RESUMEN
X-linked sideroblastic anemia (XLSA) and X-linked protoporphyria (XLPP) are uncommon diseases caused by loss-of-function and gain-of-function mutations, respectively, in the erythroid form of 5-aminolevulinic acid synthetase, ALAS2, which encodes the first enzyme in heme biosynthesis. A related sideroblastic anemia is due to mutations in SLC25A38, which supplies mitochondrial glycine for ALAS2 (SLC25A38-CSA). The lack of viable animal models has limited studies on the pathophysiology and development of therapies for these conditions. Here, using CRISPR-CAS9 gene editing technology, we have generated knock-in mouse models that recapitulate the main features of XLSA and XLPP, and, using conventional conditional gene targeting in embryonic stem cells, we also developed a faithful model of the SLC25A38-CSA. In addition to examining the phenotypes and natural history of each disease, we determine the effect of restriction or supplementation of dietary pyridoxine (vitamin B6), the essential cofactor of ALAS2, on the anemia and porphyria. In addition to the well-documented response of XLSA mutations to pyridoxine supplementation, we also demonstrate the relative insensitivity of the XLPP porphyria, severe sensitivity of the XLSA models, and an extreme hypersensitivity of the SLC25A38-CSA model to pyridoxine deficiency, a phenotype that is not shared with another mouse hereditary anemia model, Hbbth3/+ ï¢-thalassemia intermedia. Thus, in addition to generating animal models useful for examining the pathophysiology and treatment of these diseases, we have uncovered an unsuspected conditional synthetic lethality between the heme synthesis-related CSAs and pyridoxine deficiency. These findings have the potential to inform novel therapeutic paradigms for the treatment of these diseases.
RESUMEN
Genetic mutations in the isocitrate dehydrogenase (IDH) gene that result in a pathological enzymatic activity to produce oncometabolite have been detected in acute myeloid leukemia (AML) patients. While specific inhibitors that target mutant IDH enzymes and normalize intracellular oncometabolite level have been developed, refractoriness and resistance has been reported. Since acquisition of pathological enzymatic activity is accompanied by the abrogation of the crucial WT IDH enzymatic activity in IDH mutant cells, aberrant metabolism in IDH mutant cells can potentially persist even after the normalization of intracellular oncometabolite level. Comparisons of isogenic AML cell lines with and without IDH2 gene mutations revealed two mutually exclusive signalings for growth advantage of IDH2 mutant cells, STAT phosphorylation associated with intracellular oncometabolite level and phospholipid metabolic adaptation. The latter came to light after the oncometabolite normalization and increased the resistance of IDH2 mutant cells to arachidonic acid-mediated apoptosis. The release of this metabolic adaptation by FDA-approved anti-inflammatory drugs targeting the metabolism of arachidonic acid could sensitize IDH2 mutant cells to apoptosis, resulting in their eradication in vitro and in vivo. Our findings will contribute to the development of alternative therapeutic options for IDH2 mutant AML patients who do not tolerate currently available therapies.
Asunto(s)
Leucemia Mieloide Aguda , Humanos , Ácido Araquidónico/uso terapéutico , Mutación , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Isocitrato Deshidrogenasa/metabolismoRESUMEN
Hematopoietic stem cells (HSC) rarely divide, rest in quiescence, and proliferate only upon stress hematopoiesis. The cytokine thrombopoietin (Thpo) has been perplexingly described to induce quiescence and promote self-renewal divisions in HSCs. To clarify the contradictory effect of Thpo, we conducted a detailed analysis on conventional (Thpo-/-) and liver-specific (Thpofl/fl;AlbCre+/-) Thpo-deletion models. Thpo-/- HSCs exhibited profound loss of quiescence, impaired cell cycle progression, and increased apoptosis. Thpo-/- HSCs also exhibited diminished mitochondrial mass and impaired mitochondrial bioenergetics. Abnormal HSC phenotypes in Thpo-/- mice were reversible after HSC transplantation into wild-type recipients. Moreover, Thpo-/- HSCs acquired quiescence with extended administration of a Thpo receptor agonist, romiplostim, and were prone to subsequent stem cell exhaustion during competitive bone marrow transplantation. Thpofl/fl;AlbCre+/- HSCs exhibited similar stem cell phenotypes but to a lesser degree compared with Thpo-/- HSCs. HSCs that survive Thpo deficiency acquire quiescence in a dose-dependent manner through the modification of their metabolic state.
Asunto(s)
Células Madre Hematopoyéticas/citología , Trombopoyetina/deficiencia , Animales , Apoptosis , Ciclo Celular , Autorrenovación de las Células , Metabolismo Energético/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Ratones , Ratones Noqueados , Ratones Transgénicos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Estrés Oxidativo/efectos de los fármacos , Receptores Fc , Receptores de Trombopoyetina/agonistas , Proteínas Recombinantes de Fusión/farmacología , Transducción de Señal , Trombopoyetina/genética , Trombopoyetina/farmacología , TranscriptomaRESUMEN
Although an essential role for canonical Notch signaling in generation of hematopoietic stem cells in the embryo and in thymic T-cell development is well established, its role in adult bone marrow (BM) myelopoiesis remains unclear. Some studies, analyzing myeloid progenitors in adult mice with inhibited Notch signaling, implicated distinct roles of canonical Notch signaling in regulation of progenitors for the megakaryocyte, erythroid, and granulocyte-macrophage cell lineages. However, these studies might also have targeted other pathways. Therefore, we specifically deleted, in adult BM, the transcription factor recombination signal-binding protein J κ (Rbpj), through which canonical signaling from all Notch receptors converges. Notably, detailed progenitor staging established that canonical Notch signaling is fully dispensable for all investigated stages of megakaryocyte, erythroid, and myeloid progenitors in steady state unperturbed hematopoiesis, after competitive BM transplantation, and in stress-induced erythropoiesis. Moreover, expression of key regulators of these hematopoietic lineages and Notch target genes were unaffected by Rbpj deficiency in BM progenitor cells.
Asunto(s)
Médula Ósea/metabolismo , Eritropoyesis , Mielopoyesis , Receptores Notch/metabolismo , Transducción de Señal , Estrés Fisiológico , Animales , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/genética , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Ratones , Ratones Transgénicos , Receptores Notch/genéticaRESUMEN
Leukemia stem cells (LSCs) are widely believed to reside in well-characterized bone marrow (BM) niches; however, the capacity of the BM niches to accommodate LSCs is insufficient, and a significant proportion of LSCs are instead maintained in regions outside the BM. The molecular basis for this niche-independent behavior of LSCs remains elusive. Here, we show that integrin-α9 overexpression (ITGA9 OE) plays a pivotal role in the extramedullary maintenance of LSCs by molecularly mimicking the niche-interacting status, through the binding with its soluble ligand, osteopontin (OPN). Retroviral insertional mutagenesis conducted on leukemia-prone Runx-deficient mice identified Itga9 OE as a novel leukemogenic event. Itga9 OE activates Akt and p38MAPK signaling pathways. The elevated Myc expression subsequently enhances ribosomal biogenesis to overcome the cell integrity defect caused by the preexisting Runx alteration. The Itga9-Myc axis, originally discovered in mice, was further confirmed in multiple human acute myeloid leukemia (AML) subtypes, other than RUNX leukemias. In addition, ITGA9 was shown to be a functional LSC marker of the best prognostic value among 14 known LSC markers tested. Notably, the binding of ITGA9 with soluble OPN, a known negative regulator against HSC activation, induced LSC dormancy, while the disruption of ITGA9-soluble OPN interaction caused rapid cell propagation. These findings suggest that the ITGA9 OE increases both actively proliferating leukemia cells and dormant LSCs in a well-balanced manner, thereby maintaining LSCs. The ITGA9 OE would serve as a novel therapeutic target in AML.
Asunto(s)
Leucemia Mieloide Aguda , Células Madre Neoplásicas , Animales , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Humanos , Ratones , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Osteopontina/genética , Osteopontina/metabolismo , Nicho de Células Madre , Cadenas alfa de Integrinas/metabolismo , Cadenas alfa de Integrinas/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Regulación Leucémica de la Expresión Génica , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Ratones Endogámicos C57BL , Transducción de SeñalRESUMEN
The genetic lesions that drive acute megakaryoblastic leukemia (AMKL) have not been fully elucidated. To search for genetic alterations in AMKL, we performed targeted deep sequencing in 34 AMKL patient samples and 8 AMKL cell lines and detected frequent genetic mutations in the NOTCH pathway in addition to previously reported alterations in GATA-1 and the JAK-STAT pathway. Pharmacological and genetic NOTCH activation, but not inhibition, significantly suppressed AMKL cell proliferation in both in vitro and in vivo assays employing a patient-derived xenograft model. These results suggest that NOTCH inactivation underlies AMKL leukemogenesis. and NOTCH activation holds the potential for therapeutic application in AMKL.
RESUMEN
Acute myeloid leukemia (AML), the most frequent leukemia in adults, is driven by recurrent somatically acquired genetic lesions in a restricted number of genes. Treatment with tyrosine kinase inhibitors has demonstrated that targeting of prevalent FMS-related receptor tyrosine kinase 3 (FLT3) gain-of-function mutations can provide significant survival benefits for patients, although the efficacy of FLT3 inhibitors in eliminating FLT3-mutated clones is variable. We identified a T cell receptor (TCR) reactive to the recurrent D835Y driver mutation in the FLT3 tyrosine kinase domain (TCRFLT3D/Y). TCRFLT3D/Y-redirected T cells selectively eliminated primary human AML cells harboring the FLT3D835Y mutation in vitro and in vivo. TCRFLT3D/Y cells rejected both CD34+ and CD34- AML in mice engrafted with primary leukemia from patients, reaching minimal residual disease-negative levels, and eliminated primary CD34+ AML leukemia-propagating cells in vivo. Thus, T cells targeting a single shared mutation can provide efficient immunotherapy toward selective elimination of clonally involved primary AML cells in vivo.
Asunto(s)
Leucemia Mieloide Aguda , Proteínas Tirosina Quinasas , Adulto , Humanos , Animales , Ratones , Mutación , Proteínas Tirosina Quinasas/genética , Mutación con Ganancia de Función , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Receptores de Antígenos de Linfocitos T/genética , Tirosina Quinasa 3 Similar a fms/genéticaRESUMEN
Protein arginine methyltransferase 5 (PRMT5) is essential for hematopoiesis, while PRMT5 inhibition remains a promising therapeutic strategy against various cancers. Here, we demonstrate that hematopoietic stem cell (HSC) quiescence and viability are severely perturbed upon PRMT5 depletion, which also increases HSC size, PI3K/AKT/mechanistic target of rapamycin (mTOR) pathway activity, and protein synthesis rate. We uncover a critical role for PRMT5 in maintaining HSC genomic integrity by modulating splicing of genes involved in DNA repair. We found that reducing PRMT5 activity upregulates exon skipping and intron retention events that impair gene expression. Genes across multiple DNA repair pathways are affected, several of which mediate interstrand crosslink repair and homologous recombination. Consequently, loss of PRMT5 activity leads to endogenous DNA damage that triggers p53 activation, induces apoptosis, and culminates in rapid HSC exhaustion, which is significantly delayed by p53 depletion. Collectively, these findings establish the importance of cell-intrinsic PRMT5 activity in HSCs.
Asunto(s)
Células Madre Hematopoyéticas/enzimología , Proteína-Arginina N-Metiltransferasas/fisiología , Proteostasis , Empalme del ARN , Animales , Apoptosis , Línea Celular , Daño del ADN , Reparación del ADN , Genoma , Células Madre Hematopoyéticas/metabolismo , Ratones , Biosíntesis de Proteínas , Proteína-Arginina N-Metiltransferasas/genética , Proteína-Arginina N-Metiltransferasas/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Increased levels and non-telomeric roles have been reported for shelterin proteins, including RAP1 in cancers. Herein using Rap1 null mice, we provide the genetic evidence that mammalian Rap1 plays a major role in hematopoietic stem cell survival, oncogenesis and response to chemotherapy. Strikingly, this function of RAP1 is independent of its association with the telomere or with its known partner TRF2. We show that RAP1 interacts with many members of the DNA damage response (DDR) pathway. RAP1 depleted cells show reduced interaction between XRCC4/DNA Ligase IV and DNA-PK, and are impaired in DNA Ligase IV recruitment to damaged chromatin for efficient repair. Consistent with its role in DNA damage repair, RAP1 loss decreases double-strand break repair via NHEJ in vivo, and consequently reduces B cell class switch recombination. Finally, we discover that RAP1 levels are predictive of the success of chemotherapy in breast and colon cancer.
Asunto(s)
Antineoplásicos/farmacología , Carcinogénesis/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Proteínas de Unión al GTP rap1/metabolismo , Animales , Carcinogénesis/efectos de los fármacos , Carcinogénesis/patología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Daño del ADN , ADN Ligasa (ATP)/metabolismo , Reparación del ADN/efectos de los fármacos , Reparación del ADN/efectos de la radiación , Proteína Quinasa Activada por ADN/metabolismo , Fluorouracilo/farmacología , Rayos gamma , Inestabilidad Genómica/efectos de los fármacos , Inestabilidad Genómica/efectos de la radiación , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de la radiación , Humanos , Ratones Noqueados , Mutágenos/toxicidad , Unión Proteica/efectos de los fármacos , Unión Proteica/efectos de la radiación , Proteínas Proto-Oncogénicas c-myc/metabolismo , Complejo Shelterina , Análisis de SupervivenciaRESUMEN
The RUNX genes encode transcription factors involved in development and human disease. RUNX1 and RUNX3 are frequently associated with leukemias, yet the basis for their involvement in leukemogenesis is not fully understood. Here, we show that Runx1;Runx3 double-knockout (DKO) mice exhibited lethal phenotypes due to bone marrow failure and myeloproliferative disorder. These contradictory clinical manifestations are reminiscent of human inherited bone marrow failure syndromes such as Fanconi anemia (FA), caused by defective DNA repair. Indeed, Runx1;Runx3 DKO cells showed mitomycin C hypersensitivity, due to impairment of monoubiquitinated-FANCD2 recruitment to DNA damage foci, although FANCD2 monoubiquitination in the FA pathway was unaffected. RUNX1 and RUNX3 interact with FANCD2 independently of CBFß, suggesting a nontranscriptional role for RUNX in DNA repair. These findings suggest that RUNX dysfunction causes DNA repair defect, besides transcriptional misregulation, and promotes the development of leukemias and other cancers.
Asunto(s)
Médula Ósea/metabolismo , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 3 del Factor de Unión al Sitio Principal/genética , Reparación del ADN , Anemia de Fanconi/genética , Leucemia/genética , Animales , Médula Ósea/patología , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Subunidad alfa 3 del Factor de Unión al Sitio Principal/metabolismo , Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Eliminación de Gen , Predisposición Genética a la Enfermedad , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Leucemia/metabolismo , Ratones , Ratones Endogámicos C57BLRESUMEN
This study characterized human umbilical vein endothelial cell (HUVEC) adhesion, proliferation, and gene expression on bilayered polyelectrolyte coatings composed of an outermost layer of glycosaminoglycans (hyaluronan, heparin, or chondroitin sulfate), with an underlying layer of poly-L-lysine or chitosan. The proportion of cells that adhered to the various polyelectrolyte coatings after 1 and 2 h incubations was quantified by the WST-8 assay. Interchanging poly-L-lysine with chitosan resulted in significant differences in cellular adhesion to the outermost glycosaminoglycan layer after 1 h, but these differences became insignificant after 2 h. The proliferation of HUVEC on the various bilayered polyelectrolyte coatings over 10 days was characterized using the WST-8 assay. Regardless of whether the underlying layer was poly-L-lysine or chitosan, HUVEC proliferation on the hyaluronan outermost layer was significantly less than on heparin or chondroitin sulfate. Additionally, it was observed that there was more proliferation with poly-L-lysine as the underlying layer, compared to chitosan. Subsequently, real-time polymerase chain reaction was used to analyze the expression of seven genes related to adhesion, migration, and endothelial function (VWF, VEGFR, VEGFA, endoglin, integrin-α5, ICAM1, and ICAM2) by HUVEC cultured on the various bilayered polyelectrolyte coatings for 3 days. With poly-L-lysine as the underlying layer, biologically significant differences (greater than twofold) in the expression of VWF, VEGFR, VEGFA, endoglin, and ICAM1 were observed among the three glycosaminoglycans. With chitosan as the underlying layer, all three glycosaminoglycans displayed biologically significant differences in the expression of VWF and VEGFR compared to the chitosan control. CT-HA displayed the highest level of expression of VWF, whereas expression levels of VEGFR were almost similar among the three glycosaminoglycans.