The definition of initiating genetic events remains up to date the main research quest in MPN disease biology. Identification of characteristic mutational clusters has shed more light on the ...complexity of various MPN genotypes, even though no specific genetic lesion or mutational order can be exclusively linked to the different MPN subtypes. Our aim is to determine the sequence of mutational clusters that orchestrate the expansion of aberrant clones in MPN with respect to the prime hematopoietic stem cell compartment.
Genotyping for 54 MPN related gene mutations (100 MPN Patient cohort with myelofibrosis) indicated high incidence of mutations in chromatin regulator genes ASXL1 (40%) and EZH2 (14%). In 11% of the patients both ASXL1 and EZH2 gene mutations were detected, which correlated with significantly lower hemoglobin levels, higher leukocyte counts, higher numbers of additional mutations and higher risk score.
In our former work we reported the identification of an expanded Lin-CD133+CD34+ neoplastic HSC population in patient peripheral blood that reproduces PMF parameters and fibrosis in vivo. Analysis of this aberrant HSC in the single cell level revealed the heterogeneity of the malignant clones and the mutational hierarchies that propagate myeloproliferation. From this analysis we report that ASXL1 and/or EZH2 mutations precede JAK2 signalling related genetic lesions (JAK2, CALR, MPL) or other epigenetic modifier mutational groups (DMT3A, TET2). Neoplastic stem cells bearing both ASXL1/EZH2 mutations exhibit higher levels of engraftment in vivo and limited erythroid colony output in vitro .
In order to determine the exact contribution of chromatin regulator mutations with regard to clonal hematopoiesis determined by changes in prime HSC dynamics we established a sorting strategy for the isolation of early LT-HSC circulating in MF patient peripheral blood. By usage of 27 lineage specific and 6 additional HSC specific markers we sorted HSC on the apex of the hierarchy with the phenotype; 27xLin-CD34+CD38-CD90+CD93+CD49f+CD133+ and all classes of early and more committed progenitors from 11 patients with ASXL1 and/or EZH2 mutations. Our data indicate that both mutations emerge in the LT-HSC fraction (10/11 patients) dominate aberrant myelopoiesis and can be also detected in mature T cells (3/11 Patients). JAK2-V617F mutation was not detected in the early HSC fraction.
The incidence of EZH2 mutations with or without the synergistic effect of ASXL1 mutations, is correlated with the expansion of morphologically aberrant erythroid progenitors in patient peripheral blood, which are found to be arrested in G0 phase of the cell cycle.
Our results suggest that ASXL1 and EZH2 mutations are the drivers of clonal hematopoiesis in MPN and occur early at the multipotent HSC compartment. EZH2 mutations in PMF represent the first genetic cluster that correlates with aberrant erythropoiesis in these patients.
No relevant conflicts of interest to declare.
Despite progress in the development of combined antiretroviral therapies (cART), HIV infection remains a significant challenge for human health. Current problems of cART include multi-drug-resistant ...virus variants, long-term toxicity and enormous treatment costs. Therefore, the identification of novel effective drugs is urgently needed.
We developed a straightforward screening approach for simultaneously evaluating the sensitivity of multiple HIV gag-pol mutants to antiviral drugs in one assay. Our technique is based on multi-colour lentiviral self-inactivating (SIN) LeGO vector technology.
We demonstrated the successful use of this approach for screening compounds against up to four HIV gag-pol variants (wild-type and three mutants) simultaneously. Importantly, the technique was adapted to Biosafety Level 1 conditions by utilising ecotropic pseudotypes. This allowed upscaling to a large-scale screening protocol exploited by pharmaceutical companies in a successful proof-of-concept experiment.
The technology developed here facilitates fast screening for anti-HIV activity of individual agents from large compound libraries. Although drugs targeting gag-pol variants were used here, our approach permits screening compounds that target several different, key cellular and viral functions of the HIV life-cycle. The modular principle of the method also allows the easy exchange of various mutations in HIV sequences. In conclusion, the methodology presented here provides a valuable new approach for the identification of novel anti-HIV drugs.
Mice lacking transcription factor interferon consensus sequence binding protein (ICSBP) develop a syndrome similar to human chronic myeloid leukemia and are immunodeficient. In order to define the ...molecular mechanisms responsible for the cellular defects of ICSBP−/− mice, we used bone marrow‐derived macrophages (BMM) to identify genes deregulated in the absence of ICSBP. Here, we report that disabled‐2 (Dab2), a signal phosphoprotein, is transcriptionally up‐regulated and accumulates in the cytoskeleton/membrane fraction of ICSBP−/− BMM. Moreover, our results revealed Dab2 as a novel IFN‐γ‐response gene. Both ICSBP and the Ets‐transcription factor PU.1 bind to the Dab2 promoter, whereby ICSBP represses PU.1‐induced Dab2 promoter transactivation in vitro. Notably, repression of Dab2 expression by ICSBP is also found in myeloid progenitors. Overexpression of Dab2 leads to accelerated cell adhesion and spreading, accompanied by enhanced actin fiber formation. Furthermore, cell adhesion induces transient Dab2 phosphorylation and its translocation to the cytoskeletal/membrane fraction. Our results identify a novel role of Dab2 as an inducer of cell adhesion and spreading, and strongly suggest that the up‐regulation of Dab2 contributes to the hematopoietic defect seen in ICSBP−/− mice.
Primary myelofibrosis (PMF) is a hematopoietic stem cell (HSC) disease, characterized by aberrant differentiation of all myeloid lineages and profound disruption of the bone marrow niche. PMF samples ...carry several mutations, but their cell origin and hierarchy in regulating the different waves of clonal and aberrant myeloproliferation from the prime HSC compartment is poorly understood. Genotyping of >2000 colonies from CD133+HSC and progenitors from PMF patients confirmed the complex genetic heterogeneity within the neoplastic population. Notably, mutations in chromatin regulators ASXL1 and/or EZH2 were identified as the first genetic lesions, preceding both JAK2-V617F and CALR mutations, and are thus drivers of clonal myelopoiesis in a PMF subset. HSC from PMF patients with double ASXL1/EZH2 mutations exhibited significantly higher engraftment in immunodeficient mice than those from patients without histone modifier mutations. EZH2 mutations correlate with aberrant erythropoiesis in PMF patients, exemplified by impaired maturation and cell cycle arrest of erythroid progenitors. These data underscore the importance of post-transcriptional modifiers of histones in neoplastic stem cells, whose clonal growth sustains aberrant myelopoiesis and expansion of pre-leukemic clones in PMF.
Among cognitively intact patients, self- and caregiver report of patient pain has been associated with worse psychological health for both the patient and caregiver. Little existing research examines ...factors associated with patient self-report of pain and caregiver report of patient pain among community-dwelling persons with dementia.
To identify patient and caregiver factors associated with self- and caregiver report of patient pain among community-dwelling persons with dementia.
Cross-sectional analysis of a longitudinal study using structured interviews with dementia patients and their family caregivers.
Urban outpatient geriatrics clinics affiliated with a university hospital. Dyads composed of dementia patients and their family caregivers were approached prior to routine clinic visits to participate in the study.
Relevant patient measurements included self-report of pain; cognitive, functional, and comorbidity assessments; a screen for depression; and demographic information. Relevant caregiver measurements included their report of patient pain and agitation, screens for depression and strain, and demographic information.
Of 115 dyads, 37 patients (32%) and 57 caregivers (53%) reported the patient to be in pain. No patient or caregiver factors were significantly associated with patient self-report of pain. In the univariate analysis, caregiver reports of patient pain were associated with the patient not being depressed (p = .036), caregiver reports of patient agitation (p = .038), higher level of education in the caregiver (p = .029), and caregiver depression (p = .019). In multivariate logistic regression analyses, caregiver depression and patient agitation remained significantly associated with caregiver report of patient pain.
In community-dwelling persons with dementia, self-report of pain was not associated with any other variables measured, suggesting that pain should be assessed through direct self-report and treated accordingly. Caregiver report of patient pain was associated with both caregiver report of patient agitation and caregiver depression. These findings suggest that clinicians may need to routinely assess patient pain, patient agitation, and caregiver depression. More research is needed to understand the relationship between these conditions.
Neuroblastoma (NB) has a low frequency of recurrent mutations compared to other cancers, which hinders the development of targeted therapies and novel risk stratification strategies. Multikinase ...inhibitors have shown potential in treating high-risk NB, but their efficacy is likely impaired by the cancer cells' ability to adapt to these drugs through the employment of alternative signaling pathways. Based on the expression of 48 growth factor-related genes in 1189 NB tumors, we have developed a model for NB patient survival prediction. This model discriminates between stage 4 NB tumors with favorable outcomes (>80% overall survival) and very poor outcomes (<10%) independently from MYCN-amplification status. Using signaling pathway analysis and gene set enrichment methods in 60 NB patients with known therapy response, we identified signaling pathways, including EPO, NGF, and HGF, upregulated in patients with no or partial response. In a therapeutic setting, we showed that among six selected growth factors, EPO, and NGF showed the most pronounced protective effects in vitro against several promising anti-NB multikinase inhibitors: imatinib, dasatinib, crizotinib, cabozantinib, and axitinib. Mechanistically kinase inhibitors potentiated NB cells to stronger ERK activation by EPO and NGF. The protective action of these growth factors strongly correlated with ERK activation and was ERK-dependent. ERK inhibitors combined with anticancer drugs, especially with dasatinib, showed a synergistic effect on NB cell death. Consideration of growth factor signaling activity benefits NB outcome prediction and tailoring therapy regimens to treat NB.
Deliberative bodies have recommended additional protections for persons with dementia included in clinical trials. This survey of experienced dementia researchers revealed that 45 to 64% considered ...that specific ones of these recommendations would increase subject protection, and 40 to 86% considered they would make research less feasible. The real tradeoff between protection and difficulty in conducting research on devastating illnesses needs to be confronted when new regulations in this area are debated.
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