Histone chaperone HIRA, promyelocytic leukemia protein, and p62/SQSTM1 coordinate to regulate inflammation during cell senescence.

Cellular senescence, a stress-induced stable proliferation arrest associated with an inflammatory senescence-associated secretory phenotype (SASP), is a cause of aging. In senescent cells, cytoplasmic chromatin fragments (CCFs) activate SASP via the anti-viral cGAS/STING pathway. Promyelocytic leukemia (PML) protein organizes PML nuclear bodies (NBs), which are also involved in senescence and anti-viral immunity. The HIRA histone H3.3 chaperone localizes to PML NBs in senescent cells. Here, we show that HIRA and PML are essential for SASP expression, tightly linked to HIRA's localization to PML NBs. Inactivation of HIRA does not directly block expression of nuclear factor κB (NF-κB) target genes. Instead, an H3.3-independent HIRA function activates SASP through a CCF-cGAS-STING-TBK1-NF-κB pathway. HIRA physically interacts with p62/SQSTM1, an autophagy regulator and negative SASP regulator. HIRA and p62 co-localize in PML NBs, linked to their antagonistic regulation of SASP, with PML NBs controlling their spatial configuration. These results outline a role for HIRA and PML in the regulation of SASP.

Inflammation and Fibrosis in Progeria: Organ-Specific Responses in an HGPS Mouse Model.

Hutchinson-Gilford Progeria Syndrome (HGPS) is an extremely rare genetic disorder that causes accelerated aging, due to a pathogenic variant in the LMNA gene. This pathogenic results in the production of progerin, a defective protein that disrupts the nuclear lamina's structure. In our study, we conducted a histopathological analysis of various organs in the LmnaG609G/G609G mouse model, which is commonly used to study HGPS. The objective of this study was to show that progerin accumulation drives systemic but organ-specific tissue damage and accelerated aging phenotypes. Our findings show significant fibrosis, inflammation, and dysfunction in multiple organ systems, including the skin, cardiovascular system, muscles, lungs, liver, kidneys, spleen, thymus, and heart. Specifically, we observed severe vascular fibrosis, reduced muscle regeneration, lung tissue remodeling, depletion of fat in the liver, and disruptions in immune structures. These results underscore the systemic nature of the disease and suggest that chronic inflammation and fibrosis play crucial roles in the accelerated aging seen in HGPS. Additionally, our study highlights that each organ responds differently to the toxic effects of progerin, indicating that there are distinct mechanisms of tissue-specific damage.

Combining gene expression analysis of gastric cancer cell lines and tumor specimens to identify biomarkers for anti-HER therapies-the role of HAS2, SHB and HBEGF.

BACKGROUND: The standard treatment for patients with advanced HER2-positive gastric cancer is a combination of the antibody trastuzumab and platin-fluoropyrimidine chemotherapy. As some patients do not respond to trastuzumab therapy or develop resistance during treatment, the search for alternative treatment options and biomarkers to predict therapy response is the focus of research. We compared the efficacy of trastuzumab and other HER-targeting drugs such as cetuximab and afatinib. We also hypothesized that treatment-dependent regulation of a gene indicates its importance in response and that it can therefore be used as a biomarker for patient stratification. METHODS: A selection of gastric cancer cell lines (Hs746T, MKN1, MKN7 and NCI-N87) was treated with EGF, cetuximab, trastuzumab or afatinib for a period of 4 or 24 h. The effects of treatment on gene expression were measured by RNA sequencing and the resulting biomarker candidates were tested in an available cohort of gastric cancer patients from the VARIANZ trial or functionally analyzed in vitro. RESULTS: After treatment of the cell lines with afatinib, the highest number of regulated genes was observed, followed by cetuximab and trastuzumab. Although trastuzumab showed only relatively small effects on gene expression, BMF, HAS2 and SHB could be identified as candidate biomarkers for response to trastuzumab. Subsequent studies confirmed HAS2 and SHB as potential predictive markers for response to trastuzumab therapy in clinical samples from the VARIANZ trial. AREG, EREG and HBEGF were identified as candidate biomarkers for treatment with afatinib and cetuximab. Functional analysis confirmed that HBEGF is a resistance factor for cetuximab. CONCLUSION: By confirming HAS2, SHB and HBEGF as biomarkers for anti-HER therapies, we provide evidence that the regulation of gene expression after treatment can be used for biomarker discovery. TRIAL REGISTRATION: Clinical specimens of the VARIANZ study (NCT02305043) were used to test biomarker candidates.

Baricitinib, a JAK-STAT Inhibitor, Reduces the Cellular Toxicity of the Farnesyltransferase Inhibitor Lonafarnib in Progeria Cells.

Hutchinson-Gilford progeria syndrome (HGPS) is an ultra-rare multisystem premature aging disorder that leads to early death (mean age of 14.7 years) due to myocardial infarction or stroke. Most cases have a de novo point mutation at position G608G within exon 11 of the LMNA gene. This mutation leads to the production of a permanently farnesylated truncated prelamin A protein called "progerin" that is toxic to the cells. Recently, farnesyltransferase inhibitor (FTI) lonafarnib has been approved by the FDA for the treatment of patients with HGPS. While lonafarnib treatment irrefutably ameliorates HGPS disease, it is however not a cure. FTI has been shown to cause several cellular side effects, including genomic instability as well as binucleated and donut-shaped nuclei. We report that, in addition to these cellular stresses, FTI caused an increased frequency of cytosolic DNA fragment formation. These extranuclear DNA fragments colocalized with cGAs and activated the cGAS-STING-STAT1 signaling axis, upregulating the expression of proinflammatory cytokines in FTI-treated human HGPS fibroblasts. Treatment with lonafarnib and baricitinib, a JAK-STAT inhibitor, not only prevented the activation of the cGAS STING-STAT1 pathway, but also improved the overall HGPS cellular homeostasis. These ameliorations included progerin levels, nuclear shape, proteostasis, cellular ATP, proliferation, and the reduction of cellular inflammation and senescence. Thus, we suggest that combining lonafarnib with baricitinib might provide an opportunity to reduce FTI cellular toxicity and ameliorate HGPS symptoms further than lonafarnib alone.

Correction to: Determining the effects of trastuzumab, cetuximab and afatinib by phosphoprotein, gene expression and phenotypic analysis in gastric cancer cell lines.

An amendment to this paper has been published and can be accessed via the original article.

Determining the effects of trastuzumab, cetuximab and afatinib by phosphoprotein, gene expression and phenotypic analysis in gastric cancer cell lines.

BACKGROUND: Gastric cancer is the fifth most frequently diagnosed cancer and the third leading cause of cancer death worldwide. The molecular mechanisms of action for anti-HER-family drugs in gastric cancer cells are incompletely understood. We compared the molecular effects of trastuzumab and the other HER-family targeting drugs cetuximab and afatinib on phosphoprotein and gene expression level to gain insights into the regulated pathways. Moreover, we intended to identify genes involved in phenotypic effects of anti-HER therapies. METHODS: A time-resolved analysis of downstream intracellular kinases following EGF, cetuximab, trastuzumab and afatinib treatment was performed by Luminex analysis in the gastric cancer cell lines Hs746T, MKN1, MKN7 and NCI-N87. The changes in gene expression after treatment of the gastric cancer cell lines with EGF, cetuximab, trastuzumab or afatinib for 4 or 24 h were analyzed by RNA sequencing. Significantly enriched pathways and gene ontology terms were identified by functional enrichment analysis. Furthermore, effects of trastuzumab and afatinib on cell motility and apoptosis were analyzed by time-lapse microscopy and western blot for cleaved caspase 3. RESULTS: The Luminex analysis of kinase activity revealed no effects of trastuzumab, while alterations of AKT1, MAPK3, MEK1 and p70S6K1 activations were observed under cetuximab and afatinib treatment. On gene expression level, cetuximab mainly affected the signaling pathways, whereas afatinib had an effect on both signaling and cell cycle pathways. In contrast, trastuzumab had little effects on gene expression. Afatinib reduced average speed in MKN1 and MKN7 cells and induced apoptosis in NCI-N87 cells. Following treatment with afatinib, a list of 14 genes that might be involved in the decrease of cell motility and a list of 44 genes that might have a potential role in induction of apoptosis was suggested. The importance of one of these genes (HBEGF) as regulator of motility was confirmed by knockdown experiments. CONCLUSIONS: Taken together, we described the different molecular effects of trastuzumab, cetuximab and afatinib on kinase activity and gene expression. The phenotypic changes following afatinib treatment were reflected by altered biological functions indicated by overrepresentation of gene ontology terms. The importance of identified genes for cell motility was validated in case of HBEGF.

Histone chaperone HIRA, Promyelocytic Leukemia (PML) protein and p62/SQSTM1 coordinate to regulate inflammation during cell senescence.

Cellular senescence, a stress-induced stable proliferation arrest associated with an inflammatory Senescence-Associated Secretory Phenotype (SASP), is a cause of aging. In senescent cells, Cytoplasmic Chromatin Fragments (CCFs) activate SASP via the anti-viral cGAS/STING pathway. PML protein organizes PML nuclear bodies (NBs), also involved in senescence and anti-viral immunity. The HIRA histone H3.3 chaperone localizes to PML NBs in senescent cells. Here, we show that HIRA and PML are essential for SASP expression, tightly linked to HIRA's localization to PML NBs. Inactivation of HIRA does not directly block expression of NF-κB target genes. Instead, an H3.3-independent HIRA function activates SASP through a CCF-cGAS-STING-TBK1-NF-κB pathway. HIRA physically interacts with p62/SQSTM1, an autophagy regulator and negative SASP regulator. HIRA and p62 co-localize in PML NBs, linked to their antagonistic regulation of SASP, with PML NBs controlling their spatial configuration. These results outline a role for HIRA and PML in regulation of SASP.

Impact of MnTBAP and Baricitinib Treatment on Hutchinson-Gilford Progeria Fibroblasts.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare premature aging disease. It is caused by a mutation in the LMNA gene, which results in a 50-amino-acid truncation of prelamin A. The resultant truncated prelamin A (progerin) lacks the cleavage site for the zinc-metallopeptidase ZMPSTE24. Progerin is permanently farnesylated, carboxymethylated, and strongly anchored to the nuclear envelope. This leads to abnormalities, such as altered nuclear shape, mitochondrial dysfunction, and inflammation. HGPS patients display symptoms of physiological aging, including atherosclerosis, alopecia, lipodystrophy, and arthritis. Currently, no cure for HGPS exists. Here we focus on a drug combination consisting of the superoxide dismutase mimetic MnTBAP and JAK1/2 inhibitor baricitinib (Bar) to restore phenotypic alterations in HGPS fibroblasts. Treating HGPS fibroblasts with the MnTBAP/Bar combination improved mitochondrial functions and sustained Bar's positive effects on reducing progerin and pro-inflammatory factor levels. Collectively, MnTBAP/Bar combination treatment ameliorates the aberrant phenotype of HGPS fibroblasts and is a potential treatment strategy for patients with HGPS.

Nuclear Pore Complexes Cluster in Dysmorphic Nuclei of Normal and Progeria Cells during Replicative Senescence.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare premature aging disease caused by a mutation in LMNA. A G608G mutation in exon 11 of LMNA is responsible for most HGPS cases, generating a truncated protein called "progerin". Progerin is permanently farnesylated and accumulates in HGPS cells, causing multiple cellular defects such as nuclear dysmorphism, a thickened lamina, loss of heterochromatin, premature senescence, and clustering of Nuclear Pore Complexes (NPC). To identify the mechanism of NPC clustering in HGPS cells, we evaluated post-mitotic NPC assembly in control and HGPS cells and found no defects. Next, we examined the occurrence of NPC clustering in control and HGPS cells during replicative senescence. We reported that NPC clustering occurs solely in the dysmorphic nuclei of control and HGPS cells. Hence, NPC clustering occurred at a higher frequency in HGPS cells compared to control cells at early passages; however, in late cultures with similar senescence index, NPCs clustering occurred at a similar rate in both control and HGPS. Our results show that progerin does not disrupt post-mitotic reassembly of NPCs. However, NPCs frequently cluster in dysmorphic nuclei with a high progerin content. Additionally, nuclear envelope defects that arise during replicative senescence cause NPC clustering in senescent cells with dysmorphic nuclei.

Inhibition of JAK-STAT Signaling with Baricitinib Reduces Inflammation and Improves Cellular Homeostasis in Progeria Cells.

Hutchinson-Gilford progeria syndrome (HGPS), a rare premature aging disorder that leads to death at an average age of 14.7 years due to myocardial infarction or stroke, is caused by mutations in the LMNA gene. Nearly 90% of HGPS cases carry the G608G mutation within exon 11 that generates a truncated prelamin A protein "progerin". Progerin accumulates in HGPS cells and induces premature senescence at the cellular and organismal levels. Children suffering from HGPS develop numerous clinical features that overlap with normal aging, including atherosclerosis, arthritis, hair loss and lipodystrophy. To determine whether an aberrant signaling pathway might underlie the development of these four diseases (atherosclerosis, arthritis, hair loss and lipodystrophy), we performed a text mining analysis of scientific literature and databases. We found a total of 17 genes associated with all four pathologies, 14 of which were linked to the JAK1/2-STAT1/3 signaling pathway. We report that the inhibition of the JAK-STAT pathway with baricitinib, a Food and Drug Administration-approved JAK1/2 inhibitor, restored cellular homeostasis, delayed senescence and decreased proinflammatory markers in HGPS cells. Our ex vivo data using human cell models indicate that the overactivation of JAK-STAT signaling mediates premature senescence and that the inhibition of this pathway could show promise for the treatment of HGPS and age-related pathologies.