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BACKGROUND: PNT2258 is a liposomal formulation that encapsulates multiple copies of PNT100, a native, chemically unmodified, 24-base DNA oligonucleotide designed to target the regulatory region upstream of the B-cell lymphoma 2 (BCL2) gene. METHODS: This phase II, multicenter, single-arm, open-label, 2-stage design study investigated the single-agent activity of PNT2258 in patients with relapsed/refractory DLBCL. Initially, patients had to have a performance status (PS) of ≤2 and prior exposure to CD20-targeted therapy, an alkylating agent, and a steroid with no upper limit. Criteria were modified to PS of 0 or 1 and at least 1 to ≤3 prior therapies (identified as the target population) after observing an initially high frequency of rapid disease progression in patients with extensive prior therapies or poor PS. RESULTS: The study was stopped early following an interim analysis, despite surpassing the protocol predetermined futility boundary, because the ORR was below the expectations of response in an evolving DLBCL treatment landscape. The final analysis included all 45 enrolled patients and demonstrated an ORR of 11%. In the response evaluable subset (n = 26), defined as patients in the target population with exposure to ≥8 doses of PNT2258 within the first 35 days and evaluable baseline/post-baseline scans, the ORR was 19%. The most common adverse events were fatigue (44%), nausea (42%), diarrhea (40%), pyrexia (36%), anemia (32%), and vomiting (27%). CONCLUSIONS: PNT2258 was well-tolerated in a chemotherapy refractory DLBCL population. Despite demonstration of single-agent activity, ORR was lower than acceptable for further new therapy development.
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ADN/metabolismo , Linfoma de Células B Grandes Difuso/terapia , Oligonucleótidos/uso terapéutico , Adulto , Anciano , Anciano de 80 o más Años , Humanos , Persona de Mediana Edad , Oligonucleótidos/farmacología , Recurrencia , Resultado del TratamientoRESUMEN
BACKGROUND: PNT2258 consists of a native, chemically unmodified, 24-base DNA oligonucleotide designed to target the regulatory region upstream of the BCL2 gene, delivered in a protective liposome. Derangement of BCL2-regulated control mechanisms is a defining characteristic of certain malignancies, and it was hypothesized that the oligonucleotide would promote anticancer activity via suppression of BCL2 transcription. METHODS: PNT2258 was evaluated in this, multicenter, nonrandomized, open-label Phase 2 study in 13 participants with relapsed/refractory B-cell malignancies to investigate potential antitumor activity and safety. Participants with follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, or chronic lymphocytic leukemia received intravenous PNT2258 120 mg/m2 on Days 1 to 5 of a 21-day cycle for up to 8 cycles, followed by 100 mg/m2 on Days 1 to 2 of a 28-day cycle until study withdrawal. RESULTS: All 13 participants were treated with PNT2258 monotherapy and evaluable for response and safety and tolerability. The overall response rate was 53.8% (7/13; 95% confidence interval [CI], 25.1%-80.8%). Median duration of response was 23.4 months (range, 3, 31.5). The disease control rate of participants with stable disease or better was 84.6% (95% CI, 54.6%-98.1%). The most frequently (≥50%) observed adverse events (AEs) were nausea, chills, diarrhea, fatigue, headache, vomiting, and back pain. Hypertension (30.8%) and diarrhea (23.1%) were the most frequent grade ≥3 AEs. No deaths were observed. CONCLUSION: Clinically meaningful and durable activity with an acceptable safety profile was observed in participants with relapsed/refractory B-cell malignancies who received single-agent PNT2258. TRIAL REGISTRATION: NCT01733238, first posted 26-Nov-2012. https://clinicaltrials.gov/ct2/show/NCT01733238.
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Leucemia Linfocítica Crónica de Células B , Linfoma Folicular , Linfoma de Células B Grandes Difuso , Adulto , Humanos , Leucemia Linfocítica Crónica de Células B/patología , Linfoma de Células B Grandes Difuso/patología , Oligodesoxirribonucleótidos , Resultado del TratamientoRESUMEN
Ibrutinib is approved for chronic lymphocytic leukemia (CLL). However, its role in the treatment of multiple myeloma (MM) is not clear and is under investigation. We report a case of CLL that developed MM while on therapy with ibrutinib indicating that this drug may not be active against MM.
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Non-Hodgkin's lymphoma (NHL) is the most common hematological malignancy in the US. Many types remain incurable despite response to initial therapy and achievement of complete remission (CR). Advanced laboratory techniques like multicolor flow cytometry (FCM) and polymerase chain reaction (PCR) have demonstrated persistence of rare malignant cell population post therapy. However, the functional and biological characteristics of this population have not been elucidated. Established B-lymphoma cell lines (B-NHL) and patient-derived samples (PDS) were analyzed using 8-color FCM. CD34+ sub-population was enriched using in vitro exposure to 2-chlorodeoxyadenosine (2-CdA) and by CD34 magnetic beads. Genetic analysis of cell fractions was done by karyotyping and array comparative genomic hybridization (aCGH). Sensitivity to chemotherapy was assayed by short-term in vitro exposure to chemotherapy. Clonogenicity was determined by soft agar colony formation assay, and proliferation was determined using DNA staining with propidium iodide and FCM. FCM demonstrated the presence of a minute sub-clone of monotypic B-cells that express CD34 in B-NHL cell lines (3 of 3) and in PDS (8 of 8). This sub-population enriched up to 50 fold in vitro by exposure to 2-CdA and up to 80% purity by CD34 magnetic bead column isolation. Except for CD34 expression, this population expressed identical phenotype and genotype to parent cells, but was more proliferative, Hoechst 33342-positive, clonogenic, and resistant to chemotherapy compared with the CD34- population. The isolated CD34+ monotypic B-cells may contribute to resistance of certain NHL to treatment and should be targeted by potential new drugs for NHL.
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INTRODUCTION: Previous research suggested that a novel compound PNT2258 inhibits B-cell lymphoma 2 (BCL-2) transcription by DNA interference (DNAi) and demonstrated its activity in preclinical xenograft models and in a pilot Phase II clinical trial in non-Hodgkin's lymphoma (NHL). While the drug downregulates BCL-2 at the promoter, mRNA, and protein levels, there is a significant homology (13-16 bases) between PNT100 and a number of promoters of genes involved in cell cycle regulation and survival. In this study, we identify cyclin-dependent kinase-4 (CDK4) as an unintended target gene of PNT2258 and examine its relevance to NHL. METHODS: We performed a Basic Local Alignment Search Tool (BLAST) homology search using PNT100 DNAi sequences. Also, we conducted CDK4 promoter assay in K562 cells and studied the protein expression of CDK4 in Wayne State University (WSU)-follicular small cleaved cell lymphoma (FSCCL), WSU-diffuse large cell lymphoma, and WSU-Waldenström's macroglobulinemia (WM) lymphoma cells. RESULTS: BLAST homology search showed that PNT100 completely binds to BCL-2 gene as expected. However, there was 100% homology in a stretch of 14 bases (8-21) between PNT100 and CDK4. PNT2258 strongly inhibited CDK4 promoter activity in K562 cells. Moreover, CDK4 protein expression was significantly downregulated by PNT2258 in WSU-FSCCL and WSU-WM cell lines. DISCUSSION: DNAi may work not only through knocking down the intended gene but also by knocking down other genes. PNT2258 affects CDK4 expression and promoter activity. Results of the present study suggest a broader mechanism of action for DNAi targeting both intended (BCL-2) and unintended (CDK4) genes.
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BACKGROUND: The combination of rituximab and 2-CdA is an effective therapy for B-cell tumors. However, the molecular mechanisms and enzymatic pathways involved in the interaction between the two agents are not fully understood. In this study, we provide molecular evidence for positive interaction between these two agents with resultant therapeutic benefit. METHODS: Efficacy of the R-2CdA regimen was evaluated in thirteen patients with B-cell tumors (9 CLL; 3 WM and 1 FL), in vitro against 3 lymphoma cell lines and in a xenograft mouse model. Treatment-induced changes involving phenotype, kinase activity and protein expression were assessed in vitro and in the mouse xenograft tumors. The interaction between RTX and 2-CdA was analyzed using the multiple comparison method, Tukey's honestly significant difference (HSD). For the clinical and animal data, survival functions were estimated using the Kaplan-Meier method and compared by the log-rank test. P-values <0.05 were considered statistically significant. All statistical analyses were evaluated using GraphPad Prism 4 (San Diego, CA). RESULTS: 9 of 12 (75%) evaluable patients responded to the R-2-CdA regimen with median duration of response of 34 months. Median survival of patients from diagnosis and from completion of R-2-CdA treatment was 13.3 and 7.9 years, respectively. In vitro, the combination was effective in all 3 cell lines of lymphomas but with higher sensitivity in the follicular lymphoma cell line. The combination was also effective in the WSU-WM-SCID xenograft model with dose-dependent response and synergistic benefit. All animals were tumor-free for up to 120 days post 2 cycles of this regimen. Rituximab induced activation of deoxycytidine kinase (dCK), p38 mitogen activated protein kinase (p38MAPK) and glycogen synthase kinase-3ß (GSK-3ß) in the xenograft WSU-WM tumors. Chemical inhibition of p38MAPK led to inhibition of the GSK-3ß phosphorylation suggesting that GSK-3ß is regulated by p38MAPK in this model. CONCLUSION: Collectively, our studies show concordance between the activity of R-2-CdA in vitro, in human and in WSU-WM xenograft model attesting to the validity of this model in predicting clinical response. Modulation of dCK and GSK-3ß by rituximab may contribute to the positive therapeutic interaction between rituximab and 2-CdA.
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BACKGROUND: Lymphomas frequently retain wild-type (wt) p53 function but overexpress HDM2, thereby compromising p53 activity. Therefore, lymphoma is a suitable model for studying the therapeutic value of disrupting the HDM2-p53 interaction by small-molecule inhibitors (SMIs). HDM2 have been developed and are under various stages of preclinical and clinical investigation. Previously, we examined the anti-lymphoma activity of MI-319, the laboratory grade of a new class of HDM2 SMI, the spiro-oxindole, in follicular lymphoma. Since then, MI-219, the clinical grade has become readily available. This study further examines the preclinical effects and mechanisms of MI-219 in a panel of human lymphoma cell lines as well as a cohort of patient-derived B-lymphocytes for its potential clinical use. RESULTS: Preclinical assessment of MI-219 was evaluated by means of an in vitro and ex vivo approach and compared to Nutlin-3, the gold standard. Characterization of p53 activity and stability were assessed by quantitative PCR, Western blot, and immunoprecipitation. Biological outcome was measured using Trypan blue exclusion assay, Annexin V/PI, PARP and caspase-3 cleavage. Surprisingly, the overall biological effects of Nutlin-3 were more delayed (48 h) while MI-219 triggered an earlier response (12-24 h), predominantly in the form of apoptotic cell death. Using a cell free autoubiquitination assay, neither agent interfered with HDM2 E3 ligase function. MI-219 was more effective in upregulating wt-p53 stabilization compared to Nutlin-3. MI-219, but not Nutlin-3, enhanced the autoubiquitination and degradation of HDM2. CONCLUSIONS: Our data reveals unexpected differences between MI-219 and the well-studied Nutlin-3 in lymphoma cell lines and patient samples. We suggest a novel mechanism for MI-219 that alters the functional activity of HDM2 through enhanced autoubiquitination and degradation. Additionally, this mechanism appears to correspond to biological outcome. Our results provide evidence that different classes of HDM2 SMIs elicit molecular events that extend beyond HDM2-p53 dissociation which may be of biological and potentially therapeutic importance.
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Imidazoles/farmacología , Indoles/farmacología , Linfoma de Células B/tratamiento farmacológico , Linfoma de Células B/metabolismo , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Compuestos de Espiro/farmacología , Proteína p53 Supresora de Tumor/metabolismo , Anciano , Anciano de 80 o más Años , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Femenino , Humanos , Linfoma de Células B/patología , Masculino , Persona de Mediana Edad , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Ubiquitinación/efectos de los fármacosRESUMEN
BACKGROUND: Syncytial giant cell hepatitis (GCH) is an uncommon and an underreported disease entity. In two previously reported cases of GCH in patients with Chronic Lymphocytic Leukemia (CLL) liver failure ensued. Autoimmune and infective causes have been implicated but its etiology remains unclear. CASE PRESENTATION: A 60-year-old female with CLL presented with acute hepatitis with negative viral and auto-immune serologies and without any prior toxic exposure. Liver biopsy showed typical histological features of GCH. The patient was successfully treated with corticosteroids and intravenous immunoglobulin (IVIG). Her liver enzymes returned to baseline and have remained normal as of the last follow up almost 4 years later. CONCLUSIONS: Association of GCH with CLL may be under recognized. Clinical suspicion of GCH in CLL patients with serology-negative hepatitis, early liver biopsy and therapeutic intervention may influence outcome. This is the first case report of successful treatment of GCH in CLL patients. Moreover, our case also demonstrates the ability to resume effective CLL therapy post-GCH diagnosis without detriment to the liver.
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BACKGROUND: MI-319 is a synthetic small molecule designed to target the MDM2-P53 interaction. It is closely related to MDM2 antagonists MI-219 and Nutlin-3 in terms of the expected working mechanisms. The purpose of this study was to evaluate anti-lymphoma activity of MI-319 in WSU-FSCCL, a B-cell follicular lymphoma line. For comparison purpose, MI-319, MI-219 and Nutlin-3 were assessed side by side against FSCCL and three other B-cell hematological tumor cell lines in growth inhibition and gene expression profiling experiments. RESULTS: MI-319 was shown to bind to MDM2 protein with an affinity slightly higher than that of MI-219 and Nutlin-3. Nevertheless, cell growth inhibition and gene expression profiling experiments revealed that the three compounds have quite similar potency against the tumor cell lines tested in this study. In vitro, MI-319 exhibited the strongest anti-proliferation activity against FSCCL and four patient cells, which all have wild-type p53. Data obtained from Western blotting, cell cycle and apoptosis analysis experiments indicated that FSCCL exhibited strong cell cycle arrest and significant apoptotic cell death; cells with mutant p53 did not show significant apoptotic cell death with drug concentrations up to 10 muM, but displayed weaker and differential cell cycle responses. In our systemic mouse model for FSCCL, MI-319 was tolerated well by the animals, displayed effectiveness against FSCCL-lymphoma cells in blood, brain and bone marrow, and achieved significant therapeutic impact (p < 0.0001) by conferring the treatment group a > 28% (%ILS, 14.4 days) increase in median survival days. CONCLUSION: Overall, MI-319 probably has an anti-lymphoma potency equal to that of MI-219 and Nutlin-3. It is a potent agent against FSCCL in vitro and in vivo and holds the promises to be developed further for the treatment of follicular lymphoma that retains wild-type p53.
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Antineoplásicos/farmacología , Indoles/farmacología , Linfoma Folicular/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Compuestos de Espiro/farmacología , Proteína p53 Supresora de Tumor/fisiología , Administración Oral , Animales , Antineoplásicos/metabolismo , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Modelos Animales de Enfermedad , Humanos , Indoles/uso terapéutico , Ratones , Ratones SCID , Unión Proteica , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Compuestos de Espiro/uso terapéutico , Trasplante HeterólogoRESUMEN
PURPOSE: To investigate the activity of SAR3419, a novel humanized anti-CD19 antibody (huB4), conjugated to a cytotoxic maytansine derivative N(2)'-deacetyl-N(2)'-(4-mercapto-4-methyl-1-oxopentyl) maytansine, in preclinical xenograft models for non-Hodgkin's lymphoma. EXPERIMENTAL DESIGN: Antitumor activity of SAR3419 was assessed as a single agent and in comparison with conventional therapies using a subcutaneous model for diffuse large B-cell lymphoma (WSU-DLCL2) and a systemic model for follicular small cleaved cell lymphoma (WSU-FSCCL) in mice with severe combined immune deficiency. RESULTS: Our results showed that in these chemotherapy-resistant models, SAR3419 was more effective than CHOP (cyclophosphamide-Adriamycin-vincristine-prednisone) regimen or rituximab. Only treatment with SAR3419 led to survival of the whole group of animals to the end of the experiment (150-155 days) in both models. Higher doses of SAR3419 (15 and 30 mg/kg) were more effective than lower dose of 7.5 mg/kg. The immunoconjugation was necessary because neither huB4 nor DM4 alone had significant activity. Treatment with rituximab resulted in antitumor activity in both models comparable with the low dose of SAR3419. Cyclophosphamide-Adriamycin-vincristine-prednisone alone showed modest activity in both models. Necropsy and tissue staining in the WSU-FSCCL systemic model revealed that all deaths featured leptomeningeal lymphoma in the control and treated groups. Interestingly, some of the animals that survived to the end of the experiment and seemed healthy at time of euthanasia did show microscopic evidence of lymphoma. CONCLUSIONS: Overall, SAR3419 is a very active immunotoxin in preclinical models for human B-cell lymphoma and holds promise as a novel and well-tolerated therapy in B-cell non-Hodgkin's lymphoma.
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Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos Fitogénicos/uso terapéutico , Inmunoconjugados/uso terapéutico , Linfoma Folicular/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Maitansina/análogos & derivados , Maitansina/uso terapéutico , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales de Origen Murino , Antígenos CD19/metabolismo , Antineoplásicos Fitogénicos/química , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Línea Celular Tumoral , Ciclofosfamida/uso terapéutico , Doxorrubicina/uso terapéutico , Humanos , Factores Inmunológicos/uso terapéutico , Linfoma Folicular/patología , Linfoma de Células B Grandes Difuso/patología , Ratones , Ratones SCID , Prednisona/uso terapéutico , Rituximab , Vincristina/uso terapéutico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The Bcl-2 family of proteins is critical to the life and death of malignant B-lymphocytes. Interfering with their activity using small-molecule inhibitors (SMI) is being explored as a new therapeutic strategy for treating B-cell tumors. We evaluated the efficacy of TW-37, a non-peptidic SMI of Bcl-2 against a range spectrum of human B-cell lines, fresh patient samples and animal xenograft models. Multiple cytochemical and molecular approaches such as acridine orange/ethidium bromide assay for apoptosis, co-immunoprecipitation of complexes and western blot analysis, caspase luminescent activity assay and apoptotic DNA fragmentation assay were used to demonstrate the effect of TW-37 on different B-cell lines, patient derived samples, as well as in animal xenograft models. Nanomolar concentrations of TW-37 were able to induce apoptosis in both fresh samples and established cell lines with IC50 in most cases of 165-320 nM. Apoptosis was independent of proliferative status or pathological classification of B-cell tumor. TW-37 was able to block Bim-Bcl-XL and Bim-Mcl-1 heterodimerization and induced apoptosis via activation of caspases -9, -3, PARP and DNA fragmentation. TW-37 administered to tumor-bearing SCID mice led to significant tumor growth inhibition (T/C), tumor growth delay (T-C) and Log10kill, when used at its maximum tolerated dose (40 mg/kg x 3 days) via tail vein. TW-37 failed to induce changes in the Bcl-2 proteins levels suggesting that assessment of baseline Bcl-2 family proteins can be used to predict response to the drug. These findings indicate activity of TW-37 across the spectrum of human B-cell tumors and support the concept of targeting the Bcl-2 system as a therapeutic strategy regardless of the stage of B-cell differentiation.
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Benzamidas/farmacología , Diferenciación Celular , Proliferación Celular/efectos de los fármacos , Leucemia de Células B/patología , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Sulfonas/farmacología , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Benzamidas/química , Benzamidas/uso terapéutico , Diferenciación Celular/efectos de los fármacos , Progresión de la Enfermedad , Femenino , Humanos , Leucemia de Células B/tratamiento farmacológico , Ratones , Ratones Endogámicos ICR , Ratones SCID , Peso Molecular , Sulfonas/química , Sulfonas/uso terapéutico , Resultado del Tratamiento , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Pre-treatment with bryostatin 1 (bryo) has been shown to potentiate the efficacy of (2-chloro-2-deoxyadenosine, cladribine, 2-CdA) in B-cell chronic lymphocytic leukemia (B-CLL) by increasing the ratio of deoxycytidine kinase (dCK) to 5'-nucleotidase (5'-NT) activity. The bryo-induced increase in dCK/5'-NT activity alone has not been a conclusive indication of final clinical outcome. Therefore, we used an ex vivo assay to investigate factors which may affect the bryo-induced enhancement of 2-CdA efficacy in B-CLL patient-derived samples. Bryo-induced increase in dCK/5'-NT was inversely associated with Rai stage CLL (r=-0.86). Increased dCK/5'-NT activity was not correlated with increased efficacy (cell death) or percentage of cellular [8-3H]-2-CdA converted to [8-3H]-2-CdATP ex vivo. Bryo pre-treatment increased the cellular uptake of [8-3H]-2-CdA and incorporation of [8-3H]-2-CdA metabolites into the DNA fraction. Cell death from 2-CdA was inversely correlated with bryo-induced activity of the DNA repair enzyme, DNA-PKcs, (r=-0.77). Thus, the ability of B-CLL to repair damaged DNA may be a more important predictor of the response to bryo/2-CdA and eventual clinical outcome than dCK/5'-NT activity. Additional CLL patients under bryo-2-CdA therapy are needed to verify these important observations.
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2-Cloroadenosina/análogos & derivados , 2-Cloroadenosina/toxicidad , Antimetabolitos Antineoplásicos/toxicidad , Desoxiadenosinas/toxicidad , Lactonas/farmacología , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , 2-Cloroadenosina/farmacocinética , 5'-Nucleotidasa/metabolismo , Brioestatinas , Muerte Celular , Proteína Quinasa Activada por ADN , Proteínas de Unión al ADN/metabolismo , Desoxiadenosinas/farmacocinética , Desoxicitidina Quinasa/metabolismo , Resistencia a Antineoplásicos , Sinergismo Farmacológico , Humanos , Leucemia Linfocítica Crónica de Células B/metabolismo , Macrólidos , Proteínas Nucleares , Proteínas Serina-Treonina Quinasas/metabolismo , Células Tumorales Cultivadas , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
We have previously reported that XK469 (2-[4-(7-chloro-2-quinoxalinyloxyphenoxy]-propionic acid) enhances topo IIalpha expression in WSU-WM cells in vitro [E. Mensah-Osman et al., Mol. Cancer Ther., 1: 1321-1326, 2002]. To test the hypothesis that XK469-induced expression of topo IIalpha sensitizes WSU-WM cells to the topo IIalpha inhibitor etoposide (VP-16), we investigated the antitumor effects of XK469 and VP-16 in vivo, using the WSU-WM SCID xenograft model. Individual dosages of XK469 at 20-60 mg/kg/injection i.v. for a maximum-tolerated dose of 240 mg/kg were achievable in SCID mice. Simultaneous administration of a subtherapeutic dose of XK469 (20 mg/kg) and VP-16 at its maximum-tolerated dose of 15 mg/kg proved to be highly toxic and lethal. However, daily sequential treatment of XK469 given i.v. via tail vein at 20 mg/kg for a total of 120 mg/kg, followed 7 h later by VP-16 i.p. at 15 mg/kg for a total of 90 mg/kg, had no significant toxicity in SCID mice. The sequential treatment was associated with enhanced antitumor activity. Tumor growth inhibition T/C, tumor growth delay T-C, and log(10) kill for XK469 alone were 61%, 3 days and 0.46; VP-16 alone 6%, 12 days and 1.83, respectively; whereas the sequential administration of both agents gave a T/C value of 0%, T-C value of 23 days and a log(10) kill of 3.5. On the basis of these animal results, we conclude that the sequential treatment of WSU-WM tumors with XK469 and VP-16 was highly active. The study supports our in vitro observation that XK469 potentiates VP-16 activity. The sequential use of both agents resulted in clinically significant antitumor activity in the WM model.
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Etopósido/uso terapéutico , Quinoxalinas/uso terapéutico , Macroglobulinemia de Waldenström/tratamiento farmacológico , Animales , División Celular/efectos de los fármacos , Etopósido/toxicidad , Humanos , Ratones , Ratones SCID , Quinoxalinas/toxicidad , Trasplante Heterólogo , Macroglobulinemia de Waldenström/patologíaRESUMEN
The Wayne State University Waldenstrom's Macroglobulinemia xenograft model in mice with severe combined immune deficiency (WSU-WM-SCID) is the only preclinical animal model available for this disease. It is based on a permanent, EBV- IgMlambda cell line (WSU-WM) established from a patient with a 10-year history of Waldenstrom's macroglobulinemia (WM). These cells are CD5(-)CD10(+)CD19(+)CD20(+)CD22(+) and have t(8;14) (q24;32), t(12;17) (q24;q21), 2p-. WSU-WM cells also express DNA topoisomerase II (alpha and beta), and are bcl(2)(+)bcl(XL)(+)bax(-). Although the tumor has aggressive biological behavior with c-myc-IgH rearrangement, it has retained the salient features of WM. The breakpoint on 8q24 is downstream of c-myc exon 3, which is not usual for Burkitt-type breakpoints. WSU-WM cells also express both secretory (s(u)) and membrane (m(u)) IgM mRNA and secrete IgM in culture supernatant. Histiologically, WSU-WM-SCID xenograft tumors have lymphoplasmacytoid morphology. These features indicate biological, but not histological evolution. The WSU-WM-SCID is a model of a more aggressive and resistant WM usually seen toward the late stages of disease. It is, therefore, a particularly useful tool in developing new therapeutic strategies for the more aggressive WM, including targeted therapy, which exploits unique molecular characteristics of tumor cells.
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Línea Celular , Modelos Animales , Macroglobulinemia de Waldenström , Animales , Línea Celular/química , Línea Celular/inmunología , Citogenética , Humanos , Ratones , Ratones SCID , Trasplante de Neoplasias , Fenotipo , Macroglobulinemia de Waldenström/patologíaRESUMEN
We have previously reported that combretastatin-A4 prodrug (CA4P), anantitubulin/antiangiogenic agent isolated from the South African willow tree Combretum caffrum, induced cell death primarily through mitotic catastrophe in a panel of human B-lymphoid tumors. In this study, we investigated the molecular aspects of the mitotic catastrophe and whether or not it shares the same pathways of apoptosis. For this we studied the effect of CA4P on selected markers of apoptosis [caspases 9 and 3, poly(ADP-ribose) polymerase (PARP), bcl-2, and bax] and G2-M protein regulators (p53, MDM2, 14-3-3sigma, GADD45, cdc2, cdc25, chk1, wee1, p21, and cyclin B1). The chronic lymphocytic leukemia cell line WSU-CLL was used for this purpose. Western blot analysis showed that 24 h of CA4P (5 nM) exposure induces caspase 9 activation and PARP cleavage. However, the addition of Z-Val-Ala-Asp-fluoromethylketone (a general caspase inhibitor) or Z-Leu-Glu(OMe)-His-Asp(OMe)-CH2F (a caspase 9 inhibitor) before CA4P treatment did not block cell death. No change in bcl-2 or bax protein expression was observed. Exposure of WSU-CLL cells to 4 and 5 nM CA4P was associated with overproduction of total p53 and no dramatic change in MDM2, 14-3-3sigma, GADD45, the cyclin-dependent kinase cdc2, its inhibitory phosphorylation, the cdc2-inhibitory kinase (wee1), chk1, or cdc25 hyperphosphorylation. The overaccumulation of p21 and cyclin B1 protein was obvious at 24 h. Furthermore, CA4P treatment showed an increase in the expression of a marker of mitosis (mitotic protein monoclonal-2 antibody) and an overaccumulation of the cyclin B in the nucleus. Our findings suggest that CA4P induces mitotic catastrophe and arrest of WSU-CLL cells mostly in the M phase independent of p53 and independent of chk1 and cdc2 phosphorylation pathways. Apoptosis is a secondary mechanism of death in a small proportion of cells through activation of caspase 9 and PARP cleavage. The two mechanisms of cell death, i.e., mitotic catastrophe and apoptosis, are independent of each other in our model.
Asunto(s)
Biomarcadores de Tumor , Caspasas/metabolismo , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Proteínas de Neoplasias , Proteínas Nucleares , Poli(ADP-Ribosa) Polimerasas/metabolismo , Profármacos/farmacología , Estilbenos/farmacología , Proteínas 14-3-3 , Antineoplásicos Fitogénicos/farmacología , Western Blotting , Proteína Quinasa CDC2/metabolismo , Caspasa 3 , Caspasa 9 , Núcleo Celular/metabolismo , Supervivencia Celular , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/metabolismo , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Exonucleasas/metabolismo , Exorribonucleasas , Citometría de Flujo , Fase G2 , Marcadores Genéticos , Humanos , Mitosis , Fosforilación , Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-mdm2 , Factores de Tiempo , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/metabolismo , Proteína X Asociada a bcl-2RESUMEN
The role of DNA topoisomerase (Topo) IIbeta in cancer chemotherapy remains unclear, although this particular isoform has been implicated in drug resistance. In this study, we investigated Topo IIbeta as a target for 2-[4-(7-chloro-2-quinoxalinyloxy)phenoxy]-propionic acid (XK469), a novel synthetic quinoxaline phenoxypropionic acid derivative, in a Waldenstrom's macroglobulinemia (WM) model. In vitro, the WSU-WM cell line was exposed to 1.0, 2.0, 5.0, 8.0, and 10 microM XK469. Our results demonstrate a concentration-dependent cell growth inhibition with a concentration-independent inhibition of Topo IIbeta, as determined by band depletion assay. The cell growth inhibition of cells correlated well with increase in Bax:Bcl-2 ratio and poly(ADP-ribose) polymerase (PARP) cleavage. We used our established WSU-WM severe combined immunodeficient mouse xenograft model to test the efficacy and effect of XK469 on Topo IIbeta in vivo. Topo IIbeta was inhibited equally using two different dose schedules (20 and 40 mg/kg, i.v., for a total of 120 and 240 mg/kg, respectively); however, there was no significant decrease in tumor weight. Western blot analysis of cells isolated from s.c. tumors showed no induction of the Bax protein and a very low Bax:Bcl-2 ratio of approximately 0.3 in correlation with minimum PARP cleavage. Our study shows that XK469 inhibits Topo IIbeta in WSU-WM cells both in vitro and in vivo at or below the maximum tolerated dose in severe combined immunodeficient mice. However, there was no change of apoptosis-related molecules such as PARP, Bax, and Bcl-2 or reduction in tumor weight in association with Topo IIbeta inhibition. We conclude that Topo IIbeta inhibition by XK469 as a target is not sufficient for therapeutic effects in WSU-WM.
Asunto(s)
Quinoxalinas/farmacología , Inhibidores de Topoisomerasa II , Macroglobulinemia de Waldenström/patología , Animales , Western Blotting , División Celular , ADN-Topoisomerasas de Tipo II , Proteínas de Unión al ADN , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Ratones , Ratones SCID , Trasplante de Neoplasias , Poli(ADP-Ribosa) Polimerasas/metabolismo , Factores de TiempoRESUMEN
Topoisomerase (Topo) IIalpha has proven to be an adequate anticancer target for tumors expressing this enzyme. In this study, we elucidated the effect of 2-[4-(7-chloro-2-quinoxalinyloxyphenoxy]-propionic acid (XK469; a new Topo IIbeta inhibitor) in the modulation of Topo IIalpha levels and sensitivity to Topo IIalpha poisons. We demonstrate by Western blot analysis that indolent B-cell tumors express undetectable levels of this enzyme and are refractory to the effects of Topo IIalpha poisons such as VP16. Using the Waldenstrom's macroglobulinemia (WM) cell line WSU-WM, we show that XK469 induced the expression of Topo IIalpha protein by 24 h compared with control. Immunofluorescence studies by confocal microscopy using a specific monoclonal antibody against Topo IIalpha supported the immunoblot findings with high intensity staining in XK469-exposed cells. To determine the effect of up-regulating Topo IIalpha on sensitivity of Topo IIalpha-directed inhibitors, WSU-WM cells were exposed to simultaneous, sequential, and reverse order XK469 and VP16. We demonstrate that 24 h of exposure to XK469 before VP16 resulted in a maximum synergistic response. In contrast, simultaneous or reverse order exposure resulted in an antagonistic effect. A similar trend was observed with cells obtained from chronic lymphocytic leukemia patients, but not in normal lymphocytes. This increase in VP16 sensitivity after 24 h of XK469 exposure was associated with VP16-dependent DNA cleavage, as demonstrated by formation of a smeared DNA band in a SDS-KCL DNA cleavage assay. From this study, we concluded that XK469 up-regulates Topo IIalpha levels and consequently sensitizes indolent malignant B cells to the cytotoxic effect of VP16 in a schedule-dependent manner.