RESUMEN
Targeted protein degradation (TPD) has emerged as a potentially transformational therapeutic modality with considerable promise. Molecular glue degraders remodel the surface of E3 ligases inducing interactions with neosubstrates resulting in their polyubiquitination and proteasomal degradation. Molecular glues are clinically precedented and have demonstrated the ability to degrade proteins-of-interest (POIs) previously deemed undruggable due to the absence of a traditional small molecule binding pocket. Heterobifunctional proteolysis targeting chimeras (PROTACs) possess ligands for an E3 complex and the POIs, which are chemically linked together, and similarly hijack the ubiquitin machinery to deplete the target. There has been a recent surge in the number of degraders entering clinical trials, particularly directed toward cancer. Nearly all utilize CRL4CRBN as the E3 ligase, and a relatively limited diversity of POIs are currently targeted. In this review, we provide an overview of the degraders in clinical trials and provide a perspective on the lessons learned from their development and emerging human data that will be broadly useful to those working in the TPD field.
Asunto(s)
Neoplasias , Proteínas , Humanos , Proteínas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteolisis , Neoplasias/tratamiento farmacológicoRESUMEN
Thalidomide and its derivatives are molecular glues that bind cereblon (CRBN), a component of an E3 ubiquitin ligase complex, and mediate protein interactions with neosubstrates resulting in their polyubiquitination and proteasomal degradation. The structural features of neosubstrate binding have been elucidated that highlight key interactions with a ß-hairpin degron containing a glycine, which is present in a wide-range of proteins, including zinc-finger transcription factors such as IKZF1, and the translation termination factor GSPT1. Here, we profile 14 closely-related thalidomide derivatives in CRBN occupancy, and IKZF1 and GSPT1 degradation cell-based assays, and use crystal structures, computational docking and molecular dynamics to delineate subtle structure-activity relationships. Our findings will enable the rational design of CRBN modulators in the future, and help avoid the degradation of GSPT1 which is broadly cytotoxic.
RESUMEN
Electrophilic biocompatible warheads, particularly cysteine-reactive acrylamides, have enabled the development of covalent inhibitor drugs and chemical biology probes, but cysteine is rarely present in protein binding sites. Therefore, expansion of the list of targetable amino acid residues is required to augment the synthetic bology toolkit of site-selective protein modifications. This work describes the first rational targeting of a specific histidine residue in a protein binding site using sulfonyl exchange chemistry. Structure-based drug design was used to incorporate sulfonyl fluoride and triazole reactive groups into the isoindolinone thalidomide congener EM12 to yield potent covalent inhibitors of the cereblon E3 ubiquitin ligase complex through engagement of His353. Conversely, the fluorosulfate derivative EM12-FS labels His353, but degrades a novel neosubstrate, the protein N-terminal glutamine amidohydrolase NTAQ1, which is involved in the N-end rule pathway and DNA damage response. Targeted protein degradation using cereblon ligands has become an important new drug discovery modality and the chemical probes and covalent labeling strategy described here will broadly impact this exciting area of therapeutic research.
RESUMEN
Immunomodulatory drugs (IMiDs) thalidomide, lenalidomide, and pomalidomide engage cereblon and mediate a protein interface with neosubstrates such as zinc finger transcription factors promoting their polyubiquitination and degradation. The IMiDs have garnered considerable excitement in drug discovery, leading to exploration of targeted protein degradation strategies. Although the molecular modes-of-action of the IMiDs and related degraders have been the subject of intense research, their pharmacokinetics and disposition have been relatively understudied. Here, we assess the effects of physicochemistry of the IMiDs, the phthalimide EM-12, and the candidate drug CC-220 (iberdomide) on lipophilicity, solubility, metabolism, permeability, intracellular bioavailability, and cell-based potency. The insights yielded in this study will enable the rational property-based design and development of targeted protein degraders in the future.
RESUMEN
Oncofetal protein SALL4 is critical for cancer cell survival. Targeting SALL4, however, is only applicable in a fraction of cancer patients who are positive for this gene. To overcome this limitation, we propose to induce a cancer vulnerability by engineering a partial dependency upon SALL4. Following exogenous expression of SALL4, SALL4-negative cancer cells became partially dependent on SALL4. Treatment of SALL4-negative cells with the FDA-approved hypomethylating agent 5-aza-2'-deoxycytidine (DAC) resulted in transient upregulation of SALL4. DAC pretreatment sensitized SALL4-negative cancer cells to entinostat, which negatively affected SALL4 expression through a microRNA, miRNA-205, both in culture and in vivo. Moreover, SALL4 was essential for the efficiency of sequential treatment of DAC and entinostat. Overall, this proof-of-concept study provides a framework whereby the targeting pathways such as SALL4-centered therapy can be expanded, sensitizing cancer cells to treatment by transient target induction and engineering a dependency. SIGNIFICANCE: These findings provide a therapeutic approach for patients harboring no suitable target by induction of a SALL4-mediated vulnerability.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Metilación de ADN , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Factores de Transcripción/antagonistas & inhibidores , Animales , Apoptosis , Benzamidas/administración & dosificación , Proliferación Celular , Decitabina/administración & dosificación , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Neoplasias/metabolismo , Neoplasias/patología , Piridinas/administración & dosificación , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The architecture of chromatin regulates eukaryotic cell states by controlling transcription factor access to sites of gene regulation. Here we describe a dual transposase-peroxidase approach, integrative DNA and protein tagging (iDAPT), which detects both DNA (iDAPT-seq) and protein (iDAPT-MS) associated with accessible regions of chromatin. In addition to direct identification of bound transcription factors, iDAPT enables the inference of their gene regulatory networks, protein interactors and regulation of chromatin accessibility. We applied iDAPT to profile the epigenomic consequences of granulocytic differentiation of acute promyelocytic leukemia, yielding previously undescribed mechanistic insights. Our findings demonstrate the power of iDAPT as a platform for studying the dynamic epigenomic landscapes and their transcription factor components associated with biological phenomena and disease.
Asunto(s)
Cromatina/metabolismo , ADN/genética , Regulación de la Expresión Génica/genética , Histonas/metabolismo , Leucemia Promielocítica Aguda/genética , Redes Reguladoras de Genes , Humanos , Leucemia Promielocítica Aguda/patología , Factores de Transcripción/metabolismoRESUMEN
The zinc finger transcription factor SALL4 is highly expressed in embryonic stem cells, downregulated in most adult tissues, but reactivated in many aggressive cancers. This unique expression pattern makes SALL4 an attractive therapeutic target. However, whether SALL4 binds DNA directly to regulate gene expression is unclear, and many of its targets in cancer cells remain elusive. Here, through an unbiased screen of protein binding microarray (PBM) and cleavage under targets and release using nuclease (CUT&RUN) experiments, we identify and validate the DNA binding domain of SALL4 and its consensus binding sequence. Combined with RNA sequencing (RNA-seq) analyses after SALL4 knockdown, we discover hundreds of new SALL4 target genes that it directly regulates in aggressive liver cancer cells, including genes encoding a family of histone 3 lysine 9-specific demethylases (KDMs). Taken together, these results elucidate the mechanism of SALL4 DNA binding and reveal pathways and molecules to target in SALL4-dependent tumors.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Proteínas de Unión al ADN/metabolismo , Células Madre Embrionarias/metabolismo , Regulación Neoplásica de la Expresión Génica , Histona Demetilasas/metabolismo , Factores de Transcripción/metabolismo , Dedos de Zinc , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Carcinoma Hepatocelular/genética , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Técnicas de Silenciamiento del Gen , Células HEK293 , Células HeLa , Histona Demetilasas/genética , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Análisis por Matrices de Proteínas , Unión Proteica , Análisis de Secuencia de ARN , Factores de Transcripción/genéticaRESUMEN
BACKGROUND & AIMS: Some oncogenes encode transcription factors, but few drugs have been successfully developed to block their activity specifically in cancer cells. The transcription factor SALL4 is aberrantly expressed in solid tumor and leukemia cells. We developed a screen to identify compounds that reduce the viability of liver cancer cells that express high levels of SALL4, and we investigated their mechanisms. METHODS: We developed a stringent high-throughput screening platform comprising unmodified SNU-387 and SNU-398 liver cancer cell lines and SNU-387 cell lines engineered to express low and high levels of SALL4. We screened 1597 pharmacologically active small molecules and 21,575 natural product extracts from plant, bacteria, and fungal sources for those that selectively reduce the viability of cells with high levels of SALL4 (SALL4hi cells). We compared gene expression patterns of SALL4hi cells vs SALL4-knockdown cells using RNA sequencing and real-time polymerase chain reaction analyses. Xenograft tumors were grown in NOD/SCID gamma mice from SALL4hi SNU-398 or HCC26.1 cells or from SALL4lo patient-derived xenograft (PDX) cells; mice were given injections of identified compounds or sorafenib, and the effects on tumor growth were measured. RESULTS: Our screening identified 1 small molecule (PI-103) and 4 natural compound analogues (oligomycin, efrapeptin, antimycin, and leucinostatin) that selectively reduced viability of SALL4hi cells. We performed validation studies, and 4 of these compounds were found to inhibit oxidative phosphorylation. The adenosine triphosphate (ATP) synthase inhibitor oligomycin reduced the viability of SALL4hi hepatocellular carcinoma and non-small-cell lung cancer cell lines with minimal effects on SALL4lo cells. Oligomycin also reduced the growth of xenograft tumors grown from SALL4hi SNU-398 or HCC26.1 cells to a greater extent than sorafenib, but oligomycin had little effect on tumors grown from SALL4lo PDX cells. Oligomycin was not toxic to mice. Analyses of chromatin immunoprecipitation sequencing data showed that SALL4 binds approximately 50% of mitochondrial genes, including many oxidative phosphorylation genes, to activate their transcription. In comparing SALL4hi and SALL4-knockdown cells, we found SALL4 to increase oxidative phosphorylation, oxygen consumption rate, mitochondrial membrane potential, and use of oxidative phosphorylation-related metabolites to generate ATP. CONCLUSIONS: In a screening for compounds that reduce the viability of cells that express high levels of the transcription factor SALL4, we identified inhibitors of oxidative phosphorylation, which slowed the growth of xenograft tumors from SALL4hi cells in mice. SALL4 activates the transcription of genes that regulate oxidative phosphorylation to increase oxygen consumption, mitochondrial membrane potential, and ATP generation in cancer cells. Inhibitors of oxidative phosphorylation might be used for the treatment of liver tumors with high levels of SALL4.
Asunto(s)
Antineoplásicos/farmacología , Ensayos Analíticos de Alto Rendimiento/métodos , Neoplasias Hepáticas/tratamiento farmacológico , Factores de Transcripción/antagonistas & inhibidores , Animales , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Masculino , Ratones , Fosforilación Oxidativa/efectos de los fármacos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Hematopoietic stem cells are capable of self-renewal or differentiation along three main lineages: myeloid, erythroid, and lymphoid. One of the earliest lineage decisions for blood progenitor cells is whether to adopt the lymphoid or myeloid fate. Previous work had shown that myocyte enhancer factor 2C (MEF2C) is indispensable for the lymphoid fate decision, yet the specific mechanism of action remained unclear. Here, we have identified early B cell factor-1 (EBF1) as a co-regulator of gene expression with MEF2C. A genome-wide survey of MEF2C and EBF1 binding sites identified a subset of B cell-specific genes that they target. We also determined that the p38 MAPK pathway activates MEF2C to drive B cell differentiation. Mef2c knockout mice showed reduced B lymphoid-specific gene expression as well as increased myeloid gene expression, consistent with MEF2C's role as a lineage fate regulator. This is further supported by interaction between MEF2C and the histone deacetylase, HDAC7, revealing a likely mechanism to repress the myeloid transcription program. This study thus elucidates both activation and repression mechanisms, identifies regulatory partners, and downstream targets by which MEF2C regulates lymphoid-specific differentiation.
Asunto(s)
Linfocitos B/metabolismo , Factores de Transcripción MEF2/metabolismo , Transactivadores/metabolismo , Transcripción Genética , Linfocitos B/citología , Diferenciación Celular/genética , Línea Celular , Linaje de la Célula/genética , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Histona Desacetilasas/metabolismo , Humanos , Inmunoprecipitación , Células Mieloides/metabolismo , Fosforilación , Células Precursoras de Linfocitos B/metabolismo , Transporte de Proteínas , Fracciones Subcelulares/metabolismo , Activación Transcripcional , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
There is a growing body of evidence supporting that cancer cells share many similarities with embryonic stem cells (ESCs). For example, aggressive cancers and ESCs share a common gene expression signature that includes hundreds of genes. Since ESC genes are not present in most adult tissues, they could be ideal candidate targets for cancer-specific diagnosis and treatment. This is an exciting cancer-targeting model. The major hurdle to test this model is to identify the key factors/pathway(s) within ESCs that are responsible for the cancer phenotype. SALL4 is one of few genes that can establish this link. The first publication of SALL4 is on its mutation in a human inherited disorder with multiple developmental defects. Since then, over 300 papers have been published on various aspects of this gene in stem cells, development, and cancers. This review aims to summarize our current knowledge of SALL4, including a SALL4-based approach to classify and target cancers. Many questions about this important gene still remain unanswered, specifically, on how this gene regulates cell fates at a molecular level. Understanding SALL4's molecular functions will allow development of specific targeted approaches in the future.
Asunto(s)
Neoplasias/genética , Células Madre/citología , Factores de Transcripción/genética , Humanos , Neoplasias/patologíaRESUMEN
Amphibian neural development occurs as a two-step process: (1) induction specifies a neural fate in undifferentiated ectoderm; and (2) transformation induces posterior spinal cord and hindbrain. Signaling through the Fgf, retinoic acid (RA) and Wnt/ß-catenin pathways is necessary and sufficient to induce posterior fates in the neural plate, yet a mechanistic understanding of the process is lacking. Here, we screened for factors enriched in posterior neural tissue and identify spalt-like 4 (sall4), which is induced by Fgf. Knockdown of Sall4 results in loss of spinal cord marker expression and increased expression of pou5f3.2 (oct25), pou5f3.3 (oct60) and pou5f3.1 (oct91) (collectively, pou5f3 genes), the closest Xenopus homologs of mammalian stem cell factor Pou5f1 (Oct4). Overexpression of the pou5f3 genes results in the loss of spinal cord identity and knockdown of pou5f3 function restores spinal cord marker expression in Sall4 morphants. Finally, knockdown of Sall4 blocks the posteriorizing effects of Fgf and RA signaling in the neurectoderm. These results suggest that Sall4, activated by posteriorizing signals, represses the pou5f3 genes to provide a permissive environment allowing for additional Wnt/Fgf/RA signals to posteriorize the neural plate.
Asunto(s)
Tipificación del Cuerpo , Linaje de la Célula , Neuronas/citología , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Animales , Biomarcadores/metabolismo , Tipificación del Cuerpo/genética , Diferenciación Celular/genética , Linaje de la Célula/efectos de los fármacos , Linaje de la Célula/genética , Embrión no Mamífero/efectos de los fármacos , Embrión no Mamífero/metabolismo , Factores de Crecimiento de Fibroblastos/farmacología , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Pruebas Genéticas , Morfolinos/farmacología , Placa Neural/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Proteínas Represoras/genética , Rombencéfalo/metabolismo , Médula Espinal/citología , Médula Espinal/efectos de los fármacos , Médula Espinal/embriología , Factores de Transcripción/genética , Transcripción Genética , Tretinoina/farmacología , Vía de Señalización Wnt/efectos de los fármacos , Vía de Señalización Wnt/genética , Proteínas de Xenopus/genética , Xenopus laevis/embriología , Xenopus laevis/genéticaRESUMEN
BACKGROUND: Stem cell factor SALL4 is a zinc finger transcription factor. It plays vital roles in the maintenance of embryonic stem cell properties, functions as an oncogene in leukemia, and has been recently proposed to use for cord blood expansion. The mechanism(s) by which SALL4 functions in normal human hematopoiesis, including identification of its target genes, still need to be explored. STUDY DESIGN AND METHODS: Chromatin immunoprecipitation followed by microarray hybridization (ChIP-chip) was used for mapping SALL4 global gene targets in normal primary CD34+ cells. The results were then correlated with SALL4 functional studies in the CD34+ cells. RESULTS: More than 1000 potential SALL4 downstream target genes have been identified, and validation of binding by ChIP-quantitative polymerase chain reaction was performed for 5% of potential targets. These include genes that are involving in hematopoietic differentiation and self-renewal, such as HOXA9, RUNX1, CD34, and PTEN. Down regulation of SALL4 expression using small-hairpin RNA in these cells led to decreased in vitro myeloid colony-forming abilities and impaired in vivo engraftment. Furthermore, HOXA9 was identified to be a major SALL4 target in normal human hematopoiesis and the loss of either SALL4 or HOXA9 expression in CD34+ cells shared a similar phenotype. CONCLUSION: Taken together, SALL4 is a key regulator in normal human hematopoiesis and the mechanism of its function is at least in part through the HOXA9. Future study will determine whether modulating the SALL4/HOXA9 pathway can be used in cellular therapy such as cord blood expansion and/or myeloid engraftment.
Asunto(s)
Regulación de la Expresión Génica , Hematopoyesis/genética , Células Madre Hematopoyéticas/fisiología , Factores de Transcripción/genética , Animales , Antígenos CD34/metabolismo , Diferenciación Celular/genética , Inmunoprecipitación de Cromatina , Hematopoyesis/fisiología , Trasplante de Células Madre Hematopoyéticas , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/fisiología , Humanos , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Factores de Transcripción/fisiologíaRESUMEN
SALL4, a member of the SALL gene family, is one of the most important transcriptional regulators of stem cells. It is of particular interest to stem cell biologists because it is linked to the self-renewal of both embryonic stem cells (ESCs) and hematopoietic stem cells (HSCs), and it is involved in human leukemia. In ESCs, the Sall4/Oct4/Nanog core transcriptional network governs the self-renewal and pluripotent properties of human and murine ESCs. In normal HSCs and leukemic stem cells (LSCs), SALL4 is linked to three known pathways that are involved in self-renewal: Wnt/ß-catenin, Bmi-1, and Pten. Despite the important shared role of SALL4 in self-renewal of HSCs and LSCs, our recent studies obtained through correlating global downstream target genes and unique functional studies in normal versus leukemic cells have demonstrated that SALL4 has differential effects on both pro- and anti-apoptotic pathways in normal and leukemic cells. Targeting SALL4, particularly when combined with the use of ABT-737, a BCL2 antagonist, could lead to leukemic cell-specific apoptosis. This review summarizes our current knowledge on the SALL gene family development, particularly on the role of SALL4 in stem cells, as well as tumorigenesis, especially leukemogenesis.
Asunto(s)
Transformación Celular Neoplásica , Leucemia/fisiopatología , Factores de Transcripción/fisiología , Animales , Regulación del Desarrollo de la Expresión Génica , Humanos , Leucemia/genética , Ratones , Factores de Transcripción/genéticaRESUMEN
SALL4, a newly identified zinc-finger transcriptional factor important for embryonic development, is mapped to chromosome 20q13. Previously, we reported that SALL4 was constitutively expressed in acute myeloid leukemia and SALL4 transgenic mice developed acute myeloid leukemia. In this study, we aimed to survey SALL4 protein expression in benign and neoplastic hematopoietic tissues in addition to acute myeloid leukemia using immunostaining with a polyclonal anti-SALL4 antibody. Primary hematological tumors (178) and 15 benign hematopoietic tissues were examined. Reverse transcription-polymerase chain reaction was also performed to detect SALL4 mRNA expression on eight precursor B-cell lymphoblastic leukemia/lymphomas, 10 benign hematopoietic tissues, and seven hematopoietic cancer cell lines. Of the benign tissues, SALL4 expression was detectable only in CD34+ hematopoietic stem/progenitor cells (2/2 at protein level, 3/3 at RNA level). In neoplastic tissues, only precursor B-cell lymphoblastic leukemia/lymphomas had detectable SALL4 (12/16 at protein level, 7/8 at RNA level), similar to that observed in acute myeloid leukemia. Of the seven cell lines examined, only those derived from acute myeloid leukemia and precursor B-cell lymphoblastic leukemia/lymphomas were positive. To conclude, SALL4 expression is normally restricted to CD34+ hematopoietic stem/progenitor cells. The persistence of SALL4 expression in leukemic blasts in precursor B-cell lymphoblastic leukemia/lymphomas resembles to what we observed in acute myeloid leukemia, and correlates with the maturation arrest of these cells. We have shown in our previous study that the constitutive expression of SALL4 in mice can lead to acute myeloid leukemia development. The similar expression pattern of SALL4 in acute myeloid leukemia and B-cell lymphoblastic leukemia/lymphomas suggests that these two disease entities may share similar biological features and/or mechanisms of leukemogenesis. More definite studies to investigate the role of SALL4 in the pathogenesis of B-cell lymphoblastic leukemia/lymphomas are needed in the future to address this question.