RESUMEN
Increasing use of covalent and noncovalent inhibitors of Bruton's tyrosine kinase (BTK) has elucidated a series of acquired drug-resistant BTK mutations in patients with B cell malignancies. Here we identify inhibitor resistance mutations in BTK with distinct enzymatic activities, including some that impair BTK enzymatic activity while imparting novel protein-protein interactions that sustain B cell receptor (BCR) signaling. Furthermore, we describe a clinical-stage BTK and IKZF1/3 degrader, NX-2127, that can bind and proteasomally degrade each mutant BTK proteoform, resulting in potent blockade of BCR signaling. Treatment of chronic lymphocytic leukemia with NX-2127 achieves >80% degradation of BTK in patients and demonstrates proof-of-concept therapeutic benefit. These data reveal an oncogenic scaffold function of mutant BTK that confers resistance across clinically approved BTK inhibitors but is overcome by BTK degradation in patients.
Asunto(s)
Agammaglobulinemia Tirosina Quinasa , Resistencia a Antineoplásicos , Factor de Transcripción Ikaros , Leucemia Linfocítica Crónica de Células B , Inhibidores de Proteínas Quinasas , Proteolisis , Humanos , Agammaglobulinemia Tirosina Quinasa/genética , Agammaglobulinemia Tirosina Quinasa/metabolismo , Factor de Transcripción Ikaros/metabolismo , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/genética , Mutación , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Transducción de Señal , Proteolisis/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacosRESUMEN
Bruton's tyrosine kinase (BTK), a member of the TEC family of kinases, is an essential effector of B-cell receptor (BCR) signaling. Chronic activation of BTK-mediated BCR signaling is a hallmark of many hematological malignancies, which makes it an attractive therapeutic target. Pharmacological inhibition of BTK enzymatic function is now a well-proven strategy for the treatment of patients with these malignancies. We report the discovery and characterization of NX-2127, a BTK degrader with concomitant immunomodulatory activity. By design, NX-2127 mediates the degradation of transcription factors IKZF1 and IKZF3 through molecular glue interactions with the cereblon E3 ubiquitin ligase complex. NX-2127 degrades common BTK resistance mutants, including BTKC481S. NX-2127 is orally bioavailable, exhibits in vivo degradation across species, and demonstrates efficacy in preclinical oncology models. NX-2127 has advanced into first-in-human clinical trials and achieves deep and sustained degradation of BTK following daily oral dosing at 100 mg.
Asunto(s)
Inhibidores de Proteínas Quinasas , Proteínas Tirosina Quinasas , Humanos , Agammaglobulinemia Tirosina Quinasa , Inhibidores de Proteínas Quinasas/efectos adversos , Transducción de SeñalRESUMEN
There is great interest in understanding the cellular mechanisms controlling autophagy, a tightly regulated catabolic and stress-response pathway. Prior work has uncovered links between autophagy and the Golgi reassembly stacking protein of 55â kDa (GRASP55), but their precise interrelationship remains unclear. Intriguingly, both autophagy and GRASP55 have been functionally and spatially linked to the endoplasmic reticulum (ER)---Golgi interface, broaching this compartment as a site where GRASP55 and autophagy may intersect. Here, we uncover that loss of GRASP55 enhances LC3 puncta formation, indicating that GRASP55 restricts autophagosome formation. Additionally, using proximity-dependent biotinylation, we identify a GRASP55 proximal interactome highly associated with the ER-Golgi interface. Both nutrient starvation and loss of GRASP55 are associated with coalescence of early secretory pathway markers. In light of these findings, we propose that GRASP55 regulates spatial organization of the ER-Golgi interface, which suppresses early autophagosome formation.
Asunto(s)
Autofagosomas/genética , Autofagia/genética , Retículo Endoplásmico/metabolismo , Proteínas de la Matriz de Golgi/metabolismo , Transducción de Señal/genética , HumanosRESUMEN
Traditionally viewed as an autodigestive pathway, autophagy also facilitates cellular secretion; however, the mechanisms underlying these processes remain unclear. Here, we demonstrate that components of the autophagy machinery specify secretion within extracellular vesicles (EVs). Using a proximity-dependent biotinylation proteomics strategy, we identify 200 putative targets of LC3-dependent secretion. This secretome consists of a highly interconnected network enriched in RNA-binding proteins (RBPs) and EV cargoes. Proteomic and RNA profiling of EVs identifies diverse RBPs and small non-coding RNAs requiring the LC3-conjugation machinery for packaging and secretion. Focusing on two RBPs, heterogeneous nuclear ribonucleoprotein K (HNRNPK) and scaffold-attachment factor B (SAFB), we demonstrate that these proteins interact with LC3 and are secreted within EVs enriched with lipidated LC3. Furthermore, their secretion requires the LC3-conjugation machinery, neutral sphingomyelinase 2 (nSMase2) and LC3-dependent recruitment of factor associated with nSMase2 activity (FAN). Hence, the LC3-conjugation pathway controls EV cargo loading and secretion.
Asunto(s)
Autofagosomas/metabolismo , Autofagia/genética , Vesículas Extracelulares/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas de Unión al ARN/genética , Proteínas Adaptadoras del Transporte Vesicular/deficiencia , Proteínas Adaptadoras del Transporte Vesicular/genética , Animales , Autofagosomas/química , Proteína 7 Relacionada con la Autofagia/deficiencia , Proteína 7 Relacionada con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/deficiencia , Proteínas Relacionadas con la Autofagia/genética , Transporte Biológico , Biotinilación , Vesículas Extracelulares/química , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Células HEK293 , Ribonucleoproteína Heterogénea-Nuclear Grupo K/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo K/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lisosomas/química , Lisosomas/metabolismo , Proteínas de Unión a la Región de Fijación a la Matriz/genética , Proteínas de Unión a la Región de Fijación a la Matriz/metabolismo , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Matriz Nuclear/genética , Proteínas Asociadas a Matriz Nuclear/metabolismo , Proteómica/métodos , Células RAW 264.7 , ARN Pequeño no Traducido/genética , ARN Pequeño no Traducido/metabolismo , Proteínas de Unión al ARN/clasificación , Proteínas de Unión al ARN/metabolismo , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Esfingomielina Fosfodiesterasa/genética , Esfingomielina Fosfodiesterasa/metabolismoRESUMEN
Multi-subunit cullin-RING ligases (CRLs) are the largest family of ubiquitin E3 ligases in humans. CRL activity is tightly regulated to prevent unintended substrate degradation or autocatalytic degradation of CRL subunits. Using a proteomics strategy, we discovered that CRL4AMBRA1 (CRL substrate receptor denoted in superscript) targets Elongin C (ELOC), the essential adapter protein of CRL5 complexes, for polyubiquitination and degradation. We showed that the ubiquitin ligase function of CRL4AMBRA1 is required to disrupt the assembly and attenuate the ligase activity of human CRL5SOCS3 and HIV-1 CRL5VIF complexes as AMBRA1 depletion leads to hyperactivation of both CRL5 complexes. Moreover, CRL4AMBRA1 modulates interleukin-6/STAT3 signaling and HIV-1 infectivity that are regulated by CRL5SOCS3 and CRL5VIF, respectively. Thus, by discovering a substrate of CRL4AMBRA1, ELOC, the shared adapter of CRL5 ubiquitin ligases, we uncovered a novel CRL cross-regulation pathway.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Elonguina/metabolismo , Infecciones por VIH/metabolismo , VIH-1/metabolismo , Proteolisis , Transducción de Señal , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Elonguina/genética , Células HEK293 , Infecciones por VIH/genética , VIH-1/genética , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Proteína 3 Supresora de la Señalización de Citocinas/genética , Proteína 3 Supresora de la Señalización de Citocinas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Autophagy traditionally sustains metabolism in stressed cells by promoting intracellular catabolism and nutrient recycling. Here, we demonstrate that in response to stresses requiring increased glycolytic demand, the core autophagy machinery also facilitates glucose uptake and glycolytic flux by promoting cell surface expression of the glucose transporter GLUT1/Slc2a1. During metabolic stress, LC3+ autophagic compartments bind and sequester the RabGAP protein TBC1D5 away from its inhibitory interactions with the retromer complex, thereby enabling retromer recruitment to endosome membranes and GLUT1 plasma membrane translocation. In contrast, TBC1D5 inhibitory interactions with the retromer are maintained in autophagy-deficient cells, leading to GLUT1 mis-sorting into endolysosomal compartments. Furthermore, TBC1D5 depletion in autophagy-deficient cells rescues retromer recruitment to endosomal membranes and GLUT1 surface recycling. Hence, TBC1D5 shuttling to autophagosomes during metabolic stress facilitates retromer-dependent GLUT1 trafficking. Overall, our results illuminate key interconnections between the autophagy and endosomal pathways dictating GLUT1 trafficking and extracellular nutrient uptake.
Asunto(s)
Autofagia , Membrana Celular/metabolismo , Fibroblastos/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Transportador de Glucosa de Tipo 1/metabolismo , Glucosa/metabolismo , Glucólisis , Estrés Fisiológico , Animales , Autofagosomas/metabolismo , Autofagosomas/patología , Proteína 5 Relacionada con la Autofagia/genética , Proteína 5 Relacionada con la Autofagia/metabolismo , Proteína 7 Relacionada con la Autofagia/genética , Proteína 7 Relacionada con la Autofagia/metabolismo , Endosomas/metabolismo , Endosomas/patología , Femenino , Fibroblastos/patología , Proteínas Activadoras de GTPasa/genética , Transportador de Glucosa de Tipo 1/genética , Células HEK293 , Humanos , Cinética , Lisosomas/metabolismo , Lisosomas/patología , Ratones , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Transporte de Proteínas , Interferencia de ARN , Transducción de Señal , Transfección , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Synthetic protein switches controlled with user-defined inputs are powerful tools for studying and controlling dynamic cellular processes. To date, these approaches have relied primarily on intermolecular regulation. Here we report a computationally guided framework for engineering intramolecular regulation of protein function. We utilize this framework to develop chemically inducible activator of RAS (CIAR), a single-component RAS rheostat that directly activates endogenous RAS in response to a small molecule. Using CIAR, we show that direct RAS activation elicits markedly different RAS-ERK signaling dynamics from growth factor stimulation, and that these dynamics differ among cell types. We also found that the clinically approved RAF inhibitor vemurafenib potently primes cells to respond to direct wild-type RAS activation. These results demonstrate the utility of CIAR for quantitatively interrogating RAS signaling. Finally, we demonstrate the general utility of our approach in design of intramolecularly regulated protein tools by applying it to the Rho family of guanine nucleotide exchange factors.
Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Sistema de Señalización de MAP Quinasas , Ingeniería de Proteínas , Proteínas ras/química , Proteínas ras/metabolismo , Línea Celular , Humanos , Modelos MolecularesRESUMEN
The rising success of cancer immunotherapy has produced immense interest in defining the clinical contexts that may benefit from this therapeutic approach. To this end, there is a need to ascertain how the therapeutic modulation of intrinsic cancer cell programs influences the anticancer immune response. For example, the role of autophagy as a tumor cell survival and metabolic fitness pathway is being therapeutically targeted in ongoing clinical trials that combine cancer therapies with antimalarial drugs for the treatment of a broad spectrum of cancers, many of which will likely benefit from immunotherapy. However, our current understanding of the interplay between autophagy and the immune response remains incomplete. Here, we have evaluated how autophagy inhibition impacts the antitumor immune response in immune-competent mouse models of melanoma and mammary cancer. We observed equivalent levels of T cell infiltration and function within autophagy-competent and -deficient tumors, even upon treatment with the anthracycline chemotherapeutic doxorubicin. Similarly, we found equivalent T cell responses upon systemic treatment of tumor-bearing mice with antimalarial drugs. Our findings demonstrate that antitumor adaptive immunity is not adversely impaired by autophagy inhibition in these models, allowing for the future possibility of combining autophagy inhibitors with immunotherapy in certain clinical contexts.
Asunto(s)
Antimaláricos/farmacología , Autofagia/efectos de los fármacos , Inmunidad Celular/efectos de los fármacos , Neoplasias Mamarias Experimentales , Melanoma , Linfocitos T/inmunología , Animales , Autofagia/inmunología , Línea Celular Tumoral , Femenino , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/inmunología , Neoplasias Mamarias Experimentales/patología , Melanoma/tratamiento farmacológico , Melanoma/inmunología , Melanoma/patología , Ratones , Ratones Transgénicos , Linfocitos T/patologíaRESUMEN
Autophagy is a catabolic pathway involving the sequestration of cellular contents into a double-membrane vesicle, the autophagosome. Although recent studies have demonstrated that autophagy supports cell migration, the underlying mechanisms remain unknown. Using live-cell imaging, we uncover that autophagy promotes optimal migratory rate and facilitates the dynamic assembly and disassembly of cell-matrix focal adhesions (FAs), which is essential for efficient motility. Additionally, our studies reveal that autophagosomes associate with FAs primarily during disassembly, suggesting autophagy locally facilitates the destabilization of cell-matrix contact sites. Furthermore, we identify the selective autophagy cargo receptor neighbor of BRCA1 (NBR1) as a key mediator of autophagy-dependent FA remodeling. NBR1 depletion impairs FA turnover and decreases targeting of autophagosomes to FAs, whereas ectopic expression of autophagy-competent, but not autophagy-defective, NBR1 enhances FA disassembly and reduces FA lifetime during migration. Our findings provide mechanistic insight into how autophagy promotes migration by revealing a requirement for NBR1-mediated selective autophagy in enabling FA disassembly in motile cells.