-type TP53 MCL cells, the Nutlin-3/bortezomib combination caused G0/G1 cell cycle arrest followed by the increase in apoptosis induction. These findings indicate potential therapeutic efficacy of Nutlin-3/bortezomib combination for the treatment of chemorefractory MCL.

Topics: Antineoplastic Agents; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Blotting, Western; Boronic Acids; Bortezomib; Caspase 3; Caspase 9; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Drug Synergism; Humans; Imidazoles; Lymphoma, Mantle-Cell; Mitochondria; Mutation; Myeloid Cell Leukemia Sequence 1 Protein; Piperazines; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-mdm2; Pyrazines; Reverse Transcriptase Polymerase Chain Reaction; Tumor Suppressor Protein p53

Mantle cell lymphoma (MCL) has one of the poorest prognoses of the non-Hodgkin's lymphomas, and novel therapeutic approaches are needed. We wished to determine whether Nutlin-3, a novel small-molecule murine double minute 2 (MDM2) antagonist that efficiently activates TP53, might be effective in inducing cell death in MCL.. MCL cell lines with known TP53 status were treated with Nutlin-3, and biological and biochemical consequences were studied. Synergies with the prototypic genotoxic agent doxorubicin and the novel proteasome inhibitor bortezomib were assessed.. Nutlin-3 resulted in a reduction in cell proliferation/viability (IC50 < 10 micromol/L), an increase in the apoptotic fraction, and cell cycle arrest in wild-type (wt) TP53 Z-138 and Granta 519 cells. These effects were accompanied by TP53 accumulation and induction of TP53-dependent proteins p21, MDM2, Puma, and Noxa. Cell cycle arrest was characterized by suppression of S phase and an increase in the G0-G1 and G2-M fractions and accompanied by suppression of total and phosphorylated retinoblastoma protein and a decrease in G2-M-associated proteins cyclin B and CDC2. The combination of Nutlin-3 with doxorubicin or bortezomib was synergistic in wt-TP53 MCL cells. Nutlin-3 also induced cell cycle arrest and reduced cell viability in the mutant TP53 MINO cells but at a significantly higher IC50 (22.5 micromol/L). These effects were associated with induction of the TP53 homologue p73, slight increases in p21 and Noxa, and caspase activation. Nutlin-3 and bortezomib synergistically inhibited cell growth of MINO.. These findings suggest that the MDM2 antagonist Nutlin-3 may be an effective agent in the treatment of MCL with or without wt-TP53.

Topics: AMP-Activated Protein Kinases; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Boronic Acids; Bortezomib; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Doxorubicin; Humans; Imidazoles; Lymphoma, Mantle-Cell; Membrane Proteins; Nuclear Proteins; Piperazines; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; PTEN Phosphohydrolase; Pyrazines; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

Mantle cell lymphoma (MCL) is a clinically aggressive B-cell non-Hodgkin lymphoma characterized by the t(11;14)(q13;q32) and overexpression of cyclin D1. A high proportion of MCL tumors harbor wild-type (wt) and potentially functional p53 gene. We show here that stabilization and activation of wt-p53 using a recently developed potent MDM2 inhibitor, nutlin 3A, results in significant p53-dependent G1-S cell cycle arrest and apoptosis in MCL cells through regulation of p53 target genes. As mTOR signaling is activated in MCL and may control cyclin D1 levels, we show that p53 activation may downregulate the AKT/mTOR pathway through a mechanism involving AMP kinase (AMPK). Despite the non-genotoxic mode of nutlin 3A treatment, we show evidence that stabilization of p53 is associated with its phosphorylation at serine 15 residue and activation of AMPK. Stimulation of AMPK kinase activity using AICAR inhibits phosphorylation of critical downstream effectors of mTOR signaling, such as 4E-BP1 and rpS6. Pharmacologic inhibition of AMPK using compound C in nutlin-3A-treated MCL cells harboring wt-p53 did not affect the level of (ser15)p-p53, suggesting that the (ser15)p-p53 --> AMPK is the direction involved in the p53/AMPK/mTOR cross talk. These data establish a p53 --> AMPK --> mTOR mechanism in MCL and uncover a novel biologic effect of potent MDM2 inhibitors in preclinical models of MCL.

Topics: AMP-Activated Protein Kinase Kinases; Apoptosis; Down-Regulation; Imidazoles; Lymphoma, Mantle-Cell; Phosphorylation; Piperazines; Protein Kinases; Protein Stability; Proto-Oncogene Proteins c-mdm2; Receptor Cross-Talk; Signal Transduction; TOR Serine-Threonine Kinases; Tumor Cells, Cultured; Tumor Suppressor Protein p53