Patrice Dubreuil

Mast cell leukemia

Mast cell leukemia (MCL) is a very rare form of aggressive systemic mastocytosis accounting for < 1% of all mastocytosis. It may appear de novo or secondary to previous mastocytosis and shares more clinicopathologic aspects with systemic mastocytosis than with acute myeloid leukemia. Symptoms of mast cell activation-involvement of the liver, spleen, peritoneum, bones, and marrow-are frequent. Diagnosis is based on the presence of ≥ 20% atypical mast cells in the marrow or ≥ 10% in the blood; however, an aleukemic variant is frequently encountered in which the number of circulating mast cells is < 10%. The common phenotypic features of pathologic mast cells encountered in most forms of mastocytosis are unreliable in MCL. Unexpectedly, non-KIT D816V mutations are frequent and therefore, complete gene sequencing is necessary. Therapy usually fails and the median survival time is < 6 months. The role of combination therapies and bone marrow transplantation needs further investigation.

Lineage-specific enhancers activate self-renewal genes in macrophages and embryonic stem cells

Genetic programming for self-renewal Instead of repopulating themselves from tissue-resident stem cell pools like most types of differentiated cells, tissue macrophages maintain themselves by self-renewing. The underlying genetic programs that allow for this, however, are unknown. Soucie et al. now report that in macrophages at homeostasis, a pair of transcription factors (MafB and c-Maf) bind to and repress the enhancers of genes regulating self-renewal. When macrophages need to replenish their stocks, for example in response to injury, they transiently decrease MafB and c-Maf expression so they can self-renew. A parallel pathway also operates to control the self-renewal of embryonic stem cells. Science, this issue p. 10.1126/science.aad5510 Tissue macrophages and embryonic stem cells use similar genetic programs to self-renew. INTRODUCTION In many organs of the body, differentiated cells are frequently lost and need to be replaced as part of normal homeostatic tissue maintenance or in response to injury. In most cases, this regeneration is assured by differentiation from tissue-specific stem cells. Together with a few other cell types, tissue macrophages represent a rare exception to this pathway, as they can be maintained independently of blood stem cells by local proliferation. Under certain conditions, mature macrophages can also be expanded and maintained long term in culture without transformation or loss of differentiation status. The gene regulatory mechanisms that allow such differentiated cells to self-renew while maintaining cell type–specific identity have so far remained unknown. Self-renewing macrophages provide a rare opportunity to study this question. RATIONALE Molecularly, cell identity can be defined by the genomic positions of gene regulatory enhancer elements. The cell type–specific signatures and activity status of such elements have been characterized by the analysis of specific histone modifications and the binding of regulatory proteins. To identify the regulatory mechanisms that enable macrophage self-renewal capacity to be integrated into the overall program of epigenetic macrophage identity, we have compared the enhancer repertoires of quiescent and self-renewing macrophages. Based on our previous observations that deletion of MafB and c-Maf transcription factors results in an extended self-renewal capacity of macrophages, we further investigated how the absence of Maf transcription factors affects the enhancers of specific self-renewal genes and how these mechanisms activate macrophage self-renewal under homeostatic and challenge conditions in vivo. RESULTS Compared to quiescent macrophages, self-renewing macrophages showed no appreciable difference with respect to genome-wide enhancer positions but displayed an increase in the activation status of many enhancers that were also bound by the lineage-specifying transcription factor PU.1 in both cell types. This finding suggests that these poised macrophage-specific enhancers became active in self-renewing macrophages. We found activated enhancers to be associated with a network of genes, centered on Myc and Klf2, that were up-regulated and functionally important for self-renewal in these cells. The same genes were also required for embryonic stem (ES) cell self-renewal but were associated with a distinct, ES cell–specific set of enhancers. We observed that activated self-renewal–associated macrophage enhancers were directly repressed by MafB binding. The loss of MafB and c-Maf expression relieved this repression and led to activation of the self-renewal gene network in MafB and cMaf knockout macrophages, as well as in alveolar macrophages that express constitutively low levels of these transcription factors. In vivo single-cell analysis further revealed that, both in the steady state and in response to immune stimulation, proliferating resident macrophages could access this network by transient down-regulation of Maf transcription factors. CONCLUSION Our results demonstrate that self-renewal in macrophages involves down-regulation of MafB and cMaf, as well as concomitant activation of a self-renewal gene network shared with ES cells but controlled from cell type–specific enhancers. Macrophage enhancers associated with self-renewal genes are already present in quiescent cells and can become activated when direct repression by Maf transcription factors is relieved. Our findings provide a general molecular rationale for the compatibility of self-renewal and differentiated cell functions and may also be more generally relevant for the direct activation of self-renewal activity in other differentiated cell types with therapeutic potential. The self-renewal potential of both ES cells and differentiated macrophages is dependent on a shared network of self-renewal genes (left) that are controlled by distinct lineage-specific enhancers (right). In quiescent macrophages, the transcription factor MafB binds and represses these enhancers. The loss of MafB expression results in enhancer activation and enables macrophage self-renewal. At bottom left, red arrows indicate activation; blue bars represent inhibition. Circle size is a function of the number of times the target is affected by other regulators. MΦ, macrophage; E, enhancer; P, promoter. ILLUSTRATION: SERENA BIELLI Differentiated macrophages can self-renew in tissues and expand long term in culture, but the gene regulatory mechanisms that accomplish self-renewal in the differentiated state have remained unknown. Here we show that in mice, the transcription factors MafB and c-Maf repress a macrophage-specific enhancer repertoire associated with a gene network that controls self-renewal. Single-cell analysis revealed that, in vivo, proliferating resident macrophages can access this network by transient down-regulation of Maf transcription factors. The network also controls embryonic stem cell self-renewal but is associated with distinct embryonic stem cell–specific enhancers. This indicates that distinct lineage-specific enhancer platforms regulate a shared network of genes that control self-renewal potential in both stem and mature cells.

ASXL1 but Not TET2 Mutations Adversely Impact Overall Survival of Patients Suffering Systemic Mastocytosis with Associated Clonal Hematologic Non-Mast-Cell Diseases

Systemic mastocytosis with associated hematologic clonal non-mast cell disease (SM-AHNMD) is a rare and heterogeneous subtype of SM and few studies on this specific entity have been reported. Sixty two patients with Systemic mastocytosis with associated hematologic clonal non-mast cell disease (SM-AHNMD) were presented. Myeloid AHNMD was the most frequent (82%) cases. This subset of patients were older, had more cutaneous lesions, splenomegaly, liver enlargement, ascites; lower bone mineral density and hemoglobin levels and higher tryptase level than lymphoid AHNMD. Defects in KIT, TET2, ASXL1 and CBL were positive in 87%, 27%, 14%, and 11% of cases respectively. The overall survival of patients with SM-AHNMD was 85.2 months. Within the myeloid group, SM-MPN fared better than SM-MDS or SM-AML (pu200a=u200a0.044,). In univariate analysis, the presence of C-findings, the AHNMD subtypes (SM-MDS/CMML/AML versus SM-MPN/hypereosinophilia) (pu200a=u200a0.044), Neutropenia (pu200a=u200a0.015), high monocyte level (pu200a=u200a0.015) and the presence of ASXL1 mutation had detrimental effects on OS (pu200a=u200a0.007). In multivariate analysis and penalized Cox model, only the presence of ASXL1 mutation remained an independent prognostic factor that negatively affected OS (pu200a=u200a0.035). SM-AHNMD is heterogeneous with variable prognosis according to the type of the AHNMD. ASXL1 is mutated in a subset of myeloid AHNMD and adversely impact on OS.

SRSF2-p95 hotspot mutation is highly associated with advanced forms of mastocytosis and mutations in epigenetic regulator genes

Mastocytosis is a rare and chronic disease with phenotypes ranging from indolent to severe. Prognosis for this disease is variable and very few biomarkers to predict disease evolution or outcome are currently known. We have performed comprehensive screening in our large cohort of mastocytosis patients for mutations previously found in other myeloid diseases and that could serve as prognostic indicators. KIT, SRSF2-P95 and TET2 mutations were by far the most frequent, detected in 81%, 24% and 21% of patients, respectively. Where TET2 and SRSF2-P95 mutation both correlated with advanced disease phenotypes, SRSF2-P95 hotspot mutation was found almost exclusively in patients diagnosed with associated clonal hematologic non-mast cell disease. Statistically, TET2 and SRSF2-P95 mutations were highly associated, suggesting a mechanistic link between these two factors. Finally, analysis of both clonal and sorted cell populations from patients confirms the presence of these mutations in the mast cell component of the disease, suggests an ontological mutation hierarchy and provides evidence for the expansion of multiple clones. This highlights the prognostic potential of such approaches, if applied systematically, for delineating the roles of specific mutations in predisposing and/or driving distinct disease phenotypes.

Post-paralysis tyrosine kinase inhibition with masitinib abrogates neuroinflammation and slows disease progression in inherited amyotrophic lateral sclerosis

BackgroundIn the SOD1G93A mutant rat model of amyotrophic lateral sclerosis (ALS), neuronal death and rapid paralysis progression are associated with the emergence of activated aberrant glial cells that proliferate in the degenerating spinal cord. Whether pharmacological downregulation of such aberrant glial cells will decrease motor neuron death and prolong survival is unknown. We hypothesized that proliferation of aberrant glial cells is dependent on kinase receptor activation, and therefore, the tyrosine kinase inhibitor masitinib (AB1010) could potentially control neuroinflammation in the rat model of ALS.MethodsThe cellular effects of pharmacological inhibition of tyrosine kinases with masitinib were analyzed in cell cultures of microglia isolated from aged symptomatic SOD1G93A rats. To determine whether masitinib prevented the appearance of aberrant glial cells or modified post-paralysis survival, the drug was orally administered at 30xa0mg/kg/day starting after paralysis onset.ResultsWe found that masitinib selectively inhibited the tyrosine kinase receptor colony-stimulating factor 1R (CSF-1R) at nanomolar concentrations. In microglia cultures from symptomatic SOD1G93A spinal cords, masitinib prevented CSF-induced proliferation, cell migration, and the expression of inflammatory mediators. Oral administration of masitinib to SOD1G93A rats starting after paralysis onset decreased the number of aberrant glial cells, microgliosis, and motor neuron pathology in the degenerating spinal cord, relative to vehicle-treated rats. Masitinib treatment initiated 7xa0days after paralysis onset prolonged post-paralysis survival by 40xa0%.ConclusionsThese data show that masitinib is capable of controlling microgliosis and the emergence/expansion of aberrant glial cells, thus providing a strong biological rationale for its use to control neuroinflammation in ALS. Remarkably, masitinib significantly prolonged survival when delivered after paralysis onset, an unprecedented effect in preclinical models of ALS, and therefore appears well-suited for treating ALS.

Long-term efficacy and safety of cladribine (2-CdA) in adult patients with mastocytosis

Mastocytosis (M) is a clonal myeloid-disabling disorder for which no curative therapy is currently available. Cladribine (2-chlorodeoxyadenosine [2-CdA]) is a synthetic purine analog cytoreductive treatment, for which efficacy is mostly reported in advanced M. Here we report, with a long-term follow-up period (>10 years) efficacy and safety in 68 adult patients with M (36 [53%] had indolent M and 32 [47%] had advanced M) treated by 2-CdA (0.14 mg/kg in infusion or subcutaneously, days 1-5; repeated at 4-12 weeks until 1 to 9 courses). Median 2-CdA courses number was 3.7 (1-9). The overall response rate was 72% (complete remission [R]/major/partial R: 0%/47%/25%) and according to indolent/advanced M was 92% (major/partial R: 56%/36%) and 50% (major/partial R: 37.5%/12.5%), respectively. Clinical improvement was observed for 10 of 11 mediator release and 6 of 7 mast cell infiltration-related symptoms including urticaria pigmentosa and organomegaly (P < .02). Serum tryptase levels decreased (P = .01). Median durations of response were 3.71 (0.1-8) and 2.47 (0.5-8.6) years for indolent and aggressive M, respectively. The most frequent grade 3/4 toxicities were lymphopenia (82%), neutropenia (47%), and opportunistic infections (13%). 2-CdA appears to provide a significant efficacy with some toxicity in various M subtypes, mostly in indolent M, refractory to multiple symptomatic therapies.

Mast cells' involvement in inflammation pathways linked to depression: evidence in mastocytosis

Converging sources of evidence point to a role for inflammation in the development of depression, fatigue and cognitive dysfunction. More precisely, the tryptophan (TRP) catabolism is thought to play a major role in inflammation-induced depression. Mastocytosis is a rare disease in which chronic symptoms, including depression, are related to mast cell accumulation and activation. Our objectives were to study the correlations between neuropsychiatric features and the TRP catabolism pathway in mastocytosis in order to demonstrate mast cells potential involvement in inflammation-induced depression. Fifty-four patients with mastocytosis and a mean age of 50.1 years were enrolled in the study and compared healthy age-matched controls. Depression and stress were evaluated with the Beck Depression Inventory revised and the Perceived Stress Scale. All patients had measurements of TRP, serotonin (5-HT), kynurenine (KYN), indoleamine 2,3-dioxygenase 1 (IDO1) activity (ratio KYN/TRP), kynurenic acid (KA) and quinolinic acid (QA). Patients displayed significantly lower levels of TRP and 5-HT without hypoalbuminemia or malabsorption, higher IDO1 activity, and higher levels of KA and QA, with an imbalance towards the latter. High perceived stress and high depression scores were associated with low TRP and high IDO1 activity. In conclusion, TRP metabolism is altered in mastocytosis and correlates with perceived stress and depression, demonstrating mast cells involvement in inflammation pathways linked to depression.

Gastrointestinal manifestations in mastocytosis: A study of 83 patients

BACKGROUNDnMastocytosis is a heterogeneous disease characterized by mast cell accumulation in 1 or more organs. Gastrointestinal manifestations of systemic mastocytosis have been previously studied in small cohorts of patients, and no specific histologic description is available.nnnOBJECTIVEnWe sought to assess the clinical and pathologic features of gastrointestinal manifestations in patients with mastocytosis.nnnMETHODSnMedical history and gastrointestinal symptoms of patients with mastocytosis (n = 83) were compared with those of matched healthy subjects (n = 83) by means of patient questionnaire. Data were analyzed for epidemiologic, clinical, biological, and genetic factors associated with gastrointestinal symptoms for patients with mastocytosis. A comparative analysis of gastrointestinal histology from patients with mastocytosis (n = 23), control subjects with inflammatory bowel disease (n = 17), and healthy subjects (n = 19) was performed.nnnRESULTSnThe following gastrointestinal symptoms occurred more frequently and were more severe in patients with mastocytosis than in healthy subjects: bloating (33% vs 7.2%, P < .0001), abdominal pain (27.3% vs 4.8%, P < .0001), nausea (23% vs 8.4%, P = .02), and diarrhea (33.85% vs 1.2%, P < .0001). Patients with mastocytosis had a significantly higher incidence of personal history of duodenal ulcer (P = .02). Wild-type (WT) c-Kit was associated with diarrhea (P = .03). Specific histologic lesions were present in patients with mastocytosis but were not correlated with clinical symptoms.nnnCONCLUSIONnGastrointestinal manifestations in patients with mastocytosis are highly prevalent and often severe. Clinical symptoms do not correspond to histologic findings, are nonspecific, and can simulate irritable bowel syndrome.

FES kinases are required for oncogenic FLT3 signaling

The closely related non-receptor tyrosine kinases FEline Sarcoma (FES) and FEs Related (FER) are activated by cell surface receptors in hematopoietic cells. Despite the early description of oncogenic viral forms of fes, v-fes, and v-fps, the implication of FES and FER in human pathology is not known. We have recently shown that FES but not FER is necessary for oncogenic KIT receptor signaling. Here, we report that both FES and FER kinases are activated in primary acute myeloid leukemia (AML) blasts and in AML cell lines. FES and FER activation is dependent on FLT3 in cell lines harboring constitutively active FLT3 mutants. Moreover, both FES and FER proteins are critical for FLT3-internal tandem duplication (ITD) signaling and for cell proliferation in relevant AML cell lines. FER is required for cell cycle transitions, whereas FES seems necessary for cell survival. We concluded that FES and FER kinases mediate essential non-redundant functions downstream of FLT3-ITD.

Leukocyte telomere length in mastocytosis: Correlations with depression and perceived stress

BACKGROUNDnMastocytosisis a rare disease associated with chronic symptoms related to mast cell mediator release. Patients with mastocytosis display high level of negative emotionality such as depression and stress sensibility. Brain mast cells are mainly localized in the diencephalon, which is linked to emotion regulatory systems. Negative emotionality has been shown to be associated with telomere shortening. Taken together these observations led us to hypothesize that mast cells activity could be involved in both negative emotionality and telomere shortening in mastocytosis.nnnOBJECTIVEnTo demonstrate a possible relationship between negative emotionality in mastocytosis and leukocytes telomere length.nnnMETHODSnLeukocyte telomere length and telomerase activity were measured among mastocytosis patients and were correlated with perceived stress and depression assessed by the Beck Depression Inventory revised and the Perceived Stress Scale.nnnRESULTSnMild-severe depression scores were frequent (78.9%) as well as high perceived stress (42.11%). Telomere length was correlated to perceived stress (r=0.77; p=0.0001) but not to depression in our population. Patients displaying Wild-type KIT significantly presented higher perceived stress levels. Patients with the D816VC KIT mutation who had high perceived stress scores displayed significantly shorter telomere but not if they had high depression scores.nnnCONCLUSIONnThese findings suggest that high perceived stress in mastocytosis could accelerate the rate of leukocytes telomere shortening. Since mastocytosis is, by definition, a mast cell mediated disease; these cells could be involved in this phenomenon. Mechanistic causal relationships between these parameters need to be investigated.