RESUMEN
Activation of apoptosis in malignant cells is an established strategy for controlling cancer and is potentially curative. To assess the impact of concurrently inducing the extrinsic and intrinsic apoptosis-signaling pathways in acute myeloid leukemia (AML), we evaluated activity of the TRAIL receptor agonistic fusion protein eftozanermin alfa (eftoza; ABBV-621) in combination with the B-cell lymphoma protein-2 selective inhibitor venetoclax in preclinical models and human patients. Simultaneously stimulating intrinsic and extrinsic apoptosis-signaling pathways with venetoclax and eftoza, respectively, enhanced their activities in AML cell lines and patient-derived ex vivo/in vivo models. Eftoza activity alone or plus venetoclax required death receptor 4/5 (DR4/DR5) expression on the plasma membrane but was independent of TP53 or FLT3-ITD status. The safety/tolerability of eftoza as monotherapy and in combination with venetoclax was demonstrated in patients with relapsed/refractory AML in a phase 1 clinical trial. Treatment-related adverse events were reported in 2 of 4 (50%) patients treated with eftoza monotherapy and 18 of 23 (78%) treated with eftoza plus venetoclax. An overall response rate of 30% (7/23; 4 complete responses [CRs], 2 CRs with incomplete hematologic recovery, and 1 morphologic leukemia-free state) was reported in patients who received treatment with eftoza plus venetoclax and 67% (4/6) in patients with myoblasts positive for DR4/DR5 expression; no tumor responses were observed with eftoza monotherapy. These data indicate that combination therapy with eftoza plus venetoclax to simultaneously activate the extrinsic and intrinsic apoptosis-signaling pathways may improve clinical benefit compared with venetoclax monotherapy in relapsed/refractory AML with an acceptable toxicity profile. This trial was registered at www.clinicaltrials.gov as #NCT03082209.
Asunto(s)
Antineoplásicos , Leucemia Mieloide Aguda , Humanos , Antineoplásicos/uso terapéutico , Leucemia Mieloide Aguda/patología , Compuestos Bicíclicos Heterocíclicos con Puentes , Sulfonamidas , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéuticoRESUMEN
Dysregulation of apoptotic machinery is one mechanism by which acute myeloid leukemia (AML) acquires a clonal survival advantage. B-cell lymphoma protein-2 (BCL2) overexpression is a common feature in hematologic malignancies. The selective BCL2 inhibitor, venetoclax (VEN) is used in combination with azacitidine (AZA), a DNAmethyltransferase inhibitor (DNMTi), to treat patients with AML. Despite promising response rates to VEN/AZA, resistance to the agent is common. One identified mechanism of resistance is the upregulation of myeloid cell leukemia-1 protein (MCL1). Pevonedistat (PEV), a novel agent that inhibits NEDD8-activating enzyme, and AZA both upregulate NOXA (PMAIP1), a BCL2 family protein that competes with effector molecules at the BH3 binding site of MCL1. We demonstrate that PEV/AZA combination induces NOXA to a greater degree than either PEV or AZA alone, which enhances VEN-mediated apoptosis. Herein, using AML cell lines and primary AML patient samples ex vivo, including in cells with genetic alterations linked to treatment resistance, we demonstrate robust activity of the PEV/VEN/AZA triplet. These findings were corroborated in preclinical systemic engrafted models of AML. Collectively, these results provide rational for combining PEV/VEN/AZA as a novel therapeutic approach in overcoming AML resistance in current therapies.
Asunto(s)
Azacitidina , Leucemia Mieloide Aguda , Azacitidina/farmacología , Azacitidina/uso terapéutico , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéutico , Ciclopentanos , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Pirimidinas , SulfonamidasRESUMEN
The pathogenesis of acute myeloid leukemia (AML) involves serial acquisition of mutations controlling several cellular processes, requiring combination therapies affecting key downstream survival nodes in order to treat the disease effectively. The BCL2 selective inhibitor venetoclax has potent anti-leukemia efficacy; however, resistance can occur due to its inability to inhibit MCL1, which is stabilized by the MAPK pathway. In this study, we aimed to determine the anti-leukemia efficacy of concomitant targeting of the BCL2 and MAPK pathways by venetoclax and the MEK1/2 inhibitor cobimetinib, respectively. The combination demonstrated synergy in seven of 11 AML cell lines, including those resistant to single agents, and showed growth-inhibitory activity in over 60% of primary samples from patients with diverse genetic alterations. The combination markedly impaired leukemia progenitor functions, while maintaining normal progenitors. Mass cytometry data revealed that BCL2 protein is enriched in leukemia stem/progenitor cells, primarily in venetoclax-sensitive samples, and that cobimetinib suppressed cytokine-induced pERK and pS6 signaling pathways. Through proteomic profiling studies, we identified several pathways inhibited downstream of MAPK that contribute to the synergy of the combination. In OCI-AML3 cells, the combination downregulated MCL1 protein levels and disrupted both BCL2:BIM and MCL1:BIM complexes, releasing BIM to induce cell death. RNA sequencing identified several enriched pathways, including MYC, mTORC1, and p53 in cells sensitive to the drug combination. In vivo, the venetoclax-cobimetinib combination reduced leukemia burden in xenograft models using genetically engineered OCI-AML3 and MOLM13 cells. Our data thus provide a rationale for combinatorial blockade of MEK and BCL2 pathways in AML.
Asunto(s)
Leucemia Mieloide Aguda , Proteómica , Apoptosis , Azetidinas , Compuestos Bicíclicos Heterocíclicos con Puentes , Línea Celular Tumoral , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Piperidinas , Proteínas Proto-Oncogénicas c-bcl-2/genética , SulfonamidasRESUMEN
BACKGROUND: MCL-1 is a prosurvival B-cell lymphoma 2 family protein that plays a critical role in tumor maintenance and survival and can act as a resistance factor to multiple anticancer therapies. Herein, we describe the generation and characterization of the highly potent and selective MCL-1 inhibitor ABBV-467 and present findings from a first-in-human trial that included patients with relapsed/refractory multiple myeloma (NCT04178902). METHODS: Binding of ABBV-467 to human MCL-1 was assessed in multiple cell lines. The ability of ABBV-467 to induce tumor growth inhibition was investigated in xenograft models of human multiple myeloma and acute myelogenous leukemia. The first-in-human study was a multicenter, open-label, dose-escalation study assessing safety, pharmacokinetics, and efficacy of ABBV-467 monotherapy. RESULTS: Here we show that administration of ABBV-467 to MCL-1-dependent tumor cell lines triggers rapid and mechanism-based apoptosis. In vivo, intermittent dosing of ABBV-467 as monotherapy or in combination with venetoclax inhibits the growth of xenografts from human hematologic cancers. Results from a clinical trial evaluating ABBV-467 in patients with multiple myeloma based on these preclinical data indicate that treatment with ABBV-467 can result in disease control (seen in 1 patient), but may also cause increases in cardiac troponin levels in the plasma in some patients (seen in 4 of 8 patients), without other corresponding cardiac findings. CONCLUSIONS: The selectivity of ABBV-467 suggests that treatment-induced troponin release is a consequence of MCL-1 inhibition and therefore may represent a class effect of MCL-1 inhibitors in human patients.
Apoptosis is a type of cell death that removes abnormal cells from the body. Cancer cells can have increased levels of MCL-1, a protein that helps cells survive and prevents apoptosis. ABBV-467 is a new drug that blocks the action of MCL-1 (an MCL-1 inhibitor) and could promote apoptosis. In animal models, ABBV-467 led to cancer cell death and delayed tumor growth. ABBV-467 was also studied in a clinical trial in 8 patients with multiple myeloma, a blood cancer. In 1 patient, ABBV-467 treatment prevented the cancer from getting any worse for 8 months. However, in 4 out of 8 patients ABBV-467 increased the levels of troponin, a protein associated with damage to the heart. This concerning side effect may impact the future development of MCL-1 inhibitors as anticancer drugs.
RESUMEN
Colorectal cancer (CRC) is one of the most common and deadliest forms of cancer. Myeloid Cell Leukemia 1 (MCL1), a pro-survival member of the Bcl-2 protein family is associated with chemo-resistance in CRC. The ability of MCL1 to inhibit apoptosis by binding to the BH3 domains of pro-apoptotic Bcl-2 family members is a well-studied means by which this protein confers resistance to multiple anti-cancer therapies. We found that specific DNA damaging chemotherapies promote nuclear MCL1 translocation in CRC models. In p53null CRC, this process is associated with resistance to chemotherapeutic agents, the mechanism of which is distinct from the classical mitochondrial protection. We previously reported that MCL1 has a noncanonical chemoresistance capability, which requires a novel loop domain that is distinct from the BH3-binding domain associated with anti-apoptotic function. Herein we disclose that upon treatment with specific DNA-damaging chemotherapy, this loop domain binds directly to alpha-enolase which in turn binds to calmodulin; we further show these protein-protein interactions are critical in MCL1's nuclear import and chemoresistance. We additionally observed that in chemotherapy-treated p53-/- CRC models, MCL1 nuclear translocation confers sensitivity to Bcl-xL inhibitors, which has significant translational relevance given the co-expression of these proteins in CRC patient samples. Together these findings indicate that chemotherapy-induced MCL1 translocation represents a novel resistance mechanism in CRC, while also exposing an inherent and targetable Bcl-xL co-dependency in these cancers. The combination of chemotherapy and Bcl-xL inhibitors may thus represent a rational means of treating p53-/- CRC via exploitation of this unique MCL1-based chemoresistance mechanism.
Asunto(s)
Neoplasias Colorrectales , Resistencia a Antineoplásicos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Apoptosis/genética , Línea Celular Tumoral , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Humanos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteína p53 Supresora de Tumor/genéticaRESUMEN
During chronic inflammation and ageing, the increase in oxidative stress in both intracellular and extracellular compartments is likely to influence local cell functions. Redox changes alter the T-cell proteome in a quantitative and qualitative manner, and post-translational modifications to surface and cytoplasmic proteins by increased reactive species can influence T-cell function. Previously, we have shown that RA (rheumatoid arthritis) T-cells exhibit reduced ROS (reactive oxygen species) production in response to extracellular stimulation compared with age-matched controls, and basal ROS levels [measured as DCF (2',7'-dichlorofluorescein) fluorescence] are lower in RA T-cells. In contrast, exposing T-cells in vitro to different extracellular redox environments modulates intracellular signalling and enhances cytokine secretion. Together, these data suggest that a complex relationship exists between intra- and extra-cellular redox compartments which contribute to the T-cell phenotype.
Asunto(s)
Envejecimiento/fisiología , Inflamación/metabolismo , Oxidación-Reducción , Transducción de Señal/fisiología , Linfocitos T/inmunología , Linfocitos T/fisiología , Radicales Libres/metabolismo , Humanos , Estrés Oxidativo/fisiología , Especies Reactivas de Oxígeno/metabolismo , Linfocitos T/citologíaRESUMEN
BCL-XL, an antiapoptotic member of the BCL-2 family of proteins, drives tumor survival and maintenance and thus represents a key target for cancer treatment. Herein we report the rational design of a novel series of selective BCL-XL inhibitors exemplified by A-1293102. This molecule contains structural elements of selective BCL-XL inhibitor A-1155463 and the dual BCL-XL/BCL-2 inhibitors ABT-737 and navitoclax, while representing a distinct pharmacophore as assessed by an objective cheminformatic evaluation. A-1293102 exhibited picomolar binding affinity to BCL-XL and both efficiently and selectively killed BCL-XL-dependent tumor cells. X-ray crystallographic analysis demonstrated a key hydrogen bonding network in the P2 binding pocket of BCL-XL, while the bent-back moiety achieved efficient occupancy of the P4 pocket in a manner similar to that of navitoclax. A-1293102 represents one of the few distinct structural series of selective BCL-XL inhibitors, and thus serves as a useful tool for biological studies as well as a lead compound for further optimization.
RESUMEN
Cyclin-dependent kinase 9 (CDK9) is a serine/threonine kinase involved in the regulation of transcription elongation. An inhibition of CDK9 downregulates a number of short-lived proteins responsible for tumor maintenance and survival, including the antiapoptotic BCL-2 family member MCL-1. As pan-CDK inhibitors under development have faced dosing and toxicity challenges in the clinical setting, we generated selective CDK9 inhibitors that could be amenable to an oral administration. Here, we report the lead optimization of a series of azaindole-based inhibitors. To overcome early challenges with promiscuity and cardiovascular toxicity, carboxylates were introduced into the pharmacophore en route to compounds such as 14 and 16. These CDK9 inhibitors demonstrated a reduced toxicity, adequate pharmacokinetic properties, and a robust in vivo efficacy in mice upon oral dosing.
RESUMEN
TRAIL can activate cell surface death receptors, resulting in potent tumor cell death via induction of the extrinsic apoptosis pathway. Eftozanermin alfa (ABBV-621) is a second generation TRAIL receptor agonist engineered as an IgG1-Fc mutant backbone linked to two sets of trimeric native single-chain TRAIL receptor binding domain monomers. This hexavalent agonistic fusion protein binds to the death-inducing DR4 and DR5 receptors with nanomolar affinity to drive on-target biological activity with enhanced caspase-8 aggregation and death-inducing signaling complex formation independent of FcγR-mediated cross-linking, and without clinical signs or pathologic evidence of toxicity in nonrodent species. ABBV-621 induced cell death in approximately 36% (45/126) of solid cancer cell lines in vitro at subnanomolar concentrations. An in vivo patient-derived xenograft (PDX) screen of ABBV-621 activity across 15 different tumor indications resulted in an overall response (OR) of 29% (47/162). Although DR4 (TNFSFR10A) and/or DR5 (TNFSFR10B) expression levels did not predict the level of response to ABBV-621 activity in vivo, KRAS mutations were associated with elevated TNFSFR10A and TNFSFR10B and were enriched in ABBV-621-responsive colorectal carcinoma PDX models. To build upon the OR of ABBV-621 monotherapy in colorectal cancer (45%; 10/22) and pancreatic cancer (35%; 7/20), we subsequently demonstrated that inherent resistance to ABBV-621 treatment could be overcome in combination with chemotherapeutics or with selective inhibitors of BCL-XL. In summary, these data provide a preclinical rationale for the ongoing phase 1 clinical trial (NCT03082209) evaluating the activity of ABBV-621 in patients with cancer. SIGNIFICANCE: This study describes the activity of a hexavalent TRAIL-receptor agonistic fusion protein in preclinical models of solid tumors that mechanistically distinguishes this molecular entity from other TRAIL-based therapeutics.
Asunto(s)
Neoplasias Colorrectales/tratamiento farmacológico , Factor IX/farmacología , Fragmentos Fc de Inmunoglobulinas/farmacología , Neoplasias Pancreáticas/tratamiento farmacológico , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Animales , Antineoplásicos/farmacología , Apoptosis , Proliferación Celular , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Femenino , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/genética , Ligando Inductor de Apoptosis Relacionado con TNF/genética , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Since gaining approval for the treatment of chronic lymphocytic leukemia (CLL), the BCL-2 inhibitor venetoclax has transformed the treatment of this and other blood-related cancers. Reflecting the large and hydrophobic BH3-binding groove within BCL-2, venetoclax has significantly higher molecular weight and lipophilicity than most orally administered drugs, along with negligible water solubility. Although a technology-enabled formulation successfully achieves oral absorption in humans, venetoclax tablets have limited drug loading and therefore can present a substantial pill burden for patients in high-dose indications. We therefore generated a phosphate prodrug (3, ABBV-167) that confers significantly increased water solubility to venetoclax and, upon oral administration to healthy volunteers either as a solution or high drug-load immediate release tablet, extensively converts to the parent drug. Additionally, ABBV-167 demonstrated a lower food effect with respect to venetoclax tablets. These data indicate that beyond-rule-of-5 molecules can be successfully delivered to humans via a solubility-enhancing prodrug moiety to afford robust exposures of the parent drug following oral dosing.
Asunto(s)
Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéutico , Profármacos/uso terapéutico , Sulfonamidas/uso terapéutico , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Línea Celular Tumoral , Estudios Cruzados , Femenino , Voluntarios Sanos , Humanos , Profármacos/farmacología , Sulfonamidas/farmacologíaRESUMEN
Mantle cell lymphoma (MCL) is an aggressive and largely incurable subtype of non-Hodgkin's lymphoma. Venetoclax has demonstrated efficacy in MCL patients with relapsed or refractory disease, however response is variable and less durable than CLL. This may be the result of co-expression of other anti-apoptotic proteins such as MCL-1, which is associated with both intrinsic and acquired resistance to venetoclax in B-cell malignancies. One strategy for neutralizing MCL-1 and other short-lived survival factors is to inhibit CDK9, which plays a key role in transcription. Here, we report the response of MCL cell lines and primary patient samples to the combination of venetoclax and novel CDK9 inhibitors. Primary samples represented de novo patients and relapsed disease, including relapse after ibrutinib failure. Despite the diverse responses to each single agent, possibly due to variable expression of the BCL-2 family members, venetoclax plus CDK9 inhibitors synergistically induced apoptosis in MCL cells. The synergistic effect was also confirmed via venetoclax plus a direct MCL-1 inhibitor. Murine xenograft studies demonstrated potent in vivo efficacy of venetoclax plus CDK9 inhibitor that was superior to each agent alone. Together, this study supports clinical investigation of this combination in MCL, including in patients who have progressed on ibrutinib.
RESUMEN
PURPOSE: Patients with acute myeloid leukemia (AML) frequently do not respond to conventional therapies. Leukemic cell survival and treatment resistance have been attributed to the overexpression of B-cell lymphoma 2 (BCL-2) and aberrant DNA hypermethylation. In a phase Ib study in elderly patients with AML, combining the BCL-2 selective inhibitor venetoclax with hypomethylating agents 5-azacitidine (5-Aza) or decitabine resulted in 67% overall response rate; however, the underlying mechanism for this activity is unknown. EXPERIMENTAL DESIGN: We studied the consequences of combining two therapeutic agents, venetoclax and 5-Aza, in AML preclinical models and primary patient samples. We measured expression changes in the integrated stress response (ISR) and the BCL-2 family by Western blot and qPCR. Subsequently, we engineered PMAIP1 (NOXA)- and BBC3 (PUMA)-deficient AML cell lines using CRISPR-Cas9 methods to understand their respective roles in driving the venetoclax/5-Aza combinatorial activity. RESULTS: In this study, we demonstrate that venetoclax and 5-Aza act synergistically to kill AML cells in vitro and display combinatorial antitumor activity in vivo. We uncover a novel nonepigenetic mechanism for 5-Aza-induced apoptosis in AML cells through transcriptional induction of the proapoptotic BH3-only protein NOXA. This induction occurred within hours of treatment and was mediated by the ISR pathway. NOXA was detected in complex with antiapoptotic proteins, suggesting that 5-Aza may be "priming" the AML cells for venetoclax-induced apoptosis. PMAIP1 knockout confirmed its major role in driving venetoclax and 5-Aza synergy. CONCLUSIONS: These data provide a novel nonepigenetic mechanism of action for 5-Aza and its combinatorial activity with venetoclax through the ISR-mediated induction of PMAIP1.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Azacitidina/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Sulfonamidas/farmacología , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Línea Celular Tumoral , Metilación de ADN , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Leucemia Mieloide Aguda , Ratones , Proteínas Proto-Oncogénicas c-bcl-2/metabolismoRESUMEN
MCL-1 is one of the most frequently amplified genes in cancer, facilitating tumor initiation and maintenance and enabling resistance to anti-tumorigenic agents including the BCL-2 selective inhibitor venetoclax. The expression of MCL-1 is maintained via P-TEFb-mediated transcription, where the kinase CDK9 is a critical component. Consequently, we developed a series of potent small-molecule inhibitors of CDK9, exemplified by the orally active A-1592668, with CDK selectivity profiles that are distinct from related molecules that have been extensively studied clinically. Short-term treatment with A-1592668 rapidly downregulates RNA pol-II (Ser 2) phosphorylation resulting in the loss of MCL-1 protein and apoptosis in MCL-1-dependent hematologic tumor cell lines. This cell death could be attenuated by either inhibiting caspases or overexpressing BCL-2 protein. Synergistic cell killing was also observed between A-1592668 or the related analog A-1467729, and venetoclax in a number of hematologic cell lines and primary NHL patient samples. Importantly, the CDK9 inhibitor plus venetoclax combination was well tolerated in vivo and demonstrated efficacy superior to either agent alone in mouse models of lymphoma and AML. These data indicate that CDK9 inhibitors could be highly efficacious in tumors that depend on MCL-1 for survival or when used in combination with venetoclax in malignancies dependent on MCL-1 and BCL-2.
Asunto(s)
Antineoplásicos/farmacología , Quinasa 9 Dependiente de la Ciclina/antagonistas & inhibidores , Neoplasias Hematológicas , Inhibidores de Proteínas Quinasas/farmacología , Animales , Apoptosis/efectos de los fármacos , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Sinergismo Farmacológico , Humanos , Ratones , Sulfonamidas/farmacología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Herein we describe the discovery of A-1331852, a first-in-class orally active BCL-XL inhibitor that selectively and potently induces apoptosis in BCL-XL-dependent tumor cells. This molecule was generated by re-engineering our previously reported BCL-XL inhibitor A-1155463 using structure-based drug design. Key design elements included rigidification of the A-1155463 pharmacophore and introduction of sp3-rich moieties capable of generating highly productive interactions within the key P4 pocket of BCL-XL. A-1331852 has since been used as a critical tool molecule for further exploring BCL-2 family protein biology, while also representing an attractive entry into a drug discovery program.
RESUMEN
Cancer cells evade death by over-producing specific proteins that inhibit apoptosis. One such group of proteins is the Bcl-2 family, of which Bcl-x(L) is an important member. This protein binds and inhibits BAK, another protein that promotes apoptosis. While the development of chemical inhibitors that block Bcl-x(L)-BAK association have been the focus of intense research efforts, we demonstrate in this manuscript an alternative strategy to downregulate Bcl-x(L). We have identified a small molecule (Bang52) that induces apoptosis in a lymphoblast-derived cell line by lowering levels of Bcl-x(L). Since Bang52 bears no resemblance to any chemical binder of Bcl-x(L) we believe that degradation of the protein is stimulated by a new type of pathway. These findings highlight a novel approach to the development of small molecules that promote apoptosis.
Asunto(s)
Amidas/síntesis química , Antineoplásicos/síntesis química , Apoptosis , Regulación Neoplásica de la Expresión Génica , Hidrocarburos Fluorados/síntesis química , Proteína bcl-X/biosíntesis , Amidas/farmacología , Antineoplásicos/farmacología , Línea Celular Tumoral , Separación Celular , Química Farmacéutica/métodos , Regulación hacia Abajo , Diseño de Fármacos , Resistencia a Antineoplásicos , Citometría de Flujo , Humanos , Hidrocarburos Fluorados/farmacología , Modelos Químicos , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Paramount to the maintenance of normal tissue homeostasis is the induction of programmed cell death, otherwise known as apoptosis. Several disease states, including cancer, are characterized by an inability to remove unwanted cells due to a failure to commit to apoptosis. What is more, apoptosis is the central functional response behind many agents utilized in the treatment of cancer. Many of these antitumorigenic agents rely on the activation of the tumor suppressor p53. As the physiological "guardian of the genome," p53's normal function is to sense stressed or damaged cells and arrest proliferation, allowing time for cellular repair. However, if the damage is excessive, cells are removed prior to the onset of malignancy through apoptosis. Current chemotherapeutic strategies manipulate this property by damaging cells and turning on p53's transcriptional function, which consequently upregulates the expression of proapoptotic proteins such as Puma. We have also demonstrated that Puma is capable of inducing apoptosis independent of p53. In this regard, defects in the apoptotic machinery or in p53 function itself lead to a resistant phenotype that in cancer results in chemotherapeutic failure, and more often than not, poor prognosis. This chapter describes protocols for the determination of p53-dependent and -independent apoptosis utilizing primary cells from genetically altered mice.
Asunto(s)
Apoptosis , Separación Celular/métodos , Proteína p53 Supresora de Tumor/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Técnicas de Cultivo de Célula , Ciclo Celular , ADN/análisis , Citometría de Flujo/métodos , Técnicas de Silenciamiento del Gen , Ratones , Ratones Noqueados , Células Progenitoras Mieloides/citología , Células Progenitoras Mieloides/inmunología , Linfocitos T/citología , Linfocitos T/metabolismo , Timo/citología , Proteína p53 Supresora de Tumor/genética , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Sphingolipids is the collective term ascribed to components of the sphingomyelin cycle. Modulation of the cellular levels of individual sphingolipids can induce a diverse range of cellular responses including apoptosis, proliferation, and cell cycle arrest. We present data showing that rhabdomyosarcoma cell lines, independent of lineage (alveolar rhabdomyosarcoma and embryonal rhabdomyosarcoma), are particularly sensitive to the induction of apoptosis as a result of an elevation in the cellular levels of sphingosine (D-erythro-sphingosine). Sphingosine-mediated apoptosis does not require its metabolism to the related proapoptotic molecule ceramide and is stereospecific because exposure of the rhabdomyosarcoma cell line RD to the L-erythro and DL-threo isoforms of sphingosine did not induce apoptosis. Importantly, for efficient induction of apoptosis, sphingosine required Bax activation and consequential translocation to the mitochondria. This resulted in selective mitochondrial release of cytochrome c and Smac/Diablo but not other mitochondrial related factors (apoptosis-inducing factor, endonuclease G, and HtrA2/Omi). Using small interfering RNA, reduced Bax expression conferred the impaired release of mitochondrial cytochrome c to the cytoplasm following sphingosine exposure, inhibiting the induction of apoptosis. Furthermore, dissipation of the inner mitochondrial membrane potential and enhanced production of reactive oxygen species were not observed. Bax activation and cytochrome c release were independent of caspases; however, caspase-3 and caspase-9 activity distal to the mitochondria was essential for the execution of apoptosis.
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Apoptosis/efectos de los fármacos , Mitocondrias/metabolismo , Esfingosina/farmacología , Proteína X Asociada a bcl-2/metabolismo , Animales , Apoptosis/fisiología , Caspasas/metabolismo , Línea Celular Tumoral , Citocromos c/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/enzimología , Membranas Mitocondriales/efectos de los fármacos , Membranas Mitocondriales/fisiología , Especies Reactivas de Oxígeno/metabolismo , Rabdomiosarcoma Alveolar/tratamiento farmacológico , Rabdomiosarcoma Alveolar/metabolismo , Rabdomiosarcoma Alveolar/patología , Rabdomiosarcoma Embrionario/tratamiento farmacológico , Rabdomiosarcoma Embrionario/metabolismo , Rabdomiosarcoma Embrionario/patología , Transducción de Señal/efectos de los fármacos , Esfingosina/metabolismo , EstereoisomerismoRESUMEN
Cyclooxygenase-2 (COX-2) is up-regulated in human colon carcinomas, and its inhibition is associated with a reduction in tumorigenesis and a promotion of apoptosis. However, the mechanisms responsible for the antitumor effects of COX-2 inhibitors and how COX-2 modulates apoptotic signaling have not been clearly defined. We have shown that COX-2 inhibition sensitizes human colon carcinoma cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis by inducing clustering of the TRAIL receptor DR5 at the cell surface and the redistribution of the death-inducing signaling complex components (DR5, FADD, and procaspase-8) into cholesterol-rich and ceramide-rich domains known as caveolae. This process requires the accumulation of arachidonic acid and sequential activation of acid sphingomyelinase for the generation of ceramide within the plasma membrane outer leaflet. The current study highlights a novel mechanism to circumvent colorectal carcinoma cell resistance to TRAIL-mediated apoptosis using COX-2 inhibitors to manipulate the lipid metabolism within the plasma membrane.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/farmacología , Caveolas/metabolismo , Ceramidas/metabolismo , Neoplasias del Colon/patología , Inhibidores de la Ciclooxigenasa 2/farmacología , Glicoproteínas de Membrana/farmacología , Receptores del Factor de Necrosis Tumoral/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Apoptosis/efectos de los fármacos , Ácido Araquidónico/metabolismo , Caspasa 8 , Caspasas/metabolismo , Membrana Celular/metabolismo , Colesterol/metabolismo , Neoplasias del Colon/metabolismo , Neoplasias del Colon/terapia , Ciclooxigenasa 2/metabolismo , Resistencia a Antineoplásicos , Proteína de Dominio de Muerte Asociada a Fas , Humanos , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF , Transducción de Señal , Esfingomielina Fosfodiesterasa/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF , Células Tumorales CultivadasAsunto(s)
Aminopiridinas/farmacología , Antineoplásicos/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Inhibidores Enzimáticos/farmacología , Isocitrato Deshidrogenasa/genética , Leucemia Mieloide Aguda/tratamiento farmacológico , Sulfonamidas/farmacología , Triazinas/farmacología , Aminopiridinas/uso terapéutico , Animales , Antineoplásicos/uso terapéutico , Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéutico , Línea Celular Tumoral , Inhibidores Enzimáticos/uso terapéutico , Humanos , Isocitrato Deshidrogenasa/antagonistas & inhibidores , Leucemia Mieloide Aguda/genética , Mutación/efectos de los fármacos , Sulfonamidas/uso terapéutico , Triazinas/uso terapéuticoRESUMEN
The chromosomal translocation t(14;18) in follicular lymphoma (FL) is a primary oncogenic event resulting in BCL-2 over-expression. This study investigates activity of the BH3 mimetic venetoclax (ABT-199), which targets BCL-2, and mechanisms of acquired resistance in FL.The sensitivity of FL cells to venetoclax treatment correlated with BCL-2/BIM ratio. Cells with similar expression of anti-apoptotic proteins, but with higher levels of BIM were more sensitive to the treatment. Venetoclax induced dissociation of BCL-2/ BIM complex and a decrease in mitochondrial potential. Interestingly the population of cells that survived venetoclax treatment showed increased p-ERK1/2 and p-BIM (S69), as well as a decrease in total BIM levels. Venetoclax resistant cells initially showed elevated levels of p-AKT and p-Foxo1/3a, a dissociation of BIM/BCL-2/BECLIN1 complex, and a decrease in SQSTM1/p62 level (indicating increased autophagy) together with a slight decline in BIM expression. After stable resistant cell lines were established, a significant reduction of BCL-2 levels and almost total absence of BIM was observed.The acquisition of these resistance phenotypes could be prevented via selective ERK/AKT inhibition or anti-CD20 antibody treatment, thus highlighting possible combination therapies for FL patients.