Blood myeloid cells are known to be dysregulated in coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2. It is unknown whether the innate myeloid response differs with disease severity and ...whether markers of innate immunity discriminate high-risk patients. Thus, we performed high-dimensional flow cytometry and single-cell RNA sequencing of COVID-19 patient peripheral blood cells and detected disappearance of non-classical CD14LowCD16High monocytes, accumulation of HLA-DRLow classical monocytes (Human Leukocyte Antigen - DR isotype), and release of massive amounts of calprotectin (S100A8/S100A9) in severe cases. Immature CD10LowCD101−CXCR4+/− neutrophils with an immunosuppressive profile accumulated in the blood and lungs, suggesting emergency myelopoiesis. Finally, we show that calprotectin plasma level and a routine flow cytometry assay detecting decreased frequencies of non-classical monocytes could discriminate patients who develop a severe form of COVID-19, suggesting a predictive value that deserves prospective evaluation.
Display omitted
•Patients with severe COVID-19 accumulate HLA-DRLow monocytes and immature neutrophils in blood/lungs•Calprotectin level positively correlates with neutrophil count and disease severity•Loss of non-classical monocytes could identify high risk of severe COVID-19
An analysis of patients with severe COVID-19 reveals immature neutrophil and non-classical monocyte pools, with levels of the protein calprotectin linked to disease severity.
Chronic myelomonocytic leukemia (CMML) is a myelodysplastic syndrome/ myeloproliferative neoplasm whose diagnosis is currently based on the elevation of peripheral blood monocytes to >1 × 109/L, ...measured for ≥3 months. Diagnosis can be ambiguous; for example, with prefibrotic myelofibrosis or reactive monocytosis. We set up a multiparameter flow cytometry assay to distinguish CD14+/CD16− classical from CD14+/CD16+ intermediate and CD14low/CD16+ nonclassical monocyte subsets in peripheral blood mononucleated cells and in total blood samples. Compared with healthy donors and patients with reactive monocytosis or another hematologic malignancy, CMML patients demonstrate a characteristic increase in the fraction of CD14+/CD16− cells (cutoff value, 94.0%). The associated specificity and sensitivity values were 95.1% and 90.6% in the learning cohort (175 samples) and 94.1% and 91.9% in the validation cohort (307 samples), respectively. The accumulation of classical monocytes, which demonstrate a distinct gene expression pattern, is independent of the mutational background. Importantly, this increase disappears in patients who respond to hypomethylating agents. We conclude that an increase in the fraction of classical monocytes to >94.0% of total monocytes is a highly sensitive and specific diagnostic marker that rapidly and accurately distinguishes CMML from confounding diagnoses.
•An increase in the classical monocyte subset to >94% of total monocytes discriminates CMML from other monocytoses with high specificity.•This characteristic increase in classical monocytes disappears in CMML patients who respond to hypomethylating agents.
Genomic studies in chronic myeloid malignancies, including myeloproliferative neoplasms (MPN), myelodysplastic syndromes (MDS), and MPN/MDS, have identified common mutations in genes encoding ...signaling, epigenetic, transcription, and splicing factors. In the present study, we interrogated the clonal architecture by mutation-specific discrimination analysis of single-cell–derived colonies in 28 patients with chronic myelomonocytic leukemias (CMML), the most frequent MPN/MDS. This analysis reveals a linear acquisition of the studied mutations with limited branching through loss of heterozygosity. Serial analysis of untreated and treated samples demonstrates a dynamic architecture on which most current therapeutic approaches have limited effects. The main disease characteristics are early clonal dominance, arising at the CD34+/CD38− stage of hematopoiesis, and granulomonocytic differentiation skewing of multipotent and common myeloid progenitors. Comparison of clonal expansions of TET2 mutations in MDS, MPN, and CMML, together with functional invalidation of TET2 in sorted progenitors, suggests a causative link between early clonal dominance and skewed granulomonocytic differentiation. Altogether, early clonal dominance may distinguish CMML from other chronic myeloid neoplasms with similar gene mutations.
•Early clonal dominance may distinguish chronic myelomonocytic leukemia from other chronic myeloid neoplasms with similar gene mutations.•Early dominance of TET2-mutated cells in the hematopoietic tissue promotes myeloid differentiation skewing toward the granulomonocytic line.
Islands of CD123
cells have been commonly described in the bone marrow of patients with chronic myelomonocytic leukemia (CMML). Using a multiparameter flow cytometry assay, we detected an excess of ...CD123
mononucleated cells that are lineage-negative, CD45
, CD11c
, CD33
, HLA-DR
, BDCA-2
, BDCA-4
in the bone marrow of 32/159 (20%) patients. Conventional and electron microscopy, flow cytometry detection of cell surface markers, gene expression analyses, and the ability to synthesize interferon alpha in response to Toll-like receptor agonists identified these cells as bona fide plasmacytoid dendritic cells (pDCs). Whole-exome sequencing of sorted monocytes and pDCs identified somatic mutations in genes of the oncogenic RAS pathway in the two cell types of every patient. CD34
cells could generate high amount of pDCs in the absence of FMS-like tyrosine kinase 3-ligand (FLT3L). Finally, an excess of pDCs correlates with regulatory T cell accumulation and an increased risk of acute leukemia transformation. These results demonstrate the FLT3L-independent accumulation of clonal pDCs in the bone marrow of CMML patients with mutations affecting the RAS pathway, which is associated with a higher risk of disease progression.
Mutations of calreticulin (CALRm) define a subtype of myeloproliferative neoplasms (MPN). We studied the biological and genetic features of CALR-mutated essential thrombocythemia and myelofibrosis ...patients. In most cases, CALRm were found in granulocytes, monocytes, B and NK cells, but also in T cells. However, the type 1 CALRm spreads more easily than the type 2 CALRm in lymphoid cells. The CALRm were also associated with an early clonal dominance at the level of hematopoietic stem and progenitor cells (HSPC) with no significant increase during granulo/monocytic differentiation in most cases. Moreover, we found that half of type 2 CALRm patients harbors some homozygous progenitors. Those patients were associated with a higher clonal dominance during granulo/monocytic differentiation than patients with only heterozygous type 2 CALRm progenitors. When associated mutations were present, CALRm were the first genetic event suggesting that they are both the initiating and phenotypic event. In blood, type 1 CALRm led to a greater increased number of all types of progenitors compared with the type 2 CALRm. However, both types of CALRm induced an increase in megakaryocytic progenitors associated with a ruxolitinib-sensitive independent growth and with a mild constitutive signaling in megakaryocytes. At the transcriptional level, type 1 CALRm seems to deregulate more pathways than the type 2 CALRm in megakaryocytes. Altogether, our results show that CALRm modify both the HSPC and megakaryocyte biology with a stronger effect for type 1 than for type 2 CALRm.
Abstract
Somatic mutations in the calreticulin (
CALR
) gene are associated with approximately 30% of essential thrombocythemia (ET) and primary myelofibrosis (PMF).
CALR
mutations, including the two ...most frequent 52 bp deletion (
del52
) and 5 bp insertion (
ins5
), induce a frameshift to the same alternative reading frame generating new C-terminal tails. In patients,
del52
and
ins5
induce two phenotypically distinct myeloproliferative neoplasms (MPNs). They are equally found in ET, but
del52
is more frequent in PMF. We generated heterozygous and homozygous conditional inducible knock-in (KI) mice expressing a chimeric murine CALR del52 or ins5 with the human mutated C-terminal tail to investigate their pathogenic effects on hematopoiesis. Del52 induces greater phenotypic changes than ins5 including thrombocytosis, leukocytosis, splenomegaly, bone marrow hypocellularity, megakaryocytic lineage amplification, expansion and competitive advantage of the hematopoietic stem cell compartment. Homozygosity amplifies these features, suggesting a distinct contribution of homozygous clones to human MPNs. Moreover, homozygous
del52
KI mice display features of a penetrant myelofibrosis-like disorder with extramedullary hematopoiesis linked to splenomegaly, megakaryocyte hyperplasia and the presence of reticulin fibers. Overall, modeling
del52
and
ins5
mutations in mice successfully recapitulates the differences in phenotypes observed in patients.
Megakaryocytes are highly specialized precursor cells that produce platelets via cytoplasmic extensions called proplatelets. Proplatelet formation (PPF) requires profound changes in microtubule and ...actin organization. In this work, we demonstrated that DIAPH1 (mDia1), a mammalian homolog of Drosophila diaphanous that works as an effector of the small GTPase Rho, negatively regulates PPF by controlling the dynamics of the actin and microtubule cytoskeletons. Moreover, we showed that inhibition of both DIAPH1 and the Rho-associated protein kinase (Rock)/myosin pathway increased PPF via coordination of both cytoskeletons. We provide evidence that 2 major effectors of the Rho GTPase pathway (DIAPH1 and Rock/myosin II) are involved not only in Rho-mediated stress fibers assembly, but also in the regulation of microtubule stability and dynamics during PPF.
•DIAPH1 (mDia1) is involved in both Rho-mediated actin polymerization and microtubule assembly and stability during proplatelet formation.
Megakaryocytes are unique mammalian cells that undergo polyploidization (endomitosis) during differentiation, leading to an increase in cell size and protein production that precedes platelet ...production. Recent evidence demonstrates that endomitosis is a consequence of a late failure in cytokinesis associated with a contractile ring defect. Here we show that the non-muscle myosin IIB heavy chain (MYH10) is expressed in immature megakaryocytes and specifically localizes in the contractile ring. MYH10 downmodulation by short hairpin RNA increases polyploidization by inhibiting the return of 4N cells to 2N, but other regulators, such as of the G1/S transition, might regulate further polyploidization of the 4N cells. Conversely, re-expression of MYH10 in the megakaryocytes prevents polyploidization and the transition of 2N to 4N cells. During polyploidization, MYH10 expression is repressed by the major megakaryocyte transcription factor RUNX1. Thus, RUNX1-mediated silencing of MYH10 is required for the switch from mitosis to endomitosis, linking polyploidization with megakaryocyte differentiation.
Chimeric transcription factors are a hallmark of human leukemia, but the molecular mechanisms by which they block differentiation and promote aberrant self-renewal remain unclear. Here, we ...demonstrate that the ETO2-GLIS2 fusion oncoprotein, which is found in aggressive acute megakaryoblastic leukemia, confers megakaryocytic identity via the GLIS2 moiety while both ETO2 and GLIS2 domains are required to drive increased self-renewal properties. ETO2-GLIS2 directly binds DNA to control transcription of associated genes by upregulation of expression and interaction with the ETS-related ERG protein at enhancer elements. Importantly, specific interference with ETO2-GLIS2 oligomerization reverses the transcriptional activation at enhancers and promotes megakaryocytic differentiation, providing a relevant interface to target in this poor-prognosis pediatric leukemia.
Display omitted
•The GLIS2 moiety of ETO2-GLIS2 oncoprotein controls the megakaryocytic identity•Human AMKL oncogenes often cause GATA/ETS functional imbalance•ETO2-GLIS2 binds enhancer regions together with the ERG transcription factor•The NHR2 interface is essential for maintenance of ETO2-GLIS2-driven leukemia
Thirant et al. show that the ETO2-GLIS2 fusion protein found in acute megakaryoblastic leukemia confers megakaryocytic identity via the GLIS2 moiety, but requires both ETO2 and GLIS2 domains to drive self-renewal. Disruption of ETO2-GLIS2 oligomerization inhibits the maintenance of ETO2-GLIS2+ human AMKL blasts.
Macrothrombocytopenias are the most important subgroup of inherited thrombocytopenias. This subgroup is particularly heterogeneous because the affected genes are involved in various functions such as ...cell signaling, cytoskeleton organization, and gene expression. Herein we describe the clinical and hematological features of a consanguineous family with a severe autosomal recessive macrothrombocytopenia associated with a thrombocytopathy inducing a bleeding tendency in the homozygous mutated patients. Platelet activation and cytoskeleton reorganization were impaired in these homozygous patients. Exome sequencing identified a c.222C>G mutation (missense p.74Ile>Met) in PRKACG, a gene encoding the γ-catalytic subunit of the cyclic adenosine monophosphate-dependent protein kinase, the mutated allele cosegregating with the macrothrombocytopenia. We demonstrate that the p.74Ile>Met PRKACG mutation is associated with a marked defect in proplatelet formation and a low level in filamin A in megakaryocytes (MKs). The defect in proplatelet formation was rescued in vitro by lentiviral vector-mediated overexpression of wild-type PRKACG in patient MKs. We thus conclude that PRKACG is a new central actor in platelet biogenesis and a new gene involved in inherited thrombocytopenia with giant platelets associated with a thrombocytopathy.
•We identify a new type of autosomal recessive macrothrombocytopenia associated with a mutation in PRKACG, coding the PKA catalytic subunit.•The homozygous PRKACG mutation leads to a deep defect in proplatelet formation that was restored by the overexpression of wild-type PRKACG.
PDF
