RESUMO
Activated Cdc42-associated kinase (ACK) is an oncogenic nonreceptor tyrosine kinase associated with poor prognosis in several human cancers. ACK promotes proliferation, in part by contributing to the activation of Akt, the major effector of class 1A phosphoinositide 3-kinases (PI3Ks), which transduce signals via membrane phosphoinositol lipids. We now show that ACK also interacts with other key components of class 1A PI3K signaling, the PI3K regulatory subunits. We demonstrate ACK binds to all five PI3K regulatory subunit isoforms and directly phosphorylates p85α, p85ß, p50α, and p55α on Tyr607 (or analogous residues). We found that phosphorylation of p85ß promotes cell proliferation in HEK293T cells. We demonstrate that ACK interacts with p85α exclusively in nuclear-enriched cell fractions, where p85α phosphorylated at Tyr607 (pTyr607) also resides, and identify an interaction between pTyr607 and the N-terminal SH2 domain that supports dimerization of the regulatory subunits. We infer from this that ACK targets p110-independent p85 and further postulate that these regulatory subunit dimers undertake novel nuclear functions underpinning ACK activity. We conclude that these dimers represent a previously undescribed mode of regulation for the class1A PI3K regulatory subunits and potentially reveal additional avenues for therapeutic intervention.
Assuntos
Fosfatidilinositol 3-Quinases , Proteínas Tirosina Quinases , Núcleo Celular/enzimologia , Células HEK293 , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Multimerização Proteica , Proteínas Tirosina Quinases/metabolismo , Transdução de SinaisRESUMO
Low success rates during drug development are due, in part, to the difficulty of defining drug mechanism-of-action and molecular markers of therapeutic activity. Here, we integrated 199,219 drug sensitivity measurements for 397 unique anti-cancer drugs with genome-wide CRISPR loss-of-function screens in 484 cell lines to systematically investigate cellular drug mechanism-of-action. We observed an enrichment for positive associations between the profile of drug sensitivity and knockout of a drug's nominal target, and by leveraging protein-protein networks, we identified pathways underpinning drug sensitivity. This revealed an unappreciated positive association between mitochondrial E3 ubiquitin-protein ligase MARCH5 dependency and sensitivity to MCL1 inhibitors in breast cancer cell lines. We also estimated drug on-target and off-target activity, informing on specificity, potency and toxicity. Linking drug and gene dependency together with genomic data sets uncovered contexts in which molecular networks when perturbed mediate cancer cell loss-of-fitness and thereby provide independent and orthogonal evidence of biomarkers for drug development. This study illustrates how integrating cell line drug sensitivity with CRISPR loss-of-function screens can elucidate mechanism-of-action to advance drug development.
Assuntos
Antineoplásicos/farmacologia , Sistemas CRISPR-Cas , Desenvolvimento de Medicamentos/métodos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Redes Reguladoras de Genes/efeitos dos fármacos , Aptidão Genética/efeitos dos fármacos , Mapas de Interação de Proteínas/efeitos dos fármacos , Antineoplásicos/toxicidade , Biomarcadores/metabolismo , Linhagem Celular Tumoral , Técnicas de Inativação de Genes , Redes Reguladoras de Genes/genética , Aptidão Genética/genética , Genômica , Humanos , Modelos Lineares , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Preparações Farmacêuticas/metabolismo , Software , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
We explore mechanisms that enable cancer cells to tolerate PI3K or Akt inhibitors. Prolonged treatment of breast cancer cells with PI3K or Akt inhibitors leads to increased expression and activation of a kinase termed SGK3 that is related to Akt. Under these conditions, SGK3 is controlled by hVps34 that generates PtdIns(3)P, which binds to the PX domain of SGK3 promoting phosphorylation and activation by its upstream PDK1 activator. Furthermore, under conditions of prolonged PI3K/Akt pathway inhibition, SGK3 substitutes for Akt by phosphorylating TSC2 to activate mTORC1. We characterise 14h, a compound that inhibits both SGK3 activity and activation in vivo, and show that a combination of Akt and SGK inhibitors induced marked regression of BT-474 breast cancer cell-derived tumours in a xenograft model. Finally, we present the kinome-wide analysis of mRNA expression dynamics induced by PI3K/Akt inhibition. Our findings highlight the importance of the hVps34-SGK3 pathway and suggest it represents a mechanism to counteract inhibition of PI3K/Akt signalling. The data support the potential of targeting both Akt and SGK as a cancer therapeutic.
Assuntos
Carcinogênese , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Complexos Multiproteicos/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Feminino , Xenoenxertos , Humanos , Alvo Mecanístico do Complexo 1 de RapamicinaRESUMO
Activated Cdc42-associated kinase or ACK, is a non-receptor tyrosine kinase and an effector protein for the small G protein Cdc42. A substantial body of evidence has accumulated in the past few years heavily implicating ACK as a driver of oncogenic processes. Concomitantly, more is also being revealed regarding the signalling pathways involving ACK and molecular details of its modes of action. Some details are also available regarding the regulatory mechanisms of this kinase, including activation and regulation of its catalytic activity, however, a full understanding of these aspects remains elusive. This review considers the current knowledge base concerning ACK and summarizes efforts and future prospects to target ACK therapeutically in cancer.
Assuntos
Neoplasias/terapia , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Animais , Transporte Biológico , Movimento Celular , Endocitose , Ativação Enzimática , Epigênese Genética , Humanos , Neoplasias/enzimologia , Neoplasias/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Transativadores/metabolismoRESUMO
Androgen receptor (AR) signaling plays a key role in the progression of prostate cancer. This study describes the discovery and optimization of a novel series of AR PROTAC degraders that recruit the Cereblon (CRBN) E3 ligase. Having identified a series of AR ligands based on 4-(4-phenyl-1-piperidyl)-2-(trifluoromethyl)benzonitrile, our PROTAC optimization strategy focused on linker connectivity and CRBN ligand SAR to deliver potent degradation of AR in LNCaP cells. This work culminated in compounds 11 and 16 which demonstrated good rodent oral bioavailability. Subsequent SAR around the AR binding region brought in an additional desirable feature, degradation of the important treatment resistance mutation L702H. Compound 22 (AZ'3137) possessed an attractive profile showing degradation of AR and L702H mutant AR with good oral bioavailability across species. The compound also inhibited AR signaling in vitro and tumor growth in vivo in a mouse prostate cancer xenograft model.
Assuntos
Disponibilidade Biológica , Neoplasias da Próstata , Receptores Androgênicos , Masculino , Animais , Humanos , Receptores Androgênicos/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Neoplasias da Próstata/metabolismo , Administração Oral , Camundongos , Linhagem Celular Tumoral , Relação Estrutura-Atividade , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Antineoplásicos/farmacocinética , Antagonistas de Receptores de Andrógenos/farmacologia , Antagonistas de Receptores de Andrógenos/química , Antagonistas de Receptores de Andrógenos/uso terapêutico , Antagonistas de Receptores de Andrógenos/farmacocinética , Descoberta de Drogas , Ensaios Antitumorais Modelo de Xenoenxerto , RatosRESUMO
More than 50% of human tumors display hyperactivation of the serine/threonine kinase AKT. Despite evidence of clinical efficacy, the therapeutic window of the current generation of AKT inhibitors could be improved. Here, we report the development of a second-generation AKT degrader, INY-05-040, which outperformed catalytic AKT inhibition with respect to cellular suppression of AKT-dependent phenotypes in breast cancer cell lines. A growth inhibition screen with 288 cancer cell lines confirmed that INY-05-040 had a substantially higher potency than our first-generation AKT degrader (INY-03-041), with both compounds outperforming catalytic AKT inhibition by GDC-0068. Using multiomic profiling and causal network integration in breast cancer cells, we demonstrated that the enhanced efficacy of INY-05-040 was associated with sustained suppression of AKT signaling, which was followed by induction of the stress mitogen-activated protein kinase (MAPK) c-Jun N-terminal kinase (JNK). Further integration of growth inhibition assays with publicly available transcriptomic, proteomic, and reverse phase protein array (RPPA) measurements established low basal JNK signaling as a biomarker for breast cancer sensitivity to AKT degradation. Together, our study presents a framework for mapping the network-wide signaling effects of therapeutically relevant compounds and identifies INY-05-040 as a potent pharmacological suppressor of AKT signaling.
Assuntos
Neoplasias da Mama , Proteínas Quinases Ativadas por Mitógeno , Humanos , Feminino , Proteínas Proto-Oncogênicas c-akt/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Apoptose , Mitógenos , Multiômica , Proteômica , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno , Proteínas Quinases JNK Ativadas por MitógenoRESUMO
Oncology drug combinations can improve therapeutic responses and increase treatment options for patients. The number of possible combinations is vast and responses can be context-specific. Systematic screens can identify clinically relevant, actionable combinations in defined patient subtypes. We present data for 109 anticancer drug combinations from AstraZeneca's oncology small molecule portfolio screened in 755 pan-cancer cell lines. Combinations were screened in a 7 × 7 concentration matrix, with more than 4 million measurements of sensitivity, producing an exceptionally data-rich resource. We implement a new approach using combination Emax (viability effect) and highest single agent (HSA) to assess combination benefit. We designed a clinical translatability workflow to identify combinations with clearly defined patient populations, rationale for tolerability based on tumor type and combination-specific "emergent" biomarkers, and exposures relevant to clinical doses. We describe three actionable combinations in defined cancer types, confirmed in vitro and in vivo, with a focus on hematologic cancers and apoptotic targets. SIGNIFICANCE: We present the largest cancer drug combination screen published to date with 7 × 7 concentration response matrices for 109 combinations in more than 750 cell lines, complemented by multi-omics predictors of response and identification of "emergent" combination biomarkers. We prioritize hits to optimize clinical translatability, and experimentally validate novel combination hypotheses. This article is featured in Selected Articles from This Issue, p. 695.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica , Neoplasias , Humanos , Linhagem Celular Tumoral , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêuticoRESUMO
Targeting the estrogen receptor alpha (ERα) pathway is validated in the clinic as an effective means to treat ER+ breast cancers. Here we present the development of a VHL-targeting and orally bioavailable proteolysis-targeting chimera (PROTAC) degrader of ERα. In vitro studies with this PROTAC demonstrate excellent ERα degradation and ER antagonism in ER+ breast cancer cell lines. However, upon dosing the compound in vivo we observe an in vitro-in vivo disconnect. ERα degradation is lower in vivo than expected based on the in vitro data. Investigation into potential causes for the reduced maximal degradation reveals that metabolic instability of the PROTAC linker generates metabolites that compete for binding to ERα with the full PROTAC, limiting degradation. This observation highlights the requirement for metabolically stable PROTACs to ensure maximal efficacy and thus optimisation of the linker should be a key consideration when designing PROTACs.
Assuntos
Receptor alfa de Estrogênio , Proteólise , Proteína Supressora de Tumor Von Hippel-Lindau , Humanos , Receptor alfa de Estrogênio/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Feminino , Proteólise/efeitos dos fármacos , Animais , Administração Oral , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Camundongos , Antineoplásicos/farmacologia , Antineoplásicos/administração & dosagemRESUMO
We report the first disclosure of IRAK3 degraders in the scientific literature. Taking advantage of an opportune byproduct obtained during our efforts to identify IRAK4 inhibitors, we identified ready-to-use, selective IRAK3 ligands in our compound collection with the required properties for conversion into proteolysis-targeting chimera (PROTAC) degraders. This work culminated with the discovery of PROTAC 23, which we demonstrated to be a potent and selective degrader of IRAK3 after 16 h in THP1 cells. 23 induced proteasome-dependent degradation of IRAK3 and required both CRBN and IRAK3 binding for activity. We conclude that PROTAC 23 constitutes an excellent in vitro tool with which to interrogate the biology of IRAK3.
Assuntos
Quinases Associadas a Receptores de Interleucina-1/metabolismo , Ftalimidas/farmacologia , Proteólise/efeitos dos fármacos , Pirróis/farmacologia , Triazinas/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Humanos , Ligantes , Ftalimidas/síntese química , Pirróis/síntese química , Células THP-1 , Triazinas/síntese química , Ubiquitina-Proteína Ligases/metabolismoRESUMO
SGK3 is a PX domain containing protein kinase activated at endosomes downstream of class 1 and 3 PI3K family members by growth factors and oncogenic mutations. SGK3 plays a key role in mediating resistance of breast cancer cells to class 1 PI3K or Akt inhibitors, by substituting for the loss of Akt activity and restoring proliferative pathways such as mTORC1 signaling. It is therefore critical to develop tools to potently target SGK3 and obstruct its role in inhibitor resistance. Here, we describe the development of SGK3-PROTAC1, a PROTAC conjugate of the 308-R SGK inhibitor with the VH032 VHL binding ligand, targeting SGK3 for degradation. SGK3-PROTAC1 (0.3 µM) induced 50% degradation of endogenous SGK3 within 2 h, with maximal 80% degradation observed within 8 h, accompanied by a loss of phosphorylation of NDRG1, an SGK3 substrate. SGK3-PROTAC1 did not degrade closely related SGK1 and SGK2 isoforms that are nevertheless engaged and inhibited by 308-R. Proteomic analysis revealed that SGK3 was the only cellular protein whose cellular levels were significantly reduced following treatment with SGK3-PROTAC1. Low doses of SGK3-PROTAC1 (0.1-0.3 µM) restored sensitivity of SGK3 dependent ZR-75-1 and CAMA-1 breast cancer cells to Akt (AZD5363) and PI3K (GDC0941) inhibitors, whereas the cis epimer analogue incapable of binding to the VHL E3 ligase had no impact. SGK3-PROTAC1 suppressed proliferation of ZR-75-1 and CAMA-1 cancer cell lines treated with a PI3K inhibitor (GDC0941) more effectively than could be achieved by a conventional SGK isoform inhibitor (14H). This work underscores the benefit of the PROTAC approach in targeting protein kinase signaling pathways with greater efficacy and selectivity than can be achieved with conventional inhibitors. SGK3-PROTAC1 will be an important reagent to explore the roles of the SGK3 pathway.
Assuntos
Dipeptídeos/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Sulfonamidas/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Desenho de Fármacos , Humanos , Indazóis/farmacologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteólise , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirimidinas/farmacologia , Pirróis/farmacologia , Transdução de Sinais/efeitos dos fármacos , Ubiquitina-Proteína Ligases/metabolismoRESUMO
PURPOSE: In the current study, we examined the in vivo effects of AZD1152, a novel and specific inhibitor of Aurora kinase activity (with selectivity for Aurora B). EXPERIMENTAL DESIGN: The pharmacodynamic effects and efficacy of AZD1152 were determined in a panel of human tumor xenograft models. AZD1152 was dosed via several parenteral (s.c. osmotic mini-pump, i.p., and i.v.) routes. RESULTS: AZD1152 potently inhibited the growth of human colon, lung, and hematologic tumor xenografts (mean tumor growth inhibition range, 55% to > or =100%; P < 0.05) in immunodeficient mice. Detailed pharmacodynamic analysis in colorectal SW620 tumor-bearing athymic rats treated i.v. with AZD1152 revealed a temporal sequence of phenotypic events in tumors: transient suppression of histone H3 phosphorylation followed by accumulation of 4N DNA in cells (2.4-fold higher compared with controls) and then an increased proportion of polyploid cells (>4N DNA, 2.3-fold higher compared with controls). Histologic analysis showed aberrant cell division that was concurrent with an increase in apoptosis in AZD1152-treated tumors. Bone marrow analyses revealed transient myelosuppression with the drug that was fully reversible following cessation of AZD1152 treatment. CONCLUSIONS: These data suggest that selective targeting of Aurora B kinase may be a promising therapeutic approach for the treatment of a range of malignancies. In addition to the suppression of histone H3 phosphorylation, determination of tumor cell polyploidy and apoptosis may be useful biomarkers for this class of therapeutic agent. AZD1152 is currently in phase I trials.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias Experimentais/tratamento farmacológico , Organofosfatos/farmacologia , Quinazolinas/farmacologia , Animais , Aurora Quinase B , Aurora Quinases , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citometria de Fluxo , Humanos , Camundongos , Camundongos Nus , Proteínas Serina-Treonina Quinases/efeitos dos fármacos , Ratos , Transplante HeterólogoRESUMO
PI3K inhibitors with differential selectivity to distinct PI3K isoforms have been tested extensively in clinical trials, largely to target tumor epithelial cells. PI3K signaling also regulates the immune system and inhibition of PI3Kδ modulate the tumor immune microenvironment of pre-clinical mouse tumor models by relieving T-regs-mediated immunosuppression. PI3K inhibitors as a class and PI3Kδ specifically are associated with immune-related side effects. However, the impact of mixed PI3K inhibitors in tumor immunology is under-explored. Here we examine the differential effects of AZD8835, a dual PI3Kα/δ inhibitor, specifically on the tumor immune microenvironment using syngeneic models. Continuous suppression of PI3Kα/δ was not required for anti-tumor activity, as tumor growth inhibition was potentiated by an intermittent dosing/schedule in vivo. Moreover, PI3Kα/δ inhibition delivered strong single agent anti-tumor activity, which was associated with dynamic suppression of T-regs, improved CD8+ T-cell activation and memory in mouse syngeneic tumor models. Strikingly, AZD8835 promoted robust CD8+ T-cell activation dissociated from its effect on T-regs. This was associated with enhancing effector cell viability/function. Together these data reveal novel mechanisms by which PI3Kα/δ inhibitors interact with the immune system and validate the clinical compound AZD8835 as a novel immunoncology drug, independent of effects on tumor cells. These data support further clinical investigation of PI3K pathway inhibitors as immuno-oncology agents.
Assuntos
Antineoplásicos/farmacologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Classe I de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Imunomodulação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Interleucina-2/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Camundongos , Oxidiazóis/farmacologia , Piperidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The cyclin dependent kinase (CDK)-retinoblastoma (RB)-E2F pathway plays a critical role in the control of cell cycle in estrogen receptor-positive (ER+) breast cancer. Small-molecule inhibitors of CDK4/6 have shown promise in this tumor type in combination with hormonal therapies, reflecting the particular dependence of this subtype of cancer on cyclin D1 and E2F transcription factors. mTOR inhibitors have also shown potential in clinical trials in this disease setting. Recent data have suggested cooperation between the PI3K/mTOR pathway and CDK4/6 inhibition in preventing early adaptation and eliciting growth arrest, but the mechanisms of the interplay between these pathways have not been fully elucidated. Here we show that profound and durable inhibition of ER+ breast cancer growth is likely to require multiple hits on E2F-mediated transcription. We demonstrate that inhibition of mTORC1/2 does not affect ER function directly, but does cause a decrease in cyclin D1 protein, RB phosphorylation, and E2F-mediated transcription. Combination of an mTORC1/2 inhibitor with a CDK4/6 inhibitor results in more profound effects on E2F-dependent transcription, which translates into more durable growth arrest and a delay in the onset of resistance. Combined inhibition of mTORC1/2, CDK4/6, and ER delivers even more profound and durable regressions in breast cancer cell lines and xenografts. Furthermore, we show that CDK4/6 inhibitor-resistant cell lines reactivate the CDK-RB-E2F pathway, but remain sensitive to mTORC1/2 inhibition, suggesting that mTORC1/2 inhibitors may represent an option for patients that have relapsed on CDK4/6 therapy. Mol Cancer Ther; 17(5); 908-20. ©2018 AACR.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Fatores de Transcrição E2F/antagonistas & inibidores , Serina-Treonina Quinases TOR/antagonistas & inibidores , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Benzamidas , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/metabolismo , Fatores de Transcrição E2F/metabolismo , Feminino , Humanos , Células MCF-7 , Camundongos SCID , Morfolinas/administração & dosagem , Piperazinas/administração & dosagem , Piridinas/administração & dosagem , Pirimidinas , Receptores de Estrogênio/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
The Aurora kinases have been the subject of considerable interest as targets for the development of new anticancer agents. While evidence suggests inhibition of Aurora B kinase gives rise to the more pronounced antiproliferative phenotype, the most clinically advanced agents reported to date typically inhibit both Aurora A and B. We have discovered a series of pyrazoloquinazolines, some of which show greater than 1000-fold selectivity for Aurora B over Aurora A kinase activity, in recombinant enzyme assays. These compounds have been designed for parenteral administration and achieve high levels of solubility by virtue of their ability to be delivered as readily activated phosphate derivatives. The prodrugs are comprehensively converted to the des-phosphate form in vivo, and the active species have advantageous pharmacokinetic properties and safety pharmacology profiles. The compounds display striking in vivo activity, and compound 5 (AZD1152) has been selected for clinical evaluation and is currently in phase 1 clinical trials.
Assuntos
Antineoplásicos/síntese química , Organofosfatos/síntese química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirazóis/síntese química , Quinazolinas/síntese química , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Aurora Quinase A , Aurora Quinase B , Aurora Quinases , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Inibidores das Enzimas do Citocromo P-450 , Ensaios de Seleção de Medicamentos Antitumorais , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go/efeitos dos fármacos , Feminino , Histonas/metabolismo , Humanos , Masculino , Camundongos , Camundongos Nus , Organofosfatos/farmacocinética , Organofosfatos/farmacologia , Fosforilação , Pró-Fármacos/síntese química , Pró-Fármacos/farmacocinética , Pró-Fármacos/farmacologia , Ligação Proteica , Pirazóis/farmacocinética , Pirazóis/farmacologia , Quinazolinas/farmacocinética , Quinazolinas/farmacologia , Ratos , Proteínas Recombinantes/antagonistas & inibidores , Relação Estrutura-Atividade , Transplante HeterólogoRESUMO
Selective phosphoinositide 3-kinase (PI3K)/AKT/mTOR inhibitors are currently under evaluation in clinical studies. To identify tumor types that are sensitive to PI3K pathway inhibitors we screened compounds targeting PI3Kα/δ (AZD8835), PI3Kß/δ (AZD8186), AKT (AZD5363) and mTORC1/2 (AZD2014) against a cancer cell line panel (971 cell lines). There was an enrichment of hematological malignancies that were sensitive to AKT and mTOR inhibition, with the greatest degree of sensitivity observed in T-cell acute lymphoblastic leukemia (T-ALL). We found that all NOTCH mutant T-ALL cell lines were sensitive to AKT and mTORC1/2 inhibitors, with only partial sensitivity to agents that target the PI3K α, ß or δ isoforms. Induction of apoptosis only occurred following AKTi treatment in cell lines with PTEN protein loss and high levels of active AKT. In summary, we have demonstrated that T-ALL cell lines show differential sensitivity to inhibition at different nodes in the PI3K/AKT/mTOR pathway and inhibiting AKT or mTOR may have a therapeutic benefit in this disease setting.
Assuntos
Antineoplásicos/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Inibidores de Proteínas Quinases/farmacologia , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais/métodos , HumanosRESUMO
The phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signalling network is frequently de-regulated in breast cancer and has been shown to mediate resistance to anti-HER2 agents. Whilst constitutive activation of this pathway is emerging as a marker of sensitivity to various PI3K pathway inhibitors, activity of these agents in the clinic may be limited by the presence of feedback loops, leading to reactivation of receptor tyrosine kinases, such as HER2/HER3. To determine whether inhibition of HER2 could increase the efficacy of AZD5363, a novel AKT inhibitor, a panel of breast cancer cells was dosed with AZD5363 in combination with AZD8931, an inhibitor of EGFR/HER2/HER3 signalling. We show that the combined treatment resulted in synergistic growth inhibition and enhanced cell death, specifically in the HER2-amplified cell lines. Investigation of the mechanism by western blot analysis revealed that the addition of AZD8931 prevented the induction of HER2/HER3 phosphorylation induced by AZD5363 and resulted in concomitant inhibition of both the PI3K/AKT/mTOR and ERK signalling pathways and induction of apoptosis. Using the HCC1954 xenograft model, which is resistant to trastuzumab, we show that the combination of AZD5363 and AZD8931 is more efficacious than either agent alone, resulting in profound tumour regressions. We conclude that the activity of AZD5363 in HER2-amplified breast cancer cells is enhanced by the addition of AZD8931 and that dual targeting of AKT and EGFR/HER2/HER3 signalling is an attractive treatment option to be explored in the clinic.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Amplificação de Genes , Pirimidinas/administração & dosagem , Pirróis/administração & dosagem , Quinazolinas/administração & dosagem , Receptor ErbB-2/genética , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Feminino , Humanos , Células MCF-7 , Camundongos , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Pirimidinas/farmacologia , Pirróis/farmacologia , Quinazolinas/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
UNLABELLED: The phosphatidylinositol-4,5-bisphosphate 3-kinase/Akt (PI3K/Akt) pathway is a key pathway activated in castrate-resistant prostate cancer (CRPC). This preclinical study evaluates targeting of Akt with AZD5363 alone and in combination with enzalutamide (ENZ) to prevent and delay resistance. Our results demonstrate AZD5363 has significant proapoptotic, antiproliferative activity as monotherapy in ENZ-resistant cell lines in vitro and significantly decreased tumour growth in ENZ-resistant xenograft. The combination of AZD5363 and ENZ showed synergistic decreases in cell proliferation and induced cell-cycle arrest and apoptosis in prostate cancer cell lines LNCaP and C4-2. Notably, the combination of AZD5363 and ENZ resulted in an impressive regression of castrate-resistant LNCaP xenograft tumours without any recurrence demonstrated, whereas progression occurred with both monotherapies. Serum prostate-specific antigen (PSA) levels were also continuously suppressed, and nadir PSA levels were lower in the combination arm compared to ENZ alone. Combination AZD5363 and ENZ at time of castration similarly resulted in significant regression of tumours, with greater relative suppression of PSA compared to when administered to castrate-resistant xenografts. In summary, combination AZD5363 and ENZ significantly delays the development of ENZ resistance in preclinical models through synergistic increases in apoptosis and cell cycle arrest. Our results also suggest greater efficacy may be seen with earlier combination treatment. This study provides preclinical data to support evaluation of combination targeting of the PI3K/Akt pathway and the androgen-receptor axis in the clinic using AZD5363 and ENZ, respectively. PATIENT SUMMARY: Targeting of the Akt and androgen receptor pathways with AZD5363 and enzalutamide, respectively, significantly delayed the development of enzalutamide-resistant prostate cancer through increased apoptosis and cell cycle arrest. This preclinical synergy provides a strong rationale for clinical evaluation of this combination.
Assuntos
Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Feniltioidantoína/análogos & derivados , Próstata/efeitos dos fármacos , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Pirimidinas/farmacologia , Pirróis/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica , Apoptose/efeitos dos fármacos , Benzamidas , Linhagem Celular Tumoral , Humanos , Masculino , Nitrilas , Feniltioidantoína/farmacologia , Próstata/citologia , Neoplasias de Próstata Resistentes à Castração/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Androgênicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
AKT1(E17K) mutations occur at low frequency in a variety of solid tumors, including those of the breast and urinary bladder. Although this mutation has been shown to transform rodent cells in culture, it was found to be less oncogenic than PIK3CA mutations in breast epithelial cells. Moreover, the therapeutic potential of AKT inhibitors in human tumors with an endogenous AKT1(E17K) mutation is not known. Expression of exogenous copies of AKT1(E17K) in MCF10A breast epithelial cells increased phosphorylation of AKT and its substrates, induced colony formation in soft agar, and formation of lesions in the mammary fat pad of immunodeficient mice. These effects were inhibited by the allosteric and catalytic AKT inhibitors MK-2206 and AZD5363, respectively. Both AKT inhibitors caused highly significant growth inhibition of breast cancer explant models with AKT1(E17K) mutation. Furthermore, in a phase I clinical study, the catalytic Akt inhibitor AZD5363 induced partial responses in patients with breast and ovarian cancer with tumors containing AKT1(E17K) mutations. In MGH-U3 bladder cancer xenografts, which contain both AKT1(E17K) and FGFR3(Y373C) mutations, AZD5363 monotherapy did not significantly reduce tumor growth, but tumor regression was observed in combination with the FGFR inhibitor AZD4547. The data show that tumors with AKT1(E17K) mutations are rational therapeutic targets for AKT inhibitors, although combinations with other targeted agents may be required where activating oncogenic mutations of other proteins are present in the same tumor.
Assuntos
Mutação de Sentido Incorreto , Neoplasias/genética , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais/genética , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Doxiciclina/farmacologia , Feminino , Compostos Heterocíclicos com 3 Anéis/administração & dosagem , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Masculino , Camundongos Nus , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirimidinas/administração & dosagem , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Pirróis/administração & dosagem , Pirróis/farmacologia , Pirróis/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Loss of PTEN protein results in upregulation of the PI3K/AKT pathway, which appears dependent on the PI3Kß isoform. Inhibitors of PI3Kß have potential to reduce growth of tumors in which loss of PTEN drives tumor progression. We have developed a small-molecule inhibitor of PI3Kß and PI3Kδ (AZD8186) and assessed its antitumor activity across a panel of cell lines. We have then explored the antitumor effects as single agent and in combination with docetaxel in triple-negative breast (TNBC) and prostate cancer models. In vitro, AZD8186 inhibited growth of a range of cell lines. Sensitivity was associated with inhibition of the AKT pathway. Cells sensitive to AZD8186 (GI50 < 1 µmol/L) are enriched for, but not exclusively associated with, PTEN deficiency. In vivo, AZD8186 inhibits PI3K pathway biomarkers in prostate and TNBC tumors. Scheduling treatment with AZD8186 shows antitumor activity required only intermittent exposure, and that increased tumor control is achieved when AZD8186 is used in combination with docetaxel. AZD8186 is a potent inhibitor of PI3Kß with activity against PI3Kδ signaling, and has potential to reduce growth of tumors dependent on dysregulated PTEN for growth. Moreover, AZD8186 can be combined with docetaxel, a chemotherapy commonly used to treat advanced TBNC and prostate tumors. The ability to schedule AZD8186 and maintain efficacy offers opportunity to combine AZD8186 more effectively with other drugs.