RESUMO
BACKGROUND: Certain paediatric nervous system malignancies have dismal prognoses. Retinoic acid (RA) is used in neuroblastoma treatment, and preclinical data indicate potential benefit in selected paediatric brain tumour entities. However, limited single-agent efficacy necessitates combination treatment approaches. METHODS: We performed drug sensitivity profiling of 76 clinically relevant drugs in combination with RA in 16 models (including patient-derived tumouroids) of the most common paediatric nervous system tumours. Drug responses were assessed by viability assays, high-content imaging, and apoptosis assays and RA relevant pathways by RNAseq from treated models and patient samples obtained through the precision oncology programme INFORM (n = 2288). Immunoprecipitation detected BCL-2 family interactions, and zebrafish embryo xenografts were used for in vivo efficacy testing. RESULTS: Group 3 medulloblastoma (MBG3) and neuroblastoma models were highly sensitive to RA treatment. RA induced differentiation and regulated apoptotic genes. RNAseq analysis revealed high expression of BCL2L1 in MBG3 and BCL2 in neuroblastomas. Co-treatments with RA and BCL-2/XL inhibitor navitoclax synergistically decreased viability at clinically achievable concentrations. The combination of RA with navitoclax disrupted the binding of BIM to BCL-XL in MBG3 and to BCL-2 in neuroblastoma, inducing apoptosis in vitro and in vivo. CONCLUSIONS: RA treatment primes MBG3 and NB cells for apoptosis, triggered by navitoclax cotreatment.
Assuntos
Apoptose , Sinergismo Farmacológico , Meduloblastoma , Neuroblastoma , Tretinoína , Peixe-Zebra , Humanos , Animais , Tretinoína/farmacologia , Meduloblastoma/tratamento farmacológico , Meduloblastoma/patologia , Meduloblastoma/metabolismo , Meduloblastoma/genética , Apoptose/efeitos dos fármacos , Neuroblastoma/tratamento farmacológico , Neuroblastoma/patologia , Neuroblastoma/metabolismo , Neuroblastoma/genética , Linhagem Celular Tumoral , Compostos de Anilina/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Sulfonamidas/farmacologia , Proteína bcl-X/genética , Proteína bcl-X/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Camundongos , Neoplasias Cerebelares/tratamento farmacológico , Neoplasias Cerebelares/patologia , Neoplasias Cerebelares/metabolismo , Neoplasias Cerebelares/genética , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteína Proto-Oncogênica N-MycRESUMO
BACKGROUND: Neuroblastoma is a paediatric cancer that is characterised by poor prognosis for chemoresistant disease, highlighting the need for better treatment options. Here, we asked whether BH3-mimetics inhibiting BCL2 proteins may eliminate chemoresistant neuroblastoma cells. METHODS: We utilised cisplatin-adapted neuroblastoma cell lines as well as patient tissues before and after relapse to study alterations of BCL2 proteins upon chemoresistance. RESULTS: In a direct comparison of cisplatin-resistant cells we identified a prominent loss of sensitivity to BCL2/BCL-XL inhibitors that is associated with an increase in MCL1 dependency and high expression of MCL1 in patient tumour tissues. Screening of FDA-approved anti-cancer drugs in chemoresistant cells identified therapeutics that may be beneficial in combination with the clinically tested BH3-mimetic ABT263, but no synergistic drug interactions with the selective MCL1 inhibitor S63845. Further exploration of potential treatment options for chemoresistant neuroblastoma identified immunotherapy based on NK cells as highly promising, since NK cells are able to efficiently kill both parental and chemoresistant cells. CONCLUSIONS: These data highlight that the application of BH3-mimetics may differ between first line treatment and relapsed disease. Combination of NK cell-based immunotherapy with BH3-mimetics may further increase killing of chemoresistant neuroblastoma, outlining a new treatment strategy for relapsed neuroblastoma.
Assuntos
Antineoplásicos , Neuroblastoma , Criança , Humanos , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Cisplatino/farmacologia , Linhagem Celular Tumoral , Recidiva Local de Neoplasia/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Neuroblastoma/tratamento farmacológico , Antineoplásicos/farmacologia , ApoptoseRESUMO
Copy number gains and increased expression levels of cellular Inhibitor of Apoptosis protein (cIAP)1 and cIAP2 have been identified in primary diffuse large B-cell lymphoma (DLBCL) tissues. Second mitochondria-derived activator of caspases (Smac) mimetics were designed to antagonize IAP proteins. However, since their effect as single agents is limited, combination treatment represents a strategy for their clinical development. Therefore, we investigated the Smac mimetic BV6 in combination with proteasome inhibitors and analyzed the molecular mechanisms of action. We discovered that BV6 treatment sensitizes DLBCL cells to proteasome inhibition. We show a synergistic decrease in cell viability and induction of apoptosis by BV6/Carfilzomib (CFZ) treatment, which was confirmed by calculation of combination index (CI) and Bliss score. BV6 and CFZ acted together to trigger activation of BAX and BAK, which facilitated cell death, as knockdown of BAX and BAK significantly reduced BV6/CFZ-mediated cell death. Activation of BAX and BAK was accompanied by loss of mitochondrial membrane potential (MMP) and activation of caspases. Pretreatment with the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) rescued BV6/CFZ-induced cell death, confirming caspase dependency. Treatment with CFZ alone or in combination with BV6 caused accumulation of NOXA, which was required for cell death, as gene silencing by siRNA or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-mediated NOXA inactivation inhibited BV6/CFZ-induced cell death. Together, these experiments indicate that BV6 and CFZ cooperatively induce apoptotic cell death via the mitochondrial pathway. These findings emphasize the role of Smac mimetics for sensitizing DLBCL cells to proteasome inhibition with important implications for further (pre)clinical studies.
Assuntos
Antineoplásicos/farmacologia , Proteínas Reguladoras de Apoptose/farmacologia , Apoptose/efeitos dos fármacos , Linfoma Difuso de Grandes Células B/patologia , Proteínas Mitocondriais/farmacologia , Inibidores de Proteassoma/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Caspases/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sinergismo Farmacológico , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Linfoma Difuso de Grandes Células B/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Oligopeptídeos/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
BACKGROUND: Despite advances in the treatment of neuroblastoma, patients with high-risk disease still have dismal survival prognosis. Neuroblastoma cells display elevated expression of the antiapoptotic BCL-2 proteins, suggesting that BH3-mimetics may be a promising treatment option. Here, we investigated the role of BCL-2, BCL-XL and MCL-1 in neuroblastoma. METHODS: A panel of neuroblastoma cell lines and primary patient-derived cells were exposed to BH3-mimetics targeting BCL-2 (ABT-199), BCL-XL (A1331852) or MCL-1 (S63845). In addition, protein expression and interaction patterns were analysed using Western blotting and immunoprecipitation. RESULTS: All tested BH3-mimetics were able to induce apoptosis in neuroblastoma cell lines, indicating that not only BCL-2 but also BCL-XL and MCL-1 may be promising therapeutic targets. Primary patient-derived cells displayed highest sensitivity to A1331852, highlighting the important role of BCL-XL in neuroblastoma. Further analysis into the molecular mechanisms of apoptosis revealed that A1331852 and S63845 displaced proapoptotic proteins like BIM and BAK from their antiapoptotic targets, subsequently leading to the activation of BAX and BAK and caspase-dependent apoptosis. CONCLUSIONS: By using selective BH3-mimetics, this study demonstrates that BCL-2, BCL-XL, and MCL-1 are all relevant therapeutic targets in neuroblastoma. A1331852 and S63845 induce rapid apoptosis that is initiated following a displacement of BAK from BCL-XL or MCL-1, respectively.
Assuntos
Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Neuroblastoma/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteína bcl-X/genética , Apoptose/efeitos dos fármacos , Proteína 11 Semelhante a Bcl-2/genética , Biomimética , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Neuroblastoma/genética , Neuroblastoma/patologia , Fragmentos de Peptídeos/farmacologia , Proteínas Proto-Oncogênicas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Pirimidinas/farmacologia , Sulfonamidas/farmacologia , Tiofenos/farmacologia , Proteína Killer-Antagonista Homóloga a bcl-2/genéticaRESUMO
The BCL-2-specific inhibitor, ABT-199 (venetoclax) has exhibited remarkable clinical activity in nearly all cases of chronic lymphocytic leukemia. In contrast, responses are usually much less in diffuse large B-cell lymphoma (DLBCL), despite high level expression of BCL-2 in over 40% of cases, indicating that co-expression of related anti-apoptotic BCL-2 family proteins may limit the activity of ABT-199. We have investigated the roles of BCL-2 proteins in DLBCL cells using a panel of specific BCL-2 homology 3 (BH3)-mimetics and identified subgroups of these cells that exhibited marked and specific dependency on either BCL-2, BCL-XL or MCL-1 for survival. Dependency was associated with selective sequestration of the pro-apoptotic proteins BIM, BAX and BAK by the specific anti-apoptotic BCL-2 protein which was important for cellular survival. Sensitivity to BH3-mimetics was independent of genetic alterations involving the BCL-2 family and only partially correlated with protein expression levels. Treatment with ABT-199 displaced BAX and BIM from BCL-2, subsequently leading to BAK activation and apoptosis. In contrast, apoptosis induced by inhibiting BCL-XL with A1331852 was associated with a displacement of both BAX and BAK from BCL-XL and occurred independently of BIM. Finally, the MCL-1 inhibitor S63845 induced mainly BAX-dependent apoptosis mediated by a displacement of BAK, BIM and NOXA from MCL-1. In conclusion, our study indicates that in DLBCL, the heterogeneous response to BH3-mimetics is mediated by selective interactions between BAX, BAK and anti-apoptotic BCL-2 proteins.
Assuntos
Linfoma Difuso de Grandes Células B , Proteína Killer-Antagonista Homóloga a bcl-2 , Apoptose , Humanos , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína bcl-X/genéticaRESUMO
Induction of apoptosis by selective BH3-mimetics is currently investigated as a novel strategy for cancer treatment. Here, we report that selective BH3-mimetics induce apoptosis in a variety of hematological malignancies. Apoptosis is accompanied by severe mitochondrial toxicities upstream of caspase activation. Specifically, the selective BH3-mimetics ABT-199, A-1331852 and S63845, which target BCL-2, BCL-XL and MCL-1, respectively, induce comparable ultrastructural changes including mitochondrial swelling, a decrease of mitochondrial matrix density and severe loss of cristae structure. These shared effects on mitochondrial morphology indicate a similar function of these anti-apoptotic BCL-2 proteins in maintaining mitochondrial integrity and function.
Assuntos
Mitocôndrias/efeitos dos fármacos , Mimetismo Molecular , Proteína de Sequência 1 de Leucemia de Células Mieloides/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/efeitos dos fármacos , Proteína bcl-X/efeitos dos fármacos , Apoptose , Caspases/metabolismo , Ativação Enzimática , Humanos , Mitocôndrias/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína bcl-X/metabolismoRESUMO
BH3 mimetics are novel targeted drugs with remarkable specificity, potency and enormous potential to improve cancer therapy. However, acquired resistance is an emerging problem. We report the rapid development of resistance in chronic lymphocytic leukemia cells isolated from patients exposed to increasing doses of navitoclax (ABT-263), a BH3 mimetic. To mimic such rapid development of chemoresistance, we developed simple resistance models to three different BH3 mimetics, targeting BCL-2 (ABT-199), BCL-XL (A-1331852) or MCL-1 (A-1210477), in relevant hematologic cancer cell lines. In these models, resistance could not be attributed to either consistent changes in expression levels of the anti-apoptotic proteins or interactions among different pro- and anti-apoptotic BCL-2 family members. Using genetic silencing, pharmacological inhibition and metabolic supplementation, we found that targeting glutamine uptake and its downstream signaling pathways, namely glutaminolysis, reductive carboxylation, lipogenesis, cholesterogenesis and mammalian target of rapamycin signaling resulted in marked sensitization of the chemoresistant cells to BH3 mimetic-mediated apoptosis. Furthermore, our findings highlight the possibility of repurposing widely used drugs, such as statins, to target intermediary metabolism and improve the efficacy of BH3 mimetic therapy.
Assuntos
Antineoplásicos/farmacologia , Biomimética , Resistencia a Medicamentos Antineoplásicos , Glutamina/metabolismo , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Recidiva Local de Neoplasia/tratamento farmacológico , Fragmentos de Peptídeos/química , Proteínas Proto-Oncogênicas/química , Benzotiazóis/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Colesterol/biossíntese , Ensaios Clínicos Fase I como Assunto , Ensaios Clínicos Fase II como Assunto , Humanos , Indóis/farmacologia , Isoquinolinas/farmacologia , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Lipogênese/efeitos dos fármacos , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Recidiva Local de Neoplasia/metabolismo , Recidiva Local de Neoplasia/patologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Sulfonamidas/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Células Tumorais Cultivadas , Proteína bcl-X/antagonistas & inibidoresRESUMO
The B-cell lymphoma 2 (BCL2) family of proteins comprise key regulators of apoptosis and are implicated in the pathogenesis of many malignancies, including lymphomas and leukaemias. Targeting of BCL2 proteins can be directly toxic to tumour cells or render them more sensitive to chemotherapy. Inhibition of the anti-apoptotic functions of BCL2 proteins using structure-based design to produce specific inhibitors of protein-protein interactions has been achieved for BCL2, MCL1 and BCL-XL (also termed BCL2L1), providing an armamentarium of new targeted therapies called BH3-mimetics. The first BCL2-specific inhibitor, venetoclax, has shown extraordinary single agent activity in chronic lymphocytic leukaemia (CLL), with surprisingly little toxicity given the expression of BCL2 in normal tissues. Despite success in CLL, where sensitivity to BCL2 inhibition is seen in nearly all cases, key questions have not yet been addressed. For example, responses to venetoclax in other B-cell and myeloid malignancies are heterogeneous, highlighting the need to identify biomarkers that correlate with response and, secondly, to identify/develop other specific compounds that synergise with BCL2 inhibition. In this review, we summarise the biology of BCL2 proteins, the mechanism of action of BH3-mimetics and the status of their clinical development in haematological malignancies.
Assuntos
Neoplasias Hematológicas/tratamento farmacológico , Terapia de Alvo Molecular/métodos , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Compostos de Anilina/farmacologia , Compostos de Anilina/uso terapêutico , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patologia , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêuticoRESUMO
BH3-mimetics represent promising anti-cancer agents in tumors that rely on the anti-apoptotic function of B-Cell Lymphoma 2 (BCL2) proteins, particularly in leukemia and lymphoma cells primed for apoptosis. Mechanistically, BH3-mimetics may displace pro-apoptotic binding partners thus inducing BAX/BAK-mediated mitochondrial permeabilization followed by cytochrome c release, activation of the caspase cascade and apoptosis. Here, we describe a novel mode of caspase-independent cell death (CICD) induced by BH3-mimetics in a subset of diffuse large B-cell lymphoma (DLBCL) cells. Of note, rather than occurring via necroptosis, CICD induced immediately after mitochondrial permeabilization was associated with transcriptional reprogramming mediated by activation of c-Jun N-terminal Kinase (JNK) signaling and Activator Protein 1 (AP1). Thereby, CICD resulted in the JNK/AP1-mediated upregulation of inflammatory chemokines and increased migration of cytotoxic Natural Killer (NK) cells. Taken together, our study describes a novel mode of CICD triggered by BH3-mimetics that may alter the immune response towards dying cells.
Assuntos
Antineoplásicos , Linfoma Difuso de Grandes Células B , Humanos , Proteína X Associada a bcl-2/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Apoptose , Antineoplásicos/farmacologia , Caspases , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linhagem Celular , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
There is now a plethora of new precision medicines for B-cell malignancy including 'classical' kinase inhibitors, rationally designed inhibitors of anti-apoptotic proteins and antibody or antibody drug/toxin conjugates with functional properties. Some are showing spectacular single agent activity in early phase clinical studies and may reduce or, in combination, even obviate the need for chemotherapy. Nevertheless, significant problems remain if these medicines are to be introduced into routine clinical practice in a rational and affordable manner. Firstly, precision medicines must be carefully matched in a mechanistic fashion with specific subtypes of disease. Whilst sensitivity may be predicted by the detection of key mutations or by expression of target molecules, for therapies that depend on intact intracellular signalling pathways, functional assessment on viable primary malignant cells will be necessary using assays that faithfully mimic in vivo conditions. A second, but no less important challenge is to define mechanism-based synergistic combinations associated with minimal toxicities rather than simply adding new precision medicines to existing chemotherapeutic regimens. Finally, a closer, open, two-way interaction between academic medicine and the pharmaceutical industry will be necessary to achieve these aims. Implementing such changes would change radically how and where patients with B-cell malignancies are managed.
Assuntos
Antineoplásicos/farmacologia , Leucemia de Células B/tratamento farmacológico , Linfoma/tratamento farmacológico , Terapia de Alvo Molecular/métodos , Animais , Antineoplásicos/uso terapêutico , Humanos , Leucemia de Células B/metabolismo , Linfoma/metabolismoRESUMO
Apoptosis in megakaryocytes results in the formation of platelets. The role of apoptotic pathways in platelet turnover and in the apoptotic-like changes seen after platelet activation is poorly understood. ABT-263 (Navitoclax), a specific inhibitor of antiapoptotic BCL2 proteins, which is currently being evaluated in clinical trials for the treatment of leukemia and other malignancies, induces a dose-limiting thrombocytopenia. In this study, the relationship between BCL2/BCL-X(L) inhibition, apoptosis, and platelet activation was investigated. Exposure to ABT-263 induced apoptosis but repressed platelet activation by physiologic agonists. Notably, ABT-263 induced an immediate calcium response in platelets and the depletion of intracellular calcium stores, indicating that on BCL2/BCL-X(L) inhibition platelet activation is abrogated because of a diminished calcium signaling. By comparing the effects of ABT-263 and its analog ABT-737 on platelets and leukemia cells from the same donor, we show, for the first time, that these BCL2/BCL-X(L) inhibitors do not offer any selective toxicity but induce apoptosis at similar concentrations in leukemia cells and platelets. However, reticulated platelets are less sensitive to apoptosis, supporting the hypothesis that treatment with ABT-263 induces a selective loss of older platelets and providing an explanation for the transient thrombocytopenia observed on ABT-263 treatment.
Assuntos
Compostos de Anilina/farmacologia , Apoptose/efeitos dos fármacos , Plaquetas/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Ativação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Sulfonamidas/farmacologia , Proteína bcl-X/antagonistas & inibidores , Compostos de Anilina/efeitos adversos , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacologia , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/metabolismo , Compostos de Bifenilo/efeitos adversos , Compostos de Bifenilo/farmacologia , Plaquetas/metabolismo , Plaquetas/ultraestrutura , Expressão Gênica , Homeostase/efeitos dos fármacos , Humanos , Cinética , Leucemia Linfocítica Crônica de Células B/sangue , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Terapia de Alvo Molecular , Nitrofenóis/efeitos adversos , Nitrofenóis/farmacologia , Piperazinas/efeitos adversos , Piperazinas/farmacologia , Inibidores da Agregação Plaquetária/efeitos adversos , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Sulfonamidas/efeitos adversos , Trombocitopenia/induzido quimicamente , Trombopoese , Proteína bcl-X/metabolismoRESUMO
In healthy cells, pro- and anti-apoptotic BCL2 family and BH3-only proteins are expressed in a delicate equilibrium. In contrast, this homeostasis is frequently perturbed in cancer cells due to the overexpression of anti-apoptotic BCL2 family proteins. Variability in the expression and sequestration of these proteins in Diffuse Large B cell Lymphoma (DLBCL) likely contributes to variability in response to BH3-mimetics. Successful deployment of BH3-mimetics in DLBCL requires reliable predictions of which lymphoma cells will respond. Here we show that a computational systems biology approach enables accurate prediction of the sensitivity of DLBCL cells to BH3-mimetics. We found that fractional killing of DLBCL, can be explained by cell-to-cell variability in the molecular abundances of signaling proteins. Importantly, by combining protein interaction data with a knowledge of genetic lesions in DLBCL cells, our in silico models accurately predict in vitro response to BH3-mimetics. Furthermore, through virtual DLBCL cells we predict synergistic combinations of BH3-mimetics, which we then experimentally validated. These results show that computational systems biology models of apoptotic signaling, when constrained by experimental data, can facilitate the rational assignment of efficacious targeted inhibitors in B cell malignancies, paving the way for development of more personalized approaches to treatment.
Assuntos
Linfoma Difuso de Grandes Células B , Proteínas Proto-Oncogênicas c-bcl-2 , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Simulação por Computador , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/patologiaRESUMO
Although the overall survival in pediatric rhabdomyosarcoma (RMS) has increased over the last decades, the most aggressive subtype of alveolar RMS is in dire need of novel treatment strategies. RMS cells evade cell death induction and immune control by increasing the expression of inhibitors of apoptosis proteins (IAPs), which can be exploited and targeted with stimulation with Smac mimetics. Here, we used the Smac mimetic BV6 to re-sensitize RMS spheroids to cell death, which increased killing induced by natural killer (NK) cells. Single BV6 treatment of RMS spheroids did not reduce spheroidal growth. However, we observed significant spheroidal decomposition upon BV6 pre-treatment combined with NK cell co-cultivation. Molecularly, IAPs s are rapidly degraded by BV6, which activates NF-κB signal transduction pathways in RMS spheroids. RNA sequencing analysis validated NF-κB activation and identified a plethora of BV6-regulated genes. Additionally, BV6 released caspases from IAP-mediated inhibition. Here, caspase-8 might play a major role, as knockdown experiments resulted in decreased NK cell-mediated attack. Taken together, we improved the understanding of the BV6 mechanism of RMS spheroid sensitization to cytotoxic immune cells, which could be suitable for the development of novel combinatory cellular immunotherapy with Smac mimetics.
Assuntos
Apoptose , Rabdomiossarcoma , Criança , Humanos , Apoptose/fisiologia , NF-kappa B/metabolismo , Proteínas Reguladoras de Apoptose , Morte Celular , Células Matadoras Naturais/metabolismoRESUMO
Due to their physiological role in removing damaged cells, natural killer (NK) cells represent ideal candidates for cellular immunotherapy in the treatment of cancer. Thereby, the cytotoxicity of NK cells is regulated by signals on both, the NK cells as well as the targeted tumor cells, and the interplay and balance of these signals determine the killing capacity of NK cells. One promising avenue in cancer treatment is therefore the combination of NK cell therapy with agents that either help to increase the killing capacity of NK cells or sensitize tumor cells to an NK cell-mediated attack. In this mini-review, we present different strategies that can be explored to unleash the potential of NK cell immunotherapy. In particular, we summarize how modulation of apoptosis signaling within tumor cells can be exploited to sensitize tumor cells to NK cell-mediated cytotoxicity.
Assuntos
Células Matadoras Naturais , Neoplasias , Apoptose , Humanos , Imunoterapia , Imunoterapia Adotiva , Neoplasias/patologia , Neoplasias/terapiaRESUMO
The induction of apoptosis is a direct way to eliminate tumor cells and improve cancer therapy. Apoptosis is tightly controlled by the balance of pro- and antiapoptotic Bcl-2 proteins. BH3 mimetics neutralize the antiapoptotic function of Bcl-2 proteins and are highly promising compounds inducing apoptosis in several cancer entities including pediatric malignancies. However, the clinical application of BH3 mimetics in solid tumors is impeded by the frequent resistance to single BH3 mimetics and the anticipated toxicity of high concentrations or combination treatments. One potential avenue to increase the potency of BH3 mimetics is the development of immune cell-based therapies to counteract the intrinsic apoptosis resistance of tumor cells and sensitize them to immune attack. Here, we describe spheroid cultures of pediatric cancer cells that can serve as models for drug testing. In these 3D models, we were able to demonstrate that activated allogeneic Natural Killer (NK) cells migrated into tumor spheroids and displayed cytotoxicity against a wide range of pediatric cancer spheroids, highlighting their potential as anti-tumor effector cells. Next, we investigated whether treatment of tumor spheroids with subtoxic concentrations of BH3 mimetics can increase the cytotoxicity of NK cells. Notably, the cytotoxic effects of NK cells were enhanced by the addition of BH3 mimetics. Treatment with either the Bcl-XL inhibitor A1331852 or the Mcl-1 inhibitor S63845 increased the cytotoxicity of NK cells and reduced spheroid size, while the Bcl-2 inhibitor ABT-199 had no effect on NK cell-mediated killing. Taken together, this is the first study to describe the combination of BH3 mimetics targeting Bcl-XL or Mcl-1 with NK cell-based immunotherapy, highlighting the potential of BH3 mimetics in immunotherapy.
RESUMO
Inhibition of BCL2 proteins is one of the most promising new approaches to targeted cancer therapy resulting in the induction of apoptosis. Amongst the most specific BCL2-inhibitors identified are ABT-737 and ABT-263. However, targeted therapy is often only effective for a limited amount of time because of the occurrence of drug resistance. In this study, the interaction of BCL2-inhibitors with the drug efflux transporter P-glycoprotein was investigated. Using (3)H labelled ABT-263, we found that cells with high P-glycoprotein activity accumulated less drug. In addition, cells with increased P-glycoprotein expression were more resistant to apoptosis induced by either ABT-737 or ABT-263. Addition of tariquidar or verapamil sensitized the cells to BCL2-inhibitor treatment, resulting in higher apoptosis. Our data suggest that the BCL2-inhibitors ABT-737 and ABT-263 are substrates for P-glycoprotein. Over-expression of P-glycoprotein may be, at least partly, responsible for resistance to these BCL2-inhibitors.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Compostos de Anilina/metabolismo , Compostos de Bifenilo/metabolismo , Nitrofenóis/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Sulfonamidas/metabolismo , Compostos de Anilina/farmacologia , Animais , Compostos de Bifenilo/farmacologia , Linhagem Celular , Cães , Nitrofenóis/farmacologia , Piperazinas/metabolismo , Piperazinas/farmacologia , Especificidade por Substrato , Sulfonamidas/farmacologiaRESUMO
Defects in apoptosis contribute to poor outcome in pediatric acute lymphoblastic leukemia (ALL), calling for novel strategies that counter apoptosis resistance. Here, we demonstrate for the first time that small molecule inhibitors of the antiapoptotic protein XIAP cooperate with TRAIL to induce apoptosis in childhood acute leukemia cells. XIAP inhibitors at subtoxic concentrations, but not a structurally related control compound, synergize with TRAIL to trigger apoptosis and to inhibit clonogenic survival of acute leukemia cells, whereas they do not affect viability of normal peripheral blood lymphocytes, suggesting some tumor selectivity. Analysis of signaling pathways reveals that XIAP inhibitors enhance TRAIL-induced activation of caspases, loss of mitochondrial membrane potential, and cytochrome c release in a caspase-dependent manner, indicating that they promote a caspase-dependent feedback mitochondrial amplification loop. Of note, XIAP inhibitors even overcome Bcl-2-mediated resistance to TRAIL by enhancing Bcl-2 cleavage and Bak conformational change. Importantly, XIAP inhibitors kill leukemic blasts from children with ALL ex vivo and cooperate with TRAIL to induce apoptosis. In vivo, they significantly reduce leukemic burden in a mouse model of pediatric ALL engrafted in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. Thus, XIAP inhibitors present a promising novel approach for apoptosis-based therapy of childhood ALL.
Assuntos
Apoptose/fisiologia , Fragmentos de Peptídeos/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , RNA Interferente Pequeno/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidores , Animais , Caspases/metabolismo , Sobrevivência Celular/fisiologia , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Humanos , Células Jurkat , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mitocôndrias/fisiologia , Fragmentos de Peptídeos/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Receptor fas/metabolismoRESUMO
ABT-737 and its orally active analog, ABT-263, are rationally designed inhibitors of BCL2 and BCL-X(L). ABT-263 shows promising activity in early phase 1 clinical trials in B-cell malignancies, particularly chronic lymphocytic leukemia (CLL). In vitro, peripheral blood CLL cells are extremely sensitive to ABT-737 (EC(50) approximately 7 nM), with rapid induction of apoptosis in all 60 patients tested, independent of parameters associated with disease progression and chemotherapy resistance. In contrast to data from cell lines, ABT-737-induced apoptosis in CLL cells was largely MCL1-independent. Because CLL cells within lymph nodes are more resistant to apoptosis than those in peripheral blood, CLL cells were cultured on CD154-expressing fibroblasts in the presence of interleukin-4 (IL-4) to mimic the lymph node microenvironment. CLL cells thus cultured developed an approximately 1000-fold resistance to ABT-737 within 24 hours. Investigations of the underlying mechanism revealed that this resistance occurred upstream of mitochondrial perturbation and involved de novo synthesis of the antiapoptotic proteins BCL-X(L) and BCL2A1, which were responsible for resistance to low and high ABT-737 concentrations, respectively. Our data indicate that after therapy with ABT-737-related inhibitors, resistant CLL cells might develop in lymph nodes in vivo and that treatment strategies targeting multiple BCL2 antiapoptotic members simultaneously may have synergistic activity.
Assuntos
Compostos de Bifenilo/farmacologia , Resistencia a Medicamentos Antineoplásicos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/metabolismo , Nitrofenóis/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Sulfonamidas/farmacologia , Proteína bcl-X/metabolismo , Apoptose/efeitos dos fármacos , Western Blotting , Ligante de CD40/genética , Ligante de CD40/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Interleucina-4/genética , Interleucina-4/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Linfonodos/efeitos dos fármacos , Linfonodos/metabolismo , Linfonodos/patologia , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Linfócitos/patologia , Antígenos de Histocompatibilidade Menor , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides , Piperazinas/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Interferente Pequeno/farmacologia , Regulação para Cima , Proteína bcl-X/antagonistas & inibidores , Proteína bcl-X/genéticaRESUMO
The removal of cells by apoptosis is an essential process regulating tissue homeostasis. Cancer cells acquire the ability to circumvent apoptosis and survive in an unphysiological tissue context. Thereby, the Bcl-2 protein family plays a key role in the initiation of apoptosis, and overexpression of the anti-apoptotic Bcl-2 proteins is one of the molecular mechanisms protecting cancer cells from apoptosis. Recently, small molecules targeting the anti-apoptotic Bcl-2 family proteins have been identified, and with venetoclax the first of these BH3 mimetics has been approved for the treatment of leukemia. In solid tumors the anti-apoptotic Bcl-2 family proteins Mcl-1 and Bcl-xL are frequently overexpressed or genetically amplified. In this review, we summarize the role of Mcl-1 and Bcl-xL in solid tumors and compare the different BH3 mimetics targeting Mcl-1 or Bcl-xL.
Assuntos
Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Neoplasias/genética , Bibliotecas de Moléculas Pequenas/farmacologia , Proteína bcl-X/genética , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Amplificação de Genes , Regulação Neoplásica da Expressão Gênica , Humanos , Mimetismo Molecular , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Domínios Proteicos , Bibliotecas de Moléculas Pequenas/uso terapêutico , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Regulação para Cima , Proteína bcl-X/antagonistas & inibidoresRESUMO
BACKGROUND: Bortezomib has been successfully used in the treatment of multiple myeloma and has been proposed as a potential treatment for chronic lymphocytic leukemia. In this study we investigated the mechanism by which bortezomib induces apoptosis in chronic lymphocytic leukemia cells. DESIGN AND METHODS: Using western blot analysis, we monitored the regulation of BCL2 family members, proteins of the unfolded protein response (endoplasmic reticulum stress response) and activation of caspases in relation to induction of apoptosis (measured by annexin-propidium iodide staining and loss of mitochondrial membrane potential) by bortezomib in chronic lymphocytic leukemia cells. RESULTS: Bortezomib induced apoptosis through activation of the mitochondrial pathway independently of changes associated with endoplasmic reticulum stress. Perturbation of mitochondria was regulated by a rapid and transcription-independent increase of NOXA protein, which preceded release of cytochrome c, HtrA2, Smac and activation of caspase-9 and -3. NOXA had a short half life (approximately 1-2 h) and was ubiquitinated on at least three primary lysine residues, resulting in proteasomal-dependent degradation. Down-regulation of NOXA, using short interfering RNA in chronic lymphocytic leukemia cells, decreased bortezomib-induced apoptosis. Finally bortezomib when combined with seliciclib resulted in a stronger and earlier increase in NOXA protein, caspase-3 cleavage and induction of apoptosis in chronic lymphocytic leukemia cells. CONCLUSIONS: These results highlight a critical role for NOXA in bortezomib-induced apoptosis in chronic lymphocytic leukemia cells and suggest that this drug may become more efficient for the treatment of chronic lymphocytic leukemia if combined with other agents able to interfere with the basal levels of MCL1.