mk-2206 and Precursor-T-Cell-Lymphoblastic-Leukemia-Lymphoma

Calcineurin (Cn) is a calcium activated protein phosphatase involved in many aspects of normal T cell physiology, however the role of Cn and/or its downstream targets in leukemogenesis are still ill-defined. In order to identify putative downstream targets/effectors involved in the pro-oncogenic activity of Cn in T-cell acute lymphoblastic leukemia (T-ALL) we used tandem affinity chromatography, followed by mass spectrometry to purify novel Cn-interacting partners. We found the Cn-interacting proteins to be part of numerous cellular signaling pathways including eIF2 signaling and mTOR signaling. Coherently, modulation of Cn activity in T-ALL cells determined alterations in the phosphorylation status of key molecules implicated in protein translation such as eIF-2α and ribosomal protein S6. Joint targeting of PI3K-mTOR, eIF-2α and 14-3-3 signaling pathways with Cn unveiled novel synergistic pro-apoptotic drug combinations. Further analysis disclosed that the synergistic interaction between PI3K-mTOR and Cn inhibitors was prevalently due to AKT inhibition. Finally, we showed that the synergistic pro-apoptotic response determined by jointly targeting AKT and Cn pathways was linked to down-modulation of key anti-apoptotic proteins including Mcl-1, Claspin and XIAP. In conclusion, we identify AKT inhibition as a novel promising drug combination to potentiate the pro-apoptotic effects of Cn inhibitors.

Topics: Animals; Antineoplastic Agents; Apoptosis; Calcineurin; Calcineurin Inhibitors; Cell Line, Tumor; Cyclosporine; Heterocyclic Compounds, 3-Ring; Humans; Mice; Mice, Inbred NOD; Mice, SCID; NFATC Transcription Factors; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma; Proto-Oncogene Proteins c-akt; Ribosomal Protein S6 Kinases; Signal Transduction; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive neoplastic disorder in which chemotherapy resistance and refractory relapses occur, with a poorer prognostic outcome.Constitutively active PI3K/Akt/mTOR pathway is a common feature of T-ALL upregulating cell proliferation, survival and drug resistance. This pathway is currently under clinical trials with small molecules inhibitors (SMI).To verify whether a multi-inhibition treatment against Akt protein could enhance the efficacy of individual drug administration and overcome drug resistance as well as to obtain a decrease in single drug concentration, we tested on T-ALL cell lines the effects of combined treatments with three Akt inhibitors with different mode of action, GSK690693, MK-2206 and Perifosine.In cells with hyperactivated Akt, combined administration of the drugs displayed a significant synergistic and cytotoxic effect and affected PI3K/Akt/mTOR pathway at much lower concentration than single drug use. Highest synergistic effect for full inhibition of Akt was also related to the timing of every drug administration. Furthermore the triple treatment had greater efficacy in inducing cell cycle arrest in G0/G1 phase and both apoptosis and autophagy.Targeting Akt as a key protein of PI3K/Akt/mTOR pathway with multiple drugs might represent a new and promising pharmacological strategy for treatment of T-ALL patients.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Autophagy; Cell Cycle Checkpoints; Dose-Response Relationship, Drug; Drug Synergism; Enzyme Activation; Heterocyclic Compounds, 3-Ring; Humans; Jurkat Cells; Molecular Targeted Therapy; Oxadiazoles; Phosphatidylinositol 3-Kinase; Phosphorylation; Phosphorylcholine; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Signal Transduction; TOR Serine-Threonine Kinases

Clonal evolution and intratumoral heterogeneity drive cancer progression through unknown molecular mechanisms. To address this issue, functional differences between single T cell acute lymphoblastic leukemia (T-ALL) clones were assessed using a zebrafish transgenic model. Functional variation was observed within individual clones, with a minority of clones enhancing growth rate and leukemia-propagating potential with time. Akt pathway activation was acquired in a subset of these evolved clones, which increased the number of leukemia-propagating cells through activating mTORC1, elevated growth rate likely by stabilizing the Myc protein, and rendered cells resistant to dexamethasone, which was reversed by combined treatment with an Akt inhibitor. Thus, T-ALL clones spontaneously and continuously evolve to drive leukemia progression even in the absence of therapy-induced selection.

Topics: Animals; Animals, Genetically Modified; Antineoplastic Agents, Hormonal; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Clonal Evolution; Dexamethasone; Disease Progression; Drug Resistance, Neoplasm; Enzyme Activation; Genetic Variation; Heterocyclic Compounds, 3-Ring; Humans; Mechanistic Target of Rapamycin Complex 1; Molecular Sequence Data; Multiprotein Complexes; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-myc; T-Lymphocytes; TOR Serine-Threonine Kinases; Zebrafish

Topics: Animals; Dexamethasone; Disease Progression; Drug Resistance, Neoplasm; Heterocyclic Compounds, 3-Ring; Humans; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma; Proto-Oncogene Proteins c-akt; Zebrafish

Glucocorticoid resistance is a major driver of therapeutic failure in T cell acute lymphoblastic leukemia (T-ALL). Here, we identify the AKT1 kinase as a major negative regulator of the NR3C1 glucocorticoid receptor protein activity driving glucocorticoid resistance in T-ALL. Mechanistically, AKT1 impairs glucocorticoid-induced gene expression by direct phosphorylation of NR3C1 at position S134 and blocking glucocorticoid-induced NR3C1 translocation to the nucleus. Moreover, we demonstrate that loss of PTEN and consequent AKT1 activation can effectively block glucocorticoid-induced apoptosis and induce resistance to glucocorticoid therapy. Conversely, pharmacologic inhibition of AKT with MK2206 effectively restores glucocorticoid-induced NR3C1 translocation to the nucleus, increases the response of T-ALL cells to glucocorticoid therapy, and effectively reverses glucocorticoid resistance in vitro and in vivo.

Topics: Active Transport, Cell Nucleus; Animals; Dexamethasone; Drug Resistance, Neoplasm; Heterocyclic Compounds, 3-Ring; Humans; Mice; Phosphorylation; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Receptors, Glucocorticoid

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive neoplastic disorder arising from T-cell progenitors. T-ALL accounts for 15% of newly diagnosed ALL cases in children and 25% in adults. Although the prognosis of T-ALL has improved, due to the use of polychemotherapy schemes, the outcome of relapsed/chemoresistant T-ALL cases is still poor. A signaling pathway that is frequently upregulated in T-ALL, is the phosphatidylinositol 3-kinase/Akt/mTOR network. To explore whether Akt could represent a target for therapeutic intervention in T-ALL, we evaluated the effects of the novel allosteric Akt inhibitor, MK-2206, on a panel of human T-ALL cell lines and primary cells from T-ALL patients. MK-2206 decreased T-ALL cell line viability by blocking leukemic cells in the G(0)/G(1) phase of the cell cycle and inducing apoptosis. MK-2206 also induced autophagy, as demonstrated by an increase in the 14-kDa form of LC3A/B. Western blotting analysis documented a concentration-dependent dephosphorylation of Akt and its downstream targets, GSK-3α/β and FOXO3A, in response to MK-2206. MK-2206 was cytotoxic to primary T-ALL cells and induced apoptosis in a T-ALL patient cell subset (CD34(+)/CD4(-)/CD7(-)), which is enriched in leukemia-initiating cells. Taken together, our findings indicate that Akt inhibition may represent a potential therapeutic strategy in T-ALL.

Topics: Antineoplastic Agents; Apoptosis; Autophagy; Blotting, Western; Cell Cycle; Doxorubicin; Drug Synergism; Heterocyclic Compounds, 3-Ring; Humans; Phosphorylation; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Signal Transduction

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignant hematological disorder arising in the thymus from T-cell progenitors. T-ALL mainly affects children and young adults, and remains fatal in 20% of adolescents and 50% of adults, despite progress in polychemotherapy protocols. Therefore, innovative targeted therapies are desperately needed for patients with a dismal prognosis. Aberrant activation of PI3K/Akt/mTOR signaling is a common event in T-ALL patients and portends a poor prognosis. Preclinical studies have highlighted that modulators of PI3K/Akt/mTOR signaling could have a therapeutic relevance in T-ALL. However, the best strategy for inhibiting this highly complex signal transduction pathway is still unclear, as the pharmaceutical companies have disclosed an impressive array of small molecules targeting this signaling network at different levels. Here, we demonstrate that a dual PI3K/PDK1 inhibitor, NVP-BAG956, displayed the most powerful cytotoxic affects against T-ALL cell lines and primary patients samples, when compared with a pan class I PI3K inhibitor (GDC-0941), an allosteric Akt inhibitor (MK-2206), an mTORC1 allosteric inhibitor (RAD-001), or an ATP-competitive mTORC1/mTORC2 inhibitor (KU63794). Moreover, we also document that combinations of some of the aforementioned drugs strongly synergized against T-ALL cells at concentrations well below their respective IC50. This observation indicates that vertical inhibition at different levels of the PI3K/Akt/mTOR network could be considered as a future innovative strategy for treating T-ALL patients.

Topics: Adult; Caspase 3; Cell Line, Tumor; Child; Heterocyclic Compounds, 3-Ring; Humans; Imidazoles; Indazoles; Molecular Targeted Therapy; Morpholines; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Pyrimidines; Quinolines; Signal Transduction; Sulfonamides; T-Lymphocytes; TOR Serine-Threonine Kinases