RESUMEN
Reduced succinate dehydrogenase (SDH) activity resulting in adverse succinate accumulation was previously considered relevant only in 0.05 to 0.5% of kidney cancers associated with germline SDH mutations. Here, we sought to examine a broader role for SDH loss in kidney cancer pathogenesis/progression. We report that underexpression of SDH subunits resulting in accumulation of oncogenic succinate is a common feature in clear cell renal cell carcinoma (ccRCC) (â¼80% of all kidney cancers), with a marked adverse impact on survival in ccRCC patients (n = 516). We show that SDH down-regulation is a critical brake in the TCA cycle during ccRCC pathogenesis and progression. In exploring mechanisms of SDH down-regulation in ccRCC, we report that Von Hippel-Lindau loss-induced hypoxia-inducible factor-dependent up-regulation of miR-210 causes direct inhibition of the SDHD transcript. Moreover, shallow deletion of SDHB occurs in â¼20% of ccRCC. We then demonstrate that SDH loss-induced succinate accumulation contributes to adverse loss of 5-hydroxymethylcytosine, gain of 5-methylcytosine, and enhanced invasiveness in ccRCC via inhibition of ten-eleven translocation (TET)-2 activity. Intriguingly, binding affinity between the catalytic domain of recombinant TET-2 and succinate was found to be very low, suggesting that the mechanism of succinate-induced attenuation of TET-2 activity is likely via product inhibition rather than competitive inhibition. Finally, exogenous ascorbic acid, a TET-activating demethylating agent, led to reversal of the above oncogenic effects of succinate in ccRCC cells. Collectively, our study demonstrates that functional SDH deficiency is a common adverse feature of ccRCC and not just limited to the kidney cancers associated with germline SDH mutations.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Carcinoma de Células Renales/patología , Metilación de ADN , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Renales/patología , Succinato Deshidrogenasa/metabolismo , 5-Metilcitosina/química , Apoptosis , Biomarcadores de Tumor/genética , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/metabolismo , Ciclo Celular , Movimiento Celular , Proliferación Celular , Humanos , Neoplasias Renales/genética , Neoplasias Renales/metabolismo , Mutación , Invasividad Neoplásica , Pronóstico , Succinato Deshidrogenasa/genética , Tasa de Supervivencia , Células Tumorales CultivadasRESUMEN
The proton-coupled folate transporter (PCFT, SLC46A1) is required for folate intestinal absorption and transport across the choroid plexus. Recent work has identified a F392V mutation causing hereditary folate malabsorption. However, the residue properties responsible for this loss of function remains unknown. Using site-directed mutagenesis, we observed complete loss of function with charged (Lys, Asp, and Glu) and polar (Thr, Ser, and Gln) Phe-392 substitutions and minimal function with some neutral substitutions; however, F392M retained full function. Using the substituted-cysteine accessibility method (with N-biotinyl aminoethyl methanethiosulfonate labeling), Phe-392 mutations causing loss of function, although preserving membrane expression and trafficking, also resulted in loss of accessibility of the substituted cysteine in P314C-PCFT located within the aqueous translocation pathway. F392V function and accessibility of the P314C cysteine were restored by insertion of a G305L (suppressor) mutation. A S196L mutation localized in proximity to Gly-305 by homology modeling was inactive. However, when inserted into the inactive F392V scaffold, function was restored (mutually compensatory mutations), as was accessibility of the P314C cysteine residue. Reduced function, documented with F392H PCFT, was due to a 15-fold decrease in methotrexate influx Vmax, accompanied by a decreased influx Kt (4.5-fold) and Ki (3-fold). The data indicate that Phe-392 is required for rapid oscillation of the carrier among its conformational states and suggest that this is achieved by dampening affinity of the protein for its folate substrates. F392V and other inactivating Phe-392 PCFT mutations lock the protein in its inward-open conformation. Reach (length) and hydrophobicity of Phe-392 appear to be features required for full activity.
Asunto(s)
Transportador de Folato Acoplado a Protón/metabolismo , Secuencia de Aminoácidos , Animales , Transporte Biológico , Cisteína/química , Cisteína/metabolismo , Deficiencia de Ácido Fólico/patología , Células HeLa , Humanos , Cinética , Síndromes de Malabsorción/patología , Metotrexato/metabolismo , Mutagénesis Sitio-Dirigida , Estructura Terciaria de Proteína , Transportador de Folato Acoplado a Protón/química , Transportador de Folato Acoplado a Protón/genéticaRESUMEN
The proton-coupled folate transporter (PCFT) mediates intestinal absorption of folates and their transport from blood to cerebrospinal fluid across the choroid plexus. Substitutions at Asp-109 in the first intracellular loop between the first and second transmembrane domains (TMDs) abolish PCFT function, but protein expression and trafficking to the cell membrane are retained. Here, we used site-directed mutagenesis, the substituted-cysteine accessibility method, functional analyses, and homology modeling to determine whether the D109A substitution locks PCFT in one of its conformational states. Cys-substituted residues lining the PCFT aqueous translocation pathway and accessible in WT PCFT to the membrane-impermeable cysteine-biotinylation reagent, MTSEA-biotin, lost accessibility when introduced into the D109A scaffold. Substitutions at Gly-305 located exofacially within the eighth TMD, particularly with bulky residues, when introduced into the D109A scaffold largely restored function and MTSEA-biotin accessibility to Cys-substituted residues within the pathway. Likewise, Ser-196 substitution in the fifth TMD, predicted by homology modeling to be in proximity to Gly-305, also partially restored function found in solute transporters, is critical to oscillation of the carrier among its conformational states. Substitutions at Asp-109 and Gly-112 lock PCFT in an inward-open conformation, resulting in the loss of function. However, the integrity of the locked protein is preserved, indicated by the restoration of function after insertion of a second "unlocking" mutation. and accessibility. Similarly, the inactivating G112K substitution within the first intracellular loop was partially reactivated by introducing the G305L substitution. These data indicate that the first intracellular loop, with a sequence identical to "motif A" (GXXXDXXGR(R/K)).
Asunto(s)
Transportador de Folato Acoplado a Protón/metabolismo , Cisteína/química , Glicina/metabolismo , Células HeLa , Humanos , Cinética , Mutación , Conformación Proteica , Transportador de Folato Acoplado a Protón/químicaRESUMEN
The proton-coupled folate transporter (PCFT-SLC46A1) is required for folate transport across the apical membrane of the small intestine and across the choroid plexus. This study focuses on the structure/function of the 7th transmembrane domain (TMD), and its relationship to the 8th TMD as assessed by the substituted cysteine accessibility method (SCAM) and dicysteine cross-linking. Nine exofacial residues (I278C; H281C-L288C) of 23 residues in the 7th TMD were accessible to 2-((biotinoyl)amino)ethyl methanethiosulfonate (MTSEA-biotin). Pemetrexed, a high-affinity substrate for PCFT, decreased or abolished biotinylation of seven of these residues consistent with their location in or near the folate binding pocket. Homology models of PCFT based on Glut5 fructose transporter structures in both inward- and outward- open conformations were constructed and predicted that two pairs of residues (T289-I304C and Q285-Q311C) from the 7th and 8th TMDs should be in sufficiently close proximity to form a disulfide bond when substituted with cysteines. The single Cys-substituted mutants were accessible to MTSEA-biotin and functional with and without pretreatment with dithiotreitol. However, the double mutants were either not accessible at all, or accessibility was markedly reduced and function markedly impaired. This occurred spontaneously without inclusion of an oxidizing agent. Dithiotreitol restored accessibility and function consistent with disulfide bond disruption. The data establish the proximity of exofacial regions of the 7th and 8th TMDs and their role in defining the aqueous translocation pathway and suggest that these helices may be a component of an exofacial cleft through which substrates enter the protein binding pocket in its outward-open conformation.
Asunto(s)
Ácido Fólico/metabolismo , Transportador de Folato Acoplado a Protón/metabolismo , Transporte Biológico , Cisteína , Disulfuros/metabolismo , Células HeLa , Humanos , Cinética , Modelos Moleculares , Mutación , Oxidación-Reducción , Pemetrexed/metabolismo , Conformación Proteica en Hélice alfa , Dominios Proteicos , Transportador de Folato Acoplado a Protón/química , Transportador de Folato Acoplado a Protón/genética , Relación Estructura-ActividadRESUMEN
The proton-coupled folate transporter (PCFT) is ubiquitously expressed in solid tumors to which it delivers antifolates, particularly pemetrexed, into cancer cells. Studies of PCFT-mediated transport, to date, have focused exclusively on the influx of folates and antifolates. This article addresses the impact of PCFT on concentrative transport, critical to the formation of the active polyglutamate congeners, and at pH levels relevant to the tumor microenvironment. An HeLa-derived cell line was employed, in which folate-specific transport was mediated exclusively by PCFT. At pH 7.0, there was a substantial chemical gradient for methotrexate that decreased as the extracellular pH was increased. A chemical gradient was still detected at pH 7.4 in the usual HEPES-based transport buffer in contrast to what was observed in a bicarbonate/CO2-buffered medium. This antifolate gradient correlated with an alkaline intracellular pH in the former (pH 7.85), but not the latter (pH 7.39), buffer and was abolished by the protonophore carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone. The gradient in HEPES buffer at pH 7.4 was the result of the activity of Na+/H+ exchanger(s); it was eliminated by inhibitors of Na+/H+ exchanger (s) or Na+/K+ ATPase. An antifolate chemical gradient was also detected in bicarbonate buffer at pH 6.9 versus 7.4, also suppressed by carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone. When the membrane potential is considered, PCFT generates substantial transmembrane electrochemical-potential gradients at extracellular pH levels relevant to the tumor microenvironment. The augmentation of intracellular pH, when cells are in a HEPES buffer, should be taken into consideration in studies that encompass all proton-coupled transporter families.
Asunto(s)
Antagonistas del Ácido Fólico/farmacocinética , Metotrexato/farmacocinética , Transportador de Folato Acoplado a Protón/metabolismo , Transporte Biológico Activo , Tampones (Química) , HEPES/farmacología , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Ácido Poliglutámico/metabolismo , Microambiente TumoralRESUMEN
The substituted cysteine accessibility method (SCAM) is widely used to study the structure and function of channels, receptors and transporters. In its usual application, a cysteine residue is introduced into a protein which lacks native cysteines following which the accessibility of the residue to the aqueous compartment is assessed. Implicit, and generally assumed, is that if the cysteine-substituted residue is not available to react with sulfhydryl reagents it is not exposed to the extracellular compartment or within the aqueous translocation pathway. We demonstrate here, in a Hela-derived cell line, that some cysteine-substituted residues of the proton-coupled folate transporter (PCFT, SLC46A1) that are inaccessible to 2-((biotinoyl)amino)ethyl methanethiosulfonate are glutathionylated by biotinylated glutathione ethyl ester in the absence of an oxidizing agent. Intramolecular disulfide formation involving cysteine-substituted residues was also identified in some instances. These posttranslational modifications limit the accessibility of the cysteine residues to sulfhydryl-reactive reagents and can have a profound impact on the interpretation of SCAM but may not alter function. When a posttranslationally modified residue is used as a reference extracellular control, the high level of exposure required for detection on Western blot results in erroneous detection of otherwise inaccessible intracellular cysteine-substituted residues. The data indicate that in the application of SCAM, when a cysteine-substituted residue does not appear to be accessible to sulfhydryl-reactive reagents, the possibility of a posttranslational modification should be excluded. The data explain the discrepancies in the assessment, and confirm the localization, of the first intracellular loop of PCFT.
Asunto(s)
Sustitución de Aminoácidos/fisiología , Cisteína/química , Cisteína/metabolismo , Glutatión/química , Glutatión/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Sitios de Unión , Células HeLa , Humanos , Unión Proteica , Ingeniería de Proteínas/métodos , Relación Estructura-ActividadRESUMEN
The proton-coupled folate transporter (PCFT-SLC46A1) is required for intestinal folate absorption and folate transport across the choroid plexus. This report addresses the structure/function of the 8th transmembrane helix. Based upon biotinylation of cysteine-substituted residues by MTSEA-biotin, 14 contiguous exofacial residues to Leu316 were accessible to the extracellular compartment of the 23 residues in this helix (Leu303-Leu325). Pemetrexed blocked biotinylation of six Cys-substituted residues deep within the helix implicating an important role for this region in folate binding. Accessibility decreased at 4°C vs RT. The influx Kt, Ki and Vmax were markedly increased for the P314C mutant, similar to what was observed for Y315A and Y315P mutants. However, the Kt, alone, was increased for the P314Y mutant. To correlate these observations with PCFT structural changes during the transport cycle, homology models were built for PCFT based upon the recently reported structures of bovine and rodent GLUT5 fructose transporters in the inward-open and outward- open conformations, respectively. The models predict substantial structural alterations in the exofacial region of the eighth transmembrane helix as it cycles between its conformational states that can account for the extended and contiguous aqueous accessibility of this region of the helix. Further, a helix break in one of the two conformations can account for the critical roles Pro314 and Tyr315, located in this region, play in PCFT function. The data indicates that the 8th transmembrane helix of PCFT plays an important role in defining the aqueous channel and the folate binding pocket.
Asunto(s)
Membrana Celular/química , Ácido Fólico/metabolismo , Dominios y Motivos de Interacción de Proteínas , Transportador de Folato Acoplado a Protón/química , Transportador de Folato Acoplado a Protón/metabolismo , Sustitución de Aminoácidos , Sitios de Unión/genética , Membrana Celular/metabolismo , Células HeLa , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Proteínas Mutantes/química , Unión Proteica/genética , Dominios Proteicos/genética , Dominios y Motivos de Interacción de Proteínas/genética , Transporte de Proteínas/genética , Transportador de Folato Acoplado a Protón/genética , Agua/químicaRESUMEN
Most organisms possess bifunctional FolD [5,10-methylenetetrahydrofolate (5,10-CH2-THF) dehydrogenase-cyclohydrolase] to generate NADPH and 10-formyltetrahdrofolate (10-CHO-THF) required in various metabolic steps. In addition, some organisms including Clostridium perfringens possess another protein, Fhs (formyltetrahydrofolate synthetase), to synthesize 10-CHO-THF. Here, we show that unlike the bifunctional FolD of Escherichia coli (EcoFolD), and contrary to its annotated bifunctional nature, C. perfringens FolD (CpeFolD) is a monofunctional 5,10-CH2-THF dehydrogenase. The dehydrogenase activity of CpeFolD is about five times more efficient than that of EcoFolD. The 5,10-methenyltetrahydrofolate (5,10-CH+-THF) cyclohydrolase activity in C. perfringens is provided by another protein, FchA (5,10-CH+-THF cyclohydrolase), whose cyclohydrolase activity is â¼ 10 times more efficient than that of EcoFolD. Kinetic parameters for CpeFhs were also determined for utilization of all of its substrates. Both CpeFolD and CpeFchA are required to substitute for the single bifunctional FolD in E. coli. The simultaneous presence of CpeFolD and CpeFchA is also necessary to rescue an E. coli folD deletion strain (harbouring CpeFhs support) for its formate and glycine auxotrophies, and to alleviate its susceptibility to trimethoprim (an antifolate drug) or UV light. The presence of the three clostridial proteins (FolD, FchA and Fhs) is required to maintain folate homeostasis in the cell.
Asunto(s)
Clostridium perfringens/enzimología , Escherichia coli/enzimología , Formiato-Tetrahidrofolato Ligasa/metabolismo , Meteniltetrahidrofolato Ciclohidrolasa/metabolismo , Metilenotetrahidrofolato Deshidrogenasa (NADP)/metabolismo , Secuencia de Aminoácidos , Clostridium perfringens/química , Clostridium perfringens/genética , Escherichia coli/química , Escherichia coli/genética , Formiato-Tetrahidrofolato Ligasa/química , Formiato-Tetrahidrofolato Ligasa/genética , Cinética , Meteniltetrahidrofolato Ciclohidrolasa/química , Meteniltetrahidrofolato Ciclohidrolasa/genética , Metilenotetrahidrofolato Deshidrogenasa (NADP)/química , Metilenotetrahidrofolato Deshidrogenasa (NADP)/genética , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
In cells, N(10)-formyltetrahydrofolate (N(10)-fTHF) is required for formylation of eubacterial/organellar initiator tRNA and purine nucleotide biosynthesis. Biosynthesis of N(10)-fTHF is catalyzed by 5,10-methylene-tetrahydrofolate dehydrogenase/cyclohydrolase (FolD) and/or 10-formyltetrahydrofolate synthetase (Fhs). All eubacteria possess FolD, but some possess both FolD and Fhs. However, the reasons for possessing Fhs in addition to FolD have remained unclear. We used Escherichia coli, which naturally lacks fhs, as our model. We show that in E. coli, the essential function of folD could be replaced by Clostridium perfringens fhs when it was provided on a medium-copy-number plasmid or integrated as a single-copy gene in the chromosome. The fhs-supported folD deletion (ΔfolD) strains grow well in a complex medium. However, these strains require purines and glycine as supplements for growth in M9 minimal medium. The in vivo levels of N(10)-fTHF in the ΔfolD strain (supported by plasmid-borne fhs) were limiting despite the high capacity of the available Fhs to synthesize N(10)-fTHF in vitro. Auxotrophy for purines could be alleviated by supplementing formate to the medium, and that for glycine was alleviated by engineering THF import into the cells. The ΔfolD strain (harboring fhs on the chromosome) showed a high NADP(+)-to-NADPH ratio and hypersensitivity to trimethoprim. The presence of fhs in E. coli was disadvantageous for its aerobic growth. However, under hypoxia, E. coli strains harboring fhs outcompeted those lacking it. The computational analysis revealed a predominant natural occurrence of fhs in anaerobic and facultative anaerobic bacteria.
Asunto(s)
Proteínas Bacterianas/metabolismo , Evolución Biológica , Clostridium perfringens/enzimología , Escherichia coli/metabolismo , Formiato-Tetrahidrofolato Ligasa/metabolismo , Redes y Vías Metabólicas , Viabilidad Microbiana , Oxígeno/metabolismo , Proteínas Bacterianas/genética , Carbono/metabolismo , Clonación Molecular , Clostridium perfringens/genética , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Formiato-Tetrahidrofolato Ligasa/genética , Formiltetrahidrofolatos/metabolismo , Metilenotetrahidrofolato Deshidrogenasa (NADP)/genética , Metilenotetrahidrofolato Deshidrogenasa (NADP)/metabolismo , Oxígeno/análisisRESUMEN
While therapies targeting CD19 by antibodies, chimeric antigen receptor T cells (CAR-T), and T cell engagers have improved the response rates in B cell malignancies, the emergence of resistant cell populations with low CD19 expression can lead to relapsed disease. We developed an in vitro model of adaptive resistance facilitated by chronic exposure of leukemia cells to a CD19 immunotoxin. Single-cell RNA-Seq (scRNA-Seq) showed an increase in transcriptionally distinct CD19lo populations among resistant cells. Mass cytometry demonstrated that CD22 was also decreased in these CD19lo-resistant cells. An assay for transposase-accessible chromatin with sequencing (ATAC-Seq) showed decreased chromatin accessibility at promoters of both CD19 and CD22 in the resistant cell populations. Combined loss of both CD19 and CD22 antigens was validated in samples from pediatric and young adult patients with B cell acute lymphoblastic leukemia (B-ALL) that relapsed after CD19 CAR-T-targeted therapy. Functionally, resistant cells were characterized by slower growth and lower basal levels of MEK activation. CD19lo resistant cells exhibited preserved B cell receptor signaling and were more sensitive to both Bruton's tyrosine kinase (BTK) and MEK inhibition. These data demonstrate that resistance to CD19 immunotherapies can result in decreased expression of both CD19 and CD22 and can result in dependency on BTK pathways.
Asunto(s)
Antígenos CD19 , Leucemia-Linfoma Linfoblástico de Células Precursoras , Lectina 2 Similar a Ig de Unión al Ácido Siálico , Niño , Humanos , Adulto Joven , Agammaglobulinemia Tirosina Quinasa , Antígenos CD19/genética , Cromatina , Inmunoterapia Adoptiva , Quinasas de Proteína Quinasa Activadas por Mitógenos , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Receptores Quiméricos de Antígenos , Lectina 2 Similar a Ig de Unión al Ácido Siálico/genéticaRESUMEN
Acute myeloid leukemia (AML) is an aggressive hematologic malignancy that continues to have poor prognosis despite recent therapeutic advances. Venetoclax (Ven), a BCL2-inhibitor has shown a high response rate in AML; however, relapse is invariable due to mitochondrial dysregulation that includes upregulation of the antiapoptotic protein MCL1, a central mechanism of Ven resistance (Ven-res). We have previously demonstrated that the transcription factor STAT3 is upregulated in AML hematopoietic stem and progenitor cells (HSPCs) and can be effectively targeted to induce apoptosis of these aberrant cells. We now show that overexpression of STAT3 alone is sufficient to initiate a strong AML phenotype in a transgenic murine model. Phospho-proteomic data from Ven treated AML patients show a strong correlation of high total STAT3 and phospho-STAT3 [both p-STAT3(Y705) and p-STAT3(S727)] expression with worse survival and reduced remission duration. Additionally, significant upregulation of STAT3 was observed in Ven-res cell lines, in vivo models and primary patient samples. A novel and specific degrader of STAT3 demonstrated targeted reduction of total STAT3 and resulting inhibition of its active p-STAT3(Y705) and p-STAT3(S727) forms. Treatment with the STAT3 degrader induced apoptosis in parental and Ven-res AML cell lines and decreased mitochondrial depolarisation, and thereby dependency on MCL1 in Ven-res AML cell line, as observed by BH3 profiling assay. STAT3 degrader treatment also enhanced differentiation of myeloid and erythroid colonies in Ven-res peripheral blood mononuclear cells (PBMNCs). Upregulation of p-STAT3(S727) was also associated with pronounced mitochondrial structural and functional dysfunction in Ven-res cell lines, that were restored by STAT3 degradation. Treatment with a clinical-stage STAT3 degrader, KT-333 resulted in a significant reduction in STAT3 and MCL1 protein levels within two weeks of treatment in a cell derived xenograft model of Ven-res AML. Additionally, this treatment significant improvement in the survival of a Ven-res patient-derived xenograft in-vivo study. Degradation of STAT3 resulting in downregulation of MCL1 and improvements in global mitochondrial dysfunction suggests a novel mechanism of overcoming Ven-res in AML. Statement of Purpose: Five-year survival from AML is dismal at 30%. Our prior research demonstrated STAT3 over-expression in AML HSPC's to be associated with inferior survival. We now explore STAT3 over-expression in Ven-res AML, explain STAT3 mediated mitochondrial perturbations and describe a novel therapeutic strategy, STAT3 degradation to overcome Ven-res.
RESUMEN
Myelodysplastic syndrome (MDS) is a heterogeneous group of bone marrow stem cell disorders characterized by ineffective hematopoiesis and cytopenias, most commonly anemia. Red cell transfusion therapy for anemia in MDS results in iron overload, correlating with reduced overall survival. Whether the treatment of iron overload benefits MDS patients remains controversial. We evaluate underlying iron-related pathophysiology and the effect of iron chelation using deferiprone on erythropoiesis in NUP98-HOXD13 transgenic mice, a highly penetrant well-established MDS mouse model. Our results characterize an iron overload phenotype with aberrant erythropoiesis in these mice which was reversed by deferiprone-treatment. Serum erythropoietin levels decreased while erythroblast erythropoietin receptor expression increased in deferiprone-treated MDS mice. We demonstrate, for the first time, normalized expression of the iron chaperones Pcbp1 and Ncoa4 and increased ferritin stores in late-stage erythroblasts from deferiprone-treated MDS mice, evidence of aberrant iron trafficking in MDS erythroblasts. Importantly, erythroblast ferritin is increased in response to deferiprone, correlating with decreased erythroblast ROS. Finally, we confirmed increased expression of genes involved in iron uptake, sensing, and trafficking in stem and progenitor cells from MDS patients. Taken together, our findings provide evidence that erythroblast-specific iron metabolism is a novel potential therapeutic target to reverse ineffective erythropoiesis in MDS.
Asunto(s)
Anemia , Sobrecarga de Hierro , Humanos , Ratones , Animales , Eritropoyesis , Deferiprona , Sobrecarga de Hierro/complicaciones , Hierro , Ratones Transgénicos , Ferritinas , Quelantes del Hierro/farmacologíaRESUMEN
Dysregulation of the innate immune system and inflammatory-related pathways has been implicated in hematopoietic defects in the bone marrow microenvironment and associated with aging, clonal hematopoiesis, myelodysplastic syndromes (MDS), and acute myeloid leukemia (AML). As the innate immune system and its pathway regulators have been implicated in the pathogenesis of MDS/AML, novel approaches targeting these pathways have shown promising results. Variability in expression of Toll like receptors (TLRs), abnormal levels of MyD88 and subsequent activation of NF-κß, dysregulated IL1-receptor associated kinases (IRAK), alterations in TGF-ß and SMAD signaling, high levels of S100A8/A9 have all been implicated in pathogenesis of MDS/AML. In this review we not only discuss the interplay of various innate immune pathways in MDS pathogenesis but also focus on potential therapeutic targets from recent clinical trials including the use of monoclonal antibodies and small molecule inhibitors against these pathways.
RESUMEN
Malignancies can become reliant on glutamine as an alternative energy source and as a facilitator of aberrant DNA methylation, thus implicating glutaminase (GLS) as a potential therapeutic target. We demonstrate preclinical synergy of telaglenastat (CB-839), a selective GLS inhibitor, when combined with azacytidine (AZA), in vitro and in vivo, followed by a phase Ib/II study of the combination in patients with advanced MDS. Treatment with telaglenastat/AZA led to an ORR of 70% with CR/mCRs in 53% patients and a median overall survival of 11.6 months. scRNAseq and flow cytometry demonstrated a myeloid differentiation program at the stem cell level in clinical responders. Expression of non-canonical glutamine transporter, SLC38A1, was found to be overexpressed in MDS stem cells; was associated with clinical responses to telaglenastat/AZA and predictive of worse prognosis in a large MDS cohort. These data demonstrate the safety and efficacy of a combined metabolic and epigenetic approach in MDS.
RESUMEN
Hematopoietic stem and progenitor cells (HSPCs) sustain lifelong hematopoiesis. Mutations of pre-mRNA splicing machinery, especially splicing factor 3b, subunit 1 (SF3B1), are early lesions found in malignancies arising from HSPC dysfunction. However, why splicing factor deficits contribute to HSPC defects remains incompletely understood. Using zebrafish, we show that HSPC formation in sf3b1 homozygous mutants is dependent on STAT3 activation. Clinically, mutations in SF3B1 are heterozygous; thus, we explored if targeting STAT3 could be a vulnerability in these cells. We show that SF3B1 heterozygosity confers heightened sensitivity to STAT3 inhibition in zebrafish, mouse, and human HSPCs. Cells carrying mutations in other splicing factors or treated with splicing modulators are also more sensitive to STAT3 inhibition. Mechanistically, we illustrate that STAT3 inhibition exacerbates aberrant splicing in SF3B1 mutant cells. Our findings reveal a conserved vulnerability of splicing factor mutant HSPCs that could allow for their selective targeting in hematologic malignancies.
Asunto(s)
Hematopoyesis , Pez Cebra , Ratones , Humanos , Animales , Factores de Empalme de ARN/genética , Factores de Empalme de ARN/metabolismo , Pez Cebra/metabolismo , Hematopoyesis/genética , Empalme del ARN/genética , Células Madre Hematopoyéticas/metabolismo , Mutación/genética , Fosfoproteínas/metabolismo , Factor de Transcripción STAT3/genéticaRESUMEN
Advanced pancreatic ductal adenocarcinoma (PDAC) is usually an incurable malignancy that needs newer therapeutic targets. Interleukin-1 receptor accessory protein (IL1RAP) is an innate immune mediator that regulates activation of pro-inflammatory and mitogenic signaling pathways. Immunohistochemistry on tissue microarrays demonstrated expression of IL1RAP in majority of human PDAC specimens and in murine pancreatic tumors from K-RasG122D/p53R172H/PDXCre (KPC) mice. Single cell RNA-Seq analysis of human primary pre-neoplastic lesions and adenocarcinoma specimens indicated that overexpression occurs during carcinogenesis. IL1RAP overexpression was associated with worse overall survival. IL1RAP knockdown significantly reduced cell viability, invasiveness, and clonogenic growth in pancreatic cancer cell lines. Inhibition of the downstream interleukin-1 receptor-associated kinase 4 (IRAK4) using two pharmacologic inhibitors, CA-4948 and PF06650833, resulted in reduced growth in pancreatic cancer cell lines and in xenograft models.
Asunto(s)
Adenocarcinoma , Carcinoma Ductal Pancreático , Proteína Accesoria del Receptor de Interleucina-1 , Neoplasias Pancreáticas , Adenocarcinoma/patología , Animales , Carcinogénesis , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Humanos , Inmunidad Innata , Proteína Accesoria del Receptor de Interleucina-1/metabolismo , Ratones , Neoplasias Pancreáticas/patología , Neoplasias PancreáticasRESUMEN
Background: Mutations in the SF3B1 splicing factor are commonly seen in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), yet the specific oncogenic pathways activated by mis-splicing have not been fully elucidated. Inflammatory immune pathways have been shown to play roles in the pathogenesis of MDS, though the exact mechanisms of their activation in splicing mutant cases are not well understood. Methods: RNA-seq data from SF3B1 mutant samples was analyzed and functional roles of interleukin-1 receptor-associated kinase 4 (IRAK4) isoforms were determined. Efficacy of IRAK4 inhibition was evaluated in preclinical models of MDS/AML. Results: RNA-seq splicing analysis of SF3B1 mutant MDS samples revealed retention of full-length exon 6 of IRAK4, a critical downstream mediator that links the Myddosome to inflammatory NF-kB activation. Exon 6 retention leads to a longer isoform, encoding a protein (IRAK4-long) that contains the entire death domain and kinase domain, leading to maximal activation of NF-kB. Cells with wild-type SF3B1 contain smaller IRAK4 isoforms that are targeted for proteasomal degradation. Expression of IRAK4-long in SF3B1 mutant cells induces TRAF6 activation leading to K63-linked ubiquitination of CDK2, associated with a block in hematopoietic differentiation. Inhibition of IRAK4 with CA-4948, leads to reduction in NF-kB activation, inflammatory cytokine production, enhanced myeloid differentiation in vitro and reduced leukemic growth in xenograft models. Conclusions: SF3B1 mutation leads to expression of a therapeutically targetable, longer, oncogenic IRAK4 isoform in AML/MDS models. Funding: This work was supported by Cincinnati Children's Hospital Research Foundation, Leukemia Lymphoma Society, and National Institute of Health (R35HL135787, RO1HL111103, RO1DK102759, RO1HL114582), Gabrielle's Angel Foundation for Cancer Research, and Edward P. Evans Foundation grants to DTS. AV is supported by Edward P. Evans Foundation, National Institute of Health (R01HL150832, R01HL139487, R01CA275007), Leukemia and Lymphoma Society, Curis and a gift from the Jane and Myles P. Dempsey family. AP and JB are supported by Blood Cancer UK (grants 13042 and 19004). GC is supported by a training grant from NYSTEM. We acknowledge support of this research from The Einstein Training Program in Stem Cell Research from the Empire State Stem Cell Fund through New York State Department of Health Contract C34874GG. MS is supported by a National Institute of Health Research Training and Career Development Grant (F31HL132420).
Genes contain blocks of code that tell cells how to make each part of a protein. Between these blocks are sections of linking DNA, which cells remove when they are preparing to use their genes. Scientists call this process 'splicing'. Cells can splice some genes in more than one way, allowing them to make different proteins from the same genetic code. Mutations that affect the splicing process can change the way cells make their proteins, leading to disease. For example, the myelodysplastic syndromes are a group of blood cancers often caused by mutations in splicing proteins, such as SF3B1. The disorder stops blood cells from maturing and causes abnormal inflammation. So far, the link between splicing, blood cell immaturity, inflammation and cancer is not clear. To find out more, Choudhary, Pellagatti et al. looked at the spliced genetic code from people with myelodysplastic syndromes. Mutations in the splicing protein SF3B1 changed the way cells spliced an important signalling molecule known as IRAK4. Affected cells cut out less genetic code and made a longer version of this signalling protein, named IRAK4-Long. This altered protein activated inflammation and stopped blood cells from maturing. Blocking IRAK4-Long reversed the effects. It also reduced tumour formation in mice carrying affected human cells. The molecule used to block IRAK4, CA-4948 also known as Emavusertib is currently being evaluated in clinical trials for myelodysplastic syndromes and other types of blood cancer. The work of Choudhary, Pellagatti et al. could help scientists to design genetic tests to predict which patients might benefit from this treatment.
Asunto(s)
Leucemia Mieloide Aguda , Síndromes Mielodisplásicos , Fosfoproteínas/metabolismo , Factores de Empalme de ARN/metabolismo , Niño , Humanos , Quinasas Asociadas a Receptores de Interleucina-1/genética , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Mutación , Síndromes Mielodisplásicos/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Isoformas de Proteínas/metabolismo , Empalme del ARNRESUMEN
The terrorist attacks on the World Trade Center (WTC) created an unprecedented environmental exposure to aerosolized dust, gases and potential carcinogens. Clonal hematopoiesis (CH) is defined as the acquisition of somatic mutations in blood cells and is associated with smoking and exposure to genotoxic stimuli. Here we show that deep targeted sequencing of blood samples identified a significantly higher proportion of WTC-exposed first responders with CH (10%; 48 out of 481) when compared with non-WTC-exposed firefighters (6.7%; 17 out of 255; odds ratio, 3.14; 95% confidence interval, 1.64-6.03; P = 0.0006) after controlling for age, sex and race/ethnicity. The frequency of somatic mutations in WTC-exposed first responders showed an age-related increase and predominantly affected DNMT3A, TET2 and other CH-associated genes. Exposure of lymphoblastoid cells to WTC particulate matter led to dysregulation of DNA replication at common fragile sites in vitro. Moreover, mice treated with WTC particulate matter developed an increased burden of mutations in hematopoietic stem and progenitor cell compartments. In summary, the high burden of CH in WTC-exposed first responders provides a rationale for enhanced screening and preventative efforts in this population.
Asunto(s)
Desastres , Socorristas , Ataques Terroristas del 11 de Septiembre , Animales , Hematopoyesis Clonal , Polvo , Humanos , RatonesRESUMEN
Mitogen-activated protein kinases (MAPKs) are inactivated by dual-specificity phosphatases (DUSPs), the activities of which are tightly regulated during cell differentiation. Using knockdown screening and single-cell transcriptional analysis, we demonstrate that DUSP4 is the phosphatase that specifically inactivates p38 kinase to promote megakaryocyte (Mk) differentiation. Mechanistically, PRMT1-mediated methylation of DUSP4 triggers its ubiquitinylation by an E3 ligase HUWE1. Interestingly, the mechanistic axis of the DUSP4 degradation and p38 activation is also associated with a transcriptional signature of immune activation in Mk cells. In the context of thrombocytopenia observed in myelodysplastic syndrome (MDS), we demonstrate that high levels of p38 MAPK and PRMT1 are associated with low platelet counts and adverse prognosis, while pharmacological inhibition of p38 MAPK or PRMT1 stimulates megakaryopoiesis. These findings provide mechanistic insights into the role of the PRMT1-DUSP4-p38 axis on Mk differentiation and present a strategy for treatment of thrombocytopenia associated with MDS.
Asunto(s)
Diferenciación Celular , Fosfatasas de Especificidad Dual , Megacariocitos , Fosfatasas de la Proteína Quinasa Activada por Mitógenos , Adulto , Animales , Niño , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto Joven , Arginina/metabolismo , Línea Celular , Fosfatasas de Especificidad Dual/metabolismo , Estabilidad de Enzimas , Células HEK293 , Sistema de Señalización de MAP Quinasas , Megacariocitos/citología , Megacariocitos/enzimología , Metilación , Ratones Endogámicos C57BL , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/metabolismo , Síndromes Mielodisplásicos/enzimología , Síndromes Mielodisplásicos/patología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Poliubiquitina/metabolismo , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteolisis , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/metabolismo , UbiquitinaciónRESUMEN
Signaling by the TGF-ß superfamily is important in the regulation of hematopoiesis and is dysregulated in myelodysplastic syndromes (MDSs), contributing to ineffective hematopoiesis and clinical cytopenias. TGF-ß, activins, and growth differentiation factors exert inhibitory effects on red cell formation by activating canonical SMAD2/3 pathway signaling. In this Review, we summarize evidence that overactivation of SMAD2/3 signaling pathways in MDSs causes anemia due to impaired erythroid maturation. We also describe the basis for biological activity of activin receptor ligand traps, novel fusion proteins such as luspatercept that are promising as erythroid maturation agents to alleviate anemia and related comorbidities in MDSs and other conditions characterized by impaired erythroid maturation.