BRAF is a commonly mutated oncogene in various human malignancies and a target of a new class of anti-cancer agents, BRAF-inhibitors (BRAFi). The initial enthusiasm for these agents, based on the early successes in the management of metastatic melanoma, is now challenged by the mounting evidence of intrinsic BRAFi-insensitivity in many BRAF-mutated tumors, by the scarcity of complete responses, and by the inevitable emergence of drug resistance in initially responsive cases. These setbacks put an emphasis on discovering the means to increase the efficacy of BRAFi and to prevent or overcome BRAFi-resistance. We explored the role of p21-activated kinases (PAKs), in particular PAK1, in BRAFi response. BRAFi lowered the levels of active PAK1 in treated cells. An activated form of PAK1 conferred BRAFi-resistance on otherwise sensitive cells, while genetic or pharmacologic suppression of PAK1 had a sensitizing effect. While activation of AKT1 and RAC1 proto-oncogenes increased BRAFi-tolerance, the protective effect was negated in the presence of PAK inhibitors. Furthermore, combining otherwise ineffective doses of PAK- and BRAF-inhibitors synergistically affected intrinsically BRAFi-resistant cells. Considering the high incidence of PAK1 activation in cancers, our findings suggests PAK inhibition as a strategy to augment BRAFi therapy and overcome some of the well-known resistance mechanisms.
The activation of oncogenic mitogen-activated protein kinase cascade via mutations in BRAF is often observed in human melanomas. Targeted inhibitors of BRAF (BRAFi), alone or as a part of a combination therapy, offer a significant benefit to such patients. Unfortunately, some cases are initially nonresponsive to these drugs, while others become refractory in the course of treatment, underscoring the need to understand and mitigate the underlying resistance mechanisms. We report that interference with polo-like kinase 3 (PLK3) reduces the tolerance of BRAF-mutant melanoma cells to BRAFi, while increased PLK3 expression has the opposite effect.Accordingly, PLK3 expression correlates with tolerance to BRAFi in a panel of BRAFmutant cell lines and is elevated in a subset of recurring BRAFi-resistant melanomas.In PLK3-expressing cells, R406, a kinase inhibitor whose targets include PLK3, recapitulates the sensitizing effects of genetic PLK3 inhibitors. The findings support a role for PLK3 as a predictor of BRAFi efficacy and suggest suppression of PLK3 as a way to improve the efficacy of targeted therapy. K E Y W O R D SBRAF, cobimetinib, PLK3, R406, vemurafenib
Age-associated low-grade sterile inflammation, commonly referred to as inflammaging, is a recognized hallmark of aging, which contributes to many age-related diseases. While tissue-resident macrophages are innate immune cells that secrete many types of inflammatory cytokines in response to various stimuli, it is not clear whether they have a role in driving inflammaging. Here we characterized the transcriptional changes associated with physiological aging in mouse resident macrophage populations across different tissues and sexes. Although the age-related transcriptomic signatures of resident macrophages were strikingly tissuespecific, the differentially expressed genes were collectively enriched for those with important innate immune functions such as antigen presentation, cytokine production, and cell adhesion. The brain-resident microglia had the most wide-ranging age-related alterations, with compromised expression of tissue-specific genes and relatively exaggerated responses to endotoxin stimulation. Despite the tissue-specific patterns of aging transcriptomes, components of the hedgehog (Hh) signaling pathway were decreased in aged macrophages across multiple tissues. In vivo suppression of Hh signaling in young animals increased the expression of proinflammatory cytokines, while in vitro activation of Hh signaling in old macrophages, in turn, suppressed the expression of these inflammatory cytokines. This suggests that hedgehog signaling could be a potential intervention axis for mitigating age-associated inflammation and related diseases. Overall, our data represent a resourceful catalog of tissue-specific and sex-specific transcriptomic changes in resident macrophages of peritoneum, liver, and brain, during physiological aging.
Age-associated low-grade sterile inflammation, commonly referred to as inflammaging, is a recognized hallmark of aging, which contributes to many age-related diseases. While tissue-resident macrophages are innate immune cells that secrete many types of inflammatory cytokines in response to various stimuli, it is not clear whether they have a role in driving inflammaging. Here we characterized the transcriptional changes associated with physiological aging in mouse resident macrophage populations across different tissues and sexes. Although the age-related transcriptomic signatures of resident macrophages were strikingly tissue-specific, the differentially expressed genes were collectively enriched for those with important innate immune functions such as antigen presentation, cytokine production, and cell adhesion. The brain-resident microglia had the most wide-ranging age-related alterations, with compromised expression of tissue-specific genes and relatively exaggerated responses to endotoxin stimulation. Despite the tissue-specific patterns of aging transcriptomes, components of the hedgehog (Hh) signaling pathway were decreased in aged macrophages across multiple tissues. In vivo suppression of Hh signaling in young animals increased the expression of pro-inflammatory cytokines, while in vitro activation of Hh signaling in old macrophages, in turn, suppressed the expression of these inflammatory cytokines. This suggests that hedgehog signaling could be a potential intervention axis for mitigating age-associated inflammation and related diseases. Overall, our data represent a resourceful catalog of tissue-specific and sex-specific transcriptomic changes in resident macrophages of peritoneum, liver, and brain, during physiological aging.
Hyperactivity of serine-threonine kinase AKT is one of the most common molecular abnormalities in cancer, where it contributes to poor outcomes by facilitating the growth and survival of malignant cells. Despite its well-documented anti-apoptotic effects, hyperactivity of AKT is also known to be stressful to a cell. In an attempt to better elucidate this phenomenon, we observed the signs of proteotoxic stress in cells that harbor hyperactive AKT or have lost its principal negative regulator, PTEN. The activity of HSF1 was predictably elevated under these circumstances. However, such cells proved more sensitive to various regimens of heat shock, including the conditions that were well-tolerated by syngeneic cells without AKT hyperactivity. The sensitizing effect of hyperactive AKT was also seen in HSF1-deficient cells, suggesting that the phenomenon does not require the regulation of HSF1 by this kinase. Notably, the elevated activity of AKT was accompanied by increased levels of XBP1, a key component of cell defense against proteotoxic stress. Interestingly, the cells harboring hyperactive AKT were also more dependent on XBP1 for their growth. Our observations suggest that proteotoxic stress conferred by hyperactive AKT represents a targetable vulnerability, which can be exploited by either elevating the stress above the level tolerated by such cells or by eliminating the factors that enable such tolerance.
Aging is associated with changes in gene expression. Age-related changes in chromatin structure may contribute to alterations in gene expression and to aging phenotypes in human. Macrophages play essential roles in tissue homeostasis and repair. Macrophage activation by bacterial LPS leads to induction of a complex inflammatory gene program dependent on numerous transcription factor (TF) families. We previously showed that chromatin accessibility of macrophages is massively increased after LPS treatment. Similar programs may also contribute to the chronically elevated inflammatory state in aging. Therefore, it is important to understand how macrophages are epigenetically and functionally altered during aging. To address the age-associated differences in the transcriptome and chromatin landscape of macrophages, we have performed RNA-seq and ATAC-seq of tissue-resident macrophages in the presence or absence of LPS. Our results revealed that age effect in macrophages seems cell type-specific with the strongest effect in microglia. The majority of age-dependent TF footprints in macrophages showed reduced TF occupancy, but with either reduced or increased chromatin accessibility. We are investigating the functional relevance of the candidate TFs identified in our analysis. Our data highlight the tissue-specificity of aging features that occur in resident macrophages in vivo.
The High Mobility Group (HMG) proteins are ubiquitously present in the nuclei of all vertebrate cells. HMG proteins bind dynamically to chromatin without DNA sequence specificity, and they affect DNA-dependent functions such as replication, repair, and transcription, but only in the context of chromatin. HMGs are also suggested to play an important role in the immune system. Both HMGB1 and HMGN1 can act as alarmins. HMGN proteins also affect the response of B lymphocytes to bacterial LPS stimulation. HMGN proteins are typically enriched at regulatory sites such as enhancers and promoters, and have been shown to affect the chromatin landscape, suggesting a role in epigenetic regulatory processes. In LPS-stimulated bone marrow-derived macrophages (BMDMs), most HMG genes are downregulated in response to LPS (Oh KS et al, 2017 Immunity, PMID: 28801231. To understand the role of HMG proteins in the regulation of macrophage chromatin, we performed RNA-seq and ATAC-seq on BMDMs and two tissue-resident macrophage populations from mice lacking both HMGN1 and HMGN2. We identified several hundred differentially expressed genes in BMDMs with a large gene cluster showing female-specific downregulation in the double knockout. In peritoneal macrophages, the largest differentially expressed gene cluster was from the female-specific upregulated genes. The most dramatic transcriptome-wide changes were observed in microglia. Interestingly, MHC genes were commonly upregulated in macrophages from mutant mice. Together with alterations in chromatin, this raises the possibility that HMGNs maintain functional macrophage enhancers with specificity for the resident tissue.
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