sirolimus and Leukemia--Lymphocytic--Chronic--B-Cell

Mechanistic target for rapamycin (mTOR) is a serine/threonine protein kinase that forms two distinct complexes mTORC1 and mTORC2, integrating mitogen and nutrient signals to regulate cell survival and proliferation; processes which are commonly deregulated in human cancers. mTORC1 and mTORC2 have divergent molecular associations and cellular functions: mTORC1 regulates in mRNA translation and protein synthesis, while mTORC2 is involved in the regulation of cellular survival and metabolism. Through AKT phosphorylation/activation, mTORC2 has also been reported to regulate cell migration. Recent attention has focused on the aberrant activation of the PI3K/mTOR pathway in B cell malignancies and there is growing evidence for its involvement in disease pathogenesis, due to its location downstream of other established novel drug targets that intercept B cell receptor (BCR) signals. Shared pharmacological features of BCR signal inhibitors include a striking "lymphocyte redistribution" effect whereby patients experience a sharp increase in lymphocyte count on initiation of therapy followed by a steady decline. Chronic lymphocytic leukemia (CLL) serves as a paradigm for migration studies as lymphocytes are among the most widely travelled cells in the body, a product of their role in immunological surveillance. The subversion of normal lymphocyte movement in CLL is being elucidated; this review aims to describe the migration impairment which occurs as part of the wider context of cancer cell migration defects, with a focus on the role of mTOR in mediating migration effects downstream of BCR ligation and other microenvironmental signals.

Topics: B-Lymphocytes; Cell Movement; Humans; Immunosuppressive Agents; Leukemia, Lymphocytic, Chronic, B-Cell; Models, Immunological; Morpholines; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Chronic lymphocytic B-cell leukemia (B-CLL) is an incurable disease characterized by the accumulation of monoclonal mature B cells, although disease progression relies upon cycling B-CLL cells in proliferation centers in central lymph organs. Rapamycin and its analogs are immunosuppressant drugs that exert their activity by specific inhibition of the mammalian target of rapamycin (mTOR). mTOR inhibition induces cell cycle arrest not only in normal lymphocytes but also in malignant cells. Therefore, rapamycins have recently entered the field of cancer treatment. In the present review we discuss how progression through the cell cycle is regulated in B-CLL cells and how rapamycin and its analogs can be used as target therapies against proliferating B-CLL cells. We also focus on additional effects of rapamycin, such as targeting the interaction between malignant B cells and the microenvironment.

Topics: Animals; Cell Proliferation; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Protein Kinase Inhibitors; Protein Kinases; Sirolimus; TOR Serine-Threonine Kinases

Preclinical studies have shown augmented activity when combining Bruton tyrosine kinase inhibitors (BTKi) with inhibitors of mammalian target of rapamycin (mTOR) and immunomodulatory agents (IMiD). We conducted a phase 1, open-label study at five centers in USA to evaluate the safety of triplet BTKi/mTOR/IMiD therapy. Eligible patients were adults aged 18 years or older with relapsed/refractory CLL, B cell NHL, or Hodgkin lymphoma. Our dose escalation study used an accelerated titration design and moved sequentially from single agent BTKi (DTRMWXHS-12), doublet (DTRMWXHS-12 + everolimus), and then to triplet therapy (DTRMWXHS-12 + everolimus + pomalidomide). All drugs were dosed once daily on days 1-21 of each 28-day cycle. The primary goal was to establish the recommended phase 2 dose of the triplet combination. Between September 27, 2016, and July 24, 2019, a total of 32 patients with a median age of 70 years (range 46 to 94 years) were enrolled. No MTD was identified for monotherapy and the doublet combination. The MTD for the triplet combination was determined to be DTRMWXHS-12 200 mg + everolimus 5 mg + pomalidomide 2 mg. Responses across all studied cohorts were seen in 13 of 32 (41.9%). Combining DTRMWXHS-12 with everolimus and pomalidomide is tolerable and shows clinical activity. Additional trials could confirm benefit of this all-oral combination therapy for relapsed/refractory lymphomas.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Everolimus; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma; Middle Aged; Neoplasm Recurrence, Local; Protein Kinase Inhibitors; Sirolimus; TOR Serine-Threonine Kinases; Treatment Outcome; Tyrosine Kinase Inhibitors

Multiple studies have demonstrated the efficacy and safety of ibrutinib for chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) and mantle cell lymphoma (MCL). This first-in-class inhibitor of Bruton's tyrosine kinase has become a standard treatment for patients with CLL and MCL.. We conducted an integrated safety analysis to characterize the frequency, severity, natural history, and outcomes of adverse events (AEs) with ibrutinib versus comparators. Data were pooled from 4 completed randomized controlled studies that had included 756 ibrutinib-treated and 749 comparator-treated patients with CLL/SLL or relapsed/refractory MCL. Safety analyses included reporting of AEs using crude and exposure-adjusted incidence rates.. The median treatment duration was 13.3 months (maximum, 28.2 months) for ibrutinib and 5.8 months (maximum, 27.3 months) for comparators. When adjusted for exposure, diarrhea, atrial fibrillation, and hypertension were the only common grade ≥ 3 AEs more often reported with ibrutinib than with the comparators. Dose reductions (7% vs. 14%) and discontinuation (12% vs. 16%) because of AEs occurred less often with ibrutinib, and deaths due to AEs occurred at similar rates (6% vs. 7%). When adjusted for exposure, the corresponding data were all lower with ibrutinib than with the comparators (0.06 vs. 0.22, 0.11 vs. 0.22, and 0.06 vs. 0.09 patient-exposure-years, respectively). The prevalence of common grade 3/4 AEs with ibrutinib generally decreased over time, with the exception of hypertension.. These results from an integrated analysis support a favorable benefit/risk profile of ibrutinib in patients with CLL/SLL and MCL.

Topics: Adenine; Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Chlorambucil; Female; Follow-Up Studies; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, Mantle-Cell; Male; Middle Aged; Patient Safety; Piperidines; Prognosis; Pyrazoles; Pyrimidines; Rituximab; Sirolimus; Survival Rate

Patients with recurrent/refractory chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL) often have chemotherapy-resistant disease, resulting in poor prognosis. The aim of this study was to learn if inhibition of the mammalian target of rapamycin (mTOR) would produce tumor responses.. This was a phase 2 study of oral single-agent everolimus (10 mg/day) for recurrent/refractory indolent lymphoid malignancies including CLL.. Four of 22 patients with CLL (18%; 95% confidence interval, 5%-40%) achieved a partial remission to therapy. An unanticipated finding in this study was an increase in absolute lymphocyte count (ALC) associated with a decrease in lymphadenopathy in 8 (36%) patients. ALC increased a median of 4.8-fold (range, 1.9- to 25.1-fold), and the clinically measurable lymphadenopathy decreased a median of 75.5% (range, 38%-93%) compared with baseline measurements.. Everolimus has modest antitumor activity against CLL and can mobilize malignant cells from nodal masses into the peripheral circulation in a subset of CLL patients. Because CLL cells in lymphatic tissue and bone marrow can be more resistant to therapy than circulating CLL cells, the ability of everolimus to mobilize CLL cells into the circulation could be used in combination therapeutic regimens.

Topics: Aged; Aged, 80 and over; Antineoplastic Agents; Drug Administration Schedule; Drug Resistance, Neoplasm; Everolimus; Female; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Lymph Nodes; Lymphocyte Count; Male; Middle Aged; Recurrence; Sirolimus

Despite high initial remission rates, most lymphomas relapse and require further therapy. The mammalian target of rapamycin (mTOR) pathway is a validated target in mantle cell lymphoma, but has not been extensively evaluated in other lymphomas.. We performed a phase II trial of single-agent temsirolimus 25-mg weekly in patients with relapsed aggressive and indolent lymphomas. The primary objective was overall and complete response rate. Patients were stratified by histology: group A (diffuse large B-cell lymphoma, transformed follicular lymphoma), group B (follicular lymphoma), and group C (chronic lymphocytic leukemia/small lymphocytic lymphoma, and other indolent lymphomas).. Eighty-nine patients were treated, with outcome strongly dependent on histology. Group A had an overall and complete response rate of 28.1% and 12.5%, respectively, and median progression-free survival (PFS) of 2.6 months and median overall survival (OS) of 7.2 months. Group B had overall and complete response rates of 53.8% and 25.6%, respectively, and median PFS of 12.7 months; median OS has not yet been reached. Group C had a partial response rate of 11% with no complete responders. Toxicity was mainly mild and/or reversible myelosuppression and mucositis; however, four patients developed pneumonitis.. Single-agent temsirolimus has significant activity in both diffuse large B-cell lymphoma and follicular lymphoma, although the durability of responses and PFS are longer for patients with follicular lymphoma. This is the first report of substantial activity of temsirolimus in lymphomas other than mantle cell lymphoma, and supports further evaluation of mTOR as a target in these diseases.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Bone Marrow; Chicago; Disease-Free Survival; Female; Humans; Intracellular Signaling Peptides and Proteins; Kaplan-Meier Estimate; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, Follicular; Lymphoma, Large B-Cell, Diffuse; Lymphoma, Mantle-Cell; Lymphoma, Non-Hodgkin; Male; Middle Aged; Mucositis; Pneumonia; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Remission Induction; Sirolimus; TOR Serine-Threonine Kinases; Treatment Outcome

Although B-cell chronic lymphocytic leukemia (CLL) is treatable, it remains an incurable disease and most patients inevitably suffer relapse. Many therapeutic options exist for those requiring therapy, including monoclonal antibodies and stem cell transplantation, but remissions tend to last shorter in the course of the disease. Targeting the cell cycle has recently been realized to be an attractive therapeutic approach in solid and hematological malignancies, and the proliferative nature of B-CLL is increasingly accepted. Here, we report data on a phase II pilot trial with the oral mammalian target of rapamycin (mTOR) inhibitor RAD001 5 mg/daily in patients with advanced B-CLL who had progressive disease after at least two lines of treatment. After treatment of seven patients, this trial was stopped because of toxicity concerns, although some degree of activity was observed (one partial remission, three patients with stable disease). Interestingly, cyclin E expression decreased in responding patients. Further strategies of mTOR inhibition by RAD001 in B-CLL should focus on different treatment schedules, adequate anti-infectious prophylaxis, or combinations with cytotoxic drugs.

Topics: Cell Cycle; Everolimus; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Pilot Projects; Pneumonia; Protein Kinases; Sirolimus; TOR Serine-Threonine Kinases

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

Topics: Animals; Disease Progression; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Mechanistic Target of Rapamycin Complex 1; Mice; Phosphorylation; Signal Transduction; Sirolimus

Obinutuzumab is a glycoengineered tumor-targeting anti-CD20 mAb with a modified crystallizable fragment (Fc) domain designed to increase the affinity for the FcγRIIIA/CD16 receptor, which was recently approved for clinical use in CLL and follicular lymphoma. Here we extend our previous observation that, in human NK cells, the sustained CD16 ligation by obinutuzumab-opsonized targets leads to a markedly enhanced IFN-γ production upon a subsequent cytokine re-stimulation. The increased IFN-γ competence in response to IL-2 or IL-15 is attributable to post-transcriptional regulation, as it does not correlate with the upregulation of IFN-γ mRNA levels. Different from the reference molecule rituximab, we observe that the stimulation with obinutuzumab promotes the upregulation of microRNA (miR)-155 expression. A similar trend was also observed in NK cells from untreated CLL patients stimulated with obinutuzumab-opsonized autologous leukemia. miR-155 upregulation associates with reduced levels of SHIP-1 inositol phosphatase, which acts in constraining PI3K-dependent signals, by virtue of its ability to mediate phosphatidylinositol 3,4,5-trisphosphate (PIP3) de-phosphorylation. Downstream of PI3K, the phosphorylation status of mammalian target of rapamycin (mTOR) effector molecule, S6, results in amplified response to IL-2 or IL-15 stimulation in obinutuzumab-experienced cells. Importantly, NK cell treatment with the PI3K or mTOR inhibitors, idelalisib and rapamycin, respectively, prevents the enhanced cytokine responsiveness, thus, highlighting the relevance of the PI3K/mTOR axis in CD16-dependent priming. The enhanced IFN-γ competence may be envisaged to potentiate the immunoregulatory role of NK cells in a therapeutic setting.

Topics: Antibodies, Monoclonal, Humanized; Antineoplastic Agents, Immunological; Cell Line, Tumor; Cells, Cultured; Enzyme Inhibitors; Gene Expression; Humans; Interferon-gamma; Interleukin-12; Killer Cells, Natural; Leukemia, Lymphocytic, Chronic, B-Cell; MicroRNAs; Phosphatidylinositol 3-Kinases; Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases; Purines; Quinazolinones; Receptors, IgG; Sirolimus; TOR Serine-Threonine Kinases

Topics: Antineoplastic Combined Chemotherapy Protocols; Biomarkers; Biomarkers, Tumor; Disease Susceptibility; Female; Flow Cytometry; Gene Expression; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Male; Mutation; Sirolimus; Treatment Outcome; Vidarabine

Activated regulatory T cells (Tregs) suppress proliferation and differentiation of normal B cells. In our study, allogeneic polyclonal CD4 (+) CD25 (+) Tregs and CD4 (+) CD25 (+) CD127(lo)Tregs expanded in vitro in the presence of rapamycin and low dose IL-2 suppressed proliferation of 11 out of 12 established lymphoma B-cell lines. The effect of expanded CD4 (+) CD25 (+) Tregs on survival of freshly isolated lymphoma B cells maintained in culture with soluble multimeric CD40L and IL-4 was variable across lymphoma entities. The survival of freshly isolated follicular lymphoma cells usually decreased in cocultures with CD4 (+) CD25 (+) Tregs. Treg effect on chronic lymphocytic leukemia/small lymphocytic lymphoma cells ranged from suppression to help in individual patients. CD4 (+) CD25 (+) Tregs or CD4 (+) CD25 (+) CD127(lo)Tregs expanded ex vivo with rapamycin could be used to suppress regrowth of residual lymphoma after autologous hematopoietic cell transplantation (HCT), and to counteract both graft-versus-host disease and lymphoma re-growth after allogeneic HCT in select patients with lymphoma susceptible to the regulation by Tregs.

Topics: Adult; Aged; Aged, 80 and over; B-Lymphocytes; Biopsy, Fine-Needle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Coculture Techniques; Female; Flow Cytometry; Forkhead Transcription Factors; Humans; Interleukin-2; Leukemia, Lymphocytic, Chronic, B-Cell; Lymph Nodes; Lymphocyte Activation; Lymphoma, B-Cell; Male; Middle Aged; Primary Cell Culture; Sirolimus; T-Lymphocytes, Regulatory

Topics: Adenine; Antineoplastic Agents; Bendamustine Hydrochloride; Bortezomib; Chromosomes, Human, Pair 17; Cytarabine; Humans; Lenalidomide; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, Mantle-Cell; Mutation; Neoplasm Recurrence, Local; Piperidines; Pyrazoles; Pyrimidines; Rituximab; Sirolimus; Thalidomide; Tumor Suppressor Protein p53; Vidarabine

Chronic lymphocytic leukemia (CLL) remains incurable; therefore searching for new therapeutic strategies in this disease is necessary. An important mechanism of tumor development is neoangiogenesis. A potent antiangiogenic factor, bevacizumab (Avastin, AVA), has been poorly explored in CLL so far. In the current study we assessed cytotoxic activity of AVA alone or in combinations with drugs routinely used in this disease.. Cells isolated from 60 CLL patients were treated with AVA alone or in combination with anti-CD20 monoclonal antibody (MoAb), rituximab (RIT), anti-CD52 MoAb, alemtuzumab (ALT), 2-CdA (2-chlorodeoxyadenosine), FA (fludarabine), MAF (mafosfamide) or RAPA (rapamycin). Cytotoxicity was assessed by propidium iodide staining. Apoptosis was evaluated using annexin-V and TUNEL assays. Additionally, a drop of mitochondrial potential (DYm) as well as expression of apoptosis-regulating proteins Bax, Bak, Bid, Bad, Bcl-2, Mcl-2, XIAP, FLIP, Akt and Bcl-2-A1 were determined by flow cytometry.. At the dose of 40 μg/ml, after 48 hours of incubation, AVA induced significant cytotoxicity against CLL cells. The drug triggered apoptosis, with activation of caspase-3 and -9, but not caspase-8, along with a drop of DYm. Incubation with AVA induced significant overexpression of proapoptotic Bak and Bad as well as downregulation of antiapoptotic Mcl-2 and Akt proteins. Combination of AVA with RIT, ALT or RAPA significantly increased cytotoxicity when compared with the effects of single drugs.. In conclusion, this is the first report showing proapoptotic activity of AVA against CLL cells. Combination of AVA with RIT, ALT or RAPA may be a promising therapeutic strategy, which requires confirmation in further studies.

Topics: Alemtuzumab; Angiogenesis Inhibitors; Antibodies, Monoclonal, Humanized; Antibodies, Monoclonal, Murine-Derived; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Bevacizumab; Caspase 3; Caspase 9; Cladribine; Cyclophosphamide; Down-Regulation; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Proteasome Inhibitors; Rituximab; Sirolimus; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vidarabine

Alterations in plasma lipid profile and in intracellular cholesterol homoeostasis have been described in various malignancies; however, significance of these alterations, if any, in cancer biology is not clear. The aim of the present study was to investigate a possible correlation between alterations in cholesterol metabolism and expansion of leukaemia cell numbers.. Lipid profiles in plasma and in primary leukaemia cells isolated from patients with acute or chronic lymphocytic leukaemia (ALL and CLL) were studied.. Decreased levels of HDL-C were observed in plasma of leukaemic patients, levels of total cholesterol, LDL-C, triglycerides and phospholipids were unchanged or only slightly increased. As compared to normal lymphocytes, freshly isolated leukaemic cells showed increased levels of cholesterol esters and reduction in free cholesterol. Growth stimulation of ALL and CLL cells with phytohemagglutinin led to further increase in levels of cholesterol esters. Conversely, treatment with an inhibitor of cell proliferation such as the mTOR inhibitor, RAD, caused decline in population growth rate of leukaemia cells, which was preceded by sharp reduction in rate of cholesterol esterification. On the other hand, exposure of leukaemic cells to two inhibitors of cholesterol esterification, progesterone and SaH 58-035, caused 60% reduction in their proliferation rate. In addition to demonstrating tight correlation between cell number expansion and cholesterol esterification in leukaemic cells, these results suggest that pathways that control cholesterol esterification might represent a promising targets for novel anticancer strategies.

Topics: Adult; Aged; Amides; Antineoplastic Agents; Cell Proliferation; Cholesterol Esters; Cholesterol, HDL; Everolimus; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Lipid Metabolism; Lipids; Middle Aged; Organosilicon Compounds; Phytohemagglutinins; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Progesterone; Sirolimus

B chronic lymphocytic leukemia (B-CLL) cells exist in patients as slowly accumulating resting as well as proliferating B cells. In this study, we examined whether Rapamycin and Curcumin, two naturally occurring compounds shown to have apoptotic effects, could selectively induce apoptosis in resting B-CLL cells. Mononuclear cells isolated from patients with B-CLL were treated with these agents and analysed by AnnexinV/propidium iodide binding, caspase activity, and changes in bcl-2/Bax ratio. Rapamycin and curcumin significantly induced apoptosis in resting B-CLL cells obtained from patients with CLL. Furthermore, rapamycin and curcumin increased caspase 9, 3 and 7 activity, decreased anti-apoptotic bcl-2 levels, and increased the pro-apoptotic protein Bax. These data suggest rapamycin and curcumin may be an effective treatment for B-CLL and are of high clinical significance considering the growing population of patients and lack of efficient treatment for this malignant disease.

Topics: Aged; Aged, 80 and over; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Caspase 3; Caspase 7; Caspase 9; Curcumin; Female; Flow Cytometry; Humans; Immunosuppressive Agents; Leukemia, Lymphocytic, Chronic, B-Cell; Leukocytes, Mononuclear; Male; Middle Aged; Proto-Oncogene Proteins c-bcl-2; Sirolimus; Time Factors; Tumor Cells, Cultured

The mammalian target of rapamycin inhibitor rapamycin and its analogues show promising anticancer activity in various experimental tumor models and are presently evaluated in clinical trials. We, here, evaluated the in vitro activity of rapamycin with regard to tumor-type specificity and possible mechanisms of drug resistance in 97 tumor cell samples from patients and in a resistance-based cell line panel, using the fluorometric microculture cytotoxicity assay. Rapamycin was dose-dependently cytotoxic in patient tumor cells and in cell lines. In primary cells, rapamycin was more active in hematological than in solid tumor samples, with chronic lymphocytic leukemia (CLL) and acute lymphocytic leukemia being the most sensitive tumor types. Considerable inter-individual differences in sensitivity were apparent among CLL samples, but no difference was observed between IGHV mutated and unmutated CLL samples, whereas a tendency to lower rapamycin sensitivity was indicated for samples displaying poor-prognostic genomic markers. Combination experiments in CLL cells indicated that rapamycin acted synergistically with vincristine, cisplatin, chlorambucil and taxotere. These results and the clinically-experienced good tolerance to rapamycin analogues encourage clinical studies of rapamycin in CLL treatment as single agent but also in combination with, e.g., vincristine and chlorambucil.

Topics: Antibiotics, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Chlorambucil; Cisplatin; Docetaxel; Drug Screening Assays, Antitumor; Drug Synergism; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Sirolimus; Taxoids; Tumor Cells, Cultured; Vincristine

We report on a patient with relapsed chronic lymphocytic leukemia (CLL) treated with the novel mTOR inhibitor RAD001 within a phase II clinical trial. Although the patient initially responded to therapy, RAD001 was discontinued after 32 weeks due to progression and fludarabine-based chemotherapy was started. The patient subsequently developed a rapidly fatal Epstein-Barr-virus-associated lymphoproliferative disorder, clonally unrelated to the CLL. The clinical course suggests caution when using newer immunosuppressive drugs for treatment of CLL, especially in the context of additional purine analog therapy.

Topics: Aged; Epstein-Barr Virus Infections; Everolimus; Fatal Outcome; Female; Herpesvirus 4, Human; Humans; Immunosuppressive Agents; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoproliferative Disorders; Phosphoinositide-3 Kinase Inhibitors; Protein Kinases; Sirolimus; TOR Serine-Threonine Kinases; Vidarabine

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the world. The TCL1 gene, responsible for prolymphocytic T cell leukemia, is also overexpressed in human B cell malignancies and overexpression of the Tcl1 protein occurs frequently in CLL. Aging transgenic mice that overexpress TCL1 under control of the mu immunoglobulin gene enhancer, develop a CD5+ B cell lymphoproliferative disorder mimicking human CLL and implicating TCL1 in the pathogenesis of CLL. In the current study, we exploited this transgenic mouse to investigate two different CLL-related issues: potential treatment of CLL and characterization of neoplasms that accompany CLL. We successfully transplanted CLL cells into syngeneic mice that led to CLL development in the recipient mice. This approach allowed us to verify the involvement of the Tcl1/Akt/mTOR biochemical pathway in the disease by testing the ability of a specific pharmacologic agent, rapamycin, to slow CLL. We also showed that 36% of these transgenic mice were affected by solid malignancies, in which the expression of the Tcl1 protein was absent. These findings indicate that other oncogenic mechanism(s) may be involved in the development of solid tumors in Emu-TCL1 transgenic mice.

Topics: Animals; Antibiotics, Antineoplastic; Disease Models, Animal; Humans; Immunohistochemistry; Leukemia, Lymphocytic, Chronic, B-Cell; Mice; Mice, Transgenic; Proto-Oncogene Proteins; Sirolimus

1. T-cell proliferation is critical for mounting an effective adaptive immune response. It is regulated by signals through the T-cell receptor, through co-stimulation and through cytokines such as interleukin-2 (IL-2). Phosphatidylinositol 3-kinase (PI3K) lies downstream of each of these pathways and has been directly implicated in the regulation of lymphocyte proliferation. 2. In this study, we have shown that PI3K regulates cyclin D2 and cyclin D3, the first cell cycle proteins induced in T-cell proliferation, transcriptionally and post-transcriptionally. In T-lymphoblasts, LY294002, a PI3K inhibitor, prevents the induction of both D-type cyclin mRNA and protein, while rapamycin inhibits the induction of protein. Rapamycin inhibits mammalian target of rapamycin (mTOR), which lies downstream of PI3K. 3. Furthermore, our data show that the combination of LY294002 and rapamycin results in a co-operative inhibition of T-cell proliferation. This co-operation occurs in Kit225 cells stimulated with IL-2, and also in resting peripheral blood lymphocytes stimulated with antibodies to the T-cell receptor in the presence and absence of antibodies to CD28. 4. These data indicate that PI3K regulates T-cell proliferation in response to diverse stimuli, and suggest that combinations of inhibitors, perhaps isoform-selective, may be useful as alternative immunosuppressive therapies.

Topics: Cell Line, Tumor; Cell Proliferation; Cell Survival; Chromones; Drug Interactions; Enzyme Inhibitors; Flow Cytometry; Humans; Interleukin-2; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphocyte Activation; Morpholines; Phosphoinositide-3 Kinase Inhibitors; Sirolimus; T-Lymphocytes; Thymidine

Human telomerase activity is induced by Ag receptor ligation in T and B cells. However, it is unknown whether telomerase activity is increased in association with activation and proliferation of NK cells. We found that telomerase activity in a human NK cell line (NK-92), which requires IL-2 for proliferation, was increased within 24 h after stimulation with IL-2. Levels of human telomerase reverse transcriptase (hTERT) mRNA and protein correlated with telomerase activity. ERK1/2 and Akt kinase (Akt) were activated by IL-2 stimulation. LY294002, an inhibitor of PI3K, abolished expression of hTERT mRNA and protein expression and abolished hTERT activity, whereas PD98059, which inhibits MEK1/2 and thus ERK1/2, had no effect. In addition, radicicol, an inhibitor of heat shock protein 90 (Hsp90), and rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), blocked IL-2-induced hTERT activity and nuclear translocation of hTERT but not hTERT mRNA expression. hTERT was coimmunoprecipitated with Akt, Hsp90, mTOR, and p70 S6 kinase (S6K), suggesting that these molecules form a physical complex. Immunoprecipitates of Akt, Hsp90, mTOR, and S6K from IL-2-stimulated NK-92 cells contained telomerase activity. Furthermore, the findings that Hsp90 and mTOR immunoprecipitates from primary samples contained telomerase activity are consistent with the results from NK-92 cells. These results indicate that IL-2 stimulation induces hTERT activation and that the mechanism of IL-2-induced hTERT activation involves transcriptional or posttranslational regulation through the pathway including PI3K/Akt, Hsp90, mTOR, and S6K in NK cells.

Topics: Cell Line, Transformed; Chromones; DNA-Binding Proteins; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Flavonoids; HSP90 Heat-Shock Proteins; Humans; Interleukin-2; Killer Cells, Natural; Lactones; Leukemia, Lymphocytic, Chronic, B-Cell; Leukemia, Lymphoid; Lymphocyte Activation; Macrolides; Morpholines; Phosphatidylinositol 3-Kinases; Protein Kinases; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Ribosomal Protein S6 Kinases, 70-kDa; RNA-Directed DNA Polymerase; Sirolimus; Telomerase; TOR Serine-Threonine Kinases; Transcription, Genetic

In B-cell chronic lymphocytic leukemia (B-CLL), malignant cells seem to be arrested in the G(0)/early G(1) phase of the cell cycle, and defective apoptosis might be involved in disease progression. However, increasing evidence exists that B-CLL is more than a disease consisting of slowly accumulating resting B cells: a proliferating pool of cells has been described in lymph nodes and bone marrow and might feed the accumulating pool in the blood. Rapamycin has been reported to inhibit cell cycle progression in a variety of cell types, including human B cells, and has shown activity against a broad range of human tumor cell lines. Therefore, we investigated the ability of rapamycin to block cell cycle progression in proliferating B-CLL cells. We have recently demonstrated that stimulation with CpG-oligonucleotides and interleukin-2 provides a valuable model for studying cell cycle regulation in malignant B cells. In our present study, we demonstrated that rapamycin induced cell cycle arrest in proliferating B-CLL cells and inhibited phosphorylation of p70s6 kinase (p70(s6k)). In contrast to previous reports on nonmalignant B cells, the expression of the cell cycle inhibitor p27 was not changed in rapamycin-treated leukemic cells. Treatment with rapamycin prevented retinoblastoma protein (RB) phosphorylation in B-CLL cells without affecting the expression of cyclin D2, but cyclin D3 was no longer detectable in rapamycin-treated B-CLL cells. In addition, rapamycin treatment inhibited cyclin-dependent kinase 2 activity by preventing up-regulation of cyclin E and cyclin A. Interestingly, survivin, which is expressed in the proliferation centers of B-CLL patients in vivo, is not up-regulated in rapamycin-treated cells. Therefore, rapamycin interferes with the expression of many critical molecules for cell cycle regulation in cycling B-CLL cells. We conclude from our study that rapamycin might be an attractive substance for therapy for B-CLL patients by inducing a G(1) arrest in proliferating tumor cells.

Topics: Aged; Aged, 80 and over; Antibiotics, Antineoplastic; B-Lymphocytes; Cell Culture Techniques; Cell Cycle; Cyclin A; Cyclin D3; Cyclin E; Cyclins; G1 Phase; Humans; Inhibitor of Apoptosis Proteins; Leukemia, Lymphocytic, Chronic, B-Cell; Male; Microtubule-Associated Proteins; Middle Aged; Neoplasm Proteins; Sirolimus; Survivin