RESUMEN
BH3 mimetics like venetoclax target prosurvival Bcl-2 family proteins and are important therapeutics in the treatment of hematological malignancies. We demonstrate that endogenous Bfl-1 expression can render preclinical lymphoma tumor models insensitive to Mcl-1 and Bcl-2 inhibitors. However, suppression of Bfl-1 alone was insufficient to fully induce apoptosis in Bfl-1-expressing lymphomas, highlighting the need for targeting additional prosurvival proteins in this context. Importantly, we demonstrated that cyclin-dependent kinase 9 (CDK9) inhibitors rapidly downregulate both Bfl-1 and Mcl-1, inducing apoptosis in BH3-mimetic-resistant lymphoma cell lines in vitro and driving in vivo tumor regressions in diffuse large B-cell lymphoma patient-derived xenograft models expressing Bfl-1. These data underscore the need to clinically develop CDK9 inhibitors, like AZD4573, for the treatment of lymphomas using Bfl-1 as a selection biomarker.
Asunto(s)
Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Quinasa 9 Dependiente de la Ciclina/antagonistas & inhibidores , Resistencia a Antineoplásicos/efectos de los fármacos , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Compuestos Macrocíclicos/farmacología , Terapia Molecular Dirigida , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Sulfonamidas/farmacología , Animales , Antineoplásicos/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéutico , Línea Celular Tumoral , Quinasa 9 Dependiente de la Ciclina/fisiología , Cicloheximida/farmacología , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Leupeptinas/farmacología , Compuestos Macrocíclicos/uso terapéutico , Ratones , Ratones Endogámicos NOD , Ratones SCID , Antígenos de Histocompatibilidad Menor/biosíntesis , Antígenos de Histocompatibilidad Menor/genética , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/biosíntesis , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Fragmentos de Péptidos/antagonistas & inhibidores , Piperazinas/farmacología , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/genética , Piridinas/farmacología , Sulfonamidas/uso terapéutico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
There has been huge progress in the discovery of targeted cancer therapies in recent years. However, even for the most successful and impactful cancer drugs which have been approved, both innate and acquired mechanisms of resistance are commonplace. These emerging mechanisms of resistance have been studied intensively, which has enabled drug discovery scientists to learn how it may be possible to overcome such resistance in subsequent generations of treatments. In some cases, novel drug candidates have been able to supersede previously approved agents; in other cases they have been used sequentially or in combinations with existing treatments. This review summarizes the current field in terms of the challenges and opportunities that cancer resistance presents to drug discovery scientists, with a focus on small molecule therapeutics. As part of this review, common themes and approaches have been identified which have been utilized to successfully target emerging mechanisms of resistance. This includes the increase in target potency and selectivity, alternative chemical scaffolds, change of mechanism of action (covalents, PROTACs), increases in blood-brain barrier permeability (BBBP), and the targeting of allosteric pockets. Finally, wider approaches are covered such as monoclonal antibodies (mAbs), bispecific antibodies, antibody drug conjugates (ADCs), and combination therapies.
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Anticuerpos Monoclonales/química , Antineoplásicos/química , Inmunoconjugados/química , Sitio Alostérico , Animales , Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica , Barrera Hematoencefálica/metabolismo , Diseño de Fármacos , Resistencia a Antineoplásicos , Humanos , Inmunoconjugados/farmacología , Modelos Moleculares , Medicina de Precisión , Unión Proteica , Conformación Proteica , Transducción de Señal , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Antineoplásicos/farmacología , Sistemas CRISPR-Cas , Desarrollo de Medicamentos/métodos , Ensayos de Selección de Medicamentos Antitumorales/métodos , Redes Reguladoras de Genes/efectos de los fármacos , Aptitud Genética/efectos de los fármacos , Mapas de Interacción de Proteínas/efectos de los fármacos , Antineoplásicos/toxicidad , Biomarcadores/metabolismo , Línea Celular Tumoral , Técnicas de Inactivación de Genes , Redes Reguladoras de Genes/genética , Aptitud Genética/genética , Genómica , Humanos , Modelos Lineales , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Preparaciones Farmacéuticas/metabolismo , Programas Informáticos , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Spleen tyrosine kinase (SYK) is a non-receptor cytosolic kinase. Due to its pivotal role in B cell receptor and Fc-receptor signaling, inhibition of SYK has been targeted in a variety of disease areas. Herein, we report the optimization of a series of potent and selective SYK inhibitors, focusing on improving metabolic stability, pharmacokinetics and hERG inhibition. As a result, we identified 30, which exhibited no hERG activity but unfortunately was poorly absorbed in rats and mice. We also identified a SYK chemical probe, 17, which exhibits excellent potency at SYK, and an adequate rodent PK profile to support in vivo efficacy/PD studies.
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Indazoles/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Quinasa Syk/antagonistas & inhibidores , Animales , Sitios de Unión , Células CACO-2 , Cristalografía por Rayos X , Canal de Potasio ERG1/antagonistas & inhibidores , Humanos , Indazoles/síntesis química , Indazoles/metabolismo , Indazoles/farmacocinética , Ratones , Microsomas Hepáticos/metabolismo , Estructura Molecular , Unión Proteica , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacocinética , Ratas Wistar , Relación Estructura-Actividad , Quinasa Syk/química , Quinasa Syk/metabolismoRESUMEN
The stability of the Wnt pathway transcription factor beta-catenin is tightly regulated by the multi-subunit destruction complex. Deregulated Wnt pathway activity has been implicated in many cancers, making this pathway an attractive target for anticancer therapies. However, the development of targeted Wnt pathway inhibitors has been hampered by the limited number of pathway components that are amenable to small molecule inhibition. Here, we used a chemical genetic screen to identify a small molecule, XAV939, which selectively inhibits beta-catenin-mediated transcription. XAV939 stimulates beta-catenin degradation by stabilizing axin, the concentration-limiting component of the destruction complex. Using a quantitative chemical proteomic approach, we discovered that XAV939 stabilizes axin by inhibiting the poly-ADP-ribosylating enzymes tankyrase 1 and tankyrase 2. Both tankyrase isoforms interact with a highly conserved domain of axin and stimulate its degradation through the ubiquitin-proteasome pathway. Thus, our study provides new mechanistic insights into the regulation of axin protein homeostasis and presents new avenues for targeted Wnt pathway therapies.
Asunto(s)
Proteínas Represoras/metabolismo , Transducción de Señal/efectos de los fármacos , Tanquirasas/antagonistas & inhibidores , Proteínas Wnt/antagonistas & inhibidores , Proteína Axina , División Celular/efectos de los fármacos , Línea Celular , Línea Celular Tumoral , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/metabolismo , Compuestos Heterocíclicos con 3 Anillos/farmacología , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Proteómica , Proteínas Represoras/química , Tanquirasas/metabolismo , Transcripción Genética/efectos de los fármacos , Ubiquitina/metabolismo , Ubiquitinación , Proteínas Wnt/metabolismo , beta Catenina/antagonistas & inhibidores , beta Catenina/metabolismoRESUMEN
PURPOSE: We evaluated the properties and activity of AZD9574, a blood-brain barrier (BBB) penetrant selective inhibitor of PARP1, and assessed its efficacy and safety alone and in combination with temozolomide (TMZ) in preclinical models. EXPERIMENTAL DESIGN: AZD9574 was interrogated in vitro for selectivity, PARylation inhibition, PARP-DNA trapping, the ability to cross the BBB, and the potential to inhibit cancer cell proliferation. In vivo efficacy was determined using subcutaneous as well as intracranial mouse xenograft models. Mouse, rat, and monkey were used to assess AZD9574 BBB penetration and rat models were used to evaluate potential hematotoxicity for AZD9574 monotherapy and the TMZ combination. RESULTS: AZD9574 demonstrated PARP1-selectivity in fluorescence anisotropy, PARylation, and PARP-DNA trapping assays and in vivo experiments demonstrated BBB penetration. AZD9574 showed potent single agent efficacy in preclinical models with homologous recombination repair deficiency in vitro and in vivo. In an O6-methylguanine-DNA methyltransferase (MGMT)-methylated orthotopic glioma model, AZD9574 in combination with TMZ was superior in extending the survival of tumor-bearing mice compared with TMZ alone. CONCLUSIONS: The combination of three key features-PARP1 selectivity, PARP1 trapping profile, and high central nervous system penetration in a single molecule-supports the development of AZD9574 as the best-in-class PARP inhibitor for the treatment of primary and secondary brain tumors. As documented by in vitro and in vivo studies, AZD9574 shows robust anticancer efficacy as a single agent as well as in combination with TMZ. AZD9574 is currently in a phase I trial (NCT05417594). See related commentary by Lynce and Lin, p. 1217.
Asunto(s)
Neoplasias Encefálicas , Glioma , Animales , Humanos , Ratones , Ratas , Antineoplásicos Alquilantes/farmacología , Barrera Hematoencefálica/metabolismo , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/patología , Línea Celular Tumoral , ADN , Glioma/tratamiento farmacológico , Glioma/patología , O(6)-Metilguanina-ADN Metiltransferasa/genética , Poli(ADP-Ribosa) Polimerasa-1 , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Temozolomida/farmacología , Temozolomida/uso terapéutico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica , Neoplasias , Humanos , Línea Celular Tumoral , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Ensayos de Selección de Medicamentos Antitumorales/métodos , Antineoplásicos/farmacología , Antineoplásicos/uso terapéuticoRESUMEN
Disregulated Wnt/beta-catenin signaling has been linked to various human diseases, including cancers. Inhibitors of oncogenic Wnt signaling are likely to have a therapeutic effect in cancers. LRP5 and LRP6 are closely related membrane coreceptors for Wnt proteins. Using a phage-display library, we identified anti-LRP6 antibodies that either inhibit or enhance Wnt signaling. Two classes of LRP6 antagonistic antibodies were discovered: one class specifically inhibits Wnt proteins represented by Wnt1, whereas the second class specifically inhibits Wnt proteins represented by Wnt3a. Epitope-mapping experiments indicated that Wnt1 class-specific antibodies bind to the first propeller and Wnt3a class-specific antibodies bind to the third propeller of LRP6, suggesting that Wnt1- and Wnt3a-class proteins interact with distinct LRP6 propeller domains. This conclusion is further supported by the structural functional analysis of LRP5/6 and the finding that the Wnt antagonist Sclerostin interacts with the first propeller of LRP5/6 and preferentially inhibits the Wnt1-class proteins. We also show that Wnt1 or Wnt3a class-specific anti-LRP6 antibodies specifically block growth of MMTV-Wnt1 or MMTV-Wnt3 xenografts in vivo. Therapeutic application of these antibodies could be limited without knowing the type of Wnt proteins expressed in cancers. This is further complicated by our finding that bivalent LRP6 antibodies sensitize cells to the nonblocked class of Wnt proteins. The generation of a biparatopic LRP6 antibody blocks both Wnt1- and Wnt3a-mediated signaling without showing agonistic activity. Our studies provide insights into Wnt-induced LRP5/6 activation and show the potential utility of LRP6 antibodies in Wnt-driven cancer.
Asunto(s)
Anticuerpos/farmacología , Proteínas Relacionadas con Receptor de LDL/inmunología , Ligandos , Proteínas Wnt/metabolismo , Animales , Anticuerpos/inmunología , Línea Celular , Transformación Celular Viral , Femenino , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Immunoblotting , Proteínas Relacionadas con Receptor de LDL/genética , Proteínas Relacionadas con Receptor de LDL/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad , Virus del Tumor Mamario del Ratón/genética , Ratones , Ratones Desnudos , Neoplasias Experimentales/patología , Neoplasias Experimentales/prevención & control , Unión Proteica/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Carga Tumoral/efectos de los fármacos , Proteínas Wnt/genética , Proteína Wnt1/genética , Proteína Wnt1/metabolismo , Proteína Wnt3 , Proteína Wnt3A , Ensayos Antitumor por Modelo de Xenoinjerto , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Antitumor immunity can be hampered by immunosuppressive mechanisms in the tumor microenvironment, including recruitment of arginase (ARG) expressing myeloid cells that deplete l-arginine essential for optimal T-cell and natural killer cell function. Hence, ARG inhibition can reverse immunosuppression enhancing antitumor immunity. We describe AZD0011, a novel peptidic boronic acid prodrug to deliver an orally available, highly potent, ARG inhibitor payload (AZD0011-PL). We demonstrate that AZD0011-PL is unable to permeate cells, suggesting that this compound will only inhibit extracellular ARG. In vivo, AZD0011 monotherapy leads to arginine increases, immune cell activation, and tumor growth inhibition in various syngeneic models. Antitumor responses increase when AZD0011 is combined with anti-PD-L1 treatment, correlating with increases in multiple tumor immune cell populations. We demonstrate a novel triple combination of AZD0011, anti-PD-L1, and anti-NKG2A, and combination benefits with type I IFN inducers, including polyI:C and radiotherapy. Our preclinical data demonstrate AZD0011's ability to reverse tumor immunosuppression and enhance immune stimulation and antitumor responses with diverse combination partners providing potential strategies to increase immuno-oncology therapies clinically.
Asunto(s)
Arginasa , Linfocitos T , Humanos , Línea Celular Tumoral , Terapia de Inmunosupresión , Tolerancia Inmunológica , Microambiente TumoralRESUMEN
PURPOSE: We hypothesized that inhibition and trapping of PARP1 alone would be sufficient to achieve antitumor activity. In particular, we aimed to achieve selectivity over PARP2, which has been shown to play a role in the survival of hematopoietic/stem progenitor cells in animal models. We developed AZD5305 with the aim of achieving improved clinical efficacy and wider therapeutic window. This next-generation PARP inhibitor (PARPi) could provide a paradigm shift in clinical outcomes achieved by first-generation PARPi, particularly in combination. EXPERIMENTAL DESIGN: AZD5305 was tested in vitro for PARylation inhibition, PARP-DNA trapping, and antiproliferative abilities. In vivo efficacy was determined in mouse xenograft and PDX models. The potential for hematologic toxicity was evaluated in rat models, as monotherapy and combination. RESULTS: AZD5305 is a highly potent and selective inhibitor of PARP1 with 500-fold selectivity for PARP1 over PARP2. AZD5305 inhibits growth in cells with deficiencies in DNA repair, with minimal/no effects in other cells. Unlike first-generation PARPi, AZD5305 has minimal effects on hematologic parameters in a rat pre-clinical model at predicted clinically efficacious exposures. Animal models treated with AZD5305 at doses ≥0.1 mg/kg once daily achieved greater depth of tumor regression compared to olaparib 100 mg/kg once daily, and longer duration of response. CONCLUSIONS: AZD5305 potently and selectively inhibits PARP1 resulting in excellent antiproliferative activity and unprecedented selectivity for DNA repair deficient versus proficient cells. These data confirm the hypothesis that targeting only PARP1 can retain the therapeutic benefit of nonselective PARPi, while reducing potential for hematotoxicity. AZD5305 is currently in phase I trials (NCT04644068).
Asunto(s)
Antineoplásicos , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Humanos , Ratones , Ratas , Animales , Línea Celular Tumoral , Ensayos Antitumor por Modelo de Xenoinjerto , Ftalazinas/farmacología , Poli(ADP-Ribosa) Polimerasa-1 , Antineoplásicos/farmacología , Reparación del ADNRESUMEN
BACKGROUND: The Regulatory T cell (Treg) lineage is defined by the transcription factor FOXP3, which controls immune-suppressive gene expression profiles. Tregs are often recruited in high frequencies to the tumor microenvironment where they can suppress antitumor immunity. We hypothesized that pharmacological inhibition of FOXP3 by systemically delivered, unformulated constrained ethyl-modified antisense oligonucleotides could modulate the activity of Tregs and augment antitumor immunity providing therapeutic benefit in cancer models and potentially in man. METHODS: We have identified murine Foxp3 antisense oligonucleotides (ASOs) and clinical candidate human FOXP3 ASO AZD8701. Pharmacology and biological effects of FOXP3 inhibitors on Treg function and antitumor immunity were tested in cultured Tregs and mouse syngeneic tumor models. Experiments were controlled by vehicle and non-targeting control ASO groups as well as by use of multiple independent FOXP3 ASOs. Statistical significance of biological effects was evaluated by one or two-way analysis of variance with multiple comparisons. RESULTS: AZD8701 demonstrated a dose-dependent knockdown of FOXP3 in primary Tregs, reduction of suppressive function and efficient target downregulation in humanized mice at clinically relevant doses. Surrogate murine FOXP3 ASO, which efficiently downregulated Foxp3 messenger RNA and protein levels in primary Tregs, reduced Treg suppressive function in immune suppression assays in vitro. FOXP3 ASO promoted more than 70% reduction in FOXP3 levels in Tregs in vitro and in vivo, strongly modulated Treg effector molecules (eg, ICOS, CTLA-4, CD25 and 4-1BB), and augmented CD8+ T cell activation and produced antitumor activity in syngeneic tumor models. The combination of FOXP3 ASOs with immune checkpoint blockade further enhanced antitumor efficacy. CONCLUSIONS: Antisense inhibitors of FOXP3 offer a promising novel cancer immunotherapy approach. AZD8701 is being developed clinically as a first-in-class FOXP3 inhibitor for the treatment of cancer currently in Ph1a/b clinical trial (NCT04504669).
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Neoplasias , Oligonucleótidos Antisentido , Animales , Modelos Animales de Enfermedad , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Humanos , Terapia de Inmunosupresión , Inmunoterapia , Ratones , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Linfocitos T Reguladores , Microambiente TumoralRESUMEN
Suppressive myeloid cells mediate resistance to immune checkpoint blockade. PI3Kγ inhibition can target suppressive macrophages, and enhance efficacy of immune checkpoint inhibitors. However, how PI3Kγ inhibitors function in different tumor microenvironments (TME) to activate specific immune cells is underexplored. The effect of the novel PI3Kγ inhibitor AZD3458 was assessed in preclinical models. AZD3458 enhanced antitumor activity of immune checkpoint inhibitors in 4T1, CT26, and MC38 syngeneic models, increasing CD8+ T-cell activation status. Immune and TME biomarker analysis of MC38 tumors revealed that AZD3458 monotherapy or combination treatment did not repolarize the phenotype of tumor-associated macrophage cells but induced gene signatures associated with LPS and type II INF activation. The activation biomarkers were present across tumor macrophages that appear phenotypically heterogenous. AZD3458 alone or in combination with PD-1-blocking antibodies promoted an increase in antigen-presenting (MHCII+) and cytotoxic (iNOS+)-activated macrophages, as well as dendritic cell activation. AZD3458 reduced IL-10 secretion and signaling in primary human macrophages and murine tumor-associated macrophages, but did not strongly regulate IL-12 as observed in other studies. Therefore, rather than polarizing tumor macrophages, PI3Kγ inhibition with AZD3458 promotes a cytotoxic switch of macrophages into antigen-presenting activated macrophages, resulting in CD8 T-cell-mediated antitumor activity with immune checkpoint inhibitors associated with tumor and peripheral immune activation.
Asunto(s)
Fosfatidilinositol 3-Quinasa Clase Ib/metabolismo , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Macrófagos/efectos de los fármacos , RatonesRESUMEN
Dual Bcl-2/Bcl-xL inhibitors are expected to deliver therapeutic benefit in many haematological and solid malignancies, however, their use is limited by tolerability issues. AZD4320, a potent dual Bcl-2/Bcl-xL inhibitor, has shown good efficacy however had dose limiting cardiovascular toxicity in preclinical species, coupled with challenging physicochemical properties, which prevented its clinical development. Here, we describe the design and development of AZD0466, a drug-dendrimer conjugate, where AZD4320 is chemically conjugated to a PEGylated poly-lysine dendrimer. Mathematical modelling was employed to determine the optimal release rate of the drug from the dendrimer for maximal therapeutic index in terms of preclinical anti-tumour efficacy and cardiovascular tolerability. The optimised candidate is shown to be efficacious and better tolerated in preclinical models compared with AZD4320 alone. The AZD4320-dendrimer conjugate (AZD0466) identified, through mathematical modelling, has resulted in an improved therapeutic index and thus enabled progression of this promising dual Bcl-2/Bcl-xL inhibitor into clinical development.
Asunto(s)
Antineoplásicos , Dendrímeros , Neoplasias/tratamiento farmacológico , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapéutico , Dendrímeros/síntesis química , Dendrímeros/química , Dendrímeros/farmacocinética , Dendrímeros/uso terapéutico , Perros , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Neoplasias/metabolismo , Neoplasias/patología , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Ratas , Ratas Wistar , Índice Terapéutico , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína bcl-X/antagonistas & inhibidoresRESUMEN
The RAS-regulated RAF-MEK1/2-ERK1/2 (RAS/MAPK) signaling pathway is a major driver in oncogenesis and is frequently dysregulated in human cancers, primarily by mutations in BRAF or RAS genes. The clinical benefit of inhibitors of this pathway as single agents has only been realized in BRAF-mutant melanoma, with limited effect of single-agent pathway inhibitors in KRAS-mutant tumors. Combined inhibition of multiple nodes within this pathway, such as MEK1/2 and ERK1/2, may be necessary to effectively suppress pathway signaling in KRAS-mutant tumors and achieve meaningful clinical benefit. Here, we report the discovery and characterization of AZD0364, a novel, reversible, ATP-competitive ERK1/2 inhibitor with high potency and kinase selectivity. In vitro, AZD0364 treatment resulted in inhibition of proximal and distal biomarkers and reduced proliferation in sensitive BRAF-mutant and KRAS-mutant cell lines. In multiple in vivo xenograft models, AZD0364 showed dose- and time-dependent modulation of ERK1/2-dependent signaling biomarkers resulting in tumor regression in sensitive BRAF- and KRAS-mutant xenografts. We demonstrate that AZD0364 in combination with the MEK1/2 inhibitor, selumetinib (AZD6244 and ARRY142886), enhances efficacy in KRAS-mutant preclinical models that are moderately sensitive or resistant to MEK1/2 inhibition. This combination results in deeper and more durable suppression of the RAS/MAPK signaling pathway that is not achievable with single-agent treatment. The AZD0364 and selumetinib combination also results in significant tumor regressions in multiple KRAS-mutant xenograft models. The combination of ERK1/2 and MEK1/2 inhibition thereby represents a viable clinical approach to target KRAS-mutant tumors.
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Bencimidazoles/uso terapéutico , Imidazoles/uso terapéutico , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Pirazinas/uso terapéutico , Pirimidinas/uso terapéutico , Animales , Bencimidazoles/farmacología , Modelos Animales de Enfermedad , Humanos , Imidazoles/farmacología , Ratones , Ratones Desnudos , Pirazinas/farmacología , Pirimidinas/farmacologíaRESUMEN
Poly-ADP-ribose-polymerase (PARP) inhibitors have achieved regulatory approval in oncology for homologous recombination repair deficient tumors including BRCA mutation. However, some have failed in combination with first-line chemotherapies, usually due to overlapping hematological toxicities. Currently approved PARP inhibitors lack selectivity for PARP1 over PARP2 and some other 16 PARP family members, and we hypothesized that this could contribute to toxicity. Recent literature has demonstrated that PARP1 inhibition and PARP1-DNA trapping are key for driving efficacy in a BRCA mutant background. Herein, we describe the structure- and property-based design of 25 (AZD5305), a potent and selective PARP1 inhibitor and PARP1-DNA trapper with excellent in vivo efficacy in a BRCA mutant HBCx-17 PDX model. Compound 25 is highly selective for PARP1 over other PARP family members, with good secondary pharmacology and physicochemical properties and excellent pharmacokinetics in preclinical species, with reduced effects on human bone marrow progenitor cells in vitro.
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ADN , Poli(ADP-Ribosa) Polimerasa-1 , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Poli(ADP-Ribosa) Polimerasas , Humanos , Cristalografía por Rayos X , ADN/química , Poli(ADP-Ribosa) Polimerasa-1/antagonistas & inhibidores , Inhibidores de Poli(ADP-Ribosa) Polimerasas/química , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Especificidad por SustratoRESUMEN
Panobinostat (LBH589) is a highly potent deacetylase inhibitor that has demonstrated clinical efficacy in patients with advanced cutaneous T-cell lymphoma (CTCL). To gain a better understanding of the compound activity in this tumor type, we investigated the cellular and molecular effects of panobinostat using both in vitro and in vivo models of CTCL. All 4 tested CTCL cell lines exhibited very high sensitivity to panobinostat-induced growth inhibition. However, only 2 of 4 lines exhibited significant response to the cytotoxic activity of panobinostat. In a CTCL xenograft mouse tumor model, panobinostat treatment resulted in complete tumor regression. The difference in cell sensitivity to panobinostat-induced death enabled us to further investigate potential mechanisms responsible for tumor sensitivity or resistance. In CTCL cell lines that were insensitive to panobinostat-induced apoptosis, constitutively activated NF-kappaB and high levels of Bcl-2 were observed. Inhibition of Bcl-2 sensitized cells to the cytotoxic activity of panobinostat. Conversely, knockdown of Bax diminished the CTCL cell sensitivity. Interestingly, panobinostat could induce cytotoxicity in vorinostat-resistant CTCL cells by downregulating phosphorylated STAT3 and STAT5 proteins. These studies suggest distinct mechanisms responsible for resistance to different deacetylase inhibitors. We show that the intrinsic apoptotic signaling plays an essential role in mediating panobinostat anticancer activity. Moreover, cancer cell sensitivity to panobinostat treatment may be further improved by combination with inhibition of anti-apoptotic factors. These data provide preclinical support that panobinostat, as a single agent or in combination with other anticancer agents, is a promising therapy for CTCL.
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Antineoplásicos/farmacología , Inhibidores de Histona Desacetilasas/farmacología , Ácidos Hidroxámicos/uso terapéutico , Linfoma Cutáneo de Células T/tratamiento farmacológico , Neoplasias Cutáneas/tratamiento farmacológico , Animales , Antineoplásicos/uso terapéutico , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos , Femenino , Inhibidores de Histona Desacetilasas/uso terapéutico , Humanos , Ácidos Hidroxámicos/farmacología , Indoles , Ratones , Ratones SCID , Panobinostat , Interferencia de ARN , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína X Asociada a bcl-2/genéticaRESUMEN
Accumulation of extracellular adenosine within the microenvironment is a strategy exploited by tumors to escape detection by the immune system. Adenosine signaling through the adenosine 2A receptor (A2AR) on immune cells elicits a range of immunosuppressive effects which promote tumor growth and limit the efficacy of immune checkpoint inhibitors. Preclinical data with A2AR inhibitors have demonstrated tumor regressions in mouse models by rescuing T cell function; however, the mechanism and role on other immune cells has not been fully elucidated. METHODS: We report here the development of a small molecule A2AR inhibitor including characterization of binding and inhibition of A2AR function with varying amounts of a stable version of adenosine. Functional activity was tested in both mouse and human T cells and dendritic cells (DCs) in in vitro assays to understand the intrinsic role on each cell type. The role of adenosine and A2AR inhibition was tested in DC differentiation assays as well as co-culture assays to access the cross-priming function of DCs. Syngeneic models were used to assess tumor growth alone and in combination with alphaprogrammed death-ligand 1 (αPD-L1). Immunophenotyping by flow cytometry was performed to examine global immune cell changes upon A2AR inhibition. RESULTS: We provide the first report of AZD4635, a novel small molecule A2AR antagonist which inhibits downstream signaling and increases T cell function as well as a novel mechanism of enhancing antigen presentation by CD103+ DCs. The role of antigen presentation by DCs, particularly CD103+ DCs, is critical to drive antitumor immunity providing rational to combine a priming agent AZD4635 with check point blockade. We find adenosine impairs the maturation and antigen presentation function of CD103+ DCs. We show in multiple syngeneic mouse tumor models that treatment of AZD4635 alone and in combination with αPD-L1 led to decreased tumor volume correlating with enhanced CD103+ function and T cell response. We extend these studies into human DCs to show that adenosine promotes a tolerogenic phenotype that can be reversed with AZD4635 restoring antigen-specific T cell activation. Our results support the novel role of adenosine signaling as an intrinsic negative regulator of CD103+ DCs maturation and priming. We show that potent inhibition of A2AR with AZD4635 reduces tumor burden and enhances antitumor immunity. This unique mechanism of action in CD103+ DCs may contribute to clinical responses as AZD4635 is being evaluated in clinical trials with IMFINZI (durvalumab, αPD-L1) in patients with solid malignancies. CONCLUSION: We provide evidence implicating suppression of adaptive and innate immunity by adenosine as a mechanism for immune evasion by tumors. Inhibition of adenosine signaling through selective small molecule inhibition of A2AR using AZD4635 restores T cell function via an internal mechanism as well as tumor antigen cross-presentation by CD103+ DCs resulting in antitumor immunity.
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Antígenos CD/metabolismo , Antineoplásicos Inmunológicos/uso terapéutico , Células Dendríticas/inmunología , Cadenas alfa de Integrinas/metabolismo , Neoplasias/inmunología , Receptor de Adenosina A2A/metabolismo , Antineoplásicos Inmunológicos/farmacología , Línea Celular Tumoral , Femenino , Humanos , Masculino , Transducción de SeñalRESUMEN
Gastrointestinal stromal tumor (GIST) is the most common human sarcoma driven by mutations in KIT or platelet-derived growth factor α (PDGFRα). Although first-line treatment, imatinib, has revolutionized GIST treatment, drug resistance due to acquisition of secondary KIT/PDGFRα mutations develops in a majority of patients. Second- and third-line treatments, sunitinib and regorafenib, lack activity against a plethora of mutations in KIT/PDGFRα in GIST, with median time to disease progression of 4 to 6 months and inhibition of vascular endothelial growth factor receptor 2 (VEGFR2) causing high-grade hypertension. Patients with GIST have an unmet need for a well-tolerated drug that robustly inhibits a range of KIT/PDGFRα mutations. Here, we report the discovery and pharmacological characterization of AZD3229, a potent and selective small-molecule inhibitor of KIT and PDGFRα designed to inhibit a broad range of primary and imatinib-resistant secondary mutations seen in GIST. In engineered and GIST-derived cell lines, AZD3229 is 15 to 60 times more potent than imatinib in inhibiting KIT primary mutations and has low nanomolar activity against a wide spectrum of secondary mutations. AZD3229 causes durable inhibition of KIT signaling in patient-derived xenograft (PDX) models of GIST, leading to tumor regressions at doses that showed no changes in arterial blood pressure (BP) in rat telemetry studies. AZD3229 has a superior potency and selectivity profile to standard of care (SoC) agents-imatinib, sunitinib, and regorafenib, as well as investigational agents, avapritinib (BLU-285) and ripretinib (DCC-2618). AZD3229 has the potential to be a best-in-class inhibitor for clinically relevant KIT/PDGFRα mutations in GIST.
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Antineoplásicos , Tumores del Estroma Gastrointestinal , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Resistencia a Antineoplásicos , Tumores del Estroma Gastrointestinal/tratamiento farmacológico , Tumores del Estroma Gastrointestinal/genética , Humanos , Mutación , Naftiridinas , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas c-kit/genética , Pirazoles , Pirroles , Ratas , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Triazinas , Urea/análogos & derivados , Factor A de Crecimiento Endotelial VascularRESUMEN
Deregulation of the PRC2 complex, comprised of the core subunits EZH2, SUZ12, and EED, drives aberrant hypermethylation of H3K27 and tumorigenicity of many cancers. Although inhibitors of EZH2 have shown promising clinical activity, preclinical data suggest that resistance can be acquired through secondary mutations in EZH2 that abrogate drug target engagement. To address these limitations, we have designed several hetero-bifunctional PROTACs (proteolysis-targeting chimera) to efficiently target EED for elimination. Our PROTACs bind to EED (pKD â¼ 9.0) and promote ternary complex formation with the E3 ubiquitin ligase. The PROTACs potently inhibit PRC2 enzyme activity (pIC50 â¼ 8.1) and induce rapid degradation of not only EED but also EZH2 and SUZ12 within the PRC2 complex. Furthermore, the PROTACs selectively inhibit proliferation of PRC2-dependent cancer cells (half maximal growth inhibition [GI50] = 49-58 nM). In summary, our data demonstrate a therapeutic modality to target PRC2-dependent cancer through a PROTAC-mediated degradation mechanism.
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Complejo Represivo Polycomb 2/metabolismo , Proteolisis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Estructura Molecular , Complejo Represivo Polycomb 2/antagonistas & inhibidores , Relación Estructura-ActividadRESUMEN
PURPOSE: Cyclin-dependent kinase 9 (CDK9) is a transcriptional regulator and potential therapeutic target for many cancers. Multiple nonselective CDK9 inhibitors have progressed clinically but were limited by a narrow therapeutic window. This work describes a novel, potent, and highly selective CDK9 inhibitor, AZD4573. EXPERIMENTAL DESIGN: The antitumor activity of AZD4573 was determined across broad cancer cell line panels in vitro as well as cell line- and patient-derived xenograft models in vivo. Multiple approaches, including integrated transcriptomic and proteomic analyses, loss-of-function pathway interrogation, and pharmacologic comparisons, were employed to further understand the major mechanism driving AZD4573 activity and to establish an exposure/effect relationship. RESULTS: AZD4573 is a highly selective and potent CDK9 inhibitor. It demonstrated rapid induction of apoptosis and subsequent cell death broadly across hematologic cancer models in vitro, and MCL-1 depletion in a dose- and time-dependent manner was identified as a major mechanism through which AZD4573 induces cell death in tumor cells. This pharmacodynamic (PD) response was also observed in vivo, which led to regressions in both subcutaneous tumor xenografts and disseminated models at tolerated doses both as monotherapy or in combination with venetoclax. This understanding of the mechanism, exposure, and antitumor activity of AZD4573 facilitated development of a robust pharmacokinetic/PD/efficacy model used to inform the clinical trial design. CONCLUSIONS: Selective targeting of CDK9 enables the indirect inhibition of MCL-1, providing a therapeutic option for MCL-1-dependent diseases. Accordingly, AZD4573 is currently being evaluated in a phase I clinical trial for patients with hematologic malignancies (clinicaltrials.gov identifier: NCT03263637).See related commentary by Alcon et al., p. 761.