sirolimus and Leukemia--Myelogenous--Chronic--BCR-ABL-Positive

Chronic myeloid leukemia (CML) is a stem cell disease characterized by excessive accumulation of clonal myeloid (precursor) cells in hematopoietic tissues. CML cells display the translocation t(9; 22) that creates the bcr/abl oncogene. The respective oncoprotein (= BCR/ABL) exhibits constitutive tyrosine kinase activity and promotes growth and survival in CML cells. Clinically, CML can be divided into three phases: the chronic phase (CP), the accelerated phase (AP), and the blast phase (BP) that resembles acute leukemia. Progression to AP and BP is associated with occurrence of additional genetic defects that cooperate with bcr/abl in leukemogenesis and lead to resistance against antileukemic drugs. The prognosis in CML is variable depending on the phase of disease, age, and response to therapy. The only curative approach available to date is stem cell transplantation. For those who cannot be transplanted, the BCR/ABL tyrosine kinase inhibitor STI571 (Glivec, Imatinib), interferon-alpha (with or without ARAC), or other cytoreductive drugs are prescribed. Currently available data show that STI571 is a superior compound compared to other drugs in producing complete cytogenetic and molecular responses. However, despite superior initial data and high expectations for an effect on survival, long term results are not available so far, and resistance against STI571 has been reported. Forthcoming strategies are therefore attempting to prevent or counteract STI571 resistance by co-administration of other antileukemic drugs. Whether these strategies will lead to curative drug therapy in CML in the future remains at present unknown.

Topics: Adult; Aged; Aged, 80 and over; Antibiotics, Antineoplastic; Antimetabolites, Antineoplastic; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Benzamides; Bone Marrow Examination; Clinical Trials as Topic; Cytarabine; Diagnosis, Differential; Drug Resistance; Enzyme Inhibitors; Female; Fusion Proteins, bcr-abl; Humans; Imatinib Mesylate; Immunophenotyping; Interferon-alpha; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Middle Aged; Multivariate Analysis; Piperazines; Prognosis; Pyrimidines; Risk Factors; Sirolimus; Stem Cell Transplantation; Time Factors

A phase 3 clinical trial (BMT CTN 0402) comparing tacrolimus/sirolimus (Tac/Sir) vs tacrolimus/methotrexate (Tac/Mtx) as graft-versus-host disease (GVHD) prophylaxis after matched-related allogeneic hematopoietic cell transplantation (HCT) recently showed no difference between study arms in acute GVHD-free survival. Within this setting of a prospective, multicenter study with uniform GVHD prophylaxis, conditioning regimen, and donor source, we explored the correlation of 10 previously identified biomarkers with clinical outcomes after allogeneic HCT. We measured biomarkers from plasma samples collected in 211 patients using enzyme-linked immunosorbent assay (Tac/Sir = 104, Tac/Mtx = 107). High suppression of tumorigenicity-2 (ST2) and T-cell immunoglobulin mucin-3 (TIM3) at day 28 correlated with 2-year nonrelapse mortality in multivariate analysis (P = .0050, P = .0075, respectively) and in a proportional hazards model with time-dependent covariates (adjusted hazard ratio: 2.43 [1.49-3.95], P = .0038 and 4.87 [2.53-9.34], P < .0001, respectively). High ST2 and TIM3 correlated with overall survival. Chemokine (C-X-C motif) ligand 9 (CXCL9) levels above the median were associated with chronic GVHD compared with levels below the median in a time-dependent proportional hazard analysis (P = .0069). Low L-Ficolin was associated with hepatic veno-occlusive disease (P = .0053, AUC = 0.80). We confirmed the correlation of plasma-derived proteins, previously assessed in single-center cohorts, with clinical outcomes after allogeneic HCT within this prospective, multicenter study.

Topics: Adolescent; Adult; Allografts; Area Under Curve; Biomarkers, Tumor; Drug Therapy, Combination; Enzyme-Linked Immunosorbent Assay; Female; Graft vs Host Disease; Hematopoietic Stem Cell Transplantation; Humans; Immunosuppressive Agents; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid, Acute; Male; Methotrexate; Middle Aged; Myelodysplastic Syndromes; ROC Curve; Sensitivity and Specificity; Sirolimus; Tacrolimus; Transplantation, Homologous; Young Adult

Hypoxia is a critical condition that governs survival, self-renewal, quiescence, metabolic shift and refractoriness to leukemic stem cell (LSC) therapy. The present study aims to investigate the hypoxia-driven regulation of the mammalian Target of the Rapamycin-2 (mTORC2) complex to unravel it as a novel potential target in chronic myeloid leukemia (CML) therapeutic strategies. After inducing hypoxia in a CML cell line model, we investigated the activities of mTORC1 and mTORC2. Surprisingly, we detected a significant activation of mTORC2 at the expense of mTORC1, accompanied by the nuclear localization of the main substrate phospho-Akt (Ser473). Moreover, the Gene Ontology analysis of CML patients' CD34+ cells showed enrichment in the mTORC2 signature, further strengthening our data. The deregulation of mTOR complexes highlights how hypoxia could be crucial in CML development. In conclusion, we propose a mechanism by which CML cells residing under a low-oxygen tension, i.e., in the leukemia quiescent LSCs, singularly regulate the mTORC2 and its downstream effectors.

Topics: Chronic Disease; Humans; Hypoxia; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Sirolimus; Stem Cells; TOR Serine-Threonine Kinases

Topics: Angiomyolipoma; Brain; Brain Neoplasms; Female; Humans; Immunosuppressive Agents; Kidney Neoplasms; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Magnetic Resonance Imaging; Multiple Pulmonary Nodules; Neoplasms, Multiple Primary; Rhabdomyoma; Sirolimus; Skin Neoplasms; Tomography, X-Ray Computed; Tuberous Sclerosis; Tuberous Sclerosis Complex 1 Protein; Tuberous Sclerosis Complex 2 Protein; Young Adult

The gradual emerging of resistance to imatinib urgently calls for the development of new therapy for chronic myeloid leukemia (CML). The fusion protein Bcr-Abl, which promotes the malignant transformation of CML cells, is mainly located in the cytoplasm, while the c-Abl protein which is expressed in the nucleus can induce apoptosis. Based on the hetero-dimerization of FKBP (the 12-kDa FK506- and rapamycin-binding protein) and FRB (the FKBP-rapamycin binding domain of the protein kinase, mTOR) mediated by AP21967, we constructed a nuclear transport system to induce cytoplasmic Bcr-Abl into nuclear. In this study, we reported the construction of the nuclear transport system, and we demonstrated that FN3R (three nuclear localization signals were fused to FRBT2098L with a FLAG tag), HF2S (two FKBP domains were in tandem and fused to the SH2 domain of Grb2 with an HA tag) and Bcr-Abl form a complexus upon AP21967. Bcr-Abl was imported into the nucleus successfully by the nuclear transport system. The nuclear transport system inhibited CML cell proliferation through mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 5 (STAT5) pathways mainly by HF2S. It was proven that nuclear located Bcr-Abl induced CML cell (including imatinib-resistant K562G01 cells) apoptosis by activation of p73 and its downstream molecules. In summary, our study provides a new targeted therapy for the CML patients even with Tyrosine Kinase Inhibitor (TKI)-resistance.

Topics: Apoptosis; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Drug Resistance, Neoplasm; Fusion Proteins, bcr-abl; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; MAP Kinase Signaling System; Nuclear Localization Signals; Protein Transport; Sirolimus; STAT5 Transcription Factor; Tyrosine

Kindlins are FERM-containing cytoplasmic proteins that regulate integrin-mediated cell-cell and cell-extracellular matrix (ECM) attachments. Kindlin-3 is expressed in hematopoietic cells, platelets, and endothelial cells. Studies have shown that kindlin-3 stabilizes cell adhesion mediated by ß1, ß2, and ß3 integrins. Apart from integrin cytoplasmic tails, kindlins are known to interact with other cytoplasmic proteins. Here we demonstrate that kindlin-3 can associate with ribosome via the receptor for activated-C kinase 1 (RACK1) scaffold protein based on immunoprecipitation, ribosome binding, and proximity ligation assays. We show that kindlin-3 regulates c-Myc protein expression in the human chronic myeloid leukemia cell line K562. Cell proliferation was reduced following siRNA reduction of kindlin-3 expression and a significant reduction in tumor mass was observed in xenograft experiments. Mechanistically, kindlin-3 is involved in integrin α5ß1-Akt-mTOR-p70S6K signaling; however, its regulation of c-Myc protein expression could be independent of this signaling axis.

Topics: Animals; Antibiotics, Antineoplastic; Cell Proliferation; GTP-Binding Proteins; HEK293 Cells; Human Umbilical Vein Endothelial Cells; Humans; Integrin beta3; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, Knockout; Neoplasm Proteins; Protein Binding; Proto-Oncogene Proteins c-myc; Receptors for Activated C Kinase; Receptors, Cell Surface; Recombinant Proteins; Ribosomes; RNA Interference; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

A small population of cancer stem cells named the "side population" (SP) has been demonstrated to be responsible for the persistence of many solid tumors. However, the role of the SP in leukemic pathogenesis remains controversial. The resistance of leukemic stem cells to targeted therapies, such as tyrosine kinase inhibitors (TKIs), results in therapeutic failure or refractory/relapsed disease in chronic myeloid leukemia (CML). The drug pump, ATP-binding cassette sub-family G member 2 (ABCG2), is well known as a specific marker of the SP and could be controlled by several pathways, including the PI3K/Akt pathway. Our data demonstrated that compared with wild-type K562 cells, the higher percentage of ABCG2+ cells corresponded to the higher SP fraction in K562/ABCG2 (ABCG2 overexpressing) and K562/IMR (resistance to imatinib) cells, which exhibited enhanced drug resistance along with downregulated phosphatase and tensin homologue deleted on chromosome -10 (PTEN) and activated phosphorylated-Akt (p-Akt). PTEN and p-Akt downregulation could be abrogated by both the PI3K inhibitor LY294002 and the mTOR inhibitor rapamycin. Moreover, in CML patients in the accelerated phase/blastic phase (AP/BP), increased SP phenotype rather than ABCG2 expression was accompanied by the loss of PTEN protein and the up-regulation of p-Akt expression. These results suggested that the expression of ABCG2 and the SP may be regulated by PTEN through the PI3K/Akt pathway, which would be a potentially effective strategy for targeting CML stem cells.

Topics: Adult; Aged; Apoptosis; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Blotting, Western; Cell Survival; Chromones; Enzyme Inhibitors; Female; Humans; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Middle Aged; Mitoxantrone; Morpholines; Neoplasm Proteins; Neoplastic Stem Cells; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Side-Population Cells; Signal Transduction; Sirolimus; Young Adult

Chronic myelogenous leukemia is a neoplasm of myeloid progenitor cells. We recently found that rapamycin could induce G0/G1 phase arrest and apoptosis and inhibit proliferation of K562 cells through inhibiting mammalian target of rapamycin (mTOR) pathway. However, whether rapamycin has synergistic effects with other drugs in chronic myelogenous leukemia (CML) therapies remain unclear. Therefore, we examined the effect of rapamycin combined with celecoxib on K562 cells in vitro. The survival rates showed a significant decrease in rapamycin + celecoxib treatment group. The combination treatment also increased the G0/G1 phase cells as compared to rapamycin or celecoxib treatment alone (P < 0.05), accompanied with the decreased population of S phase cells. Meanwhile, the rate of apoptosis was 15.87 ± 2.21 % in rapamycin + celecoxib treatment group, significantly higher than that in mono treatment group (P < 0.05). Western blot and reverse transcription PCR (RT-PCR) analysis showed that the expressions of mTOR, 4E-BP1, and p70S6K were all significantly decreased in K562 cells after rapamycin + celecoxib treatment (P < 0.05). In conclusion, rapamycin combined with celecoxib could induce cell cycle arrest and apoptosis and decrease the expressions of mTOR, 4E-BP1, and p70S6K. It suggested that the combination could enhance the antitumor effects of mono treatment on CML cells through downregulating mTOR pathway.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Celecoxib; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Phosphorylation; Pyrazoles; Signal Transduction; Sirolimus; Sulfonamides; TOR Serine-Threonine Kinases

Topics: Adult; Benzamides; Drug Therapy, Combination; Humans; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Piperazines; Pyrimidines; Sirolimus

In chronic myelogenous leukemia, the constitutive activation of the BCR-ABL kinase transforms cells to an addicted state that requires glucose metabolism for survival. We investigated S6K1, a protein kinase that drives glycolysis in leukemia cells, as a target for counteracting glucose-dependent survival induced by BCR-ABL. BCR-ABL potently activated S6K1-dependent signaling and glycolysis. Although S6K1 knockdown or rapamycin treatment suppressed glycolysis in BCR-ABL-transformed cells, these treatments did not induce cell death. Instead, loss of S6K1 triggered compensatory activation of fatty-acid oxidation, a metabolic program that can support glucose-independent cell survival. Fatty-acid oxidation in response to S6K1 inactivation required the expression of the fatty-acid transporter carnitine palmitoyl transferase 1c, which was recently linked to rapamycin resistance in cancer. Finally, addition of an inhibitor of fatty-acid oxidation significantly enhanced cytotoxicity in response to S6K1 inactivation. These data indicate that S6K1 dictates the metabolic requirements mediating BCR-ABL survival and provide a rationale for combining targeted inhibitors of signal transduction, with strategies to interrupt oncogene-induced metabolism.

Topics: Animals; Carnitine O-Palmitoyltransferase; Cell Death; Cell Line, Tumor; Cell Survival; Fatty Acid Transport Proteins; Fatty Acids; Fusion Proteins, bcr-abl; Glucose; Glycolysis; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mice; Mice, Inbred C57BL; Oxidation-Reduction; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus

Chronic myelogenous leukemia (CML) is a neoplasm of myeloid progenitor cells expressing Bcr-Abl fusion protein. However, some patients with CML are less likely to respond to imatinib, the inhibitor of Bcr-Abl kinase. Recent studies showed that mTOR pathway can increase responses to imatinib. The analysis of mTOR pathway in CML may provide new insights into possible targets of novel therapies. Therefore, we examined the expression of mTOR pathway molecules in bone marrow cells from CML patients and effect of rapamycin on K562 cells in vitro. Western blot analysis showed the visibly higher phosphorylation of mTOR (70.6%), 4E-BP1 (76.5%) and p70S6K (73.5%) in bone marrow cells from CML patients. Moreover, treatment of CML cell line (K562) with rapamycin resulted in a decrease of phosphorylation of mTOR, 4E-BP1 and p70S6K. In vitro, the cell viability in groups with rapamycin treatment displayed a significant decrease in a dose-dependent manner by MTT. The data presented an increase of G0/G1 phase cells and decrease of S phase cells after rapamycin treatment, and the decreased expression of cyclinD1, higher expression of p21 at mRNA level was also detected in K562 with rapamycin. Treatment with 20 nmol/l or more rapamycin could increase apoptotic cells, decrease expression of bcl-2 and activate caspase-3. In conclusion, the mTOR pathway might be involved in chronic myelogenous leukemia. Inhibition of mTOR pathway could interfer with cell proliferation and increase cell apoptosis in K562 cells. It suggested that mTOR might be an important therapeutic target for myelogenous leukemia.

Topics: Adult; Aged; Antibiotics, Antineoplastic; Apoptosis; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Female; Humans; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Middle Aged; Phosphorylation; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transcription, Genetic

To explore the inhibitory effects of rapamycin on proliferation of chronic myelogenous leukemia (CML) cells and its possible mechanism.. The effects of rapamycin at various concentrations on cell proliferation of CML cell line K562 cells were analyzed by MTT. The expressions of mTOR, 4E-BP1 and p70S6K at protein and mRNA level in K562 cells with rapamycin treatment were detected by Western blot and RT-PCR. The protein expressions and phosphorylation of mTOR, 4E-BP1 and p70S6K in primary bone marrow cells from CML patients at chronic phase (CP) were also investigated by Western blot, bone marrow cells from healthy people were used as control. Data were analyzed by the χ(2) test, Fisher's exact test and one-way analysis of variance (ANOVA).. The phosphorylation of mTOR, 4E-BP1 and p70S6K were significantly increased in CML bone marrow cells compared with that of normal control (70.6% vs 30.0%, 76.5% vs 40.0%, 73.5% vs 20.0%, respectively, P < 0.05). The proliferation of K562 cells was significantly inhibited with 20 nmol/L and more rapamycin treatment. The phosphorylation of mTOR was decreased after rapamycin treatment, as well as the expressions of 4E-BP1 and p70S6K at protein and mRNA level (P < 0.05).. mTOR signaling played an important role in CML pathogenesis, and rapamycin could decrease CML cells proliferation by inhibiting the activity of mTOR signaling in vitro.

Topics: Case-Control Studies; Cell Proliferation; Humans; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Phosphorylation; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Chimeric Bcr-Abl oncoprotein is the molecular hallmark of chronic myeloid leukemia (CML) and hence a lucrative target for therapeutic intervention of CML.However, limited efficacy of current first line treatment for CML calls attention for further development of more efficient strategies. Recently, much attention has been given to nanoparticle (NP) based drug delivery systems loaded with dual drugs to improve current disease therapies by overcoming toxicity and other side effects associated with high doses of single drugs. In the present study, we document to explore an approach to simultaneously deliver two drugs at target sites (i.e. Bcr-Abl oncoprotein) using poly (lactide-co-glycolide) (PLGA) nanoparticles. Preliminary study included screening six different anticancer drugs and their nanoformulations on leukemia cells. Results confirmed superlative antileukemic activity of paclitaxel (especially in formulations) on model cell line K562, but only upon longer exposure. Thus to lower time of action of such a potent drug, different drug combination were experimented taking the advantage of synergistic action of both the drugs. Evaluation at molecular and genetic level helped to identify signaling pathways upstream and downstream of Bcr-Abl, leading to its suppression. Results helped to illustrate dynamic changes primarily involved in inducing apoptotic activities on drug exposure of leukemia cells, thereby facilitating us to integrate different drug combinations in a more specific manner in near future to study CML in clinical settings.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Curcumin; Drug Synergism; Humans; Lactic Acid; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Membrane Potential, Mitochondrial; Nanoparticles; Paclitaxel; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Polymerase Chain Reaction; Sirolimus

The oncoprotein BCR-ABL transforms myeloid progenitor cells and is responsible for the development of chronic myeloid leukemia (CML). In transformed cells, BCR-ABL suppresses apoptosis as well as autophagy, a catabolic process in which cellular components are degraded by the lysosomal machinery. The mechanism by which BCR-ABL suppresses autophagy is not known. Here we report that in both mouse and human BCR-ABL-transformed cells, activating transcription factor 5 (ATF5), a prosurvival factor, suppresses autophagy but does not affect apoptosis. We find that BCR-ABL, through PI3K/AKT/FOXO4 signaling, transcriptionally up-regulates ATF5 expression and that ATF5, in turn, stimulates transcription of mammalian target of rapamycin (mTOR; also called mechanistic target of rapamycin), a well-established master negative-regulator of autophagy. Previous studies have shown that the BCR-ABL inhibitor imatinib mesylate induces both apoptosis and autophagy, and that the resultant autophagy modulates the efficiency by which imatinib kills BCR-ABL-transformed cells. We demonstrate that imatinib-induced autophagy is because of inhibition of the BCR-ABL/PI3K/AKT/FOXO4/ATF5/mTOR pathway that we have identified in this study.

Topics: Activating Transcription Factors; Animals; Antineoplastic Agents; Autophagy; Benzamides; Blotting, Western; Cell Transformation, Neoplastic; Chromatin Immunoprecipitation; Fusion Proteins, bcr-abl; Gene Expression Regulation, Neoplastic; Humans; Imatinib Mesylate; Immunosuppressive Agents; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Luciferases; Mice; Phosphatidylinositol 3-Kinases; Phosphorylation; Piperazines; Promoter Regions, Genetic; Proto-Oncogene Proteins c-akt; Pyrimidines; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sirolimus; TOR Serine-Threonine Kinases; Transcription, Genetic

Topics: Adult; Antibiotics, Antineoplastic; Benzamides; Blast Crisis; Drug Resistance, Neoplasm; Humans; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Piperazines; Pyrimidines; Sirolimus

Constitutive tyrosine kinase (TK) activity of p210 BCR-ABL fusion protein of chronic myeloid leukemia (CML) usurps physiological functions of normal p145 c-ABL protein. Accordingly, its inhibition by imatinib mesylate (IM) lets p145 c-ABL translocate into the nuclear compartment, which drives cell growth arrest and apoptotic death. Here we show that IM and the mammalian target of rapamycin (mTOR) inhibitor RAD001 (Everolimus) have additive effects on BCR-ABL-expressing cells. Those effects are at least partly conditional upon the enhanced nuclear accumulation of p145 c-ABL through events encompassing post-translational modifications of p145 c-ABL (Thr(735) phosphorylation) precluding its nuclear export and of 14-3-3 sigma (Ser(186) phosphorylation by c-Jun N-terminal kinase [JNK]) promoting p145 c-ABL nuclear re-import.

Topics: Antineoplastic Combined Chemotherapy Protocols; Benzamides; Blotting, Western; Cell Line, Tumor; Cell Nucleus; Cell Separation; Everolimus; Flow Cytometry; Gene Expression; Humans; Imatinib Mesylate; Intracellular Signaling Peptides and Proteins; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Microscopy, Confocal; Piperazines; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Protein Transport; Proto-Oncogene Proteins c-abl; Pyrimidines; Sirolimus; TOR Serine-Threonine Kinases

The mammalian target of rapamycin (mTOR) is one target of BCR-ABL fusion gene of chronic myeloid leukemia (CML). Moreover, it drives a compensatory route to Imatinib mesylate (IM) possibly involved in the progression of leukemic progenitors towards a drug-resistant phenotype. Accordingly, mTOR inhibitors are proposed for combined therapeutic strategies in CML. The major caveat in the use of mTOR inhibitors for cancer therapy comes from the induction of an mTOR-phosphatidylinositol 3 kinase (PI3k) feedback loop driving the retrograde activation of Akt. Here we show that the rapamycin derivative RAD 001 (everolimus, Novartis Institutes for Biomedical Research) inhibits mTOR and, more importantly, revokes mTOR late re-activation in response to IM. RAD 001 interferes with the assembly of both mTOR complexes: mTORC1 and mTORC2. The inhibition of mTORC2 results in the de-phosphorylation of Akt at Ser(473) in the hydrophobic motif of C-terminal tail required for Akt full activation and precludes Akt re-phosphorylation in response to IM. Moreover, RAD 001-induced inhibition of Akt causes the de-phosphorylation of tuberous sclerosis tumor suppressor protein TSC2 at 14-3-3 binding sites, TSC2 release from 14-3-3 sigma (restoring its inhibitory function on mTORC1) and nuclear import (promoting the nuclear translocation of cyclin-dependent kinase [CDK] inhibitor p27(Kip1), the stabilization of p27(Kip1) ligand with CDK2, and the G(0)/G(1) arrest). RAD 001 cytotoxicity on cells not expressing the BCR-ABL fusion gene or its p210 protein tyrosine kinase (TK) activity suggests that the inhibition of normal hematopoiesis may represent a drug side effect.

Topics: 14-3-3 Proteins; Active Transport, Cell Nucleus; Animals; Antineoplastic Agents; Benzamides; Biomarkers, Tumor; Cell Line; Cell Proliferation; Drug Interactions; Drug Resistance; Everolimus; Exonucleases; Exoribonucleases; Fusion Proteins, bcr-abl; Hematopoiesis; Imatinib Mesylate; Intracellular Signaling Peptides and Proteins; Lethal Dose 50; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mechanistic Target of Rapamycin Complex 1; Mice; Multiprotein Complexes; Myeloid Progenitor Cells; Neoplasm Proteins; Phosphorylation; Piperazines; Protein Serine-Threonine Kinases; Proteins; Proto-Oncogene Proteins c-akt; Pyrimidines; Sirolimus; TOR Serine-Threonine Kinases; Trans-Activators; Transcription Factors; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins

mTOR-generated signals play critical roles in growth of leukemic cells by controlling mRNA translation of genes that promote mitogenic responses. Despite extensive work on the functional relevance of rapamycin-sensitive mTORC1 complexes, much less is known on the roles of rapamycin-insensitive (RI) complexes, including mTORC2 and RI-mTORC1, in BCR-ABL-leukemogenesis. We provide evidence for the presence of mTORC2 complexes in BCR-ABL-transformed cells and identify phosphorylation of 4E-BP1 on Thr37/46 and Ser65 as RI-mTORC1 signals in primary chronic myelogenous leukemia (CML) cells. Our studies establish that a unique dual mTORC2/mTORC1 inhibitor, OSI-027, induces potent suppressive effects on primitive leukemic progenitors from CML patients and generates antileukemic responses in cells expressing the T315I-BCR-ABL mutation, which is refractory to all BCR-ABL kinase inhibitors currently in clinical use. Induction of apoptosis by OSI-027 appears to negatively correlate with induction of autophagy in some types of BCR-ABL transformed cells, as shown by the induction of autophagy during OSI-027-treatment and the potentiation of apoptosis by concomitant inhibition of such autophagy. Altogether, our studies establish critical roles for mTORC2 and RI-mTORC1 complexes in survival and growth of BCR-ABL cells and suggest that dual therapeutic targeting of such complexes may provide an approach to overcome leukemic cell resistance in CML and Ph+ ALL.

Topics: Apoptosis; Cellular Structures; Fusion Proteins, bcr-abl; Humans; Intracellular Signaling Peptides and Proteins; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mutation; Phosphorylation; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Topics: Benzamides; Dasatinib; Drug Delivery Systems; ErbB Receptors; Hemorrhage; Humans; Imatinib Mesylate; Interdisciplinary Communication; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Lung; Piperazines; Protein Kinase Inhibitors; Protein Kinases; Pyrimidines; Sirolimus; Skin; Thiazoles; TOR Serine-Threonine Kinases; Vascular Endothelial Growth Factor A

Rapamycin, an inhibitor of mammalian target of rapamycin kinase, is a potent immunosuppressive drug that also displays antineoplastic properties and expands regulatory T cells. Steroid-refractory acute graft-versus-host disease (GVHD) remains a significant cause of mortality after allogeneic stem-cell transplantation and therapeutic options are not codified. We retrospectively evaluated the role of rapamycin in this setting.. In this retrospective single-center study, 22 patients were identified, from October 2004 to February 2008, as having received rapamycin for acute GVHD refractory to one or more lines of treatment. We analyzed the efficacy and tolerance of rapamycin and the outcome of these 22 patients in this setting.. Rapamycin resulted in a rapid and sustained complete remission of GVHD in 72% of heavily pretreated patients. Cytopenias were frequent but did not require treatment interruption. Thrombotic microangiopathy developed in 36% of patients when rapamycin was associated with calcineurin inhibitors and frequently resolved after interruption of one or both drugs. At a median follow-up of 13 months, overall survival was 41%. Previous treatment with high-dose steroid pulses was associated with a worse outcome (survival 12% vs. 69%). The major cause of death was infectious complications (77%).. Despite a small and heterogeneous population of patients, these results are encouraging and provide a rationale for prospective studies that use rapamycin in steroid-refractory acute GVHD as a second- or third-line agent.

Topics: Adolescent; Adult; Bacterial Infections; Graft vs Host Disease; Hematopoietic Stem Cell Transplantation; Humans; Immunosuppressive Agents; Leukemia; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Middle Aged; Retrospective Studies; Sirolimus; Survival Rate; Survivors; Transplantation, Homologous; Treatment Outcome; Young Adult

The oncogenic kinase Bcr-Abl is thought to cause chronic myelogenous leukemia (CML) by altering the transcription of specific genes with growth- and survival-promoting functions. Recently, Bcr-Abl has also been shown to activate an important regulator of protein synthesis, the mammalian target of rapamycin (mTOR), which suggests that dysregulated translation may also contribute to CML pathogenesis. In this study, we found that both Bcr-Abl and the rapamycin-sensitive mTORC1 complex contribute to the phosphorylation (inactivation) of 4E-BP1, an inhibitor of the eIF4E translation initiation factor. Experiments with rapamycin and the Bcr-Abl inhibitor, imatinib mesylate, in Bcr-Abl-expressing cell lines and primary CML cells indicated that Bcr-Abl and mTORC1 induced formation of the translation initiation complex, eIF4F. This was characterized by reduced 4E-BP1 binding and increased eIF4G binding to eIF4E, two events that lead to the assembly of eIF4F. One target transcript is cyclin D3, which is regulated in Bcr-Abl-expressing cells by both Bcr-Abl and mTORC1 in a translational manner. In addition, the combination of imatinib and rapamycin was found to act synergistically against committed CML progenitors from chronic and blast phase patients. These experiments establish a novel mechanism of action for Bcr-Abl, and they provide insights into the modes of action of imatinib mesylate and rapamycin in treatment of CML. They also suggest that aberrant cap-dependent mRNA translation may be a therapeutic target in Bcr-Abl-driven malignancies.

Topics: Adaptor Proteins, Signal Transducing; Animals; Antibiotics, Antineoplastic; Benzamides; Carrier Proteins; Cell Cycle Proteins; Cyclin D3; Cyclins; Eukaryotic Initiation Factor-4F; Eukaryotic Initiation Factor-4G; Eukaryotic Initiation Factors; Fusion Proteins, bcr-abl; Humans; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mechanistic Target of Rapamycin Complex 1; Mice; Multiprotein Complexes; Phosphoproteins; Phosphorylation; Piperazines; Protein Biosynthesis; Proteins; Pyrimidines; Sirolimus; TOR Serine-Threonine Kinases; Transcription Factors; Tumor Cells, Cultured

The mammalian target of rapamycin (mTOR) has recently been described to be constitutively activated in Bcr-Abl-transformed cells and to mediate rapamycin-induced inhibition of growth in respective cell lines. We have recently shown that rapamycin down-regulates expression of vascular endothelial growth factor (VEGF), a mediator of leukemia-associated angiogenesis, in primary CML cells. In the present study, we analyzed growth-inhibitory in vitro and in vivo effects of rapamycin on primary CML cells and asked whether rapamycin-induced suppression of VEGF in leukemic cells is related to growth inhibition. Rapamycin dose dependently inhibited growth of primary CML cells obtained from patients with imatinib-responsive or imatinib-resistant disease as well as growth of Bcr-Abl-transformed imatinib-resistant cell lines. Moreover, we observed potent cytoreductive effects of rapamycin in a patient with imatinib-resistant Bcr-Abl+ leukemia. The growth-inhibitory effects of rapamycin on CML cells were found to be associated with G1 cell cycle arrest and with induction of apoptosis. In all cell types tested, rapamycin was found to down-regulate expression of VEGF. However, exogenously added VEGF did not counteract the rapamycin-induced decrease in proliferation. In conclusion, rapamycin inhibits growth of CML cells in vitro and in vivo and, in addition, down-regulates expression of VEGF. Both effects may contribute to the antileukemic activity of the drug in CML.

Topics: Antineoplastic Agents; Apoptosis; Benzamides; Cell Cycle; Cell Division; Cell Survival; Drug Resistance, Neoplasm; Flow Cytometry; Fusion Proteins, bcr-abl; G1 Phase; Gene Expression Regulation; Humans; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Piperazines; Point Mutation; Protein Kinases; Pyrimidines; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; Sirolimus; TOR Serine-Threonine Kinases; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A

The precise mechanisms by which imatinib mesylate (STI571) and interferon alpha (IFNalpha) exhibit antileukemic effects are not known. We examined the effects of IFNs or imatinib mesylate on signaling pathways regulating initiation of mRNA translation in BCR-ABL-expressing cells. Treatment of IFN-sensitive KT-1 cells with IFNalpha resulted in phosphorylation/activation of mammalian target of rapamycin (mTOR) and downstream activation of p70 S6 kinase. The IFN-activated p70 S6 kinase was found to regulate phosphorylation of S6 ribosomal protein, which regulates translation of mRNAs with oligopyrimidine tracts in the 5'-untranslated region. In addition, IFNalpha treatment resulted in an mTOR- and/or phosphatidyl-inositol 3'(PI 3') kinase-dependent phosphorylation of 4E-BP1 repressor of mRNA translation on sites that are required for its deactivation and dissociation from the eukaryotic initiation factor-4E (eIF4E) complex. In contrast to the effects of IFNs, imatinib mesylate suppressed p70 S6 kinase activity, consistent with inhibition of BCR-ABL-mediated activation of the mTOR/p70 S6 kinase pathway. Moreover, the mTOR inhibitor rapamycin enhanced the suppressive effects of imatinib mesylate on primary leukemic granulocyte macrophage-colony-forming unit (CFU-GM) progenitors from patients with chronic myelogenous leukemia (CML). Taken altogether, our data demonstrate that IFNs and imatinib mesylate differentially regulate PI 3' kinase/mTOR-dependent signaling cascades in BCR-ABL-transformed cells, consistent with distinct effects of these agents on pathways regulating mRNA translation. They also support the concept that combined use of imatinib mesylate with mTOR inhibitors may be an appropriate future therapeutic strategy for the treatment of CML.

Topics: 5' Untranslated Regions; Androstadienes; Benzamides; Cell Line; Cell Line, Tumor; Cell Survival; Eukaryotic Initiation Factor-4E; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Granulocytes; Humans; Imatinib Mesylate; Immunoblotting; Interferon-alpha; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Phosphatidylinositol 3-Kinases; Phosphorylation; Piperazines; Protein Biosynthesis; Protein Kinases; Pyrimidines; Ribosomal Protein S6 Kinases, 70-kDa; RNA, Messenger; Signal Transduction; Sirolimus; Stem Cells; Time Factors; TOR Serine-Threonine Kinases; Wortmannin

BCR/ABL-kinase mutations frequently mediate clinical resistance to the selective tyrosine kinase inhibitor Imatinib mesylate (IM, Gleevec). However, mechanisms that promote survival of BCR/ABL-positive cells before clinically overt IM resistance occurs have poorly been defined so far. Here, we demonstrate that IM-treatment activated the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTor)-pathway in BCR/ABL-positive LAMA-cells and primary leukemia cells in vitro, as well as in a chronic phase CML patient in vivo. In fact, PI3K/Akt-activation critically mediated survival during the early phase of IM resistance development before manifestation of BCR/ABL-dependent strong IM resistance such as through a kinase mutation. Accordingly, inhibition of IM-induced Akt activation using mTor inhibitors and Akt-specific siRNA effectively antagonized development of incipient IM-resistance in vitro. In contrast, IM-resistant chronic myeloid leukemia (CML) patients with BCR/ABL kinase mutations (n=15), and IM-refractory BCR/ABL-positive acute lymphatic leukemia patients (n=2) displayed inconsistent and kinase mutation-independent autonomous patterns of Akt-pathway activation, and mTor-inhibition overcame IM resistance only if Akt was strongly activated. Together, an IM-induced compensatory Akt/mTor activation may represent a novel mechanism for the persistence of BCR/ABL-positive cells in IM-treated patients. Treatment with mTor inhibitors may thus be particularly effective in IM-sensitive patients, whereas Akt-pathway activation variably contributes to clinically overt IM resistance.

Topics: Antineoplastic Agents; Apoptosis; Benzamides; Blotting, Western; Cell Cycle; Drug Resistance, Neoplasm; Enzyme Activation; Everolimus; Fusion Proteins, bcr-abl; Humans; Imatinib Mesylate; Immunosuppressive Agents; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mutagenesis; Phosphatidylinositol 3-Kinases; Phosphorylation; Piperazines; Protein Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Pyrimidines; Reverse Transcriptase Polymerase Chain Reaction; Ribosomal Protein S6 Kinases, 70-kDa; RNA, Messenger; RNA, Small Interfering; Sirolimus; TOR Serine-Threonine Kinases; Tumor Cells, Cultured

Identification of signaling pathways downstream of Abl tyrosine kinase may increase our understanding of the pathogenesis of chronic myelogenous leukemia (CML) and suggest strategies to improve clinical treatment of the disease. By combining the use of a phosphospecific antibody recognizing a substrate motif of serine/threonine kinases with bioinformatics, we found that the translational regulators ribosomal protein S6 and 4E-BP1 are constitutively phosphorylated in CML cells. Experiments with specific inhibitors indicated the phosphorylation is downstream of Bcr-Abl kinase and the mammalian target of rapamycin (mTOR). These results suggest that Bcr-Abl may regulate translation of critical targets in CML cells via mTOR. They also provide a rationale for testing the combination of mTOR inhibitors with the Abl kinase inhibitor imatinib in patients with CML. The mTOR inhibitor rapamycin enhanced imatinib-mediated killing of CML cell lines in vitro, and it overcame imatinib resistance in cells with Bcr-Abl gene amplification.

Topics: Adaptor Proteins, Signal Transducing; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Benzamides; Carrier Proteins; Cell Cycle Proteins; Drug Synergism; Eukaryotic Initiation Factors; Fusion Proteins, bcr-abl; Hematopoietic Stem Cells; Humans; Imatinib Mesylate; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mice; Phosphatidylinositol 3-Kinases; Phosphoproteins; Phosphorylation; Piperazines; Protein Kinase Inhibitors; Protein Kinases; Protein-Tyrosine Kinases; Pyrimidines; Ribosomal Protein S6; Sirolimus; TOR Serine-Threonine Kinases