RESUMO
In the rapidly evolving landscape of medical research, the emergence of RNA-based therapeutics is paradigm shifting. It is mainly driven by the molecular adaptability and capacity to provide precision in targeting. The coronavirus disease 2019 pandemic crisis underscored the effectiveness of the mRNA therapeutic development platform and brought it to the forefront of RNA-based interventions. These RNA-based therapeutic approaches can reshape gene expression, manipulate cellular functions, and correct the aberrant molecular processes underlying various diseases. The new technologies hold the potential to engineer and deliver tailored therapeutic agents to tackle genetic disorders, cancers, and infectious diseases in a highly personalized and precisely tuned manner. The review discusses the most recent advancements in the field of mRNA therapeutics for cancer treatment, with a focus on the features of the most utilized RNA-based therapeutic interventions, current pre-clinical and clinical developments, and the remaining challenges in delivery strategies, effectiveness, and safety considerations.
RESUMO
RNA deadenylation, the process of shortening of the 3' poly(A) tail of an RNA molecule, is one of the key steps of post-transcriptional regulation of gene expression in eukaryotic cells. PAN2/3 and CCR4-NOT (CNOT) are the two dominant RNA deadenylation complexes, which play central roles in mediating mRNA decay and translation. While degradation is the final fate of virtually all RNAs in their life cycles, selection of RNA targets as well as control of the rate and timing of RNA decay, in coordination with other molecular pathways, including translation, can be modulated in certain contexts. Such regulation influences cell growth, proliferation, and differentiation at the cellular level; and contributes to establish polarity and regulate signaling at the tissue level. Dysregulation of deadenylation processes have also been implicated in human diseases ranging from cardiac diseases and neurodevelopmental disorders to cancers. In this review, we will discuss mechanisms of gene expression control mediated by the RNA deadenylation complexes and highlight relevant evidence supporting the emerging roles of RNA deadenylation and its regulatory proteins during development and in diseases. A systemic understanding of these mechanisms will be a critical foundation for development of effective strategies to therapeutically target them.
Assuntos
Exorribonucleases , RNA , Humanos , RNA/genética , Exorribonucleases/genética , Exorribonucleases/metabolismo , RNA Mensageiro/metabolismo , Fatores de Transcrição/metabolismo , Regulação da Expressão GênicaRESUMO
Double plant homeodomain finger 2 (DPF2) is a highly evolutionarily conserved member of the d4 protein family that is ubiquitously expressed in human tissues and was recently shown to inhibit the myeloid differentiation of hematopoietic stem/progenitor and acute myelogenous leukemia cells. Here, we present the crystal structure of the tandem plant homeodomain finger domain of human DPF2 at 1.6-Å resolution. We show that DPF2 interacts with the acetylated tails of both histones 3 and 4 via bipartite binding pockets on the DPF2 surface. Blocking these interactions through targeted mutagenesis of DPF2 abolishes its recruitment to target chromatin regions as well as its ability to prevent myeloid differentiation in vivo. Our findings suggest that the histone binding of DPF2 plays an important regulatory role in the transcriptional program that drives myeloid differentiation.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Histonas/química , Histonas/metabolismo , Células Mieloides/citologia , Células Mieloides/metabolismo , Acetilação , Diferenciação Celular/fisiologia , Cromatina/química , Cromatina/metabolismo , Cristalografia por Raios X , Hematopoese/fisiologia , Humanos , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Fatores de TranscriçãoRESUMO
Protein synthesis is frequently deregulated during tumorigenesis. However, the precise contexts of selective translational control and the regulators of such mechanisms in cancer is poorly understood. Here, we uncovered CNOT3, a subunit of the CCR4-NOT complex, as an essential modulator of translation in myeloid leukemia. Elevated CNOT3 expression correlates with unfavorable outcomes in patients with acute myeloid leukemia (AML). CNOT3 depletion induces differentiation and apoptosis and delayed leukemogenesis. Transcriptomic and proteomic profiling uncovers c-MYC as a critical downstream target which is translationally regulated by CNOT3. Global analysis of mRNA features demonstrates that CNOT3 selectively influences expression of target genes in a codon usage dependent manner. Furthermore, CNOT3 associates with the protein network largely consisting of ribosomal proteins and translation elongation factors in leukemia cells. Overall, our work elicits the direct requirement for translation efficiency in tumorigenesis and propose targeting the post-transcriptional circuitry via CNOT3 as a therapeutic vulnerability in AML.
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Leucemia Mieloide Aguda , Proteômica , Fatores de Transcrição , Humanos , Carcinogênese/genética , Diferenciação Celular , Leucemia Mieloide Aguda/genética , Receptores CCR4 , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Most cancers are characterized by multiple molecular alterations, but identification of the key proteins involved in these signaling pathways is currently beyond reach. We show that the inhibitor PU-H71 preferentially targets tumor-enriched Hsp90 complexes and affinity captures Hsp90-dependent oncogenic client proteins. We have used PU-H71 affinity capture to design a proteomic approach that, when combined with bioinformatic pathway analysis, identifies dysregulated signaling networks and key oncoproteins in chronic myeloid leukemia. The identified interactome overlaps with the well-characterized altered proteome in this cancer, indicating that this method can provide global insights into the biology of individual tumors, including primary patient specimens. In addition, we show that this approach can be used to identify previously uncharacterized oncoproteins and mechanisms, potentially leading to new targeted therapies. We further show that the abundance of the PU-H71-enriched Hsp90 species, which is not dictated by Hsp90 expression alone, is predictive of the cell's sensitivity to Hsp90 inhibition.
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Regulação Neoplásica da Expressão Gênica/fisiologia , Proteínas de Choque Térmico HSP90/metabolismo , Neoplasias/metabolismo , Proteômica/métodos , Animais , Antineoplásicos/farmacologia , Benzodioxóis/farmacologia , Linhagem Celular Tumoral , Biologia Computacional , Descoberta de Drogas , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/genética , Humanos , Neoplasias/genética , Purinas/farmacologia , Transdução de SinaisRESUMO
RNA modifications play an important role in various cancers including blood cancers by controlling gene expression programs critical for survival, proliferation and differentiation of cancer cells. While hundreds of RNA modifications have been identified, many have not been functionally characterized. With development of enabling technologies to identify and map RNA modifications, tremendous advancement has been made in our understanding of the biological functions of these molecular markers in diverse cellular contexts. In the last 5 years, N6-methyladenosine (m6A), the most prevalent internal mRNA modification, has been extensively implicated in many facets of leukemogenesis. Other types of RNA modifications are also involved in the regulation of cell fate decisions and tumorigenesis. Here, we summarize existing knowledge and recent discoveries regarding the role of RNA modifications in leukemia. We choose to highlight cutting-edge techniques to characterize and profile RNA modifications while discussing critical functions of key modifiers and regulatory mechanisms in the pathogenesis of hematological malignancies and touch on therapeutic strategies targeting RNA modifications. These important advancements in the field will continue to foster a strong foundation for the development of innovative treatments for hematological malignancies.
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Neoplasias Hematológicas , Leucemia , Humanos , RNA Mensageiro/genética , Diferenciação Celular , Leucemia/genética , Leucemia/terapia , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/terapiaRESUMO
Regulation of gene expression at the RNA level is an important regulatory mechanism in cancer. However, posttranscriptional molecular pathways underlying tumorigenesis remain largely unexplored. In this study, we uncovered a functional axis consisting of microRNA (miR)-148a-3p, RNA helicase DDX6, and its downstream target thioredoxin-interacting protein (TXNIP) in acute myeloid leukemia (AML). Using a DROSHA-knockout cell system to evaluate miR-mediated gene expression control, we comprehensively profiled putative transcripts regulated by miR-148a-3p and identified DDX6 as a direct target of miR-148a-3p in AML cells. DDX6 depletion induced cell cycle arrest, apoptosis, and differentiation, although delaying leukemia development in vivo. Genome-wide assessment of DDX6-binding transcripts and gene expression profiling of DDX6-depleted cells revealed TXNIP, a tumor suppressor, as the functional downstream target of DDX6. Overall, our study identified DDX6 as a posttranscriptional regulator that is required for AML survival. We proposed the regulatory link between miR-148a-3p and DDX6 as a potential therapeutic target in leukemia.
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Leucemia Mieloide Aguda , MicroRNAs , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Genes Supressores de Tumor , Leucemia Mieloide Aguda/genética , Diferenciação Celular/fisiologia , Proteínas Proto-Oncogênicas/genética , RNA Helicases DEAD-box/genéticaRESUMO
Multiple myeloma (MM) is an incurable malignancy of plasma cells. To identify targets for MM immunotherapy, we develop an integrated pipeline based on mass spectrometry analysis of seven MM cell lines and RNA sequencing (RNA-seq) from 900+ patients. Starting from 4,000+ candidates, we identify the most highly expressed cell surface proteins. We annotate candidate protein expression in many healthy tissues and validate the expression of promising targets in 30+ patient samples with relapsed/refractory MM, as well as in primary healthy hematopoietic stem cells and T cells by flow cytometry. Six candidates (ILT3, SEMA4A, CCR1, LRRC8D, FCRL3, IL12RB1) and B cell maturation antigen (BCMA) present the most favorable profile in malignant and healthy cells. We develop a bispecific T cell engager targeting ILT3 that shows potent killing effects in vitro and decreased tumor burden and prolonged mice survival in vivo, suggesting therapeutic relevance. Our study uncovers MM-associated antigens that hold great promise for immune-based therapies of MM.
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Mieloma Múltiplo , Animais , Camundongos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/patologia , Imunoterapia/métodos , Linfócitos T , Plasmócitos/metabolismoRESUMO
Tissue homeostasis is maintained after stress by engaging and activating the hematopoietic stem and progenitor compartments in the blood. Hematopoietic stem cells (HSCs) are essential for long-term repopulation after secondary transplantation. Here, using a conditional knockout mouse model, we revealed that the RNA-binding protein SYNCRIP is required for maintenance of blood homeostasis especially after regenerative stress due to defects in HSCs and progenitors. Mechanistically, we find that SYNCRIP loss results in a failure to maintain proteome homeostasis that is essential for HSC maintenance. SYNCRIP depletion results in increased protein synthesis, a dysregulated epichaperome, an accumulation of misfolded proteins and induces endoplasmic reticulum stress. Additionally, we find that SYNCRIP is required for translation of CDC42 RHO-GTPase, and loss of SYNCRIP results in defects in polarity, asymmetric segregation, and dilution of unfolded proteins. Forced expression of CDC42 recovers polarity and in vitro replating activities of HSCs. Taken together, we uncovered a post-transcriptional regulatory program that safeguards HSC self-renewal capacity and blood homeostasis.
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Células-Tronco Hematopoéticas , Ribonucleoproteínas Nucleares Heterogêneas , Proteostase , Animais , Camundongos , Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Camundongos Knockout , Proteostase/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Post-transcriptional RNA modifications determine RNA fate by influencing numerous processes such as translation, decay and localization. One of the most abundant RNA modifications is N6-methyladenoside (m6A), which has been shown to be important in healthy as well as malignant hematopoiesis. Several proteins representing key players in m6A RNA biology, such as m6A writers, erasers and readers, were recently reported to be essential for hematopoietic stem cell (HSC) function. In leukemia, expression of m6A regulators has been shown to be increased, opening up potential opportunities for therapeutic exploitation by targeting them in blood malignancies. These recent discoveries were the focus of the Fall 2021 International Society for Experimental Hematology New Investigators webinar. We review here the latest findings in the field of mRNA modifications in normal and malignant hematopoiesis and how this might open up novel therapeutic options.
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Hematopoese , Leucemia , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Humanos , Leucemia/genética , RNA/metabolismo , Processamento Pós-Transcricional do RNARESUMO
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.
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Cromatina , Leucemia Mieloide Aguda , Animais , Cromatina/genética , Regulação da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas/genética , Humanos , Leucemia Mieloide Aguda/genética , Camundongos , Proteínas de Ligação a RNA/genética , Fatores de Transcrição/genéticaRESUMO
COVID-19 has unfortunately halted lab work, conferences, and in-person networking, which is especially detrimental to researchers just starting their labs. Through social media and our reviewer networks, we met some early-career stem cell investigators impacted by the closures. Here, they introduce themselves and their research to our readers.
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COVID-19 , Pesquisadores , Feminino , Humanos , MasculinoRESUMO
The cell-context dependency for RNA binding proteins (RBPs) mediated control of stem cell fate remains to be defined. Here we adapt the HyperTRIBE method using an RBP fused to a Drosophila RNA editing enzyme (ADAR) to globally map the mRNA targets of the RBP MSI2 in mammalian adult normal and malignant stem cells. We reveal a unique MUSASHI-2 (MSI2) mRNA binding network in hematopoietic stem cells that changes during transition to multipotent progenitors. Additionally, we discover a significant increase in RNA binding activity of MSI2 in leukemic stem cells compared with normal hematopoietic stem and progenitor cells, resulting in selective regulation of MSI2's oncogenic targets. This provides a basis for MSI2 increased dependency in leukemia cells compared to normal cells. Moreover, our study provides a way to measure RBP function in rare cells and suggests that RBPs can achieve differential binding activity during cell state transition independent of gene expression.
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Diferenciação Celular/genética , Células-Tronco Hematopoéticas/patologia , Leucemia/genética , Células-Tronco Neoplásicas/patologia , Proteínas de Ligação a RNA/metabolismo , Adenosina Desaminase/genética , Animais , Sítios de Ligação/genética , Modelos Animais de Doenças , Proteínas de Drosophila/genética , Regulação Leucêmica da Expressão Gênica , Redes Reguladoras de Genes , Células HEK293 , Humanos , Leucemia/sangue , Leucemia/patologia , Camundongos , Camundongos Knockout , Cultura Primária de Células , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , RNA-Seq , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Hematopoietic development and differentiation are highly regulated processes, and recent studies focusing on m6A mRNA methylation have uncovered how this mark controls cell fate in both normal and malignant hematopoietic states. In this review, we focus on how writers, readers, and erasers of RNA methylation can mediate distinct phenotypes on mRNAs and on cells. Targeting the RNA methylation program has emerged as a potential novel therapeutic strategy, and we explore the role for these regulators in both normal and dysregulated cell contexts. SIGNIFICANCE: RNA methylation is required for cancer cell survival in solid tumors and in acute myeloid leukemia, and targeting this pathway has been proposed as a new therapeutic strategy in cancer. However, understanding the role for RNA methylation in both normal and malignant states is essential for understanding the potential consequences for therapeutic intervention.
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Leucemia Mieloide Aguda/metabolismo , RNA Mensageiro/metabolismo , Animais , Diferenciação Celular , Regulação Neoplásica da Expressão Gênica , Hematopoese , Humanos , Leucemia Mieloide Aguda/genética , Metilação , RNA Mensageiro/genéticaRESUMO
Stem cells balance cellular fates through asymmetric and symmetric divisions in order to self-renew or to generate downstream progenitors. Symmetric commitment divisions in stem cells are required for rapid regeneration during tissue damage and stress. The control of symmetric commitment remains poorly defined. Using single-cell RNA sequencing (scRNA-seq) in combination with transcriptomic profiling of HSPCs (hematopoietic stem and progenitor cells) from control and m6A methyltransferase Mettl3 conditional knockout mice, we found that m6A-deficient hematopoietic stem cells (HSCs) fail to symmetrically differentiate. Dividing HSCs are expanded and are blocked in an intermediate state that molecularly and functionally resembles multipotent progenitors. Mechanistically, RNA methylation controls Myc mRNA abundance in differentiating HSCs. We identified MYC as a marker for HSC asymmetric and symmetric commitment. Overall, our results indicate that RNA methylation controls symmetric commitment and cell identity of HSCs and may provide a general mechanism for how stem cells regulate differentiation fate choice.
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Diferenciação Celular , Linhagem da Célula , Hematopoese , Células-Tronco Hematopoéticas/citologia , Metiltransferases/fisiologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Feminino , Células-Tronco Hematopoéticas/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Masculino , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Proto-Oncogênicas c-myc/genética , Estabilidade de RNA , Análise de Célula ÚnicaRESUMO
One of the biggest challenges in treating acute myeloid leukemia (AML) is relapse of aggressive disease after treatment. In this issue of Cancer Cell, Boyd et al. characterize a molecularly distinct population of chemotherapy-induced transient leukemic regenerating cells (LRCs), which can be exploited to prevent AML recurrence.
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Antineoplásicos , Leucemia Mieloide Aguda , Humanos , RecidivaRESUMO
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 da Medula Óssea/citologia , Diferenciação Celular/fisiologia , Leucemia Mieloide Aguda/patologia , Metiltransferases/fisiologia , Adenosina/biossíntese , Células Cultivadas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Humanos , Células Tumorais CultivadasRESUMO
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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Regulação Leucêmica da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas/genética , Leucemia Mieloide/genética , Proteínas de Ligação a RNA/metabolismo , Animais , Sobrevivência Celular , Feminino , Hematopoese/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Leucemia Aguda Bifenotípica/genética , Leucemia Aguda Bifenotípica/patologia , Leucemia Mieloide/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Progenitoras Mieloides/metabolismo , Células Progenitoras Mieloides/patologia , RNA Interferente Pequeno , Proteínas de Ligação a RNA/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Epigenetic regulation of key transcriptional programs is a critical mechanism that controls hematopoietic development, and, thus, aberrant expression patterns or mutations in epigenetic regulators occur frequently in hematologic malignancies. We demonstrate that the Polycomb protein L3MBTL1, which is monoallelically deleted in 20q- myeloid malignancies, represses the ability of stem cells to drive hematopoietic-specific transcriptional programs by regulating the expression of SMAD5 and impairing its recruitment to target regulatory regions. Indeed, knockdown of L3MBTL1 promotes the development of hematopoiesis and impairs neural cell fate in human pluripotent stem cells. We also found a role for L3MBTL1 in regulating SMAD5 target gene expression in mature hematopoietic cell populations, thereby affecting erythroid differentiation. Taken together, we have identified epigenetic priming of hematopoietic-specific transcriptional networks, which may assist in the development of therapeutic approaches for patients with anemia.
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Diferenciação Celular/genética , Proteínas Cromossômicas não Histona/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Hematopoese/genética , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Proteína Smad5/metabolismo , Transcrição Gênica , Regulação para Baixo , Células Precursoras Eritroides/citologia , Células Precursoras Eritroides/metabolismo , Humanos , Imunofenotipagem , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Proteínas Repressoras , Proteína Smad5/genética , Proteínas Supressoras de TumorRESUMO
Histone-modifying enzymes have recently been shown to play a central role in the regulation of both normal and malignant hematopoiesis. Post-translational modifications of histones and non-histone proteins underlies a regulatory complexity affecting numerous processes including transcriptional regulation, RNA processing and DNA damage response. Insights into the functions of these enzymes as well as their role in the epigenetic alterations found in leukemia will guide the development of novel therapeutic approaches. This review discusses examples of the proteins that have been implicated in the pathogenesis of leukemia, that may serve as potential therapeutic targets.