Presence of mutant p53 increases stem cell frequency and is associated with reduced binding to classic TP53 binding sites in cell lines and primary AMLs

With an overall 5%-10% incidence rate in acute myeloid leukemia (AML), the occurrence of TP53 mutations is low compared with that in solid tumors. However, when focusing on high-risk groups including secondary AML (sAML) and therapy-related AMLs, the frequency of mutations reaches up to 35%. Mutations may include loss of heterozygosity (LOH) or deletion of the 17p allele, but are mostly missense substitutions that are located in the DNA-binding domain. Despite elaborate research on the effects of TP53 mutations in solid tumors, in hematological malignancies, the effects of TP53 mutations versus loss of TP53 remain unclear and under debate. Here, we compared the cellular effects of a TP53 mutant and loss of TP53 in human hematopoietic stem and progenitor cells (HSPCs). We found that when expressing TP53 mutant or loss of TP53 using siRNA, CD34+/CD38- cells have a significantly enhanced replating potential, which could not be demonstrated for the CD34+/CD38+ population. Using RNA-sequencing analysis, we found a loss of expression of p53 target genes in cells with TP53 knockdown. In contrast, an increased expression of a large number of genes was observed when expressing TP53 mutant, resulting in an increase in expression of genes involved in megakaryocytic differentiation, plasma membrane binding, and extracellular structure organization. When binding of p53 wild type and p53 mutant was compared in cell lines, we found that mutant p53 binds to a large number of binding sites genomewide, contrary to wild-type p53, for which binding is restricted to genes with a p53 binding motif. These findings were verified in primary AMLs with and without mutated TP53. In conclusion, in our models, we identified overlapping effects of TP53 mutant and loss of TP53 on in vitro stem cell properties but distinct effects on DNA binding and gene expression

Immune Responses to RHAMM in Patients with Acute Myeloid Leukemia after Chemotherapy and Allogeneic Stem Cell Transplantation

Leukemic blasts overexpress immunogenic antigens, so-called leukemia-associated antigens like the receptor for hyaluronan acid-mediated motility (RHAMM). Persistent RHAMM expression and decreasing CD8+ T-cell responses to RHAMM in the framework of allogeneic stem cell transplantation or chemotherapy alone might indicate the immune escape of leukemia cells. In the present study, we analyzed the expression of RHAMM in 48 patients suffering from acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Furthermore, we correlated transcripts with the clinical course of the disease before and after treatment. Real-time quantitative reverse transcriptase polymerase chain reaction was performed from RNA of peripheral blood mononuclear cells. T cell responses against RHAMM were assessed by tetramer staining (flow cytometry) and enzyme-linked immunospot (ELISPOT) assays. Results were correlated with the clinical outcome of patients. The results of the present study showed that almost 60% of the patients were RHAMM positive; specific T-cells recognizing RHAMM could be detected, but they were nonfunctional in terms of interferon gamma or granzyme B release as demonstrated by ELISPOT assays. Immunotherapies like peptide vaccination or adoptive transfer of RHAMM-specific T cells might improve the immune response and the outcome of AML/MDS patients

Chronic Graft versus Host Disease but not the Intensity of Conditioning has Impact on Survival after Allogeneic Hematopoietic Stem Cell Transplantation for Advanced Hematological Diseasess

Background: Allogeneic hematopoietic stem cell transplantation (alloHSCT) is often performed in cases of advanced hematological diseases, but because of the associated mortality and a high risk of relapse it is life prolonging only in some patients. Patients and Methods: A retrospective multi-center analysis of 401 patients was conducted to analyze the variables associated with outcome after alloHSCT in advanced hematological diseases. The Cox proportional hazards model was used to assess the independence of overall survival (OS) and disease-free survival (DFS) from prognostic factors in a multivariate model. Results: The 5-year OS and DFS were 27.3 and 21.1% respectively. Multivariate analysis showed that the underlying malignancy had a significant influence on OS and DFS (p < 0.001 and p < 0.011, respectively), whereas development of severe acute graft versus host disease (GvHD) had a negative impact on OS (p < 0.001). Development of chronic GvHD showed a trend to a better OS (p = 0.085) and DFS (p = 0.199). No impact was seen for the intensity of conditioning. Conclusion: Development of chronic GvHD but not the conditioning regimen improved the outcome after alloHSCT for advanced malignancies, underlining the importance of immunological rather than cytotoxic effects

Novel insights into the electrophysiology of murine cardiac macrophages: relevance of voltage-gated potassium channels

Aims Macrophages (MΦ), known for immunological roles, such as phagocytosis and antigen presentation, have been found to electrotonically couple to cardiomyocytes (CM) of the atrioventricular node via Cx43, affecting cardiac conduction in isolated mouse hearts. Here, we characterize passive and active electrophysiological properties of murine cardiac resident MΦ, and model their potential electrophysiological relevance for CM. Methods and results We combined classic electrophysiological approaches with 3D florescence imaging, RNA-sequencing, pharmacological interventions, and computer simulations. We used Cx3creYFP/+1 mice wherein cardiac MΦ are fluorescently labelled. FACS-purified fluorescent MΦ from mouse hearts were studied by whole-cell patch-clamp. MΦ electrophysiological properties include: membrane resistance 2.2±0.1 GΩ (all data mean±SEM), capacitance 18.3±0.1 pF, resting membrane potential −39.6±0.3 mV, and several voltage-activated, outward or inwardly rectifying potassium currents. Using ion channel blockers (barium, TEA, 4-AP, margatoxin, XEN-D0103, and DIDS), flow cytometry, immuno-staining, and RNA-sequencing, we identified Kv1.3, Kv1.5, and Kir2.1 as channels contributing to observed ion currents. MΦ displayed four patterns for outward and two for inward-rectifier potassium currents. Additionally, MΦ showed surface expression of Cx43, a prerequisite for homo- and/or heterotypic electrotonic coupling. Experimental results fed into development of an original computational model to describe cardiac MΦ electrophysiology. Computer simulations to quantitatively assess plausible effects of MΦ on electrotonically coupled CM showed that MΦ can depolarize resting CM, shorten early and prolong late action potential duration, with effects depending on coupling strength and individual MΦ electrophysiological properties, in particular resting membrane potential and presence/absence of Kir2.1. Conclusion Our results provide a first electrophysiological characterization of cardiac resident MΦ, and a computational model to quantitatively explore their relevance in the heterocellular heart. Future work will be focussed at distinguishing electrophysiological effects of MΦ–CM coupling on both cell types during steady-state and in patho-physiological remodelling, when immune cells change their phenotype, proliferate, and/or invade from external sources

P2Y₁₂-dependent activation of hematopoietic stem and progenitor cells promotes emergency hematopoiesis after myocardial infarction

Emergency hematopoiesis is the driving force of the inflammatory response to myocardial infarction (MI). Increased proliferation of hematopoietic stem and progenitor cells (LSK) after MI enhances cell production in the bone marrow (BM) and replenishes leukocyte supply for local cell recruitment to the infarct. Decoding the regulation of the inflammatory cascade after MI may provide new avenues to improve post-MI remodeling. In this study, we describe the influence of adenosine diphosphate (ADP)-dependent P2Y12-mediated signaling on emergency hematopoiesis and cardiac remodeling after MI. Permanent coronary ligation was performed to induce MI in a murine model. BM activation, inflammatory cell composition and cardiac function were assessed using global and platelet-specific gene knockout and pharmacological inhibition models for P2Y12. Complementary in vitro studies allowed for investigation of ADP-dependent effects on LSK cells. We found that ADP acts as a danger signal for the hematopoietic BM and fosters emergency hematopoiesis by promoting Akt phosphorylation and cell cycle progression. We were able to detect P2Y12 in LSK, implicating a direct effect of ADP on LSK via P2Y12 signaling. P2Y12 knockout and P2Y12 inhibitor treatment with prasugrel reduced emergency hematopoiesis and the excessive inflammatory response to MI, translating to lower numbers of downstream progeny and inflammatory cells in the blood and infarct. Ultimately, P2Y12 inhibition preserved cardiac function and reduced chronic adverse cardiac remodeling after MI. P2Y12-dependent signaling is involved in emergency hematopoiesis after MI and fuels post-ischemic inflammation, proposing a novel, non-canonical value for P2Y12 antagonists beyond inhibition of platelet-mediated atherothrombosis

Bone marrow transplantation modulates tissue macrophage phenotype and enhances cardiac recovery after subsequent acute myocardial infarction

AbstractBackgroundBone marrow transplantation (BMT) is commonly used in experimental studies to investigate the contribution of BM-derived circulating cells to different disease processes. During studies investigating the cardiac response to acute myocardial infarction (MI) induced by permanent coronary ligation in mice that had previously undergone BMT, we found that BMT itself affects the remodelling response.Methods and resultsCompared to matched naive mice, animals that had previously undergone BMT developed significantly less post-MI adverse remodelling, infarct thinning and contractile dysfunction as assessed by serial magnetic resonance imaging. Cardiac rupture in male mice was prevented. Histological analysis showed that the infarcts of mice that had undergone BMT had a significantly higher number of inflammatory cells, surviving cardiomyocytes and neovessels than control mice, as well as evidence of significant haemosiderin deposition. Flow cytometric and histological analyses demonstrated a higher number of alternatively activated (M2) macrophages in myocardium of the BMT group compared to control animals even before MI, and this increased further in the infarcts of the BMT mice after MI.ConclusionsThe process of BMT itself substantially alters tissue macrophage phenotype and the subsequent response to acute MI. An increase in alternatively activated macrophages in this setting appears to enhance cardiac recovery after MI

The Self-Assembly of a Cyclometalated Palladium Photosensitizer into Protein-Stabilized Nanorods Triggers Drug Uptake In Vitro and In Vivo

Enhanced passive diffusion is usually considered to be the primary cause of the enhanced cellular uptake of cyclometalated drugs because cyclometalation lowers the charge of a metal complex and increases its lipophilicity. However, in this work, monocationic cyclometalated palladium complexes [1]OAc (N^N^C^N) and [2]OAc (N^N^N^C) were found to self-assemble, in aqueous solutions, into soluble supramolecular nanorods, while their tetrapyridyl bicationic analogue [3](OAc)2 (N^N^N^N) dissolved as isolated molecules. These nanorods formed via metallophilic Pd···Pd interaction and π–π stacking and were stabilized in the cell medium by serum proteins, in the absence of which the nanorods precipitated. In cell cultures, these protein-stabilized self-assembled nanorods were responsible for the improved cellular uptake of the cyclometalated compounds, which took place via endocytosis (i.e., an active uptake pathway). In addition to triggering self-assembly, cyclometalation in [1]OAc also led to dramatically enhanced photodynamic properties under blue light irradiation. These combined penetration and photodynamic properties were observed in multicellular tumor spheroids and in a mice tumor xenograft, demonstrating that protein-stabilized nanoaggregation of cyclometalated drugs such as [1]OAc also allows efficient cellular uptake in 3D tumor models. Overall, serum proteins appear to be a major element in drug design because they strongly influence the size and bioavailability of supramolecular drug aggregates and hence their efficacy in vitro and in vivo.Supramolecular & Biomaterials Chemistr

Decision trees to characterise the roles of permeability and solubility on the prediction of oral absorption

Oral absorption of compounds depends on many physiological, physiochemical and formulation factors. Two important properties that govern oral absorption are in vitro permeability and solubility, which are commonly used as indicators of human intestinal absorption. Despite this, the nature and exact characteristics of the relationship between these parameters are not well understood. In this study a large dataset of human intestinal absorption was collated along with in vitro permeability, aqueous solubility, melting point, and maximum dose for the same compounds. The dataset allowed a permeability threshold to be established objectively to predict high or low intestinal absorption. Using this permeability threshold, classification decision trees incorporating a solubility-related parameter such as experimental or predicted solubility, or the melting point based absorption potential (MPbAP), along with structural molecular descriptors were developed and validated to predict oral absorption class. The decision trees were able to determine the individual roles of permeability and solubility in oral absorption process. Poorly permeable compounds with high solubility show low intestinal absorption, whereas poorly water soluble compounds with high or low permeability may have high intestinal absorption provided that they have certain molecular characteristics such as a small polar surface or specific topology. © 2014 Published by Elsevier Masson SAS