The obligatory intracellular bacterium Chlamydophila psittaci is the causative agent of psittacosis in birds and humans. The capability of this zoonotic pathogen to develop a persistent phase is likely to play a role in chronicity of infections, as well as in failure of antibiotic therapy and immunoprophylaxis. To elucidate three different in vitro models for transition of C. psittaci to persistence (iron depletion, penicillin G treatment, and gamma interferon [IFN-␥] exposure), a set of 27 genes was examined by mRNA expression analysis using quantitative real-time PCR. While the phenotypical characteristics were the same as in other chlamydiae, i.e., aberrant morphology of reticulate bodies, loss of cultivability, and rescue of infectivity upon removal of inducers, the transcriptional response of C. psittaci to persistence-inducing factors included several new and distinctive features. Consistent downregulation of membrane proteins, chlamydial sigma factors, cell division protein, and reticulate body-elementary body differentiation proteins from 24 h postinfection onward proved to be a general feature of C. psittaci persistence. However, other genes displayed considerable variations in response patterns from one model to another, which suggests that there is no persistence model per se. In contrast to results for Chlamydia trachomatis, late shutdown of essential genes in C. psittaci was more comprehensive with IFN-␥-induced persistence, which is probably due to the absence of a functional tryptophan synthesis operon.
The preferentially expressed antigen of melanoma (PRAME) is expressed at high levels in large fractions of human malignancies, e.g., acute myeloid leukemia. Therefore, PRAME is an important marker for diagnosis of various malignant diseases and a relevant parameter for monitoring minimal residual disease. It is supposed to be involved in tumorigenic processes. Because of these important aspects we investigated its transcriptional regulation in detail. Most relevant was a detailed DNA methylation analysis of the PRAME 5' region by genomic sequencing in correlation with PRAME expression in various human patient samples and cell lines. In combination with DNA-truncation/transfection experiments with respect to DNA methylation, we show that changes in the methylation pattern in defined parts of the regulatory regions of PRAME are sufficient for its upregulation in cells usually not expressing the gene.
Acute Myeloid Leukemia (AML) is driven by cell populations with stem cell-like characteristics, so called leukemia stem cells (LSC). The transcription factor Meis1 is one of the critical regulators of LSC and is capable to rapidly induce AML in murine models in the context of Hox gene overexpression. Despite sophisticated studies identifying Hox- and Meis1-regulated genes, the knowledge about their impact on intracellular signaling pathways and its functional consequences is still limited. Since Hox and Meis1 gene overexpression is often found in high risk AML and since both factors are currently considered as undruggable, we aimed to elucidate their role in regulating intracellular signaling and to investigate, if cells transformed by Hoxa9 and Meis1 are addicted to certain signaling processes.
To characterize the effect of Meis1 in the context of Hox gene overexpression on protein expression and intracellular signaling, we have performed a comprehensive (phospho)proteomic analysis and correlated it with transcriptome sequencing data. Our analysis revealed that Meis1 upregulates expression of spleen tyrosine kinase (Syk) without affecting its mRNA expression level. This was confirmed in patient-derived AML cells. By global analysis of microRNA expression and subsequent functional analyses, we could identify the downregulation of miR-146a, which turned out to be PU.1-dependent, as a mediator for this post-transcriptional upregulation of SYK in Hoxa9/Meis1 overexpressing cells. To further investigate, if an activation of Syk signaling can mimic Meis1 in inducing leukemia in our murine AML transplantation model, we overexpressed Syk in Hoxa9-transformed myeloid progenitors and found that this resulted in an acceleration in leukemia development comparable to the acceleration observed upon combined Hoxa9/Meis1 overexpression. We also found that Syk overexpression resulted in an increased expression of Meis1 and an induction of Meis1-dependent gene expression signatures. Notably, Hoxa9/Meis1-transformed cells also exhibited a remarkable sensitivity to Syk inhibition and SYK knockdown in vitro and in vivo, while Hoxa9-transformed cells did not.
In summary, we identified a previously unknown signaling loop between Meis1 overexpression and Syk signaling involving miR-146a as a regulator of SYK expression. Hence, we believe that Syk inhibition by small molecules might be a potential therapeutic option for AMLs particularly in the context of Hox/Meis1-overexpression.
Disclosures
Berg: Astellas: Other: Travel Funding; Alexion: Other: Travel Funding; Celgene: Other: Travel Funding.
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