RESUMEN

Identification of driver genes and pathological bone marrow diagnosis were considered essential for subclassification of MPN in the revised 4th edition of the WHO classification, and this remained nearly unchanged in the 5th edition. Pathological diagnosis of primary myelofibrosis/pre-fibrotic stage by biopsy is now feasible and is becoming standardized. Progressive myelofibrosis and blast transformation are the advanced forms of MPN, and various therapeutic interventions for these forms have been devised. Observation of activated megakaryocytes on evaluation of megakaryocyte morphology could predict the progression of myelofibrosis. This paper describes recent progress in pathological diagnosis of MPN and how it is performed in practice.

Asunto(s)

Médula Ósea , Trastornos Mieloproliferativos , Humanos , Trastornos Mieloproliferativos/diagnóstico , Trastornos Mieloproliferativos/patología , Trastornos Mieloproliferativos/genética , Médula Ósea/patología , Mielofibrosis Primaria/diagnóstico , Mielofibrosis Primaria/patología

RESUMEN

Philadelphia-Negative Myeloproliferative neoplasms (MPNs) are a diverse group of blood cancers leading to excessive production of mature blood cells. These chronic diseases, including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), can significantly impact patient quality of life and are still incurable in the vast majority of the cases. This review examines the mechanobiology within a bone marrow niche, emphasizing the role of mechanical cues and the primary cilium in the pathophysiology of MPNs. It discusses the influence of extracellular matrix components, cell-cell and cell-matrix interactions, and mechanosensitive structures on hematopoietic stem cell (HSC) behavior and disease progression. Additionally, the potential implications of the primary cilium as a chemo- and mechanosensory organelle in bone marrow cells are explored, highlighting its involvement in signaling pathways crucial for hematopoietic regulation. This review proposes future research directions to better understand the dysregulated bone marrow niche in MPNs and to identify novel therapeutic targets.

Asunto(s)

Cilios , Trastornos Mieloproliferativos , Humanos , Trastornos Mieloproliferativos/metabolismo , Trastornos Mieloproliferativos/patología , Trastornos Mieloproliferativos/fisiopatología , Cilios/metabolismo , Cilios/patología , Animales , Médula Ósea/patología , Médula Ósea/metabolismo , Células Madre Hematopoyéticas/metabolismo , Mecanotransducción Celular , Matriz Extracelular/metabolismo , Transducción de Señal , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología

RESUMEN

Chronic myeloid leukemia is defined by the presence of the Philadelphia translocation t (9; 22) resulting in the BCR::ABL1 fusion. The other myeloproliferative neoplasms (MPN) subtypes also carry typical chromosomal abnormalities, which however are not pathognomonic for a specific entity of MPN. According to the WHO classification the distinction between these entities is still based on the integration of cytological, histopathological and molecular findings. Progression of CML into accelerated and blastic phase is usually driven by additional chromosome abnormalities and ABL1 kinase mutations. In the other MPN subtypes the additional mutations besides driver gene mutations in JAK2, MPL and CALR have a decisive impact on the propensity for progression. In addition, the sequence in which the driver mutations and risk conveying additional mutations have been acquired appears to play an important role. Here, we review cytogenetic and molecular changes in CML and MPN that should be evaluated during diagnosis and disease monitoring.

Asunto(s)

Janus Quinasa 2 , Leucemia Mielógena Crónica BCR-ABL Positiva , Mutación , Trastornos Mieloproliferativos , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Leucemia Mielógena Crónica BCR-ABL Positiva/diagnóstico , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/diagnóstico , Trastornos Mieloproliferativos/patología , Janus Quinasa 2/genética , Aberraciones Cromosómicas , Genómica/métodos , Proteínas de Fusión bcr-abl/genética , Receptores de Trombopoyetina/genética , Calreticulina/genética , Translocación Genética

RESUMEN

OBJECTIVES: The International Consensus Classification (ICC) and 5th Edition of the World Health Organization Classification (WHO-5) made substantive updates to the classification of myeloid neoplasms. This study compares the systems in a series of myeloid neoplasms with increased blasts, analyzing implications for diagnostic workflow and reporting. METHODS: Bone marrow biopsies categorized as myelodysplastic syndrome with excess blasts (MDS-EB) or acute myeloid leukemia (AML) by WHO-R4 were identified. Results of morphology review, karyotype, fluorescence in situ hybridization, and next-generation sequencing were compiled. Cases were retrospectively re-classified by WHO-5 and ICC. RESULTS: 46 cases were reviewed. 28 cases (61 %) had ≥20 % blasts, with the remaining cases having 5-19.5 % blasts. The most common differences in classification were 1) the designation of MDS versus MDS/AML (10/46, 22 %) for cases with 10-19 % blasts and 2) the ICC's designation of TP53 variants as a separate classifier for AML (8/46, 17 %). Bi-allelic/multi-hit TP53 alterations were identified in 15 cases (33 %). Variants of potential germline significance were identified in 29 (63 %) cases. CONCLUSIONS: While terminology differences between WHO-5 and ICC exist, both systems invoke similar opportunities for improved reporting: standardized classification of pathogenic variants (notably TP53), streamlined systems to evaluate for potential germline variants, and integrated reporting of morphologic and genetic data.

Asunto(s)

Síndromes Mielodisplásicos , Organización Mundial de la Salud , Humanos , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/clasificación , Síndromes Mielodisplásicos/patología , Masculino , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Leucemia Mieloide Aguda/clasificación , Femenino , Estudios Retrospectivos , Persona de Mediana Edad , Anciano , Patología Molecular , Patólogos , Secuenciación de Nucleótidos de Alto Rendimiento , Adulto , Anciano de 80 o más Años , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/clasificación , Trastornos Mieloproliferativos/patología , Trastornos Mieloproliferativos/diagnóstico

Asunto(s)

Microscopía Fluorescente , Trastornos Mieloproliferativos , Humanos , Trastornos Mieloproliferativos/sangre , Trastornos Mieloproliferativos/patología , Trastornos Mieloproliferativos/diagnóstico , Microscopía Fluorescente/métodos , Femenino , Masculino , Persona de Mediana Edad , Anciano , Adulto , Anciano de 80 o más Años

RESUMEN

Increasing evidence supports the interplay between oncogenic mutations and immune escape mechanisms. Strategies to counteract the immune escape mediated by oncogenic signaling could provide improved therapeutic options for patients with various malignancies. As mutant calreticulin (CALR) is a common driver of myeloproliferative neoplasms (MPN), we analyzed the impact of oncogenic CALRdel52 on the bone marrow (BM) microenvironment in MPN. Single-cell RNA sequencing revealed that CALRdel52 led to the expansion of TGFß1-producing erythroid progenitor cells and promoted the expansion of FoxP3+ regulatory T cells (Treg) in a murine MPN model. Treatment with an anti-TGFß antibody improved mouse survival and increased the glycolytic activity in CD4+ and CD8+ T cells in vivo, whereas T-cell depletion abrogated the protective effects conferred by neutralizing TGFß. TGFß1 reduced perforin and TNFα production by T cells in vitro. TGFß1 production by CALRdel52 cells was dependent on JAK1/2, PI3K, and ERK activity, which activated the transcription factor Sp1 to induce TGFß1 expression. In four independent patient cohorts, TGFß1 expression was increased in the BM of patients with MPN compared with healthy individuals, and the BM of patients with MPN contained a higher frequency of Treg compared with healthy individuals. Together, this study identified an ERK/Sp1/TGFß1 axis in CALRdel52 MPNs as a mechanism of immunosuppression that can be targeted to elicit T-cell-mediated cytotoxicity. Significance: Targeting the mutant calreticulin/TGFß1 axis increases T-cell activity and glycolytic capacity, providing the rationale for conducting clinical trials on TGFß antagonists as an immunotherapeutic strategy in patients with myeloproliferative neoplasms.

Asunto(s)

Calreticulina , Trastornos Mieloproliferativos , Linfocitos T Reguladores , Microambiente Tumoral , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Calreticulina/metabolismo , Animales , Humanos , Ratones , Trastornos Mieloproliferativos/inmunología , Trastornos Mieloproliferativos/metabolismo , Trastornos Mieloproliferativos/patología , Microambiente Tumoral/inmunología , Factor de Crecimiento Transformador beta/metabolismo , Médula Ósea/inmunología , Médula Ósea/metabolismo , Escape del Tumor/inmunología , Ratones Endogámicos C57BL , Factor de Crecimiento Transformador beta1/metabolismo , Mutación

RESUMEN

Myeloproliferative neoplasms (MPNs), namely, polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), are clonal stem cell disorders defined by an excessive production of functionally mature and terminally differentiated myeloid cells. MPNs can transform into secondary acute myeloid leukemia (sAML/blast phase MPN) and are linked to alterations in the redox balance, i.e., elevated concentrations of reactive oxygen species and markers of oxidative stress (OS), and changes in antioxidant systems. We evaluated OS in 117 chronic phase MPNs and 21 sAML cases versus controls by measuring total antioxidant capacity (TAC) and 8-hydroxy-2'-deoxy-guanosine (8-OHdG) concentrations. TAC was higher in MPNs than controls (p = 0.03), particularly in ET (p = 0.04) and PMF (p = 0.01). MPL W515L-positive MPNs had higher TAC than controls (p = 0.002) and triple-negative MPNs (p = 0.01). PMF patients who had treatment expressed lower TAC than therapy-free subjects (p = 0.03). 8-OHdG concentrations were similar between controls and MPNs, controls and sAML, and MPNs and sAML. We noted associations between TAC and MPNs (OR = 1.82; p = 0.05), i.e., ET (OR = 2.36; p = 0.03) and PMF (OR = 2.11; p = 0.03), but not sAML. 8-OHdG concentrations were not associated with MPNs (OR = 1.73; p = 0.62) or sAML (OR = 1.89; p = 0.49). In conclusion, we detected redox imbalances in MPNs based on disease subtype, driver mutations, and treatment history.

Asunto(s)

8-Hidroxi-2'-Desoxicoguanosina , Antioxidantes , Trastornos Mieloproliferativos , Humanos , Masculino , Femenino , 8-Hidroxi-2'-Desoxicoguanosina/metabolismo , Persona de Mediana Edad , Anciano , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/metabolismo , Trastornos Mieloproliferativos/patología , Antioxidantes/metabolismo , Adulto , Estrés Oxidativo , Anciano de 80 o más Años , Crisis Blástica/metabolismo , Crisis Blástica/genética , Crisis Blástica/patología , Proteínas de Fusión bcr-abl/genética , Proteínas de Fusión bcr-abl/metabolismo , Mielofibrosis Primaria/genética , Mielofibrosis Primaria/metabolismo , Mielofibrosis Primaria/patología

Asunto(s)

ADN (Citosina-5-)-Metiltransferasas , ADN Metiltransferasa 3A , Humanos , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Animales , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , Ratones , Trastornos Mieloproliferativos/patología , Trastornos Mieloproliferativos/metabolismo , Interferones/metabolismo

RESUMEN

BACKGROUND: Neutrophils play a crucial role in inflammation and in the increased thrombotic risk in myeloproliferative neoplasms (MPNs). We have investigated how neutrophil-specific expression of JAK2-V617F or CALRdel re-programs the functions of neutrophils. METHODS: Ly6G-Cre JAK2-V617F and Ly6G-Cre CALRdel mice were generated. MPN parameters as blood counts, splenomegaly and bone marrow histology were compared to wild-type mice. Megakaryocyte differentiation was investigated using lineage-negative bone marrow cells upon in vitro incubation with TPO/IL-1ß. Cytokine concentrations in serum of mice were determined by Mouse Cytokine Array. IL-1α expression in various hematopoietic cell populations was determined by intracellular FACS analysis. RNA-seq to analyse gene expression of inflammatory cytokines was performed in isolated neutrophils from JAK2-V617F and CALR-mutated mice and patients. Bioenergetics of neutrophils were recorded on a Seahorse extracellular flux analyzer. Cell motility of neutrophils was monitored in vitro (time lapse microscopy), and in vivo (two-photon microscopy) upon creating an inflammatory environment. Cell adhesion to integrins, E-selectin and P-selection was investigated in-vitro. Statistical analysis was carried out using GraphPad Prism. Data are shown as mean ± SEM. Unpaired, two-tailed t-tests were applied. RESULTS: Strikingly, neutrophil-specific expression of JAK2-V617F, but not CALRdel, was sufficient to induce pro-inflammatory cytokines including IL-1 in serum of mice. RNA-seq analysis in neutrophils from JAK2-V617F mice and patients revealed a distinct inflammatory chemokine signature which was not expressed in CALR-mutant neutrophils. In addition, IL-1 response genes were significantly enriched in neutrophils of JAK2-V617F patients as compared to CALR-mutant patients. Thus, JAK2-V617F positive neutrophils, but not CALR-mutant neutrophils, are pathogenic drivers of inflammation in MPN. In line with this, expression of JAK2-V617F or CALRdel elicited a significant difference in the metabolic phenotype of neutrophils, suggesting a stronger inflammatory activity of JAK2-V617F cells. Furthermore, JAK2-V617F, but not CALRdel, induced a VLA4 integrin-mediated adhesive phenotype in neutrophils. This resulted in reduced neutrophil migration in vitro and in an inflamed vessel. This mechanism may contribute to the increased thrombotic risk of JAK2-V617F patients compared to CALR-mutant individuals. CONCLUSIONS: Taken together, our findings highlight genotype-specific differences in MPN-neutrophils that have implications for the differential pathophysiology of JAK2-V617F versus CALR-mutant disease.

Asunto(s)

Inflamación , Janus Quinasa 2 , Trastornos Mieloproliferativos , Neutrófilos , Animales , Neutrófilos/metabolismo , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Ratones , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/patología , Trastornos Mieloproliferativos/metabolismo , Humanos , Inflamación/genética , Inflamación/patología , Calreticulina/genética , Calreticulina/metabolismo , Ratones Transgénicos , Ratones Endogámicos C57BL , Citocinas/metabolismo

Asunto(s)

Trastornos Mieloproliferativos , FN-kappa B , Humanos , FN-kappa B/metabolismo , Trastornos Mieloproliferativos/metabolismo , Trastornos Mieloproliferativos/patología , Trastornos Mieloproliferativos/genética , Animales , Transducción de Señal

RESUMEN

CSF3R activating mutation is a genetic hallmark of chronic neutrophilic leukemia (CNL), and is also present in a subset of atypical chronic myeloid leukemia (aCML), but infrequent in other myeloid neoplasms. However, the occurrence of CSF3R mutations in various myeloid neoplasms is not well studied. Here we evaluate the spectrum of CSF3R mutations and the clinicopathologic features of CSF3R mutated myeloid neoplasms. We retrospectively identified CSF3R mutations in a variety of myeloid neoplasms: two CNL, three atypical chronic myeloid leukemia (aCML), nine acute myeloid leukemia (AML), one chronic myelomonocytic leukemia, and one myeloproliferative neoplasm. The prototypic T618I mutation was found in 50% of cases: CNL (2/2), aCML (2/3) and AML (4/9). We observed a new recurrent CSF3R mutation Q776* in 25% of cases, and a potential-germline mutation in a 20-year-old patient. Co-occurring mutations were often in epigenetic modifier and spliceosome. IDH/RUNX1 and tumor suppressor mutations were frequent in AML but absent in CNL/aCML. All CNL/aCML patients succumbed within 2-years of diagnosis. We demonstrate that CSF3R mutations are not restricted to CNL. CNL and aCML show similar clinicopathologic and molecular features, suggesting that CNL may be best classified as myelodysplastic/myeloproliferative neoplasm rather than myeloproliferative neoplasm.

Asunto(s)

Leucemia Neutrofílica Crónica , Mutación , Receptores del Factor Estimulante de Colonias , Humanos , Receptores del Factor Estimulante de Colonias/genética , Masculino , Persona de Mediana Edad , Femenino , Anciano , Leucemia Neutrofílica Crónica/genética , Leucemia Neutrofílica Crónica/patología , Estudios Retrospectivos , Adulto , Adulto Joven , Anciano de 80 o más Años , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/patología , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Análisis Mutacional de ADN , Leucemia Mieloide Crónica Atípica BCR-ABL Negativa/genética , Leucemia Mieloide Crónica Atípica BCR-ABL Negativa/patología , Predisposición Genética a la Enfermedad , Biomarcadores de Tumor/genética , Fenotipo

RESUMEN

Myeloproliferative neoplasms (MPN) are characterized by a clonal proliferation of myeloid lineage cells within the bone marrow. The classical BCR-ABL negative MPNs are comprised of polycythaemia vera, essential thrombocythaemia and primary myelofibrosis. Historically, the majority of MPNs are diagnosed in adults older than 60 years of age; however, in recent years, there has been recognition of MPNs in the adolescent and young adult (AYA) population. AYAs with MPN, typically defined as between the ages of 15 and 39 years old, may comprise up to 20% of patients diagnosed with MPN. They demonstrate unique patterns of driver mutations and thrombotic events and remain at risk for progression to more aggressive disease states. Given the likely long length of time they will live with their disease, there is a significant unmet need in identifying well-tolerated and effective treatment options for these patients, particularly with the advent of disease modification. In this review, we provide a comprehensive overview of the clinical features, disease course and management of AYA patients with MPN and, in doing so, highlight key characteristics that distinguish them from their older counterparts.

Asunto(s)

Trastornos Mieloproliferativos , Humanos , Trastornos Mieloproliferativos/diagnóstico , Trastornos Mieloproliferativos/terapia , Trastornos Mieloproliferativos/patología , Trastornos Mieloproliferativos/genética , Adolescente , Adulto , Adulto Joven , Masculino , Femenino

Asunto(s)

Médula Ósea , Janus Quinasa 2 , Trastornos Mieloproliferativos , Humanos , Masculino , Médula Ósea/patología , Janus Quinasa 2/genética , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/patología , Proteínas de Fusión Oncogénica/genética , Anciano

RESUMEN

Typical BCR::ABL1-negative myeloproliferative neoplasms (MPN) are mainly referred to as polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofbrosis (PMF). Granulocytes in MPN patients are involved in their inflammation and form an important part of the pathophysiology of MPN patients. It has been shown that the immunophenotype of granulocytes in MPN patients is altered. We used flow cytometry to explore the immunophenotype of MPN patients and correlate it with clinical parameters. The results showed that PMF patients and PV patients had higher CD15+CD11b+ granulocytes than ET patients and normal controls. When grouped by gene mutation, changes in the granulocyte immunophenotype of MPN patients were independent of the JAK2V617F and CALR mutations. There was no significant heterogeneity in immunophenotype between ET patients and Pre-PMF, and between Overt-PMF and Pre-PMF patients. Granulocytes from some MPN patients showed an abnormal CD13/CD16 phenotype with a significant increase in mature granulocytes on molecular and cytomorphological grounds, and this abnormal pattern occurred significantly more frequently in PMF patients than in ET patients. CD15-CD11b- was negatively correlated with WBC and Hb and positively correlated with DIPSS score, whereas high CD10+ granulocytes were significantly and negatively associated with prognostic system IPSS and DIPSS scores in PMF patients. In conclusion, this study demonstrates the landscape of bone marrow granulocyte immunophenotypes in MPN patients. MPN patients, especially those with PMF, have a significant granulocyte developmental overmaturation phenotype. CD10+ granulocytes may be involved in the prognosis of PMF patients.

Asunto(s)

Citometría de Flujo , Proteínas de Fusión bcr-abl , Granulocitos , Inmunofenotipificación , Trastornos Mieloproliferativos , Humanos , Masculino , Persona de Mediana Edad , Femenino , Granulocitos/patología , Adulto , Anciano , Proteínas de Fusión bcr-abl/genética , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/inmunología , Trastornos Mieloproliferativos/patología , Janus Quinasa 2/genética , Trombocitemia Esencial/genética , Trombocitemia Esencial/patología , Anciano de 80 o más Años , China , Adulto Joven , Calreticulina/genética , Antígeno CD11b/genética , Policitemia Vera/genética , Policitemia Vera/patología , Policitemia Vera/inmunología , Mutación , Pueblo Asiatico/genética , Pueblos del Este de Asia

RESUMEN

Despite the apparent complexity of the molecular genetic underpinnings of myeloid neoplasms, most myeloid mutational profiles can be understood within a simple framework. Somatic mutations accumulate in hematopoietic stem cells with aging and toxic insults, termed clonal hematopoiesis. These "old stem cells" mutations, predominantly in the epigenetic and RNA spliceosome pathways, act as "founding" driver mutations leading to a clonal myeloid neoplasm when sufficient in number and clone size. Subsequent mutations can create the genetic flavor of the myeloid neoplasm ("backseat" drivers) due to their enrichment in certain entities or act as progression events ("aggressive" drivers) during clonal evolution.

Asunto(s)

Síndromes Mielodisplásicos , Trastornos Mieloproliferativos , Humanos , Mutación , Síndromes Mielodisplásicos/diagnóstico , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/metabolismo , Síndromes Mielodisplásicos/patología , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/patología , Trastornos Mieloproliferativos/diagnóstico , Trastornos Mieloproliferativos/metabolismo

RESUMEN

Despite advances in understanding the genetic abnormalities in myeloproliferative neoplasms (MPN) and the development of JAK2 inhibitors, there is an urgent need to devise new treatment strategies, particularly for patients with triple-negative (TN) myelofibrosis (MF) who lack mutations in the JAK2 kinase pathway and have very poor clinical outcomes. Here we report that MYC copy number gain and increased MYC expression frequently occur in TN-MF and that MYC-directed activation of S100A9, an alarmin protein that plays pivotal roles in inflammation and innate immunity, is necessary and sufficient to drive development and progression of MF. Notably, the MYC-S100A9 circuit provokes a complex network of inflammatory signaling that involves numerous hematopoietic cell types in the bone marrow microenvironment. Accordingly, genetic ablation of S100A9 or treatment with small molecules targeting the MYC-S100A9 pathway effectively ameliorates MF phenotypes, highlighting the MYC-alarmin axis as a novel therapeutic vulnerability for this subgroup of MPNs. Significance: This study establishes that MYC expression is increased in TN-MPNs via trisomy 8, that a MYC-S100A9 circuit manifest in these cases is sufficient to provoke myelofibrosis and inflammation in diverse hematopoietic cell types in the BM niche, and that the MYC-S100A9 circuit is targetable in TN-MPNs.

Asunto(s)

Calgranulina B , Cromosomas Humanos Par 8 , Trastornos Mieloproliferativos , Proteínas Proto-Oncogénicas c-myc , Trisomía , Cromosomas Humanos Par 8/genética , Humanos , Trisomía/genética , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Calgranulina B/genética , Calgranulina B/metabolismo , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/metabolismo , Trastornos Mieloproliferativos/patología , Animales , Ratones , Mielofibrosis Primaria/genética , Mielofibrosis Primaria/patología , Mielofibrosis Primaria/metabolismo , Transducción de Señal/genética

RESUMEN

In somatic tissue differentiation, chromatin accessibility changes govern priming and precursor commitment towards cellular fates1-3. Therefore, somatic mutations are likely to alter chromatin accessibility patterns, as they disrupt differentiation topologies leading to abnormal clonal outgrowth. However, defining the impact of somatic mutations on the epigenome in human samples is challenging due to admixed mutated and wild-type cells. Here, to chart how somatic mutations disrupt epigenetic landscapes in human clonal outgrowths, we developed genotyping of targeted loci with single-cell chromatin accessibility (GoT-ChA). This high-throughput platform links genotypes to chromatin accessibility at single-cell resolution across thousands of cells within a single assay. We applied GoT-ChA to CD34+ cells from patients with myeloproliferative neoplasms with JAK2V617F-mutated haematopoiesis. Differential accessibility analysis between wild-type and JAK2V617F-mutant progenitors revealed both cell-intrinsic and cell-state-specific shifts within mutant haematopoietic precursors, including cell-intrinsic pro-inflammatory signatures in haematopoietic stem cells, and a distinct profibrotic inflammatory chromatin landscape in megakaryocytic progenitors. Integration of mitochondrial genome profiling and cell-surface protein expression measurement allowed expansion of genotyping onto DOGMA-seq through imputation, enabling single-cell capture of genotypes, chromatin accessibility, RNA expression and cell-surface protein expression. Collectively, we show that the JAK2V617F mutation leads to epigenetic rewiring in a cell-intrinsic and cell type-specific manner, influencing inflammation states and differentiation trajectories. We envision that GoT-ChA will empower broad future investigations of the critical link between somatic mutations and epigenetic alterations across clonal populations in malignant and non-malignant contexts.

Asunto(s)

Cromatina , Epigénesis Genética , Genotipo , Mutación , Análisis de la Célula Individual , Animales , Femenino , Humanos , Masculino , Ratones , Antígenos CD34/metabolismo , Diferenciación Celular/genética , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Epigénesis Genética/genética , Epigenoma/genética , Genoma Mitocondrial/genética , Técnicas de Genotipaje , Hematopoyesis/genética , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , Inflamación/genética , Inflamación/patología , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Megacariocitos/metabolismo , Megacariocitos/patología , Proteínas de la Membrana/genética , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/metabolismo , Trastornos Mieloproliferativos/patología , ARN/genética , Células Clonales/metabolismo

RESUMEN

Key studies in pre-leukemic disorders have linked increases in pro-inflammatory cytokines with accelerated phases of the disease, but the precise role of the cellular microenvironment in disease initiation and evolution remains poorly understood. In myeloproliferative neoplasms (MPNs), higher levels of specific cytokines have been previously correlated with increased disease severity (tumor necrosis factor-alpha [TNF-α], interferon gamma-induced protein-10 [IP-10 or CXCL10]) and decreased survival (interleukin 8 [IL-8]). Whereas TNF-α and IL-8 have been studied by numerous groups, there is a relative paucity of studies on IP-10 (CXCL10). Here we explore the relationship of IP-10 levels with detailed genomic and clinical data and undertake a complementary cytokine screen alongside functional assays in a wide range of MPN mouse models. Similar to patients, levels of IP-10 were increased in mice with more severe disease phenotypes (e.g., JAK2V617F/V617F TET2-/- double-mutant mice) compared with those with less severe phenotypes (e.g., CALRdel52 or JAK2+/V617F mice) and wild-type (WT) littermate controls. Although exposure to IP-10 did not directly alter proliferation or survival in single hematopoietic stem cells (HSCs) in vitro, IP-10-/- mice transplanted with disease-initiating HSCs developed an MPN phenotype more slowly, suggesting that the effect of IP-10 loss was noncell-autonomous. To explore the broader effects of IP-10 loss, we crossed IP-10-/- mice into a series of MPN mouse models and showed that its loss reduces the erythrocytosis observed in mice with the most severe phenotype. Together, these data point to a potential role for blocking IP-10 activity in the management of MPNs.

Asunto(s)

Quimiocina CXCL10 , Trastornos Mieloproliferativos , Policitemia , Animales , Humanos , Masculino , Ratones , Quimiocina CXCL10/genética , Quimiocina CXCL10/metabolismo , Modelos Animales de Enfermedad , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Ratones Noqueados , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/patología , Trastornos Mieloproliferativos/metabolismo , Policitemia/genética , Policitemia/patología , Policitemia/etiología , Femenino

RESUMEN

The classical BCR::ABL1-negative myeloproliferative neoplasms (MPN) form a group of bone marrow (BM) diseases with the potential to progress to acute myeloid leukemia or develop marrow fibrosis and subsequent BM failure. The mechanism by which BM fibrosis develops and the factors that drive stromal activation and fibrosis are not well understood. Cellular Communication Network 2 (CCN2), also known as CTGF (Connective Tissue Growth Factor), is a profibrotic matricellular protein functioning as an important driver and biomarker of fibrosis in a wide range of diseases outside the marrow. CCN2 can promote fibrosis directly or by acting as a factor downstream of TGF-ß, the latter already known to contribute to myelofibrosis in MPN.To study the possible involvement of CCN2 in BM fibrosis in MPN, we assessed CCN2 protein expression by immunohistochemistry in 75 BM biopsies (55 × MPN and 20 × normal controls). We found variable expression of CCN2 in megakaryocytes with significant overexpression in a subgroup of 7 (13%) MPN cases; 4 of them (3 × essential thrombocytemia and 1 × prefibrotic primary myelofibrosis) showed no fibrosis (MF-0), 2 (1 × post-polycythemic myelofibrosis and 1 × primary myelofibrosis) showed moderate fibrosis (MF-2), and 1 (primary myelofibrosis) severe fibrosis (MF-3). Remarkably, CCN2 expression did not correlate with fibrosis or other disease parameters such as platelet count or thrombovascular events, neither in this subgroup nor in the whole study group. This suggests that in BM of MPN patients other, CCN2-independent pathways (such as noncanonical TGF-ß signaling) may be more important for the development of fibrosis.

Asunto(s)

Factor de Crecimiento del Tejido Conjuntivo , Trastornos Mieloproliferativos , Mielofibrosis Primaria , Transducción de Señal , Factor de Crecimiento Transformador beta , Humanos , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Mielofibrosis Primaria/patología , Mielofibrosis Primaria/metabolismo , Persona de Mediana Edad , Masculino , Femenino , Anciano , Trastornos Mieloproliferativos/patología , Trastornos Mieloproliferativos/metabolismo , Adulto , Médula Ósea/patología , Médula Ósea/metabolismo , Anciano de 80 o más Años , Inmunohistoquímica , Fibrosis/patología

RESUMEN

A 6-year-old spayed female Scottish Fold cat presented with lethargy and anorexia. A complete blood cell count indicated severe anemia and mild thrombocytopenia. Examination of peripheral blood smears revealed marked changes in the erythroid lineage, including the presence of basophilic stippling and Howell-Jolly bodies as well as an increase in nucleated erythrocytes, polychromatophils, ovalocytes, and schistocytes. Additionally, some erythrocytes contained a ring or figure-eight shaped structure known as a Cabot ring, which were especially observed in polychromatophilic erythrocytes. Hemolytic diseases (Mycoplasma infection and IMHA) were diagnostically excluded, and the cat was treated through prednisolone administration, whole blood transfusion, and administration of vitamins (K2 and B12); however, the anemia progressively worsened. Cabot rings were observed until Day 22 and subsequently disappeared as the number of nucleated RBCs increased, and the erythrocyte lineage shifted to immature population. On Day 42, peripheral blood examination revealed further left shifting and appearance of many rubriblasts. The patient died at home on Day 43. Necropsy revealed neoplastic cells infiltrating the bone marrow and other organs, which were immunopositive to CD71 which is an erythroid lineage marker. In humans, Cabot rings have been observed in megaloblastic anemia, lead poisoning, myelodysplastic syndrome, and myelofibrosis; further, they are thought to be related to stressed bone marrow and dyserythropoiesis. This is the first case report of a cat with Cabot rings, which are suggestive of defects in erythroid lineage production.

Asunto(s)

Enfermedades de los Gatos , Trastornos Mieloproliferativos , Gatos , Femenino , Enfermedades de los Gatos/patología , Enfermedades de los Gatos/diagnóstico , Animales , Trastornos Mieloproliferativos/veterinaria , Trastornos Mieloproliferativos/patología , Trastornos Mieloproliferativos/complicaciones , Resultado Fatal , Eritrocitos Anormales/patología , Anemia/veterinaria , Anemia/patología , Eritrocitos/patología