RESUMEN
The interaction between menin and histone-lysine N-methyltransferase 2A (KMT2A) is a critical dependency for KMT2A- or nucleophosmin 1 (NPM1)-altered leukemias and an emerging opportunity for therapeutic development. JNJ-75276617 is a novel, orally bioavailable, potent, and selective protein-protein interaction inhibitor of the binding between menin and KMT2A. In KMT2A-rearranged (KMT2A-r) and NPM1-mutant (NPM1c) AML cells, JNJ-75276617 inhibited the association of the menin-KMT2A complex with chromatin at target gene promoters, resulting in reduced expression of several menin-KMT2A target genes, including MEIS1 and FLT3. JNJ-75276617 displayed potent anti-proliferative activity across several AML and ALL cell lines and patient samples harboring KMT2A- or NPM1-alterations in vitro. In xenograft models of AML and ALL, JNJ-75276617 reduced leukemic burden and provided a significant dose-dependent survival benefit accompanied by expression changes of menin-KMT2A target genes. JNJ-75276617 demonstrated synergistic effects with gilteritinib in vitro in AML cells harboring KMT2A-r. JNJ-75276617 further exhibited synergistic effects with venetoclax and azacitidine in AML cells bearing KMT2A-r in vitro, and significantly increased survival in mice. Interestingly, JNJ-75276617 showed potent anti-proliferative activity in cell lines engineered with recently discovered mutations (MEN1M327I or MEN1T349M) that developed in patients refractory to the menin-KMT2A inhibitor revumenib. A co-crystal structure of menin in complex with JNJ-75276617 indicates a unique binding mode distinct from other menin-KMT2A inhibitors, including revumenib. JNJ-75276617 is being clinically investigated for acute leukemias harboring KMT2A or NPM1 alterations, as a monotherapy for relapsed/refractory (R/R) acute leukemia (NCT04811560), or in combination with AML-directed therapies (NCT05453903).
RESUMEN
Dysregulated innate immune signaling is linked to preleukemic conditions and myeloid malignancies. However, it is unknown whether sustained innate immune signaling contributes to malignant transformation. Here we show that cell-intrinsic innate immune signaling driven by miR-146a deletion (miR-146aKO), a commonly deleted gene in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), cooperates with mutant RUNX1 (RUNX1mut) to initially induce marrow failure and features of MDS. However, miR-146aKO hematopoietic stem and/or progenitor cells (HSPCs) expressing RUNX1mut eventually progress to a fatal AML. miR-146aKO HSPCs exhaust during serial transplantation, while expression of RUNX1mut restored their hematopoietic cell function. Thus, HSPCs exhibiting dysregulated innate immune signaling require a second hit to develop AML. Inhibiting the dysregulated innate immune pathways with a TRAF6-UBE2N inhibitor suppressed leukemic miR-146aKO/RUNX1mut HSPCs, highlighting the necessity of TRAF6-dependent cell-intrinsic innate immune signaling in initiating and maintaining AML. These findings underscore the critical role of dysregulated cell-intrinsic innate immune signaling in driving preleukemic cells toward AML progression.
RESUMEN
Acute myeloid leukemia (AML) is an aggressive malignancy that requires rapid treatment with chemotherapies to reduce tumor burden. However, these chemotherapies can compromise lymphocyte function, thereby hindering normal anti-tumor immune responses and likely limiting the efficacy of subsequent immunotherapy. To better understand these negative impacts, we assessed the immunological effects of standard-of-care AML therapies on lymphocyte phenotype and function over time. When compared to healthy donors, untreated AML patients showed evidence of lymphocyte activation and exhaustion and had more prevalent CD57+NKG2C+ adaptive NK cells, which was independent of human cytomegalovirus (HCMV) status. HMA/venetoclax treatment resulted in a greater fraction of T cells with effector memory phenotype, inhibited IFN-γ secretion by CD8+ T cells, upregulated perforin expression in NK cells, downregulated PD-1 and 2B4 expression on CD4+ T cells, and stimulated Treg proliferation and CTLA-4 expression. Additionally, we showed increased expression of perforin and CD39 and enhanced IFN-γ production by T cells from pre-treatment blood samples of venetoclax-resistant AML patients. Our results provide insight into the lymphocyte status in previously untreated AML patients and the effects of standard-of-care treatments on their biology and functions. We also found novel pre-treatment characteristics of T cells that could potentially predict venetoclax resistance.
RESUMEN
Dysregulation of innate immune signaling pathways is implicated in various hematologic malignancies. However, these pathways have not been systematically examined in acute myeloid leukemia (AML). We report that AML hematopoietic stem and progenitor cells (HSPCs) exhibit a high frequency of dysregulated innate immune-related and inflammatory pathways, referred to as oncogenic immune signaling states. Through gene expression analyses and functional studies in human AML cell lines and patient-derived samples, we found that the ubiquitin-conjugating enzyme UBE2N is required for leukemic cell function in vitro and in vivo by maintaining oncogenic immune signaling states. It is known that the enzyme function of UBE2N can be inhibited by interfering with thioester formation between ubiquitin and the active site. We performed in silico structure-based and cellular-based screens and identified two related small-molecule inhibitors UC-764864/65 that targeted UBE2N at its active site. Using these small-molecule inhibitors as chemical probes, we further revealed the therapeutic efficacy of interfering with UBE2N function. This resulted in the blocking of ubiquitination of innate immune- and inflammatory-related substrates in human AML cell lines. Inhibition of UBE2N function disrupted oncogenic immune signaling by promoting cell death of leukemic HSPCs while sparing normal HSPCs in vitro. Moreover, baseline oncogenic immune signaling states in leukemic cells derived from discrete subsets of patients with AML exhibited a selective dependency on UBE2N function in vitro and in vivo. Our study reveals that interfering with UBE2N abrogates leukemic HSPC function and underscores the dependency of AML cells on UBE2N-dependent oncogenic immune signaling states.
Asunto(s)
Leucemia Mieloide Aguda , Enzimas Ubiquitina-Conjugadoras , Proliferación Celular/genética , Humanos , Leucemia Mieloide Aguda/metabolismo , Oncogenes , Transducción de Señal/genética , Enzimas Ubiquitina-Conjugadoras/antagonistas & inhibidores , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismoRESUMEN
Clonal hematopoiesis (CH) is an aging-associated condition characterized by the clonal outgrowth of pre-leukemic cells that acquire specific mutations. Although individuals with CH are healthy, they are at an increased risk of developing myeloid malignancies, suggesting that additional alterations are needed for the transition from a pre-leukemia stage to frank leukemia. To identify signaling states that cooperate with pre-leukemic cells, we used an in vivo RNAi screening approach. One of the most prominent genes identified was the ubiquitin ligase TRAF6. Loss of TRAF6 in pre-leukemic cells results in overt myeloid leukemia and is associated with MYC-dependent stem cell signatures. TRAF6 is repressed in a subset of patients with myeloid malignancies, suggesting that subversion of TRAF6 signaling can lead to acute leukemia. Mechanistically, TRAF6 ubiquitinates MYC, an event that does not affect its protein stability but rather represses its functional activity by antagonizing an acetylation modification.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Leucemia Mieloide Aguda , Trastornos Mieloproliferativos , Hematopoyesis , Humanos , Leucemia Mieloide Aguda/patología , Mutación , Factor 6 Asociado a Receptor de TNF/genética , Factor 6 Asociado a Receptor de TNF/metabolismoRESUMEN
Chronic innate immune signaling in hematopoietic cells is widely described in myelodysplastic syndromes (MDS), and innate immune pathway activation, predominantly via pattern recognition receptors, increases the risk of developing MDS. An inflammatory component to MDS has been reported for many years, but only recently has evidence supported a more direct role of chronic innate immune signaling and associated inflammatory pathways in the pathogenesis of MDS. Here we review recent findings and discuss relevant questions related to chronic immune response dysregulation in MDS.
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Inmunidad Innata , Síndromes Mielodisplásicos/inmunología , Animales , Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/patología , Humanos , Inflamación/inmunología , Inflamación/patología , Síndromes Mielodisplásicos/patologíaRESUMEN
The surface molecule interleukin-1 receptor accessory protein (IL1RAP) is consistently overexpressed across multiple genetic subtypes of acute myeloid leukemia (AML) and other myeloid malignancies, including at the stem cell level, and is emerging as a novel therapeutic target. However, the cell-intrinsic functions of IL1RAP in AML cells are largely unknown. Here, we show that targeting of IL1RAP via RNA interference, genetic deletion, or antibodies inhibits AML pathogenesis in vitro and in vivo, without perturbing healthy hematopoietic function or viability. Furthermore, we found that the role of IL1RAP is not restricted to the IL-1 receptor pathway, but that IL1RAP physically interacts with and mediates signaling and pro-proliferative effects through FLT3 and c-KIT, two receptor tyrosine kinases with known key roles in AML pathogenesis. Our study provides a new mechanistic basis for the efficacy of IL1RAP targeting in AML and reveals a novel role for this protein in the pathogenesis of the disease.
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Carcinogénesis/metabolismo , Carcinogénesis/patología , Proteína Accesoria del Receptor de Interleucina-1/metabolismo , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Transducción de Señal , Adulto , Anciano , Animales , Anticuerpos/farmacología , Apoptosis/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Femenino , Eliminación de Gen , Hematopoyesis/efectos de los fármacos , Humanos , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Persona de Mediana Edad , Modelos Biológicos , Unión Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas c-kit/metabolismo , Factor de Células Madre/metabolismoRESUMEN
Neomorphic mutations in isocitrate dehydrogenase 1 (IDH1) are driver mutations in acute myeloid leukemia (AML) and other cancers. We report the development of new allosteric inhibitors of mutant IDH1. Crystallographic and biochemical results demonstrated that compounds of this chemical series bind to an allosteric site and lock the enzyme in a catalytically inactive conformation, thereby enabling inhibition of different clinically relevant IDH1 mutants. Treatment of IDH1 mutant primary AML cells uniformly led to a decrease in intracellular 2-HG, abrogation of the myeloid differentiation block and induction of granulocytic differentiation at the level of leukemic blasts and more immature stem-like cells, in vitro and in vivo. Molecularly, treatment with the inhibitors led to a reversal of the DNA cytosine hypermethylation patterns caused by mutant IDH1 in the cells of individuals with AML. Our study provides proof of concept for the molecular and biological activity of novel allosteric inhibitors for targeting different mutant forms of IDH1 in leukemia.
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Dihidropiridinas/farmacología , Inhibidores Enzimáticos/farmacología , Isocitrato Deshidrogenasa/antagonistas & inhibidores , Leucemia Mieloide Aguda/tratamiento farmacológico , Pirazoles/farmacología , Regulación Alostérica , Sitio Alostérico , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Islas de CpG , Cristalografía por Rayos X , Citosina/química , Citosina/metabolismo , Metilación de ADN/efectos de los fármacos , Dihidropiridinas/química , Dihidropiridinas/farmacocinética , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacocinética , Granulocitos/efectos de los fármacos , Granulocitos/enzimología , Granulocitos/patología , Humanos , Isocitrato Deshidrogenasa/química , Isocitrato Deshidrogenasa/genética , Isocitrato Deshidrogenasa/metabolismo , Cinética , Leucemia Mieloide Aguda/enzimología , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Masculino , Ratones , Modelos Moleculares , Mutación , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/enzimología , Células Madre Neoplásicas/patología , Cultivo Primario de Células , Unión Proteica , Pirazoles/química , Pirazoles/farmacocinética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Modest transcriptional changes caused by genetic or epigenetic mechanisms are frequent in human cancer. Although loss or near-complete loss of the hematopoietic transcription factor PU.1 induces acute myeloid leukemia (AML) in mice, a similar degree of PU.1 impairment is exceedingly rare in human AML; yet, moderate PU.1 inhibition is common in AML patients. We assessed functional consequences of modest reductions in PU.1 expression on leukemia development in mice harboring DNA lesions resembling those acquired during human stem cell aging. Heterozygous deletion of an enhancer of PU.1, which resulted in a 35% reduction of PU.1 expression, was sufficient to induce myeloid-biased preleukemic stem cells and their subsequent transformation to AML in a DNA mismatch repair-deficient background. AML progression was mediated by inhibition of expression of a PU.1-cooperating transcription factor, Irf8. Notably, we found marked molecular similarities between the disease in these mice and human myelodysplastic syndrome and AML. This study demonstrates that minimal reduction of a key lineage-specific transcription factor, which commonly occurs in human disease, is sufficient to initiate cancer development, and it provides mechanistic insight into the formation and progression of preleukemic stem cells in AML.
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Leucemia Mieloide Aguda/metabolismo , Preleucemia/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores/metabolismo , Animales , Progresión de la Enfermedad , Humanos , Leucemia Mieloide Aguda/patología , Ratones , Preleucemia/patología , Proteínas Proto-Oncogénicas/genética , Transactivadores/genéticaRESUMEN
Overexpression of immune-related genes is widely reported in myelodysplastic syndromes (MDSs), and chronic immune stimulation increases the risk for developing MDS. Aberrant innate immune activation, such as that caused by increased toll-like receptor (TLR) signaling, in MDS can contribute to systemic effects on hematopoiesis, in addition to cell-intrinsic defects on hematopoietic stem/progenitor cell (HSPC) function. This review will deconstruct aberrant function of TLR signaling mediators within MDS HSPCs that may contribute to cell-intrinsic consequences on hematopoiesis and disease pathogenesis. We will discuss the contribution of chronic TLR signaling to the pathogenesis of MDS based on evidence from patients and mouse genetic models.
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Hematopoyesis/inmunología , Células Madre Hematopoyéticas/inmunología , Inmunidad Innata , Síndromes Mielodisplásicos/inmunología , Transducción de Señal/inmunología , Receptores Toll-Like/inmunología , Animales , Modelos Animales de Enfermedad , Hematopoyesis/genética , Células Madre Hematopoyéticas/patología , Humanos , Ratones , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/patología , Transducción de Señal/genética , Receptores Toll-Like/genéticaRESUMEN
Specific combinations of acute myeloid leukemia (AML) disease alleles, including FLT3 and TET2 mutations, confer distinct biologic features and adverse outcome. We generated mice with mutations in Tet2 and Flt3, which resulted in fully penetrant, lethal AML. Multipotent Tet2(-/-);Flt3(ITD) progenitors (LSK CD48(+)CD150(-)) propagate disease in secondary recipients and were refractory to standard AML chemotherapy and FLT3-targeted therapy. Flt3(ITD) mutations and Tet2 loss cooperatively remodeled DNA methylation and gene expression to an extent not seen with either mutant allele alone, including at the Gata2 locus. Re-expression of Gata2 induced differentiation in AML stem cells and attenuated leukemogenesis. TET2 and FLT3 mutations cooperatively induce AML, with a defined leukemia stem cell population characterized by site-specific changes in DNA methylation and gene expression.
Asunto(s)
Proteínas de Unión al ADN/genética , Epigénesis Genética , Leucemia Mieloide Aguda/genética , Proteínas Proto-Oncogénicas/genética , Tirosina Quinasa 3 Similar a fms/genética , Antineoplásicos/uso terapéutico , Diferenciación Celular/genética , Citarabina/uso terapéutico , Metilación de ADN , Proteínas de Unión al ADN/metabolismo , Dioxigenasas , Doxorrubicina/uso terapéutico , Factor de Transcripción GATA2/genética , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Haploinsuficiencia , Mutación , Proteínas Proto-Oncogénicas/metabolismo , Tirosina Quinasa 3 Similar a fms/metabolismoRESUMEN
Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) are associated with disease-initiating stem cells that are not eliminated by conventional therapies. Novel therapeutic targets against preleukemic stem cells need to be identified for potentially curative strategies. We conducted parallel transcriptional analysis of highly fractionated stem and progenitor populations in MDS, AML, and control samples and found interleukin 8 (IL8) to be consistently overexpressed in patient samples. The receptor for IL8, CXCR2, was also significantly increased in MDS CD34(+) cells from a large clinical cohort and was predictive of increased transfusion dependence. High CXCR2 expression was also an adverse prognostic factor in The Cancer Genome Atlas AML cohort, further pointing to the critical role of the IL8-CXCR2 axis in AML/MDS. Functionally, CXCR2 inhibition by knockdown and pharmacologic approaches led to a significant reduction in proliferation in several leukemic cell lines and primary MDS/AML samples via induction of G0/G1 cell cycle arrest. Importantly, inhibition of CXCR2 selectively inhibited immature hematopoietic stem cells from MDS/AML samples without an effect on healthy controls. CXCR2 knockdown also impaired leukemic growth in vivo. Together, these studies demonstrate that the IL8 receptor CXCR2 is an adverse prognostic factor in MDS/AML and is a potential therapeutic target against immature leukemic stem cell-enriched cell fractions in MDS and AML.
Asunto(s)
Células Madre Hematopoyéticas/metabolismo , Interleucina-8/metabolismo , Leucemia Mieloide Aguda/metabolismo , Síndromes Mielodisplásicos/metabolismo , Receptores de Interleucina-8B/metabolismo , Transducción de Señal , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular/efectos de los fármacos , Análisis por Conglomerados , Modelos Animales de Enfermedad , Expresión Génica , Perfilación de la Expresión Génica , Humanos , Interleucina-8/genética , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidad , Ratones , Síndromes Mielodisplásicos/tratamiento farmacológico , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/mortalidad , Células Madre Neoplásicas/metabolismo , Pronóstico , Receptores de Interleucina-8B/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Acute myeloid leukemia (AML) is characterized by disruption of HSC and progenitor cell differentiation. Frequently, AML is associated with mutations in genes encoding epigenetic modifiers. We hypothesized that analysis of alterations in DNA methylation patterns during healthy HSC commitment and differentiation would yield epigenetic signatures that could be used to identify stage-specific prognostic subgroups of AML. We performed a nano HpaII-tiny-fragment-enrichment-by-ligation-mediated-PCR (nanoHELP) assay to compare genome-wide cytosine methylation profiles between highly purified human long-term HSC, short-term HSC, common myeloid progenitors, and megakaryocyte-erythrocyte progenitors. We observed that the most striking epigenetic changes occurred during the commitment of short-term HSC to common myeloid progenitors and these alterations were predominantly characterized by loss of methylation. We developed a metric of the HSC commitmentassociated methylation pattern that proved to be highly prognostic of overall survival in 3 independent large AML patient cohorts, regardless of patient treatment and epigenetic mutations. Application of the epigenetic signature metric for AML prognosis was superior to evaluation of commitment-based gene expression signatures. Together, our data define a stem cell commitmentassociated methylome that is independently prognostic of poorer overall survival in AML.
Asunto(s)
Diferenciación Celular , Epigénesis Genética , Células Madre Hematopoyéticas/fisiología , Leucemia Mieloide Aguda/patología , Metilación de ADN , Humanos , Estimación de Kaplan-Meier , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/mortalidad , Pronóstico , Modelos de Riesgos Proporcionales , TranscriptomaRESUMEN
How hematopoietic stem cells (HSCs) coordinate the regulation of opposing cellular mechanisms such as self-renewal and differentiation commitment remains unclear. Here we identified the transcription factor and chromatin remodeler Satb1 as a critical regulator of HSC fate. HSCs lacking Satb1 had defective self-renewal, were less quiescent and showed accelerated lineage commitment, which resulted in progressive depletion of functional HSCs. The enhanced commitment was caused by less symmetric self-renewal and more symmetric differentiation divisions of Satb1-deficient HSCs. Satb1 simultaneously repressed sets of genes encoding molecules involved in HSC activation and cellular polarity, including Numb and Myc, which encode two key factors for the specification of stem-cell fate. Thus, Satb1 is a regulator that promotes HSC quiescence and represses lineage commitment.
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Células Madre Hematopoyéticas/fisiología , Proteínas de Unión a la Región de Fijación a la Matriz/metabolismo , Animales , Diferenciación Celular/genética , Linaje de la Célula/genética , Polaridad Celular/genética , Supervivencia Celular/genética , Células Cultivadas , Ensamble y Desensamble de Cromatina/genética , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas de Unión a la Región de Fijación a la Matriz/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismoRESUMEN
Recent experimental evidence has shown that acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) arise from transformed immature hematopoietic cells following the accumulation of multiple stepwise genetic and epigenetic changes in hematopoietic stem cells and committed progenitors. The series of transforming events initially gives rise to preleukemic stem cells (pre-LSC), preceding the formation of fully transformed leukemia stem cells (LSC). Despite the established use of poly-chemotherapy, relapse continues to be the most common cause of death in AML and MDS. The therapeutic elimination of all LSC, as well as pre-LSC, which provide a silent reservoir for the re-formation of LSC, will be essential for achieving lasting cures. Conventional sequencing and next-generation genome sequencing have allowed us to describe many of the recurrent mutations in the bulk cell populations in AML and MDS, and recent work has also focused on identifying the initial molecular changes contributing to leukemogenesis. Here we review recent and ongoing advances in understanding the roles of pre-LSC, and the aberrations that lead to pre-LSC formation and subsequent LSC transformation.
Asunto(s)
Transformación Celular Neoplásica/genética , Leucemia/patología , Células Madre Neoplásicas/patología , Preleucemia/patología , Regulación Leucémica de la Expresión Génica/fisiología , Humanos , Leucemia/genética , Biología Molecular , Células Madre Neoplásicas/fisiología , Preleucemia/genéticaRESUMEN
Homeobox domain-containing transcription factors are important regulators of hematopoiesis. Here, we report that increased levels of nonclustered H2.0-like homeobox (HLX) lead to loss of functional hematopoietic stem cells and formation of aberrant progenitors with unlimited serial clonogenicity and blocked differentiation. Inhibition of HLX reduces proliferation and clonogenicity of leukemia cells, overcomes the differentiation block, and leads to prolonged survival. HLX regulates a transcriptional program, including PAK1 and BTG1, that controls cellular differentiation and proliferation. HLX is overexpressed in 87% of patients with acute myeloid leukemia (AML) and independently correlates with inferior overall survival (n = 601, p = 2.3 × 10(-6)). Our study identifies HLX as a key regulator in immature hematopoietic and leukemia cells and as a prognostic marker and therapeutic target in AML.
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Hematopoyesis , Proteínas de Homeodominio/metabolismo , Leucemia Mieloide Aguda/patología , Factores de Transcripción/metabolismo , Animales , Antígenos CD34/metabolismo , Ciclo Celular/genética , Muerte Celular , Diferenciación Celular/genética , Células Clonales , Regulación hacia Abajo/genética , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica , Hematopoyesis/genética , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , Proteínas de Homeodominio/antagonistas & inhibidores , Proteínas de Homeodominio/genética , Humanos , Lentivirus/genética , Leucemia Mieloide Aguda/genética , Ratones , Monocitos/metabolismo , Monocitos/patología , Proteínas Proto-Oncogénicas c-kit/metabolismo , Análisis de Supervivencia , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Transcriptoma/genéticaRESUMEN
Even though hematopoietic stem cell (HSC) dysfunction is presumed in myelodysplastic syndrome (MDS), the exact nature of quantitative and qualitative alterations is unknown. We conducted a study of phenotypic and molecular alterations in highly fractionated stem and progenitor populations in a variety of MDS subtypes. We observed an expansion of the phenotypically primitive long-term HSCs (lineage(-)/CD34(+)/CD38(-)/CD90(+)) in MDS, which was most pronounced in higher-risk cases. These MDS HSCs demonstrated dysplastic clonogenic activity. Examination of progenitors revealed that lower-risk MDS is characterized by expansion of phenotypic common myeloid progenitors, whereas higher-risk cases revealed expansion of granulocyte-monocyte progenitors. Genome-wide analysis of sorted MDS HSCs revealed widespread methylomic and transcriptomic alterations. STAT3 was an aberrantly hypomethylated and overexpressed target that was validated in an independent cohort and found to be functionally relevant in MDS HSCs. FISH analysis demonstrated that a very high percentage of MDS HSC (92% ± 4%) carry cytogenetic abnormalities. Longitudinal analysis in a patient treated with 5-azacytidine revealed that karyotypically abnormal HSCs persist even during complete morphologic remission and that expansion of clonotypic HSCs precedes clinical relapse. This study demonstrates that stem and progenitor cells in MDS are characterized by stage-specific expansions and contain epigenetic and genetic alterations.
Asunto(s)
Aberraciones Cromosómicas , Cromosomas Humanos Par 7/genética , Células Madre Hematopoyéticas , Síndromes Mielodisplásicos/genética , Factor de Transcripción STAT3/genética , Antígenos CD/genética , Antígenos CD/metabolismo , Azacitidina/administración & dosificación , Estudios de Casos y Controles , Linaje de la Célula , Metilación de ADN , Epigénesis Genética , Citometría de Flujo , Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , Humanos , Cariotipificación , Síndromes Mielodisplásicos/diagnóstico , Síndromes Mielodisplásicos/tratamiento farmacológico , Síndromes Mielodisplásicos/patología , Cultivo Primario de Células , Recurrencia , Factor de Transcripción STAT3/metabolismoRESUMEN
Cellular and interpatient heterogeneity and the involvement of different stem and progenitor compartments in leukemogenesis are challenges for the identification of common pathways contributing to the initiation and maintenance of acute myeloid leukemia (AML). Here we used a strategy of parallel transcriptional analysis of phenotypic long-term hematopoietic stem cells (HSCs), short-term HSCs, and granulocyte-monocyte progenitors from individuals with high-risk (-7/7q-) AML and compared them with the corresponding cell populations from healthy controls. This analysis revealed dysregulated expression of 11 genes, including IL-1 receptor accessory protein (IL1RAP), in all leukemic stem and progenitor cell compartments. IL1RAP protein was found to be overexpressed on the surface of HSCs of AML patients, and marked cells with the -7/7q- anomaly. IL1RAP was also overexpressed on HSCs of patients with normal karyotype AML and high-risk myelodysplastic syndrome, suggesting a pervasive role in different disease subtypes. High IL1RAP expression was independently associated with poor overall survival in 3 independent cohorts of AML patients (P = 2.2 × 10(-7)). Knockdown of IL1RAP decreased clonogenicity and increased cell death of AML cells. Our study identified genes dysregulated in stem and progenitor cells in -7/7q- AML, and suggests that IL1RAP may be a promising therapeutic and prognostic target in AML and high-risk myelodysplastic syndrome.
Asunto(s)
Proteína Accesoria del Receptor de Interleucina-1/genética , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Síndromes Mielodisplásicos/diagnóstico , Síndromes Mielodisplásicos/genética , Células Madre Neoplásicas/metabolismo , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Biomarcadores de Tumor/fisiología , Estudios de Cohortes , Regulación Leucémica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Células HL-60 , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , Humanos , Proteína Accesoria del Receptor de Interleucina-1/metabolismo , Proteína Accesoria del Receptor de Interleucina-1/fisiología , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/mortalidad , Modelos Biológicos , Síndromes Mielodisplásicos/metabolismo , Síndromes Mielodisplásicos/mortalidad , Células Madre Neoplásicas/patología , Pronóstico , Análisis de Supervivencia , Células Tumorales Cultivadas , Regulación hacia Arriba/genéticaRESUMEN
Eltrombopag (EP) is a small-molecule, nonpeptide thrombopoietin receptor (TPO-R) agonist that has been approved recently for the treatment of thrombocytopenia in patients with chronic immune thrombocytopenic purpura. Prior studies have shown that EP stimulates megakaryopoiesis in BM cells from patients with acute myeloid leukemia and myelodysplastic syndrome, and the results also suggested that it may inhibit leukemia cell growth. In the present study, we studied the effects of EP on leukemia cell proliferation and the mechanism of its antiproliferative effects. We found that EP leads to a decreased cell division rate, a block in G(1) phase of cell cycle, and increased differentiation in human and murine leukemia cells. Because EP is species specific in that it can only bind TPO-R in human and primate cells, these findings further suggested that the antileukemic effect is independent of TPO-R. We found that treatment with EP leads to a reduction in free intracellular iron in leukemic cells in a dose-dependent manner. Experimental increase of intracellular iron abrogated the antiproliferative and differentiation-inducing effects of EP, demonstrating that its antileukemic effects are mediated through modulation of intracellular iron content. Finally, determination of EP's antileukemic activity in vivo demonstrated its ability to prolong survival in 2 mouse models of leukemia.
Asunto(s)
Benzoatos/farmacología , Hidrazinas/farmacología , Hierro/metabolismo , Leucemia/tratamiento farmacológico , Leucemia/metabolismo , Pirazoles/farmacología , Animales , Antineoplásicos/farmacología , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Células HL-60 , Humanos , Leucemia/patología , Leucemia Experimental/tratamiento farmacológico , Leucemia Experimental/metabolismo , Leucemia Experimental/patología , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Receptores de Trombopoyetina/agonistas , Células U937RESUMEN
The deposition of toxic amyloid-ß (Aß) peptide aggregates in the brain is a hallmark of Alzheimer's disease. The intramembrane proteolysis by γ-secretase of the amyloid precursor protein ß-carboxy-terminal fragment (APP-ßCTF) constitutes the final step in the production of Aß peptides. Mounting evidence suggests that APP-ßCTF is a transmembrane domain (TMD) dimer, and that dimerization might modulate the production of Aß species that are prone to aggregation and are therefore most toxic. We combined experimental and computational approaches to study the molecular determinants and thermodynamics of APP-ßCTF dimerization, and we produced a unifying structural model that reconciles much of the published data. Using a cell assay that exploits a dimerization-dependent activator of transcription, we identified specific dimerization-affecting mutations located mostly at the N-terminus of the TMD of APP-ßCTF. The ability of selected mutants to affect the dimerization of full-length APP-ßCTF was confirmed by fluorescence resonance energy transfer experiments. Free-energy estimates of the wild type and mutants of the TMD of APP-ßCTF derived from enhanced molecular dynamics simulations showed that the dimeric state is composed of different arrangements, in which either (709)GXXXA(713) or (700)GXXXG(704)GXXXG(708) interaction motifs can engage in symmetric or asymmetric associations. Mutations along the TMD of APP-ßCTF were found to modulate the relative free energy of the dimeric configurations and to differently affect the distribution of interfaces within the dimeric state. This observation might have important biological implications, since dimers with a different arrangement of the transmembrane helices are likely to be recognized differently by γ-secretase and to lead to a variation in Aß levels.