sirolimus and Leukemia--Myeloid--Acute

Differentiation therapy of acute promyelocytic leukemia with all-trans retinoic acid represents the most successful pharmacological therapy of acute myeloid leukemia (AML). Numerous studies demonstrate that drugs that inhibit mechanistic target of rapamycin (mTOR) and activate AMP-kinase (AMPK) have beneficial effects in promoting differentiation and blocking proliferation of AML. Most of these drugs are already in use for other purposes; rapalogs as immunosuppressants, biguanides as oral antidiabetics, and 5-amino-4-imidazolecarboxamide ribonucleoside (AICAr, acadesine) as an exercise mimetic. Although most of these pharmacological modulators have been widely used for decades, their mechanism of action is only partially understood. In this review, we summarize the role of AMPK and mTOR in hematological malignancies and discuss the possible role of pharmacological modulators in proliferation and differentiation of leukemia cells.

Topics: Aminoimidazole Carboxamide; AMP-Activated Protein Kinases; Clinical Trials as Topic; Humans; Leukemia, Myeloid, Acute; Metformin; Ribonucleosides; RNA, Small Interfering; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Acute myeloid leukemia (AML) continues to represent an area of critical unmet need with respect to new and effective targeted therapies. The Bcl-2 family of pro- and antiapoptotic proteins stands at the crossroads of cellular survival and death, and the expression of and interactions between these proteins determine tumor cell fate. Malignant cells, which are often primed for apoptosis, are particularly vulnerable to the simultaneous disruption of cooperative survival signaling pathways. Indeed, the single agent activity of agents such as mammalian target of rapamycin (mTOR) and mitogen-activated protein kinase kinase (MEK) inhibitors in AML has been modest. Much work in recent years has focused on strategies to enhance the therapeutic potential of the bona fide BH3-mimetic, ABT-737, which inhibits B-cell lymphoma 2 (Bcl-2) and Bcl-xL. Most of these strategies target Mcl-1, an antiapoptotic protein not inhibited by ABT-737. The phosphatidylinositol-3-kinase (PI3K)/Akt/mTOR and Ras/Raf/MEK/ERK signaling pathways are central to the growth, proliferation, and survival of AML cells, and there is much interest currently in pharmacologically interrupting these pathways. Dual inhibitors of PI3K and mTOR overcome some intrinsic disadvantages of rapamycin and its derivatives, which selectively inhibit mTOR. In this review, we discuss why combining dual PI3K/mTOR blockade with inhibition of Bcl-2 and Bcl-xL, by virtue of allowing coordinate inhibition of three mutually synergistic pathways in AML cells, may be a particularly attractive therapeutic strategy in AML, the success of which may be predicted for by basal Akt activation.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; bcl-X Protein; Biphenyl Compounds; Humans; Leukemia, Myeloid, Acute; Nitrophenols; Phosphoinositide-3 Kinase Inhibitors; Piperazines; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Sirolimus; Sulfonamides; TOR Serine-Threonine Kinases

Mammalian/mechanistic target of rapamycin (mTOR) is a highly conserved serine/threonine kinase that responds to environmental determinants such as growth factor concentration, nutrient availability, energy sufficiency and stress. mTOR forms two different complexes, called mTOR complex 1 (mTORC1) and mTORC2, and these complexes have distinct substrate molecules that function in the regulation of protein translation and cellular metabolism. Although mTOR was originally discovered as a target protein of rapamycin, a natural macrolide immunosuppressant, rapamycin mainly inhibits the kinase activity of mTORC1, whereas mTORC2 is affected to a much lesser extent. The inhibitory effects of rapamycin on mTORC1 substrates are complex, and the use of rapamycin to investigate mTORC functions may provide misleading results. In contrast to pharmacological inhibition, studies using genetic approaches to the disruption of mTORC subunits have clearly demonstrated the physiological roles of the distinct mTOR complexes in organogenesis and tumourigenesis. In this review, we provide an overview of current knowledge about the roles of the mTOR complexes in haemato-lymphopoiesis and leukemogenesis.

Topics: Animals; Carcinogenesis; Hematopoiesis; Humans; Leukemia, Myeloid, Acute; Lymphoid Progenitor Cells; Lymphopoiesis; Sirolimus; TOR Serine-Threonine Kinases

The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling axis plays a central role in cell proliferation, growth, and survival under physiological conditions. However, aberrant PI3K/Akt/mTOR signaling has been implicated in many human cancers, including acute myelogenous leukemia (AML). Therefore, the PI3K/Akt/mTOR network is considered as a validated target for innovative cancer therapy. The limit of acceptable toxicity for standard polychemotherapy has been reached in AML. Novel therapeutic strategies are therefore needed. This review highlights how the PI3K/Akt/mTOR signaling axis is constitutively active in AML patients, where it affects survival, proliferation, and drug-resistance of leukemic cells including leukemic stem cells. Effective targeting of this pathway with small molecule kinase inhibitors, employed alone or in combination with other drugs, could result in the suppression of leukemic cell growth. Furthermore, targeting the PI3K/Akt/mTOR signaling network with small pharmacological inhibitors, employed either alone or in combinations with other drugs, may result in less toxic and more efficacious treatment of AML patients. Efforts to exploit pharmacological inhibitors of the PI3K/Akt/mTOR cascade which show efficacy and safety in the clinical setting are now underway.

Topics: Antineoplastic Agents; Cell Proliferation; Drug Resistance, Neoplasm; Humans; Leukemia, Myeloid, Acute; Molecular Targeted Therapy; Neoplastic Stem Cells; Phosphatidylinositol 3-Kinase; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

The mammalian Target Of Rapamycin Complex 1 (mTORC1) pathway is commonly activated in cancer cells including acute myeloid leukemia (AML) and has been designed as a major target for cancer therapy. However, the efficacy of rapalogs (mTORC1 inhibitors) is limited in AML, due to the feedback activation of PI3K or ERK signaling pathways upon mTORC1 inhibition, which pathways should be simultaneously targeted to enhance the anti-leukemic activity of rapalogs. Moreover, the mRNA translation process is mTORC1-independent in AML, although markedly contributing to oncogenesis in this disease, and this also strongly participates to rapalogs resistance. Translation inhibition could be achieved by directly targeting the translation initiating complex using the 4EGI-1 compound, anti-eIF4E antisense oligonucleotides or the antiviral drug ribavirin or by second generation mTOR inhibitors (TORkinhibs). These new approaches represent promising perspectives for AML therapy that should have clinical development in the future.

Topics: Antibiotics, Antineoplastic; Extracellular Signal-Regulated MAP Kinases; Humans; Leukemia, Myeloid, Acute; Mechanistic Target of Rapamycin Complex 1; Multiprotein Complexes; Phosphatidylinositol 3-Kinases; Protein Biosynthesis; Proteins; RNA, Messenger; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transcription Factors

The phosphatidylinositol 3-kinase (PI3K)/Akt (protein kinase B, PKB)/mammalian Target Of Rapamycin (mTOR) signaling pathway plays a critical role in many cellular functions which are elicited by extracellular stimuli. However, constitutively active PI3K/Akt/mTOR signaling has also been firmly established as a major determinant for cell growth, proliferation, and survival in an wide array of human cancers. Thus, blocking the PI3K/AKT/mTOR signal transduction network could be an effective new strategy for targeted anticancer therapy. Pharmacological inhibitors of this signaling cascade are powerful antineoplastic agents in vitro and in xenografted models of tumors, and some of them are now being tested in clinical trials. Recent studies showed that PI3K/Akt/mTOR axis is frequently activated in acute myelogenous leukemia (AML) patient blasts and strongly contributes to proliferation, survival, and drug-resistance of these cells. Both the disease-free survival and overall survival are significantly shorter in AML cases with PI3K/Akt/mTOR upregulation. Therefore, this signal transduction cascade may represent a target for innovative therapeutic treatments of AML patients. In this review, we discuss the possible mechanisms of activation of this pathway in AML cells and the downstream molecular targets of the PI3K/Akt/mTOR signaling network which are important for blocking apoptosis, enhancing proliferation, and promoting drug-resistance of leukemic cells. We also highlight several pharmacological inhibitors which have been used to block this pathway for targeted therapy of AML. These small molecules induce apoptosis or sensitize AML cells to existing drugs, and might be used in the future for improving the outcome of this hematological disorder.

Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; Cell Cycle; Drug Resistance, Neoplasm; Humans; Leukemia, Myeloid, Acute; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

To improve the outcome of relapsed/refractory acute myeloid leukemia (AML), a randomized phase II trial of three novel regimens was conducted. Ninety patients were enrolled and were in first relapse or were refractory to induction/re-induction chemotherapy. They were randomized to the following regimens: carboplatin-topotecan (CT), each by continuous infusion for 5 days; alvocidib (formerly flavopiridol), cytarabine, and mitoxantrone (FLAM) in a timed sequential regimen; or sirolimus combined with mitoxantrone, etoposide, and cytarabine (S-MEC). The primary objective was attainment of a complete remission (CR). A Simon two-stage design was used for each of the three arms. The median age of the patients in the FLAM arm was older at 62 years compared with 55 years for the CT arm and the S-MEC arm. The overall response was 14% in the CT arm (5/35, 90% CI 7%-35%), 28% in the FLAM arm (10/36, 90% CI, 16%-43%), and 16% in the S-MEC arm (3/19, 90% CI, 4%-36%). There were nine treatment-related deaths, seven of which occurred in the FLAM arm with four of these in elderly patients. We conclude that the FLAM regimen had an encouraging response rate and should be considered for further clinical development but should be used with caution in elderly patients.

Topics: Aged; Antineoplastic Combined Chemotherapy Protocols; Carboplatin; Cytarabine; Disease-Free Survival; Etoposide; Female; Flavonoids; Follow-Up Studies; Gastrointestinal Diseases; Hematologic Diseases; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Mitoxantrone; Piperidines; Recurrence; Remission Induction; Salvage Therapy; Sirolimus; Topotecan; Tumor Lysis Syndrome

Background Mammalian Target of Rapamycin Complex 1 (mTORC1) inhibitors enhance chemotherapy response in acute myelogenous leukemia (AML) cells in vitro. However whether inhibiting mTORC1 enhances clinical response to AML chemotherapy remains controversial. We previously optimized measurement of mTORC1's kinase activity in AML blasts during clinical trials using serial phospho-specific flow cytometry of formaldehyde-fixed whole blood or marrow specimens. To validate mTORC1 as a therapeutic target in AML, we performed two clinical trials combining an mTORC1 inhibitor (sirolimus) and MEC (mitoxantrone, etoposide, cytarabine) in patients with relapsed, refractory, or untreated high-risk AML. Methods Flow cytometric measurements of ribosomal protein S6 phosphorylation (pS6) were performed before and during sirolimus treatment to determine whether mTORC1 inhibition enriched for chemotherapy response. Results In 51 evaluable subjects, the overall response rate (ORR) to the combination regimen was 47% (95% confidence interval 33-61%, 33% CR, 2% CRi, 12% PR) and similar toxicity to historic experience with MEC alone. 37 subjects had baseline pS6 measured pre-sirolimus, of whom 27 (73%) exhibited mTORC1 activity. ORR was not significantly different between subjects with and without baseline mTORC1 activity (52% vs 40%, respectively, p = 0.20). The ORR among subjects with baseline target activation and mTORC1 inhibition during therapy was 71% (12/17) compared to 20% (2/10) in subjects without target inhibition. Conclusions Fixed, whole blood pS6 by flow cytometry may be a predictive biomarker for clinical response to mTORC1 inhibitor-based regimens. These data provide clinical confirmation that mTORC1 activation mediates chemotherapy resistance in patients with AML.

Topics: Antineoplastic Combined Chemotherapy Protocols; Cytarabine; Etoposide; Female; Humans; Leukemia, Myeloid, Acute; Male; Mechanistic Target of Rapamycin Complex 1; Middle Aged; Mitoxantrone; Phosphorylation; Pilot Projects; Remission Induction; Signal Transduction; Sirolimus

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

A phase I study utilizing decitabine (DAC) followed by the mammalian target of rapamycin (mTOR) inhibitor, rapamycin, in patients with relapsed/refractory adult AML was undertaken to assess safety and feasibility. Patients received DAC 20mg/m(2) intravenously daily for 5 days followed by rapamycin from day 6 to day 25 at doses of 2 mg, 4 mg, and 6 mg/day in a standard 3+3 dose escalation design. Twelve patients completed treatment for safety evaluation. Maximum tolerated dose (MTD) was not reached, and except for grade 3 mucositis in 4 patients, no other significant unexpected non-hematologic toxicities have occurred indicating safety of this regimen. This trial is registered at clinical trials.gov as NCT00861874.

Topics: Aged; Antineoplastic Combined Chemotherapy Protocols; Azacitidine; Decitabine; Dose-Response Relationship, Drug; Drug Administration Routes; Drug Administration Schedule; Drug Resistance, Neoplasm; Female; Humans; Leukemia, Myeloid, Acute; Male; Maximum Tolerated Dose; Middle Aged; Pilot Projects; Recurrence; Sirolimus

The mTORC1 signaling pathway is constitutively activated in almost all acute myelogenous leukemia (AML) patients. We conducted a phase Ib trial combining RAD001 (everolimus), an allosteric inhibitor of mTORC1, and conventional chemotherapy, in AML patients under 65 years of age at first relapse (clinical trial NCT 01074086). Increasing doses of RAD001 from 10-70 mg were administrated orally on days 1 and 7 (d1 and d7) of a 3+7 daunorubicin+cytarabine conventional induction chemotherapy regimen. Twenty-eight patients were enrolled in this trial. The treatment was well tolerated with <10% toxicity, mainly involving the gastrointestinal tract and lungs. In this phase Ib trial, the RAD001 maximum tolerated dose was not reached at 70 mg. Sixty-eight percent of patients achieved CR, of which 14 received a double induction. Eight subsequently were intensified with allogeneic-stem cell transplant. Strong plasma inhibition of P-p70S6K was observed after RAD001 administration, still detectable at d7 (d7)at the 70 mg dosage. CR rates in patients with RAD001 areas under or above the curve median were 53% versus 85%. A 70 mg dose of RAD001 at d1 and d7 of an induction chemotherapy regimen for AML has acceptable toxicity and may improve treatment.

Topics: Adult; Aged; Antibiotics, Antineoplastic; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Cytarabine; Daunorubicin; Everolimus; Female; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Recurrence; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Treatment Outcome; Young Adult

The mammalian target of rapamycin (mTOR) signalling pathway has emerged as an important therapeutic target for acute myeloid leukaemia (AML). This study assessed the combination of temsirolimus, an mTOR inhibitor, and lower-dose clofarabine as salvage therapy in older patients with AML. Induction consisted of clofarabine 20mg/m(2) on days 1-5 and temsirolimus 25mg (flat dose) on days 1, 8 and 15. Patients achieving complete remission with (CR) or without (CRi) full haematological recovery could receive monthly temsirolimus maintenance. In 53 evaluable patients, the overall remission rate (ORR) was 21% (8% CR, 13% CRi). Median disease-free survival was 3·5months, and median overall survival was 4months (9·1months for responders). The most common non-haematological severe adverse events included infection (48%), febrile neutropenia (34%) and transaminitis (11%). The 30-d all-cause induction mortality was 13%. Laboratory data from 25 patients demonstrated that a >50%in vivo inhibition of S6 ribosomal protein phosphorylation was highly correlated with response rate (75% with inhibition versus 0% without inhibition; P=0·0001), suggesting that targeting the mTOR pathway is clinically relevant. The acceptable safety profile and the predictive value of target inhibition encourage further investigation of this novel regimen.

Topics: Adenine Nucleotides; Age Factors; Aged; Antineoplastic Combined Chemotherapy Protocols; Arabinonucleosides; Clofarabine; Disease-Free Survival; Female; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Salvage Therapy; Sirolimus; TOR Serine-Threonine Kinases

Integration of signal transduction inhibitors into chemotherapy regimens generally has generally not led to anticipated increases in response and survival. However, it remains unclear whether this is because of inadequate or inconsistent inhibition of target or other complex biology. The mTOR signaling pathway is frequently activated in acute myelogenous leukemia (AML) and we previously showed the safety of combining the mTOR inhibitor, sirolimus, with mitoxantrone, etoposide, and cytarabine (MEC) chemotherapy. However, we did not reliably determine the extent of mTOR inhibition on that study. Here, we sought to develop an assay that allowed us to serially quantify the activation state of mTOR kinase during therapy.. To provide evidence of mTOR kinase activation and inhibition, we applied a validated whole blood fixation/permeabilization technique for flow cytometry to serially monitor S6 ribosomal protein (S6) phosphorylation in immunophenotypically identified AML blasts.. With this approach, we show activation of mTOR signaling in 8 of 10 subjects' samples (80%) and conclusively show inhibition of mTOR in the majority of subjects' tumor cell during therapy. Of note, S6 phosphorylation in AML blasts is heterogeneous and, in some cases, intrinsically resistant to rapamycin at clinically achieved concentrations.. The methodology described is rapid and reproducible. We show the feasibility of real-time, direct pharmacodynamic monitoring by flow cytometry during clinical trials combining intensive chemotherapy and signal transduction inhibitors. This approach greatly clarifies pharmacokinetic/pharmacodynamic relationships and has broad application to preclinical and clinical testing of drugs whose direct or downstream effects disrupt PI3K/AKT/mTOR signaling.

Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Cytarabine; Drug Administration Schedule; Etoposide; Female; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Mitoxantrone; Phosphorylation; Pilot Projects; Ribosomal Protein S6; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

A calcineurin inhibitor combined with methotrexate is the standard prophylaxis for graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (HSCT). Everolimus, a derivative of sirolimus, seems to mediate antileukemia effects. We report on a combination of everolimus and tacrolimus in 24 patients (median age, 62 years) with either myelodysplastic syndrome (MDS; n = 17) or acute myeloid leukemia (AML; n = 7) undergoing intensive conditioning followed by HSCT from related (n = 4) or unrelated (n = 20) donors. All patients engrafted, and only 1 patient experienced grade IV mucositis. Nine patients (37%) developed acute grade II-IV GVHD, and 11 of 17 evaluable patients (64%) developed chronic extensive GVHD. Transplantation-associated microangiopathy (TMA) occurred in 7 patients (29%), with 2 cases of acute renal failure. The study was terminated prematurely because an additional 6 patients (25%) developed sinusoidal obstruction syndrome (SOS), which was fatal in 2 cases. With a median follow-up of 26 months, the 2-year overall survival rate was 47%. Although this new combination appears to be effective as a prophylactic regimen for acute GVHD, the incidence of TMA and SOS is considerably higher than seen with other regimens.

Topics: Adult; Aged; Anemia, Hemolytic; Everolimus; Female; Graft vs Host Disease; Hematopoietic Stem Cell Transplantation; Hepatic Veno-Occlusive Disease; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Myelodysplastic Syndromes; Sirolimus; Survival Rate; Tacrolimus; Transplantation, Homologous

Inhibiting mammalian target of rapamycin (mTOR) signaling in acute myelogenous leukemia (AML) blasts and leukemic stem cells may enhance their sensitivity to cytotoxic agents. We sought to determine the safety and describe the toxicity of this approach by adding the mTOR inhibitor, sirolimus (rapamycin), to intensive AML induction chemotherapy.. We performed a phase I dose escalation study of sirolimus with the chemotherapy regimen MEC (mitoxantrone, etoposide, and cytarabine) in patients with relapsed, refractory, or untreated secondary AML.. Twenty-nine subjects received sirolimus and MEC across five dose levels. Dose-limiting toxicities were irreversible marrow aplasia and multiorgan failure. The maximum tolerated dose (MTD) of sirolimus was determined to be a 12 mg loading dose on day 1 followed by 4 mg/d on days 2 to 7, concurrent with MEC chemotherapy. Complete or partial remissions occurred in 6 (22%) of the 27 subjects who completed chemotherapy, including 3 (25%) of the 12 subjects treated at the MTD. At the MTD, measured rapamycin trough levels were within the therapeutic range for solid organ transplantation. However, direct measurement of the mTOR target p70 S6 kinase phosphorylation in marrow blasts from these subjects only showed definite target inhibition in one of five evaluable samples.. Sirolimus and MEC is an active and feasible regimen. However, as administered in this study, the synergy between MEC and sirolimus was not confirmed. Future studies are planned with different schedules to clarify the clinical and biochemical effects of sirolimus in AML and to determine whether target inhibition predicts chemotherapy response.

Topics: Adult; Aged; Antibiotics, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Carboplatin; Drug Administration Schedule; Drug Resistance, Neoplasm; Etoposide; Female; Humans; Leukemia, Myeloid, Acute; Male; Maximum Tolerated Dose; Melphalan; Middle Aged; Protein Kinases; Recurrence; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

The mammalian target of rapamycin (mTOR) has recently been identified as a potential target in acute myeloid leukemia (AML).. We treated 5 patients with chemotherapy-refractory AML with the mTOR-inhibitor rapamycin at 2mg per os daily for 14 days, with dose adjustment allowed to reach a target serum rapamycin concentration of 10-20 ng/mL. Four of five patients received additional hydroxyurea at constant dose during treatment with rapamycin.. Two patients achieved a leukocyte response, in one of them, a prolonged response was seen. In the other patients, blast counts remained stable or increased during rapamycin therapy. We did not observe severe hematologic or non-hematologic side effects of rapamycin.. Rapamycin at 2mg per day acts mildly cytoreductive in a subgroup of patients with refractory AML. Higher doses and drug combinations may be required to obtain long lasting anti-leukemic effects in these patients.

Topics: Aged; Aged, 80 and over; Antibiotics, Antineoplastic; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Female; Humans; Hydroxyurea; Leukemia, Myeloid, Acute; Leukocyte Count; Male; Middle Aged; Pilot Projects; Sirolimus; Treatment Outcome

Everolimus (RAD001, Novartis), an oral derivative of rapamycin, inhibits the mammalian target of rapamycin (mTOR), which regulates many aspects of cell growth and division. A phase I/II study was done to determine safety and efficacy of everolimus in patients with relapsed or refractory hematologic malignancies.. Two dose levels (5 and 10 mg orally once daily continuously) were evaluated in the phase I portion of this study to determine the maximum tolerated dose of everolimus to be used in the phase II study.. Twenty-seven patients (9 acute myelogenous leukemia, 5 myelodysplastic syndrome, 6 B-chronic lymphocytic leukemia, 4 mantle cell lymphoma, 1 myelofibrosis, 1 natural killer cell/T-cell leukemia, and 1 T-cell prolymphocytic leukemia) received everolimus. No dose-limiting toxicities were observed. Grade 3 potentially drug-related toxicities included hyperglycemia (22%), hypophosphatemia (7%), fatigue (7%), anorexia (4%), and diarrhea (4%). One patient developed a cutaneous leukocytoclastic vasculitis requiring a skin graft. One patient with refractory anemia with excess blasts achieved a major platelet response of over 3-month duration. A second patient with refractory anemia with excess blasts showed a minor platelet response of 25-day duration. Phosphorylation of downstream targets of mTOR, eukaryotic initiation factor 4E-binding protein 1, and/or, p70 S6 kinase, was inhibited in six of nine patient samples, including those from the patient with a major platelet response.. Everolimus is well tolerated at a daily dose of 10 mg daily and may have activity in patients with myelodysplastic syndrome. Studies of everolimus in combination with therapeutic agents directed against other components of the phosphatidylinositol 3-kinase/Akt/mTOR pathway are warranted.

Topics: Adaptor Proteins, Signal Transducing; Administration, Oral; Adolescent; Adult; Aged; Cell Cycle Proteins; Dose-Response Relationship, Drug; Drug Administration Schedule; Drug-Related Side Effects and Adverse Reactions; Everolimus; Female; Humans; Killer Cells, Natural; Leukemia, Lymphocytic, Chronic, B-Cell; Leukemia, Myeloid, Acute; Leukemia, Prolymphocytic; Leukemia, T-Cell; Lymphoma, Mantle-Cell; Male; Maximum Tolerated Dose; Middle Aged; Myelodysplastic Syndromes; Phosphoproteins; Phosphorylation; Protein Kinases; Recurrence; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; T-Lymphocytes; TOR Serine-Threonine Kinases; Treatment Outcome; Vasculitis, Leukocytoclastic, Cutaneous

Patients with tuberous sclerosis complex (TSC) demonstrate disrupted lipid homeostasis before and during treatment with mammalian target of rapamycin (mTOR) inhibitor. However, few previous reports focused on if the serum lipid status at baseline would influence lipid metabolic side-effects of mTOR inhibitors for TSC associated renal angiomyolipomas (TSC-AML). The present study was designed to evaluate the predictive function of serum lipid status at baseline for hyperlipidaemia by mTOR inhibitor treatment in TSC-AML patients.. The clinical data of TSC-AML patients who took mTOR inhibitors in Department of Urology of Peking Union Medical College Hospital (PUMCH) from 1 January 2014 to 1 January 2021, were retrospectively analysed. The record of lipid parameters at baseline and the highest levels of total cholesterol (TC) and triglyceride (TG) after treatment at least ≥3 months were collected. The correlation of serum lipid parameters at baseline with incidence of hyperlipidaemia during mTOR inhibitor treatment was analysed. Receiver operating characteristic (ROC) curve analysis was conducted to evaluate the ability of the serum lipid parameters in predicting hyperlipidaemia.. 19 patients experienced hyperlipidaemia and 13 patients still had normal TC and TG levels during mTOR inhibitor treatment. The levels of high-density lipoprotein cholesterol (HDL-C) (0.98 ± 0.30 mmol/L vs. 1.23 ± 0.31 mmol/L, p = 0.030), low-density lipoprotein cholesterol (LDL-C) (2.47 ± 0.69 mmol/L vs. 1.95 ± 0.53 mmol/L, p = 0.029) and apolipoprotein B (ApoB) (0.82 ± 0.21 g/L vs. 0.65 ± 0.16 g/L, p = 0.019) are higher in the patients who experienced hyperlipidaemia during mTOR inhibition therapy. TC, TG, LDL-C, ApoB and high-sensitivity C-reactive protein (hsCRP) at baseline had positive correlation with TC after treatment; ApoB at baseline had positive correlation, while HDL-C and free fat acid (FFA) at baseline had negative correlation with TG after treatment. Therefore, ApoB concentration at baseline has statistically significant correlation with both TC (p < 0.001) and TG (p = 0.012) levels after mTOR inhibitor treatment. ROC curve and AUC revealed that ApoB with a cut-off value of 0.640g/L may be the best parameter for predicting hyperlipidaemia during mTOR inhibitor treatment in TSC-AML patients. The incidence rates of hyperlipidaemia were 27.3% and 76.2% among the patients with ApoB level ≤0.640 g/L and >0.640 g/L respectively.. Some baseline serum lipid parameters could be used for predicting incidence of hyperlipidaemia during mTOR inhibition therapy in TSC-AML patients, and ApoB with 0.640 g/L as a cut-off value may be a potentially optimal indicator, which could help for diagnosis and treatment decision-making.

Topics: Apolipoproteins B; Cholesterol, HDL; Cholesterol, LDL; Humans; Hyperlipidemias; Leukemia, Myeloid, Acute; Lipids; MTOR Inhibitors; Retrospective Studies; Sirolimus; TOR Serine-Threonine Kinases; Triglycerides; Tuberous Sclerosis

Post-transplant cyclophosphamide (PTCy) has emerged as a promising graft-versus-host disease (GvHD) prophylaxis in allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, no studies have reported the efficacy of a GvHD prophylaxis based on PTCy with sirolimus (Sir-PTCy) in patients with acute myeloid leukemia (AML). In this retrospective study, we analyze the use of sirolimus in combination with PTCy, with or without mycophenolate mofetil (MMF), on 242 consecutive adult patients with AML undergoing a myeloablative first allo-HSCT from different donor types, in three European centers between January 2017 and December 2020. Seventy-seven (32%) patients received allo-HSCT from HLA-matched sibling donor, 101 (42%) from HLA-matched and mismatched unrelated donor, and 64 (26%) from haploidentical donor. Except for neutrophil and platelet engraftment, which was slower in the haploidentical cohort, no significant differences were observed in major transplant outcomes according to donor type in univariate and multivariate analysis. GvHD prophylaxis with Sir-PTCy, with or without MMF, is safe and effective in patients with AML undergoing myeloablative allo-HSCT, resulting in low rates of transplant-related mortality, relapse/progression, and acute and chronic GvHD in all donor settings.

Topics: Adult; Cyclophosphamide; Graft vs Host Disease; Hematopoietic Stem Cell Transplantation; Humans; Leukemia, Myeloid, Acute; Mycophenolic Acid; Retrospective Studies; Sirolimus; Transplantation Conditioning; Unrelated Donors

The capacity of respiratory viruses to undergo evolution within the respiratory tract raises the possibility of evolution under the selective pressure of the host environment or drug treatment. Long-term infections in immunocompromised hosts are potential drivers of viral evolution and development of infectious variants. We showed that intrahost evolution in chronic human parainfluenza virus 3 (HPIV3) infection in immunocompromised individuals elicited mutations that favored viral entry and persistence, suggesting that similar processes may operate across enveloped respiratory viruses. We profiled longitudinal HPIV3 infections from 2 immunocompromised individuals that persisted for 278 and 98 days. Mutations accrued in the HPIV3 attachment protein hemagglutinin-neuraminidase (HN), including the first in vivo mutation in HN's receptor binding site responsible for activating the viral fusion process. Fixation of this mutation was associated with exposure to a drug that cleaves host-cell sialic acid moieties. Longitudinal adaptation of HN was associated with features that promote viral entry and persistence in cells, including greater avidity for sialic acid and more active fusion activity in vitro, but not with antibody escape. Long-term infection thus led to mutations promoting viral persistence, suggesting that host-directed therapeutics may support the evolution of viruses that alter their biophysical characteristics to persist in the face of these agents in vivo.

Topics: Adult; Binding Sites; DNA Mutational Analysis; Female; Gene Frequency; Graft vs Host Disease; HEK293 Cells; Humans; Immunocompromised Host; Leukemia, Myeloid, Acute; Lung; Lung Diseases; Mutation; Mycophenolic Acid; N-Acetylneuraminic Acid; Parainfluenza Virus 3, Human; Paramyxoviridae Infections; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Receptors, Virus; Sirolimus; Viral Fusion Proteins; Virus Internalization; Young Adult

To compare the efficacy and safety of CD34+ selected ex vivo T-cell depletion (TCD) vs post-transplant cyclophosphamide, sirolimus, and mycophenolate mofetil (PTCy-Sir-MMF) as graft-vs-host disease (GVHD) prophylaxis.. We retrospectively included patients who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT) with either TCD (n = 38) or PTCy-Sir-MMF (n = 91).. Cumulative incidence of neutrophil and platelet recovery was 92% vs 99% (P = .06) and 89% vs 97% (P = .3) in TCD and PTCy-Sir-MMF, respectively. Cumulative incidences of aGHVD grade II-IV, III-IV, and moderate to severe cGVHD were 11% vs 19% (P = .2), 3% vs 2% (P = .9), and 3% vs 36% (P < .001) in TCD and PTCy-Sir-MMF, respectively. The 2-year non-relapse mortality, relapse, disease-free and overall survival were 25% vs 8% (P = .01), 20% vs 16% (P = .2), 55% vs 76% (P = .004), 57% vs 83% (P = .004) for TCD and PTCy-Sir-MMF, respectively. Cumulative incidence of cytomegalovirus and Epstein-Barr infection requiring therapy was 76% vs 40% (P < .001) and 32% vs 0% (P < .001) in TCD and PTCy-Sir-MMF, respectively. PTCy-Sir-MMF platform showed faster T-cell reconstitution.. PTCy-Sir-MMF provides better survival outcomes but is associated with higher risk of cGVHD compared to TCD.

Topics: Adolescent; Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Biomarkers; Cyclophosphamide; Female; Graft vs Host Disease; Hematopoietic Stem Cell Transplantation; Humans; Immune Reconstitution; Leukemia, Myeloid, Acute; Leukocyte Count; Lymphocyte Depletion; Male; Middle Aged; Mycophenolic Acid; Postoperative Care; Postoperative Complications; Prognosis; Recurrence; Severity of Illness Index; Sirolimus; T-Lymphocytes; Transplantation, Homologous; Treatment Outcome; Young Adult

Ongoing clinical trials show limited efficacy for Chimeric antigen receptor (CAR) T treatment for acute myeloid leukemia (AML). The aim of this study was to identify potential causes of the reported limited efficacy from CAR-T therapies against AML.. We generated CAR-T cells targeting Epithelial cell adhesion molecule (EpCAM) and evaluated their killing activity against AML cells. We examined the impacts of modulating mTORC1 and mTORC2 signaling in CAR-T cells in terms of CXCR4 levels. We examined the effects of a rapamycin pretreatment of EpCAM CAR-T cells (during. EpCAM CAR-T exhibited killing activity against AML cells but failed to eliminate AML cells in bone marrow. Subsequent investigations revealed that aberrantly activated mTORC1 signaling in CAR-T cells results in decreased bone marrow infiltration and decreased the levels of the rapamycin target CXCR4. Attenuating mTORC1 activity with the rapamycin pretreatment increased the capacity of CAR-T cells to infiltrate bone marrow and enhanced the extent of bone marrow AML cell elimination in leukemia xenograft mouse models.. Our study reveals a potential cause for the limited efficacy of CAR-T reported from current AML clinical trials and illustrates an easy-to-implement pretreatment strategy, which enhances the anti-AML efficacy of CAR-T cells.

Topics: Animals; Bone Marrow Cells; Immunotherapy, Adoptive; Leukemia, Myeloid, Acute; Mice; Sirolimus; T-Lymphocytes; Tumor Cells, Cultured

Cyclophosphamide (Cy)/etoposide combined with fractionated total body irradiation (FTBI) or i.v. busulfan (Bu) has been the main conditioning regimens for allogeneic hematopoietic cell transplantation (alloHCT) for young patients with acute myelogenous leukemia (AML) eligible for a myeloablative conditioning (MAC) regimen. Recent data has suggested that i.v. Bu could be the preferred myeloablative regimen in patients with myeloid malignancies. However, Bu-based regimens are associated with higher rates of sinusoidal obstruction syndrome. Here we report long-term survival outcomes of patients with AML receiving FTBI combined with Cy or etoposide before undergoing alloHCT at City of Hope (COH). We obtained a retrospective review of a prospectively maintained institutional registry of clinical outcomes in 167 patients (median age, 41 years; range, 18 to 57 years) with AML in first or second complete remission who underwent alloHCT at COH between 2005 and 2015. Eligible patients received a MAC regimen with FTBI (1320 cGy) and Cy (120 mg/kg) for unrelated donor transplantation or etoposide (60 mg/kg) for related donor transplantation. Graft-versus-host disease (GVHD) prophylaxis was provided with tacrolimus and sirolimus. In this retrospective study, 6-year overall survival was 60% and nonrelapse mortality was 15%. The GRFS rate was 45% at 1 year and 39% at 2 years. We also describe late metabolic effects and report the cumulative incidence of secondary malignancies (9.5%). Overall, in this young adult patient population, our results compare favorably to chemotherapy-based (i.v. Bu) conditioning regimens without significant long-term toxicity arising from TBI-based regimens.

Topics: Adolescent; Adult; Busulfan; Cyclophosphamide; Follow-Up Studies; Graft vs Host Disease; Hematopoietic Stem Cell Transplantation; Humans; Leukemia, Myeloid, Acute; Middle Aged; Retrospective Studies; Sirolimus; Tacrolimus; Transplantation Conditioning; Whole-Body Irradiation; Young Adult

Topics: Histone Demethylases; Humans; Leukemia, Myeloid, Acute; Mechanistic Target of Rapamycin Complex 1; Signal Transduction; Sirolimus

Topics: Animals; Consolidation Chemotherapy; Everolimus; Leukemia, Myeloid, Acute; Nuts; Sirolimus; TOR Serine-Threonine Kinases; United Kingdom

Kinase inhibitors are important cancer therapeutics. Polypharmacology is commonly observed, requiring thorough target deconvolution to understand drug mechanism of action. Using chemical proteomics, we analyzed the target spectrum of 243 clinically evaluated kinase drugs. The data revealed previously unknown targets for established drugs, offered a perspective on the "druggable" kinome, highlighted (non)kinase off-targets, and suggested potential therapeutic applications. Integration of phosphoproteomic data refined drug-affected pathways, identified response markers, and strengthened rationale for combination treatments. We exemplify translational value by discovering SIK2 (salt-inducible kinase 2) inhibitors that modulate cytokine production in primary cells, by identifying drugs against the lung cancer survival marker MELK (maternal embryonic leucine zipper kinase), and by repurposing cabozantinib to treat FLT3-ITD-positive acute myeloid leukemia. This resource, available via the ProteomicsDB database, should facilitate basic, clinical, and drug discovery research and aid clinical decision-making.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Cytokines; Drug Discovery; fms-Like Tyrosine Kinase 3; Humans; Leukemia, Myeloid, Acute; Lung Neoplasms; Mice; Molecular Targeted Therapy; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Proteomics; Xenograft Model Antitumor Assays

Topics: Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Combined Modality Therapy; Female; Graft vs Host Disease; Humans; Immunosuppressive Agents; Leukemia, B-Cell; Leukemia, Myeloid, Acute; Lymphoma, Follicular; Male; Middle Aged; Mycophenolic Acid; Myelodysplastic Syndromes; Neoplasms, Second Primary; Peripheral Blood Stem Cell Transplantation; Retrospective Studies; Sirolimus

Although mTOR (mammalian target of rapamycin) activation is frequently observed in acute myeloid leukemia (AML) patients, the precise function and the downstream targets of mTOR are poorly understood. Here we revealed that PFKFB3, but not PFKFB1, PFKFB2 nor PFKFB4 was a novel downstream substrate of mTOR signaling pathway as PFKFB3 level was augmented after knocking down TSC2 in THP1 and OCI-AML3 cells. Importantly, PFKFB3 silencing suppressed glycolysis and cell proliferation of TSC2 silencing OCI-AML3 cells and activated apoptosis pathway. These results suggested that mTOR up-regulation of PFKFB3 was essential for AML cells survival. Mechanistically, Rapamycin treatment or Raptor knockdown reduced the expression of PFKFB3 in TSC2 knockdown cells, while Rictor silencing did not have such effect. Furthermore, we also revealed that mTORC1 up-regulated PFKFB3 was dependent on hypoxia-inducible factor 1α (HIF1α), a positive regulator of glycolysis. Moreover, PFKFB3 inhibitor PFK15 and rapamycin synergistically blunted the AML cell proliferation. Taken together, PFKFB3 was a promising drug target in AML patients harboring mTOR hyper-activation.

Topics: Apoptosis; Cell Line, Tumor; Cell Proliferation; Cell Survival; Enzyme Inhibitors; Gene Knockdown Techniques; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Leukemia, Myeloid, Acute; Mechanistic Target of Rapamycin Complex 1; Multiprotein Complexes; Phosphofructokinase-2; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins; Up-Regulation

Momordica anti-human immunodeficiency virus protein of 30 kDa (MAP30) has been shown to exhibit potent antitumor activities against several solid tumors. In the present investigation we demonstrated that MAP30 significantly inhibited the proliferation of acute myeloid leukemia (AML) HL-60 and THP-1 cell lines and patient AML cells through autophagy inhibition and apoptosis induction. Intriguingly, MAP30-induced cell death and apoptosis were partially rescued in combination with an autophagy activator rapamycin, and aggravated in combination with an autophagy inhibitor bafilomycin A1 in HL-60 cells, suggesting that autophagy is a pro-survival signal and its inhibition contributes to the induction of apoptosis in MAP30‑induced cell death. Further mechanism analysis demonstrated that MAP30 enhanced p300, and C646, a selective inhibitor of p300, markedly promoted autophagy and partially rescued the MAP30-induced cell death in HL-60 cells and patient AML cells. Collectively, our findings suggest that apoptosis and autophagy act cooperatively to elicit MAP30-induced cell death and MAP30 may be a potential antitumor drug candidate against AML.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Autophagy; Benzoates; Cell Line, Tumor; Cell Proliferation; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Macrolides; Momordica charantia; Nitrobenzenes; p300-CBP Transcription Factors; Plant Extracts; Pyrazoles; Pyrazolones; Ribosome Inactivating Proteins, Type 2; Sirolimus

Topics: Adolescent; Adult; Aged; Female; Follow-Up Studies; Hematopoietic Stem Cell Transplantation; Hodgkin Disease; Humans; Leukemia, Myeloid, Acute; Lymphoma, Non-Hodgkin; Male; Middle Aged; Multiple Myeloma; Remission Induction; Risk; Sirolimus; Transplantation Conditioning; Transplantation, Homologous; Treatment Outcome; Triazoles; Young Adult

Although multidrug approaches to cancer therapy are common, few strategies are based on rigorous scientific principles. Rather, drug combinations are largely dictated by empirical or clinical parameters. In the present study we developed a strategy for rational design of a regimen that selectively targets human acute myelogenous leukemia (AML) stem cells. As a starting point, we used parthenolide, an agent shown to target critical mechanisms of redox balance in primary AML cells. Next, using proteomic, genomic, and metabolomic methods, we determined that treatment with parthenolide leads to induction of compensatory mechanisms that include up-regulated NADPH production via the pentose phosphate pathway as well as activation of the Nrf2-mediated oxidative stress response pathway. Using this knowledge we identified 2-deoxyglucose and temsirolimus as agents that can be added to a parthenolide regimen as a means to inhibit such compensatory events and thereby further enhance eradication of AML cells. We demonstrate that the parthenolide, 2-deoxyglucose, temsirolimus (termed PDT) regimen is a potent means of targeting AML stem cells but has little to no effect on normal stem cells. Taken together our findings illustrate a comprehensive approach to designing combination anticancer drug regimens.

Topics: Antineoplastic Combined Chemotherapy Protocols; Deoxyglucose; Female; Gene Expression Regulation, Leukemic; Humans; Leukemia, Myeloid, Acute; Male; NADP; Neoplasm Proteins; Neoplastic Stem Cells; NF-E2-Related Factor 2; Sesquiterpenes; Sirolimus; Up-Regulation

In acute myeloid leukemia (AML), several signaling pathways such as the phosphatidylinositol-3-kinase/AKT and the mammalian target of rapamycin (PI3K/AKT/mTOR) pathway are deregulated and constitutively activated as a consequence of genetic and cytogenetic abnormalities. We tested the effectiveness of PI3K/AKT/mTOR-targeting therapies and tried to identify alterations that associate with treatment sensitivity. By analyzing primary samples and cell lines, we observed a wide range of cytotoxic activity for inhibition of AKT (MK-2206), mTORC1 (rapamycin) and PI3K/mTORC1/2 (BEZ-235) with a high sensitivity of cells carrying an MLL rearrangement. In vivo PI3K/mTOR inhibition delayed tumor progression, reduced tumor load and prolonged survival in an MLL-AF9(+)/FLT3-ITD(+) xenograft mouse model. By performing targeted amplicon sequencing in 38 MLL-AF9(+) and 125 cytogenetically normal AML patient samples, we found a high additional mutation rate for genes involved in growth factor signaling in 79% of all MLL-AF9(+) samples, which could lead to a possible benefit of this cohort. PI3K/mTOR inhibition for 24 h led to the cross-activation of the ERK pathway. Further in vitro studies combining PI3K/mTOR and ERK pathway inhibition revealed highly synergistic effects in apoptosis assays. Our data implicate a possible therapeutic benefit of PI3K/mTOR inhibition in the MLL-mutated subgroup. Inhibiting rescue pathways could improve the therapeutic efficacy of PI3K-targeted therapies in AML.

Topics: Animals; Antineoplastic Agents; Apoptosis; Drug Synergism; Gene Expression Regulation, Leukemic; Gene Rearrangement; Heterocyclic Compounds, 3-Ring; Humans; Imidazoles; Leukemia, Myeloid, Acute; Mice; Myeloid-Lymphoid Leukemia Protein; Oncogene Proteins, Fusion; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Quinolines; Signal Transduction; Sirolimus; Survival Analysis; TOR Serine-Threonine Kinases; Tumor Burden; Xenograft Model Antitumor Assays

Arsenic trioxide (ATO) has potent clinical activity in the treatment of patients with acute promyelocytic leukemia (APL), but is much less efficacious in acute myeloid leukemia (AML) lacking t(15;17) translocation. Recent studies have indicated that the addition of mammalian target of rapamycin (mTOR) inhibitors may increase the sensitivity of malignant cells to ATO. The aim of the present study was to test for possible synergistic effects of ATO and rapamycin at therapeutically achievable doses in non-APL AML cells. In HL-60 and U937 cell lines, the inhibitory effects of low concentrations of ATO and rapamycin were synergistic and more pronounced in U937 cells. The combination of drugs increased apoptosis in HL-60 cells and increased the percentage of cells in G(0)/G(1) phase in both cell lines. In U937 cells, rapamycin alone increased the activity of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and the addition of ATO decreased the level of phosphorylated ERK, Ser473 phosphorylated Akt and anti-apoptotic Mcl-1 protein. Primary AML cells show high sensitivity to growth-inhibitory effects of rapamycin alone or in combination with ATO. The results of the present study reveal the mechanism of the synergistic effects of two drugs at therapeutically achievable doses in non-APL AML cells.

Topics: Aged; Antineoplastic Agents; Apoptosis; Arsenic Trioxide; Arsenicals; Cell Cycle; Cell Line, Tumor; Cell Survival; Chromosomes, Human, Pair 15; Chromosomes, Human, Pair 17; Drug Synergism; Female; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Mitogen-Activated Protein Kinases; Oxides; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Translocation, Genetic

Sirolimus (SR) is a lipophilic macrocytic lactone with immunosuppressive properties (mTOR inhibitor) commonly used in solid organ transplantation and recently introduced in the prophylaxis and treatment of graft-versus-host disease. Its numerous side effects include: hyperlipidemia, arthralgias, noncardiac peripheral edema, thrombotic microangiopathy and interstitial pneumonitis. SR-associated pneumonitis is a rare but potentially serious complication due to its increasing utilization in transplant patients.. We report the case of a patient undergoing hematopoietic stem cell transplantation with severe respiratory distress and SR therapy.. Microbiological tests were all negative and other complications related to transplantation were discarded. The chest computed tomography of high-resolution showed pneumonitis. The SR therapy was interrupted and treatment was started with steroids with resolution of symptoms.. SR associated pneumonitis is a potentially fatal side effect. In patients treated with SR and respiratory failure, we must suspect this complication because early recognition along with drug discontinuation and steroid treatment is essential to reverse this complication.

Topics: Adrenal Cortex Hormones; Hematopoietic Stem Cell Transplantation; Humans; Immunosuppressive Agents; Leukemia, Myeloid, Acute; Male; Middle Aged; Oxygen Inhalation Therapy; Pneumonia; Respiratory Insufficiency; Sirolimus; Tomography, X-Ray Computed

Topics: AMP-Activated Protein Kinases; Animals; Autophagy; Cell Death; Humans; Imidazoles; Leukemia, Myeloid, Acute; Mechanistic Target of Rapamycin Complex 1; Mice; Molecular Targeted Therapy; Multiprotein Complexes; Pyrimidinones; Sirolimus; TOR Serine-Threonine Kinases

P21, a multifunctional cell cycle-regulatory molecule, regulates apoptotic cell death. In this study we examined the effect of altered p21 expression on the sensitivity of acute myeloid leukemia cells in response to HDAC inhibitor SAHA treatment and investigated the underlying mechanism.. Stably transfected HL60 cell lines were established in RPMI-1640 with supplementation of G-418. Cell viability was measured by MTT assay. Western blot was applied to assess the protein expression levels of target genes. Cell apoptosis was monitored by AnnexinV-PE/7AAD assay.. We showed HL60 cells that that didn't up-regulate p21 expression were more sensitive to SAHA-mediated apoptosis than NB4 and U937 cells that had increased p21 level. Enforced expression of p21 in HL60 cells reduced sensitivity to SAHA and blocked TRAIL-mediated apoptosis. Conversely, p21 silencing in NB4 cells enhanced SAHA-mediated apoptosis and lethality. Finally, we found that combined treatment with SAHA and rapamycin down-regulated p21 and enhanced apoptosis in AML cells.. We conclude that up-regulated p21 expression mediates resistance to SAHA via inhibition of TRAIL apoptotic pathway. P21 may serve as a candidate biomarker to predict responsiveness or resistance to SAHA-based therapy in AML patients. In addition, rapamycin may be an effective agent to override p21-mediated resistance to SAHA in AML patients.

Topics: Apoptosis; Base Sequence; Blotting, Western; Caspase 8; Cyclin-Dependent Kinase Inhibitor p21; Down-Regulation; Drug Resistance, Neoplasm; Histone Deacetylase Inhibitors; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; RNA Interference; Sirolimus; TNF-Related Apoptosis-Inducing Ligand; Up-Regulation

Both the occurrence and recurrence of acute leukemia (AL) might suggest the presence of leukemia stem cells. Side population (SP) cells, exhibiting stem cell-like properties, express ABCG2 (breast cancer resistance protein [BCRP]). This study revealed that over-expression of ABCG2 in Jurkat and HL60 cells led to an increased SP fraction, up-regulated levels of phosphorylated-PI3K and phosphorylated-Akt, and enhanced drug resistance, all of which could be attenuated by treatment with either the PI3K inhibitor LY294002 or the mTOR inhibitor rapamycin. ABCG2 expression and SP cell counts were further characterized in 222 adult AL patients at three disease stages: upon diagnosis, at remission and at refractory/relapse (R/R), while 10 healthy donors served as the normal controls. Only a small fraction of the ABCG2+population (0.05-12.3%) and SP cells (0.02-1.60%) were observed in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) patients. In the normal control population, the SP cell fraction represented a statistically higher percentage of total cells compared to the fraction of SP cells upon diagnosis or relapse in both AML and ALL. In addition, we demonstrated that ABCG2 expression and SP cell ratios can be upregulated by the inactivation of phosphatase and tensin homolog (PTEN) protein, achieved in this study by removing inhibition of the PI3K/Akt pathway. Collectively, this study suggests that the PTEN/PI3K/Akt pathway up-regulates ABCG2 expression and the SP cell population and is a potential AL-specific treatment target worth investigating further.

Topics: Adolescent; Adult; Aged; Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Case-Control Studies; Chromones; Female; Gene Expression Regulation, Leukemic; HL-60 Cells; Humans; Jurkat Cells; Leukemia, Myeloid, Acute; Male; Middle Aged; Mitoxantrone; Morpholines; Neoplasm Proteins; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Signal Transduction; Sirolimus; Stem Cells; Young Adult

Simultaneous inhibition of deregulated cancer kinome using rationally designed nanomedicine is an advanced therapeutic approach. Herein, we have developed a polymer-protein core-shell nanomedicine to inhibit critically aberrant pro-survival kinases (mTOR, MAPK and STAT5) in primitive (CD34(+)/CD38(-)) Acute Myeloid Leukemia (AML) cells. The nanomedicine consists of poly-lactide-co-glycolide core (~250 nm) loaded with mTOR inhibitor, everolimus, and albumin shell (~25 nm thick) loaded with MAPK/STAT5 inhibitor, sorafenib and the whole construct was surface conjugated with monoclonal antibody against CD33 receptor overexpressed in AML. Electron microscopy confirmed formation of core-shell nanostructure (~290 nm) and flow cytometry and confocal studies showed enhanced cellular uptake of targeted nanomedicine. Simultaneous inhibition of critical kinases causing synergistic lethality against leukemic cells, without affecting healthy blood cells, was demonstrated using immunoblotting, cytotoxicity and apoptosis assays. This cell receptor plus multi-kinase targeted core-shell nanomedicine was found better specific and tolerable compared to current clinical regime of cytarabine and daunorubicin.. These authors demonstrate simultaneous inhibition of critical kinases causing synergistic lethality against leukemic cells, without affecting healthy blood cells by using rationally designed polymer-protein core-shell nanomedicine, provoding an advanced method to eliminate cancer cells, with the hope of future therapeutic use.

Topics: Antineoplastic Agents; Cell Line, Tumor; Drug Delivery Systems; Everolimus; Humans; Leukemia, Myeloid, Acute; Mitogen-Activated Protein Kinases; Models, Molecular; Nanomedicine; Niacinamide; Phenylurea Compounds; Polyglactin 910; Protein Kinase Inhibitors; Sialic Acid Binding Ig-like Lectin 3; Sirolimus; Sorafenib; STAT5 Transcription Factor; TOR Serine-Threonine Kinases

Acute myeloid leukemia (AML) is a heterogeneous and aggressive malignancy with poor overall survival. Constitutive as well as cytokine-initiated activation of PI3K/Akt/mTOR signaling is a common feature of AML patients, and inhibition of this pathway is considered as a possible therapeutic strategy in AML. Human AML cells and different stromal cell populations were cultured under highly standardized in vitro conditions. We investigated the effects of mTOR inhibitors (rapamycin and temsirolimus) and PI3K inhibitors (GDC-0941 and 3-methyladenin (3-MA)) on cell proliferation and the constitutive release of angioregulatory mediators by AML and stromal cells. Primary human AML cells were heterogeneous, though most patients showed high CXCL8 levels and detectable release of CXCL10, Ang-1, HGF and MMP-9. Hierarchical clustering analysis showed that disruption of PI3K/Akt/mTOR pathways decreased AML cell release of CXCL8-11 for a large subset of patients, whereas the effects on other mediators were divergent. Various stromal cells (endothelial cells, fibroblasts, cells with osteoblastic phenotype) also showed constitutive release of angioregulatory mediators, and inhibitors of both the PI3K and mTOR pathway had anti-proliferative effects on stromal cells and resulted in decreased release of these angioregulatory mediators. PI3K and mTOR inhibitors can decrease constitutive cytokine release both by AML and stromal cells, suggesting potential direct and indirect antileukemic effects.

Topics: Adenine; Adult; Aged; Aged, 80 and over; Bone Marrow Cells; Cytogenetics; Cytokines; Female; Humans; Indazoles; Leukemia, Myeloid, Acute; Male; Middle Aged; Molecular Targeted Therapy; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; RNA, Messenger; Signal Transduction; Sirolimus; Stromal Cells; Sulfonamides; TOR Serine-Threonine Kinases; Young Adult

Aurora kinases are overexpressed in large numbers of tumors and considered as potential therapeutic targets. In this study, we found that the Aurora kinases inhibitors MK-0457 (MK) and ZM447439 (ZM) induced polyploidization in acute myeloid leukemia (AML) cell lines. The level of glycolytic metabolism was significantly increased in the polyploidy cells, which were sensitive to glycolysis inhibitor 2-deoxy-D-glucose (2DG), suggesting that polyploidy cells might be eliminated by metabolism deprivation. Indeed, inhibition of mTOR pathway by mTOR inhibitors (rapamycin and PP242) or 2DG promoted not only apoptosis but also autophagy in the polyploidy cells induced by Aurora inhibitors. Mechanically, PP242 or2DGdecreased the level of glucose uptake and lactate production in polyploidy cells as well as the expression of p62/SQSTM1. Moreover, knockdown of p62/SQSTM1 sensitized cells to the Aurora inhibitor whereas overexpression of p62/SQSTM1 reduced drug efficacy. Thus, our results revealed that inhibition of mTOR pathway decreased the glycolytic metabolism of the polyploidy cells, and increased the efficacy of Aurora kinases inhibitors, providing a novel approach of combination treatment in AML.

Topics: Adaptor Proteins, Signal Transducing; Antineoplastic Agents; Aurora Kinases; Benzamides; Cell Line, Tumor; Deoxyglucose; Gene Expression Regulation, Neoplastic; Glucose; Glycolysis; HL-60 Cells; Humans; Indoles; Lactic Acid; Leukemia, Myeloid, Acute; Piperazines; Polyploidy; Purines; Quinazolines; Sequestosome-1 Protein; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; U937 Cells

Targeted therapies are all the rage in oncology research these days. The problem remains as to how to confirm that the target is actually being hit in vivo. This report describes the application of phospho-specific flow cytometry to establish in vivo target inhibition in real time.

Topics: Antineoplastic Combined Chemotherapy Protocols; Cytarabine; Etoposide; Female; Humans; Leukemia, Myeloid, Acute; Male; Mitoxantrone; Ribosomal Protein S6; Sirolimus; TOR Serine-Threonine Kinases

Rapamycin and its derivatives have been proposed in the treatment of leukemia based on their cytostatic effects, but their possible role in differentiation therapy is less explored. The aim of the present study was to investigate the possible beneficial effects of the combination of rapamycin and dimethyl sulfoxide (DMSO) on growth arrest and differentiation of acute myelogenous leukemia (AML) cells. In myeloblastic HL-60, promyelocytic NB4, monocytic U937, immature KG-1 and erythro-megakaryocytic K562 cell lines, rapamycin alone had modest inhibitory effects, DMSO inhibited proliferation in a dose-dependent manner, and the combination of rapamycin and DMSO reduced the number of viable cells significantly more than either agent alone. In NB4 cells, rapamycin had no statistically significant effects on the DMSO-mediated increase in expression of CD11b, but increased apoptosis. These results demonstrate that rapamycin enhances DMSO-mediated growth arrest, and suggest that mTOR (mammalian target of rapamycin) inhibitors may have beneficial effects in differentiation therapy of AML.

Topics: Cell Proliferation; Dimethyl Sulfoxide; Drug Synergism; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Ribosomal Protein S6 Kinases, 70-kDa; Sirolimus; TOR Serine-Threonine Kinases

The interactions between the bone marrow (BM) microenvironment and acute myeloid leukemia (AML) is known to promote survival of AML cells. In this study, we used reverse phase-protein array (RPPA) technology to measure changes in multiple proteins induced by stroma in leukemic cells. We then investigated the potential of an mTOR kinase inhibitor, PP242, to disrupt leukemia/stroma interactions, and examined the effects of PP242 in vivo using a mouse model. Using RPPA, we confirmed that multiple survival signaling pathways, including the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR), were up-regulated in primary AML cells cocultured with stroma. PP242 effectively induced apoptosis in primary samples cultured with or without stroma. Mechanistically, PP242 attenuated the activities of mTORC1 and mTORC2, sequentially inhibited phosphorylated AKT, S6K, and 4EBP1, and concurrently suppressed chemokine receptor CXCR4 expression in primary leukemic cells and in stromal cells cultured alone or cocultured with leukemic cells. In the in vivo leukemia mouse model, PP242 inhibited mTOR signaling in leukemic cells and demonstrated a greater antileukemia effect than rapamycin. Our findings indicate that disrupting mTOR/AKT signaling with a selective mTOR kinase inhibitor can effectively target leukemic cells within the BM microenvironment.

Topics: Animals; Antibiotics, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Blotting, Western; Bone Marrow; Cell Proliferation; Coculture Techniques; Flow Cytometry; Humans; Indoles; Leukemia, Experimental; Leukemia, Myeloid, Acute; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Mesenchymal Stem Cells; Mice; Mice, SCID; Multiprotein Complexes; Phosphorylation; Protein Array Analysis; Protein Kinase Inhibitors; Purines; Real-Time Polymerase Chain Reaction; Receptors, CXCR4; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Signaling through the phosphatidylinositol 3-kinase (PI3K) pathway and its downstream effectors, Akt and mechanistic target of rapamycin (mTOR), is aberrantly activated in acute myeloid leukemia (AML) patients, where it contributes to leukemic cell proliferation, survival, and drug-resistance. Thus, inhibiting mTOR signaling in AML blasts could enhance their sensitivity to cytotoxic agents. Preclinical data also suggest that allosteric mTOR inhibition with rapamycin impaired leukemia initiating cells (LICs) function. In this study, we assessed the therapeutic potential of a combination consisting of temsirolimus [an allosteric mTOR complex 1 (mTORC1) inhibitor] with clofarabine, a nucleoside analogue with potent inhibitory effects on both ribonucleotide reductase and DNA polymerase. The drug combination (CLO-TOR) displayed synergistic cytotoxic effects against a panel of AML cell lines and primary cells from AML patients. Treatment with CLO-TOR induced a G₀/G₁-phase cell cycle arrest, apoptosis, and autophagy. CLO-TOR was pro-apoptotic in an AML patient blast subset (CD34⁺/CD38⁻/CD123⁺), which is enriched in putative leukemia initiating cells (LICs). In summary, the CLO-TOR combination could represent a novel valuable treatment for AML patients, also in light of its efficacy against LICs.

Topics: Adenine Nucleotides; ADP-ribosyl Cyclase 1; Allosteric Regulation; Antigens, CD34; Antimetabolites, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Arabinonucleosides; Autophagy; Cell Line, Tumor; Clofarabine; Dose-Response Relationship, Drug; Drug Synergism; Eukaryotic Initiation Factor-4F; Extracellular Signal-Regulated MAP Kinases; G1 Phase Cell Cycle Checkpoints; Humans; Interleukin-3 Receptor alpha Subunit; Leukemia, Myeloid, Acute; Membrane Glycoproteins; Phosphatidylinositol 3-Kinase; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-myc; Signal Transduction; Sirolimus; STAT3 Transcription Factor; Time Factors; TOR Serine-Threonine Kinases; Tumor Cells, Cultured

A novel strategy has been suggested to enhance rapamycin-based cancer therapy through combining mammalian target of rapamycin (mTOR)-inhibitors with an inhibitor of the phosphatydilinositol 3-kinase PI3K/Akt or mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway. However, recent study demonstrated the potentiating effect of rapamycin on all-trans-retinoic acid (ATRA)-mediated differentiation of acute myelogenous leukemia (AML) cells, prompting us to investigate the effects of longitudinal inhibition of PI3K/Akt/mTOR signaling pathway on both proliferation and differentiative capacity of AML. In NB4, HL-60, U937 and K562 cell lines, rapamycin exerted minimal antiproliferative effects, and combining PI3K inhibitor LY 294002 and rapamycin inhibited proliferation more than LY 294002 alone. Rapamycin potentiated differentiation of ATRA-treated NB4 cells, but the combination of rapamycin and LY 294002 inhibited the expression of CD11b in both ATRA- and phorbol myristate acetate (PMA)-stimulated cells more than PI3K inhibitor alone. These results demonstrate that, although the combination of PI3K inhibitor and rapamycin is more effective in inhibiting proliferation of AML, the concomitant inhibition of PI3K and mTOR by LY 294002 and rapamycin has more inhibitory effects on ATRA-mediated differentiation than the presence of PI3K-inhibitor alone, and diminishes positive effects of rapamycin on leukemia cell differentiation.

Topics: Blotting, Western; Cell Cycle; Cell Differentiation; Cell Proliferation; Drug Synergism; Extracellular Signal-Regulated MAP Kinases; Humans; Leukemia, Myeloid, Acute; Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tretinoin; Tumor Cells, Cultured

To determine whether mTORC2 and rapamycin-insensitive (RI)-mTORC1 complexes are present in acute myeloid leukemia (AML) cells and to examine the effects of dual mTORC2/mTORC1 inhibition on primitive AML leukemic progenitors.. Combinations of different experimental approaches were used, including immunoblotting to detect phosphorylated/activated forms of elements of the mTOR pathway in leukemic cell lines and primary AML blasts; cell-proliferation assays; direct assessment of mRNA translation in polysomal fractions of leukemic cells; and clonogenic assays in methylcellulose to evaluate leukemic progenitor-colony formation.. mTORC2 complexes are active in AML cells and play critical roles in leukemogenesis. RI-mTORC1 complexes are also formed and regulate the activity of the translational repressor 4E-BP1 in AML cells. OSI-027 blocks mTORC1 and mTORC2 activities and suppresses mRNA translation of cyclin D1 and other genes that mediate proliferative responses in AML cells. Moreover, OSI-027 acts as a potent suppressor of primitive leukemic precursors from AML patients and is much more effective than rapamycin in eliciting antileukemic effects in vitro.. Dual targeting of mTORC2 and mTORC1 results in potent suppressive effects on primitive leukemic progenitors from AML patients. Inhibition of the mTOR catalytic site with OSI-027 results in suppression of both mTORC2 and RI-mTORC1 complexes and elicits much more potent antileukemic responses than selective mTORC1 targeting with rapamycin.

Topics: Antibiotics, Antineoplastic; Apoptosis Regulatory Proteins; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Dose-Response Relationship, Drug; Gene Expression Regulation, Leukemic; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Mechanistic Target of Rapamycin Complex 1; Multiprotein Complexes; Neoplastic Stem Cells; Oncogene Protein v-akt; Phosphorylation; Proteins; Ribosomal Protein S6 Kinases, 70-kDa; RNA-Binding Proteins; Sirolimus; TOR Serine-Threonine Kinases; Transcription Factors; U937 Cells

Tyrosine kinases such as SRC family kinases (SFKs) as well as the mammalian target of rapamycin (mTOR) serine/threonine kinase are often constitutively activated in acute myeloid leukemia (AML) and hence constitute potential therapeutic targets. Here we demonstrate that the epidermal growth factor receptor (EGFR) inhibitor erlotinib, which has previously been shown to mediate antiproliferative/cytotoxic off-target effects in myelodysplastic syndrome (MDS) and AML blasts, reduces SFK overactivation. Erlotinib induced an arrest in the G 1 phase of the cell cycle that, in cells with constitutive SFK activation, could be recapitulated by chemical inhibition of SFKs with 3-(4-chlorophenyl)1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-α]pyrimidin-4-amine (PP2). Moreover, erlotinib inhibited the phosphorylation of mTOR targets like p70 (SK6) , stimulated the maturation of the autophagic marker LC3 and promoted the formation of autophagosomes. Notably, PP2 and the mTOR inhibitor rapamycin had a similar cell cycle-arresting activity to erlotinib, but neither of these compounds alone induced significant levels of cell death. Altogether, these results suggest that the therapeutic off-target effect of erlotinib may be linked to, yet cannot be entirely explained by, the inhibition of oncogenic signaling via SFKs and mTOR. Thus, combination therapies with erlotinib and rapamycin might be beneficial for MDS and AML patients.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Autophagy; Biomarkers, Tumor; Enzyme Activation; Erlotinib Hydrochloride; Flow Cytometry; G1 Phase Cell Cycle Checkpoints; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Microscopy, Electron; Microscopy, Fluorescence; Microtubule-Associated Proteins; Phosphorylation; Pyrimidines; Quinazolines; Signal Transduction; Sirolimus; src-Family Kinases; Stilbenes; TOR Serine-Threonine Kinases

Human cancers, including acute myeloid leukemia (AML), commonly display constitutive phosphoinositide 3-kinase (PI3K) AKT signaling. However, the exact role of AKT activation in leukemia and its effects on hematopoietic stem cells (HSCs) are poorly understood. Several members of the PI3K pathway, phosphatase and tensin homolog (Pten), the forkhead box, subgroup O (FOXO) transcription factors, and TSC1, have demonstrated functions in normal and leukemic stem cells but are rarely mutated in leukemia. We developed an activated allele of AKT1 that models increased signaling in normal and leukemic stem cells. In our murine bone marrow transplantation model using a myristoylated AKT1 (myr-AKT), recipients develop myeloproliferative disease, T-cell lymphoma, or AML. Analysis of the HSCs in myr-AKT mice reveals transient expansion and increased cycling, associated with impaired engraftment. myr-AKT-expressing bone marrow cells are unable to form cobblestones in long-term cocultures. Rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR) rescues cobblestone formation in myr-AKT-expressing bone marrow cells and increases the survival of myr-AKT mice. This study demonstrates that enhanced AKT activation is an important mechanism of transformation in AML and that HSCs are highly sensitive to excess AKT/mTOR signaling.

Topics: Animals; Antibiotics, Antineoplastic; Bone Marrow Cells; Bone Marrow Transplantation; Cell Division; Cell Line; Hematopoietic Stem Cells; Humans; Intracellular Signaling Peptides and Proteins; Kidney; Leukemia, Myeloid, Acute; Lymphoma, T-Cell; Mice; Mice, Inbred C57BL; Myeloproliferative Disorders; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Signal Transduction; Sirolimus; Spleen; TOR Serine-Threonine Kinases

Topics: Antibiotics, Antineoplastic; Bone Marrow Transplantation; Graft vs Host Disease; Humans; Immunosuppressive Agents; Leukemia, Myeloid, Acute; Lung Neoplasms; Male; Middle Aged; Sarcoma, Kaposi; Sirolimus; Transplantation, Homologous

Differentiation-inducing therapy by agents such as 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) represents a useful approach for the treatment of acute myelogenous leukemia (AML). We previously showed that Gemini-23-yne-26,27-hexafluoro-D(3) inhibited the proliferation of MCF-7 breast cancer cells in association with inhibition of the mammalian target of rapamycin (mTOR) signaling. This study explored the drug interaction of 1,25(OH)(2)D(3) and rapamycin analog RAD001 (everolimus) in AML cells.. Effects of RAD001 and 1,25-(OH)(2)D(3) on the proliferation and differentiation of U937 cells were assessed by colony-forming assay and quantification of CD11b cell surface antigens and their endocytic capability, respectively. Effects of RAD001 and 1,25-(OH)(2)D(3) on Akt/mTOR complex-1 (mTORC1) signaling and cell-cycle-related molecules were explored by Western blot analysis. The reporter gene and chromatin immunoprecipitation assays were employed to examine the effects of RAD001 and 1,25-(OH)(2)D(3) on the promoter of the p21(waf1) gene. U937 murine xenograft model was utilized to explore the effects of RAD001 and 1,25-(OH)(2)D(3) in vivo.. RAD001 potentiated the ability of 1,25(OH)(2)D(3) to induce growth arrest and differentiation of AML cells in parallel with downregulation of the levels of p-S6K and p-4E-BP1, substrates of mTORC1. In addition, RAD001 significantly enhanced 1,25(OH)(2)D(3)-mediated transcriptional activity of p21(waf1) in association with increased levels of the acetylated forms of histone H3 and vitamin D receptor bound to the p21(waf1) promoter in U937 cells. Moreover, RAD001 (3 mg/kg, every another day) significantly enhanced 1,25(OH)(2)D(3)-induced growth inhibition of U937 tumor xenografts in nude mice without adverse effects.. Concomitant administration of 1,25(OH)(2)D(3) and the mTORC1 inhibitor may be a promising treatment strategy for individuals with AML.

Topics: Acetylation; Animals; Calcitriol; CD11b Antigen; Cell Cycle; Cell Differentiation; Colony-Forming Units Assay; Cyclin-Dependent Kinase Inhibitor p21; Down-Regulation; Drug Synergism; Everolimus; Female; Gene Expression Regulation, Leukemic; Histones; Humans; Immunosuppressive Agents; Leukemia, Myeloid, Acute; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Inbred BALB C; Mice, Nude; Multiprotein Complexes; Promoter Regions, Genetic; Proteins; Receptors, Calcitriol; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transcription Factors; U937 Cells; Vitamins; Xenograft Model Antitumor Assays

We have previously shown that the plant-derived compound parthenolide (PTL) can impair the survival and leukemogenic activity of primary human acute myeloid leukemia (AML) stem cells. However, despite the activity of this agent, PTL also induces cellular protective responses that likely function to reduce its overall cytotoxicity. Thus, we sought to identify pharmacologic agents that enhance the antileukemic potential of PTL. Toward this goal, we used the gene expression signature of PTL to identify compounds that inhibit cytoprotective responses by performing chemical genomic screening of the Connectivity Map database. This screen identified compounds acting along the phosphatidylinositol 3-kinase and mammalian target of rapamycin pathways. Compared with single agent treatment, exposure of AML cells to the combination of PTL and phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitors significantly decreased viability of AML cells and reduced tumor burden in vitro and in murine xenotransplantation models. Taken together, our data show that rational drug combinations can be identified using chemical genomic screening strategies and that inhibition of cytoprotective functions can enhance the eradication of primary human AML cells.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Biomarkers, Tumor; Blotting, Western; Bone Marrow; Cell Proliferation; Cells, Cultured; Drug Synergism; Enzyme Inhibitors; Flow Cytometry; Gene Expression Profiling; Humans; Leukemia, Myeloid, Acute; Mice; Mice, Inbred NOD; Mice, SCID; Oligonucleotide Array Sequence Analysis; Phosphatidylinositol 3-Kinases; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sesquiterpenes; Sirolimus; TOR Serine-Threonine Kinases

In order to investigate the effect of rapamycin on the proliferation of human acute myeloid leukemia (AML) cells, the sensitive cells HL-60 and multidrug-resistant HL-60/VCR cells were chosen as research objects. The proliferation of cells was detected by growth curve method. The flow cytometer was used to analyze cell cycle. The expression of P-glycoprotein (Pgp) was determined by Western blot. The results demonstrated that there was a significant difference of cell growth inhibition rate between control group and rapamycin group (p < 0.05). The cell growth inhibition rate was dose- and time- dependent (p < 0.05). Flow cytometry detection showed that the cell percentage of G(1) phase in rapamycin group was higher than that in group without rapamycin, and that of S phase was lower. The cell growth inhibition rate in 50 nmol/L and 100 nmol/L rapamycin plus daunorubicin (DNR) group was more than that in DNR alone group (p < 0.05), especially when DNR was added at 24 hours interval after RAP. The expression of Pgp of HL-60/VCR cells was inhibited by rapamycin. It is concluded that the rapamycin can inhibit the proliferation of sensitive HL-60 and multidrug resistant HL-60/VCR cells. It can also increase sensitivity of HL-60 and HL-60/VCR cells to DNR, which provides new strategy for the therapy of refractory AML.

Topics: Antibiotics, Antineoplastic; ATP Binding Cassette Transporter, Subfamily B, Member 1; Cell Proliferation; Drug Resistance, Multiple; Drug Resistance, Neoplasm; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Sirolimus; Vincristine

The mammalian target of rapamycin (mTOR) has recently been implicated in leukaemic cell growth, tumour-associated angiogenesis and expression of vascular endothelial growth factor (VEGF). We examined whether mTOR plays a role as regulator of growth and VEGF-expression in acute myeloid leukaemia (AML). Three mTOR-targeting drugs, rapamycin, everolimus (RAD001) and CCI-779, were applied. The effects of these drugs on growth, survival, apoptosis and VEGF expression in primary AML cells and various AML cell lines were examined.. Growth of AML cells and AML-derived cell lines was assessed by (3)H-thymidine incorporation, survival was examined by light- and electron microscopy, by Tunel assay and by AnnexinV-staining, and the expression of VEGF by Northern blotting, RT-PCR and ELISA.. Rapamycin was found to counteract growth in the AML cell lines U937 and KG1a as well as in primary AML cells in 14/18 patients examined. The effects of rapamycin and its derivatives were dose-dependent (IC(50): 10 pM-100 nM). It was also found that exposure to mTOR-targeting drugs resulted in apoptosis and in decreased expression of VEGF in leukaemic cells.. mTOR-targeting drugs exert antileukaemic effects on AML cells in vitro through multiple actions, including direct inhibition of proliferation, induction of apoptosis and suppression of VEGF. Based on this study and other studies, mTOR can be regarded as a potential drug target in AML.

Topics: Adult; Aged; Antibiotics, Antineoplastic; Apoptosis; Dose-Response Relationship, Drug; Female; Gene Expression Regulation, Neoplastic; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Protein Kinases; Sirolimus; TOR Serine-Threonine Kinases; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A

The deregulation of translation markedly contributes to the malignant phenotype in cancers, and the assembly of the translation initiating complex eIF4F is the limiting step of this process. The mammalian Target of Rapamycin Complex 1 (mTORC1) is thought to positively regulate eIF4F assembly and subsequent oncogenic protein synthesis through 4E-BP1 phosphorylation. We showed here that the translation inhibitor 4EGI-1 decreased the clonogenic growth of leukemic progenitors and induced apoptosis of blast cells, with limited toxicity against normal hematopoiesis, which emphasize the importance of translation deregulation in acute myeloid leukemia (AML) biology. However, the mTORC1 inhibitor RAD001 (a rapamycin derivate) did not induce AML blast cell apoptosis. We herein demonstrated that mTORC1 disruption using raptor siRNA or RAD001 failed to inhibit 4E-BP1 phosphorylation in AML. Moreover, RAD001 failed to inhibit eIF4F assembly, to decrease the proportion of polysome-bound c-Myc mRNA, and to reduce the translation-dependent accumulation of oncogenic proteins. We identified the Pim-2 serine/threonine kinase as mainly responsible for 4E-BP1 phosphorylation on the S(65) residue and subsequent translation control in AML. Our results strongly implicate an mTORC1-independent deregulation of oncogenic proteins synthesis in human myeloid leukemogenesis. Direct inhibition of the translation initiating complex thus represents an attractive option for the development of new therapies in AML.

Topics: Adaptor Proteins, Signal Transducing; Antibiotics, Antineoplastic; Apoptosis; Cell Cycle Proteins; Cell Proliferation; Cells, Cultured; Drug Evaluation, Preclinical; Drug Resistance; Everolimus; Hematopoiesis; Humans; Hydrazones; Leukemia, Myeloid, Acute; Mechanistic Target of Rapamycin Complex 1; Multiprotein Complexes; Neoplastic Stem Cells; Nitro Compounds; Phosphoproteins; Phosphorylation; Protein Biosynthesis; Protein Synthesis Inhibitors; Proteins; Sirolimus; Thiazoles; TOR Serine-Threonine Kinases; Transcription Factors

Our study explored the drug interaction of all-trans retinoic acid (ATRA) and RAD001 (everolimus), the inhibitor of mammalian target of rapamycin complex 1 (mTORC1), in acute myelogenous leukemia (AML) NB4 and HL60 cells. RAD001 (10 nM) significantly enhanced the ATRA-induced growth arrest and differentiation of these cells, as measured by colony-forming assay and cell cycle analysis, and expression of CD11b cell surface antigen and nitroblue tetrazolium reduction, respectively. ATRA (0.1-1 microM) upregulated levels of RTP801, a negative regulator of mTORC1, and inhibited mTORC1 signaling as assessed by measurement of the levels of p-p70S6K and p-4E-BP1 in HL60 and NB4 cells. ATRA (0.1-1 microM) in combination with RAD001 (10 nM) strikingly downregulated the levels of p-70S6K and p-4E-BP1 without affecting the total amount of these proteins. Notably, RAD001 (10 nM) significantly augmented ATRA-induced expression of CCAAT/enhancer-binding protein epsilon (C/EBPepsilon) and p27(kip1) and downregulated levels of c-Myc in these cells. Furthermore, RAD001 (5 mg/kg) enhanced the ability of ATRA (10 mg/kg) to inhibit the proliferation of HL60 cells growing as tumor xenografts in immune-deficient nude mice. Taken together, concomitant blockade of the RA and mTORC1 signaling may be a promising treatment strategy for individuals with AML.

Topics: Animals; Antineoplastic Agents; Biomarkers, Tumor; Blotting, Western; CCAAT-Enhancer-Binding Proteins; CD11b Antigen; Cell Cycle; Cell Differentiation; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p27; Down-Regulation; Drug Synergism; Everolimus; Female; Gene Expression Regulation, Neoplastic; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Inbred BALB C; Mice, Nude; Multiprotein Complexes; Neoplasm Proteins; Nitroblue Tetrazolium; Proteins; Proto-Oncogene Proteins c-myc; Receptors, Retinoic Acid; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transcription Factors; Transplantation, Heterologous; Tretinoin; Tumor Stem Cell Assay; Up-Regulation

This study found that MS-275, a novel synthetic benzamide histone deacetylase inhibitor (HDACI), blocked Akt/mammalian target of rapamycin (mTOR) signaling in acute myelogenous leukemia (AML) HL60 and acute promyelocytic leukemia (APL) NB4 cells, as assessed by decreased levels of the phosphorylated (p)-Akt, p-p70 ribosomal S6 kinase (p70S6K) and p-S6K by western blot analysis. Interestingly, further inactivation of mTOR by rapamycin analog RAD001 (everolimus) significantly enhanced MS-275-mediated growth inhibition and apoptosis of these cells in parallel with enhanced upregulation of p27(kip1) and downregulation of c-Myc. In addition, RAD001 potentiated the ability of MS-275 to induce differentiation of HL60 and NB4 cells, as measured by the expression of CD11b cell surface antigens, as well as reduction of nitroblue tetrazolium. Importantly, RAD001 potentiated the ability of MS-275 to induce the expression of the myeloid differentiation-related transcription factor, CCAAT enhancer-binding protein-epsilon, in these cells in association with enhanced acetylation of histone H3 on its promoter. Furthermore, RAD001 (5 mg/kg) significantly enhanced the effects of MS-275 (10 mg/kg) to inhibit proliferation of HL60 tumor xenografts in nude mice without adverse effects. Taken together, concomitant administration of an HDACI and an mTOR inhibitor may be a promising treatment strategy for the individuals with a subset of human leukemia.

Topics: Acetylation; Animals; Apoptosis; Benzamides; CCAAT-Enhancer-Binding Proteins; Cell Differentiation; Cell Division; Drug Synergism; Everolimus; Female; Histone Deacetylase 1; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; HL-60 Cells; Humans; Immunosuppressive Agents; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Inbred BALB C; Mice, Nude; Multiprotein Complexes; Promoter Regions, Genetic; Proteins; Proto-Oncogene Proteins c-akt; Pyridines; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transcription Factors; Xenograft Model Antitumor Assays

The phosphatidylinositol 3-kinase (PI3K)/Akt and mTORC1 pathways are frequently activated, representing potential therapeutic targets in acute myeloid leukemia (AML). In 19 AML samples with constitutive PI3K/Akt activation, the rapamycin derivative inhibitor everolimus (RAD001) increased Akt phosphorylation. This mTOR C1-mediated Akt up-regulation was explained by an insulin-like growth factor-1 (IGF-1)/IGF-1 receptor autocrine loop: (1) blast cells expressed functional IGF-1 receptors, and IGF-1-induced Akt activation was increased by RAD001, (2) a neutralizing anti-IGF-1R alpha-IR3 monoclonal antibody reversed the RAD001-induced Akt phosphorylation, and (3) autocrine production of IGF-1 was detected in purified blast cells by quantitative reverse transcription-polymerase chain reaction and immunofluorescence. This RAD001-induced PI3K/Akt up-regulation was due to an up-regulated expression of the IRS2 adaptor. Finally, we observed that concomitant inhibition of mTORC1 and PI3K/Akt by RAD001 and IC87114 induced additive antiproliferative effects. Our results suggest that dual inhibition of the mTORC1 complex and the IGF-1/IGF-1R/PI3K/Akt pathway in AML may enhance the efficacy of mTOR inhibitors in treatment of this disease.

Topics: Cell Division; Everolimus; Humans; Immunosuppressive Agents; In Vitro Techniques; Leukemia, Myeloid, Acute; Mechanistic Target of Rapamycin Complex 1; Multiprotein Complexes; Phosphatidylinositol 3-Kinases; Phosphorylation; Proteins; Proto-Oncogene Proteins c-akt; Receptor, IGF Type 1; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transcription Factors; Up-Regulation

Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Cell Line, Tumor; Humans; Leukemia, Myeloid, Acute; Protein Kinases; Sirolimus; TOR Serine-Threonine Kinases

Receptor or nonreceptor tyrosine kinases (TKs) are known to play an important role in leukemogenesis. Here we studied the level of protein tyrosine phosphorylations in a series of fresh AML samples and evaluated the effect of TK inhibitors. Compared with normal hematopoietic progenitors, a high level of tyrosine phosphorylation was detected in most acute myeloid leukemia (AML) samples. The Src family kinases (SFKs) appeared constitutively activated in most cases, including in the CD34(+)CD38(-)CD123(+) compartment as revealed by the level of phosphorylated tyrosine 416. Lyn was the major SFK family member expressed in an active form in AML cells where it was abnormally distributed throughout the plasma membrane and the cytosol as opposed to normal hematopoietic progenitors. The SFK inhibitor, PP2, strongly reduced the global level of tyrosine phosphorylations, inhibited cell proliferation, and induced apoptosis in patient samples without affecting normal granulomonocytic colony forming units. Moreover, silencing Lyn expression by small interfering RNA in primary AML cells strongly inhibited proliferation. Interestingly, a link between Lyn and the mTOR pathway was observed as PP2 and a Lyn knockdown both affected the phosphorylation of mTOR targets without inhibiting Akt phosphorylation. Lyn should be considered as a novel pharmacologic target for AML therapy.

Topics: Apoptosis; Bone Marrow Cells; Cell Line, Tumor; Chromones; Enzyme Inhibitors; Flow Cytometry; Humans; Leukemia, Myeloid, Acute; Morpholines; Oncogene Proteins, Viral; Phosphotyrosine; Reference Values; RNA, Small Interfering; Sirolimus; src-Family Kinases; U937 Cells

Topics: Aged; Aged, 80 and over; Antibiotics, Antineoplastic; Humans; Leukemia, Myeloid, Acute; Myelodysplastic Syndromes; Sirolimus; Treatment Failure

The mTOR complex 2 (mTORC2) containing mTOR and rictor is thought to be rapamycin insensitive and was recently shown to regulate the prosurvival kinase AKT by phosphorylation on Ser473. We investigated the molecular effects of mTOR inhibition by the rapamycin derivatives (RDs) temsirolimus (CCI-779) and everolimus (RAD001) in acute myeloid leukemia (AML) cells. Unexpectedly, RDs not only inhibited the mTOR complex 1 (mTORC1) containing mTOR and raptor with decreased p70S6K, 4EPB1 phosphorylation, and GLUT1 mRNA, but also blocked AKT activation via inhibition of mTORC2 formation. This resulted in suppression of phosphorylation of the direct AKT substrate FKHR and decreased transcription of D-cyclins in AML cells. Similar observations were made in samples from patients with hematologic malignancies who received RDs in clinical studies. Our study provides the first evidence that rapamycin derivatives inhibit AKT signaling in primary AML cells both in vitro and in vivo, and supports the therapeutic potential of mTOR inhibition strategies in leukemias.

Topics: Adaptor Proteins, Signal Transducing; Carrier Proteins; Cyclin D; Cyclins; Drug Screening Assays, Antitumor; Enzyme Activation; Everolimus; Gene Expression Regulation, Leukemic; Glucose Transporter Type 1; Humans; Immunosuppressive Agents; Leukemia, Myeloid, Acute; Phosphorylation; Protein Kinase Inhibitors; Protein Kinases; Proteins; Proto-Oncogene Proteins c-akt; Rapamycin-Insensitive Companion of mTOR Protein; Regulatory-Associated Protein of mTOR; Ribosomal Protein S6 Kinases, 70-kDa; RNA, Messenger; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transcription, Genetic; U937 Cells

Oncogenic activation loop KIT mutations are observed in acute myeloid leukemia (AML) and systemic mastocytosis (SM); however, unlike the KIT juxtamembrane mutants, the activation loop mutants are insensitive to imatinib mesylate. Furthermore, as prior studies primarily used heterologous cell lines, the molecular mechanism(s) underlying oncogenic KIT-induced transformation in primary cells is poorly understood. We demonstrate that expression of KITD814V in primary hematopoietic stem/progenitor cells (HSC/Ps) and mast cell progenitors (MCps) induces constitutive KIT autophosphorylation, supports ligand-independent hyperproliferation, and promotes promiscuous cooperation with multiple cytokines. Genetic disruption of p85 alpha, the regulatory subunit of class IA lipid kinase phosphoinositol-3-kinase (PI3K), but not of p85 beta, or genetic disruption of the hematopoietic cell-specific Rho GTPase, Rac2, normalizes KITD814V-induced ligand-independent hyperproliferation. Additionally, deficiency of p85 alpha or Rac2 corrects the promiscuous hyperproliferation observed in response to multiple cytokines in both KITD814V-expressing HSC/Ps and MCps. Treatment of KITD814V-expressing HSC/Ps with a Rac inhibitor (NC23766) or with rapamycin showed a dose-dependent suppression in ligand-independent growth. Taken together, our results identify p85 alpha and Rac2 as potential novel therapeutic targets for the treatment of KITD814V-bearing AML and SM.

Topics: Amino Acid Substitution; Animals; Antibiotics, Antineoplastic; Benzamides; Cell Proliferation; Cell Transformation, Neoplastic; Cytokines; Drug Resistance, Neoplasm; Enzyme Inhibitors; Hematopoietic Stem Cells; Imatinib Mesylate; Leukemia, Myeloid, Acute; Mastocytosis, Systemic; Mice; Mice, Knockout; Mutation, Missense; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Piperazines; Protein Subunits; Proto-Oncogene Proteins c-kit; Pyrimidines; rac GTP-Binding Proteins; RAC2 GTP-Binding Protein; Sirolimus

The mammalian target of rapamycin (mTOR) is a key regulator of growth and survival in many cell types. Its constitutive activation has been involved in the pathogenesis of various cancers. In this study, we show that mTOR inhibition by rapamycin strongly inhibits the growth of the most immature acute myeloid leukemia (AML) cell lines through blockade in G0/G1 phase of the cell cycle. Accordingly, 2 downstream effectors of mTOR, 4E-BP1 and p70S6K, are phosphorylated in a rapamycin-sensitive manner in a series of 23 AML cases. Interestingly, the mTOR inhibitor markedly impairs the clonogenic properties of fresh AML cells while sparing normal hematopoietic progenitors. Moreover, rapamycin induces significant clinical responses in 4 of 9 patients with either refractory/relapsed de novo AML or secondary AML. Overall, our data strongly suggest that mTOR is aberrantly regulated in most AML cells and that rapamycin and analogs, by targeting the clonogenic compartment of the leukemic clone, may be used as new compounds in AML therapy.

Topics: Adaptor Proteins, Signal Transducing; Adolescent; Adult; Aged; Antibiotics, Antineoplastic; Cell Cycle Proteins; Female; G1 Phase; Hematopoietic Stem Cells; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Phosphoproteins; Phosphorylation; Protein Kinases; Protein Processing, Post-Translational; Recurrence; Resting Phase, Cell Cycle; Ribosomal Protein S6 Kinases, 70-kDa; Sirolimus; TOR Serine-Threonine Kinases; Tumor Cells, Cultured

Non-myeloablative conditioning has extended the use of allogeneic haematopoietic transplant to many previously ineligible patients. We added the immunosuppressive and antitumour agent sirolimus (rapamycin) to an established transplant regimen of fludarabine 25 mg/m(2) days -7 through -3 and cyclophosphamide 1000 mg/m(2) days -7 and -6, with tacrolimus and methotrexate immunoprophyllaxis. A total of 23 patients with acute myelogenous leukaemia (AML) were treated, with a median age of 59 years (range: 28-72) at transplant. Only seven patients in total were in complete remission prior to transplantation. Nine patients were in chemotherapy-refractory progression and seven were primarily refractory to induction therapy. Six patients received matched sibling, 11 unrelated donor, 1-5/6 matched and five haploidentical (haplo - three of six or four of six matched) transplants. The haplo-recipients also received antithymocyte globulin, all patients engrafted. Only two, both recipients of haploidentical cells, have died from transplant-related causes. Twelve of 23 patients survived at 198-1162-d post-transplant (median 578). Four of 12 survivors relapsed at 83, 88, 243 and 508 d and three were in remission after chemotherapy and donor lymphocyte infusion. Although follow up is short, this data suggests that non-myeloablative haematopoietic cell transplantation with sirolimus (rapamycin)-based immunosuppression may provide disease control over several years in some patients with advanced and poor prognosis AML.

Topics: Adult; Aged; Antibiotics, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Chronic Disease; Cyclophosphamide; Disease Progression; Follow-Up Studies; Graft Survival; Graft vs Host Disease; Hematopoietic Stem Cell Transplantation; Humans; Immunosuppressive Agents; Leukemia, Myeloid, Acute; Middle Aged; Sirolimus; Survival Analysis; Treatment Outcome; Vidarabine

Acute myeloid leukemia cells have constitutive activation of phosphatidylinositol 3(PI3) kinase and require PI3 kinase activation for survival; however, the function of the PI3 kinase pathway in the survival of leukemic cells is poorly defined. We have studied the role of one PI3 kinase substrate, mTOR (mammalian target of rapamycin), in primary leukemic cells. In initial experiments, we have defined a novel growth medium that improves survival of acute myeloid leukemia (AML) blasts in long-term suspension culture and the survival of leukemic stem cells in short-term cultures. Inhibition of mTOR using rapamycin leads to a modest decrease in cell survival after 2 days of incubation with more significant decrease in survival after 7 days of culture. However, when rapamycin is added to etoposide in 2-day cultures, there is a dramatic increase in the cytotoxicity of etoposide against AML blasts. Furthermore, etoposide consistently decreased the engraftment of AML cells in nonobese diabetic/severe combined immunodeficient (NOD/SCID) animals, and this effect was enhanced by coincubation with rapamycin, demonstrating that mTOR regulates survival of AML stem cells after etoposide treatment. These results suggest that rapamycin in combination with etoposide-based chemotherapy may be efficacious in the treatment of AML.

Topics: Animals; Cell Survival; Culture Media; Etoposide; Humans; Leukemia, Myeloid, Acute; Mice; Protein Kinases; Sirolimus; Stem Cells; Time Factors; TOR Serine-Threonine Kinases; Tumor Cells, Cultured

Rapamycin (RAPA), an inhibitor of cytokine responses, is under investigation in humans for graft-vs-host disease (GVHD) prevention. The mechanisms responsible for GVHD prevention are unknown. We show that RAPA is more effective in inhibiting CD8+ or TCR gammadelta+ than CD4+ T cell-mediated murine GVHD. To determine how RAPA inhibited GVHD, thoracic duct lymphocytes (TDL) were isolated from recipients of allogeneic donor grafts. Compared with controls, RAPA-treated recipients had a marked decrease in donor TDL T cell number between days 5 and 24 posttransplant. CD8+ T cell expansion was preferentially inhibited. RAPA inhibited Th1 or Th1 cytotoxic (Tc1) cytokines, but not Th2 or Tc2, cell generation. In situ mRNA hybridization also showed that TDL T cells from RAPA-treated mice had a lower frequency of granzyme B+ cells, indicating that RAPA inhibited the generation of CTL capable of mediating cytolysis through the release of granzyme B. In another system, RAPA was found to inhibit the GVL response of delayed donor lymphocyte infusions. Since CD8+ T cells are the primary effectors in this system, these data suggest that RAPA directly interfered with GVL effector cell expansion or function. We conclude that RAPA is effective in inhibiting Th1 or Tc1 cytokine production and CD8+ and TCRgammadelta+ T cell-mediated GVHD, but abrogates GVL.

Topics: Animals; Bone Marrow Transplantation; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Separation; Cytokines; Cytotoxicity, Immunologic; Female; Graft vs Host Disease; Histocompatibility Antigens Class II; Immunosuppressive Agents; Injections, Intraperitoneal; Leukemia, Myeloid, Acute; Lymphocyte Activation; Lymphocyte Transfusion; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Transgenic; Polyenes; Radiation Chimera; Receptors, Antigen, T-Cell, alpha-beta; Sirolimus; T-Lymphocyte Subsets; Th1 Cells; Up-Regulation