RESUMO
Severe congenital neutropenia (CN) is an inherited pre-leukemia bone marrow failure syndrome commonly caused by autosomal-dominant ELANE mutations (ELANE-CN). ELANE-CN patients are treated with daily injections of recombinant human granulocyte colony-stimulating factor (rhG-CSF). However, some patients do not respond to rhG-CSF, and approximately 15% of ELANE-CN patients develop myelodysplasia or acute myeloid leukemia. Here, we report the development of a curative therapy for ELANE-CN through inhibition of ELANE mRNA expression by introducing two single-strand DNA breaks at the opposing DNA strands of the ELANE promoter TATA box using CRISPR-Cas9D10A nickases-termed MILESTONE. This editing effectively restored defective neutrophil differentiation of ELANE-CN CD34+ hematopoietic stem and progenitor cells (HSPCs) in vitro and in vivo, without affecting the functions of the edited neutrophils. CRISPResso analysis of the edited ELANE-CN CD34+ HSPCs revealed on-target efficiencies of over 90%. Simultaneously, GUIDE-seq, CAST-Seq, and rhAmpSeq indicated a safe off-target profile with no off-target sites or chromosomal translocations. Taken together, ex vivo gene editing of ELANE-CN HSPCs using MILESTONE in the setting of autologous stem cell transplantation could be a universal, safe, and efficient gene therapy approach for ELANE-CN patients.
Assuntos
Sistemas CRISPR-Cas , Síndrome Congênita de Insuficiência da Medula Óssea , Edição de Genes , Terapia Genética , Elastase de Leucócito , Neutropenia , Regiões Promotoras Genéticas , Edição de Genes/métodos , Humanos , Neutropenia/congênito , Neutropenia/terapia , Neutropenia/genética , Terapia Genética/métodos , Síndrome Congênita de Insuficiência da Medula Óssea/terapia , Síndrome Congênita de Insuficiência da Medula Óssea/genética , Elastase de Leucócito/genética , Elastase de Leucócito/metabolismo , Animais , Camundongos , Neutrófilos/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Mutação , Modelos Animais de Doenças , Fator Estimulador de Colônias de Granulócitos/genética , Doenças Genéticas Ligadas ao Cromossomo X/terapia , Doenças Genéticas Ligadas ao Cromossomo X/genéticaRESUMO
Mutations in the ELANE gene, encoding the neutrophil elastase (NE) protein, are responsible for most CyN cases and approximately 25 % of CN cases. In CN and in CyN, a median of 2.8 % of CD34+ cells were early CD49f+ hematopoietic stem cells (eHSC) that did not express ELANE and thus escape from the unfolded protein response (UPR) caused by mutated NE. In CyN, the CD49f+ cells respond to G-CSF with a significant upregulation of the hematopoietic stem-cell-specific transcription factors, C/EBP/, MLL1, HOXA9, MEIS1, and HLF during the ascending arm of the cycle, resulting in the differentiation of myeloid cells to mature neutrophils at the cycle peak. However, NE protein released by neutrophils at the cycle's peak caused a negative feedback loop on granulopoiesis through the proteolytic digestion of G-CSF. In contrast, in CN patients, CD49f+ cells failed to express mRNA levels of HSC-specific transcription factors mentioned above. Rescue of C/EBP//expression in CN restored granulopoiesis.
RESUMO
HAX1-related congenital neutropenia (HAX1-CN) is a rare autosomal recessive disorder caused by pathogenic variants in the HAX1 gene. HAX1-CN patients suffer from bone marrow failure as assessed by a maturation arrest of the myelopoiesis revealing persistent severe neutropenia from birth. The disorder is strongly associated with severe bacterial infections and a high risk of developing myelodysplastic syndrome or acute myeloid leukaemia. This study aimed to describe the long-term course of the disease, the treatment, outcome and quality of life in patients with homozygous HAX1 mutations reported to the European branch of the Severe Chronic Neutropenia International Registry. We have analysed a total of 72 patients with different types of homozygous (n = 68), compound heterozygous (n = 3), and digenic (n = 1) HAX1 mutations. The cohort includes 56 paediatric (<18 years) and 16 adult patients. All patients were initially treated with G-CSF with a sufficient increase in absolute neutrophil counts. Twelve patients required haematopoietic stem cell transplantation for leukaemia (n = 8) and non-leukaemic indications (n = 4). While previous genotype-phenotype reports documented a striking correlation between two main transcript variants and clinical neurological phenotypes, our current analysis reveals novel mutation subtypes and clinical overlaps between all genotypes including severe secondary manifestations, e.g., high incidence of secondary ovarian insufficiency.
Assuntos
Neutropenia , Qualidade de Vida , Humanos , Proteínas/genética , Mutação , Neutropenia/congênito , Sistema de Registros , Proteínas Adaptadoras de Transdução de Sinal/genéticaRESUMO
Most studies on leukemia focus on leukemia cells as isolated objects without considering the bone marrow niche. Pal et al. have recreated the bone marrow niche using induced pluripotent stem cells (iPSCs), identifying CDH2 as a therapeutically druggable leukemia-promoting factor.1.
Assuntos
Medula Óssea , Leucemia , Humanos , Medicina RegenerativaRESUMO
Severe congenital neutropenia (CN) is a pre-leukemic bone marrow failure syndrome that can evolve to acute myeloid leukemia (AML). Mutations in CSF3R and RUNX1 are frequently observed in CN patients, although how they drive the transition from CN to AML (CN/AML) is unclear. Here we establish a model of stepwise leukemogenesis in CN/AML using CRISPR-Cas9 gene editing of CN patient-derived iPSCs. We identified BAALC upregulation and resultant phosphorylation of MK2a as a key leukemogenic event. BAALC deletion or treatment with CMPD1, a selective inhibitor of MK2a phosphorylation, blocked proliferation and induced differentiation of primary CN/AML blasts and CN/AML iPSC-derived hematopoietic stem and progenitor cells (HSPCs) without affecting healthy donor or CN iPSC-derived HSPCs. Beyond detailing a useful method for future investigation of stepwise leukemogenesis, this study suggests that targeting BAALC and/or MK2a phosphorylation may prevent leukemogenic transformation or eliminate AML blasts in CN/AML and RUNX1 mutant BAALC(hi) de novo AML.
Assuntos
Células-Tronco Pluripotentes Induzidas , Leucemia Mieloide Aguda , Proteínas de Neoplasias , Neutropenia , Síndrome Congênita de Insuficiência da Medula Óssea , Humanos , Leucemia Mieloide Aguda/genética , Mutação/genética , Proteínas de Neoplasias/genética , Neutropenia/congênito , Neutropenia/genética , OncogenesRESUMO
Patients with the pre-leukemia bone marrow failure syndrome called severe congenital neutropenia (CN) have an approximately 15% risk of developing acute myeloid leukemia (AML; called here CN/AML). Most CN/AML patients co-acquire CSF3R and RUNX1 mutations, which play cooperative roles in the development of AML. To establish an in vitro model of leukemogenesis, we utilized bone marrow lin- cells from transgenic C57BL/6-d715 Csf3r mice expressing a CN patient-mimicking truncated CSF3R mutation. We transduced these cells with vectors encoding RUNX1 wild type (WT) or RUNX1 mutant proteins carrying the R139G or R174L mutations. Cells transduced with these RUNX1 mutants showed diminished in vitro myeloid differentiation and elevated replating capacity, compared with those expressing WT RUNX1. mRNA expression analysis showed that cells transduced with the RUNX1 mutants exhibited hyperactivation of inflammatory signaling and innate immunity pathways, including IL-6, TLR, NF-kappaB, IFN, and TREM1 signaling. These data suggest that the expression of mutated RUNX1 in a CSF3R-mutated background may activate the pro-inflammatory cell state and inhibit myeloid differentiation.
Assuntos
Síndrome Congênita de Insuficiência da Medula Óssea/genética , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Células-Tronco Hematopoéticas/patologia , Células Mieloides/patologia , Mielopoese/genética , Neutropenia/congênito , Pré-Leucemia/genética , Receptores de Fator Estimulador de Colônias/genética , Animais , Divisão Celular , Ensaio de Unidades Formadoras de Colônias , Síndrome Congênita de Insuficiência da Medula Óssea/patologia , Subunidade alfa 2 de Fator de Ligação ao Core/fisiologia , Perfilação da Expressão Gênica , Imunidade Inata , Inflamação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neutropenia/genética , Neutropenia/patologia , Pré-Leucemia/patologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores de Fator Estimulador de Colônias/fisiologia , Proteínas Recombinantes/genética , Organismos Livres de Patógenos EspecíficosRESUMO
Cyclic neutropenia (CyN) is a hematologic disorder in which peripheral blood absolute neutrophil counts (ANCs) show cycles of approximately 21-day intervals. The majority of CyN patients harbor ELANE mutations, but the mechanism of ANC cycling is unclear. We performed analysis of bone marrow (BM) subpopulations in CyN patients at the peak and the nadir of the ANC cycle and detected high proportions of BM hematopoietic stem cells (HSCs) and hematopoietic stem and progenitor cells (HSPCs) at the nadir of the ANC cycle, as compared with the peak. BM HSPCs produced fewer granulocyte colony-forming unit colonies at the ANC peak. To investigate the mechanism of cycling, we found that mRNA expression levels of ELANE and unfolded protein response (UPR)-related genes (ATF6, BiP (HSPA5), CHOP (DDIT3), and PERK (EIF2AK3)) were elevated, but antiapoptotic genes (Bcl-2 (BCL2) and bcl-xL (BCL2L1)) were reduced in CD34+ cells tested at the ANC nadir. Moreover, HSPCs revealed increased levels of reactive oxygen species and gH2AX at the ANC nadir. We suggest that in CyN patients, some HSPCs escape the UPR-induced endoplasmic reticulum (ER) stress and proliferate in response to granulocyte colony-stimulating factor (G-CSF) to a certain threshold at which UPR again affects the majority of HSPCs. There is a cyclic balance between ER stress-induced apoptosis of HSPCs and compensatory G-CSF-stimulated HSPC proliferation followed by granulocytic differentiation.
Assuntos
Estresse do Retículo Endoplasmático/fisiologia , Elastase de Leucócito/genética , Neutropenia/etiologia , Resposta a Proteínas não Dobradas/fisiologia , Medula Óssea/efeitos dos fármacos , Medula Óssea/metabolismo , Medula Óssea/patologia , Células Cultivadas , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Seguimentos , Fator Estimulador de Colônias de Granulócitos/farmacologia , Fator Estimulador de Colônias de Granulócitos/uso terapêutico , Hematopoese/efeitos dos fármacos , Hematopoese/genética , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/patologia , Células-Tronco Hematopoéticas/fisiologia , Humanos , Elastase de Leucócito/fisiologia , Mutação , Neutropenia/tratamento farmacológico , Neutropenia/metabolismo , Neutropenia/patologia , Espécies Reativas de Oxigênio/metabolismo , Índice de Gravidade de Doença , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Resposta a Proteínas não Dobradas/genéticaRESUMO
In this chapter, we present an optimized CRISPR/Cas9 RNP nucleofection approach for gene knockout (KO) in hematopoietic stem and progenitor cells (HSPCs). With experimentally proved active locus-specific sgRNAs, we routinely reach over 80% gene KO in HSPCs, thus avoiding the need for cell sorting or enrichment of targeted cell population. Additionally, we provide a protocol for in vitro granulocytic differentiation of HSPCs after gene KO and detailed description of granulocyte function tests which can be applied to study the effects of a particular gene KO.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Granulócitos/citologia , Células-Tronco Hematopoéticas/citologia , Leucopoese , Células Cultivadas , Técnicas de Inativação de Genes/métodos , Granulócitos/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Humanos , RNA Guia de Cinetoplastídeos/genéticaRESUMO
A Autosomal-dominant ELANE mutations are the most common cause of severe congenital neutropenia. Although the majority of congenital neutropenia patients respond to daily granulocyte colony stimulating factor, approximately 15 % do not respond to this cytokine at doses up to 50 µg/kg/day and approximately 15 % of patients will develop myelodysplasia or acute myeloid leukemia. "Maturation arrest," the failure of the marrow myeloid progenitors to form mature neutrophils, is a consistent feature of ELANE associated congenital neutropenia. As mutant neutrophil elastase is the cause of this abnormality, we hypothesized that ELANE associated neutropenia could be treated and "maturation arrest" corrected by a CRISPR/Cas9-sgRNA ribonucleoprotein mediated ELANE knockout. To examine this hypothesis, we used induced pluripotent stem cells from two congenital neutropenia patients and primary hematopoietic stem and progenitor cells from four congenital neutropenia patients harboring ELANE mutations as well as HL60 cells expressing mutant ELANE We observed that granulocytic differentiation of ELANE knockout induced pluripotent stem cells and primary hematopoietic stem and progenitor cells were comparable to healthy individuals. Phagocytic functions, ROS production, and chemotaxis of the ELANE KO (knockout) neutrophils were also normal. Knockdown of ELANE in the mutant ELANE expressing HL60 cells also allowed full maturation and formation of abundant neutrophils. These observations suggest that ex vivo CRISPR/Cas9 RNP based ELANE knockout of patients' primary hematopoietic stem and progenitor cells followed by autologous transplantation may be an alternative therapy for congenital neutropenia.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Células-Tronco Pluripotentes Induzidas , Neutropenia , Sistemas CRISPR-Cas , Síndrome Congênita de Insuficiência da Medula Óssea , Humanos , Mutação , Neutropenia/congênito , Neutropenia/genéticaRESUMO
Hematopoietic transcription factor LIM domain only 2 (LMO2), a member of the TAL1 transcriptional complex, plays an essential role during early hematopoiesis and is frequently activated in T-cell acute lymphoblastic leukemia (T-ALL) patients. Here, we demonstrate that LMO2 is activated by deacetylation on lysine 74 and 78 via the nicotinamide phosphoribosyltransferase (NAMPT)/sirtuin 2 (SIRT2) pathway. LMO2 deacetylation enables LMO2 to interact with LIM domain binding 1 and activate the TAL1 complex. NAMPT/SIRT2-mediated activation of LMO2 by deacetylation appears to be important for hematopoietic differentiation of induced pluripotent stem cells and blood formation in zebrafish embryos. In T-ALL, deacetylated LMO2 induces expression of TAL1 complex target genes HHEX and NKX3.1 as well as LMO2 autoregulation. Consistent with this, inhibition of NAMPT or SIRT2 suppressed the in vitro growth and in vivo engraftment of T-ALL cells via diminished LMO2 deacetylation. This new molecular mechanism may provide new therapeutic possibilities in T-ALL and may contribute to the development of new methods for in vitro generation of blood cells.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Hematopoese , Proteínas com Domínio LIM/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Acetilação , Animais , Células Cultivadas , Células HEK293 , Humanos , Leucopoese , Camundongos , Modelos Moleculares , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Peixe-ZebraRESUMO
High frequency of acquired CSF3R (colony stimulating factor 3 receptor, granulocyte) mutations has been described in patients with severe congenital neutropenia (CN) at pre-leukemia stage and overt acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). Here, we report the establishment of an ultra-sensitive deep sequencing of a CSF3R segment encoding the intracellular "critical region" of the G-CSFR known to be mutated in CN-MDS/AML patients. Using this method, we achieved a mutant allele frequency (MAF) detection rate of 0.01%. We detected CSF3R mutations in CN patients with different genetic backgrounds, but not in patients with other types of bone marrow failure syndromes chronically treated with G-CSF (e.g., Shwachman-Diamond Syndrome). Comparison of CSF3R deep sequencing results of DNA and cDNA from the bone marrow and peripheral blood cells revealed the highest sensitivity of cDNA from the peripheral blood polymorphonuclear neutrophils. This approach enables the identification of low-frequency CSF3R mutant clones, increases sensitivity, and earlier detection of CSF3R mutations acquired during the course of leukemogenic evolution of pre-leukemia HSCs of CN patients. We suggest application of sequencing of the entire CSF3R gene at diagnosis to identify patients with inherited lost-of-function CSF3R mutations and annual ultra-deep sequencing of the critical region of CSF3R to monitor acquisition of CSF3R mutations.
Assuntos
Síndrome Congênita de Insuficiência da Medula Óssea/genética , Detecção Precoce de Câncer/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Leucemia Mieloide Aguda/genética , Mutação/genética , Síndromes Mielodisplásicas/genética , Neutropenia/congênito , Receptores de Fator Estimulador de Colônias/genética , Adolescente , Carcinogênese/genética , Criança , Análise Mutacional de DNA , Progressão da Doença , Feminino , Humanos , Masculino , Neutropenia/genética , Polimorfismo de Nucleotídeo Único , Índice de Gravidade de DoençaRESUMO
CRISPR/Cas9-mediated gene editing of stem cells and primary cell types has several limitations for clinical applications. The direct delivery of ribonucleoprotein (RNP) complexes consisting of Cas9 nuclease and guide RNA (gRNA) has improved DNA- and virus-free gene modifications, but it does not enable the essential enrichment of the gene-edited cells. Here, we established a protocol for the fluorescent labeling and delivery of CRISPR/Cas9-gRNA RNP in primary human hematopoietic stem and progenitor cells (HSPCs) and induced pluripotent stem cells (iPSCs). As a proof of principle for genes with low-abundance transcripts and context-dependent inducible expression, we successfully deleted growth arrest and DNA-damage-inducible ß (GADD45B). We found that GADD45B is indispensable for DNA damage protection and survival in stem cells. Thus, we describe an easy and efficient protocol of DNA-free gene editing of hard-to-target transcripts and enrichment of gene-modified cells that are generally difficult to transfect.
Assuntos
Antígenos de Diferenciação/genética , Sistemas CRISPR-Cas , Técnicas de Inativação de Genes , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Ribonucleoproteínas/metabolismo , Estresse Fisiológico/genética , Antígenos de Diferenciação/metabolismo , Linhagem Celular , Dano ao DNA , Edição de Genes/métodos , Marcação de Genes/métodos , Humanos , Substâncias Macromoleculares/metabolismo , Ligação Proteica , RNA Guia de Cinetoplastídeos/genética , Estresse Fisiológico/efeitos da radiaçãoRESUMO
We describe the establishment of an embryoid-body-based protocol for hematopoietic/myeloid differentiation of human induced pluripotent stem cells that allows the generation of CD34+ cells or mature myeloid cells in vitro. Using this model, we were able to recapitulate the defective granulocytic differentiation in patients with severe congenital neutropenia (CN), an inherited preleukemia bone marrow failure syndrome. Importantly, in vitro maturation arrest of granulopoiesis was associated with an elevated unfolded protein response (UPR) and enhanced expression of the cell cycle inhibitor p21. Consistent with this, we found that CD34+ cells of CN patients were highly susceptible to DNA damage and showed diminished DNA repair. These observations suggest that targeting the UPR pathway or inhibiting DNA damage might protect hematopoietic cells of CN patients from leukemogenic transformation, at least to some extent.
Assuntos
Transformação Celular Neoplásica/metabolismo , Dano ao DNA , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Leucemia/etiologia , Modelos Biológicos , Neutropenia/congênito , Resposta a Proteínas não Dobradas , Antígenos CD34/metabolismo , Biomarcadores , Células Cultivadas , Reprogramação Celular , Síndrome Congênita de Insuficiência da Medula Óssea , Estresse do Retículo Endoplasmático , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Imunofenotipagem , Células-Tronco Pluripotentes Induzidas/patologia , Leucemia/metabolismo , Leucemia/patologia , Neutropenia/etiologia , Neutropenia/metabolismo , Neutropenia/patologiaRESUMO
Acquired mutations in the intracellular part of CSF3R (colony stimulating factor 3 receptor, granulocyte) have been detected with a frequency of more than 30% in severe congenital neutropenia (CN) patients. CN is a preleukemic syndrome with a risk of approximately 20% to develop leukemia. More than 80% of CN patients who develop acute myeloid leukemia or myelodysplastic syndrome reveal CSF3R mutations, suggesting that they are involved in leukemogenesis. Using deep-sequencing technology, we were able to analyze large cohorts of CN patients for the entire CSF3R sequence as well as to identify cell clones carrying mutations in the intracellular part of CSF3R with very high sensitivity. Acquisition of CSF3R mutations is a CN-specific phenomenon and is associated with inherited mutations causing CN or cyclic neutropenia, such as ELANE mutations. In the group of CN patients negative for known germ-line mutations, biallelic CSF3R mutations were identified. In addition, CSF3R mutant clones are highly dynamic and may disappear and reappear during continuous granulocyte colony-stimulating factor (G-CSF) therapy. The time between the first detection of CSF3R mutations and overt leukemia is highly variable.
Assuntos
Leucemia Mieloide Aguda/genética , Mutação , Neutropenia/congênito , Neutropenia/genética , Receptores de Fator Estimulador de Colônias/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Leucemia Mieloide Aguda/etiologia , Leucemia Mieloide Aguda/metabolismo , Neutropenia/complicações , Neutropenia/metabolismo , Receptores de Fator Estimulador de Colônias/metabolismoRESUMO
A number of studies have demonstrated induction of the unfolded protein response (UPR) in patients with severe congenital neutropenia (CN) harbouring mutations of ELANE, encoding neutrophil elastase. Why UPR is not activated in patients with cyclic neutropenia (CyN) carrying the same ELANE mutations is unclear. We evaluated the effects of ELANE mutants on UPR induction in myeloid cells from CN and CyN patients, and analysed whether additional CN-specific defects contribute to the differences in UPR induction between CN and CyN patients harbouring identical ELANE mutations. We investigated CN-specific p.C71R and p.V174_C181del (NP_001963.1) and CN/CyN-shared p.S126L (NP_001963.1) ELANE mutants. We found that transduction of haematopoietic cells with p.C71R, but not with p.V174_C181del or p.S126L ELANE mutants induced expression of ATF6, and the ATF6 target genes PPP1R15A, DDIT3 and HSPA5. Recently, we found that levels of secretory leucocyte protease inhibitor (SLPI), a natural ELANE inhibitor, are diminished in myeloid cells from CN patients, but not CyN patients. Combined knockdown of SLPI by shRNA and transduction of ELANE p.S126L in myeloid cells led to elevated levels of ATF6, PPP1R15A and HSPA5 RNA, suggesting that normal levels of SLPI in CyN patients might protect them from the UPR induced by mutant ELANE. In summary, different ELANE mutants have different effects on UPR activation, and SLPI regulates the extent of ELANE-triggered UPR.