dactolisib and Precursor-T-Cell-Lymphoblastic-Leukemia-Lymphoma

The aim of our study is to evaluate the preclinical therapeutic activity and mechanism of action of BEZ235, a dual PI3K/mTOR inhibitor, in combination with dexamethasone in acute lymphoblastic leukemia (ALL).. The cytotoxic effects of BEZ235 and dexamethasone as single agents and in combination were assessed in a panel of ALL cell lines and xenograft models. The underlying mechanism of BEZ235 and dexamethasone was evaluated using immunoblotting, TaqMan RT-PCR, siRNA, immunohistochemistry, and immunoprecipitation.. Inhibition of the PI3K/AKT/mTOR pathway with the dual PI3K/mTOR inhibitor BEZ235 enhanced dexamethasone-induced anti-leukemic activity in in vitro (continuous cell lines and primary ALL cultures) and systemic in vivo models of T-ALL (including a patient-derived xenograft). Through inhibition of AKT1, BEZ235 was able to alleviate AKT1-mediated suppression of dexamethasone-induced apoptotic pathways leading to increased expression of the proapoptotic BCL-2 protein BIM. Downregulation of MCL-1 by BEZ235 further contributed to the modulation of dexamethasone resistance by increasing the amount of BIM available to induce apoptosis, especially in PTEN-null T-ALL where inhibition of AKT only partially overcame AKT-induced BIM suppression.. Our data support the further investigation of agents targeting the PI3K/mTOR pathway to modulate glucocorticoid resistance in T-ALL.

Topics: Animals; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Cell Line, Tumor; Cell Survival; Dexamethasone; Disease Models, Animal; Drug Resistance, Neoplasm; Drug Synergism; Female; Gene Expression Regulation, Neoplastic; Glucocorticoids; Humans; Imidazoles; Membrane Proteins; Myeloid Cell Leukemia Sequence 1 Protein; Phosphoinositide-3 Kinase Inhibitors; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma; Protein Kinase Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Quinolines; TOR Serine-Threonine Kinases; Tumor Burden; Xenograft Model Antitumor Assays

Constitutive activation of AKT is a frequent occurrence in the development of human T-cell acute lymphocytic leukemia/lymphomas (T-ALLs), due largely to inactivation of PTEN. Up regulation of MYC is also commonly observed in human T-ALLs. We previously demonstrated that expression of a constitutively active form of Lck-Akt2 alone is sufficient to initiate T-cell lymphoma in mice, and that tumor formation typically requires up regulation of Myc or Dlx5 caused by specific chromosomal rearrangements. Furthermore, Lck-Dlx5 mice develop T-ALLs that consistently acquire overexpression of Myc and activation of Akt, the latter due to loss of Pten expression. Proliferation of T-ALL cells from Lck-Dlx5 mice was found to be highly sensitive to the Akt pathway inhibitors BEZ235 and RAD001, as well as to JQ1, an inhibitor of bromodomain proteins, one of which (BRD4) regulates Myc transcription. Additionally, low concentrations of BEZ235 were found to cooperate with JQ1 to enhance cell cycle arrest. Higher concentrations of BEZ235 (≥0.5 µM) promoted cell death, although the addition of JQ1 did not result in a further increase in apoptosis. In contrast, the specific Myc inhibitor 10058-F4 caused apoptosis, and when combined with BEZ235 (≥0.5 µM), an enhanced effect on apoptosis was consistently observed. In addition, BEZ235 and RAD001 potentiated vincristine-induced apoptosis when the cells were treated with both drugs simultaneously, whereas pretreatment with BEZ235 antagonized the cell-killing effect of vincristine. Collectively, these experimental findings provide rationale for the design of novel combination therapies for T-ALL that includes targeting of AKT and MYC.

Topics: Animals; Apoptosis; Azepines; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cell Survival; Everolimus; Homeodomain Proteins; Imidazoles; Lymphocyte Specific Protein Tyrosine Kinase p56(lck); Mice; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-myc; Quinolines; Signal Transduction; Thiazoles; Transcription, Genetic; Triazoles; Vincristine

Treatment resistance in T-cell acute lymphoblastic leukemia (T-ALL) is associated with phosphatase and tensin homolog (PTEN) deletions and resultant phosphatidylinositol 3'-kinase (PI3K)-AKT pathway activation, as well as MYC overexpression, and these pathways repress mitochondrial apoptosis in established T-lymphoblasts through poorly defined mechanisms. Normal T-cell progenitors are hypersensitive to mitochondrial apoptosis, a phenotype that is dependent on the expression of proapoptotic BIM. In a conditional zebrafish model, MYC downregulation induced BIM expression in T-lymphoblasts, an effect that was blunted by expression of constitutively active AKT. In human T-ALL cell lines and treatment-resistant patient samples, treatment with MYC or PI3K-AKT pathway inhibitors each induced BIM upregulation and apoptosis, indicating that BIM is repressed downstream of MYC and PI3K-AKT in high-risk T-ALL. Restoring BIM function in human T-ALL cells using a stapled peptide mimetic of the BIM BH3 domain had therapeutic activity, indicating that BIM repression is required for T-ALL viability. In the zebrafish model, where MYC downregulation induces T-ALL regression via mitochondrial apoptosis, T-ALL persisted despite MYC downregulation in 10% of bim wild-type zebrafish, 18% of bim heterozygotes and in 33% of bim homozygous mutants (P=0.017). We conclude that downregulation of BIM represents a key survival signal downstream of oncogenic MYC and PI3K-AKT signaling in treatment-resistant T-ALL.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Cell Line, Tumor; Humans; Imidazoles; Membrane Proteins; MicroRNAs; Phosphatidylinositol 3-Kinases; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-myc; Quinolines; Signal Transduction; Zebrafish

Inhibition of signal transduction pathways has been successfully introduced into cancer treatment. The dual phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) inhibitor NVP-BEZ235 has antitumor activity in vitro against solid tumors. Here, we examined the activity of NVP-BEZ235 in acute lymphoblastic leukemia (ALL) cells and the best modalities for combination approaches.. ALL cell lines (SEM, RS4;11, Jurkat and MOLT4) were treated with NVP-BEZ235 alone, or in combination with cytarabine (AraC), doxorubicin (Doxo) or dexamethasone (Dexa).. NVP-BEZ235 potently inhibited the proliferation and metabolic activity of ALL cells. Antiproliferative effects were associated with G(0)/G(1) arrest and reduced levels of cyclin-dependent kinase 4 (CDK4) and cyclin D3. Inhibition of PI3K and mTOR activity was detected at 10 and 100 nM. NVP-BEZ235 combined with AraC, Doxo or Dexa synergistically enhanced the cytotoxicity compared to single-drug treatment, even in glucocorticoid-resistant cells.. NVP-BEZ235 displays pronounced antiproliferative effects in ALL cells and might therefore be a useful drug in the treatment of ALL.

Topics: Antineoplastic Combined Chemotherapy Protocols; Cell Cycle Checkpoints; Cell Growth Processes; Cell Line, Tumor; Cytarabine; Dexamethasone; Doxorubicin; Drug Resistance, Neoplasm; Drug Synergism; Humans; Imidazoles; Jurkat Cells; Leukemia, B-Cell; Oncogene Protein v-akt; Phosphatidylinositol 3-Kinase; Phosphoinositide-3 Kinase Inhibitors; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma; Quinolines; Signal Transduction; TOR Serine-Threonine Kinases

Recent findings have highlighted that constitutively active phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling is a common feature of T-cell acute lymphoblastic leukemia (T-ALL), where it upregulates cell proliferation, survival, and drug resistance. These observations lend compelling weight to the application of PI3K/Akt/mTOR inhibitors in the therapy of T-ALL. Here, we have analyzed the therapeutic potential of the novel dual PI3K/mTOR inhibitor NVP-BEZ235, an orally bioavailable imidazoquinoline derivative, which has entered clinical trials for solid tumors, on both T-ALL cell lines and patient samples. NVP-BEZ235 was cytotoxic to a panel of T-ALL cell lines as determined by MTT assays. NVP-BEZ235 treatment resulted in cell cycle arrest and apoptosis. Western blots showed a dose- and time-dependent dephosphorylation of Akt and mTORC1 downstream targets in response to NVP-BEZ235. Remarkably, NVP-BEZ235 targeted the side population of both T-ALL cell lines and patient lymphoblasts, which might correspond to leukemia-initiating cells, and synergized with chemotherapeutic agents (cyclophosphamide, cytarabine, dexamethasone) currently used for treating T-ALL patients. NVP-BEZ235 reduced chemoresistance to vincristine induced in Jurkat cells by coculturing with MS-5 stromal cells, which mimic the bone marrow microenvironment. NVP-BEZ235 was cytotoxic to T-ALL patient lymphoblasts displaying pathway activation, where the drug dephosphorylated eukaryotic initiation factor 4E-binding protein 1, at variance with rapamycin. Taken together, our findings indicate that longitudinal inhibition at two nodes of the PI3K/Akt/mTOR network with NVP-BEZ235, either alone or in combination with chemotherapeutic drugs, may be an efficient treatment of those T-ALLs that have aberrant upregulation of this signaling pathway for their proliferation and survival.

Topics: Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Autophagy; Cell Cycle; Cell Division; Cell Line, Tumor; Cell Survival; Coculture Techniques; Flow Cytometry; Humans; Imidazoles; Jurkat Cells; Mice; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma; Quinolines; Stromal Cells