RESUMEN
Single cell sequencing technologies have become a fixture in the molecular profiling of cells due to their ease, flexibility, and commercial availability. In particular, partitioning individual cells inside oil droplets via microfluidic reactions enables transcriptomic or multi-omic measurements for thousands of cells in parallel. Complementing the multitude of biological discoveries from genomics analyses, the past decade has brought new capabilities from assay baselines to enable a deeper understanding of the complex data from single cell multi-omics. Here, we highlight four innovations that have improved the reliability and understanding of droplet microfluidic assays. We emphasize new developments that further orient principles of technology development and guidelines for the design, benchmarking, and implementation of new droplet-based methodologies.
RESUMEN
The RAS-mitogen-activated protein kinase (MAPK) pathway includes KSR, RAF, MEK and the phospho-regulatory sensor 14-3-3. Specific assemblies among these components drive various diseases and likely dictate efficacy for numerous targeted therapies, including allosteric MEK inhibitors (MEKi). However, directly measuring drug interactions on physiological RAS-MAPK complexes in live cells has been inherently challenging to query and therefore remains poorly understood. Here we present a series of NanoBRET-based assays to quantify direct target engagement of MEKi on MEK1 and higher-order MEK1-bound complexes with ARAF, BRAF, CRAF, KSR1 and KSR2 in the presence and absence of 14-3-3 in living cells. We find distinct MEKi preferences among these complexes that can be compiled to generate inhibitor binding profiles. Further, these assays can report on the influence of the pathogenic BRAF-V600E mutant on MEKi binding. Taken together, these approaches can be used as a platform to screen for compounds intended to target specific complexes in the RAS-MAPK cascade.
Asunto(s)
Bioensayo , Proteínas Proto-Oncogénicas B-raf , Proteínas Proto-Oncogénicas B-raf/genética , Sistema de Señalización de MAP Quinasas , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
Pseudokinases often operate through functionally related enzymes and receptors. A prime example is the pseudokinase KSR (Kinase Suppressor of RAS), which can act as both an amplifier and inhibitor of members in the RAS-MAPK (Mitogen Activated Protein Kinase) signaling pathway. KSR is structurally related to the active RAF kinases over multiple domains; moreover, the pseudokinase domain of KSR forms physical and regulatory complexes with both RAF and MEK through distinct interfaces. Characterization of small molecule interactions on KSR has been used to uncover novel chemical tools and understand the mechanism of action of clinical drugs. Here, we elaborate on assays and structural methods for measuring binding at orthosteric and interfacial binding sites on KSR. These distinct small molecule pockets provide therapeutic paths for targeting KSR1 and KSR2 pseudokinases in disease, including in RAS and RAF mutant cancers.
Asunto(s)
Proteínas Quinasas , Proteínas Serina-Treonina Quinasas , Sitios de Unión , Conformación Molecular , Fosforilación , Proteínas Quinasas/química , Proteínas Proto-Oncogénicas c-raf/metabolismo , Transducción de SeñalRESUMEN
RNA binding proteins (RBPs) are key arbiters of post-transcriptional regulation and are found to be found dysregulated in hematological malignancies. Here, we identify the RBP RBMX and its retrogene RBMXL1 to be required for murine and human myeloid leukemogenesis. RBMX/L1 are overexpressed in acute myeloid leukemia (AML) primary patients compared to healthy individuals, and RBMX/L1 loss delayed leukemia development. RBMX/L1 loss lead to significant changes in chromatin accessibility, as well as chromosomal breaks and gaps. We found that RBMX/L1 directly bind to mRNAs, affect transcription of multiple loci, including CBX5 (HP1α), and control the nascent transcription of the CBX5 locus. Forced CBX5 expression rescued the RBMX/L1 depletion effects on cell growth and apoptosis. Overall, we determine that RBMX/L1 control leukemia cell survival by regulating chromatin state through their downstream target CBX5. These findings identify a mechanism for RBPs directly promoting transcription and suggest RBMX/L1, as well as CBX5, as potential therapeutic targets in myeloid malignancies.
Asunto(s)
Cromatina , Leucemia Mieloide Aguda , Animales , Cromatina/genética , Regulación de la Expresión Génica , Ribonucleoproteínas Nucleares Heterogéneas/genética , Humanos , Leucemia Mieloide Aguda/genética , Ratones , Proteínas de Unión al ARN/genética , Factores de Transcripción/genéticaRESUMEN
The MAPK/ERK kinase MEK is a shared effector of the frequent cancer drivers KRAS and BRAF that has long been pursued as a drug target in oncology1, and more recently in immunotherapy2,3 and ageing4. However, many MEK inhibitors are limited owing to on-target toxicities5-7 and drug resistance8-10. Accordingly, a molecular understanding of the structure and function of MEK within physiological complexes could provide a template for the design of safer and more effective therapies. Here we report X-ray crystal structures of MEK bound to the scaffold KSR (kinase suppressor of RAS) with various MEK inhibitors, including the clinical drug trametinib. The structures reveal an unexpected mode of binding in which trametinib directly engages KSR at the MEK interface. In the bound complex, KSR remodels the prototypical allosteric pocket of the MEK inhibitor, thereby affecting binding and kinetics, including the drug-residence time. Moreover, trametinib binds KSR-MEK but disrupts the related RAF-MEK complex through a mechanism that exploits evolutionarily conserved interface residues that distinguish these sub-complexes. On the basis of these insights, we created trametiglue, which limits adaptive resistance to MEK inhibition by enhancing interfacial binding. Our results reveal the plasticity of an interface pocket within MEK sub-complexes and have implications for the design of next-generation drugs that target the RAS pathway.
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Quinasas de Proteína Quinasa Activadas por Mitógenos/química , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , Piridonas/química , Piridonas/farmacología , Pirimidinonas/química , Pirimidinonas/farmacología , Secuencia de Aminoácidos , Animales , Sitios de Unión/efectos de los fármacos , Humanos , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Modelos Moleculares , Unión Proteica/efectos de los fármacos , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Especificidad por Sustrato , Quinasas raf/química , Quinasas raf/metabolismoRESUMEN
The MUSASHI (MSI) family of RNA binding proteins (MSI1 and MSI2) contribute to a wide spectrum of cancers including acute myeloid leukemia. We find that the small molecule Ro 08-2750 (Ro) binds directly and selectively to MSI2 and competes for its RNA binding in biochemical assays. Ro treatment in mouse and human myeloid leukemia cells results in an increase in differentiation and apoptosis, inhibition of known MSI-targets, and a shared global gene expression signature similar to shRNA depletion of MSI2. Ro demonstrates in vivo inhibition of c-MYC and reduces disease burden in a murine AML leukemia model. Thus, we identify a small molecule that targets MSI's oncogenic activity. Our study provides a framework for targeting RNA binding proteins in cancer.
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Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Leucemia Experimental/tratamiento farmacológico , Leucemia Mieloide Aguda/tratamiento farmacológico , Pteridinas/farmacología , Proteínas de Unión al ARN/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Flavinas , Perfilación de la Expresión Génica , Humanos , Leucemia Experimental/sangre , Leucemia Mieloide Aguda/sangre , Masculino , Ratones , Cultivo Primario de Células , Proteínas Proto-Oncogénicas c-myc/metabolismo , Pteridinas/uso terapéutico , ARN/metabolismo , Motivo de Reconocimiento de ARN/efectos de los fármacos , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Transcriptoma/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
Leukemias exhibit a dysregulated developmental program mediated through both genetic and epigenetic mechanisms. Although IKZF2 is expressed in hematopoietic stem cells (HSCs), we found that it is dispensable for mouse and human HSC function. In contrast to its role as a tumor suppressor in hypodiploid B-acute lymphoblastic leukemia, we found that IKZF2 is required for myeloid leukemia. IKZF2 is highly expressed in leukemic stem cells (LSCs), and its deficiency results in defective LSC function. IKZF2 depletion in acute myeloid leukemia (AML) cells reduced colony formation, increased differentiation and apoptosis, and delayed leukemogenesis. Gene expression, chromatin accessibility, and direct IKZF2 binding in MLL-AF9 LSCs demonstrate that IKZF2 regulates a HOXA9 self-renewal gene expression program and inhibits a C/EBP-driven differentiation program. Ectopic HOXA9 expression and CEBPE depletion rescued the effects of IKZF2 depletion. Thus, our study shows that IKZF2 regulates the AML LSC program and provides a rationale to therapeutically target IKZF2 in myeloid leukemia.
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Diferenciación Celular , Autorrenovación de las Células , Proteínas de Unión al ADN/fisiología , Regulación Leucémica de la Expresión Génica , Leucemia Experimental/patología , Leucemia Mieloide Aguda/patología , Células Madre Neoplásicas/patología , Factores de Transcripción/fisiología , Animales , Cromatina/genética , Cromatina/metabolismo , Femenino , Hematopoyesis , Leucemia Experimental/genética , Leucemia Experimental/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células Madre Neoplásicas/metabolismoRESUMEN
While Slicer activity of Argonaute is central to RNAi, conserved roles of slicing in endogenous regulatory biology are less clear, especially in mammals. Biogenesis of erythroid Dicer-independent mir-451 involves Ago2 catalysis, but mir-451-KO mice do not phenocopy Ago2 catalytic-dead (Ago2-CD) mice, suggesting other needs for slicing. Here, we reveal mir-486 as another dominant erythroid miRNA with atypical biogenesis. While it is Dicer dependent, it requires slicing to eliminate its star strand. Thus, in Ago2-CD conditions, miR-486-5p is functionally inactive due to duplex arrest. Genome-wide analyses reveal miR-486 and miR-451 as the major slicing-dependent miRNAs in the hematopoietic system. Moreover, mir-486-KO mice exhibit erythroid defects, and double knockout of mir-486/451 phenocopies the cell-autonomous effects of Ago2-CD in the hematopoietic system. Finally, we observe that Ago2 is the dominant-expressed Argonaute in maturing erythroblasts, reflecting a specialized environment for processing slicing-dependent miRNAs. Overall, the mammalian hematopoietic system has evolved multiple conserved requirements for Slicer-dependent miRNA biogenesis.
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Proteínas Argonautas/metabolismo , MicroARNs/genética , Animales , Proteínas Argonautas/genética , Proteínas Argonautas/fisiología , ARN Helicasas DEAD-box/metabolismo , Eritroblastos/metabolismo , Estudio de Asociación del Genoma Completo , Mamíferos/metabolismo , Ratones , Ratones Noqueados , MicroARNs/metabolismo , Interferencia de ARN , Ribonucleasa III/metabolismo , Análisis de Secuencia de ARN , Homología de Secuencia de Ácido NucleicoRESUMEN
N6-methyladenosine (m6A) is an abundant nucleotide modification in mRNA that is required for the differentiation of mouse embryonic stem cells. However, it remains unknown whether the m6A modification controls the differentiation of normal and/or malignant myeloid hematopoietic cells. Here we show that shRNA-mediated depletion of the m6A-forming enzyme METTL3 in human hematopoietic stem/progenitor cells (HSPCs) promotes cell differentiation, coupled with reduced cell proliferation. Conversely, overexpression of wild-type METTL3, but not of a catalytically inactive form of METTL3, inhibits cell differentiation and increases cell growth. METTL3 mRNA and protein are expressed more abundantly in acute myeloid leukemia (AML) cells than in healthy HSPCs or other types of tumor cells. Furthermore, METTL3 depletion in human myeloid leukemia cell lines induces cell differentiation and apoptosis and delays leukemia progression in recipient mice in vivo. Single-nucleotide-resolution mapping of m6A coupled with ribosome profiling reveals that m6A promotes the translation of c-MYC, BCL2 and PTEN mRNAs in the human acute myeloid leukemia MOLM-13 cell line. Moreover, loss of METTL3 leads to increased levels of phosphorylated AKT, which contributes to the differentiation-promoting effects of METTL3 depletion. Overall, these results provide a rationale for the therapeutic targeting of METTL3 in myeloid leukemia.
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Adenosina/análogos & derivados , Células de la Médula Ósea/citología , Diferenciación Celular/fisiología , Leucemia Mieloide Aguda/patología , Metiltransferasas/fisiología , Adenosina/biosíntesis , Células Cultivadas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Humanos , Células Tumorales CultivadasRESUMEN
The identity of the RNA-binding proteins (RBPs) that govern cancer stem cells remains poorly characterized. The MSI2 RBP is a central regulator of translation of cancer stem cell programs. Through proteomic analysis of the MSI2-interacting RBP network and functional shRNA screening, we identified 24 genes required for in vivo leukemia. Syncrip was the most differentially required gene between normal and myeloid leukemia cells. SYNCRIP depletion increased apoptosis and differentiation while delaying leukemogenesis. Gene expression profiling of SYNCRIP-depleted cells demonstrated a loss of the MLL and HOXA9 leukemia stem cell program. SYNCRIP and MSI2 interact indirectly though shared mRNA targets. SYNCRIP maintains HOXA9 translation, and MSI2 or HOXA9 overexpression rescued the effects of SYNCRIP depletion. Altogether, our data identify SYNCRIP as a new RBP that controls the myeloid leukemia stem cell program. We propose that targeting these RBP complexes might provide a novel therapeutic strategy in leukemia.
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Regulación Leucémica de la Expresión Génica , Ribonucleoproteínas Nucleares Heterogéneas/genética , Leucemia Mieloide/genética , Proteínas de Unión al ARN/metabolismo , Animales , Supervivencia Celular , Femenino , Hematopoyesis/genética , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Proteínas de Homeodominio/genética , Humanos , Leucemia Bifenotípica Aguda/genética , Leucemia Bifenotípica Aguda/patología , Leucemia Mieloide/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Células Progenitoras Mieloides/metabolismo , Células Progenitoras Mieloides/patología , ARN Interferente Pequeño , Proteínas de Unión al ARN/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Myeloid malignancy is increasingly viewed as a disease spectrum, comprising hematopoietic disorders that extend across a phenotypic continuum ranging from clonal hematopoiesis to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). In this study, we derived a collection of induced pluripotent stem cell (iPSC) lines capturing a range of disease stages encompassing preleukemia, low-risk MDS, high-risk MDS, and secondary AML. Upon their differentiation, we found hematopoietic phenotypes of graded severity and/or stage specificity that together delineate a phenotypic roadmap of disease progression culminating in serially transplantable leukemia. We also show that disease stage transitions, both reversal and progression, can be modeled in this system using genetic correction or introduction of mutations via CRISPR/Cas9 and that this iPSC-based approach can be used to uncover disease-stage-specific responses to drugs. Our study therefore provides insight into the cellular events demarcating the initiation and progression of myeloid transformation and a new platform for testing genetic and pharmacological interventions.
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Transformación Celular Neoplásica/patología , Progresión de la Enfermedad , Células Madre Pluripotentes Inducidas/citología , Leucemia Mieloide Aguda/patología , Animales , Antineoplásicos/farmacología , Transformación Celular Neoplásica/efectos de los fármacos , Reprogramación Celular/efectos de los fármacos , Reprogramación Celular/genética , Análisis Mutacional de ADN , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Leucemia Mieloide Aguda/genética , Ratones , Modelos Biológicos , Síndromes Mielodisplásicos/patología , Trasplante de Neoplasias , Fenotipo , Transcriptoma/efectos de los fármacos , Transcriptoma/genéticaRESUMEN
Myelodysplastic syndromes (MDS) are driven by complex genetic and epigenetic alterations. The MSI2 RNA-binding protein has been demonstrated to have a role in acute myeloid leukaemia and stem cell function, but its role in MDS is unknown. Here, we demonstrate that elevated MSI2 expression correlates with poor survival in MDS. Conditional deletion of Msi2 in a mouse model of MDS results in a rapid loss of MDS haematopoietic stem and progenitor cells (HSPCs) and reverses the clinical features of MDS. Inversely, inducible overexpression of MSI2 drives myeloid disease progression. The MDS HSPCs remain dependent on MSI2 expression after disease initiation. Furthermore, MSI2 expression expands and maintains a more activated (G1) MDS HSPC. Gene expression profiling of HSPCs from the MSI2 MDS mice identifies a signature that correlates with poor survival in MDS patients. Overall, we identify a role for MSI2 in MDS representing a therapeutic target in this disease.