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1.
Front Oncol ; 13: 1161254, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37228498

RESUMO

Introduction: Chronic lymphocytic leukemia (CLL) cells are metabolically flexible and adapt to modern anticancer treatments. Bruton tyrosine kinase (BTK) and B-cell lymphoma-2 (BCL-2) inhibitors have been widely used to treat CLL, but CLL cells become resistant to these treatments over time. CB-839 is a small-molecule glutaminase-1 (GLS-1) inhibitor that impairs glutamine use, disrupts downstream energy metabolism, and impedes the elimination of reactive oxygen species. Methods: To investigate the in vitro effects of CB-839 on CLL cells, we tested CB-839 alone and in combination with ibrutinib, venetoclax, or AZD-5991 on the HG-3 and MEC-1 CLL cell lines and on primary CLL lymphocytes. Results: We found that CB-839 caused dose-dependent decreases in GLS-1 activity and glutathione synthesis. CB-839-treated cells also showed increased mitochondrial superoxide metabolism and impaired energy metabolism, which were reflected in decreases in the oxygen consumption rate and depletion of the adenosine triphosphate pool and led to the inhibition of cell proliferation. In the cell lines, CB-839 combined with venetoclax or AZD-5991, but not with ibrutinib, demonstrated synergism with an increased apoptosis rate and cell proliferation inhibition. In the primary lymphocytes, no significant effects of CB-839 alone or in combination with venetoclax, ibrutinib, or AZD-5991 were observed. Discussion: Our findings suggest that CB-839 has limited efficacy in CLL treatment and shows limited synergy in combination with widely used CLL drugs.

2.
Blood Cancer J ; 12(5): 80, 2022 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-35595730

RESUMO

Pirtobrutinib (LOXO-305), a reversible inhibitor of Bruton's tyrosine kinase (BTK), was designed as an alternative strategy to treat ibrutinib-resistant disease that develops due to C481 kinase domain mutations. The clinical activity of pirtobrutinib has been demonstrated in CLL, but the mechanism of action has not been investigated. We evaluated pirtobrutinib in 4 model systems: first, MEC-1, a CLL cell line overexpressing BTKWT, BTKC481S, or BTKC481R; second, murine models driven by MEC-1 overexpressing BTKWT or BTKC481S; third, in vitro incubations of primary CLL cells; and finally, CLL patients during pirtobrutinib therapy (NCT03740529, ClinicalTrials.gov). Pirtobrutinib inhibited BTK activation as well as downstream signaling in MEC-1 isogenic cells overexpressing BTKWT, BTKC481S, or BTKC481R. In mice, overall survival was short due to aggressive disease. Pirtobrutinib treatment for 2 weeks led to reduction of spleen and liver weight in BTKWT and BTKC481S cells, respectively. In vitro incubations of CLL cells harboring wild-type or mutant BTK had inhibition of the BCR pathway with either ibrutinib or pirtobrutinib treatment. Pirtobrutinib therapy resulted in inhibition of BTK phosphorylation and downstream signaling initially in all cases irrespective of their BTK profile, but these effects started to revert in cases with other BCR pathway mutations such as PLCG2 or PLEKHG5. Levels of CCL3 and CCL4 in plasma were marginally higher in patients with mutated BTK; however, there was a bimodal distribution. Both chemokines were decreased at early time points and mimicked BCR pathway protein changes. Collectively, these results demonstrate that pirtobrutinib is an effective BTK inhibitor for CLL harboring wild-type or mutant BTK as observed by changes in CCL3 and CCL4 biomarkers and suggest that alterations in BCR pathway signaling are the mechanism for its clinical effects. Long-term evaluation is needed for BTK gatekeeper residue variation along with pathologic kinase substitution or mutations in other proteins in the BCR pathway.


Assuntos
Leucemia Linfocítica Crônica de Células B , Tirosina Quinase da Agamaglobulinemia , Animais , Estudos Clínicos como Assunto , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Camundongos , Mutação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Transdução de Sinais
3.
Front Oncol ; 12: 833714, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35273915

RESUMO

B-cell receptor (BCR) signaling pathway and Bcl-2 family prosurvival proteins, specifically Bcl-2 and Mcl-1, are functional in the pathobiology of chronic lymphocytic leukemia (CLL). A pivotal and apical molecule in the BCR pathway is Bruton's tyrosine kinase (BTK). Together, BTK, Bcl-2, and Mcl-1 participate in the maintenance, migration, proliferation, and survival of CLL cells. Several ongoing and published clinical trials in CLL reported high rates of remission, namely, undetectable measurable residual disease (u-MRD) status with combined BTK inhibitor ibrutinib and Bcl-2 antagonist, venetoclax. While the majority of patients achieve complete remission with undetectable-measurable residual disease, at least one third of patients do not achieve this milestone. We hypothesized that cells persistent during ibrutinib and venetoclax therapy may be sensitive to combined venetoclax and Mcl-1 inhibitor, AMG-176. To test this hypothesis, we took peripheral blood samples at baseline, after Cycle 1 and Cycle 3 of ibrutinib monotherapy, after one week and 1 cycle of ibrutinib plus venetoclax therapy. These serial samples were tested for pharmacodynamic changes and treated in vitro with AMG-176 or in combination with venetoclax. Compared to C1D1 cells, residual cells during ibrutinib and venetoclax treatment were inherently resistant to endogenous cell death. Single agent exposure induced some apoptosis but combination of 100 nM venetoclax and 100 or 300 nM of AMG-176 resulted in 40-100% cell death in baseline samples. Cells obtained after four cycles of ibrutinib and one cycle of venetoclax, when treated with such concentration of venetoclax and AMG-176, showed 10-80% cell death. BCR signaling pathway, measured as autophosphorylation of BTK was inhibited throughout therapy in all post-therapy samples. Among four anti-apoptotic proteins, Mcl-1 and Bfl-1 decreased during therapy in most samples. Proapoptotic proteins decreased during therapy. Collectively, these data provide a rationale to test Mcl-1 antagonists alone or in combination in CLL during treatment with ibrutinib and venetoclax.

4.
Mol Cancer Res ; 20(2): 280-292, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34654720

RESUMO

Loss-of-function somatic mutations of STK11, a tumor suppressor gene encoding LKB1 that contributes to the altered metabolic phenotype of cancer cells, is the second most common event in lung adenocarcinomas and often co-occurs with activating KRAS mutations. Tumor cells lacking LKB1 display an aggressive phenotype, with uncontrolled cell growth and higher energetic and redox stress due to its failure to balance ATP and NADPH levels in response to cellular stimulus. The identification of effective therapeutic regimens for patients with LKB1-deficient non-small cell lung cancer (NSCLC) remains a major clinical need. Here, we report that LKB1-deficient NSCLC tumor cells displayed reduced basal levels of ATP and to a lesser extent other nucleotides, and markedly enhanced sensitivity to 8-Cl-adenosine (8-Cl-Ado), an energy-depleting nucleoside analog. Treatment with 8-Cl-Ado depleted intracellular ATP levels, raised redox stress, and induced cell death leading to a compensatory suppression of mTOR signaling in LKB1-intact, but not LKB1-deficient, cells. Proteomic analysis revealed that the MAPK/MEK/ERK and PI3K/AKT pathways were activated in response to 8-Cl-Ado treatment and targeting these pathways enhanced the antitumor efficacy of 8-Cl-Ado. IMPLICATIONS: Together, our findings demonstrate that LKB1-deficient tumor cells are selectively sensitive to 8-Cl-Ado and suggest that therapeutic approaches targeting vulnerable energy stores combined with signaling pathway inhibitors merit further investigation for this patient population.


Assuntos
2-Cloroadenosina/análogos & derivados , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , 2-Cloroadenosina/farmacologia , 2-Cloroadenosina/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Homeostase , Humanos , Neoplasias Pulmonares/patologia , Mutação , Oxirredução , Transdução de Sinais , Transfecção
5.
Oncotarget ; 10(29): 2793-2809, 2019 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-31073371

RESUMO

The PIM1, PIM2, and PIM3 serine/threonine kinases play a role in the proliferation and survival of cancer cells. Mice lacking these three kinases were viable. Further, in human hematological malignancies, these proteins are overexpressed making them suitable targets. Several small molecule inhibitors against this enzyme were synthesized and tested. AZD1208, an orally available small-molecule drug, inhibits all three PIM kinases at a low nanomolar range. AZD1208 has been tested in clinical trials for patients with solid tumors and hematological malignancies, especially acute myelogenous leukemia. The present study evaluated the efficacy and biological actions of AZD1208 in chronic lymphocytic leukemia (CLL) cells. CLL cells had higher levels of PIM2 protein and mRNAs than did normal lymphocytes from healthy donors. Treatment of CLL lymphocytes with AZD1208 resulted in modest cell death, whereas practically no cytotoxicity was observed in healthy lymphocytes. To determine the mechanism by which AZD1208 inhibits PIM kinase function, we evaluated PIM kinase pathway and downstream substrates. Because peripheral blood CLL cells are replicationally quiescent, we analyzed substrates involved in apoptosis, transcription, and translation but not cell cycle targets. AZD1208 inhibited protein translation by decreasing phosphorylation levels of 4E-binding protein 1 (4E-BP1). AZD1208 induced autophagy in replicationally-quiescent CLL cells, which is consistent with protein translation inhibition. These data suggest that AZD1208 may elicit cytotoxicity in CLL cells through inhibiting translation and autophagy induction.

6.
Methods Mol Biol ; 1881: 19-25, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30350194

RESUMO

In vitro drug combination studies are commonly used for CLL primary lymphocytes. An advancement in this method is to perform ex vivo drug testing where the first agent is administered to patients and second drug is tested in these patients' cells in vitro. These assays have been effective in identifying novel agents that work additively or synergistically. In this chapter, we provide a step-by-step protocol for ex vivo drug testing that can be used for combination strategies.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Cultura Primária de Células/métodos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose/efeitos dos fármacos , Separação Celular/instrumentação , Separação Celular/métodos , Centrifugação com Gradiente de Concentração/instrumentação , Centrifugação com Gradiente de Concentração/métodos , Ensaios de Seleção de Medicamentos Antitumorais/instrumentação , Sinergismo Farmacológico , Humanos , Leucemia Linfocítica Crônica de Células B/sangue , Linfócitos , Masculino , Cultura Primária de Células/instrumentação , Células Tumorais Cultivadas
7.
Oncotarget ; 9(38): 24980-24991, 2018 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-29861847

RESUMO

Blood cells from patients with chronic lymphocytic leukemia (CLL) are replicationally quiescent but transcriptionally, translationally, and metabolically active. Recently, we demonstrated that oxidative phosphorylation (OxPhos) is a predominant pathway in CLL for energy production and is further augmented in the presence of the stromal microenvironment. Importantly, CLL cells from patients with poor prognostic markers showed increased OxPhos. From these data, we theorized that OxPhos can be targeted to treat CLL. IACS-010759, currently in clinical development, is a small-molecule, orally bioavailable OxPhos inhibitor that targets mitochondrial complex I. Treatment of primary CLL cells with IACS-010759 greatly inhibited OxPhos but caused only minor cell death at 24 and 48 h. In the presence of stroma, the drug successfully inhibited OxPhos and diminished intracellular ribonucleotide pools. However, glycolysis and glucose uptake were induced as compensatory mechanisms. To mitigate the upregulated glycolytic flux, we used 2-deoxy-D-glucose in combination with IACS-010759. This combination reduced both OxPhos and glycolysis and induced cell death. Consistent with these data, low-glucose culture conditions sensitized CLL cells to IACS-010759. Collectively, these data suggest that CLL cells adapt to use a different metabolic pathway when OxPhos is inhibited and that targeting both OxPhos and glycolysis pathways is necessary for biological effect.

8.
Neoplasia ; 19(10): 762-771, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28863345

RESUMO

Peripheral blood chronic lymphocytic leukemia (CLL) cells are replicationally quiescent mature B-cells. In short-term cultures, supporting stromal cells provide a survival advantage to CLL cells by inducing transcription and translation without promoting proliferation. We hypothesized that the stromal microenvironment augments malignant B cells' metabolism to enable the cells to cope with their energy demands for transcription and translation. We used extracellular flux analysis to assess the two major energy-generating pathways, mitochondrial oxidative phosphorylation (OxPhos) and glycolysis, in primary CLL cells in the presence of three different stromal cell lines. OxPhos, measured as the basal oxygen consumption rate (OCR) and maximum respiration capacity, was significantly higher in 28 patients' CLL cells cocultured with bone marrow-derived NK.Tert stromal cells than in CLL cells cultured alone (P = .004 and <.0001, respectively). Similar OCR induction was observed in CLL cells cocultured with M2-10B4 and HS-5 stromal lines. In contrast, heterogeneous changes in the extracellular acidification rate (a measure of glycolysis) were observed in CLL cells cocultured with stromal cells. Ingenuity Pathway Analysis of CLL cells' metabolomics profile indicated stroma-mediated stimulation of nucleotide synthesis. Quantitation of ribonucleotide pools showed a significant two-fold increase in CLL cells cocultured with stromal cells, indicating that the stroma may induce CLL cellular bioenergy and the RNA building blocks necessary for the transcriptional requirement of a prosurvival phenotype. The stroma did not impact the proliferation index (Ki-67 staining) of CLL cells. Collectively, these data suggest that short-term interaction (≤24 hours) with stroma increases OxPhos and bioenergy in replicationally quiescent CLL cells.


Assuntos
Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Células Estromais/metabolismo , Microambiente Tumoral , Apoptose , Comunicação Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Glicólise , Humanos , Espaço Intracelular/metabolismo , Leucemia Linfocítica Crônica de Células B/genética , Redes e Vias Metabólicas , Ribonucleotídeos/metabolismo , Células Tumorais Cultivadas
9.
Mol Cancer Res ; 15(12): 1692-1703, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28835371

RESUMO

Peripheral blood chronic lymphocytic leukemia (CLL) cells are quiescent but have active transcription and translation processes, suggesting that these lymphocytes are metabolically active. Based on this premise, the metabolic phenotype of CLL lymphocytes was investigated by evaluating the two intracellular ATP-generating pathways. Metabolic flux was assessed by measuring glycolysis as extracellular acidification rate (ECAR) and mitochondrial oxidative phosphorylation as oxygen consumption rate (OCR) and then correlated with prognostic factors. Further, the impact of B-cell receptor signaling (BCR) on metabolism was determined by genetic ablation and pharmacological inhibitors. Compared with proliferative B-cell lines, metabolic fluxes of oxygen and lactate were low in CLL cells. ECAR was consistently low, but OCR varied considerably in human patient samples (n = 45). Higher OCR was associated with poor prognostic factors such as ZAP 70 positivity, unmutated IGHV, high ß2M levels, and higher Rai stage. Consistent with the association of ZAP 70 and IGHV unmutated status with active BCR signaling, genetic ablation of BCR mitigated OCR in malignant B cells. Similarly, knocking out PI3Kδ, a critical component of the BCR pathway, decreased OCR and ECAR. In concert, PI3K pathway inhibitors dramatically reduced OCR and ECAR. In harmony with a decline in metabolic activity, the ribonucleotide pools in CLL cells were reduced with duvelisib treatment. Collectively, these data demonstrate that CLL metabolism, especially OCR, is linked to prognostic factors and is curbed by BCR and PI3K pathway inhibition.Implications: This study identifies a relationship between oxidative phosphorylation in CLL and prognostic factors providing a rationale to therapeutically target these processes. Mol Cancer Res; 15(12); 1692-703. ©2017 AACR.


Assuntos
Glicólise/genética , Leucemia Linfocítica Crônica de Células B/genética , Prognóstico , Receptores de Antígenos de Linfócitos B/genética , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Classe I de Fosfatidilinositol 3-Quinases/genética , Humanos , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Redes e Vias Metabólicas/genética , Fosforilação Oxidativa , Consumo de Oxigênio/genética , Proteína-Tirosina Quinase ZAP-70/genética
10.
Br J Haematol ; 179(2): 266-271, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28737232

RESUMO

8-chloro-adenosine (8-Cl-Ado) is currently in phase-I clinical trials for acute myeloid leukaemia and chronic lymphocytic leukaemia (CLL). Previously, we demonstrated that treatment with 8-Cl-Ado leads to diminished ATP levels. We hypothesized that AMP-activated protein kinase (AMPK) signalling would be initiated in these cells, leading to induction of autophagy. AMPK activation and induction of autophagy were demonstrated during preclinical incubations in CLL cells with the analogues. Importantly, we extended similar observations in CLL lymphocytes during an 8-Cl-Ado phase-I trial. In conclusion, 8-Cl-Ado treatment induces autophagy in CLL lymphocytes in vitro as well as in vivo during clinical trial.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Autofagia/efeitos dos fármacos , Desoxiadenosinas/farmacologia , Leucemia Linfocítica Crônica de Células B , Linfócitos , Ensaios Clínicos Fase I como Assunto , Indução Enzimática , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/enzimologia , Leucemia Linfocítica Crônica de Células B/patologia , Linfócitos/enzimologia , Linfócitos/patologia , Masculino
11.
Oncotarget ; 8(13): 22104-22112, 2017 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-27655665

RESUMO

Chemoimmunotherapy regimens have been the standard first-line therapy for patients with chronic lymphocytic leukemia (CLL). For young, fit patients the standard of care is combination of fludarabine, cyclophosphamide, and rituximab (FCR). Based on the preclinical work demonstrating that bendamustine combined with fludarabine resulted in increased DNA damage, we designed a phase I-II clinical trial with fludarabine, bendamustine, and rituximab (FBR) for patients with relapsed/refractory CLL. Treatment consisted of fludarabine 20 mg/m2 daily x 3 days and rituximab 375-500 mg/m2 x 1 day. Phase I included bendamustine at increasing doses of 20, 30, 40, or 50 mg/m2 daily x 3 days; phase II was with FR, and B at the selected dose. DNA damage response (H2AX phosphorylation) was evaluated in a subset of patients. Fifty-one patients were enrolled. The median age was 62 years; median number of prior therapies was 2; 40% had del(11q); and 41 patients had received prior FCR-based therapies. Hematologic toxicity was more common in ≥40 mg/m2 dose cohorts. Maximum tolerated dose (MTD) was not identified. Bendamustine-elicited H2AX phosphorylation was not dose-dependent, but markedly increased after fludarabine. We identified bendamustine 30 mg/m2 as the safe dose for phase II. The overall response rate (ORR) was 67% with 36% complete response (CR) / CR with incomplete count recovery (CRi). Younger patients (<65 years) had significantly higher ORR (81% vs. 50%; p=0.038). The median progression-free survival was 19 months, and the median overall survival was 52.5 months. FBR is an effective and tolerable CIT regimen for patients with relapsed CLL.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Idoso , Idoso de 80 Anos ou mais , Cloridrato de Bendamustina/administração & dosagem , Feminino , Seguimentos , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Rituximab/administração & dosagem , Taxa de Sobrevida , Vidarabina/administração & dosagem , Vidarabina/análogos & derivados
12.
Blood ; 123(24): 3780-9, 2014 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-24747434

RESUMO

The fludarabine and cyclophosphamide couplet has become the backbone of the chronic lymphocytic leukemia (CLL) standard of care. Although this is an effective treatment, it results in untoward toxicity. Bendamustine is a newly approved and better-tolerated alkylating agent. We hypothesized that similar to cyclophosphamide, bendamustine-induced DNA damage will be inhibited by fludarabine, resulting in increased cytotoxicity. To test this hypothesis and the role of the stromal microenvironment in this process, we treated CLL lymphocytes in vitro with each drug alone and in combination. Simultaneous or prior addition of fludarabine to bendamustine resulted in maximum cytotoxicity assayed by 3,3'-dihexyloxacarbocyanine iodine negativity, annexin positivity, and poly (adenosine 5'-diphosphate-ribose) polymerase cleavage. Cytotoxicity elicited by combination of both agents was similar in these malignant B cells cultured either in suspension or on marrow stroma cells. Cell death was associated with DNA damage response, which was determined by phosphorylation of H2AX and unscheduled DNA synthesis. H2AX activation was maximum with the drug combination, and unscheduled DNA synthesis induced by bendamustine was blocked by fludarabine. In parallel, ATM, Chk2, and p53 were phosphorylated and PUMA was induced. Cell death was caspase independent; however, caspases did decrease levels of Mcl-1 survival protein. These data provide a rationale for combining fludarabine with bendamustine for patients with CLL.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Compostos de Mostarda Nitrogenada/administração & dosagem , Vidarabina/análogos & derivados , Apoptose/efeitos dos fármacos , Cloridrato de Bendamustina , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/fisiologia , Células Cultivadas , Esquema de Medicação , Avaliação Pré-Clínica de Medicamentos , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Cultura Primária de Células , Células Estromais/efeitos dos fármacos , Células Estromais/fisiologia , Fatores de Tempo , Vidarabina/administração & dosagem
13.
Leuk Lymphoma ; 53(10): 2024-32, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22448923

RESUMO

Survival of chronic myelogenous leukemia (CML) cells is dependent on BCR-ABL kinase, the activity of which is contingent on the level of BCR-ABL protein and the availability of adenosine triphosphate (ATP). We hypothesized that 8-amino-adenosine (8-amino-Ado)-mediated reduction in cellular ATP level and inhibition of mRNA synthesis leading to a decrease in protein level would result in a multifaceted targeting of BCR-ABL. Using K562 cells, we demonstrated that there was a dose- and time-dependent increase in 8-amino-ATP accompanied by a > 95% decline in the endogenous ATP pool. In parallel, 8-amino-Ado inhibited RNA synthesis and resulted in a depletion of BCR-ABL transcript. Consistent with this, BCR-ABL and ABL protein levels were also decreased. These effects were associated with the initiation of cell death as visualized by poly(ADP-ribose) polymerase (PARP) cleavage, decreased clonogenicity and greater than additive interaction with imatinib. In imatinib-sensitive and -resistant KBM5 cells, 8-amino-Ado treatment augmented the imatinib effect on growth inhibition.


Assuntos
Adenosina/análogos & derivados , Proteínas de Fusão bcr-abl/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Adenosina/química , Adenosina/farmacologia , Adenosina/toxicidade , Trifosfato de Adenosina/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Benzamidas , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas de Fusão bcr-abl/genética , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Mesilato de Imatinib , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Transcrição Gênica/efeitos dos fármacos
14.
Blood ; 116(25): 5622-30, 2010 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-20844237

RESUMO

8-Aminoadenosine (8-NH(2)-Ado), a ribosyl nucleoside analog, in preclinical models of multiple myeloma inhibits phosphorylation of proteins in multiple growth and survival pathways, including Akt. Given that Akt controls the activity of mammalian target of rapamycin (mTOR), we hypothesized that 8-NH(2)-Ado would be active in mantle cell lymphoma (MCL), a hematological malignancy clinically responsive to mTOR inhibitors. In the current study, the preclinical efficacy of 8-NH(2)-Ado and its resulting effects on Akt/mTOR and extracellular-signal-regulated kinase signaling were evaluated using 4 MCL cell lines, primary MCL cells, and normal lymphocytes from healthy donors. For all MCL cell lines, 8-NH(2)-Ado inhibited growth and promoted cell death as shown by reduction of thymidine incorporation, loss of mitochondrial membrane potential, and poly (adenosine diphosphate-ribose) polymerase cleavage. The efficacy of 8-NH(2)-Ado was highly associated with intracellular accumulation of 8-NH(2)-adenosine triphosphate (ATP) and loss of endogenous ATP. Formation of 8-NH(2)-ATP was also associated with inhibition of transcription and translation accompanied by loss of phosphorylated (p-)Akt, p-mTOR, p-Erk1/2, p-phosphoprotein (p)38, p-S6, and p-4E-binding protein 1. While normal lymphocytes accumulated 8-NH(2)-ATP but maintained their viability with 8-NH(2)-Ado treatment, primary lymphoma cells accumulated higher concentrations of 8-NH(2)-ATP, had increased loss of ATP, and underwent apoptosis. We conclude that 8-NH(2)-Ado is efficacious in preclinical models of MCL and inhibits signaling of Akt/mTOR and Erk pathways.


Assuntos
Adenosina/análogos & derivados , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Linfoma de Célula do Manto/patologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/antagonistas & inibidores , Adenosina/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Citometria de Fluxo , Humanos , Linfoma de Célula do Manto/tratamento farmacológico , Linfoma de Célula do Manto/metabolismo , Fosforilação/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serina-Treonina Quinases TOR/metabolismo , Transcrição Gênica/efeitos dos fármacos , Células Tumorais Cultivadas
15.
Blood ; 116(7): 1083-91, 2010 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-20442367

RESUMO

Forodesine, a purine nucleoside phosphorylase inhibitor, displays in vitro activity in chronic lymphocytic leukemia (CLL) cells in presence of dGuo, which is the basis for an ongoing clinical trial in patients with fludarabine-refractory CLL. Initial clinical data indicate forodesine has significant activity on circulating CLL cells, but less activity in clearing CLL cells from tissues such as marrow. In tissue microenvironments, lymphocytes interact with accessory stromal cells that provide survival and drug-resistance signals, which may account for residual disease. Therefore, we investigated the impact of marrow stromal cells (MSCs) on forodesine-induced response in CLL lymphocytes. We demonstrate that spontaneous and forodesine-induced apoptosis of CLL cells was significantly inhibited by human and murine MSCs. Forodesine-promoted dGuo triphosphate (dGTP) accumulation and GTP and ATP depletion in CLL cells was inhibited by MSCs, providing a mechanism for resistance. Also, MSCs rescued CLL cells from forodesine-induced RNA- and protein-synthesis inhibition and stabilized and increased Mcl-1 transcript and protein levels. Conversely, MSC viability was not affected by forodesine and dGuo. Collectively, MSC-induced biochemical changes antagonized forodesine-induced CLL cell apoptosis. This provides a biochemical mechanism for MSC-derived resistance to forodesine and emphasizes the need to move toward combinations with agents that interfere with the microenvironment's protective role for improving current therapeutic efforts.


Assuntos
Medula Óssea/fisiologia , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/patologia , Nucleosídeos de Purina/farmacologia , Purina-Núcleosídeo Fosforilase/antagonistas & inibidores , Pirimidinonas/farmacologia , Células Estromais/fisiologia , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Humanos , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
16.
J Biol Chem ; 285(11): 8022-30, 2010 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-20064937

RESUMO

8-Chloroadenosine (8-Cl-Ado) is a ribosyl nucleoside analog currently in phase I testing for the treatment of chronic lymphocytic leukemia (CLL). 8-Cl-Ado activity is dependent on adenosine kinase and requires intracellular accumulation of 8-Cl-Ado as mono-, di-, and tri-phosphates. In the current study with four mantle cell lymphoma cell lines, we report a new major metabolic pathway for 8-Cl-Ado intracellular metabolism, the formation of succinyl-8-chloro-adenosine (S-8-Cl-Ado) and its monophosphate (S-8-Cl-AMP). 8-Cl-AMP levels were highly associated with S-8-Cl-AMP levels and reached a steady-state prior to the secondary metabolites, 8-Cl-ATP and S-8-Cl-Ado. Consistent with fumarate as a required substrate for formation of succinyl-8-Cl-adenylate metabolites, the S-8-Cl-adenylate concentrations in multiple cell lines were associated with fumarate loss. The distribution of metabolites was also altered using the energy metabolism modifiers, metformin and oligomycin. The rates of succinyl-8-Cl-adenylate metabolism were enhanced by increasing the intracellular fumarate concentrations after metformin co-treatment. In addition, the S-8-Cl-AMP concentrations were increased after acute inhibition of ATP synthase by oligomycin. We conclude that 8-Cl-Ado metabolism not only affects intracellular purine metabolism; 8-Cl-Ado conversion to succinyl analogs ties its metabolism to the citric acid cycle by reduction of the fumarate pool.


Assuntos
2-Cloroadenosina/análogos & derivados , Antineoplásicos/farmacocinética , Metabolismo Energético/efeitos dos fármacos , Fumaratos/metabolismo , Linfoma de Célula do Manto/tratamento farmacológico , Ácido Succínico/metabolismo , 2-Cloroadenosina/química , 2-Cloroadenosina/farmacocinética , 8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , 8-Bromo Monofosfato de Adenosina Cíclica/metabolismo , Antineoplásicos/química , Linhagem Celular Tumoral , Ciclo do Ácido Cítrico/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Hipoglicemiantes/farmacologia , Linfoma de Célula do Manto/metabolismo , Linfoma de Célula do Manto/patologia , Metformina/farmacologia , Oligomicinas/farmacologia , Purinas/metabolismo , Ácido Succínico/química , Desacopladores/farmacologia
17.
Biochem Pharmacol ; 78(6): 583-91, 2009 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-19477165

RESUMO

8-Chloroadenosine (8-Cl-Ado) is a ribonucleoside analogue that is currently in clinical trial for chronic lymphocytic leukemia. Based on the decline in cellular ATP pool following 8-Cl-Ado treatment, we hypothesized that 8-Cl-ADP and 8-Cl-ATP may interfere with ATP synthase, a key enzyme in ATP production. Mitochondrial ATP synthase is composed of two major parts; F(O) intermembrane base and F1 domain, containing alpha and beta subunits. Crystal structures of both alpha and beta subunits that bind to the substrate, ADP, are known in tight binding (alpha(dp)beta(dp)) and loose binding (alpha(tp)beta(tp)) states. Molecular docking demonstrated that 8-Cl-ADP/8-Cl-ATP occupied similar binding modes as ADP/ATP in the tight and loose binding sites of ATP synthase, respectively, suggesting that the chlorinated nucleotide metabolites may be functional substrates and inhibitors of the enzyme. The computational predictions were consistent with our whole cell biochemical results. Oligomycin, an established pharmacological inhibitor of ATP synthase, decreased both ATP and 8-Cl-ATP formation from exogenous substrates, however, did not affect pyrimidine nucleoside analogue triphosphate accumulation. Synthesis of ATP from ADP was inhibited in cells loaded with 8-Cl-ATP. These biochemical studies are in consent with the computational modeling; in the alpha(tp)beta(tp) state 8-Cl-ATP occupies similar binding as ANP, a non-hydrolyzable ATP mimic that is a known inhibitor. Similarly, in the substrate binding site (alpha(dp)beta(dp)) 8-Cl-ATP occupies a similar position as ATP mimic ADP-BeF(3)(-). Collectively, our current work suggests that 8-Cl-ADP may serve as a substrate and the 8-Cl-ATP may be an inhibitor of ATP synthase.


Assuntos
2-Cloroadenosina/análogos & derivados , Complexos de ATP Sintetase/efeitos dos fármacos , Adenosina/farmacologia , 2-Cloroadenosina/química , 2-Cloroadenosina/farmacologia , Complexos de ATP Sintetase/química , Adenosina/química , Antineoplásicos/química , Antineoplásicos/farmacologia , Glicólise/efeitos dos fármacos , Glicólise/fisiologia , Halogenação , Hidrólise , Modelos Moleculares , Consumo de Oxigênio/efeitos dos fármacos , Ligação Proteica , Conformação Proteica/efeitos dos fármacos
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