RESUMEN
Gene expression by RNA polymerase II (RNAPII) is tightly controlled by cyclin-dependent kinases (CDKs) at discrete checkpoints during the transcription cycle. The pausing checkpoint following transcription initiation is primarily controlled by CDK9. We discovered that CDK9-mediated, RNAPII-driven transcription is functionally opposed by a protein phosphatase 2A (PP2A) complex that is recruited to transcription sites by the Integrator complex subunit INTS6. PP2A dynamically antagonizes phosphorylation of key CDK9 substrates including DSIF and RNAPII-CTD. Loss of INTS6 results in resistance to tumor cell death mediated by CDK9 inhibition, decreased turnover of CDK9 phospho-substrates, and amplification of acute oncogenic transcriptional responses. Pharmacological PP2A activation synergizes with CDK9 inhibition to kill both leukemic and solid tumor cells, providing therapeutic benefit in vivo. These data demonstrate that fine control of gene expression relies on the balance between kinase and phosphatase activity throughout the transcription cycle, a process dysregulated in cancer that can be exploited therapeutically.
Asunto(s)
Quinasa 9 Dependiente de la Ciclina/metabolismo , Terapia Molecular Dirigida , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Proteína Fosfatasa 2/metabolismo , Proteínas de Unión al ARN/metabolismo , Transcripción Genética , Proteínas Supresoras de Tumor/metabolismo , Animales , Línea Celular Tumoral , Quinasa 9 Dependiente de la Ciclina/antagonistas & inhibidores , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones Endogámicos NOD , Fosforilación , Unión Proteica , ARN Polimerasa II/química , ARN Polimerasa II/metabolismo , Especificidad por SustratoRESUMEN
Targeted inhibitors of bromodomain and extraterminal (BET)-bromodomains and phosphatidylinositol-3-kinase (PI3K) signaling demonstrate potent but self-limited antilymphoma activity as single agents in the context of cellular Myelocytomatosis (cMYC) oncogene-dysregulation. However, combined PI3K and BET inhibition imparts synergistic anticancer activity with the potential for more sustained disease responses due to the mutual antagonism of compensatory epigenetic and signaling networks. Here, we describe the mechanistic and therapeutic validation of rationally designed dual PI3K/BET bromodomain inhibitors, built by linkage of established PI3K and BET inhibitor pharmacophores. The lead candidate demonstrates high selectivity, nanomolar range cellular potency, and compelling in vivo efficacy, including curative responses in the aggressive Eµ-Myc lymphoma model. These studies further support the therapeutic strategy of combined PI3K and BET inhibition and provide a potential step-change in approach to orthogonal MYC antagonism using optimized chimeric small-molecule technology.
Asunto(s)
Linfoma , Fosfatidilinositol 3-Quinasas , Humanos , Fosfatidilinositol 3-Quinasa , Agresión , Epigenómica , Linfoma/tratamiento farmacológico , Inhibidores de las Quinasa Fosfoinosítidos-3RESUMEN
Acetylation of histones by lysine acetyltransferases (KATs) is essential for chromatin organization and function1. Among the genes coding for the MYST family of KATs (KAT5-KAT8) are the oncogenes KAT6A (also known as MOZ) and KAT6B (also known as MORF and QKF)2,3. KAT6A has essential roles in normal haematopoietic stem cells4-6 and is the target of recurrent chromosomal translocations, causing acute myeloid leukaemia7,8. Similarly, chromosomal translocations in KAT6B have been identified in diverse cancers8. KAT6A suppresses cellular senescence through the regulation of suppressors of the CDKN2A locus9,10, a function that requires its KAT activity10. Loss of one allele of KAT6A extends the median survival of mice with MYC-induced lymphoma from 105 to 413 days11. These findings suggest that inhibition of KAT6A and KAT6B may provide a therapeutic benefit in cancer. Here we present highly potent, selective inhibitors of KAT6A and KAT6B, denoted WM-8014 and WM-1119. Biochemical and structural studies demonstrate that these compounds are reversible competitors of acetyl coenzyme A and inhibit MYST-catalysed histone acetylation. WM-8014 and WM-1119 induce cell cycle exit and cellular senescence without causing DNA damage. Senescence is INK4A/ARF-dependent and is accompanied by changes in gene expression that are typical of loss of KAT6A function. WM-8014 potentiates oncogene-induced senescence in vitro and in a zebrafish model of hepatocellular carcinoma. WM-1119, which has increased bioavailability, arrests the progression of lymphoma in mice. We anticipate that this class of inhibitors will help to accelerate the development of therapeutics that target gene transcription regulated by histone acetylation.
Asunto(s)
Bencenosulfonatos/farmacología , Senescencia Celular/efectos de los fármacos , Histona Acetiltransferasas/antagonistas & inhibidores , Hidrazinas/farmacología , Linfoma/tratamiento farmacológico , Linfoma/patología , Sulfonamidas/farmacología , Acetilación/efectos de los fármacos , Animales , Bencenosulfonatos/uso terapéutico , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Desarrollo de Medicamentos , Fibroblastos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Histona Acetiltransferasas/deficiencia , Histona Acetiltransferasas/genética , Histonas/química , Histonas/metabolismo , Hidrazinas/uso terapéutico , Linfoma/enzimología , Linfoma/genética , Lisina/química , Lisina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Sulfonamidas/uso terapéuticoRESUMEN
Pharmacological strategies capable of directly targeting MYC are elusive. Previous studies have shown that MYC-driven lymphomagenesis is associated with mammalian target of rapamycin (mTOR) activation and a MYC-evoked DNA damage response (DDR) transduced by phosphatidylinositol-3-kinase (PI3K)-related kinases (DNA-PK, ATM, and ATR). Here we report that BEZ235, a multitargeted pan-PI3K/dual-mTOR inhibitor, potently killed primary Myc-driven B-cell lymphomas and human cell lines bearing IG-cMYC translocations. Using pharmacologic and genetic dissection of PI3K/mTOR signaling, dual DDR/mTORC1 inhibition was identified as a key mediator of apoptosis. Moreover, apoptosis was initiated at drug concentrations insufficient to antagonize PI3K/mTORC2-regulated AKT phosphorylation. p53-independent induction of the proapoptotic BH3-only protein BMF was identified as a mechanism by which dual DDR/mTORC1 inhibition caused lymphoma cell death. BEZ235 treatment induced apoptotic tumor regressions in vivo that correlated with suppression of mTORC1-regulated substrates and reduced H2AX phosphorylation and also with feedback phosphorylation of AKT. These mechanistic studies hold important implications for the use of multitargeted PI3K inhibitors in the treatment of hematologic malignancies. In particular, the newly elucidated role of PI3K-related DDR kinases in response to PI3K inhibitors offers a novel therapeutic opportunity for the treatment of hematologic malignancies with an MYC-driven DDR.
Asunto(s)
Apoptosis/efectos de los fármacos , Imidazoles/farmacología , Linfoma de Células B/prevención & control , Quinolinas/farmacología , Animales , Western Blotting , Línea Celular , Daño del ADN/efectos de los fármacos , Receptor con Dominio Discoidina 1 , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Histonas/metabolismo , Humanos , Linfoma de Células B/genética , Linfoma de Células B/patología , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Complejos Multiproteicos/antagonistas & inhibidores , Complejos Multiproteicos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/metabolismo , Análisis de Supervivencia , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismo , Células Tumorales CultivadasRESUMEN
During T cell development, the first step in creating a unique T cell receptor (TCR) is genetic recombination of the TCRß chain. The quality of the new TCRß is assessed at the ß-selection checkpoint. Most cells fail this checkpoint and die, but the coordination of fate at the ß-selection checkpoint is not yet understood. We shed new light on fate determination during ß-selection using a selective inhibitor of histone deacetylase 6, ACY1215. ACY1215 disrupted the ß-selection checkpoint. Characterising the basis for this disruption revealed a new, pivotal stage in ß-selection, bookended by up-regulation of TCR co-receptors, CD28 and CD2, respectively. Within this "DN3bPre" stage, CD5 and Lef1 are up-regulated to reflect pre-TCR signalling, and their expression correlates with proliferation. These findings suggest a refined model of ß-selection in which a coordinated increase in expression of pre-TCR, CD28, CD5 and Lef1 allows for modulating TCR signalling strength and culminates in the expression of CD2 to enable exit from the ß-selection checkpoint.
Asunto(s)
Antígenos CD28 , Receptores de Antígenos de Linfocitos T alfa-beta , Antígenos CD28/genética , Antígenos CD28/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Histonas/metabolismo , Histona Desacetilasa 6/metabolismo , Linfocitos T/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismoRESUMEN
Rearrangements of the Mixed Lineage Leukemia (MLL/KMT2A) gene are present in approximately 10% of acute leukemias and characteristically define disease with poor outcome. Driven by the unmet need to develop better therapies for KMT2A-rearranged leukemia, we previously discovered that the novel anti-cancer agent, curaxin CBL0137, induces decondensation of chromatin in cancer cells, delays leukemia progression and potentiates standard of care chemotherapies in preclinical KMT2A-rearranged leukemia models. Based on the promising potential of histone deacetylase (HDAC) inhibitors as targeted anti-cancer agents for KMT2A-rearranged leukemia and the fact that HDAC inhibitors also decondense chromatin via an alternate mechanism, we investigated whether CBL0137 could potentiate the efficacy of the HDAC inhibitor panobinostat in KMT2A-rearranged leukemia models. The combination of CBL0137 and panobinostat rapidly killed KMT2A-rearranged leukemia cells by apoptosis and significantly delayed leukemia progression and extended survival in an aggressive model of MLL-AF9 (KMT2A:MLLT3) driven murine acute myeloid leukemia. The drug combination also exerted a strong anti-leukemia response in a rapidly progressing xenograft model derived from an infant with KMT2A-rearranged acute lymphoblastic leukemia, significantly extending survival compared to either monotherapy. The therapeutic enhancement between CBL0137 and panobinostat in KMT2A-r leukemia cells does not appear to be mediated through cooperative effects of the drugs on KMT2A rearrangement-associated histone modifications. Our data has identified the CBL0137/panobinostat combination as a potential novel targeted therapeutic approach to improve outcome for KMT2A-rearranged leukemia.
RESUMEN
LAQ824 and LBH589 (panobinostat) are histone deacetylase inhibitors (HDACi) developed as cancer therapeutics and we have used the Emu-myc lymphoma model to identify the molecular events required for their antitumor effects. Induction of tumor cell death was necessary for these agents to mediate therapeutic responses in vivo and both HDACi engaged the intrinsic apoptotic cascade that did not require p53. Death receptor pathway blockade had no effect on the therapeutic activities of LAQ824 and LBH589; however, overexpression of Bcl-2 or Bcl-X(L) protected lymphoma cells from HDACi-induced killing and suppressed their therapeutic activities. Deletion of Apaf-1 or Caspase-9 delayed HDACi-induced lymphoma killing in vitro and in vivo, associated with suppression of many biochemical indicators of apoptosis, but did not provide long-term resistance to these agents and failed to inhibit their therapeutic activities. Emu-myc lymphomas lacking a functional apoptosome displayed morphologic and biochemical features of autophagy after treatment with LAQ824 and LBH589, indicating that, in the absence of a complete intrinsic apoptosis pathway involving apoptosome formation, these HDACi can still mediate a therapeutic response. Our data indicate that damage to the mitochondria is the key event necessary for LAQ824 and LBH589 to mediate tumor cell death and a robust therapeutic response.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Inhibidores de Histona Desacetilasas , Ácidos Hidroxámicos/farmacología , Linfoma/metabolismo , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Histona Desacetilasas/metabolismo , Humanos , Indoles , Linfoma/patología , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica de Transmisión , Trasplante de Neoplasias , Panobinostat , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Receptores de Muerte Celular/metabolismo , Transducción de Señal/efectos de los fármacos , Tasa de Supervivencia , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The apoptotic and therapeutic activities of the histone deacetylase inhibitor (HDACi) vorinostat are blocked by overexpression of Bcl-2 or Bcl-X(L). Herein, we used the small molecule inhibitor ABT-737 to restore sensitivity of Emu-myc lymphomas overexpressing Bcl-2 or Bcl-X(L) to vorinostat and valproic acid (VPA). Combining low-dose ABT-737 with vorinostat or VPA resulted in synergistic apoptosis of these cells. ABT-737 was ineffective against Emu-myc/Mcl-1 and Emu-myc/A1 cells either as a single agent or in combination with HDACi. However, in contrast to the reported binding specificity data, Emu-myc/Bcl-w lymphomas were insensitive to ABT-737 used alone or in combination with HDACi, indicating that the regulatory activity of ABT-737 is restricted to Bcl-2 and Bcl-X(L). Emu-myc lymphomas that expressed Bcl-2 throughout the tumorigenesis process were especially sensitive to ABT-737, while those forced to overexpress Mcl-1 were not. This supports the notion that tumor cells "addicted" to ABT-737 target proteins (ie, Bcl-2 or Bcl-X(L)) are likely to be the most sensitive target cell population. Our studies provide important preclinical data on the binding specificity of ABT-737 and its usefulness against primary hematologic malignancies when used as a single agent and in combination with HDACi.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Compuestos de Bifenilo/farmacología , Inhibidores Enzimáticos/administración & dosificación , Inhibidores de Histona Desacetilasas , Linfoma/tratamiento farmacológico , Nitrofenoles/farmacología , Sulfonamidas/farmacología , Animales , Compuestos de Bifenilo/administración & dosificación , Supervivencia Celular/efectos de los fármacos , Sistemas de Liberación de Medicamentos , Evaluación Preclínica de Medicamentos , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Sinergismo Farmacológico , Genes bcl-2 , Genes myc , Ácidos Hidroxámicos/administración & dosificación , Linfoma/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Nitrofenoles/administración & dosificación , Piperazinas/administración & dosificación , Piperazinas/farmacología , Especificidad por Sustrato , Sulfonamidas/administración & dosificación , VorinostatRESUMEN
Internal tandem duplication of the FMS-like tyrosine kinase 3 gene (FLT3-ITD) occurs in 30% of all acute myeloid leukemias (AML). Limited clinical efficacy of FLT3 inhibitors highlights the need for alternative therapeutic modalities in this subset of disease. Using human and murine models of FLT3-ITD-driven AML, we demonstrate that FLT3-ITD promotes serine synthesis and uptake via ATF4-dependent transcriptional regulation of genes in the de novo serine biosynthesis pathway and neutral amino acid transport. Genetic or pharmacologic inhibition of PHGDH, the rate-limiting enzyme of de novo serine biosynthesis, selectively inhibited proliferation of FLT3-ITD AMLs in vitro and in vivo. Moreover, pharmacologic inhibition of PHGDH sensitized FLT3-ITD AMLs to the standard-of-care chemotherapeutic cytarabine. Collectively, these data reveal novel insights into FLT3-ITD-induced metabolic reprogramming and reveal a targetable vulnerability in FLT3-ITD AML. SIGNIFICANCE: FLT3-ITD mutations are common in AML and are associated with poor prognosis. We show that FLT3-ITD stimulates serine biosynthesis, thereby rendering FLT3-ITD-driven leukemias dependent upon serine for proliferation and survival. This metabolic dependency can be exploited pharmacologically to sensitize FLT3-ITD-driven AMLs to chemotherapy.This article is highlighted in the In This Issue feature, p. 1307.
Asunto(s)
Leucemia Mieloide Aguda/metabolismo , Serina/metabolismo , Tirosina Quinasa 3 Similar a fms/metabolismo , Animales , Línea Celular Tumoral/metabolismo , Modelos Animales de Enfermedad , Humanos , Ratones , Inhibidores de Proteínas QuinasasRESUMEN
PURPOSE: Targeting Bcl-2 family members upregulated in multiple cancers has emerged as an important area of cancer therapeutics. While venetoclax, a Bcl-2-selective inhibitor, has had success in the clinic, another family member, Bcl-xL, has also emerged as an important target and as a mechanism of resistance. Therefore, we developed a dual Bcl-2/Bcl-xL inhibitor that broadens the therapeutic activity while minimizing Bcl-xL-mediated thrombocytopenia. EXPERIMENTAL DESIGN: We used structure-based chemistry to design a small-molecule inhibitor of Bcl-2 and Bcl-xL and assessed the activity against in vitro cell lines, patient samples, and in vivo models. We applied pharmacokinetic/pharmacodynamic (PK/PD) modeling to integrate our understanding of on-target activity of the dual inhibitor in tumors and platelets across dose levels and over time. RESULTS: We discovered AZD4320, which has nanomolar affinity for Bcl-2 and Bcl-xL, and mechanistically drives cell death through the mitochondrial apoptotic pathway. AZD4320 demonstrates activity in both Bcl-2- and Bcl-xL-dependent hematologic cancer cell lines and enhanced activity in acute myeloid leukemia (AML) patient samples compared with the Bcl-2-selective agent venetoclax. A single intravenous bolus dose of AZD4320 induces tumor regression with transient thrombocytopenia, which recovers in less than a week, suggesting a clinical weekly schedule would enable targeting of Bcl-2/Bcl-xL-dependent tumors without incurring dose-limiting thrombocytopenia. AZD4320 demonstrates monotherapy activity in patient-derived AML and venetoclax-resistant xenograft models. CONCLUSIONS: AZD4320 is a potent molecule with manageable thrombocytopenia risk to explore the utility of a dual Bcl-2/Bcl-xL inhibitor across a broad range of tumor types with dysregulation of Bcl-2 prosurvival proteins.
Asunto(s)
Antineoplásicos/farmacología , Benzamidas/farmacología , Neoplasias Hematológicas/tratamiento farmacológico , Piperidinas/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Sulfonas/farmacología , Trombocitopenia/tratamiento farmacológico , Proteína bcl-X/antagonistas & inhibidores , Animales , Antineoplásicos/uso terapéutico , Apoptosis , Benzamidas/uso terapéutico , Proliferación Celular , Femenino , Neoplasias Hematológicas/metabolismo , Neoplasias Hematológicas/patología , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Piperidinas/uso terapéutico , Sulfonas/uso terapéutico , Trombocitopenia/metabolismo , Trombocitopenia/patología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Histone deacetylase inhibitors (HDACi) are compounds that target the epigenome and cause tumor cell-selective apoptosis. A large number of these agents that have different chemical structures and can target multiple HDACs are being testing in clinical trials and vorinostat is now an approved drug for the treatment of cutaneous T-cell lymphoma. Although these agents are showing promise for the treatment of hematologic malignancies, it is possible that different drugs may have different mechanistic, biological, and therapeutic activities. When comparing an HDACi belonging to the hydroxamic acid class of compounds (vorinostat) with a cyclic tetrapeptide (romidepsin), we showed that these agents regulate the expression of a common set of cellular genes, but certain genes specifically responded to each agent. Using the Emu-myc mouse model of B-cell lymphoma, we showed previously that overexpression of the prosurvival proteins Bcl-2 and Bcl-XL inhibited the apoptotic and therapeutic activities of the vorinostat. Herein, we compared and contrasted the apoptotic-inducing activities of the hydroxamic acid oxamflatin with romidepsin. Like vorinostat, oxamflatin was unable to kill lymphomas overexpressing Bcl-2 and Bcl-XL, indicating that these proteins can generally protect cells against this class of HDACi. In contrast, romidepsin was able to induce apoptosis in lymphomas overexpressing Bcl-2 with delayed kinetics of cell death and could mediate therapeutic responses against these lymphomas. However, romidepsin was inactive when Bcl-XL was overexpressed. These data provide strong support that HDACi of different chemical classes may have subtle yet potentially important differences in their molecular and biological activities.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Apoptosis , Depsipéptidos/farmacología , Inhibidores Enzimáticos/farmacología , Inhibidores de Histona Desacetilasas , Animales , Antibióticos Antineoplásicos/uso terapéutico , Depsipéptidos/uso terapéutico , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/uso terapéutico , Ácidos Hidroxámicos/farmacología , Linfoma de Células B/tratamiento farmacológico , Linfoma de Células B/enzimología , Ratones , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteína bcl-X/metabolismoRESUMEN
HIN-200 proteins are interferon-inducible proteins capable of regulating cell growth, senescence, differentiation and death. Using a combination of in silico analysis of NCBI EST databases and screening of murine C57BL/6 cDNA libraries we isolated novel murine HIN-200 cDNAs designated Ifi206S and Ifi206L encoding two putative mRNA splice variants. The p206S and p206L protein isoforms have a modular domain structure consisting of an N-terminal PAAD/DAPIN/Pyrin domain, a region rich in serine, threonine and proline residues and a C-terminal 200 B domain characteristic of other HIN-200 proteins. Ifi206 mRNA was detected only in the spleen and lung of BALB/c and C57BL/6 mice and expression was up-regulated by both types I and II IFN subtypes. p206 protein was predominantly expressed in the cytoplasm and addition of LMB, a CRM1 dependent nuclear export inhibitor, caused p206 to accumulate in the nucleus. Unlike other human and mouse HIN-200 proteins that contain only a single 200 amino acid domain, overexpression of p206 impaired the clonogenic growth of tumour cell lines. Thus, p206 represents the newest HIN-200 family member discovered. It has distinct and restricted pattern of expression however maintains many of the hallmarks of HIN-200 proteins including the presence of a characteristic 200 X domain, induction by interferon and an ability to suppress tumour cell growth.
Asunto(s)
Proteínas Nucleares/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Células 3T3 , Empalme Alternativo , Animales , Línea Celular Tumoral , Proliferación Celular , Clonación Molecular , Citoplasma/metabolismo , Biblioteca de Genes , Interferón Tipo I/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Proteínas Nucleares/genética , Especificidad de Órganos , Conformación Proteica , ARN Mensajero/metabolismo , Proteínas Recombinantes , Transfección , Proteínas Supresoras de Tumor/genéticaRESUMEN
It is now well recognized that mutations, deregulated expression, and aberrant recruitment of epigenetic readers, writers, and erasers are fundamentally important processes in the onset and maintenance of many human tumors. The molecular, biological, and biochemical characteristics of a particular class of epigenetic erasers, the histone deacetylases (HDACs), have been extensively studied and small-molecule HDAC inhibitors (HDACis) have now been clinically approved for the treatment of human hemopoietic malignancies. This review explores our current understanding of the biological and molecular effects on tumor cells following HDACi treatment. The predominant responses include induction of tumor cell death and inhibition of proliferation that in experimental models have been linked to therapeutic efficacy. However, tumor cell-intrinsic responses to HDACi, including modulating tumor immunogenicity have also been described and may have substantial roles in mediating the antitumor effects of HDACi. We posit that the field has failed to fully reconcile the biological consequences of exposure to HDACis with the molecular events that underpin these responses, however progress is being made. Understanding the pleiotrophic activities of HDACis on tumor cells will hopefully fast track the development of more potent and selective HDACi that may be used alone or in combination to improve patient outcomes.
Asunto(s)
Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Inhibidores de Histona Desacetilasas/uso terapéutico , Neoplasias/tratamiento farmacológico , Acetilación/efectos de los fármacos , Histona Desacetilasas/metabolismo , Histonas/metabolismo , Humanos , Neoplasias/metabolismo , Neoplasias/patologíaRESUMEN
Targeting BET bromodomain proteins using small molecules is an emerging anticancer strategy with clinical evaluation of at least six inhibitors now underway. Although MYC downregulation was initially proposed as a key mechanistic property of BET inhibitors, recent evidence suggests that additional antitumor activities are important. Using the Eµ-Myc model of B-cell lymphoma, we demonstrate that BET inhibition with JQ1 is a potent inducer of p53-independent apoptosis that occurs in the absence of effects on Myc gene expression. JQ1 skews the expression of proapoptotic (Bim) and antiapoptotic (BCL-2/BCL-xL) BCL-2 family members to directly engage the mitochondrial apoptotic pathway. Consistent with this, Bim knockout or Bcl-2 overexpression inhibited apoptosis induction by JQ1. We identified lymphomas that were either intrinsically resistant to JQ1-mediated death or acquired resistance following in vivo exposure. Strikingly, in both instances BCL-2 was strongly upregulated and was concomitant with activation of RAS pathways. Eµ-Myc lymphomas engineered to express activated Nras upregulated BCL-2 and acquired a JQ1 resistance phenotype. These studies provide important information on mechanisms of apoptosis induction and resistance to BET-inhibition, while providing further rationale for the translation of BET inhibitors in aggressive B-cell lymphomas. Mol Cancer Ther; 15(9); 2030-41. ©2016 AACR.
Asunto(s)
Apoptosis/genética , Azepinas/farmacología , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Linfoma de Células B/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Triazoles/farmacología , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Resistencia a Antineoplásicos/genética , Genes myc , Humanos , Linfoma de Células B/metabolismo , Linfoma de Células B/mortalidad , Linfoma de Células B/patología , Ratones , Familia de Multigenes , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Nutlin3a is a small-molecule antagonist of MDM2 that promotes non-genotoxic activation of p53 through p53 protein stabilization and transactivation of p53 target genes. Nutlin3a is the forerunner of a class of cancer therapeutics that have reached clinical trials. Using transgenic and gene-targeted mouse models lacking the critical p53 target genes, p21, Puma, and Noxa, we found that only loss of PUMA conferred profound protection against Nutlin3a-induced killing in both non-transformed lymphoid cells and Eµ-Myc lymphomas in vitro and in vivo. CRISPR/Cas9-mediated targeting of the PUMA gene rendered human hematopoietic cancer cell lines markedly resistant to Nutlin3a-induced cell death. These results demonstrate that PUMA-mediated apoptosis, but not p21-mediated cell-cycle arrest or senescence, is a critical determinant of the therapeutic response to non-genotoxic p53 activation by Nutlin3a. Importantly, in human cancer, PUMA expression may predict patient responses to treatment with MDM2 antagonists.
Asunto(s)
Antineoplásicos/farmacología , Proteínas Reguladoras de la Apoptosis/genética , Regulación Neoplásica de la Expresión Génica , Imidazoles/farmacología , Linfoma/tratamiento farmacológico , Piperazinas/farmacología , Proteína p53 Supresora de Tumor/genética , Proteínas Supresoras de Tumor/genética , Animales , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/metabolismo , Sistemas CRISPR-Cas , Puntos de Control del Ciclo Celular , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/genética , Humanos , Linfoma/genética , Linfoma/metabolismo , Linfoma/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Transducción de Señal , Proteína p53 Supresora de Tumor/agonistas , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
E proteins and their antagonists, the Id proteins, are transcriptional regulators important for normal hematopoiesis. We found that Id2 acts as a key regulator of leukemia stem cell (LSC) potential in MLL-rearranged acute myeloid leukemia (AML). Low endogenous Id2 expression is associated with LSC enrichment while Id2 overexpression impairs MLL-AF9-leukemia initiation and growth. Importantly, MLL-AF9 itself controls the E-protein pathway by suppressing Id2 while directly activating E2-2 expression, and E2-2 depletion phenocopies Id2 overexpression in MLL-AF9-AML cells. Remarkably, Id2 tumor-suppressive function is conserved in t(8;21) AML. Low expression of Id2 and its associated gene signature are associated with poor prognosis in MLL-rearranged and t(8;21) AML patients, identifying the Id2/E-protein axis as a promising new therapeutic target in AML.
Asunto(s)
Proteína 2 Inhibidora de la Diferenciación/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas de Fusión Oncogénica/genética , Proteína 2 Similar al Factor de Transcripción 7/genética , Translocación Genética , Animales , Proliferación Celular , Cromosomas Humanos Par 21/genética , Cromosomas Humanos Par 8/genética , Regulación Leucémica de la Expresión Génica , Humanos , Proteína 2 Inhibidora de la Diferenciación/metabolismo , Leucemia Mieloide Aguda/metabolismo , Ratones , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Neoplasias Experimentales , Proteínas de Fusión Oncogénica/metabolismo , Pronóstico , Células Madre/citología , Células Madre/metabolismo , Análisis de Supervivencia , Proteína 2 Similar al Factor de Transcripción 7/metabolismoRESUMEN
Positron emission tomography (PET) can be used to monitor the uptake of the labeled glucose analog fluorodeoxyglucose (¹8F-FDG), a process that is generally believed to reflect viable tumor cell mass. The use of ¹8F-FDG PET can be helpful in documenting over time the reduction in tumor mass volume in response to anticancer drug therapy in vivo. In this protocol, we describe how to monitor the response of murine B-cell lymphomas to an inducer of apoptosis, the anticancer drug vorinostat (a histone deacetylase inhibitor). B-cell lymphoma cells are injected into recipient mice and, on tumor formation, the mice are treated with vorinostat. The tracer ¹8F-FDG is then injected into the mice at several time points, and its uptake is monitored using PET. Because the uptake of ¹8F-FDG is not a direct measure of apoptosis, an additional direct method proving that apoptotic cells are present should also be performed.
Asunto(s)
Antineoplásicos/uso terapéutico , Fluorodesoxiglucosa F18 , Ácidos Hidroxámicos/uso terapéutico , Linfoma de Células B/diagnóstico por imagen , Linfoma de Células B/tratamiento farmacológico , Tomografía de Emisión de Positrones , Animales , Línea Celular Tumoral , Humanos , Ratones , Ratones Endogámicos C57BL , Vorinostat , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Positron emission tomography (PET) is used to monitor the uptake of the labeled glucose analogue fluorodeoxyglucose (¹8F-FDG) by solid tumor cells, a process generally believed to reflect viable tumor cell mass. The use of ¹8F-FDG exploits the high demand for glucose in tumor cells, and serves to document over time the response of a solid tumor to an inducer of apoptosis. The apoptosis inducer crizotinib is a small-molecule inhibitor of c-Met, a receptor tyrosine kinase that is often dysregulated in human tumors. In this protocol, we describe how to monitor the response of a solid tumor to crizotinib. Human gastric tumor cells (GTL-16 cells) are injected into recipient mice and, on tumor formation, the mice are treated with crizotinib. The tracer ¹8F-FDG is then injected into the mice at several time points, and its uptake is monitored using PET. Because ¹8F-FDG uptake varies widely among different tumor models, preliminary experiments should be performed with each new model to determine its basal level of ¹8F-FDG uptake. Verifying that the basal level of uptake is sufficiently above background levels will assure accurate quantitation. Because ¹8F-FDG uptake is not a direct measure of apoptosis, it is advisable to carry out an additional direct method to show the presence of apoptotic cells.
Asunto(s)
Antineoplásicos/uso terapéutico , Fluorodesoxiglucosa F18 , Tomografía de Emisión de Positrones , Pirazoles/uso terapéutico , Piridinas/uso terapéutico , Neoplasias Gástricas/diagnóstico por imagen , Neoplasias Gástricas/tratamiento farmacológico , Animales , Línea Celular Tumoral , Crizotinib , Modelos Animales de Enfermedad , Humanos , Ratones , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Immunohistochemistry is commonly used to show the presence of apoptotic cells in situ. In this protocol, B-cell lymphoma cells are injected into recipient mice and, on tumor formation, the mice are treated with the apoptosis inducer vorinostat (a histone deacetylase inhibitor). Tumor samples are fixed and sectioned, and fragmented DNA (a feature of apoptotic cells) is end-labeled by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Immunohistochemical methods are then used to detect the labeled DNA and identify B-cell lymphoma cells in the last stage of apoptosis. Because the assay can lead to false-positive results, it is advisable to carry out an additional assay (e.g., immunohistochemistry for active caspase-3) to confirm the presence of apoptotic cells.
Asunto(s)
Apoptosis , Inmunohistoquímica/métodos , Linfoma de Células B/patología , Animales , ADN/análisis , Fragmentación del ADN , Inhibidores de Histona Desacetilasas/administración & dosificación , Ácidos Hidroxámicos/administración & dosificación , Etiquetado Corte-Fin in Situ , Ratones , VorinostatRESUMEN
Flow cytometry assays are often used to detect apoptotic cells in in vitro cultures. Depending on the experimental model, these assays can also be useful in evaluating apoptosis in vivo. In this protocol, we describe a propidium iodide (PI) flow cytometry assay to evaluate B-cell lymphomas that have undergone apoptosis in vivo. B-cell lymphoma cells are injected into recipient mice and, on tumor formation, the mice are treated with the apoptosis inducer vorinostat (a histone deacetylase inhibitor). Tumor samples collected from the lymph nodes and/or the spleen are used to prepare a single-cell suspension that is exposed to a hypotonic solution containing the fluorochrome PI. The DNA content of the cells, now labeled with PI, is analyzed by flow cytometry. Nuclear DNA content is lost during apoptosis, resulting in a hypodiploid (or sub-G1) DNA profile during flow cytometry. In contrast, healthy cells display a sharp diploid DNA profile.