RESUMEN
Arp2/3 complex nucleates branched actin filaments that drive processes like endocytosis and lamellipodial protrusion. WISH/DIP/SPIN90 (WDS) proteins form a class of Arp2/3 complex activators or nucleation promoting factors (NPFs) that, unlike WASP family NPFs, activate Arp2/3 complex without requiring preformed actin filaments. Therefore, activation of Arp2/3 complex by WDS proteins is thought to produce the initial actin filaments that seed branching nucleation by WASP-bound Arp2/3 complexes. However, whether activation of Arp2/3 complex by WDS proteins is important for the initiation of branched actin assembly in cells has not been directly tested. Here, we used structure-based point mutations of the Schizosaccharomyces pombe WDS protein Dip1 to test the importance of its Arp2/3-activating activity in cells. Six of thirteen Dip1 mutants caused severe defects in Arp2/3 complex activation in vitro, and we found a strong correlation between the ability of mutants to activate Arp2/3 complex and to rescue endocytic actin assembly defects caused by deleting Dip1. These data support a model in which Dip1 activates Arp2/3 complex to produce actin filaments that initiate branched actin assembly at endocytic sites. Dip1 mutants that synergized with WASP in activating Arp2/3 complex in vitro showed milder defects in cells compared to those that did not, suggesting that in cells the two NPFs may coactivate Arp2/3 complex to initiate actin assembly. Finally, the mutational data reveal important complementary electrostatic contacts at the Dip1-Arp2/3 complex interface and corroborate the previously proposed wedge model, which describes how Dip1 binding triggers structural changes that activate Arp2/3 complex.
Asunto(s)
Citoesqueleto de Actina , Complejo 2-3 Proteico Relacionado con la Actina , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Citoesqueleto de Actina/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/genética , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Actinas/metabolismo , Seudópodos/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMEN
Cyclin-dependent kinases 4 and 6 (CDK4/6) are fundamental drivers of the cell cycle and are required for the initiation and progression of various malignancies. Pharmacological inhibitors of CDK4/6 have shown significant activity against several solid tumours. Their primary mechanism of action is thought to be the inhibition of phosphorylation of the retinoblastoma tumour suppressor, inducing G1 cell cycle arrest in tumour cells. Here we use mouse models of breast carcinoma and other solid tumours to show that selective CDK4/6 inhibitors not only induce tumour cell cycle arrest, but also promote anti-tumour immunity. We confirm this phenomenon through transcriptomic analysis of serial biopsies from a clinical trial of CDK4/6 inhibitor treatment for breast cancer. The enhanced anti-tumour immune response has two underpinnings. First, CDK4/6 inhibitors activate tumour cell expression of endogenous retroviral elements, thus increasing intracellular levels of double-stranded RNA. This in turn stimulates production of type III interferons and hence enhances tumour antigen presentation. Second, CDK4/6 inhibitors markedly suppress the proliferation of regulatory T cells. Mechanistically, the effects of CDK4/6 inhibitors both on tumour cells and on regulatory T cells are associated with reduced activity of the E2F target, DNA methyltransferase 1. Ultimately, these events promote cytotoxic T-cell-mediated clearance of tumour cells, which is further enhanced by the addition of immune checkpoint blockade. Our findings indicate that CDK4/6 inhibitors increase tumour immunogenicity and provide a rationale for new combination regimens comprising CDK4/6 inhibitors and immunotherapies as anti-cancer treatment.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/inmunología , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Animales , Presentación de Antígeno/efectos de los fármacos , Presentación de Antígeno/inmunología , Mimetismo Biológico/efectos de los fármacos , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Femenino , Humanos , Interferones/metabolismo , Ratones , Fosforilación/efectos de los fármacos , ARN Bicatenario/genética , Proteínas Represoras/biosíntesis , Transducción de Señal/efectos de los fármacos , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Transcriptoma , Virus/efectos de los fármacos , Virus/genética , Virus/inmunologíaRESUMEN
Pharmacological inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6) are now an established standard of care for patients with advanced hormone receptor-positive breast cancer. The canonical mechanism underlying CDK4/6 inhibitor activity is the suppression of phosphorylation of the retinoblastoma tumor suppressor protein, which serves to prevent cancer cell proliferation. Recent data suggest that these agents induce other diverse effects within both tumor and stromal compartments, which serve to explain aspects of their clinical activity. Here, we review these phenomena and discuss how they might be leveraged in the development of novel CDK4/6 inhibitor-containing combination treatments. We also briefly review the various known mechanisms of acquired resistance in the clinical setting.
Asunto(s)
Neoplasias de la Mama , Neoplasias de la Mama/patología , Quinasa 4 Dependiente de la Ciclina , Quinasa 6 Dependiente de la Ciclina , Femenino , Humanos , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéuticoRESUMEN
BACKGROUND: Heterotopic ossification (HO) may occur after musculoskeletal trauma, traumatic brain injury, and total joint arthroplasty. As such, HO is a compelling clinical concern in both military and civilian medicine. A possible etiology of HO involves dysregulated signals in the bone morphogenetic protein osteogenic cascade. Contemporary treatment options for HO (ie, nonsteroidal antiinflammatory drugs and radiation therapy) have adverse effects associated with their use and are not biologically engineered to abrogate the molecular mechanisms that govern osteogenic differentiation. QUESTIONS/PURPOSES: We hypothesized that (1) nanogel-mediated short interfering RNA (siRNA) delivery against Runt-related transcription factor 2 (Runx2) and osterix (Osx) genes will decrease messenger RNA expression; (2) inhibit activity of the osteogenic marker alkaline phosphatase (ALP); and (3) inhibit hydroxyapatite (HA) deposition in osteoblast cell cultures. METHODS: Nanogel nanostructured polymers delivered siRNA in 48-hour treatment cycles against master osteogenic regulators, Runx2 and Osx, in murine calvarial preosteoblasts (MC3T3-E1.4) stimulated for osteogenic differentiation by recombinant human bone morphogenetic protein (rhBMP-2). The efficacy of RNA interference (RNAi) therapeutics was determined by quantitation of messenger RNA knockdown (by quantitative reverse transcription-polymerase chain reaction), downstream protein knockdown (determined ALP enzymatic activity assay), and HA deposition (determined by OsteoImage™ assay). RESULTS: Gene expression assays demonstrated that nanogel-based RNAi treatments at 1:1 and 5:1 nanogel:short interfering RNA weight ratios reduced Runx2 expression by 48.59% ± 19.53% (p < 0.001) and 43.22% ± 18.01% (both p < 0.001). The same 1:1 and 5:1 treatments against both Runx2 and Osx reduced expression of Osx by 51.65% ± 10.85% and 47.65% ± 9.80% (both p < 0.001). Moreover, repeated 48-hour RNAi treatment cycles against Runx2 and Osx rhBMP-2 administration reduced ALP activity after 4 and 7 days. ALP reductions after 4 days in culture by nanogel 5:1 and 10:1 RNAi treatments were 32.4% ± 12.0% and 33.6% ± 13.8% (both p < 0.001). After 7 days in culture, nanogel 1:1 and 5:1 RNAi treatments produced 35.9% ± 14.0% and 47.7% ± 3.2% reductions in ALP activity. Osteoblast mineralization data after 21 days suggested that nanogel 1:1, 5:1, and 10:1 RNAi treatments decreased mineralization (ie, HA deposition) from cultures treated only with rhBMP-2 (p < 0.001). However, despite RNAi attack on Runx2 and Osx, HA deposition levels remained greater than non-rhBMP-2-treated cell cultures. CONCLUSIONS: Although mRNA and protein knockdown were confirmed as a result of RNAi treatments against Runx2 and Osx, complete elimination of mineralization processes was not achieved. RNAi targeting mid- and late-stage osteoblast differentiation markers such as ALP, osteocalcin, osteopontin, and bone sialoprotein) may produce the desired RNAi-nanogel nanostructured polymer HO prophylaxis. CLINICAL RELEVANCE: Successful HO prophylaxis should target and silence osteogenic markers critical for heterotopic bone formation processes. The identification of such markers, beyond RUNX2 and OSX, may enhance the effectiveness of RNAi prophylaxes for HO.
Asunto(s)
Calcificación Fisiológica , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Nanoestructuras , Osteoblastos/metabolismo , Ácidos Polimetacrílicos/química , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Factores de Transcripción/metabolismo , Transfección/métodos , Células 3T3 , Fosfatasa Alcalina/metabolismo , Animales , Biomarcadores/metabolismo , Proteína Morfogenética Ósea 2/farmacología , Calcificación Fisiológica/efectos de los fármacos , Cationes , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Regulación hacia Abajo , Durapatita/metabolismo , Geles , Ratones , Osteoblastos/efectos de los fármacos , ARN Interferente Pequeño/genética , Factor de Transcripción Sp7 , Factores de Tiempo , Factores de Transcripción/genéticaRESUMEN
Cyclin-dependent kinase 2 (CDK2) is thought to play an important role in driving proliferation of certain cancers, including those harboring CCNE1 amplification and breast cancers that have acquired resistance to CDK4/6 inhibitors (CDK4/6i). The precise impact of pharmacologic inhibition of CDK2 is not known due to the lack of selective CDK2 inhibitors. Here we describe INX-315, a novel and potent CDK2 inhibitor with high selectivity over other CDK family members. Using cell-based assays, patient-derived xenografts (PDX), and transgenic mouse models, we show that INX-315 (i) promotes retinoblastoma protein hypophosphorylation and therapy-induced senescence (TIS) in CCNE1-amplified tumors, leading to durable control of tumor growth; (ii) overcomes breast cancer resistance to CDK4/6i, restoring cell cycle control while reinstating the chromatin architecture of CDK4/6i-induced TIS; and (iii) delays the onset of CDK4/6i resistance in breast cancer by driving deeper suppression of E2F targets. Our results support the clinical development of selective CDK2 inhibitors. SIGNIFICANCE: INX-315 is a novel, selective inhibitor of CDK2. Our preclinical studies demonstrate activity for INX-315 in both CCNE1-amplified cancers and CDK4/6i-resistant breast cancer. In each case, CDK2 inhibition induces cell cycle arrest and a phenotype resembling cellular senescence. Our data support the development of selective CDK2 inhibitors in clinical trials. See related commentary by Watts and Spencer, p. 386. This article is featured in Selected Articles from This Issue, p. 384.
Asunto(s)
Neoplasias de la Mama , Animales , Ratones , Humanos , Femenino , Quinasa 2 Dependiente de la Ciclina/genética , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Puntos de Control del Ciclo Celular , Senescencia Celular , Cromatina , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina , Ratones TransgénicosRESUMEN
Pharmacologic inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6) were designed to induce cancer cell cycle arrest. Recent studies have suggested that these agents also exert other effects, influencing cancer cell immunogenicity, apoptotic responses, and differentiation. Using cell-based and mouse models of breast cancer together with clinical specimens, we show that CDK4/6 inhibitors induce remodeling of cancer cell chromatin characterized by widespread enhancer activation, and that this explains many of these effects. The newly activated enhancers include classical super-enhancers that drive luminal differentiation and apoptotic evasion, as well as a set of enhancers overlying endogenous retroviral elements that is enriched for proximity to interferon-driven genes. Mechanistically, CDK4/6 inhibition increases the level of several Activator Protein-1 (AP-1) transcription factor proteins, which are in turn implicated in the activity of many of the new enhancers. Our findings offer insights into CDK4/6 pathway biology and should inform the future development of CDK4/6 inhibitors.
Asunto(s)
Neoplasias de la Mama , Factor de Transcripción AP-1 , Animales , Neoplasias de la Mama/tratamiento farmacológico , Puntos de Control del Ciclo Celular , Quinasa 4 Dependiente de la Ciclina/genética , Femenino , Genes cdc , Humanos , Ratones , Factor de Transcripción AP-1/genéticaRESUMEN
Using transgenic mouse models, cell line-based functional studies, and clinical specimens, we show that cyclin D1/CDK4 mediate resistance to targeted therapy for HER2-positive breast cancer. This is overcome using CDK4/6 inhibitors. Inhibition of CDK4/6 not only suppresses Rb phosphorylation, but also reduces TSC2 phosphorylation and thus partially attenuates mTORC1 activity. This relieves feedback inhibition of upstream EGFR family kinases, resensitizing tumors to EGFR/HER2 blockade. Consequently, dual inhibition of EGFR/HER2 and CDK4/6 invokes a more potent suppression of TSC2 phosphorylation and hence mTORC1/S6K/S6RP activity. The suppression of both Rb and S6RP enhances G1 arrest and a phenotype resembling cellular senescence. In vivo, CDK4/6 inhibitors sensitize patient-derived xenograft tumors to HER2-targeted therapies and delay tumor recurrence in a transgenic model of HER2-positive breast cancer.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Resistencia a Antineoplásicos/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Receptor ErbB-2/metabolismo , Animales , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 6 Dependiente de la Ciclina/metabolismo , Modelos Animales de Enfermedad , Receptores ErbB/metabolismo , Femenino , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Desnudos , Ratones Transgénicos , Complejos Multiproteicos/metabolismo , Recurrencia Local de Neoplasia/mortalidad , Fosforilación/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
The endogenous RNA interference (RNAi) pathway enables control of pathologies caused by the dysregulation of proteins. Several biological molecules are active in RNAi including short interfering ribonucleic acid (siRNA). The effective utilization of siRNA as a therapeutic agent has been marked with distinct challenges, namely in intracellular delivery and achieving a sufficient dosage to affect protein expression. A delivery strategy we have developed to improve safety and efficacy of siRNA includes complexing siRNA with nanostructured polymers delivery systems (NSPs). These NSPs are synthesized via atom transfer radical polymerization (ATRP) and combine several important advances in polymer architecture for siRNA delivery. This includes shielding the cationic charge of the NSP with a poly(ethylene glycol) (PEG) shell to promote cell viability in MC3T3-E1.4 pre-osteoblasts, and minimize the inflammatory response in a C57BL/6 mouse model. In our gene knockdown experiments targeting glyceraldehyde 3-phosphate dehydrogenase Gapdh expression, star polymer and nanogel polyplexes suppressed Gapdh mRNA to levels comparable to cells treated with Lipofectamine RNAiMAX lipoplexes.