RESUMO
Recent studies have reported that the peroxisome proliferator-activated receptor gamma (PPARγ) pathway is activated in approximately 40% of patients with muscle-invasive bladder cancer. This led us to investigate pharmacological repression of PPARγ as a possible intervention strategy. Here, we characterize PPARγ antagonists and inverse agonists and find that the former behave as silent ligands, whereas inverse agonists (T0070907 and SR10221) repress downstream PPARγ target genes leading to growth inhibition in bladder cancer cell lines. To understand the mechanism, we determined the ternary crystal structure of PPARγ bound to T0070907 and the corepressor (co-R) peptide NCOR1. The structure shows that the AF-2 helix 12 (H12) rearranges to bind inside the ligand-binding domain, where it forms stabilizing interactions with the compound. This dramatic movement in H12 unveils a large interface for co-R binding. In contrast, the crystal structure of PPARγ bound to a SR10221 analog shows more subtle structural differences, where the compound binds and pushes H12 away from the ligand-binding domain to allow co-R binding. Interestingly, we found that both classes of compound promote recruitment of co-R proteins in biochemical assays but with distinct conformational changes in H12. We validate our structural models using both site-directed mutagenesis and chemical probes. Our findings offer new mechanistic insights into pharmacological modulation of PPARγ signaling.
Assuntos
PPAR gama , Neoplasias da Bexiga Urinária , Humanos , PPAR gama/metabolismo , Ligantes , Benzamidas/farmacologiaRESUMO
Nearly 30% of patients with relapsed breast cancer present activating mutations in estrogen receptor alpha (ERα) that confer partial resistance to existing endocrine-based therapies. We previously reported the development of H3B-5942, a covalent ERα antagonist that engages cysteine-530 (C530) to achieve potency against both wild-type (ERαWT) and mutant ERα (ERαMUT). Anticipating that the emergence of C530 mutations could promote resistance to H3B-5942, we applied structure-based drug design to improve the potency of the core scaffold to further enhance the antagonistic activity in addition to covalent engagement. This effort led to the development of the clinical candidate H3B-6545, a covalent antagonist that is potent against both ERαWT/MUT, and maintains potency even in the context of ERα C530 mutations. H3B-6545 demonstrates significant activity and superiority over standard-of-care fulvestrant across a panel of ERαWT and ERαMUT palbociclib sensitive and resistant models. In summary, the compelling preclinical activity of H3B-6545 supports its further development for the potential treatment of endocrine therapy-resistant ERα+ breast cancer harboring wild-type or mutant ESR1, as demonstrated by the ongoing clinical trials (NCT03250676, NCT04568902, NCT04288089). SUMMARY: H3B-6545 is an ERα covalent antagonist that exhibits encouraging preclinical activity against CDK4/6i naïve and resistant ERαWT and ERαMUT tumors.
Assuntos
Neoplasias da Mama , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Ensaios Clínicos como Assunto , Receptor alfa de Estrogênio/genética , Feminino , Fulvestranto/uso terapêutico , Humanos , Indazóis , Recidiva Local de Neoplasia , PiridinasRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
The development of tamoxifen and subsequent estrogen receptor alpha (ERα) antagonists represents a tremendous therapeutic breakthrough in the treatment of breast cancer. Despite the ability of ERα antagonists to increase survival rates, resistance to these therapies is an all-too-common occurrence. The majority of resistant tumors, including those with hotspot mutations in the ligand-binding domain of ERα, remain dependent on ERα signaling, indicating that either a more potent or novel class of antagonist could have clinical benefit. With this thought in mind, we developed a novel ERα antagonist that exhibits enhanced potency due to its ability to covalently target a unique cysteine in ER. This review describes the design of this antagonist, H3B-5942, and discusses opportunities for future improvements, which could reduce the risk of escape mutations to this therapeutic modality.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Antagonistas do Receptor de Estrogênio/uso terapêutico , Indazóis/uso terapêutico , Receptores de Estrogênio/antagonistas & inibidores , Animais , Feminino , HumanosRESUMO
Dysregulation of RNA splicing by spliceosome mutations or in cancer genes is increasingly recognized as a hallmark of cancer. Small molecule splicing modulators have been introduced into clinical trials to treat solid tumors or leukemia bearing recurrent spliceosome mutations. Nevertheless, further investigation of the molecular mechanisms that may enlighten therapeutic strategies for splicing modulators is highly desired. Here, using unbiased functional approaches, we report that the sensitivity to splicing modulation of the anti-apoptotic BCL2 family genes is a key mechanism underlying preferential cytotoxicity induced by the SF3b-targeting splicing modulator E7107. While BCL2A1, BCL2L2 and MCL1 are prone to splicing perturbation, BCL2L1 exhibits resistance to E7107-induced splicing modulation. Consequently, E7107 selectively induces apoptosis in BCL2A1-dependent melanoma cells and MCL1-dependent NSCLC cells. Furthermore, combination of BCLxL (BCL2L1-encoded) inhibitors and E7107 remarkably enhances cytotoxicity in cancer cells. These findings inform mechanism-based approaches to the future clinical development of splicing modulators in cancer treatment.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Melanoma/tratamento farmacológico , Antígenos de Histocompatibilidade Menor/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Splicing de RNA/efeitos dos fármacos , Proteína bcl-X/genética , Células A549 , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Doxiciclina/farmacologia , Sinergismo Farmacológico , Compostos de Epóxi/farmacologia , Feminino , Humanos , Neoplasias Pulmonares/genética , Macrolídeos/farmacologia , Melanoma/genética , Camundongos , Camundongos Nus , Interferência de RNA , Splicing de RNA/genética , RNA Interferente Pequeno/genética , Spliceossomos/efeitos dos fármacos , Spliceossomos/genética , Sequenciamento do Exoma , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Mutations in estrogen receptor alpha (ERα) that confer resistance to existing classes of endocrine therapies are detected in up to 30% of patients who have relapsed during endocrine treatments. Because a significant proportion of therapy-resistant breast cancer metastases continue to be dependent on ERα signaling, there remains a critical need to develop the next generation of ERα antagonists that can overcome aberrant ERα activity. Through our drug-discovery efforts, we identified H3B-5942, which covalently inactivates both wild-type and mutant ERα by targeting Cys530 and enforcing a unique antagonist conformation. H3B-5942 belongs to a class of ERα antagonists referred to as selective estrogen receptor covalent antagonists (SERCA). In vitro comparisons of H3B-5942 with standard-of-care (SoC) and experimental agents confirmed increased antagonist activity across a panel of ERαWT and ERαMUT cell lines. In vivo, H3B-5942 demonstrated significant single-agent antitumor activity in xenograft models representing ERαWT and ERαY537S breast cancer that was superior to fulvestrant. Lastly, H3B-5942 potency can be further improved in combination with CDK4/6 or mTOR inhibitors in both ERαWT and ERαMUT cell lines and/or tumor models. In summary, H3B-5942 belongs to a class of orally available ERα covalent antagonists with an improved profile over SoCs.Significance: Nearly 30% of endocrine therapy-resistant breast cancer metastases harbor constitutively activating mutations in ERα. SERCA H3B-5942 engages C530 of both ERαWT and ERαMUT, promotes a unique antagonist conformation, and demonstrates improved in vitro and in vivo activity over SoC agents. Importantly, single-agent efficacy can be further enhanced by combining with CDK4/6 or mTOR inhibitors. Cancer Discov; 8(9); 1176-93. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 1047.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Antagonistas do Receptor de Estrogênio/administração & dosagem , Receptor alfa de Estrogênio/antagonistas & inibidores , Indazóis/administração & dosagem , Mutação , Administração Oral , Animais , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cisteína/antagonistas & inibidores , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Antagonistas do Receptor de Estrogênio/química , Antagonistas do Receptor de Estrogênio/farmacologia , Receptor alfa de Estrogênio/química , Receptor alfa de Estrogênio/genética , Feminino , Humanos , Indazóis/química , Indazóis/farmacologia , Células MCF-7 , Camundongos , Conformação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Muscle-invasive bladder cancer (MIBC) is an aggressive disease with limited therapeutic options. Although immunotherapies are approved for MIBC, the majority of patients fail to respond, suggesting existence of complementary immune evasion mechanisms. Here, we report that the PPARγ/RXRα pathway constitutes a tumor-intrinsic mechanism underlying immune evasion in MIBC. Recurrent mutations in RXRα at serine 427 (S427F/Y), through conformational activation of the PPARγ/RXRα heterodimer, and focal amplification/overexpression of PPARγ converge to modulate PPARγ/RXRα-dependent transcription programs. Immune cell-infiltration is controlled by activated PPARγ/RXRα that inhibits expression/secretion of inflammatory cytokines. Clinical data sets and an in vivo tumor model indicate that PPARγHigh/RXRαS427F/Y impairs CD8+ T-cell infiltration and confers partial resistance to immunotherapies. Knockdown of PPARγ or RXRα and pharmacological inhibition of PPARγ significantly increase cytokine expression suggesting therapeutic approaches to reviving immunosurveillance and sensitivity to immunotherapies. Our study reveals a class of tumor cell-intrinsic "immuno-oncogenes" that modulate the immune microenvironment of cancer.Muscle-invasive bladder cancer (MIBC) is a potentially lethal disease. Here the authors characterize diverse genetic alterations in MIBC that convergently lead to constitutive activation of PPARgamma/RXRalpha and result in immunosurveillance escape by inhibiting CD8+ T-cell recruitment.
Assuntos
Evasão da Resposta Imune/imunologia , Monitorização Imunológica , PPAR gama/imunologia , Receptor X Retinoide alfa/imunologia , Neoplasias da Bexiga Urinária/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Citocinas/genética , Citocinas/imunologia , Citocinas/metabolismo , Perfilação da Expressão Gênica/métodos , Células HCT116 , Humanos , Immunoblotting , Imunoterapia/métodos , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Camundongos , Microscopia de Fluorescência , Mutação/imunologia , Invasividade Neoplásica , PPAR gama/química , PPAR gama/genética , Multimerização Proteica/imunologia , Receptor X Retinoide alfa/química , Receptor X Retinoide alfa/genética , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/terapiaRESUMO
Extension of neurites from a cell body is essential to form a functional nervous system; however, the mechanisms underlying neuritogenesis are poorly understood. Ena/VASP proteins regulate actin dynamics and modulate elaboration of cellular protrusions. We recently reported that cortical axon-tract formation is lost in Ena/VASP-null mice and Ena/VASP-null cortical neurons lack filopodia and fail to elaborate neurites. Here, we report that neuritogenesis in Ena/VASP-null neurons can be rescued by restoring filopodia formation through ectopic expression of the actin nucleating protein mDia2. Conversely, wild-type neurons in which filopodia formation is blocked fail to elaborate neurites. We also report that laminin, which promotes the formation of filopodia-like actin-rich protrusions, rescues neuritogenesis in Ena/VASP-deficient neurons. Therefore, filopodia formation is a key prerequisite for neuritogenesis in cortical neurons. Neurite initiation also requires microtubule extension into filopodia, suggesting that interactions between actin-filament bundles and dynamic microtubules within filopodia are crucial for neuritogenesis.
Assuntos
Córtex Cerebral/citologia , Neuritos/fisiologia , Neurônios/fisiologia , Pseudópodes/fisiologia , Actinas/metabolismo , Animais , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/fisiologia , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Laminina/fisiologia , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/fisiologia , Proteínas Associadas aos Microtúbulos , Microtúbulos/fisiologia , Mutação , Miosina Tipo II/antagonistas & inibidores , Miosinas/biossíntese , NADPH Desidrogenase/biossíntese , Neurônios/ultraestrutura , Fosfoproteínas/genética , Fosfoproteínas/fisiologiaRESUMO
DEF-1/ASAP1 is an ADP-ribosylation factor GTPase-activating protein (ARF GAP) that localizes to focal adhesions and is involved in cytoskeletal regulation. In this paper, we use a cell-based ARF GAP assay to demonstrate that DEF-1 functions as a GAP for ARF1 and not ARF6 in vivo. This degree of substrate preference was unique to DEF-1, as other ARF GAP proteins, ACAP1, ACAP2, and ARFGAP1, were able to function on both ARF1 and ARF6. Since transient overexpression of DEF-1 has been shown to interfere with focal adhesion formation and platelet-derived growth factor-induced membrane ruffling, we investigated whether NIH 3T3 cells stably expressing DEF-1 have altered cell motility. Here we report that ectopic DEF-1 enhances cell migration toward PDGF as well as IGF-1. This chemotactic effect appears to result from a general increase in cell motility, as DEF-1-expressing cells also exhibit enhanced levels of basal and chemokinetic motility. The increase in cell motility is dependent on DEF-1 GAP activity, since a DEF-1 mutant lacking the GAP domain failed to stimulate motility. This suggests that DEF-1 alters cell motility through the deactivation of ARF1. In contrast, the inhibition of cell spreading by DEF-1 was not dependent on GAP activity, indicating that spreading and motility are altered by DEF-1 through different pathways.