Expression Of Cytoskeletal Genes Research Articles

IL-2 delivery to CD8+ T cells during infection requires MRTF/SRF-dependent gene expression and cytoskeletal dynamics

Paracrine IL-2 signalling drives the CD8 + T cell expansion and differentiation that allow protection against viral infections, but the underlying molecular events are incompletely understood. Here we show that the transcription factor SRF, a master regulator of cytoskeletal gene expression, is required for effective IL-2 signalling during L. monocytogenes infection. Acting cell-autonomously with its actin-regulated cofactors MRTF-A and MRTF-B, SRF is dispensible for initial TCR-mediated CD8+ T cell proliferation, but is required for sustained IL-2 dependent CD8+ effector T cell expansion, and persistence of memory cells. Following TCR activation, Mrtfab-null CD8+ T cells produce IL-2 normally, but homotypic clustering is impaired both in vitro and in vivo. Expression of cytoskeletal structural and regulatory genes, most notably actins, is defective in Mrtfab-null CD8+ T cells. Activation-induced cell clustering in vitro requires F-actin assembly, and Mrtfab-null cell clusters are small, contain less F-actin, and defective in IL-2 retention. Clustering of Mrtfab-null cells can be partially restored by exogenous actin expression. IL-2 mediated CD8+ T cell proliferation during infection thus depends on the control of cytoskeletal dynamics and actin gene expression by MRTF-SRF signalling.

An integrated QTL and RNA-seq analysis revealed new petal morphology loci in Brassica napus L.

BackgroundRapeseed (Brassica napus L.) is one of the most important oil crops and a wildly cultivated horticultural crop. The petals of B. napus serve to protect the reproductive organs and attract pollinators and tourists. Understanding the genetic basis of petal morphology regulation is necessary for B. napus breeding.ResultsIn the present study, the quantitative trait locus (QTL) analysis for six B. napus petal morphology parameters in a double haploid (DH) population was conducted across six microenvironments. A total of 243 QTLs and five QTL hotspots were observed, including 232 novel QTLs and three novel QTL hotspots. The spatiotemporal transcriptomic analysis of the diversiform petals was also conducted, which indicated that the expression of plant hormone metabolic and cytoskeletal binding protein genes was variant among diversiform petals.ConclusionsThe integration of QTL and RNA-seq analysis revealed that plant hormones (including cytokinin, auxin, and gibberellin) and cytoskeleton were key regulators of the petal morphology. Subsequently, 61 high-confidence candidate genes of petal morphology regulation were identified, including Bn.SAUR10, Bn.ARF18, Bn.KIR1, Bn.NGA2, Bn.PRF1, and Bn.VLN4. The current study provided novel QTLs and candidate genes for further breeding B. napus varieties with diversiform petals.

Circadian disruption dysregulates lung gene expression associated with inflammatory lung injury.

Circadian systems drive the expression of multiple genes in nearly all cells and coordinate cellular-, tissue-, and system-level processes that are critical to innate immunity regulation. We examined the effects of circadian rhythm disorganization, produced by light shift exposure, on innate immunity-mediated inflammatory lung responses including vascular permeability and gene expression in a C57BL/6J murine model of inflammatory lung injury. A total of 32 C57BL/6J mice were assigned to circadian phase shifting (CPS) with intratracheal phosphate-buffered saline (PBS), CPS with intratracheal lipopolysaccharide (LPS), control (normal lighting) condition with intratracheal PBS, and control condition with intratracheal LPS. Bronchoalveolar lavage (BAL) protein, cell counts, tissue immunostaining, and differentially expressed genes (DEGs) were measured in lung tissues at 2 and 10 weeks. In mice exposed to both CPS and intratracheal LPS, both BAL protein and cell counts were increased at both 2 and 10 weeks compared to mice exposed to LPS alone. Multiple DEGs were identified in CPS-LPS-exposed lung tissues compared to LPS alone and were involved in transcriptional pathways associated with circadian rhythm disruption, regulation of lung permeability, inflammation with Rap1 signaling, and regulation of actin cytoskeleton. The most dysregulated pathways included myosin light chain kinase, MAP kinase, profilin 2, fibroblast growth factor receptor, integrin b4, and p21-activated kinase. Circadian rhythm disruption results in exacerbated immune response and dysregulated expression of cytoskeletal genes involved in the regulation of epithelial and vascular barrier integrity-the mechanistic underpinnings of acute lung injury. Further studies need to explore circadian disorganization as a druggable target.

SRF transcriptionally regulates the oligodendrocyte cytoskeleton during CNS myelination

Myelination of neuronal axons is essential for nervous system development. Myelination requires dramatic cytoskeletal dynamics in oligodendrocytes, but how actin is regulated during myelination is poorly understood. We recently identified serum response factor (SRF)-a transcription factor known to regulate expression of actin and actin regulators in other cell types-as a critical driver of myelination in the aged brain. Yet, a major gap remains in understanding the mechanistic role of SRF in oligodendrocyte lineage cells. Here, we show that SRF is required cell autonomously in oligodendrocytes for myelination during development. Combining ChIP-seq with RNA-seq identifies SRF-target genes in oligodendrocyte precursor cells and oligodendrocytes that include actin and other key cytoskeletal genes. Accordingly, SRF knockout oligodendrocytes exhibit dramatically reduced actin filament levels early in differentiation, consistent with its role in actin-dependent myelin sheath initiation. Surprisingly, oligodendrocyte-restricted loss of SRF results in upregulation of gene signatures associated with aging and neurodegenerative diseases. Together, our findings identify SRF as a transcriptional regulator that controls the expression of cytoskeletal genes required in oligodendrocytes for myelination. This study identifies an essential pathway regulating oligodendrocyte biology with high relevance to brain development, aging, and disease.

Enhancement of seaweed polysaccharides (fucoidan and laminarin) on the phagocytosis of macrophages via activation of intelectin in blunt snout bream (Megalobrama amblycephala)

Fucoidan and laminarin are polymolecular compounds that extracted from seaweed. As functional polysaccharides, they possess various biological functions such as immunomodulatory, anti-inflammatory, antiviral and antioxidant bioactivities. Fucoidan and laminarin modulate host immune responses by activating immune cells (macrophages, neutrophils, T lymphocytes) and inducing the expression of cytokines (TNFα, IL-1β, IL-6, IL-8, NF-κB). Intelectin is a novel lectin that plays important roles in bacterial agglutination and bactericidal effects by recognizing the polysaccharide components on the cell surface of pathogens, which can also enhance the phagocytosis of macrophages. The present study intends to explore the immunomodulatory effects of fucoidan, laminarin and intelectin on the macrophages of Megalobrama amblycephala. The results showed that the incubation of both fucoidan and laminarin at the concentration of 100 μg/mL for 4 h could enhance the phagocytic capacity of M. amblycephala macrophages, which also showed remodeled cytoskeleton with extended pseudopodia. In addition, fucoidan and laminarin could induce the expression of M. amblycephala intelectin (MaINTL) in the primary hepatocytes and macrophages, and the recombinant MaINTL (rMaINTL) could bind with fucoidan and laminarin. Furthermore, fucoidan and laminarin induced the expression of cytoskeletal remodeling related genes in the primary M. amblycephala macrophages, such as Rac1 and WASP, which should contribute to the enhancement of seaweed polysaccharides (fucoidan and laminarin) and MaINTL on the phagocytosis of M. amblycephala macrophages. However, even though fucoidan and laminarin could induced the expression of CXCL8, while the chemotaxis of M. amblycephala macrophages on neutrophils was not affected by them. These findings suggested that fucoidan and laminarin enhanced the phagocytic capacity of M. amblycephala macrophages via activation of MaINTL and up-regulation of the expression of downstream cytoskeletal remodeling related genes Rac1 and WASP. Therefore, fucoidan and laminarin could be used as immune enhancers to improve host immunity and disease resistance in M. amblycephala, which provided novel insights into the prevention and protection of fish bacterial diseases.

A distinct transcriptome characterizes neural crest-derived cells at the migratory wavefront during enteric nervous system development.

Enteric nervous system development relies on intestinal colonization by enteric neural crest-derived cells (ENCDCs). This is driven by a population of highly migratory and proliferative ENCDCs at the wavefront, but the molecular characteristics of these cells are unknown. ENCDCs from the wavefront and the trailing region were isolated and subjected to RNA-seq. Wavefront-ENCDCs were transcriptionally distinct from trailing ENCDCs, and temporal modelling confirmed their relative immaturity. This population of ENCDCs exhibited altered expression of ECM and cytoskeletal genes, consistent with a migratory phenotype. Unlike trailing ENCDCs, the wavefront lacked expression of genes related to neuronal or glial maturation. As wavefront ENCDC genes were associated with migration and developmental immaturity, the genes that remain expressed in later progenitor populations may be particularly pertinent to understanding the maintenance of ENCDC progenitor characteristics. Dusp6 expression was specifically upregulated at the wavefront. Inhibiting DUSP6 activity prevented wavefront colonization of the hindgut, and inhibited the migratory ability of post-colonized ENCDCs from midgut and postnatal neurospheres. These effects were reversed by simultaneous inhibition of ERK signaling, indicating that DUSP6-mediated ERK inhibition is required for ENCDC migration in mouse and chick.

IAG Regulates the Expression of Cytoskeletal Protein-Encoding Genes in Shrimp Testis.

Insulin-like androgenic gland hormone (IAG) is the master regulator of sexual differentiation and testis development in male crustaceans. However, the molecular mechanism on how IAG functions during testis development is still largely unknown. Here, the transcriptional changes were analyzed in the testes of shrimp after LvIAG knockdown in Litopenaeus vannamei. Differential expression analysis identified 111 differentially expressed genes (DEGs), including 48 upregulated DEGs and 63 downregulated DEGs, in testes of shrimp after LvIAG knockdown. Gene ontology (GO) analysis showed that these DEGs were apparently enriched in cytoskeleton-related GO items. Gene function analysis showed that genes enriched in these GO items mainly encoded actin, myosin, and heat shock protein. Interestingly, these genes were all downregulated in testis after LvIAG knockdown, which was confirmed by qRT-PCR detection. Furthermore, injection of LvIAG protein that was recombinantly expressed in insect cells upregulated the expression levels of these genes. The present study revealed that shrimp IAG might function in testis development through regulating the expression of cytoskeletal protein-encoding genes, which would provide new insights into understanding the functional mechanisms of IAG on male sexual development of crustaceans.

Distinct translatome changes in specific neural populations precede electroencephalographic changes in prion-infected mice

Selective vulnerability is an enigmatic feature of neurodegenerative diseases (NDs), whereby a widely expressed protein causes lesions in specific cell types and brain regions. Using the RiboTag method in mice, translational responses of five neural subtypes to acquired prion disease (PrD) were measured. Pre-onset and disease onset timepoints were chosen based on longitudinal electroencephalography (EEG) that revealed a gradual increase in theta power between 10- and 18-weeks after prion injection, resembling a clinical feature of human PrD. At disease onset, marked by significantly increased theta power and histopathological lesions, mice had pronounced translatome changes in all five cell types despite appearing normal. Remarkably, at a pre-onset stage, prior to EEG and neuropathological changes, we found that 1) translatomes of astrocytes indicated reduced synthesis of ribosomal and mitochondrial components, 2) glutamatergic neurons showed increased expression of cytoskeletal genes, and 3) GABAergic neurons revealed reduced expression of circadian rhythm genes. These data demonstrate that early translatome responses to neurodegeneration emerge prior to conventional markers of disease and are cell type-specific. Therapeutic strategies may need to target multiple pathways in specific populations of cells, early in disease.

Cell Responses to Simulated Microgravity and Hydrodynamic Stress Can Be Distinguished by Comparative Transcriptomics

The human immune system is compromised in microgravity (MG) conditions during an orbital flight and upon return to Earth. T cells are critical for the immune response and execute their functions via actin-mediated immune cell-cell interactions that could be disturbed by MG conditions. In our study, we have applied two conventional platforms to simulate MG conditions: fast rotating clinostat (CL) and random positioning machine (RPM), followed by global T cell transcriptome analysis using RNA sequencing. Noteworthily, both selected rotational simulated MG platforms employ forced cell movement in cultural medium and expose cells to shear forces, therefore inducing certain cell response to hydrodynamic stress. We demonstrate that the T cell transcriptome profile in response to simulated MG treatment was clearly distinguishable from the T cell transcriptome response to hydrodynamic stress (HS). Gene expression profiling of genes related to or involved in actin cytoskeleton networks using RT-qPCR confirmed two sets of differentially regulated genes in the T cell response to MG or to HS. Several key genes potentially involved in T cell gravisensing (Fam163b, Dnph1, Trim34, Upk-1b) were identified. A number of candidate biomarker genes of the response to MG (VAV1, VAV2, VAV3, and NFATC2) and of the response to HS (ITGAL, ITGB1, ITGB2, RAC1, and RAC2) could be used to distinguish between these processes on the gene transcription level. Together, MG induces changes in the overall transcriptome of T cells, leading to specific shifts in the expression of cytoskeletal network genes.

Host Cytoskeleton Gene Expression Is Correlated with the Formation of Ascovirus Reproductive Viral Vesicles.

Ascoviruses are large DNA viruses that primarily infect lepidopteran larvae. They differ markedly from other plant or animal viruses by initiating replication in the nucleus, then inducing nuclear lysis followed by extensive cellular hypertrophy and subsequent cleavage of the entire enlarged cell into numerous viral vesicles. Most progeny virions are assembled in these vesicles as they circulate in the hemolymph. Here, we report transcriptome studies of host cytoskeletal genes in larvae infected with ascoviruses from 6 h to 21 days post-infection (dpi). We focused on the cabbage looper, Trichoplusia ni, infected with the Trichoplusia ni ascovirus (TnAV), along with supporting studies on the fall armyworm, Spodoptera frugiperda, infected with the Spodoptera frugiperda ascovirus (SfAV). In T. ni, many cytoskeleton genes were upregulated at 48 hours post-infection (hpi), including 29 tubulins, 21 actins, 21 dyneins, and 13 kinesins. Mitochondrial genes were upregulated as much as two-fold at 48 hpi and were expressed at levels comparable to controls in both T. ni and S. frugiperda, even after 21 dpi, when several cytoskeleton genes remained upregulated. Our studies suggest a temporal correlation between increases in the expression of certain host cytoskeletal genes and viral vesicle formation. However, these results need confirmation through functional genetic studies of proteins encoded by these genes.

The Interplay of Microtubules with Mitochondria-ER Contact Sites (MERCs) in Glioblastoma.

Gliomas are heterogeneous neoplasms, classified into grade I to IV according to their malignancy and the presence of specific histological/molecular hallmarks. The higher grade of glioma is known as glioblastoma (GB). Although progress has been made in surgical and radiation treatments, its clinical outcome is still unfavorable. The invasive properties of GB cells and glioma aggressiveness are linked to the reshaping of the cytoskeleton. Recent works suggest that the different susceptibility of GB cells to antitumor immune response is also associated with the extent and function of mitochondria–ER contact sites (MERCs). The presence of MERCs alterations could also explain the mitochondrial defects observed in GB models, including abnormalities of energy metabolism and disruption of apoptotic and calcium signaling. Based on this evidence, the question arises as to whether a MERCs–cytoskeleton crosstalk exists, and whether GB progression is linked to an altered cytoskeleton–MERCs interaction. To address this possibility, in this review we performed a meta-analysis to compare grade I and grade IV GB patients. From this preliminary analysis, we found that GB samples (grade IV) are characterized by altered expression of cytoskeletal and MERCs related genes. Among them, the cytoskeleton-associated protein 4 (CKAP4 or CLIMP-63) appears particularly interesting as it encodes a MERCs protein controlling the ER anchoring to microtubules (MTs). Although further in-depth analyses remain necessary, this perspective review may provide new hints to better understand GB molecular etiopathogenesis, by suggesting that cytoskeletal and MERCs alterations cooperate to exacerbate the cellular phenotype of high-grade GB and that MERCs players can be exploited as novel biomarkers/targets to enhance the current therapy for GB.

Lamina-associated polypeptide 2\u03b1 is required for intranuclear MRTF-A activity

Myocardin-related transcription factor A (MRTF-A), a coactivator of serum response factor (SRF), regulates the expression of many cytoskeletal genes in response to cytoplasmic and nuclear actin dynamics. Here we describe a novel mechanism to regulate MRTF-A activity within the nucleus by showing that lamina-associated polypeptide 2α (Lap2α), the nucleoplasmic isoform of Lap2, is a direct binding partner of MRTF-A, and required for the efficient expression of MRTF-A/SRF target genes. Mechanistically, Lap2α is not required for MRTF-A nuclear localization, unlike most other MRTF-A regulators, but is required for efficient recruitment of MRTF-A to its target genes. This regulatory step takes place prior to MRTF-A chromatin binding, because Lap2α neither interacts with, nor specifically influences active histone marks on MRTF-A/SRF target genes. Phenotypically, Lap2α is required for serum-induced cell migration, and deregulated MRTF-A activity may also contribute to muscle and proliferation phenotypes associated with loss of Lap2α. Our studies therefore add another regulatory layer to the control of MRTF-A-SRF-mediated gene expression, and broaden the role of Lap2α in transcriptional regulation.

State of Drosophila melanogaster Ovaries after a Full Cycle of Gametogenesis under Microgravity Modeling: Cellular Respiration and the Content of Cytoskeletal Proteins.

The effect of weightlessness on gametogenesis and the functional state of female germ cells are still poorly understood. We studied the ovaries of Drosophila melanogaster, the full development cycle of which (from zygote to sexually mature adults) passed under simulated microgravity by a random positioning machine. The rate of cellular respiration was studied by polarography as a parameter reflecting the functional state of mitochondria. The content of cytoskeletal proteins and histones was determined using Western blotting. The relative content of mRNA was determined using qRT-PCR. The results obtained indicated an increase in the rate of cellular respiration under simulated microgravity conditions during the full cycle of gametogenesis in Drosophila melanogaster due to complex I of the respiratory chain. In addition, an increase in the contents of actin cytoskeleton components was observed against the background of an increase in the mRNA content of the cytoskeleton’s encoding genes. Moreover, we observed an increase in the relative content of histone H3 acetylated at Lys9 and Lys27, which may explain the increase in the expression of cytoskeletal genes. In conclusion, the formation of an adaptive pattern of functioning of the Drosophila melanogaster ovaries that developed under simulated microgravity includes structural and functional changes and epigenetic regulation.

Identification of potential chemical compounds enhancing generation of enucleated cells from immortalized human erythroid cell lines

Immortalized erythroid cell lines are expected to be a promising source of ex vivo manufactured red blood cells (RBCs), however the induction of enucleation in these cell lines is inefficient at present. We utilized an imaging-based high-throughput system to identify chemical compounds that trigger enucleation of human erythroid cell lines. Among >3,300 compounds, we identified multiple histone deacetylase inhibitors (HDACi) inducing enucleated cells from the cell line, although an increase in membrane fragility of enucleated cells was observed. Gene expression profiling revealed that HDACi treatment increased the expression of cytoskeletal genes, while an erythroid-specific cell membrane protein, SPTA1, was significantly down-regulated. Restoration of SPTA1 expression using CRISPR-activation partially rescued the fragility of cells and thereby improved the enucleation efficiency. Our observations provide a potential solution for the generation of mature cells from erythroid cell lines, contributing to the future realization of the use of immortalized cell lines for transfusion therapies.

The Role of Maternal pou5f3.3/oct60 Gene in the Regulation of Initial Stages of Tissue Differentiation during Xenopus laevis Embryogenesis

The role of maternal Pou5f3.3/Oct60 factor of Xenopus laevis, a homolog of the pluripotency regulator of mammalian stem cells, the Oct4 protein, in early embryonic development was studied. It was shown that the maximum concentration of the Pou5f3.3/Oct60 protein is observed at the blastula stage, and then its degradation, stimulated by posttranslational modifications after the genome activation of the embryo, occurs. Using an approach based on Pou5f3.3/Oct60 overexpression in embryos, it was found that this protein activates expression of early pluripotency marker genes, pou5f3.2, vent2.2/2.1, klf 4/5, but inhibits expression of cytoskeletal and cell adhesion genes: actin, claudin, zyxin, ldb3. In addition, Pou5f3.3/Oct60 inhibits expression of ag1/2 and agr2 genes involved in the regulation of regenerative blastema differentiation in amputated limbs, as well as in the regulation of early brain development. Given the conservatism of early stages of development in vertebrates, the data obtained in the embryos of Xenopus laevis can be used to better understand the early development in mammals, including humans.

The Adipocyte Acquires a Fibroblast-Like Transcriptional Signature in Response to a High Fat Diet

Visceral white adipose tissue (vWAT) expands and undergoes extensive remodeling during diet-induced obesity. Much is known about the contribution of various stromal vascular cells to the remodeling process, but less is known of the changes that occur within the adipocyte as it becomes progressively dysfunctional. Here, we performed a transcriptome analysis of isolated vWAT adipocytes to assess global pathway changes occurring in response to a chronic high fat diet (HFD). The data demonstrate that the adipocyte responds to the HFD by adopting a fibroblast-like phenotype, characterized by enhanced expression of ECM, focal adhesion and cytoskeletal genes and suppression of many adipocyte programs most notably those associated with mitochondria. This study reveals that during obesity the adipocyte progressively becomes metabolically dysfunctional due to its acquisition of fibrogenic functions. We propose that mechano-responsive transcription factors such as MRTFA and SRF contribute to both upregulation of morphological genes as well as suppression of mitochondrial programs.

LOXL2 Upregulates Phosphorylation of Ezrin to Promote Cytoskeletal Reorganization and Tumor Cell Invasion.

Lysyl oxidase-like 2 (LOXL2), a copper-dependent enzyme of the lysyl oxidase family and its nonsecreted, catalytically dead spliced isoform L2Δ13, enhance cell migration and invasion, stimulate filopodia formation, modulate the expression of cytoskeletal genes, and promote tumor development and metastasis in vivo. We previously showed that LOXL2 reorganizes the actin cytoskeleton in esophageal squamous cell carcinoma (ESCC) cells, however, the underlying molecular mechanisms were not identified. Here, using interactome analysis, we identified ezrin (EZR), fascin (FSCN1), heat shock protein beta-1 (HSPB1), and tropomodulin-3 (TMOD3) as actin-binding proteins that associate with cytoplasmic LOXL2, as well as with its L2Δ13 variant. High levels of LOXL2 and L2Δ13 and their cytoskeletal partners correlated with poor clinical outcome in patients with ESCC. To better understand the significance of these interactions, we focused on the interaction of LOXL2 with ezrin. Phosphorylation of ezrin at T567 was greatly reduced following depletion of LOXL2 and was enhanced following LOXL2/L2Δ13 reexpression. Furthermore, LOXL2 depletion inhibited the ability of ezrin to promote tumor progression. These results suggest that LOXL2-induced ezrin phosphorylation, which also requires PKCα, is critical for LOXL2-induced cytoskeletal reorganization that subsequently promotes tumor cell invasion and metastasis in ESCC. In summary, we have characterized a novel molecular mechanism that mediates, in part, the protumorigenic activity of LOXL2. These findings may enable the future development of therapeutic agents targeting cytoplasmic LOXL2. SIGNIFICANCE: LOXL2 and its spliced isoform L2Δ13 promote cytoskeletal reorganization and invasion of esophageal cancer cells by interacting with cytoplasmic actin-binding proteins such as ezrin.

Temporal changes in transcriptome profile provide insights of White Spot Syndrome Virus infection in Litopenaeus vannamei

Shrimp aquaculture is severely affected by WSSV. Despite an increasing effort to understand host/virus interaction by characterizing changes in gene expression (GE) following WSSV infection, the majority of published studies have focussed on a single time-point, providing limited insight on the development of host-pathogen interaction over the infection cycle. Using RNA-seq, we contrasted GE in gills of Litopenaeus vannamei at 1.5, 18 and 56 hours-post-infection (hpi), between WSSV-challenged and control shrimps. Time course analysis revealed 5097 differentially expressed genes: 63 DEGs were viral genes and their expression in WSSV group either peaked at 18 hpi (and decreased at 56 hpi) or increased linearly up to 56 hpi, suggesting a different role played by these genes during the course of infection. The remaining DEGs showed that WSSV altered the expression of metabolic, immune, apoptotic and cytoskeletal genes and was able to inhibit NF-κB and JAK/STAT pathways. Interestingly, GE changes were not consistent through the course of infection but were dynamic with time, suggesting the complexity of host-pathogen interaction. These data offer novel insights into the cellular functions that are affected during the course of infection and ultimately provide a valuable resource towards our understanding of the host-pathogen dynamics and its variation with time.

Abstract 3033: Uncovering adaptive mechanisms of resistance to targeted therapy in brain metastasis

Abstract Background A subset of lung adenocarcinoma (LUAD) can be effectively treated with EGFR tyrosine kinase inhibitors (TKIs). However, the incidence of brain metastasis increases in patients that relapse after front-line treatment, underscoring the central nervous system (CNS) as a unique sanctuary site for persistent disease. We sought to perform an integrated examination of the cellular and molecular causes of resistance to targeted therapies in brain metastasis. Results The efficacy of osimertinib, a brain penetrant third generation TKI, was studied in mice using two different EGFR mutant LUAD models. In the H1975 (EGFR L858R/T790M) model of second-line osimertinib treatment, subcutaneous tumors achieved complete response rates while H1975 brain metastases continued to grow despite strong drug penetrance into the CNS. Similarly, in the PC9 BrM4 (EGFR ex19del) model of first-line osimertinib treatment, most responding animals developed osimertinib resistance preferentially in the brain following significant regression of multi-organ metastatic lesions. Importantly, tumor cells isolated from progressing brain metastases did not exhibit resistance in vitro. However, these cells exhibited significantly enhanced resistant capacity in comparison to controls when serially transplanted into the brain demonstrating both that this resistant phenotype is selected for and that exposure to the brain metastatic niche is a requirement for drug tolerance in vivo. We utilized our recently optimized transcriptomic approach, referred to as Brain Metastasis Xenograft-RNA Sequencing (BMX-seq), to comprehensively distinguish the transcriptome of tumor versus stroma in intact drug resistant brain lesions. Accordingly, our analysis reveals that the stroma of drug resistant brain metastasis is characterized by activation of proinflammatory pathways implying that resistant cells induce and/or co-opt stromal inflammatory responses for their own benefit. Reciprocally, we identified stromal induced alterations in the expression of cytoskeletal and interferon response genes in drug resistant tumor cells. Interestingly, most of these genes are induced by the brain tumor microenvironment (TME) independently of drug treatment suggesting that the brain metastatic niche can precondition tumor cells for ensuing drug resistance. Conclusions Though advances have been made in the brain penetrating abilities of targeted therapies, acquired resistance in this unique metastatic niche still develops. Our results suggest that specific interactions between the tumor cells and stromal cells are required for the manifestation of this adaptive resistant phenotype. Furthermore, transcriptomic profiling has revealed potential regulators and molecular drivers that mechanistically explain how the brain TME preconditions metastatic cells for resistance. Citation Format: Sally J. Adua, Minghui Zhao, Paul Smith, Darren A. Cross, Don X. Nguyen. Uncovering adaptive mechanisms of resistance to targeted therapy in brain metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3033.

Nucleoskeletal regulation of transcription: Actin on MRTF.

Myocardin-related transcription factor A (MRTF-A) and serum response factor (SRF) form an essential transcriptional complex that regulates the expression of many cytoskeletal genes in response to dynamic changes in the actin cytoskeleton. The nucleoskeleton, a “dynamic network of networks,” consists of numerous proteins that contribute to nuclear shape and to its various functions, including gene expression. In this review, we will discuss recent work that has identified many nucleoskeletal proteins, such as nuclear lamina and lamina-associated proteins, nuclear actin, and the linker of the cytoskeleton and nucleoskeleton complex as important regulators of MRTF-A/SRF transcriptional activity, especially in the context of mechanical control of transcription.Impact statementRegulation of gene expression is a fundamental cellular process that ensures the appropriate response of a cell to its surroundings. Alongside biochemical signals, mechanical cues, such as substrate rigidity, have been recognized as key regulators of gene expression. Nucleoskeletal components play an important role in mechanoresponsive transcription, particularly in controlling the activity of MRTF-A/SRF transcription factors. This ensures that the cell can balance the internal and external mechanical forces by fine-tuning the expression of cytoskeletal genes.