Clonal haematopoiesis and risk of chronic liver disease.

Chronic liver disease is a major public health burden worldwide1. Although different aetiologies and mechanisms of liver injury exist, progression of chronic liver disease follows a common pathway of liver inflammation, injury and fibrosis2. Here we examined the association between clonal haematopoiesis of indeterminate potential (CHIP) and chronic liver disease in 214,563 individuals from 4 independent cohorts with whole-exome sequencing data (Framingham Heart Study, Atherosclerosis Risk in Communities Study, UK Biobank and Mass General Brigham Biobank). CHIP was associated with an increased risk of prevalent and incident chronic liver disease (odds ratio = 2.01, 95% confidence interval (95% CI) [1.46, 2.79]; P < 0.001). Individuals with CHIP were more likely to demonstrate liver inflammation and fibrosis detectable by magnetic resonance imaging compared to those without CHIP (odds ratio = 1.74, 95% CI [1.16, 2.60]; P = 0.007). To assess potential causality, Mendelian randomization analyses showed that genetic predisposition to CHIP was associated with a greater risk of chronic liver disease (odds ratio = 2.37, 95% CI [1.57, 3.6]; P < 0.001). In a dietary model of non-alcoholic steatohepatitis, mice transplanted with Tet2-deficient haematopoietic cells demonstrated more severe liver inflammation and fibrosis. These effects were mediated by the NLRP3 inflammasome and increased levels of expression of downstream inflammatory cytokines in Tet2-deficient macrophages. In summary, clonal haematopoiesis is associated with an elevated risk of liver inflammation and chronic liver disease progression through an aberrant inflammatory response.

Clonal Hematopoiesis of Indeterminate Potential With Loss of Tet2 Enhances Risk for Atrial Fibrillation Through Nlrp3 Inflammasome Activation.

BACKGROUND: Clonal hematopoiesis of indeterminate potential (CHIP), a common age-associated phenomenon, associates with increased risk of both hematological malignancy and cardiovascular disease. Although CHIP is known to increase the risk of myocardial infarction and heart failure, the influence of CHIP in cardiac arrhythmias, such as atrial fibrillation (AF), is less explored. METHODS: CHIP prevalence was determined in the UK Biobank, and incident AF analysis was stratified by CHIP status and clone size using Cox proportional hazard models. Lethally irradiated mice were transplanted with hematopoietic-specific loss of Tet2, hematopoietic-specific loss of Tet2 and Nlrp3, or wild-type control and fed a Western diet, compounded with or without NLRP3 (NLR [NACHT, LRR {leucine rich repeat}] family pyrin domain containing protein 3) inhibitor, NP3-361, for 6 to 9 weeks. Mice underwent in vivo invasive electrophysiology studies and ex vivo optical mapping. Cardiomyocytes from Ldlr-/- mice with hematopoietic-specific loss of Tet2 or wild-type control and fed a Western diet were isolated to evaluate calcium signaling dynamics and analysis. Cocultures of pluripotent stem cell-derived atrial cardiomyocytes were incubated with Tet2-deficient bone marrow-derived macrophages, wild-type control, or cytokines IL-1ß (interleukin 1ß) or IL-6 (interleukin 6). RESULTS: Analysis of the UK Biobank showed individuals with CHIP, in particular TET2 CHIP, have increased incident AF. Hematopoietic-specific inactivation of Tet2 increases AF propensity in atherogenic and nonatherogenic mouse models and is associated with increased Nlrp3 expression and CaMKII (Ca2+/calmodulin-dependent protein kinase II) activation, with AF susceptibility prevented by inactivation of Nlrp3. Cardiomyocytes isolated from Ldlr-/- mice with hematopoietic inactivation of Tet2 and fed a Western diet have impaired calcium release from the sarcoplasmic reticulum into the cytosol, contributing to atrial arrhythmogenesis. Abnormal sarcoplasmic reticulum calcium release was recapitulated in cocultures of cardiomyocytes with the addition of Tet2-deficient macrophages or cytokines IL-1ß or IL-6. CONCLUSIONS: We identified a modest association between CHIP, particularly TET2 CHIP, and incident AF in the UK Biobank population. In a mouse model of AF resulting from hematopoietic-specific inactivation of Tet2, we propose altered calcium handling as an arrhythmogenic mechanism, dependent on Nlrp3 inflammasome activation. Our data are in keeping with previous studies of CHIP in cardiovascular disease, and further studies into the therapeutic potential of NLRP3 inhibition for individuals with TET2 CHIP may be warranted.

TET2-mutant clonal hematopoiesis and risk of gout.

Gout is a common inflammatory arthritis caused by precipitation of monosodium urate (MSU) crystals in individuals with hyperuricemia. Acute flares are accompanied by secretion of proinflammatory cytokines, including interleukin-1ß (IL-1ß). Clonal hematopoiesis of indeterminate potential (CHIP) is an age-related condition predisposing to hematologic cancers and cardiovascular disease. CHIP is associated with elevated IL-1ß, thus we investigated CHIP as a risk factor for gout. To test the clinical association between CHIP and gout, we analyzed whole exome sequencing data from 177 824 individuals in the MGB Biobank (MGBB) and UK Biobank (UKB). In both cohorts, the frequency of gout was higher among individuals with CHIP than without CHIP (MGBB, CHIP with variant allele fraction [VAF] ≥2%: odds ratio [OR], 1.69; 95% CI, 1.09-2.61; P = .0189; UKB, CHIP with VAF ≥10%: OR, 1.25; 95% CI, 1.05-1.50; P = .0133). Moreover, individuals with CHIP and a VAF ≥10% had an increased risk of incident gout (UKB: hazard ratio [HR], 1.28; 95% CI, 1.06-1.55; P = .0107). In murine models of gout pathogenesis, animals with Tet2 knockout hematopoietic cells had exaggerated IL-1ß secretion and paw edema upon administration of MSU crystals. Tet2 knockout macrophages elaborated higher levels of IL-1ß in response to MSU crystals in vitro, which was ameliorated through genetic and pharmacologic Nlrp3 inflammasome inhibition. These studies show that TET2-mutant CHIP is associated with an increased risk of gout in humans and that MSU crystals lead to elevated IL-1ß levels in Tet2 knockout murine models. We identify CHIP as an amplifier of NLRP3-dependent inflammatory responses to MSU crystals in patients with gout.

Mechanical checkpoint regulates monocyte differentiation in fibrotic niches.

Myelofibrosis is a progressive bone marrow malignancy associated with monocytosis, and is believed to promote the pathological remodelling of the extracellular matrix. Here we show that the mechanical properties of myelofibrosis, namely the liquid-to-solid properties (viscoelasticity) of the bone marrow, contribute to aberrant differentiation of monocytes. Human monocytes cultured in stiff, elastic hydrogels show proinflammatory polarization and differentiation towards dendritic cells, as opposed to those cultured in a viscoelastic matrix. This mechanically induced cell differentiation is blocked by inhibiting a myeloid-specific isoform of phosphoinositide 3-kinase, PI3K-γ. We further show that murine bone marrow with myelofibrosis has a significantly increased stiffness and unveil a positive correlation between myelofibrosis grading and viscoelasticity. Treatment with a PI3K-γ inhibitor in vivo reduced frequencies of monocyte and dendritic cell populations in murine bone marrow with myelofibrosis. Moreover, transcriptional changes driven by viscoelasticity are consistent with transcriptional profiles of myeloid cells in other human fibrotic diseases. These results demonstrate that a fibrotic bone marrow niche can physically promote a proinflammatory microenvironment.

Hypoxia-inducible factors and the response to hypoxic stress.

Oxygen (O(2)) is an essential nutrient that serves as a key substrate in cellular metabolism and bioenergetics. In a variety of physiological and pathological states, organisms encounter insufficient O(2) availability, or hypoxia. In order to cope with this stress, evolutionarily conserved responses are engaged. In mammals, the primary transcriptional response to hypoxic stress is mediated by the hypoxia-inducible factors (HIFs). While canonically regulated by prolyl hydroxylase domain-containing enzymes (PHDs), the HIFα subunits are intricately responsive to numerous other factors, including factor-inhibiting HIF1α (FIH1), sirtuins, and metabolites. These transcription factors function in normal tissue homeostasis and impinge on critical aspects of disease progression and recovery. Insights from basic HIF biology are being translated into pharmaceuticals targeting the HIF pathway.

Alternate PAX3-FOXO1 oncogenic fusion in biphenotypic sinonasal sarcoma.

Biphenotypic sinonasal sarcoma (SNS) is a low grade spindle cell sarcoma that affects middle-aged adults, in which the PAX3-MAML3 chimeric transcription factor induces an aberrant dual myogenic and neuroectodermal phenotype. We report an alternate PAX3-FOXO1 oncogenic fusion in SNS, confirming the crucial role of PAX3 in SNS oncogenesis. The presence of PAX3-FOXO1 in SNS and alveolar rhabdomyosarcoma suggests that these two entities are genetically similar lesions arising from distinct progenitor cell pools. This finding has important implications for the molecular diagnosis of SNS and alveolar rhabdomyosarcoma, and underscores the critical contribution of the cell of origin to the phenotype induced by oncogenic transcription factor reprogramming.

Clonal haematopoiesis and dysregulation of the immune system.

Age-related diseases are frequently linked to pathological immune dysfunction, including excessive inflammation, autoreactivity and immunodeficiency. Recent analyses of human genetic data have revealed that somatic mutations and mosaic chromosomal alterations in blood cells - a condition known as clonal haematopoiesis (CH) - are associated with ageing and pathological immune dysfunction. Indeed, large-scale epidemiological studies and experimental mouse models have demonstrated that CH can promote cardiovascular disease, chronic obstructive pulmonary disease, chronic liver disease, osteoporosis and gout. The genes most frequently mutated in CH, the epigenetic regulators TET2 and DNMT3A, implicate increased chemokine expression and inflammasome hyperactivation in myeloid cells as a possible mechanistic connection between CH and age-related diseases. In addition, TET2 and DNMT3A mutations in lymphoid cells have been shown to drive methylation-dependent alterations in differentiation and function. Here we review the observational and mechanistic studies describing the connection between CH and pathological immune dysfunction, the effects of CH-associated genetic alterations on the function of myeloid and lymphoid cells, and the clinical and therapeutic implications of CH as a target for immunomodulation.

Runx1-R188Q germ line mutation induces inflammation and predisposition to hematologic malignancies in mice.

Germ line mutations in the RUNX1 gene cause familial platelet disorder (FPD), an inherited disease associated with lifetime risk to hematopoietic malignancies (HM). Patients with FPD frequently show clonal expansion of premalignant cells preceding HM onset. Despite the extensive studies on the role of RUNX1 in hematopoiesis, its function in the premalignant bone marrow (BM) is not well-understood. Here, we characterized the hematopoietic progenitor compartments using a mouse strain carrying an FPD-associated mutation, Runx1R188Q. Immunophenotypic analysis showed an increase in the number of hematopoietic stem and progenitor cells (HSPCs) in the Runx1R188Q/+ mice. However, the comparison of Sca-1 and CD86 markers suggested that Sca-1 expression may result from systemic inflammation. Cytokine profiling confirmed the dysregulation of interferon-response cytokines in the BM. Furthermore, the expression of CD48, another inflammation-response protein, was also increased in Runx1R188Q/+ HSPCs. The DNA-damage response activity of Runx1R188Q/+ hematopoietic progenitor cells was defective in vitro, suggesting that Runx1R188Q may promote genomic instability. The differentiation of long-term repopulating HSCs was reduced in Runx1R188Q/+ recipient mice. Furthermore, we found that Runx1R188Q/+ HSPCs outcompete their wild-type counterparts in bidirectional repopulation assays, and that the genetic makeup of recipient mice did not significantly affect the clonal dynamics under this setting. Finally, we demonstrate that Runx1R188Q predisposes to HM in cooperation with somatic mutations found in FPDHM, using 3 mouse models. These studies establish a novel murine FPDHM model and demonstrate that germ line Runx1 mutations induce a premalignant phenotype marked by BM inflammation, selective expansion capacity, defective DNA-damage response, and predisposition to HM.

Distinction of lymphoid and myeloid clonal hematopoiesis.

Clonal hematopoiesis (CH) results from somatic genomic alterations that drive clonal expansion of blood cells. Somatic gene mutations associated with hematologic malignancies detected in hematopoietic cells of healthy individuals, referred to as CH of indeterminate potential (CHIP), have been associated with myeloid malignancies, while mosaic chromosomal alterations (mCAs) have been associated with lymphoid malignancies. Here, we analyzed CHIP in 55,383 individuals and autosomal mCAs in 420,969 individuals with no history of hematologic malignancies in the UK Biobank and Mass General Brigham Biobank. We distinguished myeloid and lymphoid somatic gene mutations, as well as myeloid and lymphoid mCAs, and found both to be associated with risk of lineage-specific hematologic malignancies. Further, we performed an integrated analysis of somatic alterations with peripheral blood count parameters to stratify the risk of incident myeloid and lymphoid malignancies. These genetic alterations can be readily detected in clinical sequencing panels and used with blood count parameters to identify individuals at high risk of developing hematologic malignancies.

Dnmt3a-mutated clonal hematopoiesis promotes osteoporosis.

Osteoporosis is caused by an imbalance of osteoclasts and osteoblasts, occurring in close proximity to hematopoietic cells in the bone marrow. Recurrent somatic mutations that lead to an expanded population of mutant blood cells is termed clonal hematopoiesis of indeterminate potential (CHIP). Analyzing exome sequencing data from the UK Biobank, we found CHIP to be associated with increased incident osteoporosis diagnoses and decreased bone mineral density. In murine models, hematopoietic-specific mutations in Dnmt3a, the most commonly mutated gene in CHIP, decreased bone mass via increased osteoclastogenesis. Dnmt3a-/- demethylation opened chromatin and altered activity of inflammatory transcription factors. Bone loss was driven by proinflammatory cytokines, including Irf3-NF-κB-mediated IL-20 expression from Dnmt3a mutant macrophages. Increased osteoclastogenesis due to the Dnmt3a mutations was ameliorated by alendronate or IL-20 neutralization. These results demonstrate a novel source of osteoporosis-inducing inflammation.

Myeloproliferative neoplasms: Diagnostic workup of the cythemic patient.

Elevated peripheral blood (PB) cell counts, such as leukocytosis, thrombocytosis, and polycythemia, are often the presenting symptom in patients with myeloproliferative neoplasms (MPN). Because cythemias are nonspecific and may reflect either a reactive or neoplastic process, diagnostic workup of these patients is complicated and requires integration of numerous diagnostic modalities. Careful morphologic evaluation of the PB smear may provide insights into the underlying cause of the abnormal counts (such as the presence of teardrop erythrocytes in myelofibrosis or granulocytic dysplasia with left shift in atypical chronic myeloid leukemia). However, these morphologic findings need to be interpreted in concert with clinical findings and other laboratory results. In recent years, there has been a wealth of new genetic data in the field of MPN and many recurrent mutations have been identified, especially in cases lacking Philadelphia chromosome. Many of these genes impact the diagnosis and/or prognosis. Although certain mutations are preferentially enriched in specific MPN types, none of these mutations are disease defining; therefore, a thorough workup should always include a bone marrow biopsy for morphologic evaluation and diagnosis. This review will describe a comprehensive approach to the diagnosis of various MPN, with an emphasis on the diagnostic and prognostic implications of recurrent mutations in MPN.

Gene expression profiling distinguishes prefibrotic from overtly fibrotic myeloproliferative neoplasms and identifies disease subsets with distinct inflammatory signatures.

The Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) share similar molecular characteristics in that they frequently harbor hotspot mutations in JAK2, CALR or MPL, leading to activated JAK/STAT signaling. However, these MPN have distinct symptoms, morphology, and natural histories, including different tendencies to progress to fibrosis. Although the role of inflammation in tissue fibrosis is well recognized, inflammatory gene expression in bone marrows involved by MPN has been understudied. We analyzed the expression of inflammatory genes by directly measuring RNA transcript abundance in bone marrow biopsies of 108 MPN patients. Unsupervised analyses identified gene expression patterns that distinguish prefibrotic (grade 1-2) MPN from overtly fibrotic (grade 2-3) MPN with high sensitivity and specificity in two independent cohorts. Furthermore, prefibrotic and overtly fibrotic MPN are separable into subsets with different activities in biological pathways linked to inflammation, including cytokines, chemokines, interferon response, and toll-like receptor signaling. In conclusion, this study demonstrates that MPN with overt fibrosis is associated with significant inflammatory gene upregulation in the bone marrow, revealing potential roles for multiple pro-inflammatory signaling networks in the development of myelofibrosis and suggesting potential therapeutic mechanisms to alleviate this process in the bone marrow microenvironment.

Clinicopathological and molecular features of SF3B1-mutated myeloproliferative neoplasms.

The introduction of next-generation sequencing has broadened the genetic landscape of myeloproliferative neoplasms (MPNs) beyond JAK2, MPL, and CALR. However, the biological role and clinical impact of most other mutations are not well defined. We interrogated 101 genes in 143 BCR-ABL1-negative MPNs in chronic phase from 2 large institutions. We detected SF3B1 mutations in 15 cases (10%) and set to investigate the clinical, morphologic, and molecular features of SF3B1 mutated (SF3B1+) MPNs in comparison to SF3B1 wild-type (SF3B1-) cases and to identify distinctive features with myelodysplastic/myeloproliferative neoplasms with ring sideroblasts (RS) and thrombocytosis, which can show partial clinical and morphological overlap with MPNs. SF3B1+ cases were enriched in primary myelofibrosis in both prefibrotic and fibrotic stage, but mutations of SF3B1 seem to occur only as a late event in the fibrotic phase of essential thrombocythemia and polycythemia vera. SF3B1+ MPNs showed borderline lower hemoglobin but no other clinical or molecular differences compared to SF3B1- MPNs. Of note, RS were present only in a subset of SF3B1+ cases (4/10) without any other feature of erythroid or granulocytic dysplasia. Our results suggest that mutations in SF3B1 are not a rare event in MPNs, especially in primary myelofibrosis and during late fibrotic stages of essential thrombocythemia and polycythemia vera, but are not associated with myelodysplastic progression. Careful examination of bone marrow and peripheral blood for morphologic dysplasia is crucial to reach the correct diagnosis and avoid a misdiagnosis of myelodysplastic/myeloproliferative neoplasms with RS and thrombocytosis, a pitfall with potential prognostic and therapeutic implications.

Clinicopathologic and genetic characterization of nonacute NPM1-mutated myeloid neoplasms.

NPM1-mutated myeloid neoplasms (NPM1 + MNs) with <20% blood or bone marrow blasts are rare and have been previously shown in limited case series to exhibit an aggressive clinical course. We assembled the largest cohort of NPM1 + MN cases to date (n = 45) and compared it with NPM1 - MN (n = 95) and NPM1 + de novo acute myeloid leukemia (AML; n = 119) patients. Compared with NPM1 - MN, NPM1 + MN were associated with younger age (P = .007), a normal karyotype (P < .0001), more frequent mutations involving DNMT3A (P = .01) and PTPN11 (P = .03), and fewer involving ASXL1 (P = .003), RUNX1 (P = .0004), and TP53 (P = .02). Mutations involving IDH1 or IDH2 (IDH1/2) (P = .007) and FLT3 (internal tandem duplication, P < .0001; noninternal tandem duplication, P = .01) were less frequent in NPM1 + MN than in NPM1 + AML. In multivariable analyses performed in patients with myelodysplastic syndrome only, total mutation count (hazard ratio [HR], 1.3; P = .05), NPM1 mutation (HR, 3.6; P = .02), TP53 mutation (HR, 5.2; P = .01), and higher International Prognostic Scoring System-R score (HR, 1.7; P = .0003) were independently associated with shorter overall survival, whereas stem cell transplant conferred a favorable effect (HR, 0.1; P < .0001). These data suggest that NPM1 + MN are biologically distinct from NPM1 - MN. Similar to NPM1 + AML, patients with NPM1-mutated myelodysplastic syndrome may benefit from more intensive therapeutic regimens.