sirolimus and Leukemia-Lymphoma--Adult-T-Cell

Adult T-cell leukemia (ATL) has a poor prognosis as a result of severe immunosuppression and rapid tumor progression with resistance to conventional chemotherapy. Recent integrated-genome analysis has revealed mutations in many genes involved in the T-cell signaling pathway, suggesting that the aberration of this pathway is an important factor in ATL pathogenesis and ATL-cell proliferation. We screened a siRNA library to examine signaling-pathway functionality and found that the PI3K/Akt/mTOR pathway is critical to ATL-cell proliferation. We therefore investigated the effect of mammalian target of rapamycin (mTOR) inhibitors, including the dual inhibitors PP242 and AZD8055 and the mTORC1 inhibitors rapamycin and everolimus, on human T-cell leukemia virus type 1 (HTLV-1)-infected-cell and ATL-cell lines. Both dual inhibitors inhibited the proliferation of all tested cell lines by inducing G1-phase cell-cycle arrest and subsequent cell apoptosis, whereas the effects of the 2 mTORC1 inhibitors were limited, as they did not induce cell apoptosis. In the ATL-cell lines and in the primary ATL samples, both dual inhibitors inhibited phosphorylation of AKT at serine-473, a target of mTORC2, as well as that of S6K, whereas the mTORC1 inhibitors only inhibited mTORC1. Furthermore, AZD8055 more significantly inhibited the in vivo growth of the ATL-cell xenografts than did everolimus. These results indicate that the PI3K/mTOR pathway is critical to ATL-cell proliferation and might thus be a new therapeutic target in ATL.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Cell Proliferation; Cell Survival; Everolimus; Humans; Indoles; Leukemia-Lymphoma, Adult T-Cell; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Mice; Morpholines; Phosphorylation; Purines; RNA, Small Interfering; Signal Transduction; Sirolimus; Xenograft Model Antitumor Assays

Torin2 is a second-generation ATP-competitive inhibitor of the mammalian target of rapamycin (mTOR). Dysregulation of mTOR signaling pathway, consisting of mTOR complexes mTORC1 and mTORC2, is a promising therapeutic target in some human malignancies. We examined antitumor effects of Torin2 in adult T-cell leukemia/lymphoma (ATL)-related cell lines compared to those of rapamycin, a classical mTOR inhibitor.. Cell growth was monitored by detecting viable cells with Cell Counting Kit-8 or trypan blue. Cell cycle was studied by flow cytometric analysis. The phosphorylation status of proteins in the mTOR signaling pathway was examined by western blot analysis.. Torin2 exhibited greater efficacy in cell growth inhibition than rapamycin, associated with a strong reduction of phosphorylated v-akt murine thymoma viral oncogene homolog (AKT) (Ser 473), that is downstream of mTORC2.. Since mTORC2 activates AKT, Torin2 might inhibit both mTORC1 and mTORC2, resulting in stronger growth inhibition of ATL cells.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Etoposide; G1 Phase Cell Cycle Checkpoints; Humans; Leukemia-Lymphoma, Adult T-Cell; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Multiprotein Complexes; Naphthyridines; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Topics: Adult; Drug Administration Schedule; Erythromycin; Feasibility Studies; Hematopoietic Stem Cell Transplantation; Human T-lymphotropic virus 1; Humans; Leukemia-Lymphoma, Adult T-Cell; Recurrence; Sirolimus

HTLV-I-associated adult T-cell leukemia/lymphoma (ATL) and human T-cell lymphotropic virus type I (HTLV-I)-negative peripheral T-cell lymphomas carry poor prognosis mainly because of acquired resistance to chemotherapy. We have shown that this disease is responsive to the combination of zidovudine and interferon-α. However, long-term maintenance therapy with this combination is associated with side effects affecting patient quality of life and hence more tolerated alternatives are needed. In this submission, we explored the effect of the mammalian target of rapamycin (mTOR) complex-1 (mTORC1) inhibitor everolimus (RAD001) on ATL and HTLV-negative malignant T-cell lines. We demonstrate that, at clinically achievable concentrations, long-term treatment with everolimus resulted in a dramatic inhibitory effect on the growth of HTLV-I-positive and -negative malignant T-cells, while normal resting or activated T-lymphocytes were resistant. Everolimus specifically induced oncoprotein Tax degradation and senescence in ATL cells and cell cycle arrest and apoptosis in HTLV-I-negative malignant T-cells. Everolimus-mediated apoptosis was also associated with an upregulation of p53 upregulated modulator of apoptosis (PUMA-α) proteins, an increase in Bax proteins and downregulation of Bcl-x(L) proteins in all tested HTLV-I-positive and -negative malignant cell lines. These results support a therapeutic role for everolimus, particularly as long-term maintenance therapy in patients with ATL and other HTLV-I-negative peripheral T-cell lymphomas.

Topics: Adult; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Cells, Cultured; Cellular Senescence; Everolimus; G1 Phase; Human T-lymphotropic virus 1; Humans; Leukemia-Lymphoma, Adult T-Cell; Lymphoma, T-Cell, Peripheral; Proto-Oncogene Proteins; Signal Transduction; Sirolimus; T-Lymphocytes; TOR Serine-Threonine Kinases; Tumor Suppressor Protein p53

Topics: Adult; Antibiotics, Antineoplastic; Cell Line, Tumor; Cell Proliferation; Human T-lymphotropic virus 1; Humans; Jurkat Cells; Leukemia-Lymphoma, Adult T-Cell; Phosphatidylinositol 3-Kinases; Protein Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Topics: Adult; Antibiotics, Antineoplastic; Cell Line, Tumor; Cell Proliferation; Human T-lymphotropic virus 1; Humans; Jurkat Cells; Leukemia-Lymphoma, Adult T-Cell; Phosphatidylinositol 3-Kinases; Protein Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; Time Factors; TOR Serine-Threonine Kinases

This study found that phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling was activated in human T-cell lymphotropic virus type I (HTLV-1)-infected leukemia cells. Rapamycin (1-100 nM, 48h), the inhibitor of mTOR and its analog RAD001 (1-100 nM, 48 h)-induced growth inhibition and G0/G1 cell cycle arrest of these cells in association with de-phosphorylation of p70S6K and 4E-BP-1, although IC50 was not achieved. Paradoxically, rapamycin-stimulated phosphorylation of Akt at Ser473. Blockade of Akt signaling by the PI3K inhibitor LY294002 (1-20 microM, 48 h) also resulted in the growth inhibition and G0/G1 cell cycle arrest of HTLV-1-infected cells, with IC50 ranging from 5 to 20muM, and it caused de-phosphorylation of p70S6K and 4E-BP-1. Of note, when rapamycin was combined with LY294002, rapamycin-induced phosphorylation of Akt was blocked, and the ability of rapamycin to induce growth arrest of HTLV-1-infected T-cells and suppress the p-p70S6K and p-4E-BP-1 proteins was potentiated. Moreover, both LY294002 and rapamycin down-regulated the levels of c-Myc and cyclin D1 proteins in these cells, and their combination further decreased levels of these cell cycle-regulating proteins. Taken together, longitudinal inhibition of PI3K/Akt/mTOR signaling represents a promising treatment strategy for individuals with adult T-cell leukemia.

Topics: Adaptor Proteins, Signal Transducing; Cell Cycle; Cell Cycle Proteins; Chromones; Cyclin D; Cyclins; Enzyme Inhibitors; Human T-lymphotropic virus 1; Humans; Immunosuppressive Agents; Leukemia-Lymphoma, Adult T-Cell; Morpholines; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphoproteins; Phosphorylation; Protein Kinases; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-myc; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; T-Lymphocytes; TOR Serine-Threonine Kinases; Tumor Cells, Cultured

Acute lymphoblastic leukemia (ALL) is the most common hematological malignancy affecting children. Despite significant progress and success in the treatment of ALL, a significant number of children continue to relapse and for them, outcome remains poor. Therefore, the search for novel therapeutic approaches is warranted. The aim of this study was to investigate the AMP activated protein kinase (AMPK) as a potential target in childhood acute lymphoblastic leukemia (ALL) subtypes characterized by non-random translocation signature profiles. We evaluated the effects of the AMPK activator AICAR on cell growth, cell cycle regulators and apoptosis of various childhood ALL cells.. We found that treatment with AICAR inhibited cell proliferation, induced cell cycle arrest in G1-phase, and apoptosis in CCRF-CEM (T-ALL), NALM6 (Bp-ALL), REH (Bp-ALL, TEL/AML1) and SupB15 (Bp-ALL, BCR/ABL) cells. These effects were abolished by treatment with the adenosine kinase inhibitor 5'-iodotubericidin prior to addition of AICAR indicating that AICAR's cytotoxicity is mediated through AMPK activation. Moreover, we determined that growth inhibition exerted by AICAR was associated with activation of p38-MAPK and increased expression of the cell cycle regulators p27 and p53. We also demonstrated that AICAR mediated apoptosis through the mitochondrial pathway as revealed by the release of cytochrome C and cleavage of caspase 9. Additionally, AICAR treatment resulted in phosphorylation of Akt suggesting that activation of the PI3K/Akt pathway may represent a compensatory survival mechanism in response to apoptosis and/or cell cycle arrest. Combined treatment with AICAR and the mTOR inhibitor rapamycin resulted in additive anti-proliferative activity ALL cells.. AICAR-mediated AMPK activation was found to be a proficient cytotoxic agent in ALL cells and the mechanism of its anti-proliferative and apoptotic effect appear to be mediated via activation of p38-MAPK pathway, increased expression of cell cycle inhibitory proteins p27 and p53, and downstream effects on the mTOR pathway, hence exhibiting therapeutic potential as a molecular target for the treatment of childhood ALL. Therefore, activation of AMPK by AICAR represents a novel approach to targeted therapy, and suggests a role for AICAR in combination therapy with inhibitors of the PI3K/Akt/mTOR pathways for the treatment of childhood in ALL.

Topics: Aminoimidazole Carboxamide; AMP-Activated Protein Kinases; Antineoplastic Agents; Apoptosis; Cell Division; DNA Replication; Drug Delivery Systems; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Enzyme Activation; G1 Phase; Humans; Imidazoles; Leukemia-Lymphoma, Adult T-Cell; Multienzyme Complexes; Neoplasm Proteins; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Prodrugs; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Pyridines; Ribonucleotides; Sirolimus; Tubercidin; Tumor Cells, Cultured

Notch signaling is believed to promote cell survival in general. However, the mechanism is not clearly understood. Here, we show that cells expressing intracellular domain of human Notch1 (NIC-1) are chemoresistant in a wild-type p53-dependent manner. NIC-1 inhibited p53 by inhibiting its activating phosphorylations at Ser(15), Ser(20), and Ser(392) as well as nuclear localization. In addition, we found that inhibition of p53 by NIC-1 mainly occurs through mammalian target of rapamycin (mTOR) using phosphatidylinositol 3-kinase (PI3K)-Akt/protein kinase B (PKB) pathway as the mTOR inhibitor, rapamycin treatment abrogated NIC-1 inhibition of p53 and reversed the chemoresistance. Consistent with this, rapamycin failed to reverse NIC-1-induced chemoresistance in cells expressing rapamycin-resistant mTOR. Further, ectopic expression of eukaryotic initiation factor 4E (eIF4E), a translational regulator that acts downstream of mTOR, inhibited p53-induced apoptosis and conferred protection against p53-mediated cytotoxicity to similar extent as that of NIC-1 overexpression but was not reversed by rapamycin, which indicates that eIF4E is the major target of mTOR in Notch1-mediated survival signaling. Finally, we show that MCF7 (breast cancer) and MOLT4 (T-cell acute lymphoblastic leukemia) cells having aberrant Notch1 signaling are chemoresistant, which can be reversed by both PI3K and mTOR inhibitors. These results establish that Notch1 signaling confers chemoresistance by inhibiting p53 pathway through mTOR-dependent PI3K-Akt/PKB pathway and imply that p53 status perhaps is an important determinant in combination therapeutic strategies, which use mTOR inhibitors and chemotherapy.

Topics: Breast Neoplasms; Cell Nucleus; Cell Survival; Drug Resistance, Neoplasm; Eukaryotic Initiation Factor-4E; Humans; Leukemia-Lymphoma, Adult T-Cell; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Kinases; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-akt; Receptor, Notch1; RNA, Small Interfering; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transfection; Tumor Suppressor Protein p53

The immunosuppressive macrolide rapamycin is used in humans to prevent graft rejection. This drug acts by selectively repressing the translation of proteins that are encoded by an mRNA bearing a 5'-polypyrimidine tract (e.g., ribosomal proteins, elongation factors). The human immunodeficiency virus type 1 (HIV-1) carries a polypyrimidine motif that is located within the tat exon 2. Treatment of human T lymphoid cells with rapamycin resulted in a marked diminution of HIV-1 transcription when infection was performed with luciferase reporter T-tropic and macrophage-tropic viruses. Replication of fully infectious HIV-1 particles was abolished by rapamycin treatment. The rapamycin-mediated inhibitory effect on HIV-1 production was reversed by FK506. The anti-HIV-1 effect of rapamycin was also seen in primary human cells (i.e., peripheral blood lymphocytes) from different healthy donors. Rapamycin was shown to diminish basal HIV-1 long terminal repeat gene expression, and the observed effect of rapamycin on HIV-1 replication seems to be independent of the virus-specific transactivating Tat protein. A constitutive beta-actin promoter-based reporter gene vector was unaffected by rapamycin treatment. Kinetic virus infection studies and exposure to reporter viruses pseudotyped with heterologous envelope proteins (i.e., amphotropic murine leukemia virus and vesicular stomatitis virus G) suggested that rapamycin is primarily affecting the life cycle of HIV-1 at a transcriptional level. Northern blot analysis confirmed that this compound is selectively targeting HIV-1 mRNA synthesis.

Topics: Actins; Blotting, Northern; Gene Products, tat; Genes, Reporter; HIV Long Terminal Repeat; HIV-1; Humans; Immunosuppressive Agents; Indicators and Reagents; Leukemia-Lymphoma, Adult T-Cell; Plasmids; RNA, Messenger; Sirolimus; Tacrolimus; tat Gene Products, Human Immunodeficiency Virus; Transfection; Tumor Cells, Cultured; Virus Replication

In normal T-cell development interleukin-7 (IL-7) functions as an antiapoptotic factor by regulating bcl-2 expression in immature thymocytes and mature T cells. Similar to what occurs in normal immature thymocytes, prevention of spontaneous apoptosis by IL-7 in precursor T-cell acute lymphoblastic leukemia (T-ALL) cells correlates with up-regulation of bcl-2. IL-7 is also implicated in leukemogenesis because IL-7 transgenic mice develop lymphoid malignancies, suggesting that IL-7 may regulate the generation and expansion of malignant cells. This study shows that in the presence of IL-7, T-ALL cells not only up-regulated bcl-2 expression and escaped apoptosis but also progressed in the cell cycle, resulting in sequential induction of cyclin D2 and cyclin A. Down-regulation of p27kip1 was mandatory for IL-7-mediated cell cycle progression and temporally coincided with activation of cyclin-dependent kinase (cdk)4 and cdk2 and hyperphosphorylation of Rb. Strikingly, forced expression of p27kip1 in T-ALL cells not only prevented cell cycle progression but also reversed IL-7-mediated up-regulation of bcl-2 and promotion of viability. These results show for the first time that a causative link between IL-7-mediated proliferation and p27kip1 down-regulation exists in malignant T cells. Moreover, these results suggest that p27kip1 may function as a tumor suppressor gene not only because it is a negative regulator of cell cycle progression but also because it is associated with induction of apoptosis of primary malignant cells.

Topics: Apoptosis; Cell Cycle; Cell Cycle Proteins; Cell Survival; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Cyclins; Depression, Chemical; Gene Expression Regulation, Leukemic; Genes, bcl-2; Genes, Tumor Suppressor; Humans; Immunophenotyping; Interleukin-7; Leukemia-Lymphoma, Adult T-Cell; Neoplasm Proteins; Neoplastic Stem Cells; Oligonucleotides, Antisense; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Sirolimus; Tumor Cells, Cultured; Tumor Suppressor Proteins

To determine activation status of the IL-2R-associated (Jak/STAT) pathway in the HTLV-I infected cells, we examined tyrosine phosphorylation of Jak3, STAT3, and STAT5 in several HTLV-I(+) T-cell lines and in uncultured leukemic T cells isolated from patients with adult T-cell lymphoma/leukemia (ATLL). Constitutive basal phosphorylation of Jak3 and, usually, STAT3 and STAT5 was detected in all four IL-2-independent cell lines tested, but in none of the three IL-2-dependent cell lines. Similarly, there was no detectable basal phosphorylation of Jak3 and STAT5 in the leukemic cells from ATLL patients (0/8 and 0/3, respectively). However, stimulation with IL-2 resulted in Jak3 and STAT5 phosphorylation in both leukemic ATLL cells and IL-2-dependent lines. Furthermore, expression of SHP-1 phosphatase which is a negative regulator of cytokine receptor signaling, was lost in most IL-2 independent cell lines (3/4) but not in the leukemic ATLL cells (0/3). Finally, the HTLV-I(+) T-cell lines (313) but not the control, HTLV-I(-) T-cell lines were resistant to rapamycin and its novel analog RAD. We conclude that (1) HTLV-I infection per se does not result in a constitutive phosphorylation of the Jak3, STAT3, and STAT5 proteins; (2) malignant transformation in at least some cases of ATLL does not require the constitutive, but may require IL-2-induced, activation of the IL-2R Jak/STAT pathway; and (3) there are major differences in T-cell immortalization mechanism(s) which appear to involve SHP-1 and target molecules for rapamycin and RAD.

Topics: DNA-Binding Proteins; HTLV-I Infections; Human T-lymphotropic virus 1; Humans; Immunosuppressive Agents; Interleukin-2; Intracellular Signaling Peptides and Proteins; Janus Kinase 3; Leukemia-Lymphoma, Adult T-Cell; Lymphocyte Activation; Milk Proteins; Phosphorylation; Protein Tyrosine Phosphatase, Non-Receptor Type 11; Protein Tyrosine Phosphatase, Non-Receptor Type 6; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Receptors, Interleukin-2; Sirolimus; STAT3 Transcription Factor; STAT5 Transcription Factor; T-Lymphocytes; Trans-Activators; Tumor Cells, Cultured