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Lipid droplets (LDs) are organelles specialized in the storage of neutral lipids, cholesterol esters and triglycerides, thereby protecting cells from the toxicity of excess lipids while allowing for the mobilization of lipids in times of nutrient deprivation. Defects in LD function are associated with many diseases. S-acylation mediated by zDHHC acyltransferases modifies thousands of proteins, yet the physiological impact of this post-translational modification on individual proteins is poorly understood. Here, we show that zDHHC11 regulates LD catabolism by modifying adipose triacylglyceride lipase (ATGL), the rate-limiting enzyme of lipolysis, both in hepatocyte cultures and in mice. zDHHC11 S-acylates ATGL at cysteine 15. Preventing the S-acylation of ATGL renders it catalytically inactive despite proper localization. Overexpression of zDHHC11 reduces LD size, whereas its elimination enlarges LDs. Mutating ATGL cysteine 15 phenocopies zDHHC11 loss, causing LD accumulation, defective lipolysis and lipophagy. Our results reveal S-acylation as a mode of regulation of ATGL function and LD homoeostasis. Modulating this pathway may offer therapeutic potential for treating diseases linked to defective lipolysis, such as fatty liver disease.
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Targeted protein degradation (TPD) is an emerging therapeutic strategy that would benefit from new chemical entities with which to recruit a wider variety of ubiquitin E3 ligases to target proteins for proteasomal degradation. Here we describe a TPD strategy involving the recruitment of FBXO22 to induce degradation of the histone methyltransferase and oncogene NSD2. UNC8732 facilitates FBXO22-mediated degradation of NSD2 in acute lymphoblastic leukemia cells harboring the NSD2 gain-of-function mutation p.E1099K, resulting in growth suppression, apoptosis and reversal of drug resistance. The primary amine of UNC8732 is metabolized to an aldehyde species, which engages C326 of FBXO22 to recruit the SCFFBXO22 Cullin complex. We further demonstrate that a previously reported alkyl amine-containing degrader targeting XIAP is similarly dependent on SCFFBXO22. Overall, we present a potent NSD2 degrader for the exploration of NSD2 disease phenotypes and a new FBXO22-recruitment strategy for TPD.
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Acute myeloid leukemia stem cells (LSCs) are uniquely reliant on oxidative phosphorylation (OXPHOS) for survival. Moreover, maintenance of OXPHOS is dependent on BCL-2, creating a therapeutic opportunity to target LSCs using the BCL-2 inhibitor venetoclax. While venetoclax-based regimens have shown promising clinical activity, the emergence of drug resistance is prevalent. Thus, in the present study, we investigated how mitochondrial properties may influence venetoclax responsiveness. Our data show that utilization of mitochondrial calcium is fundamentally different between drug-responsive and non-responsive LSCs. By comparison, venetoclax-resistant LSCs demonstrate a more active metabolic (i.e. OXPHOS) status with relatively high levels of calcium. Consequently, we tested genetic and pharmacological approaches to target the mitochondrial calcium uniporter, MCU. We demonstrate that inhibition of calcium uptake reduces OXPHOS and leads to eradication of venetoclax-resistant LSCs. These findings demonstrate a central role for calcium signaling in LSCs and provide an avenue for clinical management of venetoclax resistance.
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R2TP is a chaperone complex consisting of the AAA+ ATPases RUVBL1 and RUVBL2, as well as RPAP3 and PIH1D1 proteins. R2TP is responsible for the assembly of macromolecular complexes mainly acting through different adaptors. Using proximity-labeling mass spectrometry, we identified deleted in primary ciliary dyskinesia (DPCD) as an adaptor of R2TP. Here, we demonstrate that R2TP-DPCD influences ciliogenesis initiation through a unique mechanism by interaction with Akt kinase to regulate its phosphorylation levels rather than its stability. We further show that DPCD is a heart-shaped monomeric protein with two domains. A highly conserved region in the cysteine- and histidine-rich domains-containing proteins and SGT1 (CS) domain of DPCD interacts with the RUVBL2 DII domain with high affinity to form a stable R2TP-DPCD complex both in cellulo and in vitro. Considering that DPCD is one among several CS-domain-containing proteins found to associate with RUVBL1/2, we propose that RUVBL1/2 are CS-domain-binding proteins that regulate complex assembly and downstream signaling.
Assuntos
Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Fosforilação , ATPases Associadas a Diversas Atividades Celulares , CogniçãoRESUMO
Pediatric high-grade gliomas (pHGG) are devastating and incurable brain tumors with recurrent mutations in histone H3.3. These mutations promote oncogenesis by dysregulating gene expression through alterations of histone modifications. We identify aberrant DNA repair as an independent mechanism, which fosters genome instability in H3.3 mutant pHGG, and opens new therapeutic options. The two most frequent H3.3 mutations in pHGG, K27M and G34R, drive aberrant repair of replication-associated damage by non-homologous end joining (NHEJ). Aberrant NHEJ is mediated by the DNA repair enzyme polynucleotide kinase 3'-phosphatase (PNKP), which shows increased association with mutant H3.3 at damaged replication forks. PNKP sustains the proliferation of cells bearing H3.3 mutations, thus conferring a molecular vulnerability, specific to mutant cells, with potential for therapeutic targeting.
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Neoplasias Encefálicas , Glioma , Histonas , Criança , Humanos , Neoplasias Encefálicas/patologia , Reparo do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Glioma/patologia , Histonas/genética , Histonas/metabolismo , Mutação , Fosfotransferases (Aceptor do Grupo Álcool)/genéticaRESUMO
Targeted protein degradation (TPD) is an emerging therapeutic strategy that would benefit from new chemical entities with which to recruit a wider variety of ubiquitin E3 ligases to target proteins for proteasomal degradation. Here, we describe a TPD strategy involving the recruitment of FBXO22 to induce degradation of the histone methyltransferase and oncogene NSD2. UNC8732 facilitates FBXO22-mediated degradation of NSD2 in acute lymphoblastic leukemia cells harboring the NSD2 gain of function mutation p.E1099K, resulting in growth suppression, apoptosis, and reversal of drug resistance. The primary amine of UNC8732 is metabolized to an aldehyde species, which engages C326 of FBXO22 in a covalent and reversible manner to recruit the SCF FBXO22 Cullin complex. We further demonstrate that a previously reported alkyl amine-containing degrader targeting XIAP is similarly dependent on SCF FBXO22 . Overall, we present a highly potent NSD2 degrader for the exploration of NSD2 disease phenotypes and a novel FBXO22-dependent TPD strategy.
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Mitochondrial and peroxisomal anchored protein ligase (MAPL) is a dual ubiquitin and small ubiquitin-like modifier (SUMO) ligase with roles in mitochondrial quality control, cell death and inflammation in cultured cells. Here, we show that MAPL function in the organismal context converges on metabolic control, as knockout mice are viable, insulin-sensitive, and protected from diet-induced obesity. MAPL loss leads to liver-specific activation of the integrated stress response, inducing secretion of stress hormone FGF21. MAPL knockout mice develop fully penetrant spontaneous hepatocellular carcinoma. Mechanistically, the peroxisomal bile acid transporter ABCD3 is a primary MAPL interacting partner and SUMOylated in a MAPL-dependent manner. MAPL knockout leads to increased bile acid production coupled with defective regulatory feedback in liver in vivo and in isolated primary hepatocytes, suggesting cell-autonomous function. Together, our findings establish MAPL function as a regulator of bile acid synthesis whose loss leads to the disruption of bile acid feedback mechanisms. The consequences of MAPL loss in liver, along with evidence of tumor suppression through regulation of cell survival pathways, ultimately lead to hepatocellular carcinogenesis.
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Bile , Proteínas Mitocondriais , Ubiquitina-Proteína Ligases , Animais , Camundongos , Bile/metabolismo , Ácidos e Sais Biliares , Fígado/metabolismo , Camundongos Knockout , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinasRESUMO
We previously reported that acute myeloid leukemia stem cells (LSCs) are uniquely reliant on oxidative phosphorylation (OXPHOS) for survival. Moreover, maintenance of OXPHOS is dependent on BCL2, creating a therapeutic opportunity to target LSCs using the BCL2 inhibitor drug venetoclax. While venetoclax-based regimens have indeed shown promising clinical activity, the emergence of drug resistance is prevalent. Thus, in the present study, we investigated how mitochondrial properties may influence mechanisms that dictate venetoclax responsiveness. Our data show that utilization of mitochondrial calcium is fundamentally different between drug responsive and non-responsive LSCs. By comparison, venetoclax-resistant LSCs demonstrate a more active metabolic (i.e., OXPHOS) status with relatively high steady-state levels of calcium. Consequently, we tested genetic and pharmacological approaches to target the mitochondrial calcium uniporter, MCU. We demonstrate that inhibition of calcium uptake sharply reduces OXPHOS and leads to eradication of venetoclax-resistant LSCs. These findings demonstrate a central role for calcium signaling in the biology of LSCs and provide a therapeutic avenue for clinical management of venetoclax resistance.
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The ERG (ETS-related gene) transcription factor is linked to various types of cancer, including leukemia. However, the specific ERG domains and co-factors contributing to leukemogenesis are poorly understood. Drug targeting a transcription factor such as ERG is challenging. Our study reveals the critical role of a conserved amino acid, proline, at position 199, located at the 3' end of the PNT (pointed) domain, in ERG's ability to induce leukemia. P199 is necessary for ERG to promote self-renewal, prevent myeloid differentiation in hematopoietic progenitor cells, and initiate leukemia in mouse models. Here we show that P199 facilitates ERG's interaction with the NCoR-HDAC3 co-repressor complex. Inhibiting HDAC3 reduces the growth of ERG-dependent leukemic and prostate cancer cells, indicating that the interaction between ERG and the NCoR-HDAC3 co-repressor complex is crucial for its oncogenic activity. Thus, targeting this interaction may offer a potential therapeutic intervention.
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Leucemia , Fatores de Transcrição , Animais , Masculino , Camundongos , Proteínas Correpressoras , Regulação da Expressão Gênica , Genes ReguladoresRESUMO
Breast cancer linked with BRCA1/2 mutations commonly recur and resist current therapies, including PARP inhibitors. Given the lack of effective targeted therapies for BRCA1-mutant cancers, we sought to identify novel targets to selectively kill these cancers. Here, we report that loss of RNF8 significantly protects Brca1-mutant mice against mammary tumorigenesis. RNF8 deficiency in human BRCA1-mutant breast cancer cells was found to promote R-loop accumulation and replication fork instability, leading to increased DNA damage, senescence, and synthetic lethality. Mechanistically, RNF8 interacts with XRN2, which is crucial for transcription termination and R-loop resolution. We report that RNF8 ubiquitylates XRN2 to facilitate its recruitment to R-loop-prone genomic loci and that RNF8 deficiency in BRCA1-mutant breast cancer cells decreases XRN2 occupancy at R-loop-prone sites, thereby promoting R-loop accumulation, transcription-replication collisions, excessive genomic instability, and cancer cell death. Collectively, our work identifies a synthetic lethal interaction between RNF8 and BRCA1, which is mediated by a pathological accumulation of R-loops.
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Proteína BRCA1 , Neoplasias da Mama , Animais , Feminino , Humanos , Camundongos , Proteína BRCA1/metabolismo , Proteína BRCA2/genética , Neoplasias da Mama/genética , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Exorribonucleases/metabolismo , Instabilidade Genômica , Recidiva Local de Neoplasia , Estruturas R-Loop , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Outcomes for patients with acute myeloid leukemia (AML) remain poor due to the inability of current therapeutic regimens to fully eradicate disease-initiating leukemia stem cells (LSC). Previous studies have demonstrated that oxidative phosphorylation (OXPHOS) is an essential process that is targetable in LSC. Sirtuin 3 (SIRT3), a mitochondrial deacetylase with a multi-faceted role in metabolic regulation, has been shown to regulate OXPHOS in cancer models; however, it has not yet been studied in the context of LSC. Thus, we sought to identify if SIRT3 is important for LSC function. Using RNAi and a SIRT3 inhibitor (YC8-02), we demonstrate that SIRT3 is a critical target for the survival of primary human LSC but is not essential for normal human hematopoietic stem and progenitor cell function. In order to elucidate the molecular mechanisms by which SIRT3 is essential in LSC we combined transcriptomic, proteomic, and lipidomic approaches, showing that SIRT3 is important for LSC function through the regulation of fatty acid oxidation (FAO) which is required to support OXPHOS and ATP production in human LSC. Further, we discovered two approaches to further sensitize LSC to SIRT3 inhibition. First, we found that LSC tolerate the toxic effects of fatty acid accumulation induced by SIRT3 inhibition by upregulating cholesterol esterification. Disruption of cholesterol homeostasis sensitizes LSC to YC8-02 and potentiates LSC death. Second, SIRT3 inhibition sensitizes LSC to the BCL-2 inhibitor venetoclax. Together, these findings establish SIRT3 as a regulator of lipid metabolism and potential therapeutic target in primitive AML cells.
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Leucemia Mieloide Aguda , Sirtuína 3 , Humanos , Sirtuína 3/genética , Sirtuína 3/metabolismo , Sirtuína 3/farmacologia , Proteômica , Células-Tronco Neoplásicas/metabolismo , Metabolismo dos Lipídeos , Homeostase , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Ácidos Graxos/metabolismo , Ácidos Graxos/farmacologia , Ácidos Graxos/uso terapêutico , ColesterolRESUMO
A proper understanding of disease etiology will require longitudinal systems-scale reconstruction of the multitiered architecture of eukaryotic signaling. Here we combine state-of-the-art data acquisition platforms and bioinformatics tools to devise PAMAF, a workflow that simultaneously examines twelve omics modalities, i.e., protein abundance from whole-cells, nucleus, exosomes, secretome and membrane; N-glycosylation, phosphorylation; metabolites; mRNA, miRNA; and, in parallel, single-cell transcriptomes. We apply PAMAF in an established in vitro model of TGFß-induced epithelial to mesenchymal transition (EMT) to quantify >61,000 molecules from 12 omics and 10 timepoints over 12 days. Bioinformatics analysis of this EMT-ExMap resource allowed us to identify; -topological coupling between omics, -four distinct cell states during EMT, -omics-specific kinetic paths, -stage-specific multi-omics characteristics, -distinct regulatory classes of genes, -ligand-receptor mediated intercellular crosstalk by integrating scRNAseq and subcellular proteomics, and -combinatorial drug targets (e.g., Hedgehog signaling and CAMK-II) to inhibit EMT, which we validate using a 3D mammary duct-on-a-chip platform. Overall, this study provides a resource on TGFß signaling and EMT.
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Transição Epitelial-Mesenquimal , Proteínas Hedgehog , Transição Epitelial-Mesenquimal/genética , Proteínas Hedgehog/metabolismo , Células Epiteliais/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismoRESUMO
Histone H3 mutations at amino acids 27 (H3K27M) and 34 (H3G34R) are recurrent drivers of pediatric-type high-grade glioma (pHGG). H3K27M mutations lead to global disruption of H3K27me3 through dominant negative PRC2 inhibition, while H3G34R mutations lead to local losses of H3K36me3 through inhibition of SETD2. However, their broader oncogenic mechanisms remain unclear. We characterized the H3.1K27M, H3.3K27M and H3.3G34R interactomes, finding that H3K27M is associated with epigenetic and transcription factor changes; in contrast H3G34R removes a break on cryptic transcription, limits DNA methyltransferase access, and alters mitochondrial metabolism. All 3 mutants had altered interactions with DNA repair proteins and H3K9 methyltransferases. H3K9me3 was reduced in H3K27M-containing nucleosomes, and cis-H3K9 methylation was required for H3K27M to exert its effect on global H3K27me3. H3K9 methyltransferase inhibition was lethal to H3.1K27M, H3.3K27M and H3.3G34R pHGG cells, underscoring the importance of H3K9 methylation for oncohistone-mutant gliomas and suggesting it as an attractive therapeutic target.
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Glioma , Histonas , Aminoácidos/genética , Criança , DNA , Glioma/genética , Glioma/metabolismo , Histonas/genética , Humanos , Mutação/genética , Nucleossomos , Fatores de Transcrição/genéticaRESUMO
The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other coronaviruses mediates host cell entry and is S-acylated on multiple phylogenetically conserved cysteine residues. Multiple protein acyltransferase enzymes have been reported to post-translationally modify spike proteins; however, strategies to exploit this modification are lacking. Using resin-assisted capture MS, we demonstrate that the spike protein is S-acylated in SARS-CoV-2-infected human and monkey epithelial cells. We further show that increased abundance of the acyltransferase ZDHHC5 associates with increased S-acylation of the spike protein, whereas ZDHHC5 knockout cells had a 40% reduction in the incorporation of an alkynyl-palmitate using click chemistry detection. We also found that the S-acylation of the spike protein is not limited to palmitate, as clickable versions of myristate and stearate were also labelled the protein. Yet, we observed that ZDHHC5 was only modified when incubated with alkyne-palmitate, suggesting it has specificity for this acyl-CoA, and that other ZDHHC enzymes may use additional fatty acids to modify the spike protein. Since multiple ZDHHC isoforms may modify the spike protein, we also examined the ability of the FASN inhibitor TVB-3166 to prevent S-acylation of the spike proteins of SARS-CoV-2 and human CoV-229E. We show that treating cells with TVB-3166 inhibited S-acylation of expressed spike proteins and attenuated the ability of SARS-CoV-2 and human CoV-229E to spread in vitro. Our findings further substantiate the necessity of CoV spike protein S-acylation and demonstrate that de novo fatty acid synthesis is critical for the proper S-acylation of the spike protein.
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COVID-19 , Glicoproteína da Espícula de Coronavírus , Acilação , Aciltransferases/metabolismo , Alcinos , Azetidinas , Coenzima A/metabolismo , Cisteína , Ácido Graxo Sintase Tipo I/metabolismo , Humanos , Miristatos , Nitrilas , Palmitatos , Pirazóis , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/metabolismo , EstearatosRESUMO
Mitochondrial metabolites regulate leukaemic and normal stem cells by affecting epigenetic marks. How mitochondrial enzymes localize to the nucleus to control stem cell function is less understood. We discovered that the mitochondrial metabolic enzyme hexokinase 2 (HK2) localizes to the nucleus in leukaemic and normal haematopoietic stem cells. Overexpression of nuclear HK2 increases leukaemic stem cell properties and decreases differentiation, whereas selective nuclear HK2 knockdown promotes differentiation and decreases stem cell function. Nuclear HK2 localization is phosphorylation-dependent, requires active import and export, and regulates differentiation independently of its enzymatic activity. HK2 interacts with nuclear proteins regulating chromatin openness, increasing chromatin accessibilities at leukaemic stem cell-positive signature and DNA-repair sites. Nuclear HK2 overexpression decreases double-strand breaks and confers chemoresistance, which may contribute to the mechanism by which leukaemic stem cells resist DNA-damaging agents. Thus, we describe a non-canonical mechanism by which mitochondrial enzymes influence stem cell function independently of their metabolic function.
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Hexoquinase , Leucemia Mieloide Aguda , Cromatina/metabolismo , DNA/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Hexoquinase/genética , Hexoquinase/metabolismo , Humanos , Leucemia Mieloide Aguda/metabolismoRESUMO
Protein degradation is critical to maintaining cellular homeostasis, and perturbation of the ubiquitin proteasome system leads to the accumulation of protein aggregates. These aggregates are either directed towards autophagy for destruction or sequestered into an inclusion, termed the aggresome, at the centrosome. Utilizing high-resolution quantitative analysis, here, we define aggresome assembly at the centrosome in human cells. Centriolar satellites are proteinaceous granules implicated in the trafficking of proteins to the centrosome. During aggresome assembly, satellites were required for the growth of the aggresomal structure from an initial ring of phosphorylated HSP27 deposited around the centrioles. The seeding of this phosphorylated HSP27 ring depended on the centrosomal proteins CP110, CEP97 and CEP290. Owing to limiting amounts of CP110, senescent cells, which are characterized by the accumulation of protein aggregates, were defective in aggresome formation. Furthermore, satellites and CP110-CEP97-CEP290 were required for the aggregation of mutant huntingtin. Together, these data reveal roles for CP110-CEP97-CEP290 and satellites in the control of cellular proteostasis and the aggregation of disease-relevant proteins.
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Centríolos , Agregados Proteicos , Antígenos de Neoplasias/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centríolos/metabolismo , Centrossomo/metabolismo , Cílios/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas de Choque Térmico HSP27/metabolismo , Humanos , Proteínas Associadas aos Microtúbulos/metabolismoRESUMO
Deletion of phenylalanine 508 (∆F508) of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) anion channel protein is the leading cause of Cystic Fibrosis (CF). Here, we report the analysis of CFTR and ∆F508-CFTR interactomes using BioID (proximity-dependent biotin identification), a technique that can also detect transient associations. We identified 474 high-confidence CFTR proximity-interactors, 57 of which have been previously validated, with the remainder representing novel interaction space. The ∆F508 interactome, comprising 626 proximity-interactors was markedly different from its wild type counterpart, with numerous alterations in protein associations categorized in membrane trafficking and cellular stress functions. Furthermore, analysis of the ∆F508 interactome in cells treated with Orkambi identified several interactions that were altered as a result of this drug therapy. We examined two candidate CFTR proximity interactors, VAPB and NOS1AP, in functional assays designed to assess surface delivery and overall chloride efflux. VAPB depletion impacted both CFTR surface delivery and chloride efflux, whereas NOS1AP depletion only affected the latter. The wild type and ∆F508-CFTR interactomes represent rich datasets that could be further mined to reveal additional candidates for the functional rescue of ∆F508-CFTR.
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Aminofenóis/farmacologia , Aminopiridinas/farmacologia , Benzodioxóis/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Mapas de Interação de Proteínas/efeitos dos fármacos , Quinolonas/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Cloretos/metabolismo , Combinação de Medicamentos , Células HEK293 , Humanos , Espectrometria de Massas , Mutação , Proteínas de Transporte Vesicular/metabolismoRESUMO
AML cells are arranged in a hierarchy with stem/progenitor cells giving rise to more differentiated bulk cells. Despite the importance of stem/progenitors in the pathogenesis of AML, the determinants of the AML stem/progenitor state are not fully understood. Through a comparison of genes that are significant for growth and viability of AML cells by way of a CRISPR screen, with genes that are differentially expressed in leukemia stem cells (LSC), we identified importin 11 (IPO11) as a novel target in AML. Importin 11 (IPO11) is a member of the importin ß family of proteins that mediate transport of proteins across the nuclear membrane. In AML, knockdown of IPO11 decreased growth, reduced engraftment potential of LSC, and induced differentiation. Mechanistically, we identified the transcription factors BZW1 and BZW2 as novel cargo of IPO11. We further show that BZW1/2 mediate a transcriptional signature that promotes stemness and survival of LSC. Thus, we demonstrate for the first time how specific cytoplasmic-nuclear regulation supports stem-like transcriptional signature in relapsed AML.
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Leucemia Mieloide Aguda , beta Carioferinas , Transporte Ativo do Núcleo Celular , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Leucemia Mieloide Aguda/patologia , Células-Tronco Neoplásicas/patologia , Células-Tronco/metabolismo , beta Carioferinas/genética , beta Carioferinas/metabolismoRESUMO
Cancer cells bearing distinct KRAS mutations exhibit variable sensitivity to SHP2 inhibitors (SHP2i). Here we show that cells harboring KRAS Q61H are uniquely resistant to SHP2i, and investigate the underlying mechanisms using biophysics, molecular dynamics, and cell-based approaches. Q61H mutation impairs intrinsic and GAP-mediated GTP hydrolysis, and impedes activation by SOS1, but does not alter tyrosyl phosphorylation. Wild-type and Q61H-mutant KRAS are both phosphorylated by Src on Tyr32 and Tyr64 and dephosphorylated by SHP2, however, SHP2i does not reduce ERK phosphorylation in KRAS Q61H cells. Phosphorylation of wild-type and Gly12-mutant KRAS, which are associated with sensitivity to SHP2i, confers resistance to regulation by GAP and GEF activities and impairs binding to RAF, whereas the near-complete GAP/GEF-resistance of KRAS Q61H remains unaltered, and high-affinity RAF interaction is retained. SHP2 can stimulate KRAS signaling by modulating GEF/GAP activities and dephosphorylating KRAS, processes that fail to regulate signaling of the Q61H mutant.