Novel genetically engineered H3.3G34R model reveals cooperation with ATRX loss in upregulation of Hoxa cluster genes and promotion of neuronal lineage.

Background: Pediatric high-grade gliomas (pHGGs) are aggressive pediatric CNS tumors and an important subset are characterized by mutations in H3F3A, the gene that encodes Histone H3.3 (H3.3). Substitution of Glycine at position 34 of H3.3 with either Arginine or Valine (H3.3G34R/V), was recently described and characterized in a large cohort of pHGG samples as occurring in 5-20% of pHGGs. Attempts to study the mechanism of H3.3G34R have proven difficult due to the lack of knowledge regarding the cell-of-origin and the requirement for co-occurring mutations for model development. We sought to develop a biologically relevant animal model of pHGG to probe the downstream effects of the H3.3G34R mutation in the context of vital co-occurring mutations. Methods: We developed a genetically engineered mouse model (GEMM) that incorporates PDGF-A activation, TP53 loss and the H3.3G34R mutation both in the presence and loss of Alpha thalassemia/mental retardation syndrome X-linked (ATRX), which is commonly mutated in H3.3G34 mutant pHGGs. Results: We demonstrated that ATRX loss significantly increases tumor latency in the absence of H3.3G34R and inhibits ependymal differentiation in the presence of H3.3G34R. Transcriptomic analysis revealed that ATRX loss in the context of H3.3G34R upregulates Hoxa cluster genes. We also found that the H3.3G34R overexpression leads to enrichment of neuronal markers but only in the context of ATRX loss. Conclusions: This study proposes a mechanism in which ATRX loss is the major contributor to many key transcriptomic changes in H3.3G34R pHGGs. Accession number: GSE197988.

Prenatal overexpression of platelet-derived growth factor receptor A results in central nervous system hypomyelination.

BACKGROUND: Platelet-derived growth factor (PDGF) signaling, through the ligand PDGF-A and its receptor PDGFRA, is important for the growth and maintenance of oligodendrocyte progenitor cells (OPCs) in the central nervous system (CNS). PDGFRA signaling is downregulated prior to OPC differentiation into mature myelinating oligodendrocytes. By contrast, PDGFRA is often genetically amplified or mutated in many types of gliomas, including diffuse midline glioma (DMG) where OPCs are considered the most likely cell-of-origin. The cellular and molecular changes that occur in OPCs in response to unregulated PDGFRA expression, however, are not known. METHODS: Here, we created a conditional knock-in (KI) mouse that overexpresses wild type (WT) human PDGFRA (hPDGFRA) in prenatal Olig2-expressing progenitors, and examined in vivo cellular and molecular consequences. RESULTS: The KI mice exhibited stunted growth, ataxia, and a severe loss of myelination in the brain and spinal cord. When combined with the loss of p53, a tumor suppressor gene whose activity is decreased in DMG, the KI mice failed to develop tumors but still exhibited hypomyelination. RNA-sequencing analysis revealed decreased myelination gene signatures, indicating a defect in oligodendroglial development. Mice overexpressing PDGFRA in prenatal GFAP-expressing progenitors, which give rise to a broader lineage of cells than Olig2-progenitors, also developed myelination defects. CONCLUSION: Our results suggest that embryonic overexpression of hPDGFRA in Olig2- or GFAP-progenitors is deleterious to OPC development and leads to CNS hypomyelination.

Differential gene expression of soluble CD8+ T-cell mediated suppression of HIV replication in three older children.

Suppression of human immunodeficiency virus (HIV) replication by CD8+ T-cells (CD8 suppression) contributes to survival in adults and children <1 year. Soluble CD8 suppression can also be seen in some older children with AIDS. The factor responsible, CD8-derived antiviral factor (CAF), acts at the level of HIV RNA transcription. Differential gene expression techniques have been used to define the gene(s) mediating this phenomenon in adults. Recently, CAF has been linked to exosomes secreted by CD8+ T-cells. To compare the gene expression profiles from pediatric patients with each other, with those reported in 2 previous studies in adults and in those reportedly related to exosomes, we used differential gene expression to study three older children with HIV infection, one who did demonstrate soluble CD-8 suppression and two who did not. Eighteen differentially expressed genes were also seen in one adult study (P = 0.002, χ(2) test), and 38 such genes (P < 0.0001, χ(2) test) in a second adult study. In addition, two exosome components and some RNA's related to exosomal proteins were also differentially expressed. In children with HIV infection, we found significant differentially expressed genes that correlated to those previously reported in two studies in adults. Our data also lends some support to the recent identification of CAF with exosomes secreted by CD8+ T-cells.

New mechanisms for PRLr action in breast cancer.

Prolactin (PRL) is a pleiotrophic hormone that contributes to the growth of normal and malignant breast tissues. PRL signals through its receptor (PRLr), a transmembrane receptor that belongs to the cytokine receptor family. The mechanism of how the PRL:PRLr interaction triggers activation of signaling networks remains enigmatic. This review examines the effect of ligand binding on PRLr and the processes that initiate receptor-associated signaling. Evidence for PRLr predimerization in the absence of ligand and the actions of the prolyl isomerase cyclophilin A in ligand-induced activation of PRLr-associated Jak2 kinase are discussed. These studies reveal that ligand-induced conformational change of the PRLr complex is necessary for its function and open avenues for therapies to inhibit PRLr action in breast cancer.

Ligand-independent dimerization of the human prolactin receptor isoforms: functional implications.

Prolactin (PRL) contributes to the growth of normal and malignant breast tissues. PRL initiates signaling by engaging the PRL receptor (PRLr), a transmembrane (TM) receptor belonging to the cytokine receptor family. The accepted view has been that PRL activates the PRLr by inducing dimerization of the receptor, but recent reports show ligand-independent dimerization of other cytokine receptors. Using coimmunoprecipitation assays, we have confirmed ligand-independent dimerization of the PRLr in T47D breast cancer and HepG2 liver carcinoma cells. In addition, mammalian cells transfected with differentially epitope-tagged isoforms of the PRLr indicated that long, intermediate, and DeltaS1 PRLrs dimerized in a ligand-independent manner. To determine the domain(s) involved in PRLr ligand-independent dimerization, we generated PRLr constructs as follows: (1) the TM-ICD, which consisted of the TM domain and the intracellular domain (ICD) but lacked the extracellular domain (ECD), and (2) the ECD-TM, which consisted of the TM domain and the ECD but lacked the ICD. These constructs dimerized in a ligand-independent manner in mammalian cells, implicating a significant role for the TM domain in this process. These truncated PRLrs were functionally inert alone or in combination in cells lacking the PRLr. However, when introduced into cells containing endogenous PRLr, the ECD-TM inhibited human PRLr signaling, whereas the TM-ICD potentiated human PRLr signaling. These studies indicate that the ECD-TM and the TM-ICD are capable of modulating PRLr function. We also demonstrated an endogenous TM-ICD in T47D cells, suggesting that these findings are relevant to PRL-signaling pathways in breast cancer.

Acetaminophen-induced proliferation of estrogen-responsive breast cancer cells is associated with increases in c-myc RNA expression and NF-kappaB activity.

Studies reported here tested the hypothesis that acetaminophen stimulates proliferation of E2-responsive cells by inducing expression of E2-regulated genes. Ribonuclease protection assays compared the effects of acetaminophen and E2 on expression of selected genes (c-myc, c-fos, cyclin D1, bcl-2, bax, gadd45, mcl-1, p53, p21(CIP1/WAF1), and bcl-xL) in E2-responsive breast cancer (MCF-7) and endometrial adenocarcinoma (Ishikawa) cells as well as in E2-nonresponsive (MDA-MB-231) breast cancer cells. Acetaminophen and E2 increased c-myc RNA levels in MCF-7 cells, consistent with a mitogenic activity of these compounds in MCF-7 cells. However, the magnitude and time course of acetaminophen and E2 induction of c-myc differed. Neither acetaminophen nor E2 induced c-myc in MDA-MB-231 cells, whereas E2, but not acetaminophen, weakly induced c-myc expression in Ishikawa cells. Furthermore, in these 3 cell types, the expression patterns of the other genes differed dramatically in response to acetaminophen and to E2, indicating that acetaminophen does not activate ER as a transcription factor in the same manner as does E2. Additionally, gel shift assays demonstrated that in MCF-7 cells, acetaminophen increased NF-kappaB activity approximately 40% and did not alter AP-1 activity, whereas E2 increased AP-1 activity approximately 50% and did not increase NF-B activity. These studies indicate that acetaminophen effects on gene expression and cell proliferation depend more on cell type/context than on the presence of ER.